Expanding the targeted protein degradation approach with small molecule chimeras directed to the 26S proteasome

Domain: biology
Published: 2026-03-28
Editorial outcome: major_revision

Abstract
Classical proteolysis targeting chimeras (Protacs) bind specific targets and E3 ubiquitin-ligases, promoting ubiquitination and
degradation of targets by the proteasome. Multiple chimeras that degrade proteins relevant in several diseases have been
developed, and the number is quickly increasing, indicating their therapeutic projection. Given the specificities of proteolytic
pathways and limitations in E3-based Protacs, alternative strategies in targeted protein degradation are pursued. Herein, using
two targets relevant in oncology as models (IMPDH2 and CERT1), we provide proof of concept for 26S-oriented compounds
based on small-molecule ligands of USP14, a 26S-associated deubiquitinase involved in substrate processing and allosteric
regulation of 26S activity. Our findings will expand the potential of targeted protein degradation.

Introduction
Targeted protein degradation (TPD) is a powerful strategy of inactivation of key players in human diseases, offering mechanistic
advantages as compared to classical inhibition1,2. The development of proteolysis targeting chimeras (Protacs) has generated
multiple compounds with drug profile1–3, with some currently in clinical trials4Despite the rise in Protacs progress, several
aspects, such as the reduced number of small molecule ligands adaptable for chimera building and the generally poor Protacs
solubility5–7 limit E3-based Protacs implementation. Proper levels and activation state of the ligase (in target tissues), productive
engagement of the substrate, and accessibility of the ligase to lysine residues on the target represent additional intrinsic
limitations that curtail the effectiveness of some E3-based Protacs5–8.The 26S proteasome is conformed of the core particle
(20S or CP), a cylindrical proteolytic complex, and the regulatory particle (19S or RP), consisting of the lid and base
subcomplexes (Fig. 1a)9–12 In the base, the hexameric ring of the AAA family of adenosine triphosphatase (ATPase) regulates
substrate unfolding and translocation. The lid and additional subunits of the base integrate substrate receptors, coordinating the
activity of proteasomal deubiquitinating enzymes and transitional states9,10,13. The removal of ubiquitin chains from substrates
by proteasomal deubiquitylating enzymes is a tightly regulated step in the 26S mechanism13–16. In this process, USP14 trims
supernumerary ubiquitin chains conjugated in substrates, and, by a sophisticated allostery, controls the steps of ubiquitin
recognition, substrate translocation initiation and ubiquitin chain recycling. Notably, USP14-26S proteasomes in exiting
conformational states dissociate trimmed ubiquitin slower, causing a delay in substrate reloading17. As a consequence, USP14
was initially identified as a delayer of proteasomal activity15,18. Additionally, chemical inhibition of USP14 with specific USP14
inhibitors (IU1 compounds)15,19,20 has an enhancement effect of proteasomal activity15 In the 26S proteasome mechanism, the
process of protein degradation can be viewed as a sequence of events in which ubiquitin tagging of substrates does not play a
strictly proteolytic role9,21,22 Although ubiquitin signaling (i) plays a pivotal role in substrate selection, recruitment, localization,
(ii) may assist 26S conformational changes and (iii) promotes the degradation of partially proteolyzed proteins retained in the
26S23–25, the event that initiates proteasomal degradation is the engagement of a flexible fragment of the substrate by the
ATPase engine; this engagement and consequent downstream steps are ubiquitin-independent9,21,22,26 Indeed, there is evidence
of ubiquitin-independent degradation of endogenous and engineered protein substrates by the 26S27–29 In these cases, the
triggers that promote degradation are adaptor factors, affinity surfaces or ligands involved in recruitment and localization of
specific substrates27–31. From this point of view, the proteasome contains multiple subunits and regions which may act as
anchoring sites with a potential in a chimera-based TPD approach10,13,17,32.

In the present work, by designing small molecule-based chimeras that directly interact with the proteasomal-associated factor
USP14 and two model targets, we have developed degraders that target proteins to the 26S in an E3-independent manner33.
This approach exploits the positioning of USP14 in the RP, and shares similarities with the recently described Rpn1-directed
chimeras34, although in that case the Rpn1 ligands were based on macrocyclic peptides. More recently, degraders using Rpn13
as recruiter have been also reported35. In our approach, we chose inosine monophosphate dehydrogenase 2 (IMPDH2), which
catalyses a rate-limiting reaction in the de novo biosynthesis of guanine nucleotides, essential for the synthesis of RNA and
DNA, and defined as a target in leukemias, lymphoma and other types of cancer35–39. Moreover, elevated levels of IMPDH2
have been correlated with cancer progression and chemoresistance37–39. Nonetheless, only one IMPDH2 degrader has been
reported, until now40. A second target for degradation was the ceramide transporter CERT1, which conveys ceramides from the
endoplasmic reticulum to the Golgi for sphingomyelin (SM) synthesis. By increasing the intracellular levels of ceramides
(proapoptotic lipids), CERT1 inhibition sensitizes cancer cells to anti-cancer drugs41,42, which sustains the interest of developing
CERT1-targeted degraders, so far unreported. In light of our data, these 26S-targeting ubiquitin-independent degraders (named
“26S-UIDs”30) appear as a versatile alternative tool for TPD-drug development (Fig. 1b).

Results
Design and synthesis of chimeras targeting the 26S proteasome regulator USP14
In order to approach a type of small-molecule chimera that binds directly to the 26S with the capacity to promote degradation of
native protein substrates, USP14 was considered a good binding candidate owing to the properties offered by this factor. First,
USP14 is localized at the base of the RP, interacting with the receptor Rpn1 and establishing contacts with ATPase subunits
(Fig. 1a)17. Second, USP14 exhibits trimming activity on ubiquitin chains attached to the substrates and adopts positions
proximal to the ATPase pore (Fig. 1a)15,17. Third, USP14 is a complex allosteric regulator of the proteasome and USP14 inhibition
enhances 26S activity15,17. Moreover, highly selective small molecule inhibitors of USP14 (IU1 and derivatives), exhibit the
capacity to increase the activity of the 26S15,19, and show affinities towards USP14 compatible with their use in a chimera
design15,19,20. Notably, IU1 compounds have not been explored as chimera ligands yet, with the exception of a recent article43.
Therefore, we hypothesized that IU1-based chimeras would interact with the proteasome via USP14 and localize targets to a
position prone to degradation, bypassing the requirement of previous ubiquitination.

Analysis of the structure of an IU1 optimized version, IU1-248 (Fig. 1c), bound to USP14 (PDB 6IIN20) showed that this ligand
has an exposed hydroxyl group (Fig. 2a) that does not seem essential for binding and which is amenable to derivatization,
constituting a convenient linker attachment point in the final 26S-UIDs. As for the targets selected (IMPDH2 and CERT1), the
structure of IMPDH2 with the bound inhibitor VX-49744 (Fig. 1c) shows that the tetrahydrofuranyl moiety at one end of the
molecule is exposed to the solvent and does not establish any significant interaction with the protein (Fig. 2b), thus seeming an
appropriate region for linker attachment.

Concerning CERT1, several structures with bound ligands exist in the PDB. A particular family of CERT1 ligands (HPA family)
that bind to the CERT1-START domain has been described45. HPA members contain a polar head that binds deeply in the
binding site cavity, and a flexible alkyl chain, which is closer to the surface of the protein (PDB codes 3H3Q, 3H3R, 3H3S and
3H3T45). Modeling one of these ligands derivatized with a PEG chain (HPA-14_PEG3, Supplementary Fig. 1) suggested that
this linker would be exposed to the bulk solvent and it would not perturb the structure of the protein or the binding of the ligand
(Fig. 2c). Therefore, these ligands were considered a valid starting point for the design of CERT1-targeting 26S-UIDs.

A preliminary in silico study was performed to determine whether the structures of potential 26S-UIDs based on the above could
participate on the formation of ternary complexes with USP14 and the respective protein targets. For that purpose, the software
workflow PROTAC-Model46 was used to challenge the structures of three chimeras (300, 330 and 329, containing different linker
lengths, Fig. 1c) against the pair USP14/IMPDH2. The results obtained showed that they were compatible with different relative
dispositions of the two proteins (Fig. 2d and Supplementary Figs. 2a-c). Furthermore, alignment of the structures of some of
these ternary complexes with the structure of USP14 in the context of 26S (PDB 5GJQ47) made evident that they could be
superposed without showing significant clashes with the closer 26S-partners (Fig. 2e).

Prompted by the above results, the synthesis of 26S-UIDs targeting IMPDH2 and CERT1 was undertaken using VX-497 and
an analog of the HPA-family (366) (Fig. 1c and Supplementary Figs. 3 to 12), respectively, as target ligands. At the other end,
IU1-248 was initially chosen as USP14 binder, although we switched to a distinct USP14 binder (i.e. 317, Fig. 1c) based on the
results obtained with the IMPDH2 chimeras (vide infra). Each pair of ligands was connected through PEG based linkers
conveniently attached to groups mostly exposed to the bulk solvent (Fig. 2a-c).

Functional characterization of 26S-UIDs targeting IMPDH2
Compounds 300, 329 and 330, which feature the USP14 inhibitor IU1-248 and only differ in the PEG linker length (Fig. 1c), were
prepared as putative IMPDH2 degraders (Supplementary Figs. 3, 5, 7 and 8).The compounds were first tested as IMPDH2
inhibitors using inosine 5'-monophosphate (IMP) as a substrate (Supplementary Fig. 13) and as USP14 inhibitors in Ub-AMC
processing assays18 (Supplementary Fig. 14). These assays showed that the chimeras containing the VX-497 and IU1-248
moieties could inhibit both IMPDH2 and USP14. A quantitative measurement of chimera binding was obtained by SPR, which
revealed KD values for 300, 329 and 330 towards USP14 and IMPDH2 in the 18-36 μM range (Supplementary Table 2 and
Supplementary Fig. 15). Together, these results show that chimeras can productively interact with USP14 and IMPDH2 in a
functional context, suggesting the feasibility of our approach.

After the above assays in cell-free systems, chimeras were tested in HeLa cells. This cell line was selected after a study reporting
that mycophenolic acid, an IMPDH2 selective inhibitor, was more effective at inhibiting cancer cell proliferation in fast-growing
cell lines, such as HeLa and Caco2, than in slow-growing cells48, suggesting that HeLa cell viability would be sensitive to
IMPDH2 degradation.

Treatments with compounds 300, 329 and 330 revealed that 300 was able to induce the degradation of IMPDH2 at low μM
concentrations, whereas 330 was less potent and 329 was inactive. However, in time-course experiments ranging from 4 h to
96 h, degradation was observed only at long times (72-96 h) after a single treatment with different chimera concentrations (Figs.
3a,b and Supplementary Fig. 16), which denotes a time-consuming process as compared to those reported for E3-based
Protacs.

Compound 354 (Fig. 1c) featuring ligand 317, a derivative of IU1c, was also prepared (Supplementary Figs. 4, 5 and 9) and
tested as USP14 ligand. In contrast to IU1-248, IU1c was reported not to inhibit USP14, but its capacity to bind USP14 was not
determined15. Similar to IU1c, 317 and its derivative 352 (Fig. 1c) were also poor USP14 inhibitors (Supplementary Fig. 14), but
the final chimera 354 showed a higher capacity to inhibit USP14 (Supplementary Fig. 14). However, quantification of the direct
interaction of 354 with USP14 by SPR revealed a KD value of 11 µM, better than the values corresponding to IU1-248-based
chimeras 300, 330 and 329 (18, 30 and 24 µM, respectively, Supplementary Table 2). Furthermore, comparison of the measured
KD of 317 (12 µM) to that of IU1-248 (13 µM), suggested that the IU1c moiety present in 317 could indeed bind to USP14 with
similar affinity as IU1-248. Therefore, 317 appeared to offer an optimized profile as ligand, exhibiting high affinity but low
inhibition towards USP14, properties that are apparently also conferred to its derivative 354. Computational modeling suggested
that compounds IU1c and 317 can bind to USP14 at the same site as IU1-248, but with an orientation which would pose a lower
steric impediment to ubiquitin binding and processing, and which could explain the above experimental observations (Additional
Molecular Modeling Results, Supplementary Information) .

Notably, when 354 was used in HeLa cells, potent IMPDH2 degradation was observed at sub-micromolar range, with a complete
depletion of the target at 500-1000 nM (Figs. 3a,b), outperforming 300. However, like with 300, IMPDH2 degradation occurred
at long incubation times (72-96 h) (Figs. 3a,b and Supplementary Figs. 16a-c,h), at which 354 was still present in the medium
as shown by LC/MS analyses (Supplementary Fig. 17). Treatments distributed in multiple additions of 354 did not remarkably
change the degradation time pattern (Supplementary Fig. 16i). Of note, in agreement with their mechanisms of action, cell
treatments with 354 and VX-497 caused opposite effects in steady state levels of IMPDH2: whereas 354 decreased or totally
depleted IMPDH2, VX-497 caused a dose-dependent increase of IMPDH2 levels, by a feedback response of the purine
synthesis pathway49–51 (Fig. 3a,b and Supplementary Figs. 16e,g).

Mechanistic studies using proteasome CP blockers (bortezomib and marizomib) were performed in HeLa and in HeLa cells
stably overexpressing IMPDH2 (clone 522-4) to counteract toxicity due to IMPDH2 degradation. Indeed, these cells were more
resistant to 354 than the parental HeLa cells (Fig. 3c, left). As shown in Fig. 3d, the proteasome inhibitors bortezomib and
marizomib rescued IMPDH2 degradation induced by 300 (in HeLa) and by 354 (in 522-4). In the latter case, IMPDH2
overexpression protected cells from massive death, but a higher degrader concentration was required for depletion of the
overexpressed protein. Conversely, the E1-Ub activating enzyme inhibitor MLN7243 did not prevent the target degradation (Fig.
3e). Furthermore, IMPDH2 depletion was counteracted dose dependently by preincubation with the IMPDH2 ligands VX-497
and 352 (Fig. 3f). This effect could only be partially observed since VX-497 at concentrations higher than 10 µM abrogated cell
growth (Fig. 3c, right). These results show that 354 provokes ubiquitination-independent IMPDH2 degradation by interacting
with the target and the 26S-proteasome.

To show that IMPDH2 degradation occurs in a USP14-dependent manner, the effect of 354 on IMPDH2 degradation in MEFs
Usp14‾/‾ versus wild-type was tested. We observed that IMPDH2 degradation was induced in MEFs-WT, whereas in MEFs
Usp14‾/‾ degradation was not only prevented (Fig. 3g), but the IMPDH2/Actin ratios were significantly higher in
354-treated cells that in controls. This resembles the effect observed with VX-497 (see above), indicating that in the absence of
USP14, the chimera acts as an IMPDH2 inhibitor and IMPDH2 levels increase by a feedback response of the purine synthesis
pathway.

In agreement with the involvement of IMPDH2 in favoring cancer progression52–54, IMPDH2 degrader 354 provoked a reduction
in HeLa cell viability, which was significantly higher in WT than in IMPDH2-overexpressing cells (HeLa 522-4), proving the
involvement of the target in 354 cytotoxicity (Fig. 3c, left). On the other hand, VX-497 was less toxic than 354 and the truncated
USP14 interactor 352 (Fig. 1c) did not affect cell viability (Fig. 3c, right). Since neither VX-497 nor 352 induce IMPDH2
degradation, this result supports that 354-promoted IMPDH2 depletion is involved in cell killing.

Functional characterization of 26S-UIDs targeting CERT1
Having observed good IMPDH2 degradation activity using USP14-directed chimeras, in particular with 354, and to assess
whether long incubation times were required for degradation with USP14-recruited 26S-UIDs, we turned our attention to CERT1,
a target in cancer42. For that purpose, we considered to use 317 as USP14 ligand to develop a small series of CERT1-targeting
26S-UIDs (395, 413, 386, 368 and 419), leveraging the CERT1 interaction with HPA-antagonist derivatives (Fig. 1c and
Supplementary Figs. 10 and 11). Since triple negative breast cancer MDA-MB-231 cells were used by Swanton et al. to prove
the interest of CERT1 as a therapeutic target in oncology42, the CERT1-targeting 26S-UIDs were first tested in this cell line. In
time course assays, 368 showed a degradation effect in 16-24 h, with a maximum degradation observed at 24 h (Fig. 4a), in
contrast with the long treatment times required for IMPDH2 degradation. After 24 h of incubation, dose-dependent degradation
of CERT1 was observed for all five compounds, each featuring distinct linker lengths (Figs. 4b,c). To compare their effectiveness
as degraders, a two-way ANOVA was performed to assess how CERT1 degradation (quantified via densitometric analysis)
varied with 26S-UIDs structure and concentration, treated as independent variables. Post-hoc Tukey’s tests indicated that none
of the 26S-UIDs induced significantly greater degradation than the others at the concentrations tested (Supplementary Fig. 18).
A similar pattern was observed in HERT2-positive breast cancer cell lines MDA-MB-453 and BT-474 (Supplementary Fig. 19).
However, in these cases, some statistically significant differences emerged at 40 µM for certain compounds, consistent with a
potential hook effect (Supplementary Fig. 18). Mechanistic studies showed that the proteasome inhibitor marizomib rescued
CERT1 degradation induced by 368 in MDA-MB-453 cells (both used as models), while E1-Ub activating enzyme inhibitor
MLN7243 did not prevent CERT1 degradation (Fig. 5a). The accumulation of K48-linked ubiquitinated proteins confirmed
proteasome inhibition by marizomib, while the absence of ubiquitinated histone H2A/H2B proved the effectiveness of MLN724355
Furthermore, CERT1 depletion was abrogated dose-dependently by preincubation with the CERT1 ligand 366 (Fig. 5b) and the
USP14 ligand 352 (Fig. 5c). Additionally, incubations with ligands 317 and 366 did not induce changes on CERT1 levels
(Supplementary Fig. 20). Since marizomib did not rescue CERT1 degradation to the control levels, the possibility of induction
of autophagosomal degradation of CERT1 by the USP14-based degraders was investigated. To this end, the levels of LC3-II,
a hallmark for autophagy, were, determined in MDA-MB-453 treated with 386 in the presence or absence of autophagy inhibitors.
As shown in Fig. 5d, 386 induced CERT1 degradation to similar extents in both the absence and the presence of lysosomal
protease inhibitors (lanes 3 vs 7), which block the last step of autophagic degradation, leading to accumulation of LC3-II.
Moreover, in both cases, marizomib rescued CERT1 degradation to similar levels (lanes 4 vs 8). Similar results were found with
chloroquine (Fig. 5e, lanes 3 vs 7 and lanes 4 vs 8), which inhibits the autophagy flux by impairing the autophagosome–lysosome
fusion. Overall, these results indicate that CERT1 degradation involves the proteasome and that the contribution of autophagy
is negligible. To show that CERT1 degradation is USP14-dependent, 368 effect was tested in MEFs, Usp14‾/‾ versus wild-type.
It was observed that CERT1 degradation was promoted in MEFs-WT, but not in MEFs Usp14‾/‾ (Fig. 5f). These results show
that chimeras containing 317 and 366 induce CERT1 degradation by interacting with CERT1 and the 26S-proteasome, in a
ubiquitination-independent and USP14-dependent process.

Since CERT1 transports ceramides from the ER to the Golgi apparatus for SM synthesis, CERT1 degradation was expected to
induce an increase in ceramides and a decrease in SM levels. In agreement with the former, treatment of MDA-MB-231 cells
with 368 induced an increase in ceramide and this increase was significantly higher for the C14-C20 ceramide species than for
the C22-C24 ceramides (Fig. 6a). Similar results were found with 413 in HER2+ MDA-MB-453 cells and with 395 in BT-474 cells
(Figs. 6b,c). This is consistent with the report that CERT1 efficiently transfers ceramides having C14, C16, C18, and C20 chains
(C14-C20Cers), while longer acyl chains (C22-24Cers) are more poorly transported58. Intriguingly, the expected decrease in
SM amounts upon treatment was not observed in any cell line (Supplementary Fig. 21), which is likely due to SM replenishment
by uptake from the serum present in the cell culture medium.

Modeling of 26S-UIDs ternary complexes
The formation of a USP14/26S-UID/POI ternary complex is the minimal requirement for POI degradation. In order to
experimentally validate ternary complex formation, we carried out pulldown assays based on USP14 immobilized on beads, in
parallel assessments of IMPDH2 and CERT1 interactions with USP14, in the presence of representative 354 and 386 chimeras.
The assays revealed that 354 (1 and 10 M) or 386 (5 and 30 M) could induce ternary interactions with both endogenous and
recombinant proteins. Control pulldown assays confirmed the specificity of the ternary complex formation. Compounds binding
only one protein—VX-497 (IMPDH2) and 366 (CERT1)— failed to pull down interacting partners, indicating that stable
interactions require all three components. Beads lacking USP14 also failed to pull down chimeras or targets, confirming that
both did not bind beads unspecifically. Robust pulldown signals were only observed when all components were present, and
negative controls further validated interaction specificity (Supplementary Fig. 22).

To elucidate the molecular basis of ternary complex formation, we employed the PROTAC-Model workflow, previously validated
for modeling other Protac-mediated assemblies46. This approach generates ensembles of structural models grouped into
clusters, each ranked according to protein–protein interface energy. Ternary complexes of USP14/IMPDH2 with chimeras 300,
330, and 329—all containing IU1-248 as the USP14-binding moiety—were modeled as described in the Computational Methods
section. The simulations yielded 21 models (9 clusters) for 300, 86 models (11 clusters) for 330, and 150 models (12 clusters)
for 329 (Supplementary Figs. 2a–c). Consistent with previous observations for other Protac systems58, multiple distinct protein–
protein arrangements, rather than a single preferred binding mode, met the conditions of shape complementarity, as well as the
geometric and energetic requirements of the chimeras. Remarkably, the lowest-energy cluster for all three 26S-UIDs
corresponded to the same overall protein arrangement, suggesting that each chimera can adopt conformations compatible with
this configuration (Supplementary Fig. 23a). This arrangement also exhibited the largest buried surface area (Supplementary
Fig. 2), indicative of extensive interprotein contacts. Superposition of USP14 from these complexes onto the proteasome-bound
USP14 subunit (PDB 5GJQ) revealed that the modeled configurations are sterically compatible with the 26S proteasome,
fulfilling a key structural prerequisite for 26S-UID activity (Fig. 2e). Similarly, modeling of the 317-based chimera
(USP14/354/IMPDH2) (Supplementary Fig. 2d) produced a top-ranked structure closely resembling that of 300, with only minor
differences in binding geometry (Supplementary Fig. 23a). Ternary complex models were also generated for USP14/CERT1-
START bound to chimeras 368, 386, 413, 395, and 419 (Supplementary Fig. 24). In this case, a broader diversity of binding
modes and interaction energies was observed, primarily attributable to differences in PEG–carbamate linker length. Chimeras
368 (3 PEG units), 386 (2 PEG units), and 413 (1 PEG unit) exhibited similar binding modes, whereas 419 (5 PEG units) and
395 (no linker) adopted distinct, less favorable configurations with higher interface energies, particularly pronounced for 395
(Supplementary Figs. 23b). Despite these suboptimal geometries, both chimeras retained potent degrader activity (see previous
section), suggesting that degradation efficacy is not strictly dependent on interface energy or complex geometry. Structural
analysis of the modeled ternary complexes indicated that stabilization arises from multiple intermolecular interactions between
the 26S-UID molecules and their binding partners (Figs. 7a,b), as well as between each pair of proteins (Supplementary Fig.
25). Notably, high surface complementarity was observed between protein pairs that have not naturally co-evolved to interact.
This complementarity is partly explained by the consistent occupation of USP14’s concave surface region by the target protein—
a region that corresponds to USP14’s native ubiquitin-binding site (residues 183–213, 265–278, 294–302, 330–342, and 428–
434) (Figs. 7c,d)46,58.

Methods
Computational Methods
The package Schrödinger Suite 2022-470, through its graphical interface Maestro71, was used to perform most modeling and
visualization tasks. The program Macromodel72 with the OPLS4 force field73 and GB/SA water solvation conditions74 were used
for energy minimization. The Protein Preparation Wizard75 included in Maestro was used to prepare protein structures by
removing solvent molecules and ions, filling incomplete residues and gaps, adding hydrogens, setting protonation states and
running a restrained minimization using the OPLS4 force-field. This preparation protocol was applied to all proteins studied
(USP14, IMPDH2 and CERT1), with some variations depending on the case, as next described. Chain A of structure 6IIN20,
including one molecule of inhibitor IU1-248 bound to an allosteric cavity close to the active site, was used as initial model of
USP14. This structure has several gaps (loops 220-239, 334-339 and 378-400) and incompletely defined residues, which are
located mostly away from the inhibitor binding site, except for loop 334-339. Thus, the Homology Modeling module of the
Schrodinger Suite, with the Energy-based method and default settings, was used to generate a model of the full sequence
between residues 101-494 of USP14 with the bound IU1-248 inhibitor. This structure was also used to dock a molecule of IU1c
and its derivative 317, first using the software Glide76–78 with default settings and then the best poses were redocked with the
Induced Fit Docking protocol79,80 of the Schrodinger Suite. Similarly, chain A of PDB 6UDO81 was used to obtain a model of the
full sequence between residues 12-514 of IMPDH2 with a docked molecule of inhibitor VX-49744.The structure of this inhibitor
was then modified to generate a capped version, VX-497_cap (Supplementary Fig. 1), that was later used as starting point to
model the corresponding ternary complexes (see below). Finally, PDB 2E3Q45 was used as model of the START domain of
CERT1, to which ligand HPA-14_PEG3 (Supplementary Fig. 1) was docked, as described above. The stability of this complex
(HPA-14_PEG3/CERT1) was assessed by running a 500 ns molecular dynamics simulation, as described below. This docked
ligand was further modified to introduce a triazine group (HPA-9_trz, Supplementary Fig. 1) which was later used to model the
corresponding chimeras in the ternary complexes with CERT1 and USP14.

Molecular Dynamics of USP14 complexes
Models of ligands IU1-248, IU1c and 317 docked to USP14 and of HPA-14_PEG3 docked to CERT1 were submitted to molecular
dynamics with the program Desmond 7.282,83, as included in the Schrödinger Suite 2022-4, using the OPLS4 force field. Three
simulations were performed for each USP14-ligand complex, starting from different docked poses. These systems were set up
using the System Builder of the Maestro-Desmond interface84. Solutes were placed in the center of a truncated octahedron
periodic box that extended 15 Å away from any solute atom, Na+ and Cl- ions were added to achieve neutrality and to reach a
150 mM salt concentration, and systems were solvated with TIP3P water molecules. Thus, each simulation system consisted
of one USP14 monomer and one bound-ligand molecule, 40 chloride and 48/49 sodium ions (depending on ligand protonation
state), and approximately 14330 TIP3P waters. The systems were subjected to the default relaxation protocol consisting of: 100
ps Brownian Dynamics at 10 K in the NVT ensemble, with 50 kcal mol-1 Å-2 restraints on solute heavy atoms; 12 ps molecular
dynamics at 10 K (Lagevin thermostat) in the NVT ensemble, keeping the restraints; 12 ps MD at 10 K and 1.0 atm (Lagevin
thermostat and barostat) in the NPT ensemble, keeping the restraints; 12 ps MD keeping previous conditions except for
temperature of 300 K; 24 ps MD keeping previous conditions but without restraints. Production MD simulations (500 ns, 2 fs
timestep) were performed under the same conditions (PBC, NPT ensemble, 300 K and 1.0 bar) using the Nose-Hoover
thermostat method85 with a relaxation time of 1.0 ps and the Martyna-Tobias-Klein barostat method86 with isotropic coupling and
a relaxation time of 2 ps. Integration was carried out with the RESPA integrator87 using time steps of 2.0, 2.0, and 6.0 fs for the
bonded van der Waals and short range and long range electrostatic interactions, respectively. A cut-off of 9.0 Å was applied to
van der Waals and short-range electrostatic interactions, while long-range electrostatic interactions were computed using the
smooth particle mesh Ewald method with a 10-9 Ewald tolerance88,89. Bond lengths to hydrogen atoms were constrained using
the Shake algorithm90. Coordinates were saved every 250 ps, hence 2,000 snapshots (frames) were obtained from each MD
run. The Simulation Interactions application included in the Desmond-Maestro interface was used to analyze the simulations
results.

Modeling of ternary complexes with 26S-UIDs
Structural models of the ternary complexes of 26S-UIDs (300, 330, 329, 354, 368, 386, 413, 395 and 419) with their
corresponding protein partners were generated with the software workflow PROTAC-Model46. This software combines
FRODOCK91,92 for local docking under docking site constraints, RosettaDock93 for structural refinement94, for modeling the 26S-
UIDs conformations, Open Babel95 for evaluating the 26S-UIDs conformations, AutoDock Vina96 for assessing the 26S-UIDs
binding modes, VoroMQA97,98 for reranking the protein−protein complexes, and the fraction of common contact (FCC) clustering
method99 to cluster the final results. Briefly, PROTAC-Model starts with the structures of the two proteins, in our case USP14
and IMPDH2 or CERT1, complexed to their respective ligands. In some cases, different conformations of the bound ligands
were assayed. These ligand structures must have anchor atoms where the 26S-UID linker can be attached. Thus, in most cases,
for the USP14 ligands IU1-248 and 317, their hydroxyl group was defined as anchor atom, while for the IMPDH2 and CERT1
ligands the anchor atoms were conveniently defined, as shown in Supplementary Fig. 1 (VX-497_cap and HPA-9_trz,
respectively). Chimera 395 was an exception due to the lack of a PEG-carbamate linker, thus in this case structures 317a and
HPA-2 (Supplementary Fig. 1) bound to USP14 and CERT1 were used as input for PROTAC-Model. The first step of the protocol
was the docking with FRODOCK of the target proteins to USP14, each containing its own bound ligand, using the ligand anchor
atoms as docking sites. In this way, about 25,000 complexes were generated for the USP14/IMPDH2 pairs and about 16,000
for USP14/CERT1. These complexes were then filtered, first by removing those where the two binding sites were too distant.
Then RDKit was used to model the full 26S-UIDs into the docked conformations, removing those complexes that yielded
unreasonable structures. For the remaining ones, the 26S-UIDs energy was calculated using Obenergy, and the models with
unfavorable values were removed. A further elimination criterion was that of the Vina score: only those complexes with a Vina
energy score lower than 0 kcal/mol were kept. The remaining models were reranked and refiltered by calculating the protein-
protein interface energy with VoroMQA (a unit-less value derived from pseudo-energy potentials97,98) and discarding those
complexes with positive scores. The resulting complexes were clustered with the FCC method and the clusters were ranked by
the best model in each cluster. A further refinement was performed by using RosettaDock to construct 400 models from the best
structure of each of the clusters previously generated, and reapplying the filtering, reranking and clustering steps as above.
Additional details about this protocol can be found in the original publication46. In this way, a number of clusters were generated
for each of the ternary complexes studied and representative models of each cluster were obtained (Supplementary Figs. 2 and
22), which were then minimized with MacroModel. Buried protein surface area was calculated for selected ternary complexes
with COCOMAPS100.

Reagents and general methods for synthesis
All chemicals were purchased from commercial sources and used as received unless otherwise noted. Dry solvents, obtained
from a PureSolv dispenser and subsequently degassed with inert gas, were used in most reactions. Synthesis grade or HPLC–
grade solvents were used for extractions and purifications. Unless otherwise specified all reactions were performed under Ar
inert atmosphere. Progression of the reactions was controlled by thin layer chromatography (TLC), using ALUGRAM® SIL
G/UV254 (Macherey–Nagel) silica gel pre–coated aluminum sheets (Layer: 0.2 mm, silica gel 60). Compounds were detected
by using UV light (λ=254 nm) and a stain solution of phosphomolibdic acid (5.7% in EtOH). Compounds were purified by flash
column chromatography, using silica gel (Chromatogel 60 Å, 35–75 μm) as stationary phase. Mobile phases and gradients are
specified in each case. 1H and 13C Nuclear Magnetic Resonance spectra were recorded on a Varian – Mercury 400 (1H NMR
at 400 MHz and 13C NMR at 100.6 MHz) spectrometer using CDCl3 or CD3OD as solvent. Chemical shifts of deuterated solvents
were used as internal standards. Chemical shifts are given in parts per million (ppm) and coupling constants (J) in Hertz (Hz).
Splitting patterns have been described as singlet (s), broad singlet (br s), doublet (d), triplet (t), quartet (q), quintuplet (p),
multiplet (m), apparent (app) or combinations of these descriptive names. Specific optical rotations were recorded on a digital
Perkin–Elmer 34 polarimeter at 25 ºC in a 1-dm 1-mL cell, using a sodium light lamp (λ=589 nm). Specific optical rotation values
([α]D) are expressed in deg–1·cm3·g–1, and concentrations (c) are reported in g/100 mL of solvent. High–resolution mass
spectra were recorded Waters Acquity UPLC system connected to a Waters LCT Premier Orthogonal Accelerated Time of Flight
Mass Spectrometer (Waters,Milford, MA, USA) operated in positive electrospray ionisation mode. Samples were analyzed by
FIA (Flow Injection Analysis), using ACN/water (70:30) as mobile phase. Samples were analyzed using a 10 μL volume injection.
m/z ratios are reported as values in atomic mass units. Detailed methods are given in the Supplementary Information.

Cellular and Molecular Biology Methods
Expression and purification of GST-IMPDH2 protein in E. coli
In E.coli, glutathione S-transferase fusion vector (pGEX4T3) was used to express and purify GST-IMPDH2 (ALI494 vector; see
Supplementary Table 1). Bacterial cultures were grown to an OD600 of 0.7, induced with 500 μM isopropylthiogalactoside (IPTG)
overnight at 16 °C. Cells were harvested, resuspended with 5 volumes of lysis buffer containing 50 mM NaH2PO4 pH 7.4, 150
mM NaCl, 5% glycerol and 1X concentration of protease complete inhibitor cocktail EDTA free (Roche), and lysed sonicating
with 30% amplitude during 2 effective minutes (10''ON/15''OFF). The lysate was clarified at 15000 g for 45 min, filtered using
cheese cloth, and the supernatant was applied to Glutathione agarose (Merck) at a ratio of 1 mL beads per 2 gr cell pellet. The
binding was done in end-over rotation at 4 ºC for 3 h. Beads were washed with 30 bed volumes of 50 mM NaH2PO4 pH 7.4, 150
mM NaCl, 5% glycerol. Proteins were eluted with a reduced glutathione buffer of 50 mM NaH2PO4 pH 7.4, 5% glycerol and 30
mM reduced glutathione. Reactions were incubated at 4 ºC overnight. The efficiency of elution was determined by SDS-PAGE
and Coomassie Brilliant Blue staining analysis.

Expression and purification of 6His-USP14 protein in E. coli
In E.coli, 6-His fusion vector (pET28a) was used to express and purify 6His-USP14 (BCO454 vector; see Supplementary Table
1). Bacterial cultures were grown to an OD600 of 0.7, induced with 500 μM isopropylthiogalactoside (IPTG) over night at 16 °C.
Cells were harvested, resuspended with 5 volumes of lysis buffer containing 50 mM NaH2PO4 pH 7.4, 10 mM imidazole, 150
mM NaCl, 5% glycerol and 1X concentration of protease complete inhibitor cocktail EDTA free (Roche), and lysed sonicating
with 30% amplitude during 2 effective minutes (10 s ON/15 s OFF). The lysate was clarified at 15000 g for 45 min, filtered using
cheese cloth, and the supernatant was applied to Ni-NTA beads (Invitrogen) at a ratio of 1 mL of beads per 2 g cell pellet. The
binding was done in end-over rotation at 4 ºC for 3 h. Beads were washed with 15 bed volumes of 50 mM NaH2PO4 pH 7.4, 25
mM imidazole, 150 mM NaCl, 5% glycerol followed by 15 bed volumes of 50 mM NaH2PO4 pH 7.4, 25 mM imidazole, 300 mM
NaCl, 5% glycerol. 6His-USP14 protein was eluted with a buffer containing 50 mM NaH2PO4 pH 7.4, 200 mM imidazole, 150
mM NaCl, 5% glycerol. The efficiency of elution was determined by SDS-PAGE and Coomassie Brilliant Blue staining analysis.

Ub-AMC processing assays in 26S-USP14
Assays were performed in a buffer containing 50 mM Tris pH 7.4, 1 mg/mL BSA, 1 mM DTT, 1 mM ATP/MgCl2, premixed VS-
proteasome/USP14 (Ubiquitin–vinylsulphone [VS] treated 26S proteasome reconstituted with active USP14, with a ratio of
1.25:20 nM final concentration; Ubiquigent). VS blocks the 26S proteasome endogenous DUB activity by forming an adduct
with the active site cysteine in DUBs of thiol proteases. In this way, only the USP14 DUB activity is measured in the assays).
VS-proteasome/USP14 were mixed with Ub-AMC and chimeras with a final concentration of 0.5 µM and 50 µM respectively.
30 µL of the mix were dispensed into 384-well low volume plates in triplicate. Ub-AMC hydrolysis was measured at
Ex380/Em460 using a Tecan Infinite Nano+. Fluorescence intensity was recorded every minute during 40 min.

Inosine monophosphate dehydrogenase activity assays
Activity of recombinant IMPDH2 was measured by means of enzymatic reactions carried out in the presence of 17.5 µg/ml
IMPDH2, 1 mM IMP, 2 mM NAD and 5 µM chimeras or ligands. Formation of NAD(H) was recorded for 3 min at 340 nm in
quartz cuvettes, in a Jasco V650 spectrophotometer. Three independent assays were performed for each condition.

Surface plasmon resonance (SPR)
The affinities of the synthesized compounds (here referred to as ‘analytes’) vs. different recombinant proteins were determined
using a BIACORETM T200 from Cytiva. All experiments were performed at 25 °C and data were collected at a rate of 10 Hz.
Recombinant proteins [USP14, IMPDH2, and CERT1 (amino acids 347–598; Abcam 95897), here all three referred as to
‘ligands’] were immobilized on a CM5 sensor chip series S (Cytiva) using the amine-coupling method described by the
manufacturer. In details, USP14 was immobilized in channel 2, IMPDH2 in channel 3 and CERT1 in channel 4, leaving channel
1 empty as reference. The channels were activated with EDC-NHI (at final concentration of 200 mM and 50 mM, respectively)
during 7 min, and blocked with ethanolamine-HCl (1M pH 8.5) during the same time, as the activation. For the immobilization,
the ligands were diluted with 10 mM sodium acetate buffer at pH 5.0, for USP14 and CERT1, and pH 5.5 for IMPDH2. The
density of the coupling was calculated to achieve a theoretical maximum analyte binding capacity (Rmax) within the range of 20-
150 RUs, with an injection time between 50 and 75 seconds depending on the ligand. The running buffer during the
immobilization was PBS with 0.05% Tween20 at a flow rate of 15 μL min-1 and 5 µl min-1 for the injection of the ligand. The
analyses of the analytes binding to the ligands were performed in buffer PBS with 0.05% Tween 20 and 5% DMSO at a flow
rate of 60 μL min-1. Each of the synthesized compounds was injected in the running buffer over the immobilized USP14, IMPDH2
or CERT1. For all analytes, samples at 100 µM concentration, and a 2-fold dilution series to 6.25 µM concentration, were
injected, by duplicates (1,1; 2,2; 3,3; etc.), for 60 s in the association phase, which is followed by 360 s of dissociation phase.
Equilibrium-based affinity analysis was performed using report points set 10 s before the end of the injection, within a 5-s window
frame. Data were analyzed using Biacore T200 Evaluation Software v3.1 (Cytiva). In brief: (1) solvent correction was applied,
(2) the sensorgrams were double-referenced, and (3) a 1:1 steady state affinity model was applied selecting KD fit global, Rmax
and offset fit local.

Generation of IMPDH2-GFP-overexpressing HeLa cells (clone 522-4)
In order to generate cell lines expressing stable IMPDH2-GFP, HEK293T cells were transfected with the vector BC522
(Cilantro2-IMPDH2), which contain LTR sequences that facilitate the integration of the vector into the host genome, along with
the vectors necessary to create the viral particles (vectors BC512, BC513, BC514; see Supplementary Table 1). After 48 hours,
medium with the viral particles was collected and transferred to HeLa cells for infection. This process was repeated 72 hours
post-transfection. HeLa cells were cultured with the medium with the viral particles for 48 hours, when the medium was changed.
After 24 hours, puromycin was added to the medium, and cells were kept growing with antibiotic during the following days. The
cells were observed under the microscope to evaluate the fluorescence levels by GFP and, after 8 days, single cell sorting was
done, on 96-well plates, using a BD FacsAria Fusion cell sorter (Beckton Dickinson, San Jose, California), using the BD
FACSDiva software version 8.0.1. Live cells were gated using DAPI. The cells were cultured until they grew enough to obtain a
protein extract to perform a western blot and check IMPDH2-GFP expression.

Cell cultures
HeLa and MDA-MB-231 cell lines and MEFs were grown in DMEM media (Thermo Fisher) supplemented with 10% fetal bovine
serum (FBS) (Thermo Fisher), 2 mM L-glutamine (Thermo Fisher) and 100 U/mL penicillin/100 µg/mL streptomycin (Thermo
Fisher). MDA-MB-453 and BT-474 cells were grown in DMEM/F12 media (Gibco) supplemented with 10% fetal bovine serum
(FBS) (Thermo Fisher), 2 mM L-glutamine (Thermo Fisher), amphotericin B 2.5 µg/mL (Gibco), 100 U/mL penicillin/100 µg/mL
streptomycin (Thermo Fisher) and, only with the BT-474 cell lines, 10 µg/ml insulin (Merck) were added. IMPDH2 degradation
experiments were performed in media containing 5% FBS.

Cell treatments
For IMPDH2 degradation assays, HeLa cells were seeded in 6 well-plates at 150000 cells/mL (HeLa cells and wild type
or Usp14-/- MEFs). 26S-UIDs at the indicated final concentrations were added from stock DMSO solutions 24 h after seeding
and cells were collected at the specified times in the figure legends. In mechanistic studies, bortezomib (Signalway Antibody)
and marizomib (MedChemExpress) were added 16 h before cell collection (80 h after 26S-UID addition), while MLN7243
(Selleckchem) was added 8 h before cell harvest (64 h after 26S-UID addition). At the specified times of treatment, both
adhered (trypsinizaton) and floating cells were collected and processed for WB analysis. For CERT1 degradation assays (WB protein
analysis) and lipidomics, cells were seeded in 6 well-plates at 200000 cells/mL (MDA-MB-231), 250000 cells/mL (MEF) 300000
cells/mL (MDA-MB-453 cell line) or 400000 cells/mL (BT-474 cell line). 26S-UIDs at the indicated final concentrations were
added from stock DMSO solutions (10 mM) 24 h (MEF cells) or 48 h (MDA-MB-231, MDA-MB-453 and BT-474 cell lines) after
seeding. In experiments with lapatinib (Selleckchem), the drug was added together with 26S-UIDs. In experiments with
marizomib (MedChemExpress), cells were preincubated for 3 h with the inhibitor, the medium was removed and fresh medium
containing 26S-UIDs at the concentrations shown was added. In experiments with MLN7243 (Selleckchem) and competitor
ligands (366 and 352), the compounds were added 3 h prior addition of 26S-UIDs to the cells. DMSO was added as control in
all cases. In experiments to assess the role of autophagy in CERT1 degradation, proteasome inhibitors (PI, which contain E64D,
5 µg/mL and pepstatin A, 5 µg/mL) were added together with marizomib and then again with the CERT1 degrader after medium
renewal. In the case of chloroquine (Chl), it was added 4 h before cells collection. At the specified times of treatment, both
adhered (trypsinizaton) and floating cells were collected and processed for WB analysis or lipidomics. For cell viability
determination, cells were plated at a density of: HeLa, 10000 cells/mL; MDA-MB-453, 100000 cells/mL and BT-474, 35000
cells/mL in a 96-well plate and allowed to adhere for 24 h (HeLa cells), 48 h (MDA-MB-231 cells and MDA-MB-453 cells) or 72
h (BT-474 cells). Then cells were treated (single addition) with the indicated doses of compounds for the specified times.
Lapatinib was added together with 26S-UIDs Detailed schematic representations of all the assays performed is found in
Supplementary Fig.28.

Cell viability analysis
After the treatments, the MTT reagent (Sigma) was added to the cell culture media and absorbance at 570 nm was quantified
after 3 h incubation with a SYNERGY H1 Absorbance microplate reader (BioTek). In Fig. 3c, curves adjustment with the four-
parameter logistic equation (variable slope) (GraphPad Prism 8, version 8.0.2) afforded the following CC50 values (95%
Confidence Intervals). In Fig. 3c, left: 354: 1.19 µM (0.61 to 2.32 µM) in parental HeLa; 34.5 µM (12.61 to 94.19 µM) in clone
522-4; in Fig. 3c, right: 354: 3.9 µM (1.68 to 9.37 µM); VX-497: 11.5 µM (4.15 to 32.15 µM); 352: > 100 µM.

Protein analysis
Cell pellets were resuspended in the appropriate volume of a 0.25 M saccharose solution. The suspension was submitted to
three cycles of 5 s sonication (probe)/5 s resting on ice. The cell lysate was centrifuged at 2500 g for 5 min. The supernatant
was collected and protein concentration was determined (BSA as a standard) using a BCA protein determination kit (Thermo
Scientific) according to the manufacturer’s instructions.

Samples were loaded onto a 10% polyacrylamide gel, separated by electrophoresis at 100 V/2.5-3.0 h and transferred onto a
PVDF membrane (100 V/2 h). Unspecific binding sites were then blocked with 5% milk in TBST. Anti-LC3 (Ab48394), anti-
IMPDH2 (Ab131158-1001), anti-CERT (Ab72536-1001), anti-USP14 (CS-11931), anti-GAPDH (SC47724), anti-K-48
polyubiquitin (CS-8081) and anti-H2B-Ub (CS-5546) antibodies were diluted 1:1000 in 5% milk in TBST. Anti-actin antibody
(Ab5694) was diluted 1:2000 in 5% milk in TBST. Membranes were incubated overnight at 4 ºC under gentle agitation. After
washing with TBST, membranes were probed with the correspondent secondary antibody for 1 h at room temperature (IMPDH2,
LC3, CERT, K-48 polyubiquitin, H2B-Ub and USP-14: anti-rabbit [NA934V], diluted 1:10000 in 5% milk in TBST; Actin and
GAPDH: anti-mouse [170-6516], diluted 1:10000 in 5% milk in TBST). Antibody excess was eliminated by washing with TBST
and protein detection was carried out using ECL and membrane scanning with LI-COR C-DiGit® Blot Scanner. Band intensities
were quantified by LI-COR Image Studio Lite Software.

Lipidomics
Sphingolipid extracts, fortified with internal standards (N-dodecanoylsphingosine, N-dodecanoylglucosylsphingosine, and N-
dodecanoylsphingosylphosphorylcholine, 0.5 nmol each), were prepared by mixing 100 μL of cell lysates with 0.75 mL of
methanol and chloroform (2:1 v/v) containing 0.01% BHT. The samples were sonicated in a bath-type sonicator until evenly
dispersed, then incubated overnight at 48°C in a water bath. After cooling, 75 μL of 1M KOH in methanol was added, and the
samples were sonicated and incubated for 2 hours at 37°C. Solvents were removed using a SpeedVac concentrator, and the
samples were redissolved in methanol and transferred to a new tube. Solvents were removed again with the SpeedVac
concentrator. The dried samples were redissolved in methanol, centrifuged, and the supernatants were transferred to a glass
vial for UPLC analysis.

Liquid chromatography-mass spectrometry was performed using a Waters Acquity Premier UPLC system coupled to a SELECT
SERIES Cyclic IMS, which combines a Q-Tof (Quadrupole-Time of Flight) mass spectrometer with cIM (Cyclic Ion Mobility)
(Waters, Milford, MA), operated in positive electrospray ionization mode. Full scan spectra from 50 to 1200 Da were acquired,
and individual spectra were summed to generate data points every 0.5 s. Mass accuracy and reproducibility were maintained
using an independent reference spray via the LockSpray interference. The analytical column was a 100 mm × 2.1 mm i.d., 1.7
μm C8 Acquity UPLC BEH column (Waters). The data was analyzed using MassLynx v4.2 (Waters). The two mobile phases
were: phase A: water with 2 mM ammonium formate; phase B: methanol with 1 mM ammonium formate, both containing 0.2%
formic acid. A linear gradient was programmed as follows: 0.0 min: 80% B; 3 min: 90% B; 6 min: 90% B; 15 min: 99% B; 18 min:
99% B; 20 min: 80% B; 22 min: 80% B. The flow rate was 0.3 mL/min, and the column was held at 30°C. Quantification was
performed using the extracted ion chromatogram of each compound with 50 mDa windows. The linear dynamic range was
determined by injecting standard mixtures. Positive identification of compounds was based on accurate mass measurement
with an error of <5 ppm and their LC retention time, compared to that of a standard.

Analysis of 354 by UPLC-MS
HeLa cells were seeded at the appropriate density to collect 1x106 cells per sample at the end of the experiment and allowed
to adhere for 24 h. Then, 354 was added (1 µL of a 10 mM stock solution in DMSO; 10 µM final concentration, 10 nmol). 24 or
96 h after 354’s addition, the media (containing floating cells) was collected and centrifuged (9000 g, 3 min). Media and pelleted
cells were separated. Adhered cells were trypsinized, mixed with the pelleted cells from the media, counted, centrifuged, washed
with PBS and spun down again. Pelleted cells were extracted with acetonitrile (300 μL per million cells), sonicated and analyzed
by LC/MS as indicated in the previous section. Two-hundred µL of media were diluted with 800 μL of MeOH and kept at 4 °C
overnight to precipitate proteins. Then the suspension was centrifuged (9000 g, 3 min) and the supernatant was evaporated
under vacuum. The residue was dissolved in 150 μL of acetonitrile, sonicated, centrifuged (9000 g, 3 min) and the supernatant
(150 µL) was injected into the mass spectrometer after addition of 200 pmol of C17-sphinganine as internal standard.

Pull-down assays
Immobilized His-USP14 was obtained as described above in “Expression and purification of 6His-USP14 protein in E. coli.”
methods, avoiding the elution step. After His-USP14 immobilization, beads were washed thrice with 50 mM NaH2PO4, 100 mM
NaCl, pH 8 buffer and then incubated in blocking buffer (10% BSA in 50 mM NaH2PO4, 100 mM NaCl, pH 8 buffer) for one hour
at room temperature. The beads were then washed again thrice with 50 mM NaH2PO4, 100 mM NaCl, pH 8 buffer and once
with washing buffer (50 mM NaH2PO4, 100 mM NaCl, 1 mM DTT, 10% Glycerol, protease inhibitor cocktail, pH 7.5). Empty Ni-
NTA control beads were washed and blocked with BSA in the same manner. Then, purified GST-IMPDH2 (see above) or CERT1
(95897 Abcam; digested with enterokinase to remove the His-tag; see below) proteins were added at 500nM final concentration.
The beads-substrate mixture was then aliquoted to separate tubes and 386, 366, 354 or VX497 were added at the indicated
concentration and incubated at 4ºC for two hours. The beads were washed thrice with 10 column volumes of washing buffer
and then eluted with SDS loading buffer.

For experiments in which the input substrate is a whole cell extract (WCE), the sample was prepared as follows. Ten 100 mm
dishes of confluent HeLa cells were washed with TBS and then scraped into a 15 mL falcon tube. The cells were pelleted,
resuspended in wash buffer, and then lysed by sonication (Branson sonicator microtip, 30% amplitude, 3 cycles of 1 effective
minute (3 s ON/3 s OFF). The lysate was cleared by centrifugation and then added to the beads as an input substrate, as above.
The digestion of His-CERT1 (95897 Abcam) with enterokinase, in order to remove the His-tag, was carried out as follows: His-
CERT1 protein (supplied in 20 mM Tris HCl pH 8, NaCl 100 mM by the manufacturer) was incubated during 16 hours at 22ºC
with enterokinase (His-tagged; GenScript, Z03004-100), added in a 1.2 units of EK/10 ug of protein substrate ratio. Released
His-tag and His-enterokinase were removed by adding 5 µL Ni-NTA beads in 100 µL protein solution, 2 hours of incubation at
4°C followed by centrifugation. In Suppl. Fig. 22, western blots were detected with: panel A, anti-CERT (Ab72536-1001); Panel
B, anti-CERT (PA5-90421); Panels C and D, anti-IMPDH2 (Ab131158-1001); All panels, anti-USP14 (CS-11931).

RNA extraction and reverse transcriptase (RT)-qPCR
Total RNA was extracted using the NZY Total RNA Isolation Kit (NZYtech). Then, cDNA was generated from 100 ng of RNA
using the SuperScript® VILO™ (Invitrogen). Gene products were analyzed by qPCR, using NZYSupreme qPCT Green Master
Mix 2x (NZYtech) and specific oligonucleotides in a QuantStudio5 machine (AppliedBioSystems), running in the QuantStudio™
Design & Analysis Software (AppliedBioSystems). Each value was corrected by human Actine and represented as relative units.
Each experiment was performed in duplicate. qPCR oligonucleotide sequences used, are: IMPDH2 forward: 5’-
GTGAAGCTGACAAAATCAAAGTGGC-3’, IMPDH2 reverse: 5’-CTCTGGGCACCAATGTCCTGGC-3’, Actin forward: 5’-
CACCATTGGCAATGAGCGGTT-3’, Actin reverse: 5’-AGGTCTTTGCGGATGTCCACGT-3’.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Crosas and colleagues reported two series of novel type of TPD or PROTAC compounds, named as 26S-UIDs, which are featured in Ub-independent, 26S proteasome directing mode of target protein degradation. For proof-of-concept studies, the authors employed USP14, one of major DUBs on the proteasome, as the docking site of 26S-UID at one end, and selected two targets (IMPDH2 and CERT1) for designing each recognizing ligand at the other end, creating new TPD chimeras by linking them for inducing direct proteasomal degradation simply thru proximity endowment. With some choice of synthesized compounds that were also validated by molecular simulation, they proved that these 26S-UIDs indeed are able to induce degradation of IMPDH2 and CERT1 in cancer cell lines. The authors further tested CERT1 degraders in HER2+ breast cancer cell lines (WT and lapatinib resistant), and showed that some selection of the compounds display the lapatinib drug sensitization when co-treated, which is also consistent with concomitant increase of ceramide species by degraders treatment in those cancer cell lines. This study reports a number of novel classes of TPD compounds by infusing an interesting idea of Ub-independent, USP14 employing and direct recruitment to the 26S proteasome for fullfilling substrate degradation. On the other hand, this research seems to demand significant amout of extra works one way and another to be reconsidered for publication. Please find the comments as follows.\n1. Above all, the authors need to confirm their IMPDH2 and CERT1 degraders in USP14 knockout or depleted cells with WT cells as control. These experiments will strongly demonstrate the mode of action of 26S-UIDs to be bona fide USP14-mediated and 26S directing for POI degradation.\n2. It is intriguing why USP14 was selected as 26S docking site over others. Although the authors listed four reasons for its consideration (page 3), those are still not super convincing especially given that USP14 exists as a free form as well (also stated in Discussion, page 11). This means that depending on the population of USP14 as a free form, the 26S-UIDs may not be maximally productive. Also the possible influence on endogenous USP14 function when treated with these degraders should be described, in terms of native Ub conjugates processing by USP14 or other physiological impacts. BTW, the second reason for USP14 consideration (page 3; “….USP14 exhibits activity on recruited proteasomal substrates as they translocate through….”) sounds like a wrong statement, thus needs to be corrected.\nThe rationale for selecting IMPDH2 and CERT1 as target substrates is even more ambiguous. This is also because E3-based IMPDH2 and CERT1 PROTACs were not successfully made and thus cannot be fairly compared to the 26S-UIDs for evaluating them as good proteasomal substrates. In fact, quite curious why the authors selected those two POIs over the well established targets that have been repeatedly employed in generating canonical PROTACs.\nSo, overall, the rationale for selecting USP14 and those two POIs should be more convincingly delivered.\n3. As the authors also argued, the degradation behavior of IMPDH2 by 26S-UIDs seems to be odd because only long time treatment was able to induce degradation of the POI (72-96 hr). This can be an issue or even concern in interpreting the data, so should be more thoroughly addressed. For example, more time points degradation assays need to be done in parallel since the current scheme for the chimeras treatment is not systematically investigated or mostly end point assay (i.e. at one time point). The authors also need to consider whether 26S-UID-mediated IMPDH2 degradation is coming from any secondary effect. In the similar context, the authors should find optimal conditions to treat proteasome inhibitor and MLN7243 inhibitor during the course of treatment to prove Ub-independent but proteasome-dependent degradation, although they in fact stated that those studies can be hampered due to the cytotoxicity by long time incubation. Besides, it is not clear how the authors treated the IMPDH2 degraders for 72 or 96 hr, in other words did they treat initially one time till the end or multiple times by changing the compound-containing media during the course of incubation? If the former is the case, how stable the compounds are expected and how then the intial one time treatment suddenly starts working at 72-96 hr? In any case, the detailed experimental scheme should be also given in Method section.\n4. It is intriguing to see no effects on degradation by E3-based IMPDH2 and CERT1 PROTACs. Since these PROTACs are not capable and thus does not serve as any good comparison, how the experiments done with these compounds are fit into this story (unless the authors argue for their 26S-UID superiority over the E3-based PROTACs at least on IMPDH2 and CERT1)? On the other hand, did the authors fully optimize the conditions for the treatment of these PROTACs? CRBN- and VHL-based PROTACs often work quite well even at nanomolar concentrations but show adverse, so called ‘hook’ effect at high doses. In this study, the authors routinely treated the E3 PROTACs at uM ~ tens of uM, so curious whether the titrations of nM to tens of nM make any difference. In fact, to my eyes, the lowest conc of CRBN-directed IMPDH2 PROTAC was 10 nM (Fig. S4B), in there the POI seems to be less in level. In any case, the authors should test a serial concentrations of E3-based PROTACs starting from low nM or sub nM, and also provide the rationale for using these canonical PROTACs in this study.\n5. In Fig. 6B, the marizomib (Mar) effect seems to be partial (0.6). Did the authors also test the autophagy inhibitors, such as bafilomycin? I am asking this question because recently there are number of papers that described the role or effect of USP14 and its inhibitors on autophagic pathways. By utilizing USP14 ligands in making 26S-UIDs, wondering if the partial recovery by Mar treatment could be due to any possible contribution also coming from autophagic degradation.\n6. In Fig. 7, it is puzzling that those lapatinib resistant cancer cell lines are not really lapatinib resistant compared to WT counterparts. Also what is the point of using this lapatinib resistant cell lines in this study? The authors need to address these questions.\n7. Most of phenotypic and readout assays were centered on CERT1 but not IMPDH2. Probably from this reason, Discussion section was also far more allocated to CERT1 part. The authors should show any kind of functional readouts from their IMPDH2 degraders, not just the protein level.\n8. In Figs. 6 to 8, Fig. S15, as the authors described, the experiments done with these CERT1 degarders seem to contain self-conflicting results that are not well resolved in the manuscript. All CERT1 degraders (395, 413, 386, 368 & 419) work fairly well for POI degradation and 368 might be the best (Fig. 6B). However, only 395 and 413 showed good lapatinib sensitization but not 368 or others (Figs. 7 & S15). Differential increase of ceramide species depending on degraders and cell lines was also confusing (Fig. 8). Does this mean that the degradation capacity of CERT1 26S-UIDs is not necessarily correlated with its physiological impact? The authors should try to resolve this apparent discrepancy.\nAdditionally, the authors should test 26S-UIDs alone for the drug sensitization experiments (i.e. dose-dependent manner). And the Method for measuring the level of ceramide species (for Fig. 8) was not given anywhere.\n9. At Fig. S1, given that all compounds were treated at 50 uM, the inhibitory activites of IU1 and IU1-248 in there are not well matching compared to the previous reports (#32, Lee et al. & #34, Wang et al.), in which IU1 had 4 ~ 12 uM of IC50 and IU1-248 had 0.83 uM of IC50.\n10. Lastly, this manuscript needs significant reworks, in terms of fine writing, proper citation, good ordering/arrangement, and so on. There are many things that should be pointed out including some trivial ones and I cannot list all of them. So, it is in order that the authors should thoroughly reassess to improve the manuscript. Below I only enumerate several points as examples.\n- For introducing the 26S-UIDs, the authors should also cite the work by Ali et al. (ByeTAC paper; doi: 10.1101/2024.01.20.576376). At the part of mentioning ‘a flexible fragment’, it will be good to cite the work(s) by Andreas Matouschek group as well.\n- TPD should be defined by targeted protein degradation, not by target protein degradation.\n- I see reference matching problems in many spots. The authors should try to find the most optimal references for citation (e.g. #19 at page 2, right reference? #25 at page 2 & 14 as a patent? Not full info? and so on).\n- There are also Supple Fig ordering problems. Ideally, the Supple Figs also should appear in order but Fig. S17A is the first one appearing (page 3) and thereafter, the Supple Figs are all shuffled up in ordering and very hard to follow.\n- Page 3, 4, the two sections of design of chimeras and synthesis of chimeras can be combined and compacted.\n- At multiple spots, it is said, (see SI). This is not good way to ask attention considering the SI file contains content over 130 pages. Should be more specifically designated.", "reviewer_2": "In this study, Casasampere and co-workers present a novel approach to protein degradation by designing small molecule chimeras that target the 26S proteasome via USP14, a deubiquitinating enzyme associated with the proteasome's regulatory particle. This strategy circumvents the traditional E3 ligase-dependent mechanisms used in classical PROTACs. The authors provide proof of concept for this new type of chimera, termed 26S-UIDs, by targeting two proteins of high relevance in cancer, IMPDH2 and CERT1. They present robust experimental data, including synthesis, in vitro assays, and cell-based studies, demonstrating degradation of the target proteins. Docking and molecular dynamics simulations were employed to explore the ternary complexes between USP14, the chimera, and the target protein, highlighting the potential complementarity between the binding partners. Overall, the manuscript is well-written and experimentally robust, with detailed results provided both in the main paper and supplementary materials. The authors showed careful consideration of many details, such as the consistency of the ternary complex in the proteasome context. I would recommend the editor accept this publication in Nature Communications after the authors address some points related to the molecular dynamics aspects that require attention. These points are as follows:\n1) Considering the current guidelines recommended for studies involving molecular dynamics simulations (e.g., JCIM, 10.1021/acs.jcim.3c00599), at least three replicas of each system studied, ideally starting from different coordinates and velocities with a discussion of the statistical variance observed in the calculations, should be included in the paper. The authors only use a single trajectory for all cases.\n2) For the sake of reproducibility and following the FAIR principles, it is recommended that the authors share their input files for the docking and atomistic MD simulations. This dataset could also include scripts used for the calculations and analysis. While sharing trajectories may be challenging, it could still be considered through repositories like Zenodo.\n3) The clustering analysis used to select the representative ternary complexes from the most populated clusters of the MD trajectories is not clearly explained. The only information provided on the methods states, “Trajectory analysis and hierarchical clustering were performed with CPPTRAJ.” Visualization of the cluster distributions and populations, along with more details on how the calculations were performed, would be beneficial.\n4) The authors mention that ternary complexes predicted via the Protac-model software workflow are mostly stable during the MD simulations. However, the RMSD plots in Figure S23, although showing some plateaus, indicate values around 6 to 8 Å in several cases. These values are quite high, indicating meaningful deviations from the initial ternary complex, not necessarily limited to loop changes. Direct comparisons of snapshots from the beginning and end of the simulations would be helpful. Additionally, addressing the first point (use of replicas) is crucial, as some observed structural changes may not be fully converged given that only one trajectory was analyzed.\n5) Lastly, and perhaps more importantly, I question whether the simulation length or the use of unbiased MD simulations is adequate for studying the ternary complexes like the ones shown here. Unlike stability MD simulation tests typically performed for relatively rigid protein-small molecule interactions in medicinal chemistry, PROTAC-like ternary complexes are known to be dynamic and plastic, as shown by prior experimental and simulation work in Nature journals (e.g., DOI:10.1038/s41589-018-0055-y and DOI:10.1038/s41467-022-33575-4). Obtaining meaningful data from MD simulations in these cases likely requires much more sampling than the authors have considered. I would ask the authors to explain why they believe simple/relatively short simulations are sufficient in their study.", "reviewer_3": "The manuscript by Casasampere et al entitled, “Expanding the target protein degradation approach with small molecule chimeras directed to the 26S proteasome”, presents data aimed to demonstrate how small-molecule ligands to the 26S proteasome can be used to facilitate targeted protein degradation in a ubiquitin-independent manner. These 26S-targeting ubiquitin independent degraders (26S-UIDs) aim to bypass limitations of traditional PROTACs, which facilitate co-localization of targets with E3 ligases for ubiquitin-mediated degradation, such as the efficiency of ubiquitination, E3 abundance in specific cell types, or the presence of modifiable lysine residues on the target protein of interest (POI). Moreover, 26S-UIDs would potentially be more versatile in the range of POIs that they could degrade than the initial, proof-of-concept, direct-proteasome chimeric degrader previously reported (Bashore et al Nat Chem Bio 2023) that utilized a large, macrocyclic peptide as a proteasome ligand as opposed to a small molecule. The authors seek to show this by developing and characterizing degraders towards two interesting oncology-relevant targets, instead of a standard proof-of-concept target in this field (e.g. BRD4), and further evaluate the cellular consequences of the engineered target degradation. Thus, the authors pursue a compelling area of research that seeks to expand the utility of direct 26S degradation strategies (although the claims of novelty are rather overstated given the earlier proof-of-concept study cited above, especially given that USP14 docks onto RPN1, the 26S component used in the previous study). Moreover, I think that the current characterization of the reported compounds and interpretation of the conducted experiments is limiting and I am not convinced that the data warrants the conclusions reported by the authors (see bullet points below). Based on these concerns, I cannot recommend this manuscript for publication in Nature Communications unless major revisions (including key degradation controls) are achieved.\nMajor points that require attention:\n1. While the authors show dose-dependent, chimera-mediated degradation of both CERT1 and IMPDH2, they do not conclusively show that this is due to direct 26S recruitment via USP14. First, the only evidence for a Ub-independent degradation mechanism in the paper is shown in Fig 6B, where pre-incubation with proteasome inhibitor appears to block CERT1 degradation while the E1 inhibitor has no effect on 368-mediated degradation at 24 hours. No such data is available for IMPDH2 because of the very long timeframes needed to observe degradation of this target (72-96 hours). Fig 6B also shows a single-point competition-binding experiment in which 100uM of free CERT1 ligand, 366 (although labeled on the blot as HPA) is used to block 10uM 368-mediated degradation of CERT1. Again, no such data is shown for free ligand blocking chimera-driven degradation of IMPDH2. Typically, chimera-based degraders are more thoroughly validated by titrating in either of the free ligands to compete for binding and thus disrupt ternary complex formation and degradation (not just a single competitor concentration). Besides the molecular modeling results, there is no evidence of compound-mediated ternary complex formation for either target. Also, there is no evidence that chimeric compounds bind to USP14 (and the targets) in the context of the assembled 26S proteasome, or that these proposed interactions mediate the observed target degradation. USP14 knock out or USP14 mutations that block compound binding to USP14 (or binding of USP14 to the 26S proteasome) would help provide important evidence to validate that direct 26S engagement via USP14 mediates the observed degradation as hypothesized. Alternatively, inactive stereoisomers of the chimeras could also provide appropriate controls in this regard.\n2. The authors state in the discussion that “the specificity and accurate behavior of the generated constructs were evaluated in cells, in vitro assays and by SPR”. It is unclear what is meant by “accurate behavior”, however the specificity of the chimeras did not appear to be well characterized in cells. The chimeras were shown to bind USP14 or their intended target using purified, recombinant protein stocks, but potential off-target binding in cells was not probed.\n3. While I commend the authors for exploring the downstream effects of CERT1 degradation on ceramide levels and drug sensitization, it is confusing why chimeras that achieve similar levels of CERT1 degradation show disparate effects on these readouts. There are also so many compounds and cell lines tested (all shown in separate supporting figures) that it is difficult to compare these results across these different conditions. Given the inconclusive aspects of these findings it is difficult to warrant such a large portion of the manuscript detailing these experiments—especially when the critical experiments in point #1 need to be addressed to validate the MOA of the targeted degradation.\n4. The authors must address the exceptionally poor organization of the manuscript. While the main text followed a relatively structured narrative, the haphazard curation of the figures, schemes and supporting information made the manuscript exceedingly and unnecessarily difficult to read. For example, here is the order of the first 10 figures/schemes/SI as they appeared in the main text of the manuscript: Fig1, scheme 1, Fig S17A, Fig S17B, Fig S16, Fig S17C, Fig 2A, Fig S20A-C, scheme S5 and scheme S6! This is unacceptable and should be resolved before any further review. Furthermore, the lack of organization is compounded by the sheer volume of data presented in the manuscript (135 pages of Supporting Information). Considerable editing is needed to trim the manuscript to the essential data. For example, there are 7 pages of molecular modeling results associated with 7 supporting figures, 12 supporting movies, and 9 supporting Tables all in the Supporting Information. This material is briefly touched upon in the main results section and yet seems to constitute what would be a separate companion paper given. The authors should either add the essential modeling data into the main results of the manuscript (as a substitute for the lack of biophysical ternary complex measurements), or make it a separate paper. Making this manuscript a repository for every experiment done on these systems does not enhance the paper and, in fact, detracts from the main ideas of the manuscript.\n5. There are several instances in the manuscript where conclusions are stated based on “data not shown”. While I am loath to add even more data to this manuscript given point #4, conclusions need to be supported by data that the reader can assess for themselves.\n6. It is not clear whether the CRBN- and VHL-directed chimeras described here (320, 343, 339, 360, 369 and 373) are really relevant to this work. Without evidence that these chimeras can actually mediate productive ternary complex between the targets and these E3 ligases, comparisons with the 26S-UIDs seems somewhat hollow. Linker length often requires optimization to produce effective degraders, so the fact that linkers that appear to work for the 26-UIDs don’t work for the ligase-directed chimeras is not surprising. However, if these ligase-directed chimeras were shown to mediate productive ternary complexes and target ubiquitination that was insufficient to cause target degradation, whereas direct engagement with the 26S efficiently degraded these targets, that would be a very interesting result and would further elevate the utility of direct 26S targeting degraders.\n7. The authors do not comment on the clear “hook effect” that they see with some of the 26S-UIDs. Do these make sense given the cellular levels of these targets and/or the high abundance of the 26S proteasome?\n8. I am also very curious to understand what forms of IMPDH2 are degraded given that we are told in the discussion that it forms various oligomeric complexes that are difficult to degrade. Are oligomers being targeted to the 26S and does engagement with the unfoldase machinery facilitate more efficient degradation than ubiquitination of the target? Does this “clog” the 26S such that degradation of other targets is impaired?\nMinor issues\n1. Some of the figures (including Fig 1, Scheme 1) have poor resolution when zoomed in on the digital version, which is needed given that the font is often too small (e,g Scheme 1).\n2. The yellow chimeras are very difficult to see in many of the structural figures (e.g. Fig 2A and Fig 4B) and red/green combinations should be avoided in light of red/green colorblind readers.\n3. It is unclear what degradation time point is being used in Figure 5B—24 hrs?\n4. In Fig 6B, HPA is used as a blot label instead of compound #366 as stated in the figure legend.\n5. Where are the raw SPR data used to generate the values in Table S2? Representative sensorgrams should be provided to evaluate the quality of the data. Do the weak affinities preclude using SPR to measure ternary complex formation?\n6. In Fig S1, can you better describe what 26S[VS]-USP14 is? Also compounds 352 and 354 are not introduced in the main text at this point, but are included in this analysis. Similar issue for compound 320 in Fig S2 and 360, 320, 339 and 343 in Fig S4.\n7. Ratios in Fig S3 are listed with commas instead of decimal points like the rest of the ratios shown throughout the manuscript. Please change to be consistent. Also, why does 300 increase IMPDH2 levels?\n8. Fig S5 is not listed in the main text, only in the figure legend of Fig 3.\n9. 386 should be changed to 368 in Fig S15K.\n10. The term “n” is not defined or described in the figure legend of Fig S16.\n11. Please add arrows to denote proposed attachment vectors in Fig S17A and B."}, "author_response": ""}, {"round": 2, "reviewer_comments": {"reviewer_1": "In the revised version, I recognize that the authors put substantial efforts to improve their work, which indeed forms the manuscript to be in a better shape. However, some critical issues still remain to be not well resolved, and also I have to raise a few of extra questions, some of which are based on newly added results.\n1. Even after the authors’ rebuttal, the reason for idiosyncratic degradation of IMPDH2 by 26S-UID (i.e. it takes long time up to 72-96 hr before suddenly being degraded) has not been convincingly delivered. Current statement may not be satisfactory or should not be said like “unknown”. Performing additional experiments is in order to retrieve some hints about the properties of IMPDH2 turnover by the degrader. Below are some opinions or thoughts about point #1.\n- The authors discussed like “…dynamic tetrameric, octameric and filamentous complexes,[69] making this protein difficult to inhibit and to degrade”, “…the association status of targets has been proposed as a possible cause for time-consuming degradations[70–72]”, but these statements are quite ambiguous until demonstrated to some extent.\n- Besides, the authors cited Refs 70-72 as examples of time-consuming degradation analogous to IMPDH2 degradation, but in Ref 70 (Zheng M et al., 2021), SHP2 degradation by SHP2 PROTAC can be still observed as early as 48 hr and this paper may not be anything about “the association status of targets” since there are also many other SHP2 PROTACs available nicely working in shorter time points. Ref 71 (Zeng S et al., 2021) shows EGFR degradation by EGFR PROTAC, a membrane protein probably not ideal comparison to IMPDH2. Ref 72 (Cyrus K et al., 2011) is about ERα degradation by PROTAC, in which for longer incubation (72-96 hr), medium change by twice treatment leads to better degradation, yet a different case from the IMPDH2. In any case, all these are not good examples to support the authors’ argument. Furthermore the PROTACs in there are still working at earlier time points, just gradually reaching degradation saturation when longer treated, which is distinct from abrupt IMPDH2 degradation at longer time points in this study.\n- The mode of delayed IMPDH2 degradation is bizarre but very intriguing. The most potent IMPDH2 degrader is 354, working at sub-μM range, and only get to such potency for longer 96 hr incubation. On the other hand, CERT1 degraders work as early as 24 hr but none of them show sub-μM conc of potency. How mechanically this observation can be explained? For shorter time points treatment, only 300 and 330 were shown (Supple Fig. 6), so wondering if 354 displays similar behavior as others.\n- At lines 35-36, page 3, the authors quoted solely reported IMPDH2 PROTAC (Ref 41). Curiously, this PROTAC works even for 24 hr treatment although at μM level. So, then the delayed degradation by IMPDH2 degrader may not be necessarily the nature of the target protein, rather due to the nature of degrader?\n- In the similar context of above point and also as the authors described, Supple Fig. 8 shows the cellular amounts of 354 at 96 hr as ~4 times higher than at 24 hr. Thus, apart from the nature of IMPDH2 protein, would be there any chance of the delayed degradation kinetics also to be attributed to the actual working conc of IMPDH2 degrader in cells over time? Then, what will happen if the degrader would be treated every 24 hr for total 96 hr compared to one time treatment for 96 hr as it is now?\n2. I see one of the major revisions as the addition of ‘Modeling of 26S-UIDs ternary complexes’ part. Indeed for this, the authors assigned rather extensive amount of writing with one spacious main Figure (Fig. 7). However, all these arguments are limited by degrader modeling, not real data or resolved 3D complex structures. So, unless experimentally validated, they are just nothing but speculation no matter how reasonable it sounds like.\n3. In relation to my point #2 above, I have some concerns on the argument that the authors deduced from the degrader modeling data. At lines 62 to 66, page 9, it is said, “…These findings suggest that the structures of chimeras 419 and 395 constrain the proteins to bind in a less optimal manner, which correlates with the relatively lower potency as degraders of these chimeras…”. However, although 395 and 419 are less potent than others in MDA-MB-231 cells, they are no worse than others in MDA-MB-453 and BT-474 cells. Then, I tend to think that these cell type-dependent differences in responsiveness cannot be explained simply by the ternary complex modeling. Also, apart from this modeling issue, in terms of the responsiveness, I am wondering why different cell lines show inconsistent degree of sensitivities among the CERT1 degraders? For example, as aforementioned, 368, 386, and 413 seem to be the most potent over 395 and 419 but again only in MDA-MB-231. In MDA-MB-453, both 395 and 419 look superior to 386 and probably even 413. Also in BT-474, 368 does not look that better compared to 395 and 419. How the authors could explain this conundrum?\n4. In the similar context of above point, in Fig. 6, what is the reason to show each graph derived from each different cell line?\n5. At Fig. 3f, the authors performed the competition assays with VX-497. Since they did the same kind of assays also with USP14 ligand in case of CERT1 degrader (Fig. 5c), please do it as well for IMPDH2 degarder. The authors commented the partial recovery by VX-497 due to its toxicity (page 6, lines 1-4), so the non-toxic 352 would be expected to be good competitor for IMPDH2 degrader as well.\n6. At page 5, lines 19-22, it is said, “Together, these results show that chimeras can productively interact with proteasomal USP14 and IMPDH2 in a functional context, suggesting the feasibility of our approach.” Regarding SPR data, the SPR was performed with USP14 in the absence of proteasome, so apart from the inhibitory acivity, the binding affinities of all tested chimeric compounds may just reflect their interaction with free USP14, not “proteasomal USP14”.\n7. At page 2, lines 32-35, the sentence of “USP14 is a deubiquitinating enzyme that associates …. in the regulation of the ubiquitin-proteasome pathway, with an impact in multiple cellular processes” seems to be somewhat redundant with the paragraph starting from line 45. Also, it would be better to introduce the proteasome first. So, please relocate this sentence to the back or combine with the latter by compacting and rephrasing or can just delete this sentence.\n8. The methods for IMPDH2 enzymatic assays should be described in details.\n9. Other miscellaneous points\n- Page 2, Ref 4 is not a good citation for PROTACs in clinical trials.\n- All the Figures are presented as A, B, C… but in the text, they are all a, b, c…..\n- Page 3, line 28, Inosine -> inosine\n- Page 3, line 78, inhibitor 77 -> inhibitor VX-497?\n- At Fig. 3f, last lane (VX-497, 50 μM), what happens here? I do not see any band in both blots.\n- USP14(-/-), multiple spots, the correct gene nomenclature for mouse and for KO should be used.\n- Page 7, line 7, that -> than\n- Page 9, line 68, 26STAC -> 26S-UID?\n- Page 11, lines 28 to 31, the sentence is not well matching with given references (Refs [9,21,22,65–67]).\n- In Acknowledgements, I bet Usp14-/- & WT MEF cell lines were originally generated by Scott Wilson group (U of Alabama). MJ Lee could be a donor but this person probably is not an original creator. I am kind of concerned that the original creator might not be properly acknowledged in this way. Please check it out and indicate rightful credit.", "reviewer_2": "This is my second review of the manuscript, and first of all, I recognize the efforts of Casasampere and co-workers to improve the study. However, I am concerned about the complete removal of the molecular dynamics simulation data related to the ternary complexes, as this alters the scope of the original work and prevents a full evaluation now.\nRather than refining or clarifying the methodological concerns raised, removing this section changes the scientific contribution initially presented. If every critique were addressed by eliminating data instead of improving it, it could set a concerning precedent for scientific discourse.\nThat said, I defer to the editor’s judgment on the overall suitability of the revised manuscript but wanted to share my perspective directly.", "reviewer_3": "The authors have made valiant efforts to address the many issues raised by reviewers. In particular, the USP14 KO experiments and competitor titrations go a long way in strengthening the authors’ claims of USP14-mediated recruitment to the 26S proteasome for ligase-independent degradation. However, I am still perplexed by the lack of direct ternary complex formation studies in this work and instead the reliance on molecular modeling of the proposed ternary complexes.\n1. Given that the necessary molecules and protein reagents are available for individual SPR binding experiments, why not measure compound-mediated ternary complex formation? The SPR data that is shown has some issues (see point 2) and the relatively weak double-digit micromolar (or higher) affinities may limit the technical feasibility of performing these kind of SPR-based, ternary complex measurements, but simple pulldown experiments with the recombinant proteins ± compound would easily confirm compound-mediated ternary complex formation. Because the cellular activity of these compounds is often at concentrations 10-fold lower than the measured Kd values for either USP14 or target it suggests that these compounds can act as molecular glues with USP14 and targets forming protein-protein interactions (as is suggested in the modeling). It should be straight forward to directly observe USP14-compound-target ternary complexes and measurements of the lifetime of these complexes would go a long way in explaining their cellular potency. Confirming that these complexes can also pull down 26S components in a compound-dependent manner would provide further critical validation of the MOA proposed for these compounds.\n2. The representative SPR sensorgrams shown in Supplementary Figure 5 are not conducive to kinetic fitting as described in the methods and should instead be fit using equilibrium measurements to better quantify the Kd values for these compounds binding to USP14 and either target. This kind of analysis is common for weak micromolar binding as is evident in the raw sensorgrams."}, "author_response": "Point-by-point answer to reviewers of manuscript\nWe deeply thank the referees for their thoughtful, rigorous and constructive comments, which we think have\nbeen of enormous help for us to write a better manuscript. All the reviewers’ suggestions, concerns, and\ncriticisms have been addressed in the revision as outlined in the following pages (comments, and specific\nanswers to each one, are numbered and abbreviated as C and A, respectively). To facilitate the reviewers’ work,\nwe submit a version of the main text (file “NCOMMS-Revised Main Text with marks”) in which changes related to\ncomments by Reviewers 1, 2 and 3 are in blue, green and orange, respectively. In the case of Reviewer 3,\nchanges associated to comments already raised by Referee 1 are in blue. On the other hand, with the aim to\nease the reviewers’ evaluation, please note that this document contains links to external files and to internal\nparts of the document.\nWe hope that the three referees will now find the article suitable for publication in Nat. Comm.\nReviewer 1\nC1. Above all, the authors need to confirm their IMPDH2 and CERT1 degraders in USP14 knockout or depleted\ncells with WT cells as control. These experiments will strongly demonstrate the mode of action of 26S-UIDs to be\nbona fide USP14-mediated and 26S directing for POI degradation.\nA1. We thank the reviewer for this comment, to which we completely agree. We have performed assays with\nMEFs wild-type and USP14-/- (from Dr. Min Jae Lee; Kim et al. Cell Rep. 2018, 24, 732). Cultured MEFs were\ntreated with 368 and 354, as representative compounds for CERT1 and IMPDH2 targeting, and have observed\nthat target degradation occurred in WT, but not in USP14(-/-) MEFs. We have included these results in Fig. 3g\n(IMPDH2) and Fig. 5f (CERT1) and added details in the corresponding legends. The results are discussed in the\nfollowing parts of the text:\n-Page 6, line 10: “To show that IMPDH2 degradation occurs in a USP14-dependent manner, the effect of 354\non IMPDH2 degradation in MEFs USP14(-/-) versus wild-type was tested. We observed that IMPDH2\ndegradation was induced in MEFs-WT, whereas in MEFs USP14(-/-) degradation was not only prevented (Fig.\n3g), but the IMPDH2/Actin ratios were significantly higher in 354-treated cells that in controls. This resembles\nthe effect observed with VX-497 (see above), indicating that in the absence of USP14, the chimera acts as an\nIMPDH2 inhibitor and IMPDH2 levels increase by a feedback response of the purine synthesis pathway.\n-Page 8, line 25: “To show that CERT1 degradation is USP14-dependent, 368 effect was tested in MEFs,\nUSP14(-/-) versus wild-type. It was observed that CERT1 degradation was promoted in MEFs-WT, but not in\nMEFs USP14(-/-) (Fig. 5f).\n-Page 11, line 57: “Importantly, the USP14-dependence of target degradation was demonstrated in USP14(-/-)\ncells, in which degradation of both targets was prevented (Figs. 3g and 5f).”\n-Supplementary information: MEFs have been added to the “Cell culture” Section.\nC2a. It is intriguing [C2a-1] why USP14 was selected as 26S docking site over others. Although the authors\nlisted four reasons for its consideration (Page 3), those are still not super convincing especially given that USP14\nexists as a free form as well (also stated in Discussion, Page 11). This means that depending on the population\nof USP14 as a free form, the 26S-UIDs may not be maximally productive. Also [C2a-2] the possible influence on\nendogenous USP14 function when treated with these degraders should be described, in terms of native Ub\nconjugates processing by USP14 or other physiological impacts. BTW, [C2a-3] the second reason for USP14\nconsideration (Page 3; “….USP14 exhibits activity on recruited proteasomal substrates as they translocate\nthrough….”) sounds like a wrong statement, thus needs to be corrected.\nA2a-1. Regarding the first part of the comment ([C2a-1]), we have slightly modified (new text in blue) the\narguments given in the original version (first paragraph of the “Design and synthesis of chimeras targeting the\n26S proteasome regulator USP14” section of the revised version: …”First, USP14 is localized at the base of the\nRP, interacting with the receptor Rpn1 and establishing contacts with ATPase subunits (New Fig. 1a).[13]\nSecond, USP14 exhibits trimming activity on ubiquitin chains attached to the substrates and adopts positions\nproximal to the ATPase pore.[13,16] Third,…), which we humbly think are strong enough to sustain USP14\nselection as recruiter. To reinforce the arguments deployed in the text, we have included the panel “A” in Fig. 1,\nwhich illustrates the position of USP14 in the context of the RP. However, we agree that the existence of USP14\n1\nas a free form as possible interactor with IU1-based chimeras may apparently weaken the approach. However,\nthe published data and our observations strongly suggest that this interaction is secondary and not detrimental\nfor the degradative action of IU1-based chimeras. Thus:\n1.- That the proteasome is the main interactor of USP14 is proved in affinity purification experiments showing\nthat USP14 copurifies with the proteasome and that 26S copurifies with USP14 isolated from human cells. In Liu\net al., (Nat Commun. 2018, 9:4770), USP14 pulldowns showing USP14-associated proteins are reported (in the\nSupplementary Dataset 5). It is shown that the entire 26S proteasome complex is the main interactor of USP14\n(linked file).\n2.- According to data from Lee et al. (Nature 2010, 467, 179–184), the main target of IU1 family compounds is\nthe proteasome-bound USP14. Furthermore, using cycloheximide (CHX) sensitivity assays in both S. cerevisiae\nand HeLa cells, we have observed that IU1 and IU1-derived compounds exert an action on proteasome-bound\nUSP14. This assay is based on a conserved phenotype linked to the proteasomal functions of USP14: the CHX\nsensitivity of cells in the absence of USP14 activity (Hanna et al., Mol Cell Biol. 2003, 23:9251-61). Using a yeast\nstrain that expresses human USP14 in a double gene deletion background (ubp6Δpdr5Δ), where ubp6 is the\nUSP14 yeast ortholog and pdr5 is a multidrug resistance pump (its deletion prevents drug efflux), we have\nobserved that (1) human USP14 rescues CHX sensitivity of ubp6Δpdr5Δ yeast strains (Fig. Rev 1A,B) and (2)\nUSP14 inhibitor IU1-248 and its derived active IMPDH2 degraders 300 and 329 induced CHX sensitivity (lower\ncolony formation than in controls). In contrast, IU1-c, which does not inhibit USP14 (Lee et al. Nature 2010, 467,\n179–184) and 317 and 354, which are a poorer USP14 inhibitors than the IU1-248 family (see supplementary\nFig. 4), did not induce CHX sensitivity (Fig. Rev 1C,D. Colonies were formed as in the DMSO control). Similar\nresults were found in HeLa cells, where all compounds, except for 317, with very poor USP14 inhibitory activity\n(see supplementary Fig. 4), induced CHX sensitivity (Fig. Rev 1E, compare number of colonies in plates with\nCHX 0.2 g/L and 5 M of compound).\nThese observations show the capacity if IU1-based compounds to interact with cellular proteasome-bound\nUSP14, and that putative alternative intracellular interactions of IU1 compounds do not prevent their binding to\nproteasomal USP14.\n3.- We have added a set of experiments showing that degradation by means of IU1-based 26S-UIDs is USP14-\ndependent, proteasome-dependent and ubiquitination-independent (See answers A1 and A3c to Referee #1,\nwhich are the same as answers A4 and A7 to Referee #3).\nA2a-2. Regarding the second part of the comment ([C2a-2], “Also the possible influence on endogenous USP14\nfunction when treated with these degraders should be described, in terms of native Ub conjugates processing by\nUSP14 or other physiological impacts”), this is indeed a very important aspect and we thank the reviewer for\nraising it. In order to assess the physiological impacts of 26S-IUDs degraders in proteasome activity, we\nanalyzed the formation of K48-ubiquitinated conjugates in cell extracts after the different treatments shown in\nFig. 5a. WB images of all replicates are shown in Fig. Rev 2. As shown, 24 h treatments with either 395 or 368\ndid not alter the formation of K48-Ub, indicating that 26S-UIDs did not cause a strong alteration in the status of\nthe UPS. On the contrary, the proteasome inhibitor marizomib caused a strong effect, with high accumulation of\nubiquitin conjugates. Remarkably, marizomib-induced accumulation of ubiquitin conjugates appeared to be lower\nin PROTAC’s treated cells than in treatments with marizomib alone (Fig. Rev 2, compare left, “DMSO” vs\nPROTAC lanes). This difference correlates with enhancement of proteasomal activity induced by IU1\ncompounds, and validates that these compounds interact with proteasomal USP14 in cells. On the other hand,\nthe sensitivity to cycloheximide discussed point 2 of A2a-1 can be considered a readout of the interaction of IU1-\ncompunds with proteasomal USP14 and of the impact of IU1-based compounds in proteasome function (Fig.\nRev 1.)\nWhether this increased proteasomal activity results in unspecific degradation of other proteins (off-target effects)\nis a possibility that needs to be examined (global proteomic experiments are planned for the near future). In any\ncase, we humbly think that the occurrence of off-target effects does not invalidate the proof-of-concept that\ntargeted protein degradation can be achieved with USP14-directed chimeras.\n2\nexhibits activity on recruited proteasomal substrates as they translocate through….”) sounds like a wrong\nstatement, thus needs to be corrected”), we thank the reviewer for raising this point. We agree that the sentence\nwas not properly formulated and it has been changed to “USP14 exhibits trimming activity on ubiquitin chains\nattached to the substrates and adopts positions proximal to the ATPase pore.[13,16]“ (Page 3, line 57).\nIn order to provide a more extended explanation of the rationale for selecting USP14 without overloading the\nmanuscript with additional data, we have made the following changes:\n-Page 2, line 32: “USP14 is a deubiquitinating enzyme that associates and allosterically controls the 26S\nproteasome, and is involved in the regulation of the ubiquitin-proteasome pathway, with an impact in multiple\ncellular processes.”\n-Page 2, line 45: “The removal of Ubiquitin chains from substrates by proteasomal deubiquitylating enzymes is\na tightly regulated step in the 26S mechanism [14–17]. In this process, USP14 trims supernumerary ubiquitin\nchains conjugated in substrates, and, by a sophisticated allostery, controls the steps of ubiquitin recognition,\nsubstrate translocation initiation and ubiquitin chain recycling. Notably, USP14-26S proteasomes in exiting\nconformational states dissociate trimmed ubiquitin slower, causing a delay in substrate reloading[13]. As a\nconsequence, USP14 was initially identified as a delayer of proteasomal activity[16,18]. Additionally, chemical\ninhibition of USP14 with specific USP14 inhibitors (IU1 compounds)[16,19,20], has an enhancement effect of\nproteasomal activity[16].”\n-Page 3, line 22: “This approach exploits the positioning of USP14 in the RP, and shares similarities with the\nrecently described Rpn1-directed chimeras,[33] although in that case the Rpn1 ligands were based on\nmacrocyclic peptides. More recently, Protacs using Rpn13 as recruiter have been also reported,[34]”.\n-Page 3, line 56: “Second, USP14 exhibits trimming activity on ubiquitin chains attached to the substrates and\nadopts positions proximal to the ATPase pore.[13,16]”\n-Page 3, line 65: “Notably, IU1 compounds have not been explored as chimera ligands yet. Therefore, we\nhypothesized that IU1-based chimeras would interact with the proteasome via USP14 and localize targets to\na position prone to degradation, bypassing the requirement of previous ubiquitination”\nC2b.The rationale for selecting IMPDH2 and CERT1 as target substrates is even more ambiguous. This is also\nbecause E3-based IMPDH2 and CERT1 PROTACs were not successfully made and thus cannot be fairly\ncompared to the 26S-UIDs for evaluating them as good proteasomal substrates. In fact, quite curious why the\nauthors selected those two POIs over the well-established targets that have been repeatedly employed in\ngenerating canonical PROTACs.\nA2b. The aim of this study is to unveil novel degradation ligands and a distinct mechanism, and to apply them in\nthe degradation of therapeutically relevant and poorly explored targets in the TPD field. For that reason, we\nselected two relevant cancer targets that had raised the interest of the team. First, IMPDH2 was the focus in the\ncontext of a collaboration with the leukemia expert Prof. Ferrando (initially at Columbia U., now at Regeneron\nGenetics Center). IMPDH2 is well-known regulator of purine metabolism and a target in DNA synthesis. IMPDH2\ninhibition has been widely investigated in leukemia and lymphoma treatments, and in the last years it is\nemerging as a promising strategy against multiple cancer types (lung, esophageal, colorectal, prostate, breast,\novarian, glioblastoma, osteosarcoma, melanoma, pediatric hepatoblastoma, pancreas) (a reference list is\nincluded here). Additionally, IMPDH2 is overexpressed in cancer cells, which has been linked with proliferation\nand chemoresistance (here). Therefore, for these reasons, we considered IMPDH2 a good candidate in our\napproach. Furthermore, as we mention in the manuscript, IMPDH2 targeted degradation with chimeras is still\npoorly explored. Second, the relevance of the ceramide transporter protein (CERT1) in ceramide metabolism\nand its role in cancer were mentioned and discussed in the original version of the manuscript. The identification\nof therapeutic targets within sphingolipid metabolism proteins has been a matter of investigation in Prof. Fabriàs’\nlaboratory for years. The lack of degraders in this field prompted us to select CERT1 as a proof-of-concept target\nin this study.\nBeyond that, we totally agree to the reviewer’s comment. In fact, we initially prepared two USP14-based BrD4\ndegraders featuring the BrD4 ligand JQ1 and the USP14 ligands IU1-248 and 317 bound with a PEG3 linker\n(compounds 351 and 362 in the Figure below), and tested their degradative properties along with the reported\nVHL-based degrader MZ1. Unfortunately, cell viability was severely compromised by both chimeras at the high\nconcentrations and times required for degradation (>40 and 60 µM, 16 h). In contrast, MZ1 was a more potent\ndegrader (complete degradation at 10 µM, 16 h), although not devoid of cytotoxicity in the assay conditions.\nInstead of dedicating further medicinal chemistry efforts to obtain better USP14-based BRD4 degraders, we\ndecided to proceed with IMPDH2 and CERT1, for which better USP14-based degraders were being achieved.\ntargets. Changes made in the main text include:\n-Page 3, line 32: “lymphoma and other types of cancer. Moreover, elevated levels of IMPDH2 have been\ncorrelated cancer progression and chemoresistance.”\nC3. As the authors also argued, the degradation behavior of IMPDH2 by 26S-UIDs seems to be odd because\nonly long time treatment was able to induce degradation of the POI (72-96 hr). This can be an issue or even\nconcern in interpreting the data, so should be more thoroughly addressed. For example, [C3a] more time points\ndegradation assays need to be done in parallel since the current scheme for the chimeras treatment is not\nsystematically investigated or mostly end point assay (i.e. at one time point). [C3b] The authors also need to\nconsider whether 26S-UID-mediated IMPDH2 degradation is coming from any secondary effect.[C3c]In the\nsimilar context, the authors should find optimal conditions to treat proteasome inhibitor and MLN7243 inhibitor\nduring the course of treatment to prove Ub-independent but proteasome-dependent degradation, although they\nin fact stated that those studies can be hampered due to the cytotoxicity by long time incubation. [C3d]Besides,\nit is not clear how the authors treated the IMPDH2 degraders for 72 or 96 h, in other words did they treat initially\none time till the end or multiple times by changing the compound-containing media during the course of\nincubation? If the former is the case, how stable the compounds are expected and how then the initial one time\ntreatment suddenly starts working at 72-96 hr? In any case, the detailed experimental scheme should be also\ngiven in Method section.\nA3a. We include the results addressing this important point in Supplementary Fig. 6. In the initial steps of the\nfunctional characterization of IMPDH2-targeting 26S-UIDs, the target degradation by 300 and 330 was analyzed\nin a wide range of times and concentrations spanning from 0.1 nM to 100 nM (4 h and 20 h treatments) and from\n0.1 nM to 3 M (8 h treatments) and no changes on IMPDH2 levels were observed (Supplementary Fig. 6A and\n6B). Additionally, treatments for 24 h in a concentration range from 1 nM to 10 M did not induce degradation,\nbut a slight increase in IMPDH2 levels (Supplementary Fig. 6C). Similarly, 48 h treatments with compound 300\nshowed increase in IMPDH2, but not degradation (Supplementary Fig. 6D).\nWhen treatments were performed for 72 h, degradation of IMPDH2 was observed in the presence of 300 10 M\n(Supplementary Fig.6D, right panels). In parallel assays, VX-497 at 48 and 72 h caused the same IMPDH2\ninduction effect observed for 300 and 330 at 24 and 48 h (Supplementary Fig. 6E). In the same conditions, IU1-\n248 caused not effect on IMPDH2 levels (Supplementary Fig. 6F).\nThe increase in IMPDH2 in the presence of chemical inhibitors is not unprecedented and it has been described\nin several cell types in the following articles (which have been included as references [50-52] in Page 5, line 77):\n-Chiarelli L. et al., Inosine monophosphate dehydrogenase variability in renal transplant patients on long-term\nmycophenolate mofetil therapy. Br J Clin Pharmacol. 2010, 69, 38.\n-Jain J. et al., Regulation of inosine monophosphate dehydrogenase type I and type II isoforms in human\nlymphocytes. Biochem Pharmacol. 2004, 67, 767.\n-Sanquer S. et al., Induction of inosine monophosphate dehydrogenase activity after long-term treatment with\nmycophenolate mofetil. Clin Pharmacol Ther. 1999, 65, 640.\nThus, by performing assays at different incubation times and compound concentrations, we could determine that\nIMPDH2 degradation by means of 26S-UIDs is a time-demanding process (observed at 72-96 h range), and that\nin the 24-48 h range, the 26S-UIDs induce an increase in IMPDH2 proteins levels, consistent with the literature,\nindicating the primary targeting of IMPDH2 in cells.\nConcerning the treatment times that IMPDH2 degradation requires, there are examples of degradation of targets\nby Protacs in 72 or 96 h in the literature (Cyrus et al., Impact of linker length on the activity of PROTACs. Mol\nBiosyst. 2011, 7, 359; Zheng et al., Novel PROTACs for degradation of SHP2 protein. Bioorg. Chem. 2021, 110,\n104788; Zeng et al., Proteolysis targeting chimera (PROTAC) in drug discovery paradigm: Recent progress and\nfuture challenges. Eur. J. Med. Chem. 2021, 210, 112981). Moreover, in the latter article, Zheng et al.,\nhighlighted that proteins showing time-demanding degradation by PROTACs are associated to cellular structures\n(e. gr., membranes), linking slow degradation with physical association of a target. These evidences underline\n5\nthe possibility that the time-demanding degradation of IMPDH2 could be linked to intrinsic properties and\nassociation status of IMPDH2, as we mentioned in:\n-Page 5, line 31: “300 was able to induce the degradation of IMPDH2 at low μM concentrations, whereas 330\nwas less potent and 329 was inactive. However, in time-course experiments ranging from 4 h to 96 h,\ndegradation was observed only at long times (72-96 h) after a single treatment with different chimera\nconcentrations (Figs. 3a, b and Supplementary Fig. 6), which denotes a time-consuming process as compared\nto those reported for E3-based Protacs”.\nA3b. Besides all the above, additional proofs that IMPDH2 degradation is unlikely coming from a secondary\neffect include:\n1. In the IMPDH2 enzymatic assays, VX-497 and VX-497-derived compounds are competent in IMPDH2 binding\nand inhibition (Supplementary Fig. 3), indicating the targeting of IMPDH2 in vitro.\n2. The IMPDH2 26S-UIDs bind (Supplementary Table S2) and inhibit (Supplementary Fig. 4) USP14.\nA3c. To cope with the cytotoxicity issue occurring at long incubation times we added the inhibitors at the end of\nthe assay instead of performing preincubations with the inhibitors at the initial hours of the culture. Moreover, we\nprepared a HeLa derived clone stably overexpressing IMPDH2-GFP (clone 522-4). As expected, these cells\nwere more resistant to 354 than the parental HeLa cells (Fig. 3c). Gratifyingly, with these adjustments,\nincubation conditions could be found in which the proteasome inhibitors (bortezomib and marizomib) and\nMLN7243 could be used without provoking rupture of the plasma membrane and extensive cell death despite\ncausing cell detachment. The precise experimental conditions are given in the section “Cell treatments” of the\nSupplementary Information. The new results showing that proteasome inhibitors (bortezomib and marizomib)\navoided 354-induced IMPDH2 degradation while MLN7243 did not have been added to the manuscript and are\nshown in Fig. 3d and e. Changes in the text are:\n-Page 5, line 79: “Mechanistic studies using proteasome CP blockers (bortezomib and marizomib) were\nperformed in HeLa and in HeLa cells stably overexpressing IMPDH2 (clone 522-4), more resistant to 354\nthan the parental HeLa cells (Fig. 3c). As shown in Fig. 3d, the proteasome inhibitors bortezomib and\nmarizomib rescued IMPDH2 degradation induced 300 (in HeLa) and by 354 (in 522-4). In the latter case,\nIMPDH2 overexpression protected cells from massive death, but a higher degrader concentration was\nrequired for depletion of the overexpressed protein. Conversely, the E1-Ub activating enzyme inhibitor\nMLN7243 did not prevent the target degradation (Fig. 3e).”\nA3d. We thank the reviewer for this thoughtful comment. The compounds were added only once. The\nexperimental procedure is more detailed in the revised Supplementary Materials, Section “Cell treatments”,\nwhere specific conditions for each experiment are given and illustrated. Regarding the compounds’ stability, we\nanalyzed (LC/MS) the amounts of 354 present in both HeLa cells and medium at 24 h and 96 h after the\ncompound’ addition and confirmed its occurrence in both matrices. The analyses showed that the normalized\namounts of 354 given as pmols/106 cells were about 4 times higher at 96 h than at 24 h. This indicates that the\ncompound’s uptake increases over incubation time and that it is stable for at least 96 h. The results are\npresented in Supplementary Fig. 8 and mentioned in:\n-Page 5, line 68: “However, like with 300, IMPDH2 degradation occurred at long incubation times (72-96 h),\nat which it was stably present in the medium as shown by LC/MS analyses (Supplementary Fig. 8).”\nAs for the question why the treatment starts working at 72-96 h, we mentioned above the occurrence of\nexamples in the literature of degradation of targets by Protacs in 72 or 96 h time frames and the finding that\ntime-demanding degradation by Protacs are associated to physical association of the target. These evidences\nunderline the possibility that the time-demanding degradation of IMPDH2 could be linked to intrinsic properties\nand association status of IMPDH2, as we discuss in the revised manuscript:\n-Page 11, line 79: “Degradation of ER and SHP2 in 96-h treatment has been reported, and the association\nstatus of targets has been proposed as a possible cause for time-consuming degradations[70–72].At this point,\nthe IMPDH2 form subjected to degradation is unknown.”\nC4. It is intriguing to see no effects on degradation by E3-based IMPDH2 and CERT1 PROTACs. Since these\nPROTACs are not capable and thus do not serve as any good comparison, how the experiments done with\nthese compounds are fit into this story (unless the authors argue for their 26S-UID superiority over the E3-based\nPROTACs at least on IMPDH2 and CERT1)? On the other hand, did the authors fully optimize the conditions for\nthe treatment of these PROTACs? CRBN- and VHL-based PROTACs often work quite well even at nanomolar\nconcentrations but show adverse, so called ‘hook’ effect at high doses. In this study, the authors routinely\ntreated the E3 PROTACs at uM ~ tens of uM, so curious whether the titrations of nM to tens of nM make any\ndifference. In fact, to my eyes, the lowest conc of CRBN-directed IMPDH2 PROTAC was 10 nM (Fig. S4B), in\nthere the POI seems to be less in level. In any case, the authors should test a serial concentrations of E3-based\nPROTACs starting from low nM or sub nM, and also provide the rationale for using these canonical PROTACs in\nthis study.\nA4. Although we did not observe IMPDH2 and CERT1 degradation with different concentrations (from low nM to\ntwo-digit µM) and incubation times (from 4 to 96 h) of E3-based Protacs, we fully agree that further structural\nmodifications of the linker would likely have provided active E3-based degraders. To simplify the manuscript,\nmeaningless data about these chimeras have been removed in the revised version.\nC5. In Fig. 6B, the marizomib (Mar) effect seems to be partial (0.6). Did the authors also test the autophagy\ninhibitors, such as bafilomycin? I am asking this question because recently there are number of papers that\ndescribed the role or effect of USP14 and its inhibitors on autophagic pathways. By utilizing USP14 ligands in\nmaking 26S-UIDs, wondering if the partial recovery by Mar treatment could be due to any possible contribution\nalso coming from autophagic degradation.\nA5. This is a very interesting point and we thank the referee for raising it. We tested the possibility of autophagic\ndegradation of the target in experiments combining the uses of marizomib and autophagy inhibitors (lysosomal\nprotease inhibitors and chloroquine) and the results indicated that the contribution of autophagy, if any, was\nnegligible. The results are shown in Fig. 5d, e (details in the legend). Additions to the text include:\n-Page 7, line 55: “Since marizomib did not rescue CERT1 degradation to the control levels, the possibility of\ninduction of autophagosomal degradation of CERT1 by the USP14-based degraders was investigated. To\nthis end, the levels of LC3-II, a hallmark for autophagy, were determined in MDA-MB-453 treated with 386 in\nthe presence or absence of autophagy inhibitors. As shown in Fig. 5d, 386 induced CERT1 degradation to\nsimilar extents in both the absence and the presence of lysosomal protease inhibitors (lanes 3 vs 7), which\nblock the last step of autophagic degradation, leading to accumulation of LC3-II. Moreover, in both cases,\nmarizomib rescued CERT1 degradation to similar levels (lanes 4 vs 8). Similar results were found with\nchloroquine (Fig. 5e, lanes 3 vs 7 and lanes 4 vs 8), which inhibits the autophagy flux by impairing the\nautophagosome–lysosome fusion. Overall, these results indicate that CERT1 degradation involves the\nproteasome and that the contribution of autophagy is negligible.”\n-Page 11, line 84. “It is worth to mention that proteasome inhibitors, although notably decreasing target\ndegradation, did not reestablish target levels to those observed in the controls (Figs 3d and 5a, d, e). The fact\nthat USP14 exists in an extraproteasomal pool and that USP14 plays a role in autophagic flux regulation[21,22]\nmakes conceivable the participation of additional proteolytic pathways under certain conditions. In this regard,\nour studies with the CERT1 degrader 386, which brought about similar CERT1 depletion in absence and\npresence of blockers of the autophagic flux rules out the participation of autophagy, at least in our\nexperimental conditions.”\nC6. In Fig. 7, it is puzzling that those lapatinib resistant cancer cell lines are not really lapatinib resistant\ncompared to WT counterparts. Also what is the point of using this lapatinib resistant cell lines in this study?\nThe authors need to address these questions.\nA6. The reviewer is absolutely right. In fact, resistance is minor and observed at 72 h, but not at earlier time\npoints. To simplify the manuscript, we have removed all data obtained with lapatinib resistant cells. See also\nanswer A8.\nC7. Most of phenotypic and readout assays were centered on CERT1 but not IMPDH2. Probably from this\nreason, Discussion section was also far more allocated to CERT1 part. The authors should show any kind of\nfunctional readouts from their IMPDH2 degraders, not just the protein level.\nA7. We thank the referee for this suggestion. Since IMPDH2 inhibition has been reported to reduce cell viability,\nwe tested the effect of degrader 354, the IMPDH2 inhibitor VX-497 and the truncated chimera 352 on HeLa cells\nviability. The dose-response curves are shown in Fig. 3c, right. The effect of 354 on cell viability of both wild type\nand IMPDH2-overexpressing clone 522-4 was also determined and shown in Fig. 3c, left. We found that\nIMPDH2-overexpressing HeLa cells are more resistant to 354 that the parental HeLa cells and that 354 provoked\na reduction in HeLa cell viability, which was significantly higher than that brought about by VX-497 or the\ntruncated chimera 352, which was not toxic. Additions to the text include:\n-Page 5, line 23: “After the above assays in cell-free systems, chimeras were tested in HeLa cells. This cell\nline was selected after a study reporting that mycophenolic acid, an IMPDH2 selective inhibitor, was more\neffective at inhibiting cancer cell proliferation in fast-growing cell lines, such as HeLa and Caco2, than in\nslow-growing cells[48], suggesting that HeLa cell viability would be sensitive to IMPDH2 degradation.\n-Page 7, line 5: “Next, we evaluated the functional consequences of IMPDH2 degradation. In agreement with\nthe involvement of IMPDH2 in favouring cancer progression[53–55], IMPDH2 degrader 354 provoked a\nreduction in HeLa cell viability, which was significantly higher in WT that in IMPDH2-overexpressing cells,\nproving the involvement of the target in 354 cytotoxicity. On the other hand, VX-497 was less toxic than 354\nand the truncated USP14 interactor 352 (Scheme 1) did not affect cell viability (Fig. 3c, right). Since neither\nVX-497 nor 352 induce IMPDH2 degradation, this result supports that 354-promoted IMPDH2 depletion is\ninvolved in cell killing.”\nC8. In Figs. 6 to 8, and Fig. S15, as the authors described, the experiments done with these CERT1 degraders\nseem to contain self-conflicting results that are not well resolved in the manuscript. All CERT1 degraders (395,\n413, 386, 368 & 419) work fairly well for POI degradation and 368 might be the best (Fig. 6B). However, only\n395 and 413 showed good lapatinib sensitization but not 368 or others (Fig. 7 & Supplementary Fig. 15).\nDifferential increase of ceramide species depending on degraders and cell lines was also confusing (Fig. 8).\nDoes this mean that the degradation capacity of CERT1 26S-UIDs is not necessarily correlated with its\nphysiological impact? The authors should try to resolve this apparent discrepancy.\nA8. We totally agree to this comment. With the aim to resolve this apparent discrepancy, we tried the 5 CERT1\n26S-UIDs in MDA-MB-231, MDA-MB-453 and BT-474 at times shorter than 24 h to assess for possible\ndegradation differences, but in no conditions were 395 and 413 the best degraders. Furthermore, to our regret,\nwe detected a mistake in the quantification of ceramides in MDA-MB-453/WT cells treated with 413 (original Fig.\n8D) and, therefore, what we mentioned in the original version (Page 10, line 2): “In general, in the case of the\nHER2+ cell lines, the fold increase of ceramides over controls is higher for the lapatinib sensitizing degraders\n(395 and 413) (4 to 5 times-fold) than for the non-sensitizing compound 368 (2 to 3 times-fold)” is no longer true\nfor 413 in MDA-MB-453 cells. Furthermore, in the discussion, our sentence “These data suggest that the\naccumulation of C14-C20Cers beyond a critical level may be involved in the sensitization of Her2+ cells to\nlapatinib by 395 and 413” is not supported by the revised data. Our first thought was to remove the lapatinib\nsensitization results from the manuscript, but then we thought that they are interesting from a translational\nperspective and decided to keep them without providing a mechanistic explanation. In the revised version, the\nsensitization results are only marginally mentioned in the discussion:\n-Page 12, line 36: “Further experiments to examine the translational impact of the CERT1 degraders reported\nhere are underway. Interestingly, chimeras 395 and 413 at concentrations that reduced cell viability by a 20\n% induced sensitization to lapatinib in the HER2+ cell lines MDA-MB-453 and BT-474 (Supplementary Fig.\n14). Whether this effect is mediated by CERT1 degradation and the molecular mechanisms involved in the\nsensitization are under investigation.” As requested by the referee, the dose-response effect of 395 and 413\non cell viability is now shownin Supplementary Figs. 14B and 14D.\n-Page 12, line 69: “We have shown that compounds 395 and 413 induce the accumulation of pro-death lipids\nceramides and sensitize HER2+ breast cancer cells to lapatinib. Although confirmation awaits further\ninvestigation, these results suggest that the CERT1 degradation induced by the degraders may be involved in\nthe sensitization.”\n-Page 8, line 41: In the lipidomics results, the paragraph has been modified by introducing the sentence.\n“Similar results were found with 413 in HER2+ MDA-MB-543 cells and with 395 in BT-474 cells (Fig. 6b, c).\nThis is consistent with the report that CERT1 efficiently transfers ceramides having C14, C16, C18, and C20\nchains (C14-C20Cers), while longer acyl chains (C22-24Cers) are more poorly transported.”\nC9. And the Method for measuring the level of ceramide species (for Fig. 8) was not given anywhere.\nA9. Sorry that we forgot to include this. In the revised version we have added these methods in the new section\n“Lipidomics”.\nC10.At Fig. S1, given that all compounds were treated at 50 uM, the inhibitory activites of IU1 and IU1-248 in\nthere are not well matching compared to the previous reports (#32, Lee et al. & #34, Wang et al.), in which IU1\nhad 4 ~ 12 uM of IC50 and IU1-248 had 0.83 uM of IC50.\nA10. We thank the reviewer for raising this point, which is completely right. We have reanalyzed the data\nacquired in these assays and performed more measurements with recently prepared biological materials. We\nhave observed that IU1 and IU1-248 could inhibit more efficiently proteasomal USP14 than in previous\nmeasurements, and we have included the new values to Supplementary Fig. 4. The new values obtained are\nmore consistent with the works mentioned.\nC11. Lastly, this manuscript needs significant reworks, in terms of fine writing, proper citation, good\nordering/arrangement, and so on. There are many things that should be pointed out including some trivial ones\nand I cannot list all of them. So, it is in order that the authors should thoroughly reassess to improve the\nmanuscript. Below I only enumerate several points as examples.\nA11. We are very sorry about the poor formal aspects of the original manuscript. We have revised the final text\nin terms of fine writing and proper citation. Moreover, we have rearranged the numbering and order of Figures in\norder to show data with more clarity.\nC12. For introducing the 26S-UIDs, the authors should also cite the work by Ali et al. (ByeTAC paper; doi:\n10.1101/2024.01.20.576376). At the part of mentioning ‘a flexible fragment’, it will be good to cite the work(s) by\nAndreas Matouschek group as well.\nA12. We thank the reviewer for bringing up our attention to these works, which we have cited in the revised\nversion.\nC13. TPD should be defined by targeted protein degradation, not by target protein degradation.\nA13. We have corrected this term in main and supplementary texts.\nC14. I see reference matching problems in many spots. The authors should try to find the most optimal\nreferences for citation (e.g. #19 at Page 2, right reference? #25 at Page 2 & 14 as a patent? Not full info? and so\non).\nA14. Reference [19] (C. Bashore, C. M. Dambacher, E. A. Goodall, M. E. Matyskiela, G. C. Lander, A. Martin,\nNat Struct Mol Biol 2015, 22, 712–719.) shows the Ssp2-Rpt2 fusion, which is able to recognize ssrA-tagged\nproteins as substrates. The degradation of ssrA-targets is ubiquitin-independent, as shown in the paper. For this\nreason, we have included this reference as an example of recruiting of substrates to the proteasome in a\nubiquitin-independent manner.\nReference [25] is a patent: we have cited it correctly in the new version: A. Testa, S. Hughes, S. P. Butcher,\nAlessio. Ciulli, Preparation of Bifunctional Molecules for Targeting USP14., 2019-12-19. WO2019238886A1. The\nresults shown have been not published and that is why the patent is cited.\nC15.There are also Supple Fig ordering problems. Ideally, the Supple Figs also should appear in order but\nFigure S17A is the first one appearing (Page 3) and thereafter, the Supple Figs are all shuffled up in ordering\nand very hard to follow.\nA15. We have revised the order of supplementary Figures and now they appear numerically ordered.\nC16. Page 3, 4, the two sections of design of chimeras and synthesis of chimeras can be combined and\ncompacted.\nA16. Design of chimeras and synthesis of chimeras have been combined and compacted.\nC17. At multiple spots, it is said, (see SI). This is not good way to ask attention considering the SI file contains\ncontent over 130 Pages. Should be more specifically designated.\nA17. We have corrected this problem in the new version.\nReviewer #2:\nIn this study, Casasampere and co-workers present a novel approach to protein degradation by designing small\nmolecule chimeras that target the 26S proteasome via USP14, a deubiquitinating enzyme associated with the\nproteasome's regulatory particle. This strategy circumvents the traditional E3 ligase-dependent mechanisms\nused in classical PROTACs. The authors provide proof of concept for this new type of chimera, termed 26S-\nUIDs, by targeting two proteins of high relevance in cancer, IMPDH2 and CERT1. They present robust\nexperimental data, including synthesis, in vitro assays, and cell-based studies, demonstrating degradation of the\ntarget proteins. Docking and molecular dynamics simulations were employed to explore the ternary complexes\nbetween USP14, the chimera, and the target protein, highlighting the potential complementarity between the\nbinding partners. Overall, the manuscript is well-written and experimentally robust, with detailed results provided\nboth in the main paper and supplementary materials. The authors showed careful consideration of many details,\nsuch as the consistency of the ternary complex in the proteasome context. I would recommend the editor\naccept this publication in Nature Communications after the authors address some points related to the molecular\ndynamics aspects that require attention. These points are as follows:\nC1. Considering the current guidelines recommended for studies involving molecular dynamics simulations (e.g.,\nJCIM, 10.1021/acs.jcim.3c00599), at least three replicas of each system studied, ideally starting from different\ncoordinates and velocities with a discussion of the statistical variance observed in the calculations, should be\nincluded in the paper. The authors only use a single trajectory for all cases.\nA1. We acknowledge the reviewer concerns relative to the MD simulation results included in the original version\nof the paper. Despite we agree with these concerns, we consider that extending the length of the computational\npart of the paper with longer simulations of the ternary complexes, more replicas and a more in-depth study of\nthe results is beyond the scope of this particular paper. Taking also into account the comments of reviewer 3\nwho suggested to make a separate paper with the computational results, we have decided to remove all that is\nrelated to the molecular dynamics simulations of the ternary complexes, leaving only the results relative to the\nmodeling of the ternary complexes based on protein-protein docking methods (ie. PROTAC-Model software).\nThe only simulations left in the paper are those of the complexes of USP14 with ligands IU1-248, IU1c and 317.\nFor these, we have performed additional simulations and we have included the results of three independent MD\nreplicas for each complex, as detailed in the Supplementary sections 1 and 2 and Supplementary Fig. 8.\nC2. For the sake of reproducibility and following the FAIR principles, it is recommended that the authors share\ntheir input files for the docking and atomistic MD simulations. This dataset could also include scripts used for the\ncalculations and analysis. While sharing trajectories may be challenging, it could still be considered through\nrepositories like Zenodo.\nA2. We are in the process of uploading the inputs and results of all the computational work reported in the paper\nin a public repository, as suggested.\nC3. The clustering analysis used to select the representative ternary complexes from the most populated\nclusters of the MD trajectories is not clearly explained. The only information provided on the methods states,\n“Trajectory analysis and hierarchical clustering were performed with CPPTRAJ.” Visualization of the cluster\ndistributions and populations, along with more details on how the calculations were performed, would be\nbeneficial.\nA3. After removing the MD simulations mentioned above, there are no clustering calculations on MD results left\nin the paper.\nC4. The authors mention that ternary complexes predicted via the Protac-model software workflow are mostly\nstable during the MD simulations. However, the RMSD plots in Figure S23, although showing some plateaus,\nindicate values around 6 to 8 Å in several cases. These values are quite high, indicating meaningful deviations\nfrom the initial ternary complex, not necessarily limited to loop changes. Direct comparisons of snapshots from\nthe beginning and end of the simulations would be helpful. Additionally, addressing the first point (use of\nreplicas) is crucial, as some observed structural changes may not be fully converged given that only one\ntrajectory was analyzed.\nA4. As mentioned above, most of these conflicting MD results have been removed. For the MD simulations that\nare still in the paper, we have included RMSF plots that illustrate that the main variability during the simulations\nis on the regions that were undefined in the original USP14 crystal structure used to perform all the modeling.\nSince these regions are mostly away from the ligand binding site, we believe that this variability does not affect\nmuch to ligand binding.\nC5. Lastly, and perhaps more importantly, I question whether the simulation length or the use of unbiased MD\nsimulations is adequate for studying the ternary complexes like the ones shown here. Unlike stability MD\nsimulation tests typically performed for relatively rigid protein-small molecule interactions in medicinal chemistry,\nPROTAC-like ternary complexes are known to be dynamic and plastic, as shown by prior experimental and\nsimulation work in Nature journals (e.g., DOI:10.1038/s41589-018-0055-y and DOI:10.1038/s41467-022-33575-\n4). Obtaining meaningful data from MD simulations in these cases likely requires much more sampling than the\nauthors have considered. I would ask the authors to explain why they believe simple/relatively short simulations are sufficient in their study.\nA5. The only simulations left in the paper are those of standard protein-ligand complexes, which are quite often\nsimulated for times in the 10’s to 100’s of nanoseconds scale. Here we report the results of three simulations for\neach complex, which represent a total accumulated time of 1.5 microseconds, Besides, analysis of the RMSD\nplot of each independent replica (Supplementary Fig. 8) shows that all of them reach stability, at most, after the\ninitial half of the simulation, suggesting that this simulation time is appropriate to assess these protein-ligand\ncomplexes studied.\nReviewer #3\nThe manuscript by Casasampere et al entitled, “Expanding the target protein degradation approach with small\nmolecule chimeras directed to the 26S proteasome”, presents data aimed to demonstrate how small-molecule\nligands to the 26S proteasome can be used to facilitate targeted protein degradation in a ubiquitin-independent\nmanner. These 26S-targeting ubiquitin independent degraders (26S-UIDs) aim to bypass limitations of traditional\nPROTACs, which facilitate co-localization of targets with E3 ligases for ubiquitin-mediated degradation, such as\nthe efficiency of ubiquitination, E3 abundance in specific cell types, or the presence of modifiable lysine residues\non the target protein of interest (POI). Moreover, 26S-UIDs would potentially be more versatile in the range of\nPOIs that they could degrade than the initial, proof-of-concept, direct-proteasome chimeric degrader previously\nreported (Bashore et al Nat Chem Bio 2023) that utilized a large, macrocyclic peptide as a proteasome ligand as\nopposed to a small molecule.\nC1. The authors seek to show this by developing and characterizing degraders towards two interesting\noncology-relevant targets, instead of a standard proof-of-concept target in this field (e.g. BRD4), and further\nevaluate the cellular consequences of the engineered target degradation.\nA1. The same criticism (not using a standard proof-of-concept target) was raised by reviewer 1 (C2b) and the\nanswer (A2b) is reproduced here. In fact, we initially prepared the USP14-based degraders 351 and 362,\nboth featuring the BrD4 ligand JQ1, and tested their degradative properties along with the reported\nVHL-based degrader MZ1. Unfortunately, cell viability was severely compromised by both 351 and 362 at the high\nconcentrations and times required for degradation (16 h). In contrast, MZ1 was a more potent degrader,\nalthough no devoid of cytotoxicity at 16 h. Instead of dedicating further medicinal chemistry efforts to obtain\nbetter USP14-based BRD4 degraders, we decided to proceed with IMPDH2 and CERT1, for which more potent\nUSP14-based degraders were being achieved.\nC2. Thus, the authors pursue a compelling area of research that seeks to expand the utility of direct 26S\ndegradation strategies (although the claims of novelty are rather overstated given the earlier proof-of-concept\nstudy cited above, especially given that USP14 docks onto RPN1, the 26S component used in the previous\nstudy).\nA2. We agree with the referee that this is not the first report on proteasome-based Protacs. The two previous\narticles are cited in the manuscript (one of them is a preprint), specifically in:\n-Page 3, line 22: “This approach exploits the positioning of USP14 in the RP, and shares similarities with the\nrecently described Rpn1-directed chimeras, however in that case the Rpn1 ligands were based on\nmacrocyclic peptides. More recently, Protacs using Rpn13 as recruiter have been also reported.”\n-Page 11, line 27: “Additionally, chimeras recruiting Rpn13 have been show to degrade BRD4. As shown in\nthese articles and in our work, 26S-UIDs don't hijack E3 ligases in order to promote proteasomal signaling of\nthe targets dependent on ubiquitination.”\nOn the other hand, to de-emphasize the claims for novelty, the following changes have been made in the revised\nmanuscript:\n-Abstract. The original text fragment “Herein we report a novel type of chimera based on ligands of USP14, a\n26S-associated deubiquitinating enzyme involved in substrate processing and allosteric regulation of 26S\nactivity. Using two targets relevant in oncology as models (IMPDH2 and CERT1), we provide proof of concept\nof a novel type of 26S-oriented Protacs that avoid targeting E3-ligases, 26S-UIDs, which will expand the\npotential of targeted protein degradation.” has been changed into: “Herein, using two targets relevant in\noncology as models (IMPDH2 and CERT1), we provide proof of concept for a new type of 26S-oriented Protacs\nbased on ligands of USP14, a 26S-associated deubiquitinase involved in substrate processing and allosteric\nregulation of 26S activity. Our findings will expand the potential of targeted protein degradation.”\n-Introduction, final paragraph. “novel” has been removed from the sentence “In light of our data, these novel\n26S-targeting ubiquitin-independent degraders (named “26S-UIDs”) appear as a versatile alternative tool for\nTPD-drug development (Fig. 1).”\n-Conclusion. The word “novel” has been replaced by “additional” in the first sentence of the Conclusions (“An\nadditional class of UIDs that induce degradation by directing targets to the 26S proteasome using USP14 as\nthe anchoring site are reported”.)\nC3. Moreover, I think that the current characterization of the reported compounds and interpretation of the\nconducted experiments is limiting and I am not convinced that the data warrants the conclusions reported by the\nauthors (see bullet points below). Based on these concerns, I cannot recommend this manuscript for publication\nin Nature Communications unless major revisions (including key degradation controls) are achieved.\nA3. We hope that the revised manuscript, which addresses almost all the comments made by the 3 reviewers,\nwill be more convincing.\nMajor points that require attention:\nC4. While the authors show dose-dependent, chimera-mediated degradation of both CERT1 and IMPDH2,\nthey do not conclusively show that this is due to direct 26S recruitment via USP14.\nFirst, the only evidence for a Ub-independent degradation mechanism in the paper is shown in Fig 6B, where\npre-incubation with proteasome inhibitor appears to block CERT1 degradation while the E1 inhibitor has no\neffect on 368-mediated degradation at 24 hours. No such data is available for IMPDH2 because of the very long\ntimeframes needed to observe degradation of this target (72-96 hours)\nA4. The same criticism was raised by Reviewer 1 (C3) and the answer is reproduced here. To cope with the\ncytotoxicity issue occurring at long incubation times we added the inhibitors at the end of the assay instead of\nperforming preincubations with the inhibitors at the initial hours of the culture. Moreover, we prepared a HeLa\nderived clone stably overexpressing IMPDH2-GFP (clone 522-4). As expected, these cells were more resistant\nto 354 than the parental HeLa cells (See Fig. 3c, left of the revised version). Gratifyingly, with these adjustments,\nincubation conditions could be found in which the proteasome inhibitors (bortezomib and marizomib) and\nMLN7243 could be used without provoking rupture of the plasma membrane and extensive cell death despite\ncausing cell detachment. The precise experimental conditions are given in the section “Cell treatments” of the\nSupplementary Information. The new results showing that proteasome inhibitors (bortezomib and marizomib)\navoided 354-induced IMPDH2 degradation while MLN7243 did not have been added to the manuscript and are\nshown in Figs. 3d and e. Changes in the text are:\n-Page 5, line 79: “Mechanistic studies using proteasome CP blockers (bortezomib and marizomib) were\nperformed in HeLa and in HeLa cells stably overexpressing IMPDH2 (clone 522-4), more resistant to 354\nthan the parental HeLa cells (Fig. 3c, left). As shown in Fig. 3d, the proteasome inhibitors bortezomib and\nmarizomib rescued IMPDH2 degradation induced 300 (in HeLa) and by 354 (in 522-4). In the latter case,\nIMPDH2 overexpression protected cells from massive death, but a higher degrader concentration was\nrequired for depletion of the overexpressed protein. Conversely, the E1-Ub activating enzyme inhibitor\nMLN7243 did not prevent the target degradation (Fig. 3e).”\nC5. Fig 6B also shows a single-point competition-binding experiment in which 100 uM of free CERT1 ligand,\n366 (although labeled on the blot as HPA) is used to block 10uM 368-mediated degradation of CERT1. Again,\nno such data is shown for free ligand blocking chimera-driven degradation of IMPDH2. Typically,\nchimera-based degraders are more thoroughly validated by titrating in either of the free ligands to compete for\nbinding and thus disrupt ternary complex formation and degradation (not just a single competitor concentration).\nA5. We have carried out the titration experiments mentioned by the referee with the competitors shown in the\nFigure below. The results of competition of 354 with VX-497 at inducing IMPDH2 degradation are shown in Fig.\n3f. As shown, 354-induced degradation of IMPDH2 started to be rescued with 5 µM VX-497, with maximum\ndegradation prevention at 10 µM, but the DMSO levels were not attained because higher VX-497 concentrations\nwere very toxic to cells (see Fig. 3c, right of the revised version). The results of competition between 386 and the\ntruncated USP14 ligand 352 or the CERT1 ligand 366 for CERT1 degradation are depicted in Figs. 5b, c.\nChanges in the text include:\n-Page 6, line 2: “Furthermore, IMPDH2 depletion was counteracted by preincubation with the IMPDH2 ligand\nVX-497 in a dose dependent manner (Fig. 3f). This effect could only be partially observed since VX-497 at\nconcentrations higher than 10 µM abrogated cell growth (Fig. 3c, right).”\n-Page 11, line 63: “Furthermore, the target ligands (VX-497 for IMPDH2 and 366 for CERT1) prevented\ndegradation (Figs. 3f and 5b), proving the interaction of 26S-UIDs with targetsin cellulo.”\nIMPDH2 and CERT1 degraders and their competitors used in the experiments\nformation for either target. Also, there is no evidence that chimeric compounds bind to USP14 (and the targets)\nin the context of the assembled 26S proteasome, or that these proposed interactions mediate the observed\ntarget degradation.\nA6. We think that 4 sets of new data support that chimeric compounds bind to both USP14 and the targets in the\ncontext of the assembled 26S proteasome and that these interactions mediate the observed target degradation.\nRegarding USP14:\n1. The results of experiments with USP14(-/-) cells provide evidence that direct 26S engagement via USP14\nmediates the observed degradation of both targets (See answer to C7 below).\n2. The results of competition assays with the USP14 ligand 352 showing that it prevents CERT1 mediated\ndegradation induced by 386 in a concentration-dependent manner (See answer to C5 above) is another\nproof that direct 26S engagement via USP14 mediates the target degradation.\n3. Another evidence for the 26S-UIDsinteraction with USP14 in the context of the assembled 26S was\nobtained using cycloheximide (CHX) sensitivity assays in both S. cerevisiae and HeLa cells. This assay is\nbased on a conserved phenotype linked to the proteasomal functions of USP14: the CHX sensitivity of cells\nin the absence of USP14 activity (Hanna et al., Mol Cell Biol. 2003, 23:9251-61). Using a yeast strain that\nexpresses human USP14 in a double gene deletion background (ubp6Δ/pdr5Δ), where ubp6 is the USP14\nyeast ortholog and pdr5 is a multidrug resistance pump (its deletion prevents drug efflux), we have\nobserved that (1) human USP14 rescues CHX sensitivity of ubp6Δ/pdr5Δ yeast strains (Fig. Rev 1A,B) and\n(2) USP14 inhibitor IU1-248 and its derived active IMPDH2 degraders 300 and 329 induced CHX sensitivity\n(lower colony formation than in controls). In contrast, IU1-c, which does not inhibit USP14 (Lee et al. Nature\n2010, 467, 179–184) and 317 and 354, which are a poorer USP14 inhibitors than the IU1-248 family (see\nsupplementary Fig. 4), did not induce CHX sensitivity (Fig. Rev 1C,D). Colonies were formed as in the\nDMSO control). Similar results were found in HeLa cells, where all compounds, except for 317, with very\npoor USP14 inhibitory activity (see supplementary Fig. 4), induced CHX sensitivity (Fig. Rev 1E, compare\nnumber of colonies in plates with CHX 0.2 g/L and 5 M of compound). It is true that, although the results\nof these experiments confirm the 26S-UIDs interaction with USP14 in the context of the assembled 26S,\nthey do not prove that this interaction mediates the degradation of the targets.\nRegarding the targets:\n1. Competition experiments with VX497 (IMPDH2) and 366 and 352 (CERT1) support the 26S-UIDs-target\ninteraction in the cellular context (an assembled 26S) (See answer to C5).\nWe hope that we could convince the reviewer that these observations show the capacity if IU1-based\ncompounds to interact with both the cellular proteasome-bound USP14 and the protein targets.\n26S proteasome) would help provide important evidence to validate that direct 26S engagement via USP14\nmediates the observed degradation as hypothesized. Alternatively, inactive stereoisomers of the chimeras could\nalso provide appropriate controls in this regard.\nA7. (Reproduced from answer 1 to reviewer 1, who raised the same comment). We thank the reviewer for this\ncomment, to which we completely agree. We have performed assays with MEFs wild-type and USP14-/- (from\nDr. Min Jae Lee; Kim et al. Cell Rep. 2018, 24, 732). Cultured MEFs were treated with 368 and 354, as\nrepresentative compounds for CERT1 and IMPDH2 targeting, and have observed that target degradation\noccurred in WT, but not in USP14(-/-) MEFs. We have included these results in Fig. 3g (IMPDH2) and Fig. 5f\n(CERT1) and added details in the corresponding legends. The results are discussed in the following parts of the\ntext:\n14\n-Page 6, line 10: “To show that IMPDH2 degradation occurs in a USP14-dependent manner, the effect of 354\non IMPDH2 degradation in MEFs USP14(-/-) versus wild-type was tested. We observed that IMPDH2\ndegradation was induced in MEFs-WT, whereas in MEFs USP14(-/-) degradation was not only prevented (Fig.\n3g), but the IMPDH2/Actin ratios were significantly higher in 354-treated cells that in controls. This resembles\nthe effect observed with VX-497 (see above), indicating that in the absence of USP14, the chimera acts as an\nIMPDH2 inhibitor and IMPDH2 levels increase by a feedback response of the purine synthesis pathway.\n-Page 8, line 25: “To show that CERT1 degradation is USP14-dependent, 368 effect was tested in MEFs,\nUSP14(-/-) versus wild-type. It was observed that CERT1 degradation was promoted in MEFs-WT, but not in\nMEFs USP14(-/-) (Fig. 5f).\n-Page 11, line 57: “Importantly, the USP14-dependence of target degradation was demonstrated in USP14(-/-)\ncells, in which degradation of both targets was prevented (Figs. 3g and 5f).”\n-Supplementary information: MEFs have been added to the “Cell culture” Section.\nRegarding the use of inactive stereoisomers of the chimeras as controls, this is what we aimed at when prepared\nchimeras featuring 317, but, as discussed in the manuscript, this ligand afforded better degraders than those\nbearing IU1-248. We do not have inactive stereoisomers and we hope that the reviewer is satisfied with the\nresults obtained in USP14 knock out cells.\nC8. The authors state in the discussion that “the specificity and accurate behavior of the generated constructs\nwere evaluated in cells, in vitro assays and by SPR”. It is unclear what is meant by “accurate behavior”, however\nthe specificity of the chimeras did not appear to be well characterized in cells. The chimeras were shown to bind\nUSP14 or their intended target using purified, recombinant protein stocks, but potential off-target binding in cells\nwas not probed.\nA8. We agree that“accurate behavior” is not meaningful and we modified the sentence to read “The generated\nconstructs were evaluated in cells, in vitro assays and by SPR”. We also agree that, although in the new version\nof the manuscript we have provided evidence that target proteins degradation induced by the chimeras in cells\ninvolves their binding to both, targets (competition experiments) and USP14 (experiments with knock out cells\nand cycloheximide sensitivity assays in yeast and HeLa cells), potential off-target binding of chimeras in a\ncellular context has not been explored. We plan to conduct an in depth analysis by proteomics in the coming\nmonths, but the results will take time and, in our opinion, their inclusion in this article is beyond its scope, which\nis to provide proof of concept that USP14 can be used as a novel recruiter for targeted protein degradation.\nC9. While I commend the authors for exploring the downstream effects of CERT1 degradation on ceramide\nlevels and drug sensitization, it is confusing why chimeras that achieve similar levels of CERT1 degradation\nshow disparate effects on these readouts. There are also so many compounds and cell lines tested (all shown in\nseparate supporting Figures) that it is difficult to compare these results across these different conditions. Given\nthe inconclusive aspects of these findings it is difficult to warrant such a large portion of the manuscript detailing\nthese experiments—especially when the critical experiments in point #1 need to be addressed to validate the\nMOA of the targeted degradation.\nA9. We completely agree to this comment and want to apologize for the confusing and incomplete way to\npresent these data. The same criticism was raised by reviewer 1 and we reproduce here the answer given:\nWe totally agree to this comment. With the aim to resolve this apparent discrepancy, we tried the 5 CERT1\n26S-UIDs in MDA-MB-231, MDA-MB-453 and BT-474 at times shorter than 24 h to assess for possible\ndegradation differences, but in no conditions were 395 and 413 the best degraders. Furthermore, to our regret,\nwe detected a mistake in the quantification of ceramides in MDA-MB-453/WT cells treated with 413 (original Fig.\n8D) and, therefore, what we mentioned in the original version (Page 10, line 2): “In general, in the case of the\nHER2+ cell lines, the fold increase of ceramides over controls is higher for the lapatinib sensitizing degraders\n(395 and 413) (4 to 5 times-fold) than for the non-sensitizing compound 368 (2 to 3 times-fold)” is no longer true\nfor 413 in MDA-MB-453 cells. Furthermore, in the discussion, our sentence “These data suggest that the\naccumulation of C14-C20Cers beyond a critical level may be involved in the sensitization of Her2+ cells to\nlapatinib by 395 and 413” is not supported by the revised data. Our first thought was to remove the lapatinib\nsensitization results from the manuscript, but then we thought that they are interesting from a translational\nperspective and decided to keep them without providing a mechanistic explanation. In the revised version, the\nsensitization results are only marginally mentioned in the discussion:\n-Page 12, line 36: “Further experiments to examine the translational impact of the CERT1 degraders reported\nhere are underway. Interestingly, chimeras 395 and 413 at concentrations that reduced cell viability by a 20\n% induced sensitization to lapatinib in the HER2+ cell lines MDA-MB-453 and BT-474 (Supplementary Fig.\n15\n14). Whether this effect is mediated by CERT1 degradation and the molecular mechanisms involved in the\nsensitization are under investigation.” As requested by the referee, the dose-response effect of 395 and 413\non cell viability is now shownin Supplementary Figs. 14B and 14D.\n-Page 12, line 69: “We have shown that compounds 395 and 413 induce the accumulation of pro-death lipids\nceramides and sensitize HER2+ breast cancer cells to lapatinib. Although confirmation awaits further\ninvestigation, these results suggest that the CERT1 degradation induced by the degraders may be involved in\nthe sensitization.”\n-Page 8, line 41: In the lipidomics results, the paragraph has been modified by introducing the sentence.\n“Similar results were found with 413 in HER2+ MDA-MB-543 cells and with 395 in BT-474 cells (Fig. 6b, c).\nThis is consistent with the report that CERT1 efficiently transfers ceramides having C14, C16, C18, and C20\nchains (C14-C20Cers), while longer acyl chains (C22-24Cers) are more poorly transported.”\nC10. The authors must address the exceptionally poor organization of the manuscript. While the main text\nfollowed a relatively structured narrative, the haphazard curation of the Figures, schemes and supporting\ninformation made the manuscript exceedingly and unnecessarily difficult to read. For example, here is the order\nof the first 10 Figures/schemes/SI as they appeared in the main text of the manuscript: Fig1, scheme 1, Fig\nS17A, Fig S17B, Fig S16, Fig S17C, Fig 2A, Fig S20A-C, scheme S5 and scheme S6! This is unacceptable and\nshould be resolved before any further review. Furthermore, the lack of organization is compounded by the sheer\nvolume of data presented in the manuscript (135 Pages of Supporting Information). Considerable editing is\nneeded to trim the manuscript to the essential data. For example, there are 7 Pages of molecular modeling\nresults associated with 7 supporting Figures, 12 supporting movies, and 9 supporting Tables all in the\nSupporting Information. This material is briefly touched upon in the main results section and yet seems to\nconstitute what would be a separate companion paper given. The authors should either add the essential\nmodeling data into the main results of the manuscript (as a substitute for the lack of biophysical ternary complex\nmeasurements), or make it a separate paper. Making this manuscript a repository for every experiment done on\nthese systems does not enhance the paper and, in fact, detracts from the main ideas of the manuscript.\nA10. We fully agree to the referee’s comments and want to apologize for the poor organization of the\nmanuscript. We have done our best to improve it and we hope that the reviewer will be satisfied with the new\nversion. Specifically, the molecular modeling results have been extensively reduced by removing all the\nmolecular dynamics results on the ternary complexes. Besides, a new section has been included in the main text\nthat contains the remaining modeling results, leaving only a much shorter section in the SI. Finally, the SI has\nbeen also substantially reduced by removing most of the computational material: Tables S3 to S11 have been\nremoved; computational Figures have gone from 11 to only 5; most of the movies have been eliminated, leaving\nonly 3; and we have also removed most of the PDB structures, leaving only 2. Please take into account that 36\nPages correspond to NMR spectra, which we consider important to provide to readers.\nC11. There are several instances in the manuscript where conclusions are stated based on “data not shown”.\nWhile I am loath to add even more data to this manuscript given point #4, conclusions need to be supported by\ndata that the reader can assess for themselves.\nA11. We thank the referee for this comment. The results referred to as “data not shown” have now been\nincluded as follows:\n1. “the expected decrease in SM amounts upon treatment was not observed in any cell line (data not shown)”.\nAnswer. These data is shown in Supplementary Fig. 11\n2. Legend Fig. 3. “USP14 remained stable in the assays, as assessed by immunoblotting (not shown)”. Answer.\nIn our opinion, adding all the WBs showing these data will unnecessarily increase the length of the\nsupplementary materials files and we would like to keep it as “not shown”. However, if the referee thinks that\nit should be added, we will happily do so.\n3. “However, no sensitization to paclitaxel and doxorubicin was observed in our hands (data not shown)”.\nAnswer. These data has been removed from the original manuscript\n4. “However, for a so far unknown reason, no sensitization of triple negative breast cancer MDA-MB-231 cells to\npaclitaxel and doxorubicin by the compounds was observed (data not shown).” Answer. These data have\nbeen removed from the original manuscript\n5. “In contrast, two CERT1 degraders, 395 and 413, induced sensitization to lapatinib, a dual EGFR and HER2\ninhibitor, in HER2+ MDA-MB-453 and BT-474 cell lines. Consistently with HER2 as a molecular target for\nlapatinib, no sensitization to this drug by any CERT1 degrader was observed in the triple negative breast\ncancer MDA-MB-231 cell model (data not shown).” Answer. The data on MDA-MB-231 has been removed\nfrom the original manuscript.\nand 373) are really relevant to this work. Without evidence that these chimeras can actually mediate productive\nternary complex between the targets and these E3 ligases, comparisons with the 26S-UIDs seems somewhat\nhollow. Linker length often requires optimization to produce effective degraders, so the fact that linkers that\nappear to work for the 26-UIDs don’t work for the ligase-directed chimeras is not surprising. However, if these\nligase-directed chimeras were shown to mediate productive ternary complexes and target ubiquitination that was\ninsufficient to cause target degradation, whereas direct engagement with the 26S efficiently degraded these\ntargets, that would be a very interesting result and would further elevate the utility of direct 26S targeting\ndegraders.\nA12. We absolutely agree to this criticism, which was also raised by Referee 1. We reproduce here the answer\ngiven to him/her: Although we did not observe IMPDH2 and CERT1 degradation with different concentrations\n(from low nM to two-digit µM) and incubation times (from 4 to 96 h) of E3-based Protacs, we agree that further\nstructural modifications of the linker might have provided active E3-based degraders. To simplify the manuscript,\nmeaningless data about these chimeras have been removed from the manuscript in the revised version.\nC13. The authors do not comment on the clear “hook effect” that they see with some of the 26S-UIDs. Do these\nmake sense given the cellular levels of these targets and/or the high abundance of the 26S proteasome?\nA13. Thanks so much for bringing our attention to this point, which we forgot to comment in the original version.\nAs the referee knows, the hook effect is related to binding affinities and relative concentrations of target protein,\nPROTAC and recruiter element (USP14 here). It is caused by competition of ineffective binary complexes (POI-\nProtac and recruiter-Protac) with effective ternary complexes, which result in diminished PROTAC's degradation\npotency. Therefore, the binding cooperativity of the three elements is a key design point to modulate the hook\neffect. In our work, although no hook effect was observed with USP14-based IMPDH2 degraders, it was found at\n40 M for some CERT1-targeted molecules: compounds 368 and 386 in MDA-MB-231 cells (Fig. 4b) and BT-\n474 cells (Supplementary Fig. 9c,d), and compounds 413, 368 and 386 in MDA-MB- 453 cells (Supplementary\nFig. 9a,b). Since the proteasome is an abundant particle, it is plausible that the limiting factor for the hook effect\nis the proteasome concentration rather than that of CERT1. The concentration of cytosolic proteasome is\nestimated to be around 150 nM (lower than nuclear proteasome, 900 nM) (Pack, C.G. et al. Quantitative live-cell\nimaging reveals spatio-temporal dynamics and cytoplasmic assembly of the 26S proteasome. Nat. Comm. 2014,\n5:3396). Since CERT1 is a cytosolic target, it is likely that both CERT1 and cytosolic proteasomal saturations by\nchimeras are reached at the high concentrations tested, causing the hook effect. Changes in the text include:\n-Page 11, line 65: “Of note, in treatments with 413, 368 and 386 (40 M), a slight hook effect was observed in\ndifferent cell lines (Figs. 4b, c and Supplementary Fig. 9). Since CERT1 is a cytosolic target and cytosolic\nproteasome is estimated to be less abundant than nuclear proteasome (170 nM versus 900 nM), it is likely\nthat both CERT1 and cytosolic proteasomal saturations by chimeras are responsible for the observed effect.”\nC14. I am also very curious to understand what forms of IMPDH2 are degraded given that we are told in the\ndiscussion that it forms various oligomeric complexes that are difficult to degrade. [C14a] Are oligomers being\ntargeted to the 26S and does engagement with the unfoldase machinery facilitate more efficient degradation\nthan ubiquitination of the target? [C14b] Does this “clog” the 26S such that degradation of other targets is\nimpaired?\nA14a. This is a very interesting and complex question that probably should be answered with specific\ninformation of each target. Said that, looking at the mechanism of the proteasome, engaging the unfoldase\nmachinery could be more efficient than engaging ubiquitin receptors, for a substrate to be degraded, because\nthe unfoldase step involves complete commitment of the substrate and its translocation to the proteolytic 20S\ncore particle. Instead, when a substrate interacts with ubiquitin receptor it is not fully committed. In fact, not all\nubiquitinated proteins that bind the proteasome are engaged by the ATPase.\nA14b. This is also a very interesting yet complex point. Toxic oligomers may impair 20S activity (for example\noligomers frequent in Alzheimer's, Parkinson's, and Huntington's diseases) (Thibaudeau et al., A common\nmechanism of proteasome impairment by neurodegenerative disease-associated oligomers. Nat Commun.\n2018, 9, 1097). However, IMPDH2 forms nontoxic reversible oligomers (Johnson et al. Cryo-EM structures\ndemonstrate human IMPDH2 filament assembly tunes allosteric regulation. Elife. 2020, 9, e53243), in which no\nclogging effect should be observed.\nMinor issues\n1. Some of the Figures (including Fig 1, Scheme 1) have poor resolution when zoomed in on the digital version,\nwhich is needed given that the font is often too small (e,g Scheme 1). Answer. We apologize for this issue. All\nFigures have high-resolution in the revised manuscript.\n17\n2. The yellow chimeras are very difficult to see in many of the structural Figures (e.g. Fig 2A and Fig 4B) and\nred/green combinations should be avoided in light of red/green colorblind readers. Answer. We apologize for\nthis issue. All the structural Figures have been modified to remove red-green combinations, and Figures have\nbeen made bigger to make the chimeras easier to see.\n3. It is unclear what degradation time point is being used in Figure 5B—24 hrs? Answer. We apologize for this\nissue, which has been clarified in the Figure (Fig. 4 in the revised manuscript) legend (“(B) CERT1 degradation\nby the indicated chimeras (1-40 µM, 24 h)”).\n4. In Fig 6B, HPA is used as a blot label instead of compound #366 as stated in the Figure legend. Answer. We\napologize for this issue, which has been corrected. Fig. 6B in now Fig. 5A.\n5.[5a] Where are the raw SPR data used to generate the values in Table S2? Representative sensorgrams\nshould be provided to evaluate the quality of the data. Answer. We have added the Supplementary Fig. 5\nincluding sensograms of 300 and 368, representative of IU1-248 – USP14, VX-497 – IMPDH2 (using 300),\nIU1c(317) – USP14 and HPA12(366) – CERT1 (using 368) interactions. [5b]Do the weak affinities preclude\nusing SPR to measure ternary complex formation? Answer: Since the KD values of binary interactions obtained\nwere in the range of two digits micromolar, we did not attempt estimations of ternary interactions.\n6. In Fig S1, can you better describe what 26S[VS]-USP14 is? Also compounds 352 and 354 are not introduced\nin the main text at this point, but are included in this analysis. Similar issue for compound 320 in Fig S2 and 360,\n320, 339 and 343 in Fig S4. Answer. We apologize for this issue. 26S[VS]-USP14 has been defined in the\nSupporting information file, Section 4 – Cellular and Molecular Biology Methods/Ub-AMC assays in 26S-USP14:\n“… premixed VS-proteasome/USP14 (Ubiquitin–vinylsulphone [VS] treated 26S proteasome reconstituted with\nactive USP14, with a ratio of 1.25:20 nM final concentration; Ubiquigent). VS blocks the 26S proteasome\nendogenous DUB activity by forming an adduct with the active site cysteine in DUBs of thiol proteases. In this\nway, only the USP14 DUB activity is measured in the assays).”\n7. Ratios in Fig S3 are listed with commas instead of decimal points like the rest of the ratios shown throughout\nthe manuscript. Please change to be consistent. Answer. We apologize for this issue, which has been\ncorrected. Also, why does 300 increase IMPDH2 levels? Answer. 300 contains VX-497 in its structure, which is\nan IMPDH2 inhibitor. The increase in IMPDH2 in the presence of chemical inhibitors is been described in several\ncell types in the following articles (cited in page 5, line 77 as refs. 50-52):\n- Chiarelli L. et al., Inosine monophosphate dehydrogenase variability in renal transplant patients on long-\nterm mycophenolate mofetil therapy. Br J Clin Pharmacol. 2010, 69, 38.\n- Jain J. et al., Regulation of inosine monophosphate dehydrogenase type I and type II isoforms in human\nlymphocytes. Biochem Pharmacol. 2004, 67, 767.\n- Sanquer S. et al., Induction of inosine monophosphate dehydrogenase activity after long-term treatment\nwith mycophenolate mofetil. Clin Pharmacol Ther. 1999, 65, 640.\nWe have also observed IMPDH2 increases in treatments with 300 for 24 or 48 h, in which degradation was not\ninduced, and in MEFs USP14(-/-). (See Fig. 3 g), where, in the absence of USP14, the chimera likely acts as an\nIMPDH2 inhibitor. This effect is coherent with a primary effect of VX-497-containing compounds (such as 300)\non IMPDH2 in cells. The MEFs USP14(-/-) results have been mentioned in page 6, line 33: “We observed that\nIMPDH2 degradation was induced in MEFs-WT, whereas in MEFs USP14(-/-) degradation was not only prevented\n(Fig. 3g), but the IMPDH2/Actin ratios were significantly higher in 354-treated cells that in controls. This\nresembles the effect observed with VX-497 (see above), indicating that in the absence of USP14, the chimera\nacts as an IMPDH2 inhibitor and IMPDH2 levels increase by a feedback response of the purine synthesis\npathway.”\n8. Fig S5 is not listed in the main text, only in the Figure legend of Fig 3. Answer. We apologize for this issue.\nFig. S5 corresponded to histograms showing the mean ± SD of densitometric analyses for replicates of the\nassays, which have now been moved to Fig. 3b.\n9. 386 should be changed to 368 in Fig S15K. Answer. We apologize for this issue. Fig.S15 is no longer\nincluded in the revised version.\n10. The term “n” is not defined or described in the Figure legend of Fig S16. Answer. We apologize for this\nissue. “n” is now defined in the legend to Supplementary Fig. 1 (formerly, Fig. S16): “…. n refers to the number\nof CH2 units in the chain.”\n11. Please add arrows to denote proposed attachment vectors in Fig S17A and B. Answer: The Figure has\nbeen moved to the main text (Fig. 2a-c) and red arrows and circles have been added to denote the attachment\npoints to build the chimera molecules.\nOther major changes introduced in the revised version include:\n18\n― Fig. 1. The proteasome structure has been added showing the region close to USP14 and highlighting\nproximal subunits and relevant regions. The legend has been modified accordingly.\n― Fig. 2. has been modified to include (panels A-C) what was on Supplementary Fig. 17 of the previous version\nof the paper. In addition, panels D and E have been modified according to suggestions of referee 3 to\nimprove visualization.\n― Fig. 5. Panels A and B of Fig. 6 of the original version have been moved to Supplementary Fig. 9. The\nlegends have been modified accordingly.\n― Fig. 7. We have included a new figure that summarizes the in silico results concerning the 26S-UIDs ternary\ncomplexes and that replaces Fig. 4 of the previous version. This figure illustrates which residues of each\nprotein interact with the ligands (panels A, B) and which ones establish protein-protein interactions (panels C,\nD) that help to stabilize the ternary complex. In addition, panels E, F show also the location occupied by the\nPOIs on the USP14 surface. We believe that all of these will help the reader to the understanding of the\ncorresponding section of the paper.\n― Histograms of densitometric analysis, which were in the Supplementary Information file, have been moved to\neach corresponding figure below the representative western blots. The legends have been modified\naccordingly.\nWe appreciated the reviewers’ constructive comments that helped us improve the manuscript.\nPlease find below the point-by-point responses to the reviewers’ individual comments.\nIn the main text and Supporting information documents:\nTo ease the review by the referees, this copy is marked as follows:\n• Text related to comments of Reviewers are in magenta.\n• All other relevant changes made in the new version are in red.\n• Line numbers are given in green on the left margins of the columns.\n• Quotation to figures, schemes and tables are highlighted\nREVIEWER COMMENTS\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nIn the revised version, I recognize that the authors put substantial efforts to improve their work,\nwhich indeed forms the manuscript to be in a better shape. However, some critical issues still\nremain to be not well resolved, and also, I have to raise a few of extra questions, some of which\nare based on newly added results.\nComment 1a\n1. Even after the authors’ rebuttal, the reason for idiosyncratic degradation of IMPDH2 by 26S-UID\n(i. e. it takes long time up to 72-96 hr before suddenly being degraded) has not been convincingly\ndelivered. Current statement may not be satisfactory or should not be said like “unknown”.\nPerforming additional experiments is in order to retrieve some hints about the properties of\nIMPDH2 turnover by the degrader. Below are some opinions or thoughts about point #1.\n- The authors discussed like “…dynamic tetrameric, octameric and filamentous complexes,[69]\nmaking this protein difficult to inhibit and to degrade”, “…the association status of targets has been\nproposed as a possible cause for time-consuming degradations[70–72]”, but these statements are\nquite ambiguous until demonstrated to some extent.\n- Besides, the authors cited Refs 70-72 as examples of time-consuming degradation analogous to\nIMPDH2 degradation, but in Ref 70 (Zheng M et al., 2021), SHP2 degradation by SHP2 PROTAC\ncan be still observed as early as 48 hr and this paper may not be anything about “the association\nstatus of targets” since there are also many other SHP2 PROTACs available nicely working in\nshorter time points. Ref 71 (Zeng S et al., 2021) shows EGFR degradation by EGFR PROTAC,\na membrane protein probably not ideal comparison to IMPDH2. Ref 72 (Cyrus K et al., 2011) is\nabout ERα degradation by PROTAC, in which for longer incubation (72-96 hr), medium change by\ntwice treatment leads to better degradation, yet a different case from the IMPDH2. In any case, all\nthese are not good examples to support the authors’ argument. Furthermore the PROTACs in\nthere are still working at earlier time points, just gradually reaching degradation saturation when\nlonger treated, which is distinct from abrupt IMPDH2 degradation at longer time points in this study.\nAnswer\nWe thank the reviewer for the thoughtful comments and we regret not giving good examples to\ncompare with the pattern of IMPDH2 degradation by our compounds. We agree that the examples\nprovided are not identical to IMPDH2, even though they illustrate the idea that certain chimeras\nmay exhibit degradation times beyond 24-48 hours. Indeed, we could not find cases of target\ndegradation with the behavior of IMPDH2. Moreover, we agree that the link between IMPDH2\ndegradation with its association status (monomeric, dimeric or polymeric) is hypothetical, but we\nrespectfully consider that it is not the focus of this work. On this point, since IMPDH2 appears to be\nefficiently depleted, it is plausible that the compounds may act equally on the different forms of\nIMPDH2. Considering that all these aspects and references were included as part of the\ndiscussion, we agree in suppressing them.\nContinuing with the question of the degradation times of IMPDH2, we admit that we don’t\nhave a conclusive answer for this point. However, there’s an aspect that may be linked to the\nobservation of delayed degradation. As shown in Supplementary Fig. 6C, D and G, prior to its\ndegradation, IMPDH2 protein levels are notably increased in cells, in treatments with 300, in the\ntime range of 24 and 48 hours. A plausible explanation to this observation is that, in a context of\ninhibition of IMPDH2 function, a feedback mechanism induces a boost in IMPDH2 gene\nexpression, and thus new pools of IMPDH2 protein are generated. This mechanism would explain\nthe observations that the treatment with compound 300 causes an increase in IMPDH2 protein in\nthe 24-48 range, which is followed by a sudden degradation of the target (Supplementary Fig. 6C,\nD and G). Consistently, the pattern of IMPDH2 increase is also observed in VX497 treatment,\nwhich does not induce degradation of IMPDH2, thus, IMPDH2 levels do not suddenly decrease in\nVX497 treatments (Supplementary Fig. 6E and 6G). In order to test the hypothesis of a transient\nIMPDH2 gene induction, quantitative PCR assays were carried out. Cell cultures treated with\ncompounds 354 (representative of IMPDH2 degraders), 317 (as a Usp14 binder, with no expected\ninfluence on IMPDH2 function) and VX497 (a binder and inhibitor of IMPDH2), at different\nincubation times, were analyzed by qPCR using a set oligos designed to estimate IMPDH2 and\nActin mRNA levels, as shown in Supplementary Fig. 17A (copied below) and in the Methods\nsection (page 31). It was observed that, upon 24 hours of incubation with 354 and VX497, IMPDH2\nmRNA levels exhibited a strong increase (8-to-9-fold). At 48 hours, the increase was still observed,\nalthough not to the magnitude of that at 24 hours (~3.7-4.5-fold).\n0.05\n0.04\n0.03\n0.02\n0.01\n0.00\nM\nS\nO 354\nV\nX\nM\nS\nO 354\nV\nX 317\nM\nS\nO 354\nV\nX\nD D D\nTherefore, in 24-48 hours of treatment, there’s a correlative increase in IMPDH2 mRNA and\nprotein levels in the presence of IMPDH2 inhibitor VX497 and tested degraders based on VX497\n(354 and 300). This increase may counteract the effect of the degraders, which commonly induce\nan observable decrease in the levels of IMPDH2 within the 72-96 h incubation times.\nTo corroborate this notion, we performed assays including the translation inhibitor\ncycloheximide (CHX). Even though cycloheximide induced high cell mortality in treatments longer\nthan 2 hours, exacerbated in the presence of additional drugs such as VX497 or derivative\nchimeras, we could find that short cycloheximide cell treatments with 300 significantly reduced the\nIMPDH2 buildup over cells treated with 300 in the absence of CHX (see Supplementary Fig. 17B,\nwhich has been added to the second revised version).\nTherefore, our observations suggest that in the first hours of cell treatment with VX497-\nbased chimeras, there is a balance between the increased IMPDH2 gene expression and induced\nnitcA/2HDPMI\n8 h\n24 h\n48 h\nIMPDH2 degradation that maintains steady-state levels of IMPDH2 protein at apparent stability\n(or increase).\nOverall, we have modified the discussion section by eliminating the paragraphs mentioned\nabove and adding in page 11, line 68: “MOA-wise, the time-consuming depletion of IMPDH2 is an\nintriguing observation. However, a possible explanation could be linked to the observed increase in\nboth IMPDH2 mRNA levels, assessed by qPCR (Supplementary Fig. 17) and protein levels\n(Supplementary Fig. 6c,d,g), induced by VX-497 and VX-497-based compounds (e.gr., 300 and\n354), in the initial 24-48 h of treatment, which may counteract the effect of degradation.”\nComment 1b\n- The mode of delayed IMPDH2 degradation is bizarre but very intriguing. The most potent\nIMPDH2 degrader is 354, working at sub-μM range, and only get to such potency for longer 96 hr\nincubation. On the other hand, CERT1 degraders work as early as 24 hr but none of them show\nsub-μM conc of potency. How mechanically this observation can be explained? For shorter time\npoints treatment, only 300 and 330 were shown (Supple Fig. 6), so wondering if 354 displays\nsimilar behavior as others.\nAnswer\nTo test this important point, we carried out assays in the presence of 354 in time points in the 8-72\nhours range. We observed that the increase of 354 (from 2 μM to 10 μM and 20 μM) did not cause\na faster degradation of IMPDH2. We have included these results in Suppl. Fig. 6H.\nWe have included these results in page 5, line 57, within the following text: “However, like with 300,\nIMPDH2 degradation occurred at long incubation times (72-96 h) (Fig. 3a,b and Supplementary\nFig. 6a-c,h), at which 354 was…”\nComment 1c\n- At lines 35-36, page 3, the authors quoted solely reported IMPDH2 PROTAC (Ref 41). Curiously,\nthis PROTAC works even for 24 hr treatment although at μM level. So, then the delayed\ndegradation by IMPDH2 degrader may not be necessarily the nature of the target protein, rather\ndue to the nature of degrader?\nAnswer\nSince the publication Sohbati et al. shows a chimera including mycophenolic acid (as IMPDH2\nbinder) and pomalidomide (as a CRBN recruiter), and the mechanism of action is based on\ndifferent actors, we do not have a good base to compare their compounds with ours. Sohbati et al.\ndo not provide data on distinct incubation times and on any possible effect of mycophenolic acid on\nIMPDH2 levels. Nevertheless, due to their mechanistic differences, it is possible that the different\ncompounds (Sohbati et al. degraders and our degraders) exhibit distinct degradation behaviors\ntowards IMPDH2.\nComment 1d\n- In the similar context of above point and also as the authors described, Supple Fig. 8 shows the\ncellular amounts of 354 at 96 hr as ~4 times higher than at 24 hr. Thus, apart from the nature of\nIMPDH2 protein, would be there any chance of the delayed degradation kinetics also to be\nattributed to the actual working conc of IMPDH2 degrader in cells over time? Then, what will\nhappen if the degrader would be treated every 24 hr for total 96 hr compared to one time treatment\nfor 96 hr as it is now?\nAnswer\nThis is an interesting question, and we acknowledge the reviewer for raising it. We observed that\ndegradation could be induced in the range of 1-2 µM upon 64-68 h of treatment. When assays\nbased on multiple additions of the compound or single initial additions (0.5 and 1µM) were\nperformed, we observed that the pattern of addition did not accelerate the degradation.\nWe have introduced these new data in Supplementary Fig. 6I, copied below, and included the\nfollowing text in page 5, line 61 “Treatments distributed in multiple additions of 354 did not\nremarkably change the degradation time pattern (Supplementary Fig. 6i).”\nI\n354 354 (64h total treatment)\n64h 68h 72h T=0h - 0.5 0.5 1 1 2 µM\n+24h +0.5 +1 µM\n- 1 2 - 1 2 - 1 2 µM\nIMPDH2\nIMPDH2\nGAPDH\nGAPDH\nComment 2\n2. I see one of the major revisions as the addition of ‘Modeling of 26S-UIDs ternary complexes’\npart. Indeed for this, the authors assigned rather extensive amount of writing with one spacious\nmain Figure (Fig. 7). However, all these arguments are limited by degrader modeling, not real data\nor resolved 3D complex structures. So, unless experimentally validated, they are just nothing but\nspeculation no matter how reasonable it sounds like.\nAnswer\nWe acknowledge the comments of the reviewer. In the first revision of the paper, one of the\nreferee’s comments was “The authors should either add the essential modeling data into the main\nresults of the manuscript (as a substitute for the lack of biophysical ternary complex\nmeasurements), or make it a separate paper.” therefore we decided to include this section. Now\nwe present new results that support the formation of ternary complexes of USP14/386/CERT1 and\nUSP14/354/IMPDH (see new paragraph in main text at page 9) and in Supplementary Fig. 13). In\norder to experimentally validate the formation of ternary complexes in the presence of chimeras,\nwe have performed pulldown assays by immobilizing Usp14 on beads, and incubating distinct\nsoluble fractions including CERT1 or IMPDH2, in the presence of representative compounds. We\nhave observed that 354 (1 and 10 µM) or 386 (5 and 30 µM) could induce a ternary interaction,\nwhereas compounds binding solely one protein (VX497, binding IMPDH2, and 366, binding\nCERT1) did not. Therefore, the chimeras induce interactions, which are mechanistically required\nfor the promoted degradation. The corresponding figures are included in the supplementary\ndocument as Supplementary Fig. 13 and methods are described in the Methods section of the\nSupplementary document (page 30). In the main text, we have added the following paragraph at\npage 9, line 23:\n“In order to experimentally validate ternary complex formation, we carried out pulldown assays\nbased on USP14 immobilized on beads, in parallel assessments of IMPDH2 and CERT1\ninteractions with USP14, in the presence of representative 354 and 386 chimeras. The assays\nrevealed that 354 (1 and 10 µM) or 386 (5 and 30 µM) could induce ternary interactions with both\nendogenous and recombinant proteins. Control pulldown assays confirmed the specificity of the\nternary complex formation. Compounds binding only one protein—VX-497 (IMPDH2) and 366\n(CERT1)— failed to pull down interacting partners, indicating that stable interactions require all\nthree components. Beads lacking USP14 also failed to pull down chimeras or targets, confirming\nthat both did not bind beads unspecifically. Robust pulldown signals were only observed when all\ncomponents were present, and negative controls further validated interaction specificity\n(Supplementary Fig. 13).“\nIn addition, we have reduced the contents of Fig. 7, moving two panels to the Supplementary\nMaterial (Supplementary Fig. 16), and described the computational results in a more concise\nmanner (page 9, line 41 until the end of section): “To elucidate the molecular basis of ternary\ncomplex formation, we employed the Protac-Model workflow, previously validated for modeling\nother Protac-mediated assemblies.[46] This approach generates ensembles of structural models\ngrouped into clusters, each ranked according to protein–protein interface energy. Ternary\ncomplexes of USP14/IMPDH2 with chimeras 300, 330, and 329—all containing IU1-248 as the\nUSP14-binding moiety—were modeled as described in the Computational Methods section\n(Supplementary Information, Section 2). The simulations yielded 21 models (9 clusters) for 300,\n86\nmodels (11 clusters) for 330, and 150 models (12 clusters) for 329 (Supplementary Fig. 2a–c).\nConsistent with previous observations for other Protac systems,[58] multiple distinct protein–protein\narrangements, rather than a single preferred binding mode, met the conditions of shape\ncomplementarity, as well as the geometric and energetic requirements of the chimeras.\nRemarkably, the lowest-energy cluster for all three 26S-UIDs corresponded to the same overall\nprotein arrangement, suggesting that each chimera can adopt conformations compatible with this\nconfiguration (Supplementary Fig. 14a). This arrangement also exhibited the largest buried surface\narea (Supplementary Fig. 2), indicative of extensive interprotein contacts. Superposition of USP14\nfrom these complexes onto the proteasome-bound USP14 subunit (PDB 5GJQ) revealed that the\nmodeled configurations are sterically compatible with the 26S proteasome, fulfilling a key structural\nprerequisite for 26S-UID activity (Fig. 2e). Similarly, modeling of the 317-based chimera\n(USP14/354/IMPDH2) (Supplementary Fig. 2d) produced a top-ranked structure closely\nresembling that of 300, with only minor differences in binding geometry (Supplementary Fig. 14a).\nTernary complex models were also generated for USP14/CERT1-START bound to chimeras 368,\n386, 413, 395, and 419 (Supplementary Fig. 15). In this case, a broader diversity of binding modes\nand interaction energies was observed, primarily attributable to differences in PEG–carbamate\nlinker length. Chimeras 368 (3 PEG units), 386 (2 PEG units), and 413 (1 PEG unit) exhibited\nsimilar binding modes, whereas 419 (5 PEG units) and 395 (no linker) adopted distinct, less\nfavorable configurations with higher interface energies, particularly pronounced for 395\n(Supplementary Fig. 14b). Despite these suboptimal geometries, both chimeras retained potent\ndegrader activity (see previous section), suggesting that degradation efficacy is not strictly\ndependent on interface energy or complex geometry. Structural analysis of the modeled ternary\ncomplexes indicated that stabilization arises from multiple intermolecular interactions between the\n26S-UID molecules and their binding partners (Fig. 7a,b), as well as between each pair of proteins\n(Supplementary Fig. 16). Notably, high surface complementarity was observed between protein\npairs that have not naturally co-evolved to interact. This complementarity is partly explained by the\nconsistent occupation of USP14’s concave surface region by the target protein—a region that\ncorresponds to USP14’s native ubiquitin-binding site (residues 183–213, 265–278, 294–302, 330–\n342, and 428–434) (Fig. 7c,d).[46][58”\nWith the above new results in hand and since the resolution of the 3D structures of the\nternary complexes is beyond the scope of this paper, we think that this modeling section provides\nreasonable clues about the structure of these complexes and the interactions that stabilize them.\nTherefore, despite speculative (as most theoretical and modeling results published in the\nliterature), we believe it is justified. We hope that the reviewer will agree to this and find this section\nuseful for the understanding of the mechanism of action of our compounds.\nComment 3\n3. In relation to my point #2 above, I have some concerns on the argument that the authors\ndeduced from the degrader modeling data. At lines 62 to 66, page 9, it is said, “…These findings\nsuggest that the structures of chimeras 419 and 395 constrain the proteins to bind in a less optimal\nmanner, which correlates with the relatively lower potency as degraders of these chimeras…”.\nHowever, although 395 and 419 are less potent than others in MDA-MB-231 cells, they are no\nworse than others in MDA-MB-453 and BT-474 cells. Then, I tend to think that these cell type-\ndependent differences in responsiveness cannot be explained simply by the ternary complex\nmodeling. Also, apart from this modeling issue, in terms of the responsiveness, I am wondering\nwhy different cell lines show inconsistent degree of sensitivities among the CERT1 degraders?\nFor example, as aforementioned, 368, 386, and 413 seem to be the most potent over 395 and 419 but\nagain only in MDA-MB-231. In MDA-MB-453, both 395 and 419 look superior to 386 and probably\neven 413. Also in BT-474, 368 does not look that better compared to 395 and 419. How the\nauthors could explain this conundrum?\nAnswer\nWe thank the reviewer for this comment, which we agree it needs clarification. To compare the\npotency of each Protac across the different cell lines, we performed a two-way ANOVA to evaluate\nhow the mean CERT1 degradation (based on densitometric data) varied with respect to the Protac\nstructure and concentration, treated as two independent variables. The two-way ANOVA showed\nno significant interaction between concentration and Protac, indicating that the differences between\nProtacs are consistent across concentrations. While there was a significant overall effect of Protac\ntype, post-hoc Tukey’s tests did not find any Protac to be significantly more potent than the others\nat the tested concentrations. Therefore, the data do not support that any individual Protac is more\neffective than the rest at the tested concentrations. The fact that there are significant differences\nbetween the Protacs at 40 μM is due to the possibility of a hook effect at high concentrations, and\ntherefore the impact on degradation is not being properly assessed. To cope with this comment,\nwe have added Supplementary Fig. 9, showing the results of the statistical analysis.\nFurthermore, the following changes have been introduced in the text:\nPage 7, line 33, The sentence “At 24 h incubation, CERT1 degradation was also observed\nusing the additional compounds carrying distinct linker lengths (Fig. 4b, c), with compounds 413,\n386 and 368 seeming the most potent ones.” has been replaced by “After 24 h of incubation, dose-\ndependent degradation of CERT1 was observed for all five compounds, each featuring distinct\nlinker lengths (Fig. 4b, c). To compare their effectiveness as degraders, a two-way ANOVA was\nperformed to assess how CERT1 degradation (quantified via densitometric analysis) varied with\nProtacs structure and concentration, treated as independent variables. Post-hoc Tukey’s tests\nindicated that none of the Protacs induced significantly greater degradation than the others at the\nconcentrations tested. A similar pattern was observed in HER2-positive breast cancer cell lines\nMDA-MB-453 and BT-474 (Supplementary Fig. 9). However, in these cases, some statistically\nsignificant differences emerged at 40 µM for certain compounds, consistent with a potential hook\neffect.”.\nComment 4\n4. In the similar context of above point, in Fig. 6, what is the reason to show each graph derived\nfrom each different cell line?\nAnswer\nWe acknowledge that the most comprehensive approach would have been to test all five Protacs\nacross the three cell lines. However, given that each condition requires at least three independent\nexperiments performed in triplicate, the number of samples quickly becomes substantial. Due to\nlimitations in access to LC/HRMS instrumentation, it was not feasible to analyse such a large\nsample set in a reasonable time. While testing all five Protacs in a single cell line was an\nalternative, we chose instead to evaluate one selected Protac in each of the three cell lines. We\nbelieved this approach would provide more meaningful insights into the broader applicability of the\ntreatment across different cellular contexts. Although the data did not show statistically significant\ndifferences (see answer to previous comment), we selected the following compounds and\nconcentrations based on observed trends: compound 268 (10 µM) for MDA-MB-231, compound\n413 (10 µM) for MDA-MB-453, and compound 395 (20 µM) for BT-474.\nWe have included the following paragraph in the discussion (page 12, line 28): “It is intriguing\nthat all five CERT1-targeted Protacs exhibit similar potency at the tested concentrations. While this\nmight suggest that the linker length does not significantly influence the degradative activity, the role\nof membrane permeability cannot be ruled out. It's possible that the compounds differ in their\nintrinsic potency, but these differences may be obscured by variations in cellular permeability.”.\nComment 5\n5. At Fig. 3f, the authors performed the competition assays with VX-497. Since they did the same\nkind of assays also with USP14 ligand in case of CERT1 degrader (Fig. 5c), please do it as well for\nIMPDH2 degarder. The authors commented the partial recovery by VX-497 due to its toxicity (page\n6, lines 1-4), so the non-toxic 352 would be expected to be good competitor for IMPDH2 degrader\nas well.\nAnswer\nIn order to approach this point, we have carried out cell treatments with 354 2 µM in the presence\nof different 352 concentrations (1, 2, 5, and 10 µM). We have observed that, at 5 and 10 µM, 352\ncould rescue the degradation induced by 354. The result has been included in Fig. 3f.\nThe result has been included in the following sentence in page 5, line 84: “Furthermore, IMPDH2\ndepletion was counteracted dose dependently by preincubation with the IMPDH2 ligands VX-497\nand 352 (Fig. 3f).\nComment 6\n6. At page 5, lines 19-22, it is said, “Together, these results show that chimeras can productively\ninteract with proteasomal USP14 and IMPDH2 in a functional context, suggesting the feasibility of\nour approach.” Regarding SPR data, the SPR was performed with USP14 in the absence of\nproteasome, so apart from the inhibitory acivity, the binding affinities of all tested chimeric\ncompounds may just reflect their interaction with free USP14, not “proteasomal USP14”.\nAnswer\nWe have deleted “proteasomal” from the sentence.\nComment 7\n7. At page 2, lines 32-35, the sentence of “USP14 is a deubiquitinating enzyme that associates ….\nin the regulation of the ubiquitin-proteasome pathway, with an impact in multiple cellular\nprocesses” seems to be somewhat redundant with the paragraph starting from line 45. Also, it\nwould be better to introduce the proteasome first. So, please relocate this sentence to the back or\ncombine with the latter by compacting and rephrasing or can just delete this sentence.\nAnswer\nWe have deleted the sentence.\nComment 8\n8. The methods for IMPDH2 enzymatic assays should be described in details.\nAnswer\nWe have explained the methods in the section: “Inosine monophosphate dehydrogenase\nactivity assays\" in the Supplementary file, page 26.\nComment 9\n. Other miscellaneous points\n- Page 2, Ref 4 is not a good citation for PROTACs in clinical trials.\nAnswer. we have changed the reference for a review on trials and patents (Cai et al. 2025)\n- All the Figures are presented as A, B, C… but in the text, they are all a, b, c…..\nAnswer. In order to be consistent, we have changed the lettering in all main figures to “a”, “b”,\netc,\n- Page 3, line 28, Inosine -> inosine\nAnswer. Done\n- Page 3, line 78, inhibitor 77 -> inhibitor VX-497\nAnswer. Done\n- At Fig. 3f, last lane (VX-497, 50 μM), what happens here? I do not see any band in both blots.\nAnswer. We have removed this lane because 50 μM caused strong cell lethality in these\nassays.\n- USP14(-/-), multiple spots, the correct gene nomenclature for mouse and for KO should be\nused.\nAnswer. Done\n- Page 7, line 7, that -> than\nAnswer. Done\n- Page 9, line 68, 26STAC -> 26S-UID\nAnswer. Done\n- Page 11, lines 28 to 31, the sentence is not well matching with given references (Refs\n[9,21,22,65–67]).\nAnswer. We have removed the references and included refs 1-3, which introduced E3-based\nprotacs.\n- In Acknowledgements, I bet Usp14-/- & WT MEF cell lines were originally generated by Scott\nWilson group (U of Alabama). MJ Lee could be a donor but this person probably is not an\noriginal creator. I am kind of concerned that the original creator might not be properly\nacknowledged in this way. Please check it out and indicate rightful credit.\nAnswer. Done\nReviewer #2 (Remarks to the Author):\nComment\nThis is my second review of the manuscript, and first of all, I recognize the efforts of Casasampere\nand co-workers to improve the study. However, I am concerned about the complete removal of the\nmolecular dynamics simulation data related to the ternary complexes, as this alters the scope of\nthe original work and prevents a full evaluation now.\nRather than refining or clarifying the methodological concerns raised, removing this section\nchanges the scientific contribution initially presented. If every critique were addressed by\neliminating data instead of improving it, it could set a concerning precedent for scientific discourse.\nThat said, I defer to the editor’s judgment on the overall suitability of the revised manuscript\nbut wanted to share my perspective directly.\nAnswer\nWe acknowledge and appreciate the reviewer opinion. However, we would like to recall that in the\nfirst revision of the manuscript he questioned “whether the simulation length or the use of unbiased\nMD simulations is adequate for studying the ternary complexes like the ones shown here.” and he\nadded that “Obtaining meaningful data from MD simulations in these cases likely requires much\nmore sampling than the authors have considered.” In addition, referee 3 said “Considerable editing\nis needed to trim the manuscript to the essential data. For example, there are 7 pages of molecular\nmodeling results associated with 7 supporting figures, 12 supporting movies, and 9 supporting\nTables all in the Supporting Information. This material is briefly touched upon in the main results\nsection and yet seems to constitute what would be a separate companion paper given. The\nauthors should either add the essential modeling data into the main results of the manuscript (as a\nsubstitute for the lack of biophysical ternary complex measurements), or make it a separate paper.”\nTaking this into account, we decided to remove the MD results on the ternary complexes\nsince we agreed that our results could only be considered as preliminary and that a more in depth\ncomputational study (with more starting structures, longer simulation times, more repeats,…)\nwould\nbe required to achieve more solid conclusions. We considered that this study is beyond the scope\nof this work since this is not a computational paper. Therefore, we introduced a new section in the\nmain text, as suggested, that includes only the results relative to the modeling of the ternary\ncomplexes based on protein-protein docking methods (ie. PROTAC-Model software), which have\nbeen used in the literature as a tool to predict the structure of ternary complexes of Protacs with\nreasonable accuracy (see for example: Nucleic Acids Res 2023, 51(D1), D1367-D1372; J Am\nChem Soc 2023, 145, 7123-7135; J Chem Inf Model 2020, 60, 4894-4903; Bioorganic Chemistry\n2025, 165, 109017; ACS Med Chem Lett 2025, 16, 420-427). We do not believe that this change\nhas modified the scope of the original work because all the removed information was originally\nplaced in the Supporting Information, meaning that it was not central for the interpretation of the\nexperimental results and the development of the argumentation and conclusions.\nGiven all these considerations, we hope, as mentioned above, that the referee and the editor\nwill agree that the inclusion of a modeling section that provides reasonable clues about the\nstructure of these complexes and the interactions that stabilize them is justified. We hope that they\nwill approve this and find this section useful for the understanding of the mechanism of action of\nour compounds.\nReviewer #3 (Remarks to the Author):\nThe authors have made valiant efforts to address the many issues raised by reviewers. In\nparticular, the USP14 KO experiments and competitor titrations go a long way in strengthening the\nauthors’ claims of USP14-mediated recruitment to the 26S proteasome for ligase-independent\ndegradation. However, I am still perplexed by the lack of direct ternary complex formation studies\nin this work and instead the reliance on molecular modeling of the proposed ternary complexes.\nComment 1.\n1. Given that the necessary molecules and protein reagents are available for individual SPR\nbinding experiments, why not measure compound-mediated ternary complex formation? The SPR\ndata that is shown has some issues (see point 2) and the relatively weak double-digit micromolar\n(or higher) affinities may limit the technical feasibility of performing this kind of SPR-based, ternary\ncomplex measurements, but simple pulldown experiments with the recombinant proteins ±\ncompound would easily confirm compound-mediated ternary complex formation. Because the\ncellular activity of these compounds is often at concentrations 10-fold lower than the measured Kd\nvalues for either USP14 or target it suggests that these compounds can act as molecular glues\nwith USP14 and targets forming protein-protein interactions (as is suggested in the modeling). It\nshould be straight forward to directly observe USP14-compound-target ternary complexes and\nmeasurements of the lifetime of these complexes would go a long way in explaining their cellular\npotency. Confirming that these complexes can also pull down 26S components in a compound-\ndependent manner would provide further critical validation of the MOA proposed for these\ncompounds.\nAnswer.\nWe thank the reviewer for these accurate observations. We agree that evidence of ternary\ninteractions would strengthen the work and provide a better description of the MOA. We have\napproached ternary complexes by means of pulldown assays, which are shown in Supplementary\nFig. 13. We have observed that targets (IMPDH2 and CERT1, both recombinant and present in\nwhole cell abstracts) are pulled down by immobilized Usp14 in the presence of the corresponding\nchimera.\nConcerning the Usp14-386-CERT1 pulldowns, we have observed pulled down CERT1\nprotein in assays including 386 at 5 and 30 µM. However, a big difference in signal is observed\nwhen comparing 30 and 5 µM assays. This suggests a strong difference on the effect in CERT1\ntargeting and degradation in this concentration range. This observation is consistent with the data\nobtained in cellular assays. As for the Usp14-354-IMPDH2 pulldowns, we have observed the\ndifference in range 354 1-10 µM. Moreover, in recombinant IMPDH2 (lanes 9-10), the difference is\nnot that strong. This suggests that the range of ternary targeting could be in the low 1-10 µM\nrange. The data is also compatible with the observations obtained in cell cultures.\nTaken together, these observations reinforce the idea of a ternary interaction, necessary for\ncorrect recruitment of the target with Usp14. We modestly think that the new observations\nobtained, in addition to the previous mechanistic dissections of target degradation (Fig. 3-5 and\nSupplementary Fig. 6) and the in-silico data analyzing the ternary interactions (Fig. 7 and Suppl.\nFig. 14 and 15), describe a very interesting mechanism of drug-induced target degradation, not\ndescribed in the literature. Due to the time consumed to perform the assays and the clarity of the\npulldowns based on immobilized Usp14, we have not performed the opposite pulldown\nexperiments, with immobilized IMPDH2 and CERT1, but if the reviewer considers that it is\nnecessary, we will certainly carry them out.\nWe have included the following paragraph in the main text (page 9, line 23): “In order to\nexperimentally validate ternary complex formation, we carried out pulldown assays based on\nUSP14 immobilized on beads, in parallel assessments of IMPDH2 and CERT1 interactions with\nUSP14, in the presence of representative 354 and 386 chimeras. The assays revealed that 354 (1\nand 10 µM) or 386 (5 and 30 µM) could induce ternary interactions with both endogenous and\nrecombinant proteins. Control pulldown assays confirmed the specificity of the ternary complex\nformation. Compounds binding only one protein—VX-497 (IMPDH2) and 366 (CERT1)— failed to\npull down interacting partners, indicating that stable interactions require all three components.\nBeads lacking USP14 also failed to pull down chimeras or targets, confirming that both did not bind\nbeads unspecifically. Robust pulldown signals were only observed when all components were\npresent, and negative controls further validated interaction specificity\n(Supplementary Fig. 13).“\nComment 2.\n2. The representative SPR sensorgrams shown in Supplementary Figure 5 are not conducive to\nkinetic fitting as described in the methods and should instead be fit using equilibrium\nmeasurements to better quantify the Kd values for these compounds binding to USP14 and either\ntarget. This kind of analysis is common for weak micromolar binding as is evident in the raw\nsensorgrams.\nAnswer.\nWe thank the reviewer raising this point, since there was a misunderstanding in the SPR analysis\ndata used. The sensorgrams were not used for kinetic fitting, but instead, for affinity fitting\n(constants given were calculated at the equilibrium, as mentioned by the reviewer above).\nTherefore, we have not changed the methods section since it should be correct as it is."}, {"round": 3, "reviewer_comments": {"reviewer_1": "I see that the authors put quite considerable efforts to make their study better and explainable. Although some of previously raised key issues, such as IMPDH2 degradation behavior and CERT1 degraders’ potency, still did not seem to be fully addressed, these could be intrisically challenging to be resolved at this stage. Since the merit of this manuscript is reporting new modality of 26S-UID degraders and showing them as proof-of-concept–which at least I can be more or less assured, in principle now I am in favor of this study to be published. Just for better completion of the work, I ask the authors to make some points a bit clearer.\n- For the authors’ answer to “Comment 1d”, I appreciate that they tried multi-treatments vs single treatment comparison, leading to little difference. However, I was also pointing out that the cellular amount of 354 at 96 hr is ~4 times higher than the one at 24 hr (Supple Fig. 8), while its amount in medium decreases over time. Hence, I was asking if the delayed or abrupt degradation by IMPDH2 26S-UID could be also due to this ~4-fold higher actual working concentration within cells at ~96 hr (apart from apparent transcriptional feedback response at earlier time points that the authors newly found). Is this something to be discussed as well? And in terms of retention, stability, excretion and/or metabolism of a given chemical compound (even single-treated at initial time point) in the medium or within cells, I am curious if the detection amount like this case here can be taken as common or what the authors think?\n- Based on the authors’ answers to “Comment 3” & “Comment 4”, Now I see that their original argument–i.e. 395 and 419 are less potent and 368, 386, and 413 are more potent–was largely negated. So, it would be fair to say that the authors should have been more careful in interpreting their original data. Anyway, I want to point out one minor thing in the revised text (the one described in Answers in response to above two Comments). I suggest to change Protacs to 26S-UIDs, CERT1-degraders or the compounds because the terminology of Protacs seemed to be mostly dedicated to (E3-based) Protacs in the rest of the text or in general.\n- Between lines 40 to 58, “Despite the fact that ubiquitin signal …….. downstream steps are ubiquitin-independent[9,21,22,26]”, this sentence is not well read, hard to understand, and may be structurally broken. Please rephrase for clarity.\n- For gene name in mouse MEFs, Usp14-/-, not USP14-/-.", "reviewer_2": "The authors have addressed the prior comments and provided their rationale for the changes made. Some methodological questions remain, as certain points were not directly answered. This is understandable, as the entire MD simulation section was removed, although further refinement could have been possible by only partially omitting those results. Overall, the revised manuscript presents a coherent and internally consistent study. I am satisfied that it meets the journal’s publication standards.", "reviewer_3": "The authors have sought to address the additional concerns expressed by reviewers in the re-review of the manuscript.\nHere, I will limit my third review of the manuscript to the authors' responses to my two specific comments/concerns from the last review of the material.\n1. The first issue may be easily resolved with simple rewording and/or replotting to improve clarity. Specifically, the authors' rebuttal states:\n\"We thank the reviewer raising this point, since there was a misunderstanding in the SPR analysis data used. The sensorgrams were not used for kinetic fitting, but instead, for affinity fitting (constants given were calculated at the equilibrium, as mentioned by the reviewer above). Therefore, we have not changed the methods section since it should be correct as it is.\"\nHowever, the analyses of these data are not clear and do require attention. In the methods it states \" kinetic analyses of the ligand binding were performed\". While they later say \"fitting of a 1:1 binding model by affinity was performed\" they do not clarify that this is an equilibrium analysis and what time frame of the sensorgrams were used for this analysis. Moreover, in the SPR sensorgrams a second line is apparent in several of the traces. Is this a kinetic fit on the plot, or is a second experimental trace overlayed for a given concentration (which would be odd)? Taken together, I do not feel that the paper is clear on how the SPR analyses were performed and this needs to be better clarified.\n2. The second issue raised is regarding the lack of ternary complex formation data, which the authors attempt to address with new pull-downs experiments. In this most recent revision, His-tagged USP14 is shown to pull down targets in a compound-dependent manner both in whole-cell lysates and with recombinant targets. I have several criticisms of this work:\n• First, it is not sound practice to perform a pull-down where the target and the bait share the same affinity tag. In panel A, His-tagged USP14 is used to pull-down recombinant His-tagged CERT1.\n• The authors go on to show that Ni-NTA beads alone, after blocking with BSA and washing 3x, do not pull down any His-CERT1, to but this is hard to wrap my head around. The very weak binding of BSA to Ni-NTA should not be enough to block His-tag CERT1 binding, especially after the beads were washed 3x. This does not make any sense to me. While the whole-cell extract data are not encumbered with this issue, the results of the recombinant protein pulldowns make me question the entire experiment.\n• Finally, one minor issue is that both panels A and B should each be split into 2 distinct panels for the separate WCE and Rec Protein experiments. Even if they were run on the same gel, it is a bit confusing to have them in the same figure. It would be more clear to readers if they were separate panels."}, "author_response": "We appreciate the reviewers’ addi0onal comments, which helped us improve the manuscript.\nBelow are our point-by-point responses to the comments from Reviewers #1 and #3.\nIn the main text and Supporting information documents:\nTo ease the review by the referees, this copy is marked as follows:\n• Text related to comments of Reviewers are in magenta.\n• All other relevant changes made in the new version are in red.\n• Line numbers are given in green on the left margins of the columns.\n• Quotation to figures, schemes and tables are highlighted\nREVIEWER COMMENTS\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nI see that the authors put quite considerable efforts to make their study better and explainable. Although\nsome of previously raised key issues, such as IMPDH2 degradation behavior and CERT1 degraders’\npotency, still did not seem to be fully addressed, these could be intrinsically challenging to be resolved at\nthis stage. Since the merit of this manuscript is reporting new modality of 26S-UID degraders and showing\nthem as proof-of-concept–which at least I can be more or less assured, in principle now I am in favor of\nthis study to be published. Just for better completion of the work, I ask the authors to make some points a\nbit clearer.\nComment 1:\n- For the authors’ answer to “Comment 1d”, I appreciate that they tried multi-treatments vs single treatment\ncomparison, leading to little difference. However, I was also pointing out that the cellular amount of 354 at\n96 hr is ~4 times higher than the one at 24 hr (Supple Fig. 8), while its amount in medium decreases over\ntime. Hence, I was asking if the delayed or abrupt degradation by IMPDH2 26S-UID could be also due to\nthis ~4-fold higher actual working concentration within cells at ~96 hr (apart from apparent transcriptional\nfeedback response at earlier time points that the authors newly found).\nIs this something to be discussed as well? And in terms of retention, stability, excretion and/or metabolism\nof a given chemical compound (even single-treated at initial time point) in the medium or within cells, I am\ncurious if the detection amount like this case here can be taken as common or what the authors think?\nAnswer 1:\nWe are sorry we forgot to answer this comment. We agree that the compound's uptake and intracellular\naccumulation likely contribute—together with other factors—to the delayed IMPDH2 degradation we\nobserve. Several studies have reported time-dependent increases in the apparent potency of small\nmolecules in cellular assays, with higher activity at later time points (e.g., 96 h vs. 24 h). These effects\nhave been attributed to progressive intracellular accumulation. Examples include doxorubicin (e.g., doi:\n10.3390/cells10071717; doi: 10.3389/fbioe.2020.00005) and pemetrexed (e.g., doi:\n10.3390/pharmaceutics17081044). More broadly, numerous nanoparticle-based or prodrug delivery\nsystems exhibit delayed uptake kinetics (e.g., doi: 10.3389/fbioe.2020.00005). E3-based PROTACs (and\nlikely 26S-PROTACs as well) can enter cells via passive diffusion and/or endocytic pathways, depending\non their physicochemical properties. In the case of our IMPDH2-targeting 26S-UIDs, these properties may\nlead to relatively inefficient cellular uptake, making their behaviour more reminiscent of nanoparticles than\nclassical small molecules. This could plausibly explain why sufficient intracellular concentrations to trigger\nrobust IMPDH2 degradation are not reached until later time points (e.g., ~96 h).\nIn the discussion we have added the following text:\n“An additional explanation could be the kinetics of cellular internalization and response to compounds.\nThe observation that the uptake of 354 was 4 times higher at 96 h than at 24 h (Supplementary Fig. 8),\nis aligned with a delayed response in IMPDH2 degradation. The quicker transcriptional activation observed\n(Supplementary Fig. 17) could indicate that binary interactions (IMPDH2-VX-497 moiety) are initially favored,\nversus ternary engagements. Further research would be required to validate these hypotheses.”\nComment 2:\n- Based on the authors’ answers to “Comment 3” & “Comment 4”, Now I see that their original argument–\ni.e. 395 and 419 are less potent and 368, 386, and 413 are more potent–was largely negated. So, it would\nbe fair to say that the authors should have been more careful in interpreting their original data.\nAnswer 2:\nWe fully agree with this well-founded comment and acknowledge that greater rigor was warranted in the\ninitial version. We sincerely apologize for the oversight and have taken care to address it thoroughly in the\nrevision 2.\nMinor point 1:\nAnyway, I want to point out one minor thing in the revised text (the one described in Answers in response\nto above two Comments).\nI suggest to change Protacs to 26S-UIDs, CERT1-degraders or the compounds because the terminology\nof Protacs seemed to be mostly dedicated to (E3-based) Protacs in the rest of the text or in general.\nAnswer: We have substituted all the referred “Protacs”. We only kept the references to E3-based protacs\nand to software licenses.\nMinor point 1:\n- Between lines 40 to 58, “Despite the fact that ubiquitin signal …….. downstream steps are ubiquitin-\nindependent [9,21,22,26]”, this sentence is not well read, hard to understand, and may be structurally\nbroken. Please rephrase for clarity.\nAnswer: We have rewritten this section.\nMinor point 2:\n- For gene name in mouse MEFs, Usp14-/-, not USP14-/-.\nAnswer: Fixed.\nReviewer #2 (Remarks to the Author):\nComment:\nThe authors have addressed the prior comments and provided their rationale for the changes made. Some\nmethodological questions remain, as certain points were not directly answered. This is understandable, as\nthe entire MD simulation section was removed, although further refinement could have been possible by\nonly partially omitting those results. Overall, the revised manuscript presents a coherent and internally\nconsistent study. I am satisfied that it meets the journal’s publication standards.\nAnswer:\nWe thank the referee for his/her comments and understanding of our reasoning, as well as the acceptance\nof the modifications introduced into the computational part.\nReviewer #3 (Remarks to the Author):\nThe authors have sought to address the additional concerns expressed by reviewers in the re-review of\nthe manuscript.\nHere, I will limit my third review of the manuscript to the authors' responses to my two specific\ncomments/concerns from the last review of the material.\n1. The first issue may be easily resolved with simple rewording and/or replotting to improve clarity.\nSpecifically, the authors' rebuttal states:\n\"We thank the reviewer raising this point, since there was a misunderstanding in the SPR analysis data\nused. The sensorgrams were not used for kinetic fitting, but instead, for affinity fitting (constants given\nwere calculated at the equilibrium, as mentioned by the reviewer above). Therefore, we have not changed\nthe methods section since it should be correct as it is.\"\nHowever, the analyses of these data are not clear and do require attention. In the methods it states \"\nkinetic analyses of the ligand binding were performed\". While they later say \"fitting of a 1:1 binding model\nby affinity was performed\" they do not clarify that this is an equilibrium analysis and what time frame of the\nsensorgrams were used for this analysis. Moreover, in the SPR sensorgrams a second line is apparent in\nseveral of the traces. Is this a kinetic fit on the plot, or is a second experimental trace overlayed for a given\nconcentration (which would be odd)? Taken together, I do not feel that the paper is clear on how the SPR\nanalyses were performed and this needs to be better clarified.\nAnswer 1:\nWe thank the reviewer and really apologize for not being clear enough in the answer. The reviewer is right\nin that the paragraph should clarify additional aspects of the experiment, and avoid the term ‘kinetics’.\nBelow, we show an improved version of the methods, in which we aim to better explain the affinity\napproach, including descriptions of the technical duplicates shown in the graphs, and the frame for the\nequilibrium analysis. Moreover, in order to provide a complete description of SPR methodology, we have\nfollowed the Nature Communications guidelines for SPR methods, kindly provided by the editors.\nRegarding the technical duplicates, they were injected on a 1,1; 2,2; 3,3…basis, which could explain the\nhigh reproducibility. We have additionally given consistence to the SPR terminology, in which ‘analytes’ are\nthe mobile synthesized compound (354, 386, etc), and ‘ligands’ define the immobilized protein (CERT,\nUSP14, etc).\nImproved section as included in methods:\nSurface plasmon resonance (SPR).\nThe affinities of the synthesized compounds (here referred to as ‘analytes’) vs. different recombinant proteins were\ndetermined using a BIACORETM T200 from Cytiva. All experiments were performed at 25 °C and data were\ncollected at a rate of 10 Hz. Recombinant proteins [USP14, IMPDH2, and CERT1 (amino acids 347–598; Abcam\n95897), here all three referred as to ‘ligands’] were immobilized on a CM5 sensor chip series S (Cytiva) using the\namine-coupling method described by the manufacturer. In details, USP14 was immobilized in channel 2, IMPDH2 in\nchannel 3 and CERT1 in channel 4, leaving channel 1 empty as reference. The channels were activated with EDC-\nNHI (at final concentration of 200 mM and 50 mM, respectively) during 7 min, and blocked with ethanolamine-HCl\n(1M pH 8.5) during the same time, as the activation. For the immobilization, the ligands were diluted with 10 mM\nsodium acetate buffer at pH 5.0, for USP14 and CERT1, and pH 5.5 for IMPDH2. The density of the coupling was\ncalculated to achieve a theoretical maximum analyte binding capacity (R ) within the range of 20-150 RUs, with\nmax\nan injection time between 50 and 75 seconds depending on the ligand. The running buffer during the immobilization\nwas PBS with 0.05% Tween20 at a flow rate of 15 μL min-1 and 5 µl min-1 for the injection of the ligand. The\nanalyses of the analytes binding to the ligands were performed in buffer PBS with 0.05% Tween 20 and 5% DMSO\nat a flow rate of 60 μL min-1. Each of the synthesized compounds was injected in the running buffer over the\nimmobilized USP14, IMPDH2 or CERT1. For all analytes, samples at 100 µM concentration, and a 2-fold dilution\nseries to 6.25 µM concentration, were injected, by duplicates (1,1; 2,2; 3,3; etc.), for 60 s in the association phase,\nwhich is followed by 360 s of dissociation phase. Equilibrium-based affinity analysis was performed using report\npoints set 10 s before the end of the injection, within a 5-s window frame. Data were analyzed using Biacore T200\nEvaluation Software v3.1 (Cytiva). In brief: (1) solvent correction was applied, (2) the sensorgrams were double-\nreferenced, and (3) a 1:1 steady state affinity model was applied selecting KD fit global, R and offset fit local.\nmax\nComment 2:\n2. The second issue raised is regarding the lack of ternary complex formation data, which the authors\nattempt to address with new pull-downs experiments. In this most recent revision, His-tagged USP14 is\nshown to pull down targets in a compound-dependent manner both in whole-cell lysates and with\nrecombinant targets. I have several criticisms of this work:\n• First, it is not sound practice to perform a pull-down where the target and the bait share the same affinity\ntag. In panel A, His-tagged USP14 is used to pull-down recombinant His-tagged CERT1.\n• The authors go on to show that Ni-NTA beads alone, after blocking with BSA and washing 3x, do not pull\ndown any His-CERT1, to but this is hard to wrap my head around. The very weak binding of BSA to Ni-\nNTA should not be enough to block His-tag CERT1 binding, especially after the beads were washed 3x. This\ndoes not make any sense to me. While the whole-cell extract data are not encumbered with this\nissue, the results of the recombinant protein pulldowns make me question the entire experiment.\n• Finally, one minor issue is that both panels A and B should each be split into 2 distinct panels for the\nseparate WCE and Rec Protein experiments. Even if they were run on the same gel, it is a bit confusing to\nhave them in the same figure. It would be more clear to readers if they were separate panels.\nAnswer 2:\nWe really acknowledge the reviewer for raising these issues. We realized that we inexplicably omitted one\nsentence in the description of the preparation of the empty beads for Suppl Fig. 13B (described in next\nlines). We deeply apologize for that. We agree with the referee that is not common to design a pulldown\nassay concurrently using the same tags. We have observed that fully occupied Ni-NTA beads by His-\ntagged proteins do not bind additional His-tags. Furthermore, for the preparation of control empty beads,\nwe commonly can prevent specific His-tag binding with preincubation of beads with 250 mM imidazole and\nthis way generate a context similar to that of complete Ni-NTA occupancy by His-tagged proteins (as in\nUSP14-loaded beads). In our previous submission, by mistake, we did not include these explanations. In\nany case, in the current submission, in order to provide a more appropriate experiment, we have\neliminated the previous assay in Suppl Fig. 13B, and performed a new one.\nIn the new assay we have used the same version of CERT (95897 Abcam), but we have removed, in a\npreparative step previous to the pulldown, the His-tag by enterokinase digestion, taking advantage of the\nenterokinase cleavage site present between the His-tag and CERT (a descriptive figure is shown below).\nThus, we have generated a version of CERT free of His-tag, to be used in a pulldown assay with His-\nUSP14 immobilized in Nickel-beads. In the new assays we have been able to reproduce our previous\nobservations, this time with a more accurate approach, which we have included in the new Suppl. Fig. 13,\npanel B. We thank the reviewer for this important comment.\nHIS-Enterokinase(EK)-CERT1 (95897 Abcam; START domain; amino acids 347–598)\nSequence (with EK cleavage site):\nProcessing of HIS-EK-CERT1 (95897 Abcam) with EK\nfor Pulldown assay (Suppl. Fig. 13B):\nCleavage with EK\nM + - + -\nKDa\n100\n55\n40\n(PA5-90421\nThermofisher)\nFurthermore, we have divided Supplementary Fig. 13 in four panels, which certainly makes the whole\nexperiment more understandable. Changes in the figure legend were also introduced.\nThe new methodological paragraph is:\nPull-down assays\nImmobilized His-Usp14 was obtained as described above in “Expression and purification of 6His-Usp14 protein in E.\ncoli.” methods, avoiding the elution step. After His-Usp14 immobilization, beads were washed thrice with 50 mM\nNaHPO, 100 mM NaCl, pH 8 buffer and then incubated in blocking buffer (10% BSA in 50 mM NaHPO, 100\n2 4 2 4\nmM NaCl, pH 8 buffer) for one hour at room temperature. The beads were then washed again thrice with 50 mM\nNaHPO, 100 mM NaCl, pH 8 buffer and once with washing buffer (50 mM NaHPO, 100 mM NaCl, 1 mM DTT,\n2 4 2 4\n10% Glycerol, protease inhibitor cocktail, pH 7.5). Empty Ni-NTA control beads were washed and blocked with BSA\nin the same manner. Then, purified GST-IMPDH2 (see above) or CERT (95897 Abcam; digested with enterokinase to\nremove the His-tag; see below) proteins were added at 500nM final concentration. The beads-substrate mixture was\nthen aliquoted to separate tubes and 386, 366, 354 or VX497 were added at the indicated concentration and incubated\nat 4ºC for two hours. The beads were washed thrice with 10 column volumes of washing buffer and then eluted with\nSDS loading buffer. For experiments in which the input substrate is a whole cell extract (WCE), the sample was\nprepared as follows. Ten 100 mm dishes of confluent HeLa cells were washed with TBS and then scraped into a 15\nmL falcon tube. The cells were pelleted, resuspended in wash buffer, and then lysed by sonication (Branson sonicator\nmicrotip, 30% amplitude, 3 cycles of 1 effective minute (3 s ON/3 s OFF). The lysate was cleared by centrifugation\nand then added to the beads as an input substrate, as above. The digestion of His-CERT (95897 Abcam) with\nenterokinase, in order to remove the His-tag, was carried out as follows: His-CERT protein (supplied in 20 mM Tris\nHCl pH 8, NaCl 100 mM by the manufacturer) was incubated during 16 hours at 22ºC with enterokinase (His-tagged;\nGenScript, Z03004-100), added in a 1.2 units of EK/10 ug of protein substrate ratio. Released His-tag and His-\nenterokinase were removed by adding 5 µL Ni-NTA beads in 100 µL protein solution, 2 hours of incubation at 4°C\nfollowed by centrifugation. In Suppl. Fig. 13, western blots were detected with: panel A, anti-CERT (Ab72536-1001);\nPanel B, anti-CERT (PA5-90421); Panels C and D, anti-IMPDH2 (Ab131158-1001); All panels, anti-USP14 (CS-\n11931) or anti-His (Ab18184)."}, {"round": 4, "reviewer_comments": {"reviewer_3": "The authors have now addressed the issues raised by reviewers to my satisfaction and I support publication."}, "author_response": ""}]

Decision letter
Point-by-point answer to reviewers of manuscript
We deeply thank the referees for their thoughtful, rigorous and constructive comments, which we think have
been of enormous help for us to write a better manuscript. All the reviewers’ suggestions, concerns, and
criticisms have been addressed in the revision as outlined in the following pages (comments, and specific
answers to each one, are numbered and abbreviated as C and A, respectively). To facilitate the reviewers’ work,
we submit a version of the main text (file “NCOMMS-Revised Main Text with marks”) in which changes related to
comments by Reviewers 1, 2 and 3 are in blue, green and orange, respectively. In the case of Reviewer 3,
changes associated to comments already raised by Referee 1 are in blue. On the other hand, with the aim to
ease the reviewers’ evaluation, please note that this document contains links to external files and to internal
parts of the document.
We hope that the three referees will now find the article suitable for publication in Nat. Comm.