WRN structural flexibility showcased through fragment-based lead discovery of inhibitors

Domain: chemistry
Published: 2026-01-03
Editorial outcome: major_revision

Abstract
WRN helicase is an established synthetic lethal target for inhibition in the treatment of microsatellite instability-high (MSI-H) and mismatch repair deficient (MMRd) cancers. The identification of helicase inhibitors is challenging as high-throughput biochemical screening campaigns typically return few validated hits that are often inactive in cell-based assays. Herein, we highlight the power of non-covalent fragment-based lead discovery in locating new druggable allosteric sites on WRN, enabling us to bypass the challenging behavior of WRN during high-throughput screening hampering hit identification. During the fragment optimization process, structures of WRN with key prioritized fragments reveal multiple conformations of WRN with significant domain rotations up to 180°, including a WRN conformation not previously described. Rooted in a combination of biochemical, biophysical, and structural approaches, we present the detailed analyses of optimized chemical matter evolved from screening hits and the unique ability of WRN to accommodate diverse conformations as detailed by structural characterization.

Introduction
Synthetic lethal vulnerabilities have rapidly gained interest as therapeutic targets for cancer. A term coined in 19461, synthetic lethality occurs when disruptions in two genes cause cell or organism death, but single mutations in either of these genes spare the cell or organism. The ability to selectively inhibit targets of interest while minimizing deleterious effects on healthy cells has led to the development of new therapies to treat cancer2,3. Notable examples of this type of drug therapy are PARP inhibitors for the treatment of cancers with mutations in BRCA1/2, which are DNA damage repair proteins4,5.

Cancer cells often exhibit at least partial impairment of specific DNA repair pathways, which may cause dependence on the actions of distinct repair proteins for cell survival6. Werner syndrome helicase (WRN) is known as a guardian of genome integrity, due to its critical roles in numerous aspects of nucleic acid metabolism, including genome surveillance, maintenance, and stability7. Multiple studies identified a synthetic lethal relationship between loss of WRN and microsatellite instability (MSI), which arises from deficiencies in the mismatch repair (MMR) machinery8–10. These studies showed that WRN is essential in MSI-high (MSI-H) cells, which are MMR-deficient, as the depletion of WRN induces double-stranded DNA breaks and promotes apoptosis and cell cycle arrest9,10. In contrast, deletion of WRN in microsatellite stable (MSS) cells shows no adverse events. This reliance on WRN by MMRd cells suggests a high therapeutic index for agents that disrupt WRN function in the treatment of MSI-H tumors.

WRN belongs to the five-member RecQ family of superfamily 2 (SF2) helicases, including RECQL1, Bloom syndrome protein (BLM), RECQL4, and RECQL5, all of which unwind DNA in a 3’−5’ direction11. Despite their DNA substrate dependence, the distributive RecQ helicase core has significant structural similarities to DEAD-Box RNA helicases rather than processive DNA replicative helicases12,13. Each RecQ family member features a helicase core domain consisting of two RecA-like ATPase subdomains, D1 and D2. Additionally, WRN contains a RecQ C-terminal (RQC) domain that consists of a winged helix (WH) domain, a zinc-binding domain (ZBD), and an HRDC (helicase and RNaseD C-terminal) domain11. Notably, WRN–along with its Xenopus ortholog FFA-114—is distinct within the RecQ family as the only protein that possesses an N-terminal exonuclease domain exhibiting 3’−5’ exonuclease activity15.

Helicases, in general, have proven to be challenging drug targets. Common problems include a lack of protein specificity arising from the highly conserved ATP pocket, low hit rates in high-throughput screens in standard in vitro helicase assays, and non-specific binding or activity of identified hits16. Various research groups have identified potential WRN inhibitors17–21, however no successful developments of discovered chemical matter binding the ATP pocket has been reported. This lack of success has recently been thoroughly evaluated by Hauser et al., showing that non-specific interactions can be attributed to impurities in the test samples (ML216) or covalent labeling of the WRN protein (NSC compounds)22. In addition to these challenges, the ATP-binding site in helicases is predicted to be less likely to bind a drug-like molecule than enzymes like kinases, complicating the search for potent ATP-competitive inhibitors23. These considerations indicate that allosteric inhibition of WRN might facilitate the identification of selective leads among RECQ helicases.

Recently, literature has disclosed the discovery of two conformational states of WRN24,25. Prior to those studies, only one WRN helicase structure was available in the Protein Data Bank (PDB: 6YHR)7 which featured WRN with bound ADP and resembled all other known RecQ family helicase structures. We refer to this as WRN Form A, as it was the first structure to be characterized. In one of these two recent studies24, a noncovalent compound currently under clinical development was shown to bind to an allosteric site that occupies the cleft between the two ATPase domains, effectively locking WRN in an inactive conformation. This mechanism of action is rationalized by the observation that, in this conformation, the RecQ ATPase domains D1 and D2 are rotated by approximately 180° compared to the ATP-bound WRN. This led us to label this WRN arrangement as Form B. A contemporaneous paper described a third form of WRN (Form C) in which WRN can bind both ADP and a covalent compound in the allosteric pocket simultaneously25. This structure displays smaller movements between the ATPase domains compared to Form B but nevertheless stabilizes compact conformations that lack the dynamic flexibility required for proper helicase function26–30.

Given the challenges of identifying high-quality WRN inhibitors through high-throughput screens based on the biochemical assays formerly described in literature22, we proposed screening approaches rooted in biophysics, such as fragment-based lead discovery (FBLD) or DNA-Encoded Library (DEL), as the next rational starting points for targeting this enzyme. FBLD workflows are particularly effective in identifying alternative binding sites, albeit often a mix of functional and non-functional ones, and expediting the development of novel chemical matter. These workflows leverage biomolecular Nuclear Magnetic Resonance (bioNMR) and Surface Plasmon Resonance (SPR) techniques to rapidly identify small molecules (molecular weight <300 Da) with low affinities that can be optimized toward more ligand-efficient leads. To navigate this process successfully, it is crucial to concurrently generate structural data that can elucidate the atomic interactions between the fragment hit and the target protein.

In this study, we employ FBLD to identify WRN inhibitors through a combination of 19F ligand-based NMR and SPR screening of internal fragment libraries. The prioritized hits are further analyzed using X-ray crystallography, which reveals the specific binding mode of these compounds at the known binding site of WRN in Form B, identifies an additional allosteric pocket, and uncovers a previously uncharacterized structural conformation of the WRN helicase domain with unique orientations of the ATPase domains. Ultimately, despite the apparently highly dynamic nature of WRN, which poses challenges to structure-based drug discovery, we advance one of the fragment series to achieve low µM inhibitory activity.

Results
Fragment-based screens identify allosteric binders to WRN
The integration of fragment-based screening with structure-based drug design, enabled by crystallography, provides a powerful approach for discovering and developing compounds that bind to small-molecule hotspots on the target’s surface31, including the successful application of fragment screening to identify additional allosteric binding pockets32. To identify further binding sites on WRN, we employed our FBLD platform, which relies on screening our internal fragment collections that were a priori curated to only include compounds with sufficient solubility and purity. Given the highly dynamic nature of helicases26–30, we opted to perform our fragment screen using the apo form of WRN to identify the most druggable allosteric site, avoiding any bias toward ATP-blocked conformations or other potential WRN conformations present in the solution.

We implemented two parallel screening workflows: bioNMR- and SPR-based approaches (Figure SI 1) against WRN (residues 500–942 and 500–946, respectively; Figure SI 2, Table SI1). In the bioNMR studies, we screened an internal collection of fluorine-containing fragments in mixtures using T2 relaxation CPMG experiments to identify primary hits. To address fluorine’s heightened sensitivity to changes in the chemical environment, which can lead to an increased false-positive rate, each primary hit underwent orthogonal validation in singleton mode using 19F T2 CPMG, 1H T2 CPMG, STD (Saturation-Transfer Difference), and waterLOGSY NMR experiments, enhancing the confidence in the identified fragment hits.

Based on the results (Fig. 1, Figure SI 3), hits were categorized into three ranks: Rank 1 for those showing binding with all four methods, Rank 2 for those showing binding with any three methods, and Rank 3 for those showing binding with two or fewer methods33. Each experiment provided insights into different aspects of protein-ligand interaction—STD assessed the transfer of saturation from the protein directly to bound ligands; waterLOGSY measured the transfer of magnetization from excited water molecules directly to the compounds; and 1H CPMG evaluated the signal attenuation of small molecules bound to a slowly tumbling protein. We hypothesized that compounds binding across all four methods were interacting with the most druggable binding cavity on the protein and prioritized these for crystallization efforts.

Notably, the three fragments that crystallized were bound within the Form B allosteric binding pocket25,34. Structure-based chemical expansion of two of those overlapping validated fragment hits (fragment 1 and fragment 2) is discussed. Finally, we conducted a biochemical assessment to evaluate the inhibition of WRN ATPase activity for each compound of interest by measuring ADP accumulation during steady-state turnover (using the ADP-Glo assay) in the presence of 1X Km ATP (Table SI2, Figures SI2a, 4). However, no parental fragments were able to significantly inhibit WRN, even at compound concentrations up to 100 µM.

To further investigate the chemical space accessible through our non-fluorine fragment library, we conducted a screen using an SPR binding assay performed with the apo form of WRN. A selected set of 500 fragments was tested in a single-concentration format (1 mM), and each fragment was ranked according to its binding occupancy level and binding profile (Fig. 2). The top hits from the SPR assay (Rank 1) were further evaluated with a 5-point concentration response assay and subjected to orthogonal validation in singleton mode using 1H T2 CPMG, STD, waterLOGSY NMR, and biochemical ATPase experiments.

Since none of the parental fragments significantly inhibited WRN at concentrations up to 100 µM (Table SI2), we intentionally selected compounds exhibiting either Rank 1 or Rank 3 binding profiles from the ligand-observed NMR experiments. While Rank 1 compounds provided the highest confidence in their interactions with the protein, fragments with differential binding profiles could still represent putative binders interacting with WRN in different ways. Notably, two compounds from the SPR screen yielded X-ray structures: fragment 3, which ranked highly in both SPR and NMR, occupied a different binding site, while fragment 4, which ranked high in SPR but showed no binding by NMR, occupied the residual ATP binding site. Sensorgrams for these fragments are displayed in Figure SI 5.

Analysis of all identified binding sites led us to prioritize the Form B allosteric binding site and fragment 1 for further optimization. We hypothesized that the larger volume of the Form B allosteric site would make it more amenable for structure-activity relationship (SAR) exploration as described in more detail below and in Fig. 3.

Ligand bound structures showcase WRN flexibility and an additional allosteric pocket
For these studies, a truncated construct of WRN (residues 500–942; Figure SI 2A) facilitated a reproducible crystallization system. This construct encompasses the two ATPase domains—D1 and D2—along with the zinc-binding domain (ZBD). Our initial structure of wild-type human WRN (500–942) complexed with AMP-PNP exhibits a root mean square deviation (RMSD) of 0.52 Å (Cα backbone atoms)24 when aligned with PDB ID 6YHR, a longer WRN construct consisting of residues 517–10937, despite lacking the HDRC domain (Figure SI 6). Additionally, both recent papers on WRN drug discovery utilized truncated WRN constructs—WRN (517-946) with six point mutations24 and wild type WRN (517–945)25—to identify and develop inhibitors that showed both in vivo and cellular potency. Together, this data provides confidence that the WRN (500–942) construct is appropriate for structure-based drug design and feasible for structure-activity relationship (SAR) studies. All Fo-Fc omit map ligand densities are shown in Fig. 4.

The structure of WRN with bound fragment 1 was determined to 1.6 Å resolution revealing that fragment 1 binds to the recently identified allosteric pocket where the two ATPase domains—D1 and D2—are rotated 180° relative to one another (Fig. 5A). Additionally, a loop composed of residues Val570-Ser578 obstructs the ATP binding pocket (Fig. 5B). Upon closer examination, we determined that while our structure with fragment 1 resembled Form B, it was not identical as the conformations of the D1 and D2 domains shift approximately 9 Å in relation to each other (Fig. 5C; see Discussion). Due to these conformational differences compared to Form B, we have designated this distinct conformation as WRN Form D. Fragment 1 makes direct interactions with Arg732, Tyr849, and Arg910, with its trifluoromethyl group situated within a hydrophobic pocket (Fig. 5D). Fragment 2 binds to WRN in this same pocket, with its trifluoro groups similarly positioned to interact with Tyr849 and Arg857 (Figs. 5E, F; structure resolution at 2.6 Å).

Notably, Form D can also be obtained in apo form without the presence of any ligand or inhibitor (Figure SI 7). In this apo Form D, there are no significant changes in the main chain atoms of the protein, and the allosteric pocket remains accessible, while the ATP binding pocket is blocked by the movement of the same Val570-Ser578 loop. In fact, the structures of nearly all ligands presented here were obtained by soaking the compound into apo Form D crystals.

A second allosteric pocket was identified in the 2.0 Å resolution structure of WRN Form D complexed with the SPR-discovered fragment 3 (Fig. 6A). Fragment 3 binds to a surface cleft that is approximately 21 Å from the fragment 1 binding site, positioned at the interface of the D1 and D2 domains. The movement of an N-terminal loop composed of residues Asp523-Ala532 forms this pocket (Fig. 6B), where fragment 3 interacts directly with residues Phe527, Leu528, and Lys887 (Fig. 6C).

Structural studies involving SPR fragment 4 (1.7 Å resolution) revealed two distinct patches of electron density (Fig. 4) within the remnants of the disrupted ATP binding pocket (Fig. 6D), indicating the presence of two separate molecules at this site. One region of density was confidently assigned to a single molecule of fragment 4, while the second region was ambiguous. After analyzing all buffer components and compound byproducts, we chose to model a byproduct of the compound into this space, as it best fit the initial Fo-Fc density (Fig. 6E). Although we cannot determine the exact molecule occupying this second region of density, it is evident that the pocket can accommodate this additional chemical matter. The adjacent loop composed of residues Val570-Lys577, which partially occupies the ATP pocket in Form D, is displaced by approximately 3.5 Å upon the binding of fragment 4, aligning more closely with the loop’s conformation when AMP-PNP is bound. Notably, the electron density surrounding the second molecule is somewhat unclear, and the backbone residues of the loop also show faint average electron density, suggesting weak binding of this additional copy and a correspondingly faint average density of the loop. The complete fragment 4 ligand makes direct interactions with Thr573, Gly574, and Asp668, while the carboxy group of the hypothesized byproduct product interacts with Gln605 (Fig. 6F).

Structure guided evolution of fragment 1 series leads to functional inhibition and discovery of a WRN Form E
We advanced the development of fragment 1 by maintaining its interactions with key residues Arg732, Tyr849, and Arg910, while also incorporating insights gained from the X-ray structure of fragment 2 complexed with WRN. Notably, the carboxylic acid of fragment 2 forms an important ionic interaction with Arg857, which informed the evolution of fragment 1. To design ligands that combined the structural features present in fragment 1 and fragment 2, we generated a virtual library by coupling trifluoroaniline with various proprietary acids from our corporate collection. Virtual ligands were assessed based on their docked poses, focusing on key interactions identified in the crystal structures of WRN with bound fragment 1 and fragment 2. Specifically, we looked for hydrogen bonds with Arg732 from fragment 1, hydrogen bonds with Arg857 from fragment 2, and π-stacking interactions with Tyr849 observed in both fragments. A total of 17 compounds that satisfied all three criteria were selected for synthesis. Among these, we identified several promising molecules, including compound 1 and its thiophene analog, compound 2 (Table SI2, Figure SI8).

A 1.6 Å resolution X-ray structure of WRN in complex with compound 1 revealed that two complete copies of this ligand are bound in the Form D allosteric site (Fig. 4). The trifluorophenyl aniline moiety of the first copy of compound 1 aligned well with that of fragment 1, displaying a similar H bond interaction with the backbone carbonyl of Tyr849 (Fig. 7A). Additionally, the carboxylic acid group attached to the furan ring via a one-carbon spacer effectively interacts with Arg857, as initially designed. Intriguingly, the trifluorophenyl moiety of the second copy of compound 1 was found occupying a hydrophobic groove formed by Phe917, Tyr849, and Val552. These observations prompted us to undertake several approaches, using different 5 and 6-membered heterocycles and spacers (cf. 3, 4 and 5) to explore this hydrophobic pocket, using the first copy of compound 1 as an anchor. This investigation resulted in the synthesis of compound 3, which exhibited more than a 10-fold improvement in ATPase IC50 against WRN, as well as initial evidence of selectivity over BLM (see Table SI2, Figure SI8). It was confirmed that compound 3 binds to the Form D allosteric site. An overlay of the 1.7 Å resolution compound 3-bound structure with that of compound 1 showcases the trajectory and overall binding pattern within this series, with the terminal phenyl occupying a positively charged region of the allosteric pocket (Fig. 7B). The trifluoro moiety of compound 3 binds in the same anchoring pocket as fragment 1, while the remainder of the molecule interacts with nearby protein residues Gln850, Arg732, Tyr849, and Arg857. We additionally observed that Lys555 was in proximity to the terminal phenyl moiety of compound 3. Given the flexibility of Lys555, we explored both the meta and para positions of the terminal phenyl of compound 3, as well as a previously discovered compound featuring a central pyrazole ring.

From this investigation, we discovered compound 4, which exhibited a superior profile within chemical matter made with an IC50 of 1.2 µM, a comparable binding affinity of 1.6 ± 0.65 µM, and almost 100-fold selectivity over BLM (Table SI2, Figure SI8; Docked pose Figure SI9). In a parallel effort aimed at forming a hydrogen bond while exploring the binding pocket occupied by the distal phenyl of compound 3, we identified both compounds 6 and 7 (Table SI2, Figure SI8). The 2.1 Å resolution X-ray structure of compound 6 in complex with WRN demonstrated that the portion of the molecule covering the thiazole to the anchoring trifluoro group aligns with compound 3, while the extended phenyl amide moiety of compound 6 penetrated into allosteric pocket, surrounded by residues varying in charge states including Pro551, Val552, Lys555, Thr726, Cys727, Glu846, and Phe917 (Fig. 7C). All hydrogen bonds, ionic interactions, and pi-pi interactions between compound 3 and WRN are maintained in the binding of compound 6, including the interaction to Arg857, with additional pi-pi π-stacking interactions observed between the extended terminal phenyl amide in compound 6 and the side chain of Phe917. To further explore the compound 3 distal phenyl subpocket, we explored various modifications to the central ring and its substituents. For synthetic ease, we replaced the central thiazole ring of compound 3 with a pyrimidine moiety and explored various linkers to reach the subpocket of interest. This effort yielded compound 5, which was predicted by docking to form a hydrogen bond with Arg854. Figure 3, Table SI2 and Figure SI8, Docked pose Figure SI9).

Remarkably, the structural data for WRN with bound compound 5 at 1.9 Å resolution obtained by co-crystallization revealed yet another conformation of WRN, which we will call WRN Form E (Fig. 8A). This is notable despite a nearly identical binding mode observed for compound 5 and its parent compound 3. In Form E, the ATPase domains exhibit a rotation relative to each other around the linker region consisting of residues Thr728-Asn734, with the ATP pocket occluded by the loop comprised of residues Val570-Ser578, as illustrated in the overlay with the WRN-AMP-PNP structure (Fig. 8B). Despite the significant change in overall conformation, the binding pocket itself in Form E shares 16 of the 18 residues of that in the biologically-validated Form B. Compound 5 binds with the trifluoro moiety located in the anchoring fragment pocket, interacting with Thr728, Phe730, Arg732, and Arg857 (Fig. 8C). The electron density corresponding to the terminal pyrimidine is weak (Fig. 4), suggesting that this ring may adopt two different orientations, rotating around the bond between the carbonyl carbon atom and the piperazine nitrogen atom; it is modeled here in both conformations. As compound 5 forms a hydrogen bond with Arg857 which also interacts with AMP-PNP, we hypothesize that ATP and compound 5 cannot bind WRN simultaneously.

The transition of the WRN domains from the ATP-bound Form A to Form B/D involves an approximate 180° rotation around the linker (Fig. 9A). In Form E, determined by co-crystallization of WRN with compound 5, the configuration of D2 relative to D1 also results in an approximate 180° rotation from Form A, but this rotation occurs along a different axis than that from Form A to Form D (Fig. 9B). The conformations of the domains in Forms D and E reveal an approximate 90° rotation between these two forms (Fig. 9C).

Methods
Gene expression and protein purification
The helicase domain of Human Werner-Syndrome (WRN) protein UniProt ID Q14191 for SPR, X-ray, biochemistry, and bioNMR was expressed using the residues and tags as described in the Supporting Table SI1.

For SPR measurements, the N-terminal GST-PRE and C-terminal AVI-tagged human WRN (500–946) was cloned into pFastBac1. Bacmids from this vector were transfected into Sf9 insect cells to generate baculovirus. Protein from baculovirus was expressed in Sf21 cells in Sf-900™ II SFM (ThermoFisher Scientific—Cat #11497013)+10% FCS at 26°C for 64 h in a 5 L ReadyToProcess WAVE™ 25 bioreactor system. Cells were pelleted down and stored at −80°C for future use. All purifications were carried out on ice or at 4°C unless stated.

For purification, frozen cells were thawed and resuspended in buffer A: 50 mL of 2x PBS pH 7.4, 500 mM NaCl, 10% Glycerol, 5 mM DTT, 0.1% CHAPS, supplemented with 1 Protease Inhibitor Tablet (Roche) and 20 U/mL Benzonase. Cells were lysed and homogenized prior to a centrifugation step for 40 min and 75,000 xg. The lysate was loaded onto a 15 mL Glutathione Sepharose column in batch mode and washed with 4 CV of buffer A. WRN protein was eluted with a step increase of GSH concentration to 25 mM, followed by 4 CV wash with buffer B; 50 mL of 2x PBS pH 7.4, 500 mM NaCl, 10% Glycerol, 5 mM DTT, 0.1% CHAPS, 25 mM GSH. Fractions with the highest content and purity of WRN protein were pooled and dialyzed overnight using Spectra/Por 1 Dialysis Tubing 6–8 kDa against buffer C (10 mM HEPES-NaOH, pH 7.5, 50 mM NaCl, 1 mM DTT, 5% glycerol, 0.1% CHAPS) in the presence of GST-HRV3C protease for tag cleavage. Cleaved protein sample from the initial purification step was reloaded onto a 5 mL Heparin-column (2×5 mL and washed with buffer C containing 150 mM NaCl. WRN was eluted in a 20 CV gradient to buffer D (10 mM HEPES/NaOH, pH 7.5, 1 M NaCl, 1 mM DTT, 5% glycerol, 0.1% CHAPS). Collected protein fractions were concentrated to 12.4 mg/mL in an Amicon ultra concentrator (50 kDa cut-off) and loaded at 1 mL/min onto a HiLoad 26/60 Superdex 200 column pre-equilibrated in buffer E (10 mM HEPES-NaOH pH 7.5, 500 mM NaCl, 5% Glycerol, 1 mM TCEP).

The sizing exclusion chromatogram (SEC) showed one main peak, which corresponded to monomeric, cleaved WRN protein. Suitable fractions were pooled and concentrated as above to a final concentration of 5.2 mg/mL (Figure SI 2B). Aliquots were snap frozen in liquid nitrogen and stored at −80°C.

The expression and purification workflow described above for the WRN construct encompassing residues 500–946 delivered high-quality material for SPR studies; however, further adjustment of the construct and workflow was necessary to support bioNMR and X-ray studies. Hence, for X-ray, biochemistry, and bioNMR applications, the His-FLAG tag followed by a Thrombin cleavage site was fused to the N-terminus of the helicase domain and cloned into the pFastBac1 vector for baculovirus production and large-scale expression performed in Sf21 insect cells in Sf-900 II SFM medium + 10% FCS at 26°C for 64 h in a 5 L ReadyToProcess WAVE™ 25 bioreactor system. Cells were harvested by centrifugation and stored at −80°C for future use.

All purifications were carried out on ice or at 4°C unless stated.

For purification, frozen cells were removed from −80°C storage, thawed and resuspended in lysis buffer F (50 mL 2x PBS pH 7.5, 500 mM NaCl, 1 mM TCEP, 10% glycerol, 40 mM imidazole, 0.1% CHAPS) supplemented with DNase I and Protease Inhibitor Tablet (Roche). Cells were lysed and homogenized prior to a centrifugation step for 40 min and 75,000 xg. The cleared lysate was mixed with 20 mL Ni-Sepharose beads and incubated for 1.5 h while stirring. Beads were washed with 5 column volumes of buffer F and filled into an XK column. The XK column was washed with lysis buffer containing 50 mM imidazole and eluted with a 10 CV gradient to buffer G (2x PBS pH 7.5, 500 mM NaCl, 1 mM TCEP, 10% glycerol, 500 mM imidazole, 0.1% CHAPS).

WRN eluted in imidazole gradient was incubated with 10 U thrombin/mg protein in dialysis in buffer E overnight to remove His-FLAG tag. The dialyzed protein was passed through a Ni-Sepharose column again to remove residual fusion protein. Flow-through was concentrated, diluted 6-fold with heparin buffer C (10 mM HEPES-NaOH, pH 7.5, 50 mM NaCl, 1 mM TCEP, 5% glycerol, 0.1% CHAPS), applied on heparin column (2×5 mL) and washed with heparin buffer C containing 100 mM NaCl. WRN was eluted in a 20 CV gradient to buffer D (10 mM HEPES-NaOH, pH 7.5, 1 M NaCl, 1 mM DTT, 5% glycerol, 0.1% CHAPS). Elution fractions were pooled, concentrated to 31.1 mg/mL, and loaded on HiLoad 26/60 Superdex 200 column equilibrated in SEC buffer E (10 mM HEPES-NaOH, pH 7.5, 500 mM NaCl, 5% Glycerol, 1 mM TCEP). Elution fractions were analyzed through Tricine-SDS-PAGE, and relevant fractions were pooled, concentrated, flash-frozen in liquid nitrogen and stored at −80°C (Figure SI 2A).

Human RecQ-like DNA helicase (BLM), Uniprot ID P54132, protein was designed as an N-terminally truncated variant (642-1296) and modified by fusing a 6xHis-Tev tag to the N-terminus of the selected BLM boundary (MGHHHHHHENLYFQG). The sequence was cloned into pET28b vector for expression in BL21 (DE3) cells obtained from NEB). Transformed BL21 (DE3) cells were grown in LB medium (Teknova) in the presence of 50 μg/mL Kanamycin and 150 μM ZnCl2. Expression of BLM was initiated by induction of the cells with 0.3 mM IPTG at an OD600 between 0.5-0.6. Cells were grown for 18 h at 37°C, shaking at 200 rpm in a Multitron shaker (Infors HT) and harvested via centrifugation at 4000 xg for 20 min at 4°C. Pellets were frozen and stored at −80°C.

A cell pellet representing an expression volume of 10 L was defrosted, and a weight of app. 50 g cell pellet was resuspended in 250 mL lysis buffer (50 mM Tris pH 7.5, 500 mM NaCl, 10% glycerol, 1 mM TCEP, 0.1% Triton X-100, 20 mM imidazole, 10 U/mL Benzonase and 5 protease inhibitor cocktail tablets (Roche)) and sonicated for 150 s in 5 s on/30 s off intervals with an amplitude of 40%. The lysate was cleared by a centrifugation step (230,000 xg for 45 min at 4°C). 250 mL of cleared lysate was mixed with 10 mL HisPur resin and incubated in batch for 2 h at 4°C while stirring before being packed into a column. The column was washed with 4 CV at 4 mL/min in buffer C (50 mM Tris pH 7.5, 500 mM NaCl, 10% glycerol, 1 mM TCEP, 0.1% Triton X-100, 20 mM imidazole, protease inhibitor tablets at 1/50 mL). The following gradient steps were applied: 6 CV with 5% buffer D (buffer C with an imidazole concentration increased to 250 mM), 15 CV gradient from 0% B to 100% B, followed by 3 CV of 100% buffer B. 2 mL fractions were collected with a final volume of 122 mL at 1.24 mg/mL. The pooled fractions were diluted with 50 mM Tris pH 7.5, 5% glycerol to a final NaCl concentration of 250 mM and loaded onto a 5 mL Hi Trap Heparin HP column at 4 mL/min. The column was washed with 5 CV buffer C (20 mM Tris, pH 7.5 5% glycerol, 250 mM NaCl). Protein elution was conducted using a 20 CV gradient from 0%-100% buffer D (20 mM Tris pH 7.5, 5% glycerol, 800 mM NaCl). Followed by 5 CV of 100% buffer D. 30 mL fraction volume at 1.1 mg/mL was pooled and split equally into two sub-pools (Pool 1 and 2).

Pool 1 (approximately 25 mg protein in 15 mL) was loaded onto a HiLoad 26/60 Superdex 200 column pre-equilibrated in 20 mM Tris, pH 7.5, 5% glycerol, 500 mM NaCl. 18 mL total volume was collected at 0.72 mg/mL and concentrated to approximately 6 mL in an Amicon ultra concentrator (50 kDa cut-off) with a final concentration of 2.2 mg/mL. 50 µL aliquots were frozen and stored at −80°C. Pool 2 (approximately 25.5 mg protein) was mixed with 1.28 mg His-tagged Tev protease in approximately 15 mL volume and incubated for 17 h at 4°C. Cleavage was confirmed by SDS-PAGE and anti-His Western Blot, using anti-His6-Peroxidase from mouse IgG1 (Roche 11965085001) in a 1:500 dilution, before being further processed. The cleaved sample was loaded onto a Hi Load 26/60 Superdex 200 column pre-equilibrated in 20 mM Tris pH 7.5, 5% glycerol, 500 mM NaCl. 18 mL total volume was collected at 0.72 mg/mL and concentrated to approximately 4.5 mL in an Amicon ultra concentrator (50 kDa cut-off) with a final concentration of 2.7 mg/mL. 50 μL aliquots were frozen and stored at −80°C.

BioNMR 19F fragment screening
The 1020 fragments from internal fluorine-containing collection were selected based on their measured 19F chemical shifts, purity >90%, solubility >250 µM, and lack of aggregation at 50 µM in the CPMG experiments. Fragments were pooled into the mixtures of up to twenty-one compounds covering a range of 19F chemical shifts, ensuring at least 1 ppm separation between mixture components. The control samples were prepared at 50 µM of each mixture component, with DMSO-D6 concentration always matched to 2.5% final volume. Samples to evaluate binding were prepared in 25 mM HEPES-NaOH, 50 mM NaCl, 2 mM MgCl2, pH 7.4 buffer with protein concentration at 10 µM and each mixture component at 50 µM, with DMSO-D6 concentration matched to 2.5% final volume in each sample. The 19F CPMG relaxation experiments were recorded at 295 K on a Bruker AVANCE III 600 MHz NMR spectrometer equipped with a 5 mm QCI-F cryoprobe. Bruker CPMG T2 pulse program (19Fcpmg_screen) was implemented employing ca-WURST-20 adiabatic refocusing pulse, 20 ms and 160 ms total relaxation time, with 112 scans and proton decoupling during acquisition. The NMR spectra were analyzed using the Fragment-Based Screening (FBS) module in Topspin 3.5pl7. Hits were identified by signal intensity changes and selected for singleton confirmation when the quotient Qbind was less than 0.545.

IntensityRatio+target
Qbind = ð1Þ
IntensityRatio (cid:1)target

where

PeakIntegralCPMGat160ms
IntensityRatio= ð2Þ
PeakIntegralCPMGat20ms

Because each fragment in the mixture had a known unique chemical shift, the hits could be simply identified by matching the chemical shift of the hit to that defined in the mixture reference spectra.

Each hit identified in the mixture was further submitted to a set of NMR experiments in single-molecule mode. First, the 19F CPMG experiments were performed for all hits in isolation under conditions identical to the screening setup. Next, confirmed binders were subjected to a proton-based suite of experiments to gain confidence in the binding: 1H T2 CPMG, STD, and waterLOGSY. All three experiments were acquired on a Bruker AVANCE III 600 MHz NMR spectrometer equipped with a 5 mm QCI-F cryoprobe with the same experimental conditions, where for compound-bound samples, the WRN protein was reconstituted at 10 µM in the 25 mM HEPES-NaOH, 50 mM NaCl, 2 mM MgCl2, pH 7.4 buffer. Compounds were added at a final concentration of 200 µM, and DMSO-D6 was always matched to 2.5% final sample volume. In compound QC samples, fragments were reconstituted at 200 µM in the 25 mM HEPES-NaOH, 50 mM NaCl, 2 mM MgCl2, pH 7.4 buffer with 2.5% DMSO-D6. The STD (stddiffesgp.3) experiments were performed with a train of 40 Gaussian bell-shaped selective pulses of 50 ms length, and 2 s recycle delay; the on- and off-resonance frequency for selective irradiation of protein were set to 0 ppm and −40 ppm, respectively. The number of transients was set to 256. Data were automatically processed using the proc_std Bruker AU program. The 1H T2 CPMG (cpmg_esgp2d) experiments were implemented with pulse sequence cpmg_esgp2d and CPMG filters set to 20 and 400 ms. The number of scans was 128. In water-LOGSY (ephogsygpno.2) experiments were acquired using the pulse sequence as described by Dalvit et al.46,47. In summary, experiment parameters were: 256 number of transients, water excitation performed using a 180 degree selective Gaus1_180r.1000 pulse followed by a 2 s mixing time and 5 s relaxation delay. Water suppression was achieved with a 2-ms 180-degree selective Sinc1.1000 pulse at the H2O frequency. Protein resonances were suppressed using CLEANEX spinlock with mixing time set to 30 ms. For STD, waterLOGSY, and 1H T2 CPMG experiments, the peak intestines retrieved from spectra in the presence of protein were compared with the reference spectra for the individual compounds in the buffer. Compounds were considered binders when the STD double difference was more than 10% of the reference signal, water-LOGSY gave a positive signal, and Qbind was less than 0.5 in the 1H T2 CPMG experiment.

SPR binding assays
The diversity set of 500 fragments from our internal non-fluorine containing collection was subjected to a series of SPR binding assays. The SPR binding assays were carried out using a high-throughput Biacore 8K+ SPR system (Cytiva 29283382), with biotinylated WRN protein immobilized to the biosensor chip surface using a proprietary biotin capture method (Biotin Capture Kit; Cytiva 28920234). All reagents were kept at 8°C in the reagent compartment. Briefly, the kit Series S CAP Sensor chip was docked to the instrument and prepared according to the manufacturer's protocol. A solution of 100 µg/mL WRN (diluted in 10 mM HEPES-NaOH pH 7.4, 500 mM NaCl, 5 mM MgCl2, 1 mM TCEP, 0.01% Tween 20) was injected into flow cell 2 (all 8 channels) for 2 min at a flow rate of 10 uL/min to achieve an immobilization density of ~3000 RU. Flow cell 1 was reserved as the reference cell used for reference cell correction. The sensor chip was subsequently equilibrated at 20°C in running buffer (10 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM TCEP, 0.01% Tween 20, 1% DMSO) prior to analysis of test compounds. For the primary single-point screening of the non-fluorinated fragment collection, compound stocks of 100 mM in 100% DMSO were diluted 100-fold in assay buffer to a 1 mM final test concentration with 1% DMSO in the final 100 ul sample volume. This was carried out using an automated liquid handler (Echo 655). For the 4-point concentration response test, multiple concentrations were prepared using the Echo 655, generating 4 concentration points for each compound, including 25 µM, 8.25 µM, 2.75 µM, 1.0 µM, 0.25 µM, 0.125 µM, 0.031 µM, and 0.016 µM, or 500 µM, 250 µM, 125 µM and 62.5 µM. This was performed with the appropriate back-fill to reach a final 1% DMSO in the final 100 µl sample volume. Each test compound was injected for 120 s at a 20 µL/min flow rate (association phase). This was followed by a 300 s dissociation phase using running buffer. To account for refractive index mismatch due to slight differences in the amount of DMSO in each test injection, a DMSO solvent correction was applied to all datasets. DMSO standard curve was prepared using 5 concentrations (0.5% increments from 0% DMSO to 2% DMSO.

All data were analyzed using Biacore Insight evaluation software, version 5.0.18.22102. The sensorgrams were fit with a 1:1 steady-state affinity binding model to obtain Kd, when applicable.

ATPase assay
To measure WRN ATPase activity, the accumulation of ADP was quantitated via ADP-Glo Assay kit (Promega) in all black NBS low-volume 384-well microplates (Corning, 3820). For the construction of assay-ready plates, 200 nL of each compound was dispensed in DMSO via three-fold serial dilution (100 µM, the Top highest concentration) into assay plates using an Echo acoustic dispenser. All assay reaction solutions were prepared in 1X assay buffer (25 mM HEPES-NaOH, pH 7.3, 50 mM NaCl, 2 mM MgCl2, 0.01% Tween-20, 0.5 mM TCEP, 1% DMSO). All WRN compound inhibition reactions were assembled in a final volume of 20 µL by preincubating (250 pM, final) WRN helicase core (500–942) with the respective compound (Figure SI 4), for 30 min in the presence of (100 nM, final) single stranded DNA (IDT; 5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTCGTACCCGATGTGTTCGTTC-‘3), which is required for ATP hydrolysis, prior to reaction initiation by the 1:1 addition of 1X Km ATP (50 µM, final). All BLM compound inhibition reactions were assembled in a final volume of 20 µL by preincubating (500 pM, final) BLM helicase core (642–1296) with the respective compound (Figure SI 4), for 30 min in the presence of (30 nM, final) single-stranded DNA and initiated by the 1:1 addition of 1X Km ATP (50 µM, final). Both BLM and WRN ATPase reactions were run for 1 h prior to quenching by the addition of the ADP-Glo kit reagents. High luminescence (enzyme+substrates+DMSO) represents the signal from max ATP hydrolysis (no ATPase inhibition), while low luminescence (substrates+DMSO or WRN+Substrates+20 mM positive inhibition control) represents no ATPase activity/complete inhibition. Compound concentration response curves were fit by the 4P (Spotfire) inhibition model to extract IC50, Hill slope, and % maximum inhibition. Reported errors represent the nonlinear least squares 95% confidence interval of the extracted parameter.

Ligand docking
Crystal structures were prepared using the Protein Preparation Wizard panel in Maestro 13.1 (Schrodinger Suite 2022-1). Grids of these structures were generated using the receptor grid generation panel of Maestro 13.1. The centroid of co-crystallized ligands was used to define the binding site. Ligands were prepared using the LigPrep panel of Maestro 13.1. All possible stereoisomers were generated. All docking calculations were performed using Glide (Schrodinger Suite 2022-1) using standard precision and flexible ligand sampling.

Crystal growth and preparation
AMP-PNP structure. Co-crystals of hWRN were grown in the presence of AMP-PNP by sitting drop vapor diffusion at 4°C. Prior to drop set-up, hWRN was incubated with 2 mM AMP-PNP for 2 h on ice. The drops were set in a 1:1 ratio (0.1 µL:0.1 µL) of protein mix (10 mg/mL in 1 mM TCEP, 5% (v/v) glycerol, 10 mM HEPES-NaOH pH 7.5, and 500 mM NaCl) to mother liquor containing 30% Jeffamine ED-2003, 0.2 M NaCl, 0.2 M NDSB-201, and 0.1 M MES/NaOH pH 6.0. Crystals were obtained after 3 weeks. Crystals were flash-vitrified directly from the drop and measured at temperature of 95 K.

Fragment 1
Co-crystals of hWRN were grown in the presence of ligand via hanging drop vapor diffusion at 4°C. Prior to drop set-up, hWRN was incubated with 20 mM fragment 1 for 16 h on ice. The drops were set in a 1:1 ratio (0.5 µL:0.5 µL) of protein mix (10 mg/mL in 1 mM TCEP, 5% (v/v) glycerol, 10 mM HEPES-NaOH pH 7.5, and 500 mM NaCl) to mother liquor containing 20% (w/v) PEG 6000, 0.1 M HEPES-HCl pH 6.25-6.5. Crystals were obtained after 1 week. Crystals were flash-vitrified in a solution of mother liquor + ligand with 20% (v/v) ethylene glycol and measured at a temperature of 95 K.

Apo Form D, fragment 2, fragment 3, fragment 4, compound 1, compound 3, and compound 6
Apo crystals in Form D were grown by hanging drop vapor diffusion at 4°C. The drops were set in a 1:1 ratio (0.5 µL:0.5 µL) of protein (10 mg/mL in 1 mM TCEP, 5% (v/v) glycerol, 10 mM HEPES-NaOH pH 7.5, and 500 mM NaCl) to mother liquor containing 21%–27.5% (w/v) PEG 1500 and 0.1 M MMT pH 5.75–6.25. Crystals were obtained after 1 week and were soaked at pH 7.0–9.0 with 50 mM ligand present between 12 h and three days. Crystals were flash-vitrified in a solution of mother liquor with 20% (v/v) ethylene glycol and measured at a temperature of 95 K.

Compound 5
Co-crystals of hWRN in Form E were grown in the presence of compound 5 by sitting drop vapor diffusion at 4°C. Prior to drop set-up, hWRN was incubated with 2 mM compound 5 for 16 h on ice. The drops were set in a 1:1 ratio (0.1 µL:0.1 µL) of protein mix (10 mg/mL in 1 mM TCEP, 5% (v/v) glycerol, 10 mM HEPES-NaOH pH 7.5, and 500 mM NaCl) to mother liquor containing 20% (w/v) PEG 6000, 0.2 M CaCl2, and 0.1 M MES-NaOH pH 6.0. Crystals were obtained after 3 days and were flash-vitrified in a solution of mother liquor with 20% (v/v) ethylene glycol and measured at a temperature of 95 K.

Crystal data collection
AMP-PNP structure. X-ray diffraction data were collected from crystals under cryogenic conditions at the SWISS LIGHT SOURCE PXII/X10SA (SLS, Villigen, Switzerland) at a wavelength of 1.0000 Å using a Dectris EIGER2 S 16M detector. The crystals belong to space group C2. For all structures, data were processed using the programs autoPROC (1.1.7)48, XDS (Jan 31, 2020)49, and AIMLESS (0.7.7)50.

Fragment 1, apo form D, fragment 2, fragment 3, fragment 4, and compound 1
X-ray diffractions were collected from crystals under cryogenic conditions at the SWISS LIGHT SOURCE PXII/X10SA (SLS, Villigen, Switzerland) at wavelengths between 0.9999 Å to 1.0000 Å using a Dectris EIGER2 S 16M detector. The crystals belong to space group P2221.

Compound 3
X-ray diffraction data were collected from crystals under cryogenic conditions at the DIAMOND LIGHT SOURCE IO4 (DLS, Oxford, England) at a wavelength of 0.9537 Å using a Dectris EIGER2 XE 16M detector. The crystals belong to space group P2221.

Compound 5
The X-ray diffractions were collected from complexed crystals under cryogenic conditions at the SWISS LIGHT SOURCE PXII/X10SA (SLS, Villigen, Switzerland) at a wavelength of 0.9999 Å using a Dectris EIGER2 S 16M detector. The crystals belong to space group I222.

Compound 6
X-ray diffraction data were collected from complexed crystals under cryogenic conditions at the EUROPEAN SYNCHROTRON RADIATION FACILITY ID23-1 (ESRF, Grenoble, France) at a wavelength of 0.8856 Å using a Dectris EIGER2 CdTe 16M detector. The crystals belong to space group P2221.

Structure determination and refinement
The chosen cut-off criterium for all structures is I/σI ≥ 1.2 in the last shell with reflexes equally distributed over all shells. All datasets have been checked for twinning and anisotropy using Phenix.Xtriage (1.21.1–4487)51. No indication for twinning is observable in any dataset. The datasets with compound 5 and AMP-PNP are moderately anisotropic. Processing of these datasets with STARANISO (from autoPROC 1.1.7)52 and refinement against these data did not improve the electron density map, and thus, all structures were refined against isotropically processed data.

Crystal structures of hWRN were determined by molecular replacement using Phaser (from CCP4 7.1.015 and 8.0.005)53. Proprietary models have been used as search models. For hWRN in distinct conformations, it was crucial to replace the RecA1 and the RecA2+ Zinc-binding domain independently of each other for the first replacement. For all structures, model building and refinement were performed using Coot (0.9.8.91)54 and the CCP4 software package (7.1.015, 8.0.004, 8.0.005, and 8.0.016)55, respectively. Compound geometrical restraints were prepared using CORINA (4.4.0002612.08.2021)56, and refinements were carried out with REFMAC (5.8.0267 and 5.8.0352)57. Figures were prepared using PyMOL (2.5.5)34. Crystal structures of hWRN were determined by molecular replacement using Phaser (from CCP4 7.1.015 and 8.0.005)53. Model quality was assessed, among others, with MolProbity (from CCP4 7.1.015 and 8.0.005)58. Proprietary models have been used as search models. For hWRN in distinct conformations, it was crucial to replace the RecA1 and the RecA2+ Zinc-binding domain independently of each other for the first replacement. Data collection and refinement statistics for all structures can be found in Table SI3.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Palte et al. presents a fragment-based approach to identify ligands that bind to the Werner helicase (WRN). WRN is a validated but challenging target for the development of certain cancers. Previous efforts to identify inhibitors of WRN using high throughput screening (HTS) have been hampered by the finding that many compounds that show activity in the functional assays used to identify hits, have failed to validate and show activity in cell-based assays. A central hypothesis of the current work is that by screening fragments using biophysical binding assays, validating their binding using X-ray crystallography and applying a structure-guided approach to fragment elaboration, it may be possible to circumvent some of the problems encountered in the HTS assays.\n\nThe authors describe two separate hit-finding approaches – one based on NMR experiments and the other using SPR. Each yielded hit fragments that were validated as binders using X-ray crystallography. The crystallographic analysis provided some insight into the challenges presented by WRN as a target – with multiple different conformations of the protein being observed. The authors conclude that the conformational changes observed in the structural data are relevant in the biological activity of WRN and present snapshots of the structural flexibility that exists in the protein. Ultimately, fragments that bound at distinct allosteric sites on WRN were identified, and fragments that bound at one of these sites were selected for further elaboration. This led to compounds with low micromolar activity in a biochemical assay.\n\nOverall, this is an interesting story and presents a nice example of the application of a biophysical and structural screening cascade against an extremely challenging target. In my opinion it would be of interest to a wide audience. However, I believe that there are some key omissions from the data as presented that need to be addressed. There are also several minor suggestions that I believe would strengthen the manuscript.\n\nMajor points.\n1. Lack of biophysical data in the manuscript files.\nThe SPR data presented for fragment 3 and fragment 4 show evidence of significant overbinding. The dose-response data are essentially linear up to the maximum tested concentration of 1 mM – at which point the occupancy is already > 100%. This sort of behaviour is not uncommon for weakly binding fragments – but if it were to persist with higher affinity compounds it would lead me to question the reliability of the SPR assay. However, there are no SPR data presented for the higher affinity compounds. I would suggest that SPR data for the compounds showing affinity in the biochemical assay are necessary to support the central hypothesis of the study that the biophysical assays present a means to weed out badly behaved compounds.\n2. Compound characterisation\nMany of the purified intermediates lack NMR. There is no 13C-NMR data presented for any compound. There is no HRMS or purity data. It is unclear how many of the compounds are literature compounds. If they are then a statement that the 1H-NMR is consistent with literature and a reference is required. If not, proper characterisation must be reported.\n\nMinor points\nThe authors show two examples of fragments that were identified from the NMR screen, including concentration-dependent broadening of 19F resonances in the CPMG spectra as well as evidence of binding in 1H-NMR STD, CPMG and WaterLOGSY. They make the point that binding in multiple experiments increases confidence in the identification of binders. In the SPR assay, data are presented for fragment 3 and fragment 4. Both show evidence of overbinding in the SPR assay with almost linear responses up to the maximum tested concentration of 1 mM. Fragment 3 is validated as a hit in the NMR assay whereas fragment 4 is not. Both give crystal structures. Given the central hypothesis of the work I suggest that this warrants comment. Where does the degradation product in the X-ray come from? Is it observed in the stock of the fragment? If not, is it somehow formed due to irradiation at the synchrotron?\n\nI found the order of the figures confusing. The first panel in Figure 1 shows the strategy for chemical elaboration. Figure 2 shows structures and density for fragments and elaborated compounds. Some of the structural data that underpin the elaboration strategy are not presented until Figure 5.\n\nThe abstract contains abbreviations that are not defined or explained until later in the text. Likewise, PARP, BRCA1/2 are used at the beginning of the introduction in a manner that I suggest makes it difficult for a non-expert in the field to follow. ML216 is mentioned in the introduction – but the context of the sentence is not provided until the discussion section. Druggability is used without a definition of what it mean in the introduction and in the selection of fragment 1 for further work. P5 – the sentence beginning “internal structure of WRN” may be missing something?\n\nThe confidence of the authors in the suitability of the construct is not really explained.\nWhy is this structure-guided HTE? It seems to be a pretty standard protocol of compound enumeration and docking.\nSolubility > 250 mM? In what? How is purity assessed?\nHow is non-saturable binding assessed from single concentration SPR experiments?\nFinal assay volume of 20 mL?\nHill (hill) slope.\nThere are several instances where capitalisation, sub/superscripts appear to be incorrectly formatted\nThe reference format is inconsistent as are the citation markers\nSeveral of the ESI-MS peaks are quoted to one decimal place with the final digit as 0. Eg. 422.0 – where this number is inconsistent with the expected mass. Is this really the observed mass (with an error)?\nThere are a number of 19F couplings that I would have expected to see in the 1H NMR that are not reported.\nCompound 46 seems to have an in-house code in the experimental.", "reviewer_2": "Manuscript NCOMMS-25-07608, reviewed by Markus Rudolph and Moritz Classen\nThe manuscript by Palte et al. describes the discovery and initial SAR of a number of allosteric WRN inhibitors and binders, found by conformation-agnostic pure binding assays. Fragment screens were conducted using SPR and (19F-)NMR, a nice alternative to previous HTS using enzymatic activities. Structural biology showed that the same allosteric site as discussed earlier in the Baltgalvis and Ferretti studies was found, plus additional binder locations. Molecules at these positions, however, need development into inhibitors. The best inhibitor has a comparatively large IC50 of 1.2 microM, which indicates a low affinity for a SAR-derived compound. A novel WRN conformation is present in the crystal structures presented.\nGiven the fact that no helicase-inhibitor has yet achieved approvement, these results are significant to the field and warrant publication. The manuscript would benefit from more comparison with RNA helicases, as for this structurally-related class of non-processive helicases there ample literature on conformational flexibility. For the flexibly-linked RNA helicases it is clear that the conformations \"trapped\" in crystals are very versatile and may or may not be populated in solution. Domain rotations of \"up to 180°\" are to be expected in the absence of conformational restrictions. Nonetheless, any conformation that binds an inhibitor, is worthwhile investigating.\n\nA drawback of the manuscript compared to earlier WRN inhibitor studies is the lack of cellular or in vivo data. From a biophysical standpoint, the data appear overall sound and are not over-interpreted. The NMR and SPR experiments are conducted well, and conclusions are drawn cautiously, e.g. by categorizing the SPR traces into different classes. A pity that kinetic information could not be extracted from the SPR data, as a small koff value is often indicative for a good inhibitor. The analysis of binding interfaces gleaned from crystal structures, however, appears too speculative and is not backed up by experimental (e.g. mutational) data. We acknowledge that the authors depict 2Fo-Fc electron density, but more informative to judge the quality of the ligand binding poses are omit maps, as Fourier coefficients for these are usually not deposited in the PDB.\n\nMinor points:\nThe Methods would benefit from some more details as in our opinion the experiments cannot be reproduced with the information give - see the annotated .pdf files. At times lab jargon is prominent, formatting style is inhomogeneous and some Methods parts have poor written style.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "This paper provides a textbook quality hit-to-lead story using fragment-based drug discovery approaches for a challenging target, WRN. The hit-finding strategy and data are very well executed and explained, especially in enumerating the different fragment binding profiles for NMR and SPR. I really liked the rigorous clarity and honesty of this section. The structural data is solid and will be a valuable contribution to the field, providing researchers and the industry with new potential starting points for hit-to-lead efforts on an important target. Certainly, the work merits publication.\n\nHowever, I’m not convinced this paper is of high enough impact for Nature in its current form. There are a few limitations with the work. First, given the recent literature, the novelty of the allosteric pocket is no longer a major selling point. Second, the reported potency of their compounds is limited in low uM range and no cellular activity is reported. Third, no mechanistic or selectivity data is reported for the compounds. Finally, while new and interesting conformational states of WRN are reported, likely highlighting the dynamics/plasticity of the protein, it’s not clear if these findings have biological relevance or if they are merely crystal packing artifacts. If the authors can address some of these concerns, then it would strengthen the case for publication in a high impact journal like Nature Comm. Otherwise, I would suggest a more specialized journal may be more appropriate.\n\nMajor Gaps\n(1) In terms of data presentation, the authors should really include a SAR summary table in the main figures with the key data summarized for the fragments and compounds. The key data is scattered around in various supplementary figures/tables and is too hard to track, analyze, and compare. A summary table will greatly simplify the key findings for the readership. Format can be something like this:\nCompound ID; Chemical Structure; MW & LE; NMR binder (or KD); SPR binder (or KD); Docking Score; EC50 WRN; EC50 BLM; PDB Code\n1\n(2) One of the major challenges of a fragment-based screening campaign is distinguishing true hits from non-specific aggregators. Please elaborate in the manuscript what was done to remove bad actors and where you incorporated this into the workflow. Was solubility a concern? Please include a brief sentence or two describing how the Merck fragment libraries are curated in terms of properties (MW, solubility, etc). (I see some description in the methods, but I think this point is important enough to elevate to main text).\n(3) I was surprised that there is no counter-screening / selectivity data presented in this manuscript. Please include relevant counter-screen data for BLM or whatever helicase you used to assess selectivity.\n(4) The “structure guided high throughput experimentation” section (page 6 results) isn’t explained well enough. The readership will be very interested to know how Merck, with its state-of-the-art resources, is using computational methods to streamline discovery. Please elaborate this section and methods explaining minimally how the model was validated and a figure showing the predicted docking activities and how those docking scores compared to experimental data. Are we to understand that of 17 compounds predicted by the algorithm, only 2 showed activity (11% success rate and 89% false positive)? This is about what I’d expect and I do believe the transparency and honesty around the persistent challenges with docking is very useful to point out and for the industry to acknowledge with all the current hype around AI.\n(5) The mechanistic characterization work is non-existent. Are the compounds competitive with respect to ATP or DNA? Do they show any evidence of cooperativity as was observed with VVD-133214? Novartis published a ATP-gamma-S displacement assay. Do the fragments show any activity in this assay? Please review the existing literature on MOA analyses. These are very easy assays to run and there’s no reason to not include those data to better characterize these compounds. Especially for a Nature audience.\n(6) The claim about the second bound molecule being a fragment 4 degradation product seems very unlikely. An alternative interpretation of the data could be that a molecule of malonate is bound (a component of MMT crystallization buffer). Can the authors please try modeling malonate into the density and see if this could explain the electron density? Otherwise, please provide relevant stability data on fragment 4 in their crystallization buffer to support their claim.\n(7) Please provide some text on whether or not the compounds have any cellular activity. You can use the Novartis compound or Vividion compounds as positive controls. Please address any lack of cellular activity in the text as potency limited or properties limited?\n(8) I do like the presentation of the different conformations of WRN. But it’s difficult to know if these have any biological relevance. Have the authors done any cryo-EM studies to try and understand at a population level the relative disposition of the two helicase domains in a more native state? Have the authors done any cryo-EM in the presence of DNA? Have the authors done any crystallography in the presence of DNA? Any supporting data would greatly bolster their claims and make their findings more impactful.\n\nMinor\n(1) Please move Figure 1 and the electron density Figure 2 to the supplement. Please move the bioNMR and SPR workflows to the main figures. Please move representative NMR data like S3 A&B and representative SPR data to main figures. These are the key data that informed which fragments to call out as hits. This is more important than the chemistry schematic, which is less informative and barely discussed in the main text.\n(2) Did the authors profile their compounds in the published DNA unwinding assay? Please report these data for comparison to their ATPase data.\n(3) For pymol figures, please repeat rendering with no shadows. For Figure 4, is hot pink the best color scheme? As a general rule, structure figures are easier to understand when they leverage color and contrast to help highlight the important message. For instance, if the compound binding mode is the “message”, color the compound with a brighter color than the protein so it will better stand out. As currently rendered, the compounds are camouflaged with the same color scheme as the rest of the protein. For example, in Figure 3A, GMPPNP adenosine ring is gray and the compound is blue and are so perfectly camouflaged with the protein as to be almost invisible. This would be the time to use a hot pink rendering on the compound so it can be seen against the blue protein background. Same comments apply to Figure 4A and D. Don’t camouflage what’s important!\n(4) For Figure 3 D and F, is it possible to find the same view to show the same set of residues for each compound, fragment 1 and fragment 2? It looks like the figures were made in isolation or autogenerated with a script without considering the importance of cross-comparison.\n(5) Page 5: I think there’s a missing word: “internal structure of WRN…” Please edit the sentence.\n(6) Please provide some further explanation in the text why fragment 1 was advanced and not the others? The current explanation about favorable SAR is a bit vague.\n(7) The authors performed a fragment growing strategy in their hit-to-lead progression. Did the authors ever consider a fragment linking approach? This would necessitate screening for a second fragment binder in the presence of one of their preferred/validated fragments? Some comments in the text about their strategic decisions around growth vs linking would be nice."}, "author_response": "We want to thank the reviewer for the comments. We find them to be constructive critiques of the science we share. We think the comments were addressed as follow and hope that the improved manuscript will be re-considered for publication.\n\nMajor points.\n1. Lack of biophysical data in the manuscript files.\nThe SPR data presented for fragment 3 and fragment 4 show evidence of significant overbinding. The dose-response data are essentially linear up to the maximum tested concentration of 1 mM – at which point the occupancy is already > 100%. This sort of behavior is not uncommon for weakly binding fragments – but if it were to persist with higher affinity compounds it would lead me to question the reliability of the SPR assay. However, there are no SPR data presented for the higher affinity compounds. I would suggest that SPR data for the compounds showing affinity in the biochemical assay are necessary to support the central hypothesis of the study that the biophysical assays present a means to weed out badly behaved compounds.\nResponse: To address reviewer concerns around final chemical matter we have added SPR data for the higher a\\inity compound 4 (Figure SI 9). We have also added a statement about compound a\\inity into the main text.\n2. Compound characterization. Many of the purified intermediates lack NMR. There is no 13C-NMR data presented for any compound. There is no HRMS or purity data. It is unclear how many of the compounds are literature compounds. If they are then a statement that the 1H-NMR is consistent with literature and a reference is required. If not, proper characterization must be reported.\nResponse: We have included the HRMS data for all compounds, including both fragments and newly synthesized final compounds. Additionally, we have provided the 1H NMR spectra. These data, along with the X-ray structures, unequivocally characterize the compounds and confirm their identities.\n\nMinor points\n1) The authors show two examples of fragments that were identified from the NMR screen, including concentration-dependent broadening of 19F resonances in the CPMG spectra as well as evidence of binding in 1H-NMR STD, CPMG and WaterLOGSY. They make the point that binding in multiple experiments increases confidence in the identification of binders. In the SPR assay, data are presented for fragment 3 and fragment 4. Both show evidence of overbinding in the SPR assay with almost linear responses up to the maximum tested concentration of 1 mM. Fragment 3 is validated as a hit in the NMR assay whereas fragment 4 is not. Both give crystal structures. Given the central hypothesis of the work I suggest that this warrants comment.\nResponse: We appreciate the reviewer’s comment to explain further the relationship of our hypothesis and our protocol. The optimal output of a fragment screen is chemical matter with structural information, enabling a structure-based drug design paradigm. We observed, like others iwhen conducting fragment screens, that chemical matter with the largest degree of overlap between parallel methods increases the chances of success for its structure determination by X-ray crystallography In our NMR screening paradigm and follow-up, we often prioritize fragments from Rank 1. We do not consider SPR as a pass/fail criterion as our 19F library includes compounds with solubility around 250 uM, making it di\\icult to acquire good quality SPR data for compounds in the 1 mM a\\inity range.\nRegarding the SPR results, we purposefully selected compounds with di\\erential bioNMR profiles to test if we could identify di\\erent binding sites. Indeed, we found di\\erent binding sites, but they are shallower and potentially more challenging to advance chemical matter in them. As this is the first project in which we performed this type of approach, we don’t feel comfortable making more general statements about the implementation of this workflow to find di\\erent binding sites.\n\n2) Where does the degradation product in the X-ray come from? Is it observed in the stock of the fragment? If not, is it somehow formed due to irradiation at the synchrotron?\nResponse: Apo WRN was crystallized using a bu\\er comprised of a combination of MES, DL-malic acid, and Tris, referred to here as MMT. These crystals were subjected to soaking experiments, including one with Fragment 4. It is important to note that this bu\\er does not include malonate. We attempted to model malate in the corresponding density peak; however, this did not fully account for the density observed, and a positive di\\erence density remains after refinement. Additionally, none of the other components of the crystallization bu\\er could be successfully fitted to the identified electron density. While one might speculate that malate from the buffer or another fragment/ligand could be present in the apo structure, this is not supported by the data. Given these factors, we conclude that malate is not bound, but rather a degradation product from Fragment 4 may be present. Since the compound was soaked at a concentration of 50 mM, there is a possibility that trace contaminants are present at sufficient concentrations to occupy the binding pocket. We do not attribute this to irradiation effects during data collection, as the density is consistently clear for one of the compounds across the crystal, making it improbable that only one of the two molecules is subject to degradation from the X-ray exposure. We have included density images here for the reviewer’s further reference below.\nWithin the main text we have updated the paragraph containing this information to read: “One region of density was assigned to a single molecule of fragment 4 with high confidence, while the second region was interpreted as a possible degrada'on product of a second fragment 4 molecule rather than buffer components. This interpretation was based on best fit to initial Fo-Fc density (Figure 4) and was modeled accordingly (Figures 6D-6F).”\n\n3) I found the order of the figures confusing. The first panel in Figure 1 shows the strategy for chemical elaboration. Figure 2 shows structures and density for fragments and elaborated compounds. Some of the structural data that underpin the elaboration strategy are not presented until Figure 5.\nResponse: We appreciate the comment of the reviewer. Since figures are numbered and shown in order of their description within the manuscript, we will take suggestions of what reordering would help with the presentation and readability. At another reviewer’s request, we have also moved NMR and SPR figures into the main text as Figures 1 and 2, respectively, so please be aware that overall numbering has changed.\n\n4) The abstract contains abbreviations that are not defined or explained until later in the text.\nResponse: The definitions for MSI-H (Microsatellite instability-high) and MMRd (Mismatch repair deficiency) have been added to the abstract.\n\n5) Likewise, PARP, BRCA1/2 are used at the beginning of the introduction in a manner that I suggest makes it difficult for a non-expert in the field to follow.\nResponse: The sentence has been reworded for clarity as requested. It now reads, “Notable examples of this type of drug therapy are PARP inhibitors for the treatment of cancers with mutations in BRCA1/2 which are DNA damage repair proteins4,5.“\n6) ML216 is mentioned in the introduction – but the context of the sentence is not provided until the discussion section.\nResponse: The sentence was modified to address the reviewer suggestion: “This lack of success was recently very thoroughly evaluated by Hauser et al. showing that non-specific interactions can be attributed to impurities in the test samples (ML216) or covalent labeling of the WRN protein (NSC compounds).”\n7) Druggability is used without a definition of what it means in the introduction and in the selection of fragment 1 for further work.\nResponse: We have edited these sections to more clearly describe the qualities we were looking for in a binding site. The text now reads: “In addition to these challenges, the ATP binding site in helicases is predicted to be less likely to bind a drug-like molecule than enzymes like kinases, complicating the search for potent ATP-competitive inhibitors23. These considerations indicate that allosteric inhibition of WRN might facilitate the identification of selective leads among RECQ helicases. “\n\n8) The sentence beginning “internal structure of WRN” may be missing something?\nResponse: Indeed, the sentence was incomplete upon initial submission. This has been remedied, and the wording of the sentence has changed slightly to further enhance the understanding. It now reads: “Our initial structure of wild type human WRN (500-942) complexed with AMP-PNP exhibits a root mean square deviation (RMSD) of 0.52 Å (Ca backbone atoms)34 when aligned with PDB ID 6YHR, a longer WRN construct consisting of residues 517-1093,7 despite lacking the HDRC domain.”\n\n9) The confidence of the authors in the suitability of the construct is not really explained.\nResponse: We have added additional descriptions to this section in this regard. Our initial overlay with the at-the-time only WRN structure available in the PDB lent us confidence in our construct’s suitability for SAR development. This was further bolstered by the use of similarly – though not identically – truncated WRN constructs in both 2024 papers regarding clinically-relevant WRN inhibitors where they drive SAR/SBDD with these constructs to develop inhibitors with in vivo and cellular activity.\n\n10) Why is this structure-guided HTE? It seems to be a pretty standard protocol of compound enumeration and docking.\nResponse: We thank the reviewer for bringing this to our attention to provide a better subtitle. We have modified the heading of the paragraph in the main text. Please see below the extracted paragraph from the main text.\n“Structure guided evolution of fragment 1 series leads to functional inhibition and discovery of a new WRN form\nWe advanced the development of fragment 1 by maintaining its interactions with key residues Arg732, Tyr849, and Arg910, while also incorporating insights gained from the X-ray structure of fragment 2 complexed with WRN. Notably, the carboxylic acid of fragment 2 forms an important ionic interaction with Arg857, which informed the evolution of fragment 1. To design ligands that combined the structural features present in fragment 1 and fragment 2, we generated a virtual library by coupling trifluoroaniline with various proprietary acids from our corporate collection. Virtual ligands were assessed based on their docked poses, focusing on key interactions identified in the crystal structures of WRN with bound fragment 1 and fragment 2. Specifically, we looked for hydrogen bonds with Arg732 from fragment 1, hydrogen bonds with Arg857 from fragment 2, and p-stacking interactions with Tyr849 observed in both fragments. A total of 17 compounds that satisfied all three criteria were selected for synthesis. Among these, we identified several promising molecules, including compound 1 and its thiophene analog, compound 2”\n\n11) Solubility > 250 mM? In what? How is purity assessed?\nResponse: We thank the reviewer for bringing this to our attention. The solubility was corrected to 250 µM. The solubility was assessed by 1H NMR in the presence of 25 µM TSP in the standard PBS buffer. LC-MS assessed the purity, and fragments with a purity of more than 90% were selected to be library members. In addition, the library is routinely tested for compound stability, and new mixtures are prepared when compound degradation is observed.\n\n12) How is non-saturable binding assessed from single concentration SPR experiments?\nResponse: To screen the fragments by SPR, we had to limit our data acquisition to single-point concentration. At this step, we resorted to clustering compounds based on the sensorgram profile. Indeed, the analysis is not perfect as it is only a single point but using the ratio of observed binding RU (at the end of each test fragment injection) and the theoretical maximum occupancy based on a 1:1 binding model allowed us to deprioritize bad actors (fragments with non-saturable /super-stoichiometric profile) efficiently.\n\n13) Final assay volume of 20 mL?\nResponse: We thank the reviewer for bringing this to our attention. This should be in micromolar, not millimolar. We have edited the document to use the proper micro symbol “µ”\n\n14) Hill (hill) slope.\nResponse: The capitalization has been corrected.\n\n15) There are several instances where capitalization, sub/superscripts appear to be incorrectly formatted.\nResponse: We thank you for your attention to detail. We have read through the manuscript carefully and believe we have made all necessary changes.\n\n16) The reference format is inconsistent as are the citation markers.\nResponse: We appreciate you bringing this to our attention. We have carefully checked for consistency and made all necessary changes.\n\n17) Several of the ESI-MS peaks are quoted to one decimal place with the final digit as 0. Eg. 422.0 – where this number is inconsistent with the expected mass. Is this really the observed mass (with an error)?\nResponse: We thank the reviewer for allowing us to clarify. These are observed mass with an error. These are from the low resolution mass spectrometry. We have added HRMS data for all of the fragments and final compounds.\n\n18) There are a number of 19F couplings that I would have expected to see in the 1H NMR that are not reported.\nResponse: We thank the reviewer for allowing us to further explain. Fluorine in “CF ” is 4 bonds away from the nearest aromatic C-H. In our opinion the lack of observed coupling between F and H is likely due to this distance. Furthermore, we have added the 1H NMR spectra of fragment 1, where this coupling is also absent.\n\n19) Compound 46 seems to have an in-house code in the experimental.\nResponse: We would like to thank the reviewer for pointing out this typo. We have renumbered the compounds and fixed this typo.\n\nReviewer #2 (Remarks to the Author):\n1) Given the fact that no helicase-inhibitor has yet achieved approvement, these results are significant to the field and warrant publication. The manuscript would benefit from more comparison with RNA helicases, as for this structurally-related class of non-processive helicases there ample literature on conformational flexibility. For the flexibly-linked RNA helicases it is clear that the conformations \"trapped\" in crystals are very versatile and may or may not be populated in solution. Domain rotations of \"up to 180°\" are to be expected in the absence of conformational restrictions. Nonetheless, any conformation that binds an inhibitor, is worthwhile investigating.\nResponse: Thank you for your suggestions. We have included additional information and citations around the known flexible nature of helicases in general and have highlighted both the SF2 superfamily and RecQ family. We do find significance in the full 180° rotation of these domains as, to our knowledge, no structures apart from recent studies have shown this significant of a conformational change, especially within the RecQ family of helicases and BLM in particular. There are several papers discussing a flexible aromatic loop connecting the HDRC helicase domain to the winged helix (WH) domain of RecQ, but the flexible linker we describe here is between the two ATPase domains themselves. Reviewer 5 brought up similar points, and we have included additional comments in our response there.\n\n2) A drawback of the manuscript compared to earlier WRN inhibitor studies is the lack of cellular or in vivo data. From a biophysical standpoint, the data appear overall sound and are not over-interpreted. The NMR and SPR experiments are conducted well, and conclusions are drawn cautiously, e.g. by categorizing the SPR traces into different classes. A pity that kinetic information could not be extracted from the SPR data, as a small koff value is often indicative for a good inhibitor.\nResponse: We thank the reviewer for the opportunity to explain our point of view. The lack of cellular activity reported for our evolved Fragment Series was also pointed out by Reviewer #5. We thank Reviewer #2 for recognizing the quality of our biophysical work. While we now have included the SPR sensorgrams for the highest binding affinity compound 4 from our work in Figure SI 9 (Kd = 1.2 uM), kinetic information cannot be derived as correctly stated by the Reviewer. The goal of our biophysical work was to provide multiple non-covalent fragment screening hits as potential starting points for lead discovery against this challenging drug target, which is similar in scope to the excellent paper recently reported in Nature Communications by Hommel, et al. (Discovery of a selective and biologically active low-molecular weight antagonist of human interleukin-1β | Nature Communications). Given the status of our reported series, cellular or in vivo data are out of scope.\n\n3) The analysis of binding interfaces gleaned from crystal structures, however, appears too speculative and is not backed up by experimental (e.g. mutational) data. We acknowledge that the authors depict 2Fo-Fc electron density, but more informative to judge the quality of the ligand binding poses are omit maps, as Fourier coefficients for these are usually not deposited in the PDB.\nResponse: We have updated all density images to show the Fo-Fc “omit map” densities for ligands, including the addition of a Fo-Fc map for the AMP-PNP-bound structure. Regarding the binding interfaces, the data statistics from these structures including the resolution and B-factors corroborates our interpretations. In our opinion the general binding sites of compounds analyzed during compound lead discovery efforts are not addressed with mutational data.\nWe have added a paragraph in results that address why not mutational data: Although inclusion of mutagenesis experiments would provide valuable insights into the importance of improving binding affinity through ionic interactions. In our experience, this approach is particularly beneficial when working with highly potent compounds, allowing for the modification or removal of undesired functionalities to enhance drug-like properties or mitigate off-target liabilities. However, in the relatively weaker affinity space we are currently exploring, mutagenesis of key residues linked to potency improvements via productive interactions may not not be detected. Consequently, this approach was not prioritized at this phase of our optimization. Nevertheless, three orthologous methods, X-ray structures, biophysical and biochemical data, along with the presence of trifluorophenyl in both our fragment and HRO761, provide substantial evidence supporting the validity of these compounds for further development.\n\nMinor points:\n1) The Methods would benefit from some more details as in our opinion the experiments cannot be reproduced with the information give - see the annotated .pdf files. At times lab jargon is prominent, formatting style is inhomogeneous, and some Methods parts have poor written style.\nResponse: We appreciate the request for more information on our methods and have included additional information throughout the methods sections. We have edited the manuscript to remove any jargon and increase the quality of writing overall.\n\nReviewer #5 (Remarks to the Author):\n1) First, given the recent literature, the novelty of the allosteric pocket is no longer a major selling point.\nResponse: We are delighted that the reviewer highlights that the manuscript is a textbook quality hit-to-lead story using fragment-based drug discovery approaches for a challenging target, WRN, and so merits publication. We also appreciate the opportunity to elaborate why the data presented is a good fit for publication in Nature Communications. In our view, the novelty of our work lies not only in the discovery of an allosteric pocket but also in our ability to identify multiple validated non-covalent hits, a feat that has proven challenging for others in the field (doi: 10.1002/cmdc.202300613). As the reviewer pointed out previous work identified a single hit (doi.org/10.1038/s41586-024-07350-y), such scarcity is not a robust path forward. Moreover, our fragment-based lead discovery workflow has revealed several binding sites, reinforcing the manuscript's innovative contributions to the field. Therefore, the novelty of this work is significant in at least three key aspects.\n\n2) Second, the reported potency of their compounds is limited in low uM range and no cellular activity is reported.\nResponse: The lack of cellular activity reported for our evolved Fragment Series was also pointed out by Reviewer #2. Please refer to our answer to that Reviewer vide supra for additional details. Briefly, the workflow and data presented in the manuscript is a fragment-based lead discovery e\\ort which by definition would identify small chemical fragments that may bind only weakly to the target, before growing them or combining them to produce a lead with a higher a\\inity. In the early phase of FBLD, libraries with a few thousand compounds with molecular weights of around 200 Da may be screened, and millimolar a\\inities can be considered useful. In this range of low a\\inity binding, low microM, assessing cellular activity is not conducive. In this sense this request is beyond the scope of the paper. Nature communication articles included in the Fragment collection are also examples that lack cellular activity (https://www.nature.com/articles/s41467-023-41190-0#Sec10)\n\n3) Third, no mechanistic or selectivity data is reported for the compounds.\nResponse: We appreciate the opportunity o\\ered by the reviewer to include the selectivity data. We have incorporated the selectivity data into Table SI 2.\nAs we explained before, attempting to answer mechanistic questions with compounds in the uM a\\inity range is not a customary workflow, and requested assays were not established.\n\n4) Finally, while new and interesting conformational states of WRN are reported, likely highlighting the dynamics/plasticity of the protein, it’s not clear if these findings have biological relevance or if they are merely crystal packing rs.\nResponse: We acknowledge that the identification of protein conformations and interactions may be influenced by crystallization condition. On the other hand, for WRN, previous work by Ferretti (https://doi.org/10.1038/s41586-024-07350-y) and Balgatvis (https://doi.org/10.1038/s41586-024-07318-y) on FORMS A-D have shown the biological relevance. Furthermore, given that compounds that bind to the allosteric pocket in Forms B-D were developed into clinical inhibitors, we are confident that these conformations are populated and relevant in biological contexts and could contribute to enzyme inhibition. Form E, which is being described for the first time here, further emphasizes the flexibility of the D1-D2 linker region. While we cannot definitively prove the presence of this form in a genuine biological environment, based on our understanding of helicase flexibility and the differences seen with WRN as compared to BLM, for instance, we hypothesize that this conformation is accessible. Furthermore, we agree with reviewer #2 whom states that: ‘any conformation trapped is worthwhile investigating’.\n\nMajor Gaps\n(1) In terms of data presentation, the authors should really include a SAR summary table in the main figures with the key data summarized for the fragments and compounds. The key data is scattered around in various supplementary figures/tables and is too hard to track, analyze, and compare. A summary table will greatly simplify the key findings for the readership. Format can be something like this:\nCompound ID; Chemical Structure; MW & LE; NMR binder (or KD); SPR binder (or KD); Docking Score; EC50 WRN; EC50 BLM; PDB Code\nResponse: We thank the reviewer for this suggestion that allows a better presentation of our data. To accommodate Reviewer’s suggestions, we modified Table SI 2 and included the structure, MW & LE; IC50 WRN; IC50 BLM; and PDB Code. Please see the table below. We have discussed more about docking scores in answer #4 following this and we refer the reviewer to read more there.\n\n(2) One of the major challenges of a fragment-based screening campaign is distinguishing true hits from non-specific aggregators. Please elaborate in the manuscript what was done to remove bad actors and where you incorporated this into the workflow. Was solubility a concern? Please include a brief sentence or two describing how the Merck fragment libraries are curated in terms of properties (MW, solubility, etc). (I see some description in the methods, but I think this point is important enough to elevate to main text).\nResponse: We incorporated the suggestions in the first results subsection (entitled Fragment-Based Screens iden3fy allosteric binders to WRN) by adding the following text: “To identify new binding sites on WRN, we employed our FBLD platform which relies on screening our internal fragment collections that were a priori curated to only include compounds with su\\icient solubility and purity.” To the Reviewer: To decrease the number of false positives we prioritized compounds that showed binding with the combination of 19F T CPMG, 1H T CPMG, STD, and waterLOGSY NMR experiments.\n\n(3) I was surprised that there is no counter-screening / selectivity data presented in this manuscript. Please include relevant counter-screen data for BLM or whatever helicase you used to assess selectivity.\nResponse: As we mentioned in our earlier response for point #3 under limitations, addressing mechanistic and selectivity data, we appreciated the opportunity o\\ered by the reviewer to include the selectivity data. We have incorporated the selectivity data to Table SI 2.\n\n(4) The “structure guided high throughput experimentation” section (page 6 results) isn’t explained well enough. The readership will be very interested to know how Merck, with its state-of-the-art resources, is using computational methods to streamline discovery. Please elaborate this section and methods explaining minimally how the model was validated and a figure showing the predicted docking activities and how those docking scores compared to experimental data. Are we to understand that of 17 compounds predicted by the algorithm, only 2 showed activity (11% success rate and 89% false positive)? This is about what I’d expect, and I do believe the transparency and honesty around the persistent challenges with docking is very useful to point out and for the industry to acknowledge with all the current hype around AI.\nResponse: We thank the reviewer for allowing us to expand and clarify. Following the suggestion we changed the title from, ‘structure guided high throughput experimentation’, to ‘Structure guided evolution of fragment 1 series leads to functional inhibition and discovery of a new WRN form’. The new text attempts to provide clarity regarding our methods for selecting compounds. For example, instead of using docking scores to predict binding or inhibitory activity, our focus was on identifying which geometries would facilitate the polar interactions observed in the crystal structures of fragments 1 and 2 bound to WRN. We concur with the reviewer opinion that an 11% hit rate aligns with the anticipated success for this type of approach.\n\n(5) The mechanistic characterization work is non-existent. Are the compounds competitive with respect to ATP or DNA? Do they show any evidence of cooperativity as was observed with VVD-133214? Novartis published an ATP-gamma-S displacement assay. Do the fragments show any activity in this assay? Please review the existing literature on MOA analyses. These are very easy assays to run and there’s no reason to not include those data to better characterize these compounds. Especially for a Nature audience.\nResponse: is a significant potency right shift for WRN inhibitors when going from ATPase inhibition to DNA unwinding inhibition. This suggests that a high degree of ATPase inhibition is required to significantly reduce observed DNA unwinding rates for WRN. Consequently, the threshold for helicase inhibition is not easily overcome for initial fragment screening derived small molecule hits. This consideration also applies to accurate execution of MOA studies, where weaker compounds with High IC50s present challenges in the execution of precise and informative MOA studies, which require use of much higher compound concentrations close to or exceeding their solubility limits. As a discovery campaign progresses and potency of the molecules continue to improve down to hundreds of nM, MOA studies become more feasible, and translatability to the helicase assay more accessible. We have incorporated these considerations into our discovery plan. We did, however, conduct selectivity analysis for our compounds, comparing WRN and BLM ATPase activity. This data has been included in the manuscript (Table S2) to facilitate better interpretation of the compounds.\n\n(6) The claim about the second bound molecule being a fragment 4 degradation product seems very unlikely. An alternative interpretation of the data could be that a molecule of malonate is bound (a component of MMT crystallization buffer). Can the authors please try modeling malonate into the density and see if this could explain the electron density? Otherwise, please provide relevant stability data on fragment 4 in their crystallization buffer to support their claim.\nResponse: We refer the reviewer to see our detailed response to Reviewer #1 who shared similar concerns regarding the degradation product of fragment 4. In short, we provide detailed information for reviewers to look over during the review process that we believe substantiates our hypothesis around a degradation product and have simultaneously edited the manuscript to address this concern. (from above) In our opinion since, Apo WRN was crystallized using a bu\\er comprised of a combination of MES, DL-malic acid, and Tris, referred to here as MMT. These crystals were subjected to soaking experiments, including one with Fragment 4. We would like to highlight that the bu\\er has Malid acid and not malonate acid. So we attempted to model malate in the corresponding density peak; however, this did not fully account for the density observed, and a positive di\\erence density remains after refinement. Additionally, none of the other components of the crystallization bu\\er could be successfully fitted to the identified electron density. While one might speculate that malate from the buffer or another fragment/ligand could be present in the apo structure, this is not supported by the data. Given these factors, we conclude that malate is not bound, but rather a degradation product from Fragment 4 may be present. Since the compound was soaked at a concentration of 50 mM, there is a possibility that trace contaminants are present at sufficient concentrations to occupy the binding pocket. We do not attribute this to irradiation effects during data collection, as the density is consistently clear for one of the compounds across the crystal, making it improbable that only one of the two molecules is subject to degradation from the X-ray exposure. We have included density images here for the reviewer’s further reference below.\nWithin the main text we have updated the paragraph containing this information to read: “One region of density was assigned to a single molecule of fragment 4 with high confidence, while the second region was interpreted as a possible degrada'on product of a second fragment 4 molecule rather than buffer components. This interpretation was based on best fit to initial Fo-Fc density (Figure 4) and was modeled accordingly (Figures 6D-6F).”\n\n(7) Please provide some text on whether or not the compounds have any cellular activity. You can use the Novartis compound or Vividion compounds as positive controls. Please address any lack of cellular activity in the text as potency limited or properties limited?\nResponse: As this is a FBLD campaign no cellular activity was assessed since it is assumed that the low molecular weight, low a\\inity, low potency would not yield substantive results in a cellular assay.\n\n(8) I do like the presentation of the different conformations of WRN. But it’s difficult to know if these have any biological relevance. Have the authors done any cryo-EM studies to try and understand at a population level the relative disposition of the two helicase domains in a more native state? Have the authors done any cryo-EM in the presence of DNA? Have the authors done any crystallography in the presence of DNA? Any supporting data would greatly bolster their claims and make their findings more impactful.\nResponse: We acknowledge that the determination of protein conformations and interactions may be influenced by crystallization artifacts. Given the development of clinical inhibitors that bind to the allosteric pocket in Forms B-D, we are confident that these conformations are relevant in biological contexts and contribute to enzyme inhibition. Form E, which is being described for the first time here, further emphasizes the flexibility of the D1-D2 linker region. While we cannot definitively prove the presence of this form in a biologically relevant environment, based on our understanding of helicase flexibility and the differences seen with WRN as compared to BLM, for instance, we hypothesize that this conformation is accessible. We have not conducted cryoEM studies as they extend beyond the scope of this paper.\nTo address this within the manuscript itself we have superimposed the public structure of BLM with bound DNA (PDB 4CGZ) with WRN Form D and WRN Form E in a new figure in the Supplementary Information, Fig SI 10. We aligned on the D1 and the D2 domains. In all cases the DNA binding region was accessible. We have updated the section to read: “Alignments of the ATPase D1 and D2 domains of fragment 1 and compound 5 with the structure of BLM helicase with bound DNA (PDB 4CGZ)40 indicate that DNA is likely able to bind to the two additional conformations of WRN, Form D and Form E, respectively (Figure SI 10). Consequently, the disruption of the ATP binding pocket or the prevention of domain rearrangement to achieve an ATP-binding competent conformation are both plausible mechanisms of inhibition.”\n\nMinor\n(1) Please move Figure 1 and the electron density Figure 2 to the supplement. Please move the bioNMR and SPR workflows to the main figures. Please move representative NMR data like S3 A&B and representative SPR data to main figures. These are the key data that informed which fragments to call out as hits. This is more important than the chemistry schematic, which is less informative and barely discussed in the main text.\nResponse: We appreciate the suggestions to move the figures to make the manuscript better. Key NMR and SPR figures have been moved to the main text as Figures 1 and 2, respectively. The remaining NMR and SPR data do not fit within the figure limit of the paper and as such are found in the Supplementary Information file. We have decided to display the Fo-Fc electron density maps of all ligands for transparency, so we have retained them as Figure 4.\n\n(2) Did the authors profile their compounds in the published DNA unwinding assay? Please report these data for comparison to their ATPase data.\nResponse: There is a significant potency right shift for WRN inhibitors when going from ATPase inhibition to DNA unwinding inhibition. Suggestive that a high degree of ATPase inhibition is necessary to significantly reduce observed DNA unwinding rates for WRN. As such, the threshold for helicase inhibition is not easily overcome for initial fragment screening derived small molecule hits. This consideration also applies to accurate execution of MOA studies, where weaker compounds with High IC50s present challenges in the execution of precise and informative MOA studies, which require use of much higher compound concentrations close to or exceeding their solubility limits. As a discovery campaign progresses and potency of the molecules continue to improve down to hundreds of nM, MOA studies become viable and translatability to the helicase assay more accessible; we would have incorporated both into our discovery plan. We did, however, conduct selectivity analysis for our compounds, comparing WRN and BLM ATPase activity. This data has been incorporated into the manuscript (Table S2) for better interpretation of the compounds.\n\n(3) For pymol figures, please repeat rendering with no shadows. For Figure 4, is hot pink the best color scheme? As a general rule, structure figures are easier to understand when they leverage color and contrast to help highlight the important message. For instance, if the compound binding mode is the “message”, color the compound with a brighter color than the protein so it will better stand out. As currently rendered, the compounds are camouflaged with the same color scheme as the rest of the protein. For example, in Figure 3A, GMPPNP adenosine ring is gray and the compound is blue and are so perfectly camouflaged with the protein as to be almost invisible. This would be the time to use a hot pink rendering on the compound so it can be seen against the blue protein background. Same comments apply to Figure 4A and D. Don’t camouflage what’s important!\nResponse: Thank you for the advice. We have removed shadows, change colors and remade most figures for better clarity. We have added ‘boxes’ to the ligands in the overall structure. Figure numbers have shifted due to the addition of NMR and SPR figures to the main text. The example noted here is now Figure 6.\n\n(4) For Figure 3 D and F, is it possible to find the same view to show the same set of residues for each compound, fragment 1 and fragment 2? It looks like the figures were made in isolation or autogenerated with a script without considering the importance of cross-comparison.\nResponse: The two figures were originally made to highlight only residues of importance to that specific ligand, but we agree that highlighting the same residues between these two structures can add to the story of fragment development. The images have been updated to reflect this suggestion. Please note that Figure numbers have shifted with the addition of NMR and SPR figures to the main text. The example noted here is now Figure 4.\n\n(5) Page 5: I think there’s a missing word: “internal structure of WRN…” Please edit the sentence.\nResponse: Thank you for your attention to detail. Indeed, the sentence was incomplete in the initial submission. This has been remedied, and the wording of the sentence has changed slightly to further enhance the understanding. It now reads “Our initial structure of wild type human WRN (500-942) complexed with AMP-PNP exhibits a root mean square deviation (RMSD) of 0.52 Å (Ca backbone atoms)34 when aligned with PDB ID 6YHR, a longer WRN construct consisting of residues 517-1093,7 despite lacking the HDRC domain.”\n\n(6) Please provide some further explanation in the text why fragment 1 was advanced and not the others? The current explanation about favorable SAR is a bit vague.\nResponse: We have combined this answer with the one below as they partially overlap. In prioritizing the chemical matter, we first evaluate the confidence in binding interaction using biophysical approaches. The second layer of prioritization is x-ray crystallography, and all compounds that pass that bar undergo SIMS expansion to deprioritize fragments with steep SAR. In parallel, the merging and growing approaches may be triggered to advance the fragments. The fragments 1 and 2 growth vectors highly overlapped, and merging their features allowed us to e\\iciently identify compounds 1 and 2 that further evolved to higher a\\inity chemical matter. We did not perform the fragment linking as we saw a viable path forward with the growing/merging approach. However, if this was unsuccessful, one could consider another screen for the linking approach.\n\n(7) The authors performed a fragment growing strategy in their hit-to-lead progression. Did the authors ever consider a fragment linking approach? This would necessitate screening for a second fragment binder in the presence of one of their preferred/validated fragments? Some comments in the text about their strategic decisions around growth vs linking would be nice.\nResponse: The answer was combined with point above."}, {"round": 2, "reviewer_comments": {"reviewer_1": "As I highlighted in my original review, I believe that the work presented in this manuscript will be of general interest. My original comments on the significance and data interpretation stand, and I comment here on the revisions based on the initial reviews.\n\nThe authors appear to have attempted to address most of the comments received on review. I remain highly confused by the apparent reluctance to show the primary data on which the manuscript is based. This is a manuscript that seeks to demonstrate the advantages presented by biophysical screening cascades in identifying chemical matter that binds to challenging targets such as this and is developable into higher affinity compounds. And yet only three exmaples of the SPR data are shown, none of which are in the main manuscript. Two of these are for very weak compounds and are not ideal examples of SPR binding characterisation for the reasons that I detailed in my previous review. The final compound shows saturable binding with clear dose-response. However, the Rmax in the dataset for compound 4 (MW 437) is 7 RU. In contrast, the two fragments (MW 246 & 217, respectively) have Rmax of ~18 and 16 RU and display significant overbinding. The methods do not report any differences in the loading of the protein for the SPR assay - and perhaps the authors have overlooked updating the methods section to reflect inclusion of the new data. However, I would have expected that compounds 2, 3, 4, 5, 6 and 7 should all show saturable binding in the SPR assay - and a demonstration of consistent responses and reproducible protein activity on the surface would lend a great deal more confidence in the robustness of the assay. I would regard the robustness of the SPR assay as important as this seems to be one of the major messages of the paper.\n\nNotwithstanding the addition of the HRMS data, I remain a little surprised by the lack of compound characterisation data. (No 13C NMR, no stated purities). This places quite an emphasis on analysis of the 1H NMR data. The authors state in the response letter that the 1H NMR spectra have been provided - but I could not see them in the resubmission package. Moreover, there are several compounds where there is extensive overlap in the 1H NMR data, and where intensities and couplings could not be resolved or are not reported in the manuscript. I fear that based on my experience I do not share the authors level of confidence that this represents unequivocal characterisation of all compounds.\n\nFor example - in the characterisation of compound 4, the three protons on the fluorophenol ring are reported as 7.13 (dd, J 11.2, 8.3, 1H) 6.85 (dd J 8.5, 2.0), and 6.74 (ddd J 8.3, 4.2, 2.1). Presumably the 11.2 Hz is a 19F-1H coupling, the ddd is has two 1H-1H couplings and a 4J to the 19F, which makes the dd the proton next to the OH. This would mean that there is a 4J coupling of 8.5 Hz between the proton and the 19F. That is very unusual - particularly given that the other 4J coupling has a more typical value of 4.2 Hz. That is not to say that the spectra have been reported incorrectly - just that in the absence of other data, I would be very uncomfortable reporting this as unequivocal characterisation of the compound's identity.\n\nMy other minor gripe is the description of the additional density observed in the crystals structure of fragment 4 as a degradation product. When it is first described the conclusion is that it is probably a degradation product as there is nothing that would be expected to be present in the crystallisation condition that is consistent with the density. And therefore one possible explanation is that it is a degradation product. Thereafter it is simply referred to as \"the degradation product\". I don't think that the authors have shown definitively that this is what it is - and therefore I suggest that it is inappropriate to refer to it as such.", "reviewer_2": "While evading the time-consuming task to provide additional mutational/cellular data, the authors did a good job in responding to the issues raised, imo.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "This revised version is much improved and overall, I'm supportive for publication. Congratulations on the nice work!\nMinor points, in the merged PDF, lines 313-323 appear to be intended for rebuttal letter and not the main text.\nRegarding fragment 4 degradation, in my experience, interpreting odd bits of density is always subjective. The language of the main text is appropriately qualified. But I would encourage the authors to also look at the QC of their DMSO stock solution (LCMS) to confirm if such a degradation fragment exists and/or look at the stability of their fragment in crystallization buffer. This would be helpful supporting evidence for their claim. In the absence of such data, one must consider other possibilities like a partially ordered / partial occupancy fragment 4. Or one could imagine a component of the buffer that has more than one conformation that is creating a confusing density blur. My general inclination is to be more conservative in these situations given the uncertainties."}, "author_response": "Reviewer #1 (Remarks to the Author):\nAs I highlighted in my original review, I believe that the work presented in this manuscript will be of general interest. My original comments on the significance and data interpretation stand, and I comment here on the revisions based on the initial reviews.\nThe authors appear to have attempted to address most of the comments received on review.\nWe thank the reviewer for highlighting the general interest and significance of these data.\nI remain highly confused by the apparent reluctance to show the primary data on which the manuscript is based. This is a manuscript that seeks to demonstrate the advantages presented by biophysical screening cascades in identifying chemical matter that binds to challenging targets such as this and is developable into higher affinity compounds. And yet only three exmaples of the SPR data are shown, none of which are in the main manuscript. Two of these are for very weak compounds and are not ideal examples of SPR binding characterisation for the reasons that I detailed in my previous review. The final compound shows saturable binding with clear dose-response. However, the Rmax in the dataset for compound 4 (MW 437) is 7 RU. In contrast, the two fragments (MW 246 & 217, respectively) have Rmax of ~18 and 16 RU and display significant overbinding. The methods do not report any differences in the loading of the protein for the SPR assay - and perhaps the authors have overlooked updating the methods section to reflect inclusion of the new data. However, I would have expected that compounds 2, 3, 4, 5, 6 and 7 should all show saturable binding in the SPR assay - and a demonstration of consistent responses and reproducible protein activity on the surface would lend a great deal more confidence in the robustness of the assay. I would regard the robustness of the SPR assay as important as this seems to be one of the major messages of the paper.\nResponse: Reviewer 1 makes a good point, and as we settled to center the publication around structural data, we pushed biophysical workflows to the second tier. That said, we did routinely evaluate compounds using biophysical approaches to ensure that we observed the same trend of activity/aXinity gain with biochemical and biophysical methods. We now include additional data for compounds 2, 3, 4, 5, and 7 (unfortunately, we do not have data for compound 6 and cannot acquire it as we no longer have the material).\nRegarding the comment about lower occupancy and method section, the experimental data are now updated, and indeed we observed that for all advanced chemical matter, occupancy plateaued on average around 50-60%. For compound 5, binding to WRN Form E, we could never even reach 20% occupancy. We also re-acquired data for compounds 3, 4, 5, and 7 at a lower temperature and observed comparable binding profiles and occupancies. Overall, we never saw an increase above 70% (please see the data at 10°C below). In the sensorgrams below, the temperature and occupancy are shown on the top. Please note that there was a slightly lower density of immobilization at 10˚C, which is why we see lower RU signal, but higher occupancy.\nWe added Figure SI 8. to the Supporting Information in this Revision, to show a representative SPR sensorgram, and where applicable an aXinity plot, for compounds 2, 3, 4, 5, and 7.\nNotwithstanding the addition of the HRMS data, I remain a little surprised by the lack of compound characterisation data. (No 13C NMR, no stated purities). This places quite an emphasis on analysis of the 1H NMR data. The authors state in the response letter that the 1H NMR spectra have been provided - but I could not see them in the resubmission package. Moreover, there are several compounds where there is extensive overlap in the 1H NMR data, and where intensities and couplings could not be resolved or are not reported in the manuscript. I fear that based on my experience I do not share the authors level of confidence that this represents unequivocal characterisation of all compounds.\nFor example - in the characterisation of compound 4, the three protons on the fluorophenol ring are reported as 7.13 (dd, J 11.2, 8.3, 1H) 6.85 (dd J 8.5, 2.0), and 6.74 (ddd J 8.3, 4.2, 2.1). Presumably the 11.2 Hz is a 19F-1H coupling, the ddd is has two 1H-1H couplings and a 4J to the 19F, which makes the dd the proton next to the OH. This would mean that there is a 4J coupling of 8.5 Hz between the proton and the 19F. That is very unusual - particularly given that the other 4J coupling has a more typical value of 4.2 Hz. That is not to say that the spectra have been reported incorrectly - just that in the absence of other data, I would be very uncomfortable reporting this as unequivocal characterisation of the compound's identity.\nResponse: We appreciate the thoroughness of the reviewer’s examination of our compound characterization work. In the revised manuscript, we are providing 1H and 13C spectra for all compounds along with estimations of their purities (>95%) in a revised pdf file.\nSpecifically, we include the complete characterization of compound 4, performed using 2D NMR spectroscopy. Copies of the COSY, NOESY, HSQC, and HMBC spectra are provided. All homonuclear and heteronuclear correlations were consistent with the proposed structure. Regarding the concerns about the proton-fluorine couplings in compound 4, we were slightly surprised by the somewhat higher than expected value of the 4-bond J(H,F) couplings, which are usually smaller than 6 Hz. However, the DFT computations of the J-couplings (B3LYP/6-31+G(d,p), nmr=mixed) indicate that the observed coupling is consistent with the experimental value.\nDFT values: 4J(H21,F)=8.0 Hz, 3J(24H,F)=9.8 Hz, 4J(H,F)=4.0 Hz\nExper. Values: 4J(H21,F)=8.5 Hz, 3J(24H,F)=11.3 Hz, 4J(H,F)=4.2 Hz\nA relatively high value of the 4J(H,F) coupling in this case is likely the result of the electron-withdrawing effect of the hydroxyl group positioned between the interacting nuclei.\nMy other minor gripe is the description of the additional density observed in the crystals structure of fragment 4 as a degradation product. When it is first described the conclusion is that it is probably a degradation product as there is nothing that would be expected to be present in the crystallisation condition that is consistent with the density. And therefore one possible explanation is that it is a degradation product. Thereafter it is simply referred to as \"the degradation product\". I don't think that the authors have shown definitively that this is what it is - and therefore I suggest that it is inappropriate to refer to it as such.\nResponse: We have reworded these sentences to better address this ambiguity.\n“…indicating the presence of two separate molecules at this site. One region of density was confidently assigned to a single molecule of fragment 4, while the second region was ambiguous. After analyzing all buffer components and compound byproducts, we chose to model a byproduct of the compound into this space, as it best fit the initial Fo-Fc density (Figure 6E). Although we cannot determine the exact molecule occupying this second region of density, it is evident that the pocket can accommodate this additional chemical matter.… “while the carboxy group of the hypothesized byproduct”\nAnd later\n“Furthermore, the structure featuring fragment 4 shows a clear density region for fragment 4, along with another density region that we modeled as a ligand byproduct, within the remnants of the ATP pocket.”\nReviewer #2 (Remarks to the Author):\nWhile evading the time-consuming task to provide additional mutational/cellular data, the authors did a good job in responding to the issues raised, imo.\nWe thank the reviewer for highlighting the effort in addressing the comments.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nReviewer #5 (Remarks to the Author):\nThis revised version is much improved and overall, I'm supportive for publication. Congratulations on the nice work!\nWe thank the reviewer for highlighting the effort in addressing the comments.\nMinor points, in the merged PDF, lines 313-323 appear to be intended for rebuttal letter and not the main text.\nResponse: We thank this Reviewer for catching this unintended addition to the Main Text. This section was indeed intended only for the rebuttal letter. We have not included it in the new Main Text currently submitted.\nRegarding fragment 4 degradation, in my experience, interpreting odd bits of density is always subjective. The language of the main text is appropriately qualified. But I would encourage the authors to also look at the QC of their DMSO stock solution (LCMS) to confirm if such a degradation fragment exists and/or look at the stability of their fragment in crystallization buffer. This would be helpful supporting evidence for their claim. In the absence of such data, one must consider other possibilities like a partially ordered / partial occupancy fragment 4. Or one could imagine a component of the buffer that has more than one conformation that is creating a confusing density blur. My general inclination is to be more conservative in these situations given the uncertainties.\nResponse: We agree that the density is ambiguous enough that many different interpretations could be of the same validity. We adjusted the text to touch on this, rewording the first explanation of this density as follows:\n“…indicating the presence of two separate molecules at this site. One region of density was confidently assigned to a single molecule of fragment 4, while the second region was ambiguous. After analyzing all buffer components and compound byproducts, we chose to model a byproduct of the compound into this space, as it best fit the initial Fo-Fc density (Figure 6E). Although we cannot determine the exact molecule occupying this second region of density, it is evident that the pocket can accommodate this additional chemical matter.… “while the carboxy group of the hypothesized byproduct”\nAnd later\n“Furthermore, the structure featuring fragment 4 shows a clear density region for fragment 4, along with another density region that we modeled as a ligand byproduct, within the remnants of the ATP pocket.”"}, {"round": 3, "reviewer_comments": {"reviewer_1": "I am satisfied that the authors have suitably addressed all of the commnents in my prior reviews", "reviewer_5": "The authors have addressed my concerns and I have no further comments. I am supportive for publication."}, "author_response": ""}]

Decision letter
[Editorial Note: See attachment]