# Cell-Permeable Peptide Inhibitors of the p53-hDM2 Interaction via Foldamer Helix Mimicry and Bis-Thioether Stapling

Maxime Neuville, Mathieu Bourgeais, Bo Li, Laetitia Varajao, François Hallé, Sébastien Goudreau, Emmanuelle Thinon, Morgane Pasco, Abdel-Majid Khatib, Gilles Guichard

► To cite this version:

Maxime Neuville, Mathieu Bourgeais, Bo Li, Laetitia Varajao, François Hallé, et al., Cell-Permeable Peptide Inhibitors of the p53-hDM2 Interaction via Foldamer Helix Mimicry and Bis-Thioether Stapling. Journal of Medicinal Chemistry, 2024, 68 (1), pp.236-246. 10.1021/acs.jmedchem.4c01762. hal-04931235

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Distributed under a Creative Commons Attribution 4.0 International License
Cell-Permeable Peptide Inhibitors of the p53-hDM2 Interaction via Foldamer Helix Mimicry and Bis-
Thioether Stapling

Maxime Neuville,ᵃ,ᵇ,ᶜ Mathieu Bourgeais,ᵃ,bc,† Bo Li,ª Laetitia Varajao,ª François Hallé,ª,‡ Sébastien R.

Goudreau,ᵇ Emmanuelle Thinon,ª Morgane Pasco,ª Abdel-Majid Khatib,ᶜ,∗ Gilles Guichardª,∗

ªUniv. Bordeaux, CNRS, Bordeaux INP, CBMN, UMR 5248, IECB, F-33607 Pessac, France

ᵇIMMUPHARMA BIOTECH SAS, 15 rue de Bruxelles, 75009 Paris, France

ᶜUniv. Bordeaux, INSERM, BRIC, U 1312, F-33000 Bordeaux, France

Corresponding Authors:

Abdel-Majid Khatib (majid.khatib@u-bordeaux.fr) & Gilles Guichard (g.guichard@iecb.u-bordeaux.fr)
ABSTRACT

Combining helical foldamers with α-peptides can produce α-helix mimetics with a reduced peptide character and enhanced resistance to proteolysis. Previously, we engineered a hybrid peptide-oligourea sequence replicating the N-terminal α-helical domain of p53 to achieve high affinity binding to hDM2. Here, we further advance this strategy by combining the foldamer approach with side chain crosslinking to create more constrained cell-permeable inhibitors capable of effectively engaging the target within cells. Starting from the crystal structure of the foldamer-hDM2 complex, we identified specific sites suitable for stapling, and generated a small library of macrocyclic foldamer-peptide hybrids. The most promising binders were subsequently optimized for cellular uptake and tested in a cellular assay. We observed that the introduction of a short segment of positively charged residues at the N-terminus of the sequence led to inhibitors that exhibited cytotoxic activity independently of p53. In contrast, neutral acetylated peptide-foldamer macrocycles demonstrated activity in a p53-dependent manner.

KEYWORDS

Peptide, Foldamer, Protein-protein interaction, Inhibitor, Helix, Macrocyclization, Thioether, p53-hDM2 Interaction, Cell viability
INTRODUCTION

α-Helices are frequently involved in protein-protein interactions (PPIs) and represent, in many situations, an excellent starting point to generate PPI inhibitors and pharmacological tools against targets of therapeutic interest.¹ In this process, selection of peptide binders with high affinity and/or selectivity for protein targets is facilitated by the high sequence diversity and ease of synthesis of peptides. However, short linear peptides show some inherent limitations such as poorly defined conformations, susceptibility to degradation by circulating enzymes - which may shorten their biological lifetime and duration of action - and poor cellular uptake. It has been recognized that chemically stabilized helices and helix mimics may overcome some of these drawbacks.²⁻⁴ The process of macrocyclization, such as side chain crosslinking, is widely recognized as one of the most efficient techniques for introducing constraints into peptides.⁵⁻⁷ This method is particularly effective in stabilizing helical conformations. It has been successfully utilized to produce high-affinity peptide binders capable of penetrating cell membranes.⁸⁻¹⁵ Concurrently, backbone modifications and foldamer chemistry have been used to reduce the peptide character of peptide-based PPI inhibitors and generate non-peptide inhibitors with increased stability to proteolysis.¹⁶⁻²¹ Recently, we introduced a strategy based on urea foldamers²² as α-helix mimics, whereby an α-helical peptide segment is replaced by an oligourea insert in known peptide binders.²³⁻²⁴ Starting from peptide ligands of E3 ubiquitin ligase hDM2, vitamin D receptor (VDR) and histone chaperone ASF-1, and by using structure-guided design, we generated foldamer-peptide hybrids that retain high affinity for the target proteins while showing improved resistance to proteolysis.²⁵⁻²⁶ X-ray structure analyses of several of these hybrid oligomers complexed to their respective protein targets confirmed that oligourea/peptide ligands adopt a helical conformation upon binding to the protein surface, thus suggesting that this foldamer approach could also be interfaced with macrocyclization chemistries to further enhance overall helical content and potency. Foldamer and macrocyclization chemistries have only been combined in rare cases.²⁷⁻³¹ To further explore this possibility, we thought to start from a high affinity foldamer-based inhibitor of the p53-hDM2 interaction (1) recently disclosed by our
group.²⁵ The tumor suppressor p53 is a transcription factor that regulates cellular responses by activating cell-death in response to stress conditions. The cell basal quantity of p53 is regulated by hDM2, an E3 ubiquitin ligase, but overexpression of hDM2 in cancer cells leads to uncontrolled cell growth and tumor progression. Thus, restoring the quantity and the activity of wild-type p53 (p53wt) by using inhibitors of hDM2-p53 interaction may constitute a potential therapeutic strategy for cancer treatment.³²⁻³³ Previously, ligands of hDM2 E3 ubiquitin ligase have also been proposed as components of proteolysis targeting chimera (PROTAC) for the degradation of hDM2³⁴⁻³⁵ and by extension as PROTAC degraders for other proteins of interest (POIs) involved in cancer.³⁶

The sequence of oligomer 1 consists of a peptide chain of seven residues prolonged by a short
oligourea segment at its C-terminus. Compound 1 was designed starting from PMI (TSFAE⁵YWNLL¹⁰SP),
a known high affinity peptide binder of hDM2 selected from phage display libraries,³⁷⁻³⁸ and replacing
the five C-terminal residues 8-12 by a triurea cap.²⁵ The capacity of oligomer 1 to disrupt the hDM2-
p53 interaction at low concentration (IC₅₀ = 24 ± 0.6 nM), similar to the cognate peptide³⁷ and to (-)-
Nutlin-3a³⁹ confirmed that a high degree of α-helix mimicry was achieved with the designed foldamer
insert. Herein, starting from the crystal structure of foldamer 1 bound to hDM2 (PDB ID: 6HFA, Fig. 1A),
which shows that 1 adopts a helical conformation spanning both peptide and oligourea segments, we
considered different macrocyclization schemes (Fig. 1B). These include i,i+3 and i,i+4 macrocycles at
the N-terminus of the peptide segment to promote helix nucleation,⁴⁰ as well as more central i,i+4 and
i,i+8 crosslinks between an α-amino acid side chain and a side chain in the oligourea segment. In this
work, we selected thiol bis-alkylation as cyclization chemistry because it enables both late stage
macrocyclization and the introduction of a wide array of crosslinks.¹²⁻¹³ Based on this approach, we
thus generated a small collection of stapled peptide-foldamer hybrids that we screened for inhibition
of p53-hDM2 and p53-hDM4 (also named hDMX) interactions in a biochemical assay. This led to the
identification of a series of stapled peptide-foldamer hybrid analogues of 1 with high p53-hDM2
interaction inhibitory potency, some of which demonstrated enhanced inhibition of the p53-hDM4
interaction. To further increase the therapeutic potential of these inhibitors, several sequences were
optimized for cellular uptake by modulating the formal charge from neutral to +4, and the resulting compounds were tested for their ability to reactivate the p53-dependent pathway. Noteworthy, our results demonstrate that neutral, or even negatively charged, *N*-acetylated macrocyclic foldamers effectively reactivate the p53 signaling pathway, while sequences bearing a formal charge between +2 and +4 exhibit a cell toxicity that is independent of p53 activation.

<figure><img src="image_2.png" /><figcaption>**Fig. 1.** (A) X-ray structure of peptide-oligourea hybrid **1** in complex with hDM2 (PDB: 6HFA) (left). The oligourea segment is shown in orange. Overlay of the structures of bound **1** and bound PMI (light blue, PDB: 3EQS) (right). (B) Representation of all five side-chain crosslinking schemes considered in this work and the related distances between the first carbon atoms of the side chains. The standard three or one letter code is used for α-residues, and a superscript “U” is added for the corresponding urea units (orange).</figcaption></figure>

## RESULTS AND DISCUSSION

**Synthesis.** A library of 24 stapled foldamers derived from **1** was designed by varying both the position of thiol containing residues (Cys and Cys<sup>U</sup>) in the sequence and the nature of the bis-electrophile (**a-h**, Fig. 2).
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /><figcaption>**Fig. 2. (A)** Sequences of linear peptide-oligourea hybrid **1** and cognate oligomers **2-6** prepared in this work incorporating thiol containing residues (Cys and Cys<sup>U</sup>) with different spacings for different macrocyclization schemes. **(B)** List of bis-electrophiles used in this work for thiol bis-alkylation.</figcaption></figure>

Macrocyclization was performed in solution by dithiol alkylation of linear precursors **2-6** at the late stage of the synthesis. The linear precursors derived from **1**, and incorporating either two cysteine residues to allow N-terminal macrocyclization (**2,3**) or a Cys residue and a Cys<sup>U</sup> residue (**4-6**), were synthesized on solid-support starting from a Rink Amide resin as previously described.<sup>25, 41</sup> The linear peptide-oligourea hybrids were obtained in good yield and purity after cleavage from the resin. For this reason, the linear foldamers were engaged in the macrocyclization reaction with the bis-electrophiles **a-h** without further purification and the reaction was conducted in a 1:1 (v/v) mixture of CH<sub>3</sub>CN and ammonium bicarbonate buffer (pH = 8, 0.1 M). Cyclic foldamers were obtained in yields ranging from 2-62% after final purification by C<sub>18</sub>-RP HPLC (Table S1). Two additional cyclic foldamers containing a disulfide bridge were obtained from sequences **2** and **3** following oxidation of the thiol groups.

**Primary Screening of the Library for Disruption of p53-hDM2 and p53-hDMX Interactions.** We first screened the library of 23 macrocyclized peptide-oligourea foldamers for their ability to inhibit the interaction between recombinant p53 and hDM2 (or hDMX) using a time resolved fluorescence energy transfer (TR-FRET) assay at two concentrations (100 nM and 1 µM). Most compounds already exhibit substantial hDM2 inhibition at 100 nM, hence only results at 100 nM are reported in Fig. 3 and supplementary Table S1.
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Primary screening of all macrocyclic peptide-foldamer hybrids at 100 nM for their capacity to inhibit (A) p53-hDM2 interaction and (B) p53-hDMX interaction in the TR-FRET assay. nd = not determined.</figcaption></figure>

Twelve sequences were found to give more than 50% inhibition of the p53-hDM2 interaction at 100 nM out of which six gave > 85% inhibition. The six sequences with the best inhibition profiles, namely **2b, 2c, 4b, 4e, 4g** and **5e**, correspond to peptide-oligourea hybrids with crosslinks between thiol side chains in *i*,*i*+4 positions. In the case of foldamers derived from sequence **2** bearing a staple at the *N*-terminus, short crosslinks (i.e. *n*-propyl and *trans*-butene) gave the best results whereas for sequences with a more central staple, longer crosslinks (*n*-hexyl and bis-amide) were found to give the best results. In contrast, only a fraction of the hDM2 binders (6 out of 12) was identified to inhibit more than 50% of the interaction between p53 and hDMX. This finding is however consistent with the lower ability of foldamer **1** to disrupt the interaction between p53 and hDMX (IC₅₀ = 82 nM ± 5), as compared to p53-hDM2. Noteworthy, one stapled compound (**4g**) displays a high capacity to inhibit both p53-hDM2 interaction (99% inhibition) and p53-hDMX interaction (91% inhibition) at 100 nM.

**Determination of IC₅₀ Values of the Best hDM2 Inhibitors.** We then determined the concentration-dependent effect of five of the most active peptides, namely **2b, 4b, 4e, 4g** and **5e** (Fig. 4 and Fig. S1-S16). Half-inhibition effective concentration (IC₅₀) values are reported in Table S1. Both *N*-terminal and central *i*,*i*+4 crosslinking strategies led to p53-hDM2 interaction inhibitors (IC₅₀ in the range 10-30 nM) that retain or slightly improve (i.e. **4g**, IC₅₀ = 10.1 ± 0.6 nM) the inhibitory activity of the cognate linear peptide-oligourea sequence **1** (IC₅₀ = 23.8 ± 0.6 nM). IC₅₀ determination for inhibition of the p53-hDMX
interaction confirmed that compound **4g** is also the most efficient binder to hDMX (IC₅₀ = 28 ± 1 nM) compared to **1** (IC₅₀ = 82 ± 5 nM).

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** (A, B) hDM2 and hDMX inhibition by cyclic peptide-oligourea foldamers **2b**, **4b**, **4e**, and **4g** as assessed by the TR-FRET assay. Concentration-response curves (9 points) were performed in quadruples (1, 2b, 4e) or duplicates (4b, 4g) and the data are presented as mean ± SD. (C) Molecular formulas of the representative high affinity binders **4e** and **4g** and IC₅₀ values (± SEM).</figcaption></figure>

**Initial optimization of the charge of compounds 4e and 4g to enhance cellular uptake.** Preliminary evaluation of **4e** and **4g** in a cellular viability assay at 100 µM revealed that the two compounds were not active (Fig. S17). The nature of the staple⁴² and the formal charge⁴³⁻⁴⁴ of the peptide are two parameters that can substantially influence the cell permeability and cellular activity of short helical peptides. In particular, it has been found that in many situations, sequences with a positive net charge (+1 to +7) tend to demonstrate higher cellular uptake compared to sequences with neutral or negative net charge.⁴³,⁴⁵ Since the net charge of the compounds in the **4** series is zero, we initially thought to increase the positive net charge of these foldamers first by substituting a neutral (Gln or Ala) or a positively charged residue (Arg) for Glu5 (**7-9**). However, these replacements led to a substantial loss
of binding to hDM2 and hDMX, as exemplified for the macrocycles **7e-9e** derived from **4e** (Fig. S12), and were not studied further. We next considered the introduction of one or more positively charged residues at the *N*-terminus of p53 mimetics. We first prepared two analogues of **1** in which either Thr1 was replaced by a Lys residue (**10**) or the *N*-terminus was extended by three Arg residues (**11**). Both modifications were found to be well tolerated with almost complete inhibition of the p53-hDM2 interaction at 100 nM (Fig. S19). To maximize the positive net charge of **4e** and **4g**, we next combined these two modifications and prepared compounds **12e** and **12g**, which thus have a formal charge of +4. Interestingly, the determination of IC₅₀ values revealed a more potent inhibition of p53-hDM2 and p53-hDMX interactions by both charge-optimized foldamers **12e** (IC₅₀ = 4.6 ± 0.4 nM (hDM2) and 22 ± 1 nM (hDMX)) and **12g** (IC₅₀ = 3.6 ± 0.2 nM (hDM2) and 10.3 ± 0.8 nM (hDMX)) in comparison to cognate **4e** and **4g** (Fig. 5).

We next evaluated the cell permeability of charge-optimized foldamers using the chloroalkane penetration assay (CAPA) based on the HaloTag system, developed by the Kritzer group.<sup>46-47</sup> Analogues of compounds **12e** and **12g** bearing the required chloroalkane tag (i.e. **13e** and **13g** in Fig. 20A) were synthesized and the assay was conducted on HeLa cells stably transfected with Halo-GFP-Mito (HGM). Both compounds were found to be cell permeable and to accumulate in the cytosol with measured CP<sub>50</sub> values (concentration at which 50% of the maximal cell penetration was achieved) of 1.9 ± 0.4 µM and 3.3 ± 0.6 µM respectively, suggesting a slightly better uptake of **13e** (Fig. S20B). The two macrocyclic foldamers essentially differ by the nature of their crosslink and our results suggests that the diamide crosslink may be less favorable for cell uptake compared to the hydrocarbon staple. This result is consistent with recent observation made by the Fairlie group<sup>42</sup> that macrocyclic peptides with hydrophilic linkers such as lactams translocate less readily into cells, notwithstanding their superiority for helix induction and high hDM2 affinity.
<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /><figcaption>**Fig. 5.** (A) Chemical structures of the compounds **12e** and **12g**. Concentration dependent inhibition of (B) p53-hDM2 interaction and (C) p53-hDMX interaction by cyclic peptide-oligourea foldamers **4e**, **4g**, **12e** and **12g** assessed by TR-FRET assay. Data are presented as mean ± SD (n = 2).</figcaption></figure>

**Determination of the Cellular Activity of 12e and 12g.** We next assessed the cellular activity of macrocyclic foldamers **12e** and **12g** by measuring their ability to reduce the viability of p53wt cancer cell lines overexpressing either hDM2 (HCT116 colorectal carcinoma) or hDMX (MCF-7 breast cancer cells) upon treatment with increasing concentrations of these compounds during 72 h. In this assay (Fig. 6A), the foldamer **12e** was found to manifest a strong effect at low micromolar concentration (IC₅₀ = 3.86 and 3.98 µM) in both cell lines. In contrast, compound **12g** was only poorly active even at high concentrations, despite its high potency in the TR-FRET assay (on both p53-hDM2 and p53-hDMX interactions) and similar charge distribution to **12e**. We also tested the compound **4e** to evaluate the importance of the positive net charge on the ability of **12e** to reduce the viability of HCT116 and MCF-7. The result, which shows no loss of viability of these cells, confirms the key contribution of the RRRK extension to the cellular activity of the macrocyclic foldamer **12e**. To investigate the mode of action of **12e**, we evaluated its effect on p53-mutant cancer cell lines SW480 as well as in HeLa cells, which express low levels of hDM2 (Fig. 6B), and tested its ability to restore the p53 pathway by western blot
(Fig. 6C) and Real-Time PCR (Fig. S21) analysis in comparison with the positive control Nutlin-3a. Surprisingly, western blot and Real-Time PCR analysis of HCT116 cells treated with compound **12e** did not show the expected dose-dependent increase of p53 protein levels, and RNA levels of targeted genes p21 and hDM2. Moreover, treatment of SW480 cells also led to the loss of cell viability. These results suggest that compound **12e**, despite its high affinity for the target and cell penetration capacity acts through a p53-independent pathway. Activity of **12e** in the LDH assay, to determine cytotoxicity, may suggest that cellular activity of **12e** derives from membrane disruption (Fig. 6D).

<figure><img src="image_11.png" /></figure>

**Fig. 6.** Compounds **12e** and **12g** are not inducing restauration of the wild-type p53 protein in cells and **12e** is cytotoxic. (A) Cell viability was measured by the MTS assay on human colon cancer cell line HCT116 expressing p53wt protein or human breast cancer cell line MCF-7 overexpressing hDMX with p53wt protein after 3 days of incubation with a range of concentrations from 0.1 to 40 µM of **12e** or **12g** or **4e** or Nutlin-3a. Data are presented as mean ± SD (n = 3) vs. untreated control cells. (B) Viability was measured by MTS experiment on the human colorectal cancer cell line SW480 expressing p53mut protein and the human cervical cancer cell line HeLa expressing lower levels of hDM2, after 3 days of incubation with a range of concentrations from 0.1 to 40 µM of **12e**. Data are presented as mean ± SD (n = 3) vs. untreated control cells. (C) HCT116 cells were incubated with 10 µM of **12e** or **12g** or Nutlin-3a for 6 h and the levels of p53wt protein were determined by western blotting. **12e** and **12g** are not inducing the restauration of the p53wt protein compared to the positive control Nutlin-3a. (D) Cytotoxicity was assessed by measuring LDH levels released from plasma membrane damaged HCT116 cells after 2 h of incubation. **12e** is damaging the cellular plasma membrane but **12g** is not cytotoxic. Data are presented as mean ± SD (n = 1) vs. lysed control cells.

**Neutral N-acetylated Macrocyclic Foldamer Inhibitors Enter Cells and Reactivate the p53-signaling Pathway.** We therefore decided to reconsider the design of our inhibitors at the stage of optimizing cellular uptake and to study new sequences with a reduced number of positive charges (formal charge
of +2) as well as neutral and negatively charged versions (Fig. 7). We also realized that when we conducted CAPA, all compounds were *N*-acylated with the chloroalkane tag, and we thus decided to consider *N*-acetylated derivatives during this optimization phase. Consequently, we prepared and assessed four new analogues of **4e** by introducing sequence variations at the *N*-terminus: **14e** and **15e** both having a formal charge of +2, and two acetylated versions **16e** (neutral net charge) and **17e** (negative net charge). The Leu-Thr N-terminal residues in **17e** correspond to those found in the sequence of pDI, a hDM2 inhibitor identified by phage display and of other potent peptide-based hDM2 inhibitors.<sup>15, 48</sup>

<figure><img src="image_12.png" /><figcaption>**Fig. 7.** Sequences of macrocyclic peptide-oligourea hybrids **14e-18e** derived from **4e** as well as linear derivatives **19** and **20**. The \* sign above Cys and Cys<sup>U</sup> residues indicates the presence of the *n*-hexyl crosslink between the two thiol side chains in *i*,*i*+4 positions.</figcaption></figure>

All compounds were found to bind hDM2 and hDMX in the same range as compound **4e** (see Table S1 for IC<sub>50</sub> values). We examined their effect on HCT116 cell viability and their ability to activate the p53 pathway. Compounds with a formal charge of +2 (**14e** and **15e**) exhibited lower toxicity compared to **12e**, but were not able to reactivate the p53 signaling pathway (Fig. S22). In contrast, western blotting of p53wt HCT116 cells with **16e** and **17e** resulted in a dose-dependent increase of p53 levels (Fig. 8A
and Fig. S23), similar to Nutlin-3a. In this assay, the compound **16e** was found to be slightly more potent than the compound **17e**, being able to increase p53 levels at doses as low as 0.5 µM. These effects were not observed in p53 mutant SW480 cancer cells (Fig. S24A). Activation of the p53 pathway by **16e** and **17e** in HCT116 cells was further supported by the dose-dependent induction of mRNA of target genes p21 and hDM2 (Fig. 8B), and loss of cell viability (Fig. 8C). Moreover, the compounds did not induce LDH release in the range of concentrations used in these assays (Fig. 24B), indicating the absence of a direct effect on cell membranes. Evaluation of **18e** (the chloroalkane tagged version of **16e**) in CAPA demonstrated effective cell-permeability and cytosolic localization (CP<sub>50</sub> = 0.51 ± 0.07 µM, Fig. 8D). These results are consistent with the conclusion made by the authors of two recent studies on the optimization of stapled peptide inhibitors of p53-hDM2 and hDMX interactions,<sup>15,49</sup> that neutral or negatively charged sequences yield superior cell potency and specificity without compromising cell membrane integrity compared to positively charged analogues. Interestingly, although macrocyclization with the hydrocarbon crosslink does not enhance the binding affinity of the cognate linear peptide-foldamer hybrid for hDM2 (see IC<sub>50</sub> of **16e** and **19**, Table S1), it was found to significantly improve cell permeability (see CAPA of **18e** and **20**, Fig. 8D).
<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

**Fig. 8.** Compounds **16e** and **17e** reactivate the p53 signaling pathway and induce loss of viability in wild-type p53 cell line but not in a mutant p53 cell line. (A) HCT116 cells were treated with a concentration range of 0.1 to 40 µM of **16e** or **17e** or 10 µM of Nutlin-3a for 6 h, and p53wt protein levels were assessed through western blotting (biological replicates are shown in SI). (B) HCT116 cells were treated with a concentration range of 0.1 to 10 µM of **16e** or **17e** and mRNA levels were determined by RT-qPCR. Compounds **16e** and **17e** induce a dose-dependent increase in the expression of p53 target genes, p21 and hDM2, expressed as a fold change compared to the control without treatment and normalized to GAPDH. Each data point represents a biological replicate, the error bar represents the SD of biological replicates (n = 3) and * represents p-values < 0.05. (C) Viability measurements using the MTS assay on various cell lines after 3 days of incubation with a concentration range from 0.1 to 40 µM of **16e** or **17e**, or Nutlin-3a. Results are expressed as mean ± SD (n = 3) vs. untreated control cells. HCT116 cells express p53wt protein, and SW480 cells express p53mut protein. (D) Cell-permeability of compound **18e**, the chloroalkane tagged version of **16e**; and of **20**, the linear analogue of **19**. CP₅₀ represent the concentration at which 50% cell penetration is observed. Data are presented as mean ± SD (n = 2) vs. untreated control cells.
CONCLUSION

Interfacing oligourea foldamers with peptides allows to reduce the peptide character of α-helical bioactive peptides, therefore potentiating their resistance to proteolytic degradation while preserving the initial recognition properties of the natural peptide for its biological target.²⁴⁻²⁶ Here, we demonstrate that such foldamer-based helix mimics can be further optimized in terms of binding to the POIs (hDM2 and hDMX) and cellular activity through the combination of complementary techniques, namely macrocyclization and careful charge optimization. Starting from a foldamer-based p53 mimic featuring a tri-urea insert at the C-terminus as well as its co-crystal structure with hDM2,²⁵ we considered several macrocyclization patterns involving either two amino acid side chains or the side chains of one amino acid and one urea monomer at different locations in the sequence. Using a TR-FRET assay, we identified several bis-thioether macrocycles, such as **4e** and **4g**, containing a central *i*,*i*+4 linker connecting peptide and urea segments that maintain high affinity for hDM2. Our initial efforts to enhance cellular uptake by increasing the formal charge to +4 led to compounds (**12e** and **12g**) displaying higher inhibition potency in the FRET assay against both hDM2 and hDMX. Intriguingly, only compound **12e** exhibited cellular activity at low µM concentration. However, western blot, Real-Time PCR and LDH assays unveiled a cytotoxic effect and a mechanism of action independent of p53. Subsequently, we found that *N*-acetylated and neutral foldamer sequences (e.g. **16e**) had the unique ability to reactivate the p53 pathway at low µM concentration in p53wt cancer cell lines. Overall, this study, in line with other reports on the design of peptide-based inhibitors of the p53-hDM2 interaction,⁵⁰⁻⁵² also highlights the need for caution when incorporating patches of cationic residues at the ends of some peptide-based hydrophobic sequences to target intracellular POIs. The approach reported here, uniting foldamer and macrocyclization chemistries, appears versatile enough to be readily extended to other targets, facilitating the generation of more effective foldamer-based disruptors of PPIs in the context of peptide lead optimization.
# EXPERIMENTAL SECTION

**Oligomer Synthesis.** All steps were performed under inert atmosphere and microwave irradiation on the Liberty Blue or Discover Bio system (CEM). The temperature was maintained by modulation of power and controlled with a fiber optic sensor. All compounds were obtained with a final purity > 95% by HPLC.

*General procedure A for the oligourea synthesis.* The Rink Amide resin (loading 0.51, 0.42 or 0.35 mmol/g) was swollen in DMF and the Fmoc protecting group was removed with 20% piperidine in DMF (3 mL) under MW irradiation at 70 °C, 30 W, 5 min, twice. To the resin were added the desired succinimidyl (2-azidoethyl)carbamate monomers (N₃-XaaU-Osu, 1.5 equiv.) and DIEA (3 equiv.) in 2 mL DMF under MW irradiation at 75 °C, 30 W, 20 min, twice. The introduction of first urea residue was difficult, 3 times coupling or prolonged coupling time was needed. The resin was filtered, washed with DMF and with a mixture of 1,4-dioxane/H₂O (7:3, v/v). The azide reduction was performed by adding PMe₃ (1 M in THF, 10 equiv.) in dioxane/H₂O (7:3, v/v) at 50 °C, 50 W, 15 min, twice, to the resin. The resin was filtered, washed with a mixture of 1,4-dioxane/H₂O (7:3, v/v, 1 × 2 mL) and DMF (4 × 2 mL). The coupling and reduction efficiencies were monitored with the chloranil test.

*General Procedure B for Peptide Synthesis.* To the resin were added Fmoc-Xaa-OH (5 equiv.), DIC (5 equiv.) and Oxyma (5 equiv.) in DMF at 75 °C, 70 W, 15 sec and 90 °C, 35 W, 110 sec. Except for Fmoc-Arg(Pbf)-OH, with DIC (5 equiv.) and Oxyma (5 equiv.) in DMF at 25 °C for 20 min followed by 75 °C, 5 min. Fmoc deprotection was done by adding 20% piperidine in DMF to the resin at 75 °C, 155 W, 15 sec and 90 °C, 35 W, 50 sec.

*General Procedure C for Cleavage.* When needed, the final acetylation was performed using a mixture of acetic anhydride (0.5 mL) and DIEA (10 equiv.) in 2 mL DMF at rt during 1 h. The linear sequence was cleaved from resin and side chain deprotected using TFA/TIS/H₂O/DODT (92.5:2.5:2.5:2.5, v/v/v/v) at rt for 2.5 h. The cleavage solution was evaporated and the residue was precipitated in cold ether. The
obtained precipitate was redissolved in H₂O (CH₃CN was added if needed) and lyophilized to give crude linear sequence which was directly used for stapling.

**General Procedure D for Cyclisation with Bis-electrophile Linker.** The crude linear sequence was dissolved in 1:1 (v/v) mixture of 0.1 M NH₄HCO₃ (pH = 8) and CH₃CN (0.5 mg/mL) and TCEP (3-5 equiv.) was added. The solution was stirred at rt for 10-30 min, followed by addition of the desired bis-electrophile linker (10 equiv.). The mixture was stirred at rt and the reaction was followed by HPLC and LC-MS until the disappearance of the starting oligomer. Then, the reaction was quenched with an aqueous solution of 5% TFA and lyophilized. The crude was purified by RP-HPLC using the appropriate gradient and lyophilized to afford the desired compounds.

**General Procedure E for Disulfide Bridge Cyclisation.** The linear peptide was dissolved in a DMSO/NH₄HCO₃ (pH 7-8 0.1 M) 1:10 v/v solution and stirred at rt during 4-24 h. The reaction was followed by HPLC and LC-MS until the disappearance of the starting oligomer. The crude was purified by RP-HPLC using appropriate gradient and lyophilized to afford the desired compounds.

**General procedure F for Chloroalkane Tag Coupling.** To the resin were added ct-COOH (2.5 equiv.), PyBOP (2.5 equiv.) and DIEA (5.0 equiv.) in DMF at rt during 2 h.

**HTRF Assay.** Evaluation of the inhibitory activity of compounds on p53-hDM2 interaction was determined by measuring the loss of interaction using a time resolved fluorescence energy transfer (TR-FRET) detection method (Hybrigenics Services). The concentration of the peptides and analogues was determined by dosing UV absorbance of the indole side chain (λ = 280 nm). hDM2-GST protein (Hybrigenics Services; GST-Flag-89v5; batch #6) and p53-His protein (Hybrigenics Services; YHX-His-1509v3; batch #7) were produced in *E. coli* and were purified by affinity columns. Fluorescence donor (anti-GST antibody labelled with europium cryptate -Cisbio; ref 61GSTKLA, batch 101A) was mixed with hDM2-GST protein in a reaction buffer (PBS pH 7.4, DTT 2.5 mM, Tween 0.075% + KF 400 mM). Fluorescence acceptor (anti-His antibody labelled with terbium cryptate; Cisbio; ref 61HISXLA; batch 61A) was mixed with p53-His protein in the same reaction buffer. Solutions of proteins/antibodies
were mixed in microplates at 25 nM (1:1 ratio) per well. The mixture was incubated overnight at 4 °C with reaction buffer (basal control), tested compounds or the reference inhibitor. A microplate reader PHERAstar (BMG) was used to measure the fluorescence transfer at λ<sub>ex</sub> = 337 nm and λ<sub>em</sub> = 620 nm and 665 nm. The ratio of the signal measured at 665 nm on the signal measured at 620 nm is used to determine the loss of p53-hDM2 interaction. The inhibitors have been tested at several concentrations to generate a concentration-response curve from which IC<sub>50</sub> value and SE are calculated using GraphPad Prism.

**Cell Culture.** Cell cultures were established for the human colon cancer cell lines p53wt HCT116 and p53mut SW480, using McCoy's 5A medium (Gibco) and DMEM (PAN BIOTECH), respectively. The human breast cancer cell line MCF-7 and the human cervical cancer cell line HeLa were cultured in EMEM (Gibco) and DMEM (PAN BIOTECH), respectively. Halo-GFP-Mito HeLa cells were cultured in DMEM (Gibco). All media were supplemented with 10% heat-inactivated FCS and 1% penicillin-streptomycin. The cells were maintained under fully humidified conditions at 37 °C with 5% CO<sub>2</sub>.

**MTS Cell Viability Assay.** Cells (3×10<sup>3</sup> cells/well for HCT116, SW480 and HeLa; 5×10<sup>3</sup> cells/well for MCF-7) were seeded in 96-well plates with the indicated inhibitors at various concentrations in serum-free media for 8 hours, followed by addition of serum (10%) and 64 hours of culture. Cell viability was assessed using the CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega), as previously described<sup>53</sup> and according to the manufacturer's instructions. At the end of the experiment, absorbance was measured at 490 nm using the Tecan Infinite 200 PRO (Life Sciences) plate reader. The percentage of viable cells was calculated by comparing treated cells to non-treated cells. The data were plotted using GraphPad Prism.

**Western blots.** Immunoblot analysis was conducted as previously described<sup>54</sup> with minor adjustments. HCT116 and SW480 cells in the exponential growth phase were seeded in 6-well plates one day before the experiment. Subsequently, they were exposed to specified concentrations for 6 hours without serum and then lysed using 100 µL of radioimmunoprecipitation assay (RIPA) buffer. Denaturing
polyacrylamide gel electrophoresis was employed, and the proteins were transferred onto nitrocellulose membranes using a wet transfer process. Following this, the membranes underwent a blocking step with 5% BSA in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) for 1 hour at room temperature. Primary antibodies, such as anti-p53 (Santa Cruz, sc-71817) and anti-GAPDH (ThermoScientific, MA5-15738), were applied overnight at 4 °C. After washing with TBS-T, the membranes were exposed to an anti-mouse infrared dye secondary antibody (LI-COR, 926-32212) at room temperature for 1.5 hours. The signal was detected using a LI-COR scanner.

**RNA Extraction and RT-qPCR.** Total RNA was isolated using TRIzol (MRC, TR 118), following the manufacturer's guidelines. Subsequently, RNA underwent reverse transcription using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368814) and T100 Thermal Cycler (Bio-Rad). This resulting cDNA was utilized for real-time PCR with specific primers and SYBR Green PCR Master Mix (Takyon, UF-RSMT-B0701), as previously described.⁵⁵ Quantitative PCR data were collected using the AriaMx Real-time PCR System (Agilent). Expression levels were normalized to GAPDH. The primers used in this study are p53 (Merck), hDM2 (Sigma-Aldrich), p21 (Merck) and GAPDH (Eurogentec).

**LDH Assay.** Lactate dehydrogenase (LDH) assay was carried out to quantify the release of LDH from damaged cells, using the CyQUANT LDH Cytotoxicity Assay Kit (Invitrogen, C20300). HCT116 cells were plated at a density of 6.5 × 10⁴ cells/well in 96-well plates 24 hours before the experiment, and compounds were introduced at specified concentrations in McCoy's 5A medium without serum. Some controls were added: wells with no cells were treated with the inhibitor, wells with cells were not treated with the inhibitor to measure maximum LDH release. Following a 1.25 hours incubation, lysis buffer was added to the "maximum LDH release" condition. Two hours after the experiment's initiation, 50 µL of supernatant for each well were transferred to a new plate. 50 µL of reaction mixture buffer were added to each well and the plate was incubated at room temperature, protected from light, for 30 minutes. Subsequently, 50 µL of stop solution were added, and the Optical Density (OD) was measured using a Tecan Infinite 200 PRO (Life Sciences) plate reader at 490 nm.
**CAPA.** The Halo-GFP-Mito cells used for CAPA are HeLa cells stably expressing a fusion of HaloTag, GFP, and a mitochondria-targeting peptide from the C-terminal domain of the ActA protein from *Listeria monocytogenes*.⁴⁶ This fusion anchors HaloTag to the outer mitochondrial membrane, oriented in the cytosol. Cell populations expressing HGM were isolated to obtain individual colonies. CAPA was performed as previously described with slight modifications.⁴⁷ The day before starting the experiment, Halo-GFP-Mito HeLa cells were seeded in a 96-well plate. To begin the experiment, media was aspirated and 100 µL of DMEM (Gibco) without serum was added. 25 µL of compounds were added at the desired concentration to the cells and were incubated at 37 °C for 4 hours. Supernatants were aspirated and cells were washed with Opti-MEM (Gibco) for 15 minutes. Opti-MEM was aspirated and 50 µL Opti-MEM with 5 µM ct-TAMRA dye was added to the cells for 15 minutes. The medium was aspirated and cells were washed again for 15 minutes with Opti-MEM without phenol red. This washing step was repeated a second time. The supernatant was aspirated, cells were trypsinized using Trypsin 0.05% - EDTA 0.1% solution without phenol red and resuspended in 150 µL ice-cold PBS(-Ca²⁺-Mg²⁺)/FCS 50%. 80 µL of cell suspension were typically analysed per well using BD LSRFortessa™ Cell Analyzer flow cytometer. Cells were gated, data were processed with FlowJo software and plotted using GraphPad Prism.

**Statistical Analysis.** The statistical analyses were performed using Prism software (GraphPad). The Student t-test was employed. A significance level of p-values < 0.05 was considered, and the experimental data were presented as the mean ± standard deviation of the mean.

**ASSOCIATED CONTENT**

**Supporting Information**

List of all Peptide-foldamer hybrid sequences and characterization; Primary screening of compounds **7-9** and **10-11** by TR-FRET; $IC_{50}$ (hDM2 and hDMX) determination by TR-FRET for compounds **1**, **2b-4b**, **4e**, **4g**, **5e**, **12e**, **12g**, **14e-17e**, and **19**; MTT assay on HCT116 cells with compounds **4e** and **4g** at 100
µM; CAPA of chloroalkane tagged compounds **13e** and **13g**; RT-qPCR of Nutlin 3a, **12e** and **12g**; Western blots, RT-qPCR, cell viability and LDH assay with compounds **14e** and **15e**; Western blot on HCT116 cells with compounds **16e** (biological replicate) and **17e**; Western blot on SW480 cell line and LDH assay with compounds **16e** and **17e**; ESI-MS spectra and RP-HPLC chromatograms for all compounds.

Molecular formula strings (CSV)

**AUTHOR INFORMATION**

**Corresponding Author Information**

Abdel-Majid Khatib, [majid.khatib@u-bordeaux.fr](mailto:majid.khatib@u-bordeaux.fr)

Gilles Guichard, [g.guichard@iecb.u-bordeaux.fr](mailto:g.guichard@iecb.u-bordeaux.fr)

**Present/Current Author Addresses**

†Present address: Université de Lyon, Université Lyon 1, CNRS UMR 5246 Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Faculté de Pharmacie-ISPB, 8 Avenue Rockefeller, FR-69373 Lyon Cedex 08, France.

**Author Contributions**

†M.N. and M.B. contributed equally.

M.N., B.L., M.B. and G.G. designed and synthesized stapled foldamer ligands. F.H. synthesized the protected Cys-derived monomer for incorporating Cys<sup>u</sup> units. M.B. and AM.K. designed and performed cellular assays and analyzed the results. L.V., M.B. and E.T. performed CAPA. M.P., E.T., AM.K. and G.G. conceived and supervised the project. M.N., M.B., M.P., S.R.G., AM.K. and G.G. analyzed the data. The manuscript was written through contributions of all authors. All authors read and approved the manuscript.

**Note**
The authors declare no competing interests.

## ACKNOWLEDGEMENTS

This work was funded in part by the ANR (ANR-15-CE07-0010), the Conseil Régional de Nouvelle-Aquitaine (2017-1R10115), and SIRIC Brio. A CIFRE support from IMMUPHARMA BIOTECH SAS and ANRT to M.N. and a doctoral scholarship from Univ. Bordeaux (Graduate Research School) to M.B. are gratefully acknowledged. We thank Prof. Joshua Kritzer for providing the plasmid encoding the viral vector for producing the HaloTag in mammalian cells. We thank Malo Le Chevalier for his contribution to the measurement of cell viability of 12e on MCF-7 cells. This work has benefited from the facilities and expertise of IECB Biophysical and Structural Chemistry platform (BPCS), CNRS UMS3033, Inserm US001, Univ. Bordeaux.

## ABBREVIATIONS USED

CAPA, chloroalkane penetration assay; DIC, N,N'-Diisopropylcarbodiimide; DODT, 3,6-dioxa-1,8-octanedithiol; HGM, Halo-GFP-Mito; LDH, Lactate dehydrogenase; PyBOP, benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate; RIPA, radioimmunoprecipitation assay; TIS, triisopropylsilane; VDR, vitamin D receptor;

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