Downsizing the Histone H3–H4 Quaternary Structure Into Foldamer Mimetics Yields High‐Affinity and Cell‐Permeable Ligands of ASF1

ABSTRACT

Mimicking complex protein–protein interfaces with small, well‐defined molecular scaffolds remains a major challenge in chemical biology. Here, we report a foldamer‐based downsizing strategy that compresses the quaternary architecture of the histone H3–H4 dimer into compact peptide‐oligourea hybrids acting as high‐affinity ligands of the histone chaperone Anti‐Silencing Function 1 (ASF1). Guided by multiple high‐resolution co‐crystal structures, we designed a series of foldamer mimetics that accurately reproduce both the H3 α‐helix and the H4 β‐strand epitopes. Systematic optimization of linker geometry, β‐strand mimicry, formal charge, and selective backbone N‐methylation yielded highly stable ligands with nanomolar affinities, enhanced proteolytic resistance, and robust cytosolic penetration. Notably, the optimized constructs and their N‐methylated analogues recapitulate the binding mode of the native H3–H4 dimer on ASF1 with high fidelity and engage endogenous ASF1 in cell extracts, demonstrating effective intracellular target recognition. Together, these results show that peptide‐oligourea foldamers can reproduce the structural features of a protein quaternary structure surface, combining high affinity, high stability and cell permeability.

We present a foldamer strategy to downsize the histone H3‐H4 quaternary structure into high‐affinity, medium‐sized binders (∼2 kDa) for the histone chaperone ASF1. Our peptide‐oligourea chimeras, designed through structure‐guided optimization and foldamer chemistry, recapitulate the natural H3‐H4 dimer binding mode while demonstrating plasma stability and cytosolic entry.

1. Introduction

Strategies utilizing peptidomimetic‐ and foldamer‐based methodologies to interfere with protein interactions generally focus on mimicking and/or stabilizing individual protein secondary structure elements, such as α‐helices or β‐sheet structures. Notably, stapled peptides, α‐helix mimetics, and helical foldamers have found widespread use as scaffolds to target protein‐protein interactions (PPIs) [1, 2, 3, 4, 5, 6, 7, 8, 9]. These methodologies are particularly effective for targeting clearly defined and relatively simple protein‐protein interfaces, where individual α‐helices are the central recognition elements. However, many protein interfaces exhibit greater complexity, spanning broader and less defined surface areas, requiring the replication of multiple structural elements to achieve tight binding [10]. It suggests the potential for larger foldamer and macrocyclic architectures comprising two or more secondary structure elements arranged in space [10, 11]. An advantage of non‐natural scaffolds is their likely enhanced stability against proteolytic degradation. A significant challenge related to the design of protein mimics, apart from the design itself, is the intracellular location of the target, requiring these large molecules to penetrate cells. This challenge necessitates conjugation to cell‐penetrating sequences or further engineering of the protein mimic to optimize overall charge and facilitate cell entry. These challenges contribute to the limited application of such strategies despite successful examples of such foldamer protein tertiary or quaternary structure mimics as modulators of protein‐protein or protein‐DNA/RNA interactions [12, 13, 14, 15, 16, 17].

Histone chaperones regulate the dynamics of histones within chromatin, and contribute to the establishment of numerous epigenetic markers. The two homologous human proteins ASF1A and ASF1B (named ASF1 in the following) are known to play a crucial role in histone handling coupled to DNA replication, DNA repair and gene expression regulation [18, 19]. ASF1B overexpression promotes tumor cell proliferation and is a good marker of poor prognosis in breast cancer [20, 21]. Moreover, siRNA‐mediated depletion of ASF1B markedly impairs breast cancer cell proliferation, demonstrating its functional role in this process [20]. It was proposed to be a marker of bad prognosis for about 20 other cancers [22], while ASF1A plays a role in cancer progression in hepatocellular carcinoma and adenocarcinoma [23, 24]. Inhibiting ASF1 function may be possible by targeting the shared histone H3‐H4 dimer binding interface of both proteins with competitive molecules, offering a promising strategy for the development of chemical probes and drugs. However, designing such inhibitors remains challenging due to several factors: the intracellular localization of ASF1, a binding affinity for the H3‐H4 dimer in the low nanomolar range, and a rather flat and extensive binding interface (1415 Å²) with two shallow interaction pockets located 40 Å apart, recognizing binding epitopes from both H3 and H4 (Figure 1a) [25, 26]. The C‐terminal helix of histone H3 binds a conserved concave face of ASF1, forming a buried surface area of 909 Ų. For histone H4, the direct interaction with ASF1 mainly comes from the C‐terminal residues 95‐100, which form an anti‐parallel β‐strand with ASF1, generating a buried surface area of 506 Å². Importantly, the side chain of residue F100 in H4 inserts into a hydrophobic pocket on the ASF1 surface, contributing nearly one‐third of the buried surface area of ASF1‐H4 interaction (Figure 1a). Remarkably, residues interacting with histones are 100% conserved between ASF1A and ASF1B. Previous studies using peptide inhibitors have validated the histone H3‐H4 binding interface as a druggable site, supporting ASF1 as an emerging epigenetic target in oncology and the development of complementary inhibitory scaffolds such as foldamers [27].

FIGURE 1.

(a) Structure of the complex between ASF1 and H3‐H4 dimer highlighting the two major epitopes derived from H3 and H4 (H3 in blue and H4 in green). (b) Scheme highlighting the foldamer strategy used in this study starting from a short H3 α‐helix mimic (Ch2) [29] to recapitulate the full H3‐H4 dimer. (c) Sequence of ip4 and Ch2 and general scheme of urea‐peptide chimeras; (d) chemical structures of the linker with different variations of the residue X9; (e) dissociation constants of urea‐peptide chimeras as measured by ITC. Dap stands for 1,3‐diaminopropane.

Thus far, peptidomimetic chemistry approaches to identify ASF1 ligands have largely focused on the H3 C‐terminal helix [28, 29, 30]. Recently, we used oligourea helical foldamers to reduce the peptidic character of PPI inhibitors [31, 32], and reported a rationally designed H3 helix mimetic (Ch2) that binds both ASF1A and ASF1B with a K _D in the low µM range while exhibiting improved stability in human plasma [29]. However, this early design underlines the need for additional strategies to enhance ASF1 binding affinity (Figure 1b). In this work, we demonstrate that we can expand our foldamer approach to generate cell‐permeable, medium‐size (M _w < 2.5 kDa) H3‐H4 quaternary structure mimetics with high affinity (K _D in the single‐digit nanomolar range) for ASF1. Starting from our previously described H3 helix mimetic and guided by the co‐crystal structure with ASF1, we have implemented the following workflow: (i) introduction and optimization of a linker to connect the H3 helix mimetic to the H4‐derived β‐strand; (ii) partial replacement of the native H4 sequence by a β‐strand mimic to further stabilize the bioactive conformation of the molecule and its stability in biological fluids; (iii) enhancing the cell permeability by optimizing the formal charge of the ligands, and evaluating target engagement. Throughout this process, we validated our design by careful biophysical investigation and high‐resolution structural characterization of the resulting ASF1 complexes. This workflow ensures that our foldamer‐based H3‐H4 mimetics not only exhibit high affinity for ASF1, but also possess favorable properties for cellular uptake and biological activity.

2. Results and Discussion

2.1. Foldamer Quaternary Structure Mimetics With Optimal Linker Display Enhanced ASF1 Binding Affinity

In earlier work, we used molecular modeling and iterative methods to discover ip4, an α‐peptide with high affinity for ASF1, in which H3 and H4 sequences were optimized and tethered by the GAGG α‐tetrapeptide linker (TPL) designed to optimize contacts with the protein surface (Figure 1c) [27]. Despite its good binding properties, this peptide displayed poor membrane permeability without conjugation to a cell‐penetrating peptide (CPP) and low resistance to proteolysis, thus limiting its practical utility. Drawing inspiration from this peptide, we initially employed TPL to tether Ch2 and the H4 C‐terminal β‐strand, and synthesized the first oligourea‐peptide chimera cr1 (See Table S1 and Figure S1). Surprisingly, isothermal titration calorimetry (ITC) revealed no substantial enhancement in binding affinity (K _D =  1.16 µM for cr1 vs. 2.70 µM for Ch2), despite a 100‐fold improvement observed upon tethering in the case of ip4 (Table S2 and Figure S2) [27, 29]. This result may be explained by the fact that the position of the residue forming the amide bond with the linker in Ch2 (i.e., A9) is slightly offset (by 1.9 Å) compared to the equivalent residue (i.e., A15) in ip4, indicating that the optimal linker for ip4 may not be suitable for the chimera sequence (Figure S3). To enhance the connection between the two epitopes, we increased linker flexibility by reducing the number of amide bonds, while preserving the total number of main‐chain atoms. The new aliphatic linker ‐5Ava‐Dap^U‐ (AL) also contains a urea bond to reinforce possible H‐bond formation with the surface of ASF1 and create a three‐centered H‐bond with the main‐chain CO of Arg148. This linker design, which reduces the number of amide bonds of the final molecule, may also contribute to enhancing resistance to proteolytic degradation as well as cell permeability. To explore a larger connecting space, we replaced the α‐residue A9 with diversely homologated derivatives of increasing size and prepared five new chimeras cr2‐cr6 (Figures 1d and 1e, Table S1, Figure S1). Notably, compound cr5 with a γ⁴Ala in position 9 and the ‐5Ava‐Dap^U‐ linker exhibited a K _D of 0.14 µmol, corresponding to a 20‐fold increase in affinity compared to Ch2 (Figure 1e, Table S2, Figure S2). We succeeded in growing co‐crystals and solved the crystal structure of the H3‐H4 protein mimetic cr5 in complex with ASF1 at 1.6 Å resolution (Table S3). The extended oligourea‐peptide cr5 recapitulates the binding mode of the H3‐H4 dimer effectively, with complete occupation of the two binding sites at the surface of ASF1 (Figure 2a and Figure S4a). The terminal helix is not affected by the molecule extension and superimposes well with that of Ch2 at the linker junction (I8 and A9) (Figure S4b). As with Ch2, a water molecule is inserted in the urea H‐bond network, forming a water bridge with Tyr112 of ASF1 and the carbonyl of L^U5 (Figure 2a). Note that in the linker region, the two urea NHs of Dap^U form a hydrogen bond with the CO of ASF1 Arg148, as anticipated (Figure 2b). The C‐terminal residues of cr5 from V12 to F17 form the expected β‐strand, and the side chain of F17 is buried into the desired hydrophobic pocket.

FIGURE 2.

(a) Crystal structure of cr5 in complex with ASF1: cr5 is represented as sticks using the color code indicated in Figure 1, with ASF1 as a surface (white). A selection of water molecules making direct contact with cr5 is represented as spheres. The positively charged residues in cr5 are labeled with a blue background, and the negatively charged residues with a red background. (b) Details of representative H‐bond interactions between the linker cr5 and ASF1. (c) Formal charges and K_D values as measured by ITC for compounds cr5, cr7, and cr8; d) Co‐crystal structure of cr7 with ASF1 and details showing how the Arg in position 1 of cr7 interacts with negatively charged residues of ASF1 through a water relay.

2.2. Formal Charge Modification to Improve Cell Permeability

Among strategies to improve the membrane permeability of bioactive peptides, CPPs can be conjugated to their N‐terminus or C‐terminus. However, this comes at the expense of both increased molecular weight and cost of production. An alternative solution involves introducing mutations in the bioactive sequence using cationic residues, such as arginine or lysine, to increase the net positive charge and potentially the cellular uptake [33]. Due to its side chain composition, cr5 diplays a net charge of 0 (Figure 2a). The crystal structure of cr5 with ASF1 guided the design of two new hybrid sequences, cr7 and cr8, exhibiting a positive net charge of +4 and +5, respectively. We substituted residue E1, whose side chain points towards a patch of carboxylate residues, for a cationic residue, aiming to form additional electrostatic interactions with proximal ASF1 residues. The C‐terminal carboxylate of G18, which does not make any specific interactions with ASF1, was replaced by its corresponding carboxamide. In addition, the neutral solvent‐facing residue Q^U6 was mutated to K^U. Remarkably, with a K _D of 38 nM, the corresponding sequence cr7 (+4) showed a 3.5‐fold gain in binding affinity (Figures 2c, S2, and Table S2). We also mutated the residue V12 on the β‐strand region to R in cr8. However, this replacement caused a substantial drop in affinity, suggesting that V12 contributes to the stability of the desired β‐strand structure.

To better understand the binding affinity gain resulting from charge optimization, cr7 was co‐crystallized with ASF1, and the crystal structure of the complex was solved at 2.0Å (Table S3). As shown in Figure S5a, the bound conformations of cr7 and cr5 are almost identical, with very good superimposition of the main chains and side chains, except for the N‐terminal residue. Interestingly, the R1 guanidinium moiety of cr7 is now hydrogen‐bonded to Glu49 and Asp88 through a water bridge, which may positively influence the ASF1/cr7 interaction (Figure 2d).

2.3. H4 β‐Strand Mimicry and Backbone N‐Methylation

We next investigated how to reduce the peptidic character of the H4‐derived β‐strand in cr7, in order to further enhance the resistance of the proteinomimetics to proteolytic degradation (Figure 3a). When examining the structure of cr7 in its bound state, we identified the D15‐G16 pair for replacement by a simplified dipeptide mimetic such as Dap^U. In fact, the side chain of D15 is not involved in any contacts with the ASF1 surface. One potential advantage of this modification is that removing D15 would also increase the overall positive charge of the molecule. Moreover, it would simplify the chemical synthesis of chimeras by reducing the risk of aspartimide formation [34, 35]. We also thought about combining this modification with the deletion of the C‐terminal Gly residue, which makes no contact with the protein surface. Gratifyingly, the new compound (cr9) was found to bind ASF1 with the same affinity as cr7 (Table S2 and Figure S2).

FIGURE 3.

(a) H4 epitope simplification and optimization strategy with binding affinities as measured by ITC; (b) Co‐crystal structure of cr17 with ASF1 and details of the interaction between the C‐terminal H4 mimetic and ASF1. For the chimeras in which Asp‐Gly is replaced by Dap^U, the terminal Phe residue is renumbered and referred to as F16.

We then considered modifying the sequence of cr9 by replacing the side chains of flanking residues L14 and F16 to enhance its affinity for ASF1. Initially, we simplified L14 to Gly (cr10), but this change nearly halved the binding affinity. The observed decrease in binding affinity may be related to the loss of a chiral center at this position, which may affect the ligand pre‐organization and binding. To address this problem, we prepared additional sequences (cr11‐cr13) with different substitutions at L14, including branched residues like Val and Thr, which could further stabilize the strand conformation. Notably, the Leu to Thr replacement resulted in a substantial increase in affinity, with cr13 exhibiting a K _D of 10 nM.

The benzyl side chain of Phe is buried deep within a hydrophobic pocket of ASF1, contributing to the binding of the H4 epitope to ASF1 significantly. Upon examining the crystal structures of the ligand‐ASF1 complexes, it became apparent that the bottom of the pocket is not fully occupied. Therefore, adding a well‐positioned substituent on the phenyl ring could enhance the binding affinity. Whereas ligands incorporating a 4‐/fluorophenylalanine (cr14 and cr15) were less effective than their unsubstituted counterparts, introduction of the 3‐fluoro derivative improved the binding affinity, as exemplified by compounds cr16 (K _D = 22 nM) and cr17 (K _D = 6 nM), which were derived from cr7 and cr13, respectively (Table S2 and Figure S2).

We solved the crystal structure of cr13 and cr17 in complex with ASF1 at a resolution of 1.65 Å and 1.70 Å, respectively (Table S3). These two additional structures confirmed our hypotheses and demonstrated how all four modifications (DG to Dap^U15 replacement, C‐terminal Gly deletion, L14T substitution, and 3‐fluorinated F16) introduced in the H4‐derived epitope contribute to the interaction with ASF1(Figures 3b and S5b). Moreover, they highlight the role of the T14 hydroxyl group, which forms hydrogen bonds with ASF1 Val146 and Arg148 residues, thereby further stabilizing the interaction between the peptidomimetic and ASF1. Notably, cr17, with a molecular weight of 2189 Da, fully recapitulates the binding efficiency and mode of the histone H3‐H4 heterodimer. It is currently the most potent binder to ASF1 identified so far, with a K _D in the single‐digit nM range.

N‐methylation of the amide backbone is another strategy to enhance the cell permeability of peptides [36, 37, 38] and their stability in biological fluids [39]. Based on the crystal structures of ASF1 in complex with our foldamer ligands, we identified four backbone NH amides oriented towards the solvent that could be considered for N‐methylation: R1, 5Ava10, T13, and F16 (Figure S6). The N‐methylation scan was first carried out on cr10. N‐methylated derivatives were synthesized using commercially available N‐methylated amino acids or by direct N‐methylation on resin [40]. As a result, we successfully obtained three methylated sequences (cr18‐c20). However, the coupling of Dap^U to the N‐methylated F16 residue produced a trisubstituted urea group that proved prone to cyclization during trifluoroacetic acid (TFA) cleavage from solid support, yielding a hydantoin derivative [41]. It is also worth noting that upon N‐methylation of R1, the recovered crude product following TFA cleavage always contained a fragment resulting from the scission of Ac‐N‐Me‐Arg‐OH, as confirmed by high‐performance liquid chromatography (HPLC) and liquid chromatography‐mass Spectrometry (LC‐MS) analysis [42, 43]. ITC measurements (Tables 1, S2, and Figure S2) revealed that only N‐methylation of 5‐Ava10 (cr19) was tolerated, with cr19 retaining the binding affinity of its non‐methylated counterpart. Based on this observation, we synthesized the 5‐Ava10 N‐methylated versions of cr9 and cr17 (cr21 and cr22, respectively). Overall, the binding affinity to ASF1 was only slightly affected by N‐methylation, and cr22 emerged as the strongest binder in this subset, with a K _D of 27 nM.

TABLE 1.

K_D values of N‐methylated sequences cr18‐cr22.

	N‐Methylation	K D (nM)
cr18 a	N‐Me‐R1	146 ± 16
cr19 a	N‐Me‐5Ava10	59 ± 5
cr20 a	N‐Me‐T13	531 ± 122
cr21 b	N‐Me‐5Ava10	38 ± 6
cr22 c	N‐Me‐5Ava10	27 ± 3

^a cr18‐cr20 are cr10 analogs.

^b cr21 is a cr9 analog.

^c cr22 is a cr17 analog.

2.4. H3‐H4 Mimetics Show Enhanced Stability in Human Plasma

We evaluated the proteolytic stability of four H3‐H4 mimetics in human plasma. While the reference peptide ip4 showed rapid degradation, with only 10% remaining after 6 h of incubation, the foldamer‐based ligands retained about 80% of integrity. Remarkably, even after 24 h of incubation, more than 50% of cr17 and cr22 remained intact in plasma solution, suggesting that these two sequences have excellent resistance to enzymatic degradation (Figure 4).

FIGURE 4.

Percentage of remaining intact peptide after incubation in human plasma.

2.5. H3‐H4 Mimetics Show Cell Permeability and Cytosolic Localization

Next, we evaluated the ability of ASF1 foldamer ligands to translocate into cells. We prepared a series of six fluorescein isothiocyanate (FITC)‐labeled ligands corresponding to the key steps of our design strategy: F‐cr5, F‐cr7, F‐cr9, F‐cr17, F‐cr21, and F‐cr22 (see Table S1 and Supporting Information). U2OS cells were incubated with these compounds at a final concentration of 10 µM for 4 h. The fluorescence of individual cells was quantified by confocal microscopy and corrected for background by subtracting the intrinsic fluorescence of untreated cells (Figures 5a and S7). The neutral compound F‐cr5 exhibited very low cellular uptake. In contrast, the closely related cationic compounds F‐cr7 and F‐cr9 (net charge +4) showed approximately 10‐fold higher fluorescence, underscoring the importance of positive charge for efficient internalization. No significant difference in cellular fluorescence was observed between F‐cr7 and F‐cr9, suggesting that reducing the peptidic character of the H4‐derived β‐strand does not impair uptake. The incorporation of a fluorine atom on F16 (F‐cr17) resulted in a ∼2‐fold increase in fluorescence compared to F‐cr9, pointing to a possible role for increased hydrophobicity. Finally, N‐methylation of the 5‐Ava10 group led to a further enhancement of cell penetration, with uptake increasing by 1.5‐ to 4‐fold when comparing F‐cr17 to F‐cr22 and F‐cr9 to F‐cr21, respectively, thus highlighting the contribution of backbone modifications to membrane permeability.

FIGURE 5.

Cell penetration assessment of foldamers (a) left panel: scheme of the experiment conducted to evaluate cell penetration of FITC‐labeled foldamers by confocal microscopy. Right panel: quantification of the cell fluorescence for the 6 compounds. The fluorescence for each individual cell, corrected by the mean fluorescence background of untreated cells, is plotted as dots, the mean value as bars. 1 way ANOVA multiple comparisons were performed. *** show adjusted p‐value <0.0001, NS: non‐significant (n = 3). (b) Left panel: scheme of the CAPA assay. Right panel: chloroalkane‐tagged versions of cr17 and cr22, ct‐cr17 and ct‐cr22 tested by CAPA are cell‐permeable. CP₅₀: the concentration at which 50% of cell penetration is observed (n = 3).

To further confirm these findings and quantify cytosolic penetration for the best‐performing ligands, we evaluated the permeability of the charge‐optimized and N‐methylated foldamers cr17 and cr22 using the chloroalkane penetration assay (CAPA) developed by the Kritzer group, based on the HaloTag system [44]. Analogues of compounds cr17 and cr22 bearing the required chloroalkane tag (i.e., ct‐cr17 and ct‐cr22) were synthesized (see Table S1 and Supporting Information), 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 (Figure 5b) with measured CP₅₀ values (concentration at which 50% of the maximal cytosolic penetration was achieved) of 2.5 and 0.2 µM, respectively. The CP₅₀ of ct‐cr22 compared favorably with that of ct‐Trp, a reference molecule known to enter readily into the cells [44]. This result confirms the added benefit of the single N‐methylation of the amide backbone in ct‐cr22.

Confocal microscopy experiments (Figure S7) also confirmed a nuclear localization of F‐cr17, F‐cr21, and F‐cr22. These images show that the compounds are distributed relatively homogeneously throughout the cells, with a strong and clearly visible fluorescence signal in both compartments. This result is consistent with previous reports, including using CAPA, showing that peptides of the size of our ASF1 ligands may diffuse passively into the nucleus through the nuclear pore complex (NPC) [44]. The cytoplasmic and nuclear localization of the foldamers thus matches that of ASF1, which is actually located in both the cytoplasm and nucleus. In the nucleus, ASF1 acts as a histone H3‐H4 chaperone, facilitating histone deposition during DNA replication and repair, while in the cytoplasm, it binds newly synthesized histones to prevent aggregation and promote their transport to sites of chromatin assembly. Thus, the foldamers may potentially act on ASF1 in either one or both compartments.

2.6. H3‐H4 Mimetics Are Able to Engage ASF1 in Cell Lysates

We next tested the compound's ability to interact with ASF1 in cell extracts. We synthesized a biotinylated variant of cr17 (Bt‐cr17) and a scrambled sequence as a negative control (Bt‐sfl‐cr17) ‒ which displayed no ASF1 binding by ITC (Table S2 and Figure S2). After incubation with cell extracts, compounds were captured on Strep‐Tactin beads, analyzed and quantified by immunoblotting for ASF1A and ASF1B (Figures 6a and S8). Bt‐ip4 served as a positive control, pulling down both ASF1 isoforms. Bt‐cr17 behaved like Bt‐ip4, immunoprecipitating both ASF1A and ASF1B efficiently. In contrast, the scrambled foldamer failed to retain either protein. Thus, the modified, protease‐resistant compounds maintain their ability to engage ASF1 in a cellular extract.

FIGURE 6.

Functional assessment of foldamers (a) Left panel: Pull‐down of biotinylated peptides Bt‐ip4 and foldamers Bt‐cr17 and Bt‐Sfl‐cr17 from U2OS total cell extracts on streptavidin‐functionalized sepharose beads. Proteins were revealed with a mix of anti‐ASF1A and ASF1B antibodies. Ctrl corresponds to no peptide. Two volumes of proteins eluted with an excess of biotin were analyzed (10 and 15 µL, respectively): see materials and methods for more details. Experiment was conducted in triplicate. Right panel: Western Blot quantification. Fold enrichment was calculated by the band intensity of the IP (1,5X) divided by the band intensity of the input. (b) Left panel: scheme of the cell death assay. Right panel: Cell death (% of PI positive cells) for U2OS and Hs578T cell lines. 1 way ANOVA multiple comparisons were performed. **** show adjusted p‐value < 0,0001, ***p < 0.001, **p < 0005. NS: non‐significative.

2.7. H3‐H4 Mimetics Reduce Cell Proliferation

Finally, we assessed whether these foldamers could induce cell death in U2OS cells and triple‐negative breast cancer Hs578T cells. They were added to the culture medium, and propidium iodide (PI) incorporation was quantified by fluorescence microscopy after 48 h of cell growth. Results obtained for U2OS cells treated with cr5, cr7, cr9, cr17, cr21, cr22, and scrambled controls (sfl‐cr17, sfl‐cr22) were compared to untreated cells and are presented in Figure 6b. In Hs578T cells, the analysis was restricted to cr17, cr22, and their respective scrambled controls, sfl‐cr17 and sfl‐cr22. As expected, the scrambled controls, which do not bind ASF1 (Table S2 and Figure S2), displayed no significant cell mortality in either cell line. Only cr22 significantly increased PI‐positive cells compared to untreated and sfl‐cr22‐treated conditions. No significant effects were observed for the other compounds in both U2OS and Hs578T cells. These results indicate that cr22, combining protease resistance, enhanced cell penetration, and strong ASF1 affinity, induces specific cellular effects in both osteosarcoma and triple‐negative breast cancer cells. However, the observed cytotoxic effects remain relatively modest in both cell models.

3. Conclusion

In conclusion, we have developed an effective strategy using biotic foldamers to mimic and streamline the quaternary structure of the histone H3‐H4 dimer. This approach led to the discovery of medium‐sized, high‐affinity binders for the histone chaperone ASF1. The peptide‐oligourea chimeric ligands we report have a molecular weight slightly above two kDa, yet they effectively recapitulate the binding mode of the natural protein dimer on this flat, extended protein surface. Through a combination of structure‐guided design, foldamer chemistry, careful amino acid substitutions for formal positive charge optimization, and backbone N‐methylation, we identified high‐affinity binders that effectively enter the cytosol and show promising stability in human plasma. Notably, both cr17 and cr22 demonstrate remarkably high affinity for ASF1 in vitro and show significant cell permeability and cytosolic localization. Oligomer cr17 engages cellular ASF1 similarly to the previously designed high‐affinity peptide ip4. Finally, cr22 exhibits significant, although modest, cellular toxicity in two cancer cell lines.

Overall, our findings show that the strategic incorporation of urea residues at selected positions within a peptide sequence can preserve essential structural features, mimic dipeptide motifs, and stabilize peptide epitopes against proteolysis. This strategy enables the design of stable, low‐molecular weight linear peptidomimetics capable of engaging difficult protein–protein interfaces. Applied to ASF1, such molecules provide powerful tools to interrogate its function and suggest new directions for therapeutic development in chromatin biology.

Author Contributions

Bo Li: methodology, writing – original draft, investigation, visualization. Marie E. Perrin: investigation, methodology, validation, writing – original draft, writing – review and editing, formal analysis, visualization. Emma Maillard: methodology, investigation, validation, writing – review and editing, formal analysis. Arthur Vitard: methodology, investigation, validation, writing – review and editing. Gwenaëlle Moal: methodology, investigation, formal analysis. Johanne Mbianda: methodology, investigation. Christophe André: methodology, investigation. Magda Teixeira Nunes: methodology, investigation. Marie Vandamme: methodology, investigation, formal analysis. Guillaume Pinna: methodology, investigation, conceptualization, supervision, formal analysis. Céline Douat: investigation, methodology, conceptualization, supervision, writing – review and editing. Emmanuelle Thinon: methodology, investigation, writing – review and editing, validation. Raphaël Guerois: investigation, methodology, conceptualization, writing – review and editing, visualization. Pierre Legrand: conceptualization, investigation, methodology, validation, software, supervision. Gilles Guichard: conceptualization, investigation, funding acquisition, writing – original draft, writing – review and editing, validation, methodology, supervision, project administration, visualization. Francoise Ochsenbein: writing – original draft, funding acquisition, investigation, conceptualization, validation, methodology, writing – review and editing, supervision, project administration, visualization.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: anie74158‐sup‐0001‐SuppMat.pdf.

Data Availability Statement

The structural data that support the findings of this study are openly available in RCSB Protein Data Bank (RCSB PDB) at https://www.rcsb.org/, reference numbers 9SVO, 9SS3, 9TRB, 9SQK.