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. 2025 Sep 5;58(18):2840–2851. doi: 10.1021/acs.accounts.5c00441

Stabilization of Native Protein–Protein Interactions with Molecular Glues: A 14-3‑3 Case Study

Markella Konstantinidou 1, Johanna M Virta 1, Michelle R Arkin 1,*
PMCID: PMC12444995  PMID: 40910885

Conspectus

Protein–protein interactions (PPIs) play a key role in homeostasis and are often dysregulated in disease. PPIs were traditionally considered “undruggable” due to their flat surfaces and disordered domains. Recently, the identification of PPI stabilizers, or molecular glues (MGs), compounds that bind cooperatively to PPI interfaces, has provided a new direction for the field. MGs offer exciting opportunities for chemical biology and drug discovery, particularly for intrinsically disordered domains. To date, many of the fascinating MGs were discovered serendipitously, and their molecular glue mechanism of action was understood retrospectively. Our collaborative contribution has been the development of systematic, rational approaches for the identification, optimization, and validation of MGs.

This Account focuses on the modulation of the native PPIs between the hub protein 14-3-3 and its client proteins. 14-3-3 recognizes specific phospho-serine/threonine motifs on disordered domains of hundreds of clients and, depending on the phospho site, can activate or inhibit signaling pathways. Until recently, only the natural product fusicoccin A and its analogs were known to bind at the structured 14-3-3/client interfaces and modulate cellular pathways. The complexity of the natural products significantly hindered chemical biology approaches and did not provide sufficient insight into the systematic, selective targeting of the client of interest.

Inspired by the natural products, we used fragment-based screens to identify new chemical matter for 14-3-3/client PPIs. Using disulfide-tethering technology, we targeted either engineered cysteines on 14-3-3 or the native cysteine (C38) on 14-3-3σ. Five clients (ERα, C-RAF, FOXO1, USP8, and SOS1), representing varying sequences, binding modes, and physiological roles, were included in the initial screens. We identified both selective and nonselective fragments suitable for medicinal chemistry optimization.

Starting from a fragment that stabilized two 14-3-3 clients, estrogen receptor α (ERα) and C-RAF, we developed cell-active MGs selective for ERα. ERα is a well-validated target in breast cancer, and 14-3-3 is a negative regulator that blocks ERα transcriptional activity. We used structure-guided design to optimize ligand–protein interactions at the composite PPI surface. The molecular glues were validated in biophysical assays, including intact mass spectrometry (MS) and fluorescence anisotropy (FA) assays, allowing the quantification of binding, kinetics, and cooperativity.

We explored alternative strategies for the identification and optimization of MGs. For the 14-3-3/ERα complex, we demonstrated fragment linking to generate non-covalent stabilizers and a scaffold-hopping approach using multicomponent reaction chemistry. For the 14-3-3/C-RAF complex, we used a fragment-merging approach to selectively stabilize the inhibited state of C-RAF. Binding of 14-3-3 to the inhibitory phospho-S259 site prevents C-RAF dimerization and activation, offering an alternative mechanism to block the MAPK pathway. Finally, we validated compounds in cells using pathway-specific assays and a series of proximity-based NanoBRET assays to measure cellular PPIs.

These approaches led to first-in-class MGs for the 14-3-3/ERα and 14-3-3/C-RAF targets. Overall, we have developed a systematic platform for the identification of molecular glues for native PPIs applicable to the broad 14-3-3 interactome and beyond.

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Key References

  • Kenanova, D. N. ; Visser, E. J. ; Virta, J. M. ; Sijbesma, E. ; Centorrino, F. ; Vickery, H. R. ; Zhong, M. ; Neitz, R. J. ; Brunsveld, L. ; Ottmann, C. ; Arkin, M. R. . A Systematic Approach to the Discovery of Protein–Protein Interaction Stabilizers. ACS Cent. Sci. 2023, 9 (5), 937–946 . This work introduced a systematic fragment-based screening approach. Five 14-3-3-client-derived peptides were screened with mass spectrometry. Selective and nonselective fragment hits were identified and validated with mass spectrometry, fluorescence anisotropy, and crystallography. The workflow is readily applicable to protein–protein interactions.

  • Konstantinidou, M. ; Visser, E. J. ; Vandenboorn, E. ; Chen, S. ; Jaishankar, P. ; Overmans, M. ; Dutta, S. ; Neitz, R. J. ; Renslo, A. R. ; Ottmann, C. ; Brunsveld, L. ; Arkin, M. R. . Structure-Based Optimization of Covalent, Small-Molecule Stabilizers of the 14-3-3σ/ERα Protein–Protein Interaction from Nonselective Fragments. J. Am. Chem. Soc. 2023, 145 (37), 20328–20343 . This article described the rational structure-guided medicinal chemistry optimization of a nonselective fragment hit toward cell active 14-3-3/ERα stabilizers. Structure–activity relationships were established with biophysical assays. This work showcased first-in-class covalent 14-3-3/client stabilizers.

  • Konstantinidou, M. ; Zingiridis, M. ; Pennings, M. A. M. ; Fragkiadakis, M. ; Virta, J. M. ; Revalde, J. L. ; Visser, E. J. ; Ottmann, C. ; Brunsveld, L. ; Neochoritis, C. G. ; Arkin, M. R. . Scaffold-Hopping for Molecular Glues Targeting the 14-3-3/ERα Complex. Nat. Commun. 2025, 16 (1), 6467 . This work showed a computational scaffold-hopping approach for novel molecular glues stabilizing the 14-3-3/ERα complex. The scaffold was based on multicomponent reaction chemistry, which allowed rapid development of structure–activity relationships.

  • Vickery, H. R. ; Virta, J. M. ; Konstantinidou, M. ; Arkin, M. R. . Development of a NanoBRET Assay for Evaluation of 14-3-3σ Molecular Glues. SLAS Discovery 2024, 29 (5), 100165 . This article described the development and optimization of a proximity cell-based assay for the validation of 14-3-3/client molecular glues. In contrast to the biophysical assays that utilized phospho-peptides that mimicked the 14-3-3 client, here we used full-length proteins in living cells.

Introduction

Protein–protein interactions (PPIs) play a fundamental role in maintaining homeostasis and controlling multiple cellular processes under normal conditions. , The dysregulation of native PPI networks through mutations, altered post-translational modifications, or disrupted signaling leads to various diseases from malignancies to developmental disorders. , From a drug discovery perspective, the typically flat, large, hydrophobic surfaces of PPIs and the presence of intrinsically disordered domains led to the common characterization of these systems as “undruggable”. , In the past 25 years, there have been beautiful examples of PPI inhibition, often taking advantage of transient or “cryptic” pockets in otherwise shallow sites. By contrast, PPIs formed from intrinsically disordered proteins have been harder to address. This situation has begun to change with the discovery of molecular glues (MGs), compounds that bind cooperatively at PPI interfaces thatas individual proteinslacked well-defined pockets. ,

MGs include various types of compounds ranging from natural products to small molecules, with the CRBN E3 ligase modulatory drugs (CELMoDs) − being one of the most famous examples. Most of these fascinating compounds were discovered serendipitously, and the understanding of their mechanism of action was retrospective. The challenge of how to rationally discover MGs for a PPI of interest remains a fundamental goal for advancing the field. To date, very few examples have been reported for the prospective discovery of MGs, including stabilization of β-catenin and its cognate E3 ligase SCFβ‑TrCP, the development of FKBP12-mTOR MGs, and the tricomplex CYPA:KRASG12C glues based on the chemical remodeling of cyclophilin A (CYPA).

To address this issue, we have developed systematic approaches for the discovery and optimization of MGs. Our contributions are particularly focused on stabilizing native PPIs. Our main focus is on the hub protein 14-3-3, a scaffolding protein that recognizes specific phospho-S/T motifs on disordered domains of its clients. The interactome of 14-3-3 includes hundreds of clients involved in many signaling pathways, thus offering exciting opportunities for chemical biology and drug discovery. To date, many 14-3-3 clients are still considered “undruggable” due to their intrinsically disordered domains. However, upon binding to 14-3-3, a structured binary interface is formed that can be rationally targeted by an MG with the appropriate features of shape complementarity and molecular recognition.

In this Account, we will elaborate on three major aspects of our work on 14-3-3/client MGs:

  • (1)

    screening approaches and techniques we have used for the identification of new chemical matter

  • (2)

    rational, structure-guided medicinal chemistry optimization and the key principles

  • (3)

    the development of cell-based assays to validate MGs

The results of these studies are readily applicable to native PPIs and will be useful to experts and non-experts interested in molecular glues.

Screening Approaches

When we started working on these projects, the only cell-active compounds known to stabilize 14-3-3/client interactions were the natural products fusicoccin A (FC-A) and cotylenin A (CN-A) and their semisynthetic analogs , (Figure A–C). The cocrystal structures of the natural products bound to the 14-3-3/client interfaces provided valuable proof for the ligandability of the site; however, their complex structures were difficult to translate toward rational, client-selective glues. At the same time, traditional high-throughput screening had met with limited success, and while screening hits provided proof of concept, − they were not advanced to cell-active chemistry. To address the need to identify new, advanceable chemical matter, we turned to fragment-based screening approaches, which have been successful for challenging drug targets such as PPI inhibitors. − Fragment-based drug discovery is an established philosophy for discovering ligands with low molecular weight and complexity, followed by optimization toward a drug-like molecule. − In support, crystallography-based fragment discovery methods have identified diverse chemical structures that bind to 14-3-3/client complexes near the natural-product binding site. −

1.

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Chemical structures, crystal structures of 14-3-3/client interactions, and overview of biophysical assays. (A) Chemical structures of natural products and fragments. (B, C) Crystal structures of the natural products with 14-3-3, ERα, and C-RAF pS259. The targeted cysteine for MGs is highlighted in yellow. (D) Overview of MS tethering screen. (E) Crystal structures of 14-3-3/C-RAF with selective fragments. (F) Overview of biophysical assays for validation of hits.

Utilizing a site-directed disulfide tethering screen, we identified cysteine-reactive fragments that bind in the natural-product binding site and stabilize 14-3-3/client interactions. , The principle behind disulfide tethering is that, because the disulfide bond is rapidly reversible, fragment hits are selected based on thermodynamic principles. Our in-house disulfide library comprises ∼2000 fragments spanning chemical space. These fragments contain a mixed disulfide, where one side contains a fragment-like molecule attached to a variable linker and the other side contains a solubilizing group such as 2-(dimethylamino)­ethane-1-thiol (Figure A).

While tethering had been used in the discovery of diverse inhibitors, − it had not previously been employed for discovery of MGs. Of the seven highly homologous 14-3-3 isoforms in humans, only the σ isoform contained a cysteine residue (C38) positioned at one end of the client-binding groove (Figure B–C). Screening therefore used the 14-3-3σ protein and client phospho-peptides that mimicked the 14-3-3 binding motif. The phospho-peptides were considered reasonable models for the full-length client protein because the 14-3-3-binding domains containing the phosphorylation site were already intrinsically disordered. The peptide–protein interaction was incubated with a reducing agent (typically BME) and a single dose of fragment (200 μM) for 3 h to allow the reaction to reach equilibrium. Fragment binding was measured by the mass shift via intact protein mass spectrometry (MS). To identify fragments that stabilized a PPI cooperatively, screens were run in the presence and absence of the client phospho-peptide; hits were classified as stabilizers (cooperative fragments) if they preferentially bound in the presence versus the absence of the client peptide (“apo”). Neutral binders showed similar percent tethered in both screens, whereas inhibitory fragments had high percent tethered in the apo screen but not in the presence of the phospho-peptides. This distinction allowed the classification of fragments based on their potential binding cooperativity (Figure D).

The first examples of tethering for 14-3-3/client interactions discovered cooperative fragments tethered either to the native or engineered cysteine residues near the FC-A binding site (Figure ). To evaluate the scope and limitations of the approach, we then screened 14-3-3σ against the native cysteine (C38) with five diverse client peptides, derived from two transcription factors (ERα and FOXO1), a kinase (C-RAF), a RAS guanine nucleotide exchange factor (SOS1), and a deubiquitinating enzyme (USP8). The clients encompassed different binding motifs with unique sequences surrounding the phospho residue to form different composite binding pockets. We identified both selective and nonselective hits for the five clients. Effective hits were validated for four of the clients, with selective fragments discovered for FOXO1 and C-RAF. Fragment 1 (see below) was a top hit for three clients: ERα, C-RAF, and USP8. Fragments 5 and 7 were identified as selective stabilizing hits for C-RAF (Figure A,E).

We continued to characterize 1, 5, and 7 across a series of biophysical and structural assays. In addition to measuring dose responses with MS, we used fluorescence anisotropy (FA) to measure the effect of compound binding on the affinity of the peptide–protein complex. FA assays included a fluorescein-labeled phospho-peptide client at a concentration of peptide and 14-3-3 near the EC20 for the 14-3-3/peptide interaction. Binding of the MG then induced peptide binding to 14-3-3, leading to an increase in the anisotropy. Varying the compounds’ concentration yielded an EC50 value; titrating 14-3-3 at constant compound concentration yielded a ΔApp K d for the 14-3-3/client complex (Figure F). In these validation assays, 1 acted as an MG for both 14-3-3/ERα and 14-3-3/C-RAF, with a similar EC50 value (1 μM) for both targets but higher efficacy of stabilization for C-RAF (81-fold vs 19-fold for ERα). 5 and 7 were validated as C-RAF-selective hits with EC50 values of 0.58 μM (5) and 12.2 μM (7) and efficacies of 246-fold (5) and 77-fold (7).

The validated hits were crystallized with 14-3-3σ/phospho-peptide complexes to elucidate their key interactions with the composite interface. The structural observations were consistent with the compounds’ biophysical activities. For example, comparing multiple fragment hits provided insight into how different scaffolds stabilized C-RAF’s interaction with 14-3-3σ (Figure E). Fragment 1 and the C-RAF-selective fragments 5 and 7 represented different chemical scaffolds that uniquely altered the conformation of the C-RAF peptide, particularly around the +4 Val (V263) residue. FOXO1, by contrast, was rigidly oriented in the 14-3-3σ pocket, but the side chain of the +1 Trp (W25) rotated to interact with two distinct fragment structures. Thus, specific interactions with residues C-terminal to the phospho-S/T in the client were critical for the formation of 14-3-3/client/MG complexes. Furthermore, characteristics of the clients, such as peptide flexibility, facilitated the identification of selective MGs from the disulfide screen. Altogether, disulfide tethering uncovered MG chemotypes for diverse targets with diverse 14-3-3/client structures.

Medicinal Chemistry Strategies

Structure-Guided Optimization from a Disulfide Fragment to an Irreversible MG

The crystal structures of 1 with the 14-3-3/ERα and 14-3-3/C-RAF complexes served as a starting point for medicinal chemistry optimization (Figure A). The fragment lacked selectivity and preferentially stabilized C-RAF; however, in the crystal structures, it adopted a similar binding mode with both clients. While the interactions between the fragment and 14-3-3 were similar, distinct interactions were formed between the fragment and the client phospho-peptides. On ERα, the phosphorylation occurred on the penultimate residue on the C-terminus (T594), and the ultimate residue (+1) was hydrophobic (V595). This created an open, solvent-exposed interface where the fragment primarily formed hydrophobic interactions with V595. In stark contrast, the C-RAF phosphorylation occurred as part of an internal sequence (S259), the +1 residue was polar (T260), and the rest of the sequence created a pocket that was shielded from the solvent. The bound fragment was oriented toward T260, and the rest of the peptide was in proximity but did not form favorable interactions. Based on the crystallographic insight, we made structure-guided chemical modifications in five parts of the molecule, aiming to tune the selectivity toward the 14-3-3/ERα complex by optimizing favorable interactions at the PPI interface and at the same time reducing the binding to the 14-3-3/C-RAF complex via increased steric hindrance (Figure A).

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Overview of medicinal chemistry strategies for MG optimization. Chemical structures, structures of MG/14-3-3/client complexes, and biophysical data for the optimized compounds.

The initial fragment had a reversible disulfide linker, which interacted with C38 on 14-3-3σ. The reversible nature of the bond was advantageous for the tethering screen (see above) but was unsuitable for cell assays. Therefore, the first two modifications focused on replacing the disulfide with irreversible warheads with varying linker lengths. This change required adaptation of the previously described MS assay, which was now run as a time course to evaluate binding kinetics. For most compounds, both the MS and the FA assay required overnight incubation to achieve a maximum increase in signal. Diverse electrophiles varied significantly in their observed binding affinity and kinetics. The chloroacetamide warhead demonstrated good binding affinity and moderate reactivity and was therefore selected for further SAR optimization.

The next two modifications aimed at optimizing the interactions between the compounds and V595 on ERα. Since the residue was hydrophobic, we replaced the gem-dimethyl group with linear or cyclic hydrophobic groups. Increasing the ring size and introducing heteroatoms on the rings (−F, −O, −N) improved both the potency and selectivity toward the 14-3-3/ERα complex. Replacing the ether group with an aniline allowed the compounds to adopt a different conformation, oriented toward the front of the pocket. For both ethers and anilines, the presence of a p-chlorophenyl-substituted ring in the K122 pocket was crucial for the stabilization effect. These modifications successfully allowed the formation of specific compound–peptide interactions, both with V595’s hydrophobic side chain and the terminal carboxylic acid.

The last modification, at the rim of the PPI interface, focused on optimizing the warhead linker length and restricting its conformation by introducing spirocycles. The best analogue of the series, compound 181, showed favorable interactions both with 14-3-3 and ERα, shape complementarity, and a favorable conformation in the composite 14-3-3/ERα interface (Figure A). The key interactions included a halogen bond with K122 (14-3-3), multiple hydrophobic interactions from the 2,6-dimethyl tetrahydropyran (L218, I219, L222 of 14-3-3 and V595 of ERα), and a water-mediated hydrogen bond between the aniline nitrogen and the terminal carboxylic acid of V595 (ERα). Additionally, it showed fast kinetics in the MS assay and cooperative binding in the FA assay, leading to a compound EC50 value of 1 μM for 14-3-3/ERα, with AppK d = 18 nM (116-fold stabilization). The compound also showed selectivity when tested against a small panel of eight 14-3-3 clients, including C-RAF. A comparison between 181 and the natural product FC-A showed similar cooperativity in the FA and isothermal calorimetry assays, although 181 binding was more strongly driven by enthalpy (ΔH = −8.6 kcal/mol vs −5.8 kcal/mol for FC-A).

Fragment Linking toward Non-covalent MGs

Structural information from two series of fragments was also a useful starting point for optimization. We ran a screen with an engineered cysteine on 14-3-3 (C42) in the presence of the ERα phospho-peptide and crystallized the hits, including the one shown in Figure B (C42 fragment). It had the same chlorophenyl binding element as seen in 1, but it was tethered to C42 with a shorter linker, putting the chlorophenyl group in the same location and leading to a similar degree of 14-3-3/ERα stabilization. A separate crystallographic screen for the 14-3-3/p53 complex had identified a non-covalent amidine fragment, which was bound at the rim of the interface, forming a salt bridge with E14 (14-3-3). The amidine fragment was positioned far from the client phospho-peptide and lacked interactions with it. Cocrystallization of the two fragments provided the structural insight for a fragment-linking campaign to yield non-covalent MGs for the 14-3-3/ERα complex. In the optimized linked analogs, the original fragment–protein interactions were largely conserved. The best compounds of the series showed 25-fold stabilization of the 14-3-3/ERα complex in the FA protein titration assay. Notably, similarities in the SAR were observed between the covalent and non-covalent series. In both cases, the p-chlorophenyl-substituted rings in the K122 pocket and the bulky cyclic rings with heteroatoms (−O or −N) proximal to V595 were necessary for stabilization of the 14-3-3/ERα complex. To summarize, functionally active disulfides, coupled with structurally adjacent but inactive fragments, provided a remarkably effective optimization strategy and highlighted a potentially systematic approach to targeted MGs.

A Scaffold Hopping Approach Using Multicomponent Reaction Chemistry

We recently disclosed a computational scaffold-hopping approach using pharmacophore-based docking of virtual libraries based on multicomponent reaction chemistry (MCR). , MCRs combine at least three starting materials in a single step to form a complex scaffold, thus allowing fast synthesis of analogs and rapid development of SAR. As a starting point, we used compound 127, an intermediate analogue of our first series of 14-3-3/ERα MGs. The compound had favorable interactions in the composite surface and was one of the first aniline analogs that showed the optimal ligand conformation (Figure C).

Our aim in the scaffold-hopping approach was to explore new chemical space by designing a rigid, drug-like scaffold that would maintain the 3D shape and the ideal shape complementarity in the 14-3-3/ERα interface. Pharmacophore-based queries using AnchorQuery revealed that the most suitable scaffolds derived from the Groebke–Blackburn–Bienaymé reaction (GBB), a three-component reaction using aldehydes, 2-aminopyridines, and isocyanides to form to imidazo­[1,2-a]­pyridines. To target C38, we kept the chloroacetamide warhead constant in most of the analogs since in our previous experience it had moderate, tunable reactivity.

Using structure-guided design, we varied one building block at a time, starting with the isocyanide position, which according to our design was expected to occupy the K122 (14-3-3) pocket, in close proximity to the ERα peptide. For this position, we observed different SAR compared to our previous series: the p-chlorophenyl analogs did not result in improved binding/stabilization. Instead, the ortho position had a more favorable effect, especially in the case of double-o-methyl-substituted rings, which correlated with rapid cooperative binding. We made additional changes in the other two components of the GBB reaction. First, we added hydrophobic substitutions on the 2-aminopyridines, which led to extensive hydrophobic interactions with 14-3-3 (L218, I219, L222). We also modified the aldehyde building blocks and to a smaller extent the electrophilic warheadsboth positioned at the rim of the interface.

Based on MS data and crystal structures of ternary complexes, we combined the best modifications in compound 41, which included the 2,6-dimethylaniline in the isocyanide position, an additional methyl group on the imidazopyridine ring, and an o-F group on the aryl ring at the rim of the interface (Figure C). Structurally, the 2,6-dimethylaniline groups in the K122 pocket formed favorable hydrophobic interactions. The first o-Me group was oriented in the back of the 14-3-3 pocket, which was primarily hydrophobic, forming hydrophobic interactions with I219 (3.2 Å) and facing V595 of ERα; the second o-Me group was oriented in the front of the pocket and formed hydrophobic interactions with F119 of 14-3-3 (3.8 Å). The imidazopyridine ring occupied the hydrophobic pocket of 14-3-3 formed by L218 and I219. The additional methyl group on the imidazopyridine ring formed hydrophobic interactions with L222 of 14-3-3. The o-F group on the second aryl ring was oriented “outward” and made multiple stabilizing interactions, both intermolecular (N42 of 14-3-3) and intramolecular with the o-Me substituent and aniline nitrogen of the compound itself. These additional interactions favorably restricted the rotational bonds, resulting in fast cooperative binding. Finally, the aniline nitrogen formed a water-mediated hydrogen bond with the terminal carboxylic acid of V595 (ERα).

The optimized covalent analogs from the two series, 181 and 41, had similar potencies in biophysical assays. Both compounds showed fast, cooperative binding in the MS assay and yielded a low-nanomolar AppK d for 14-3-3/ERα in a TR-FRET assay (5–8 nM). That article also described a surface plasmon resonance (SPR) method to quantify AppK d for 14-3-3/client complexes stabilized by a covalent MG. First, the covalent compounds were preincubated with the 14-3-3/ERα complex, followed by immobilization of 14-3-3 on the SPR surface and extensive washing to remove bound peptide. Peptides were then flowed over the SPR surface, and the binding kinetics and K d values were determined. In this format, 181 and 41 yielded AppK d values of 3.9 and 10.1 nM, respectively, and increased the half-lives from 3.4 to 62.5 and 47.6 s, respectively.

Fragment Merging for Selective MGs Stabilizing the 14-3-3/C-RAF Complex

In contrast to the C-terminus of ERα, the C-RAF pS259-containing peptide afforded diverse interactions with the MGs. Crystal structures of 1 and an analog with a chloroacetamide warhead (1074202, Figure D) revealed unfavorable interactions between the hydrophobic part of the fragment and T260 of C-RAF. We used a fragment-merging approach to combine parts of 1074202 with an aldehyde aryl sulfonamide fragment (TC-521) that was reported to bind to 14-3-3/p65 and could position the hydrophilic sulfonamide near T260 (C-RAF). The aldehyde, TC-521, bound reversibly to K122 (14-3-3) via an imine bond. We merged the two fragments and tested a doubly covalent analogue (compound 21), which included both a cysteine- and lysine-targeting warhead, and a series of only cysteine-targeting analogs, such as 23. We thus sought to improve the interactions with T260 and additional C-RAF residues that extended into the MG-binding site. Crystallography showed that 21 was indeed doubly covalently bound; however, it lacked specific interactions with the C-RAF peptide.

On the other hand, 23 adopted a surprising, unique, upward conformation, where the C-RAF peptide “wrapped” around the main core of the compound, thus significantly covering the binding groove. The key interactions were formed from the sulfonyl group and included a charge-assisted hydrogen bond with K122 (14-3-3) and a hydrogen bond with T260 (CRAF). The iodoaryl group was positioned between the hydrophobic pocket formed by L218 and I219 (14-3-3), V263 (C-RAF), and M265 (C-RAF). The chloroacetamide warhead formed two hydrogen bonds with R41 and N42 (14-3-3) in addition to the covalent bond with C38. The binding mode of 23 correlated with fast, cooperative binding in the MS assay and very little binding to 14-3-3 (apo), whereas 21 showed significant binding to 14-3-3 (apo). In FA protein titrations, 23 yielded an AppK d of 30 nM for the 14-3-3/C-RAF complexa 280-fold stabilizationwhereas 21 showed only 9-fold stabilization. Additionally, 23 showed remarkable selectivity when tested in a panel consisting of 80 14-3-3/client complexes in FA format. Only four clients were stabilized by 23; all showed weaker stabilization than C-RAF pS259, indicating that the compound’s multiple interactions with C-RAF favorably correlated with selectivity.

Cell Assays

In general, properly phosphorylated full-length 14-3-3 client proteins are challenging to isolate. Thus, our biophysical assays have utilized phospho-peptides derived from clients, and it is particularly critical to verify the efficacy of MGs toward full-length PPIs. We have therefore developed cellular assays to assess PPI stabilization and its downstream effects in target-specific functional assays (Figure A).

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Overview of cell assays. (A) General workflow for MG validation in cells. (B) 14-3-3/ERα ΝanoBRET with MGs and FC-A. (C) 14-3-3/C-RAF NanoBRET. (D) Effects on C-RAF phosphorylation upon treatment with MGs (23 and derivative 23′) compared to DMSO and inactive 8.

Characterization of Full-Length PPIs via NanoBRET

To evaluate MG stabilization of the full-length PPIs, we developed a bioluminescence resonance energy transfer (NanoBRET) assay with HaloTag (HT) fused to the C-terminus of 14-3-3σ and nanoluciferase (Nluc) fused to the client’s N- or C-terminus. The constructs were transfected into HEK293T cells, and fluorescent HT ligand was added. Interaction between the tagged proteins resulted in BRET and HT-ligand fluorescence. The assay was developed to detect a basal level of the BRET signal that could be increased upon compound treatment. The assay sensitivity was also enhanced by using HEK293T cells, which have a low expression of the endogenous proteins; thus, the transfected proteins did not compete with their endogenous counterparts.

NanoBRET assays validated the ability of MGs to stabilize 14-3-3/client complexes with overnight MG treatment (Figure B,C). Compounds were ranked based on their EC50 values and their maximum fold-increase in normalized BRET signals. Chemically similar inactive compounds (ERα, 85; C-RAF, 8) resulted in no change in the signal. The EC50 values of the compounds in the NanoBRET assays showed a 1–5-fold shift between the values obtained from FA. For example, 181 had an EC50 value of 5.2 μM in the 14-3-3σ/ERα NanoBRET versus 1 μM in FA. The MCR scaffold 41 had a similar BRET EC50 of 5 μM (Figure B); interestingly, 41 resulted in a higher fold increase in the BRET signal (2.6 vs 1.7 for 181). For C-RAF, 23 was the most potent, with the highest maximum BRET signal and an EC50 of 0.18 μM (Figure C). These results indicated that phospho-peptides served as reasonable mimics for the 14-3-3 client proteins and that covalent MGs were cell-permeable and selective enough to bind their targets in the presence of other cellular proteins and glutathione.

Biological Effects of 14-3-3/Client MGs

We then designed orthogonal cell-based assays to evaluate the downstream effects of 14-3-3/client stabilization. For example, 14-3-3 binding to ERα pT594 was known to negatively regulate ERα, blocking binding to chromatin. As expected, in the ER+ breast cancer cell line MCF7, the basal state of ERα was primarily non-phosphorylated at T594, leading to minimal native interaction with 14-3-3 and high ERα-mediated transcription. We expected that, upon binding to 14-3-3, the client phospho residue would be shielded from dephosphorylation (Figure A). Indeed, upon treatment with 181, we saw that pT594 was strongly increased, in agreement with increased 14-3-3/ERα complex formation observed by NanoBRET. Our collaborators then generated a reporter assay in MCF7 cells in which luciferase expression was dependent on ERα binding to the estrogen response element (ERE). In this assay, 181 (1 μM) strongly inhibited luciferase expression compared with DMSO or the inactive compound. Additional assay formats supported the on-target activity for 181. For instance, we observed a reduction in proliferation in ER+ cells, no change in proliferation in ER– cells, activity in patient-derived organoids, and complex formation using proximity-ligation assays.

We used a similar workflow to characterize MGs that stabilized 14-3-3/C-RAF. Compound 23 and related MGs increased pS259 levels up to 3.2-fold (Figure D) and increased the 14-3-3σ/C-RAF interaction seen via co-immunoprecipitation, corroborating the NanoBRET results. We also developed NanoBRET assays to evaluate how 14-3-3/C-RAF stabilization prevented the formation of downstream C-RAF PPIs. C-RAF/NRAS and C-RAF/C-RAF were known critical steps in the activation mechanism of C-RAF; − 23 inhibited formation of both complexes, with IC50 values comparable to the EC50 for 14-3-3/C-RAF stabilization. We then monitored dose-dependent reduction in ERK phosphorylation, indicating inhibition of C-RAF kinase activity.

Strong upregulation of the RAS/RAF/MAPK pathway leads to cancer, but modest upregulation by activating mutations in the pathway can lead to developmental “RASopathies” such as Noonan syndrome (NS). , We have demonstrated that mutations upstream of 14-3-3/C-RAF modestly lower C-RAF pS259 and that our MGs protect pS259 in these contexts. More challenging are NS mutations in C-RAF near the pS259 site. These mutations, including S257L, impair C-RAF’s inhibitory interaction with 14-3-3 and can lead to debilitating infant cardiac hypertrophic myopathy. Compound 23 and related MGs restore the 14-3-3/C-RAF interaction, protecting phosphorylation of pS259 and reducing levels of pERK (unpublished).

Conclusions and Perspective

We are entering a golden age of chemical biology. Many heretofore undruggable targets can now be addressed using chemistries, including covalent ligands and beyond-rule-of-five molecules, that were once verboten but have demonstrated translation from bench to bedside. Leading the way are bifunctional molecules and molecular glues that induce biology by enhancing the proximity between two biomolecules.

The highlighted papers showcase our progress toward the identification, optimization, and validation of MGs for the 14-3-3 interactome. MS-based fragment screens have enabled the systematic identification of new chemical matter for diverse 14-3-3/client interactions. Disulfide fragments provided multiple starting points for medicinal chemistry by optimizing, merging, scaffold-hopping, or linking fragments; both covalent and non-covalent strategies have been demonstrated. We adapted or developed new biophysical assay formats to validate the unique features of MGs such as cooperativity. The extraordinary availability of high-resolution, ternary crystal structures from the Ottmann and Brunsveld laboratories provided the opportunity for rational optimization of client-specific interactions at the 14-3-3/client composite interface, affording high selectivity for the PPI of interest. These strategies resulted in cell-active MGs for two 14-3-3/client interactions, allowing us to study disordered clients via an unprecedented mechanism.

In addition to MS tethering screens, 14-3-3-binding fragments have been identified using different assay readouts and screening libraries. Fluorescence polarization (FP) screens have identified fragments covalently bound to cysteine on 14-3-3 or on the phosphopeptide client. Libraries containing aldehydes have targeted imine formation with K122, a deeply buried residue conserved across 14-3-3 isoforms. Stabilizing fragments were identified for 14-3-3/p65, and analogs from this screen were further optimized toward 14-3-3/Pin1. Several non-covalent fragment libraries have also been screened using X-ray crystallography, differential scanning fluorimetry, NMR spectroscopy, and TR-FRET. Traditional libraries have also been screened. Two structurally distinct hits were identified for 14-3-3/PMA2 from a 37,000-member library using an ELISA-like assay format. The 14-3-3/SLP76 complex was screened using an HTRF assay and a chemically diverse library of 20,000 compounds. Additionally, macrocycles have been reported to restore the impaired activity of chloride channel CFTR by stabilizing the 14-3-3/CFTR interaction. The macrocycles were identified in an FP screen of 5760 compounds of Cyclenium’s proprietary small-molecule macrocycle library and bound to a novel site on the complex.

Unique 14-3-3/client interfaces have enabled unique chemical approaches. A particularly powerful example is ChREBP, one of the few 14-3-3 clients that is not phosphorylated. Our collaborators reported a virtual screening strategy that included phosphate- and phosphonate-based compounds that bind in the peptide-phosphate site and selectively stabilize the 14-3-3/ChREBP complex. Structure-guided optimization yielded cell-active MGs that protect beta cells from glucolipotoxicity and therefore show promise for treatment of type 2 diabetes. ,

From these combined efforts, we have encountered thematic challenges in stabilizing native PPIs. For covalent glues, selected warheads must not only bind the reactive residue but also glue/stabilize the complex. Our data with 14-3-3 so far suggest that a limited number of warheads bind weakly to 14-3-3 alone but strongly to the 14-3-3/client complex; thus, low-reactivity warheads and high context-dependent binding are required. To predict whether a native PPI can be stabilized by MGs and to what extent, two main aspects must be considered: complex affinity and subcellular localization. From our work, high-affinity complexes, such as 14-3-3σ/FOXO1 pT24, posed greater difficulty in improving the binding affinity or stabilizing the complex. Furthermore, the extent of stabilization necessary to achieve a pharmacological effect might also vary; for instance, significant differences are observed between oncology targets and re-establishing homeostasis in developmental disorders. Additionally, the chosen client protein and the unique features of the composite interface can also contribute to the maximum degree of stabilization achieved by MGs; for example, in the case of 14-3-3σ/ERα MGs, where the interactions occur only with the C-terminus of ERα, a smaller number of glue/complex interactions are possible compared to 14-3-3σ/CRAF MGs that interact with an internal region of CRAF. Manipulating subcellular localization can enhance PPI formation; however, it can lead to saturation of the PPI, which prevents further stabilization via MGs. In general, these MGs are not expected to be effective at substochiometric concentrations unless native 14-3-3/client biology leads to degradation (for example). Nevertheless, we observe cellular efficacy with compounds that have moderate EC50 values and high fold stabilization. Thus far, we have disclosed only hit identification and hit-to-lead optimization. In future work, we will expand ADME/PK and in vivo studies; since the cysteine-reactive warhead is present in our best hits to date, we will continue evaluating new warheads to establish translatability.

Taken together, there is a wealth of chemical starting points that can be combined and dissected in various ways to develop a suite of MGs and 14-3-3-binding ligands for a variety of applications. Since 14-3-3 is an abundant protein, targeting the σ isoform has the advantage of modulating specific interactions without disrupting the whole 14-3-3 “pool”. On the other hand, pan-14-3-3-isoform ligands are needed for tissues that do not express 14-3-3σ. The seven 14-3-3 isoforms have a broadly conserved client-binding groove and share overlapping functions in most cases. Emerging proteomics data have started defining the isoforms’ interactomes, confirming common clients among them.

In general, the use of reversible covalent ligand discovery has been remarkably effective for identifying 14-3-3/client MGs and could also be applied to other native PPIs that share key features. For instance, the presence of a nearby reactive residue that might be targeted by reversible chemistry (Cys or Lys) facilitates the use of site-directed screening. The possibility of using a peptide to mimic one of the proteins could facilitate biophysical screening and crystallization. Additionally, structural characterization of the PPI is game-changing for structure-guided drug design, especially for hub proteins, where selectivity is of the utmost importance. Many of these features are found in chaperones and E3 ligases, such as β-TrCP, KEAP1, and DCAFs; DCAF16, for instance, has a cysteine that has been successfully targeted for degradative MGs.

We have focused on stabilizing native PPIs because these systems invite systematic discovery and predictable biological outcomes. Native interactions also lay a foundation for learning how to stabilize PPIs more generally. Our 14-3-3 experiences suggest that (1) reversible covalent fragments provide site-directed screens and reduce the three-body problem intrinsic to highly cooperative ternary complex formation, (2) biophysical and structural analysis provides a critical mechanistic understanding of cooperative binding, (3) highly selective MGs are made by optimizing interactions with both sides of the PPI, even if one member of the complex is intrinsically promiscuous, and (4) high selectivity leads to small shifts in EC50 values between biophysical and cell-based measurements of PPI formation. We propose that these strategies and techniques will be broadly applicable to other native PPIs and to the even broader domain of neomorphic interactions.

Acknowledgments

We thank Professors Luc Brunsveld, Jeff Neitz, Christian Ottmann, and Adam Renslo and the many creative lab members from our groups who contributed ideas and experiments to the articles reviewed here and to other projects in our long-standing collaboration.

Glossary

Abbreviations

PPI

protein–protein interaction

MG

molecular glue

MS

mass spectrometry

FA

fluorescence anisotropy

FP

fluorescence polarization

SPR

surface plasmon resonance

MCR

multicomponent reaction

TR-FRET

time-resolved fluorescence resonance energy transfer

BRET

bioluminescence resonance energy transfer

Biographies

Markella Konstantinidou obtained a degree in Pharmacy and an M.Sc. in Medicinal Chemistry from the Aristotle University of Thessaloniki in Greece. She received her Ph.D. from the University of Groningen in the Netherlands in 2020, focusing on multicomponent reactions, their application in medicinal chemistry, and new modalities. She joined the lab of Michelle Arkin at UCSF as a postdoctoral scholar. Currently she is a staff scientist in the same lab. Her work focuses on the development of molecular glues, fragment-based screening, and biophysics for protein–protein interactions.

Johanna M. Virta attended the University of Wisconsin, Madison, where she obtained her B.S. degree in Biochemistry and Statistics in 2019. As an undergraduate researcher, she studied RNA-binding proteins. As a Ph.D. candidate in Chemistry and Chemical Biology at the University of California, San Francisco, her research focuses on drug discovery for protein–protein interactions in cancer and developmental disorders.

Michelle R. Arkin earned her Ph.D. in Chemistry at Caltech in the laboratory of Professor Jacqueline Barton. She then held a Damon Runyon Cancer Research Postdoctoral Fellowship in the Department of Protein Engineering at Genentech under the mentorship of Jim Wells. She worked for 9 years at Sunesis Pharmaceuticals, developing inhibitors for protein–protein interactions and leading translational programs for clinical-stage assets in oncology. In 2007 she moved to UCSF, where she is currently a Professor of Pharmaceutical Chemistry, Executive Director of the Small Molecule Discovery Center, and Vice Dean for Research Technology and Entrepreneurship. Her chemical biology laboratory develops methodologies and molecules to study protein–protein interaction networks.

‡.

M.K. and J.M.V. contributed equally to this work. The manuscript was written through contributions of all authors. All of the authors approved the final version of the manuscript.

This work was supported by an Ono Pharma Foundation Breakthrough Science Initiative Award and NIH (1R01 GM147696).

The authors declare the following competing financial interest(s): M.R.A. is a co-founder of Ambagon Therapeutics.

Published as part of Accounts of Chemical Research special issue “Proximity-Induced Chemical Biology”.

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