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. 2026 Aug 17;17(9):2006–2016. doi: 10.1021/acsmedchemlett.6c00325

Covalent Stabilizers of the Interaction Between 14-3-3σ and Estrogen Receptor‑α

Rama Jain †, Mark Knapp †, Sarah E Kochanek †, Adam Lewis †, Lynn M McGregor §, Vanja Stojkovic †, Jenny Tang †, Tiffany Tsang †, Kelly Yan †, Aregahegn Yifru †, Qingming Zhu †, Francesca Fabbiani ‡, Gu Feng †, Andreas O Frank †, John Fuller †, Wolfgang Jahnke ‡, Johanna M Jansen †, Jihye Jo †, Min Li †, Jennifer Lipps §, Yipin Lu †, Tim Mladenovic †, Philippe Piechon ‡, Phuong Rogemoser §, Feng Wang †, Shengtian Yang †, Colin K Skepper †,*
PMCID: PMC13573265  PMID: 42741630

Abstract

Selective stabilization of complexes formed by the hub protein 14-3-3 represents an emerging mechanism for the modulation of therapeutically relevant targets. In this letter, we describe a hit-finding campaign designed to identify small molecule stabilizers of the interaction between 14-3-3σ and the estrogen receptor alpha (ERα). Four structurally distinct hits were identified and validated using a combination of biochemical assays and biophysical techniques. Ternary complex crystal structures revealed that all four hit compounds form a covalent bond with Cys38 of 14-3-3σ via four different electrophilic warheads. Structure-based optimization of the most promising hit compound 9 led to dramatic improvements in stabilization activity and selectivity that exceeded the complex natural product fusicoccin A.

Keywords: 14-3-3, protein−protein interaction, PPI stabilization, molecular glue, ternary complex, estrogen receptor

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The 14-3-3 family of adapter proteins consists of seven isoforms that are widely expressed in human tissues. All seven isoforms share a common dimeric architecture characterized by a large central cavity that accommodates the binding of client proteins. Complex formation generally requires phosphorylation of key serine or threonine residues in the partner protein that often, but not always, reside within canonical 14-3-3 recognition motifs. The biological consequence of 14-3-3 binding depends on the client protein in question and may be context-dependent, but can include effects on cellular localization, activity, stability, trafficking and protein–protein interactions (PPIs).

The 14-3-3 interactome is remarkably large and diverse, including many proteins of therapeutic relevance. , However, 14-3-3 client proteins frequently contain regions of intrinsic disorder and can be challenging to target by conventional means. As a result, stabilization of 14-3-3 PPIs via molecular glues represents an intriguing therapeutic modality that has received increased attention in recent years. −

Breast cancer is one of the leading causes of cancer mortality worldwide. Most newly diagnosed cases are estrogen receptor-positive (ER+), and in recent decades tremendous progress has been made in the development of therapeutics that target the estrogen receptor (ER). Widely used examples include the selective estrogen receptor modulator (SERM) tamoxifen and the selective estrogen receptor degrader (SERD) fulvestrant. More recent additions to the pharmacological armamentarium include next-generation oral SERDS and bifunctional degraders. Despite this progress, significant unmet medical need remains for ER+ breast cancer, in part due to the emergence of target-based resistance (e.g., mutations in the ER ligand binding domain (LBD) or protein fusions). Novel approaches to inhibit the activity of the estrogen receptor are urgently needed, and this remains the subject of intensive research in academic and industrial laboratories.

In 2013, De Vries-van Leeuwen et al. described the interaction between 14-3-3 and the estrogen receptor-α (ERα). It was demonstrated that phosphorylation of ERα Thr594 in the C-terminal F domain generated a 14-3-3 recognition site, and that binding of 14-3-3 to ERα inhibited receptor dimerization and transcriptional activity. The natural product fusicoccin A (1) was shown to stabilize this PPI by binding to a composite pocket present in the 14-3-3/ERα complex, thereby potentiating the inhibitory effect of 14-3-3 on ERα activity. This discovery raised the intriguing possibility that such molecular glues could offer a new strategy for targeting ERα in the context of breast cancer.

Several groups have since published efforts to discover novel small molecule stabilizers of the 14-3-3/ERα interaction (Chart ). The Ottmann, Arkin and Brunsveld groups reported an elegant disulfide tethering approach that resulted in identification of stabilizers that engage a non-native cysteine residue at position 42 or 45 of 14-3-3σ. , This approach was later extended to target the native Cys38 of 14-3-3σ, yielding a hit compound (2) that was optimized to give chloroacetamide 3. This compound forms an irreversible covalent linkage with 14-3-3σ and stabilizes the ERα complex with efficacy comparable to fusicoccin A. , The scope of this chemical matter has since been further expanded through structure-based design and scaffold hopping (e.g., 4). , The Nomura group recently published an acrylamide-based compound (5) that was discovered through screening cysteine-reactive compounds for their ability to stabilize the interaction between 14-3-3σ and ERα using a fluorescence polarization (FP) assay. Compound 5 targets both Cys38 and Cys96 of 14-3-3σ to potentiate the binding of ERα, YAP and TAZ. Acrylamide-containing analogues based on the fusicoccin scaffold have also been described by the Ohkanda group, who showed that such compounds can be utilized for isoform-selective fluorescent labeling of 14-3-3σ. Finally, Doveston, Roversi and co-workers have shown that the linear aminothiol WR-1065 covalently modifies Cys38 of 14-3-3σ, thereby potentiating the binding of ERα, and to a lesser extent, p53. Intriguingly, WR-1065 acts cooperatively with fusicoccin A to enhance client protein binding.

1. Structures of Previously Reported Stabilizers of the Interaction between 14-3-3 and ERα.

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As part of a program exploring the stabilization of 14-3-3 complexes, we initiated our own effort to identify novel small molecules that can enhance the interaction between 14-3-3 and ERα. Despite increased attention in recent years, the prospective discovery of PPI stabilizers, and molecular glues generally, remains challenging. With this in mind, we elected to pursue two different hit-finding strategies in parallel to maximize our chances of finding tractable chemical matter for optimization. This included biochemical screening of diversity libraries using a FP assay, and a targeted covalent ligand finding approach that utilized a competitive chemosensor assay.

To enable the first approach (biochemical diversity screening), an FP assay was developed using 14-3-3σ (NP_006133.1) and a 15-mer pERα peptide (KYY­ITG­EAE­GFP­A­(pT594)­V) labeled with either a Cy5 or FAM fluorophore. The Cy5-based FP assay was used to screen a subset of the Novartis compound archive in single point format. Two distinct compound sets were screened: 1) a diversity set of 75,000 compounds at a concentration of 25 μM, and 2) a 10,000-member Scope library at a concentration of 100 μM. Concentrations of 14-3-3σ and pERα peptide were kept constant at 11 nM and 10 nM respectively. Primary hits were defined as compounds with activity ≥ 3 standard deviations above the DMSO mean.

Hit rates for both compound sets were ∼0.8%. Primary hits were rescreened in the absence of 14-3-3σ ([14-3-3σ] = 0 nM) to remove compounds exhibiting a signal >3 standard deviations above the DMSO mean, indicating protein-independent assay interference. The remaining hits were tested at 25 μM using an 8-point protein dose–response with the Cy5-labeled pERα peptide. This format provides a measure of the apparent affinity of the pERα peptide for 14-3-3σ (appK D). Ten compounds from the 75K diversity set and nine compounds from the 10K Scope set exhibited ≥2-fold enhancement of appK D. The relatively high affinity of the Cy5-labeled pERα peptide for 14-3-3σ (appK D ∼ 75 nM), however, made it challenging to discriminate between more effective stabilizers. Compounds of interest were subsequently evaluated in 16-point protein dose–response format using the FAM-labeled pERα peptide. Concentrations of pERα peptide and compounds were held constant at 10 nM and 25 μM respectively, and the top concentration of 14-3-3σ was 90 μM. FP assay readouts were recorded after 2 and 24 h incubation. Three of the most robust hits from this campaign are shown in Table (compounds 6–8). Compound 6 exhibited marginal (∼1.5-fold) stabilization with no significant differences observed between 2- and 24 h incubation. Compounds 7 and 8 exhibited much more pronounced stabilization (8.8-fold and 12-fold at 24 h). Like 6, compounds 7 and 8 did not show a significant difference in stabilization activity between 2 and 24 h incubation.

1. Biochemical Characterization of Compounds 1 and 6–9 .

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  DMSO 1 6 7 8 9
FAM FP
appK D (nM, 2 h) 1780 ± 360 27 ± 1 1010 ± 21 178 ± 20 127 ± 5 690 ± 36
Fold stabilization   66 1.8 10 14 2.6
appK D (nM, 24 h) 1420 ± 176 27 ± 1 1060 ± 77 161 ± 25 115 ± 2 87 ± 5
Fold stabilization   53 1.3 8.8 12 16
SPR
K D (nM) 994 NT NT 106 113 69
Fold stabilization       9.4 8.8 14
Kinetic Chemosensor
14-3-3 k inact/K I (/M/s)   NT NT 4.6 5.1 0.3
14-3-3 + pERα k inact/K I (/M/s)   NT NT 12.2 7.8 6.5
Fold increase       2.7 1.5 21.7
Glutathione (GSH) Stability
GSH t 1/2 (min)   NT 43.3 <5 <5 34.2
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a

FP assay appK D values represent the apparent affinity of the pERα peptide for 14-3-3σ in the absence (DMSO) or presence of compound at 25 μM. Fold stabilization = FP appK D (DMSO)/ FP appK D (with compound).

b

SPR K D values represent the affinity of the pERα peptide for unlabeled 14-3-3σ (DMSO) or 14-3-3σ labeled quantitatively with compounds 7–9. Fold stabilization = SPR K D (unlabeled 14-3-3σ)/SPR K D (labeled 14-3-3σ). K D values for compounds 7 and 8 are the result of a single experiment. The K D value for compound 9 is the average of two experiments.

c

k inact/K I values represent the labeling rate of compounds 7–9 for 14-3-3σ in the absence or presence of pERα peptide as measured by the chemosensor assay.

In parallel to the FP-based diversity screens described above, we carried out an orthogonal screen that was designed to identify electrophilic ligands of Cys38 in 14-3-3σ. A chemosensor assay, which measures the ability of covalent ligands to prevent labeling by a fluorogenic probe (7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM)), was miniaturized to a 1536-well format. Due to the low reactivity of 14-3-3σ Cys96 toward CPM, wild-type 14-3-3σ is suitable for such a screen (see Supplementary Figure 7). A solution containing 1 μM 14-3-3σ and ± 2 μM pERα peptide was incubated with 25 μM of each library member for 24 h prior to addition of 2.5 μM CPM to quench the labeling reaction and quantify the amount of remaining free 14-3-3σ Cys38. We screened a library of 6,500 electrophilic compounds and identified 35 molecules that exhibited >70% labeling of 14-3-3σ + pERα peptide and <70% labeling of 14-3-3σ alone. The ability of 20 of these hits to give at least 30% covalent modification of 14-3-3σ was validated using intact protein mass spectrometry in the presence or absence of pERα peptide. We next asked whether these compounds could increase the apparent affinity of pERα peptide for 14-3-3σ using the FP assay described above. We were pleased to find that 8 compounds produced an increase in the affinity of pERα peptide for 14-3-3σ. However, only one compound (9) could be robustly validated using orthogonal techniques (described below). Compound 9 exhibited similar levels of stabilization in the FP assay as 7 and 8 after 24 h, however it demonstrated a more pronounced difference in stabilization between the 2 and 24 h time points (2.6-fold stabilization at 2 h vs 16-fold stabilization at 24 h).

With four confirmed hits in hand, we used a range of biochemical and biophysical techniques to characterize and validate the activity of compounds 6–9. While the screening approach that gave rise to compound 9 was designed to identify covalent ligands, we noted that compounds 6–8 also possessed electrophilic functional groups with potential to react with nucleophilic amino acids. Indeed, intact MS experiments confirmed that compounds 7, 8 and 9 irreversibly labeled 14-3-3σ in the absence of pERα peptide (see Supplementary Table 5 and Supplementary Figure 10). Based on literature precedent and the lack of reactivity observed for Cys96 (Supplementary Figure 7), we suspected that compounds 7–9 likely targeted Cys38. Compound 6 showed no evidence of labeling under the same conditions; however, we could not rule out the potential for a covalent-reversible mechanism involving reaction of Cys38 with the cyanopyrimidine group.

In order to further characterize the stabilization activity of irreversible covalent compounds 7–9, we developed an SPR workflow in which avi-tagged 14-3-3σ was labeled quantitatively with compound and then captured on a streptavidin-coated SPR chip surface. The affinity of the pERα peptide for the modified protein was determined and compared directly to the affinity determined with unlabeled protein (SPR stabilization = K D (unlabeled 14-3-3σ)/K D labeled 14-3-3σ)). The measured dissociation constant (K D) of the pERα peptide for unlabeled 14-3-3σ was ∼1 μM, in good agreement with the FAM-pERα FP assay. Compounds 7–9 yielded stabilization factors ranging from ∼9–14, consistent with the stabilization observed in the FAM-FP assay after a 24-h incubation period.

Having demonstrated that compounds 7–9 irreversibly label 14-3-3σ in the absence of pERα peptide, we next designed experiments to understand whether the presence of pERα peptide would influence the rate of labeling. To that end, we performed a dose and time-dependence study using the chemosensor assay, and the resulting labeling data were fitted to obtain the ratio of k inact/K I (Table and Supplementary Figure 8). Among this set of compounds 9 exhibited a unique profile, reacting with 14-3-3σ more rapidly in the presence of pERα peptide (21.7-fold increase in k inact/K I in the presence of pERα peptide compared to 14-3-3σ alone). The rates of labeling of compounds 7 and 8 showed less dependence on the presence or absence of pERα peptide. This suggests that 9 exhibits greater potential for cooperative binding and labeling of the 14-3-3σ/pERα peptide complex compared to 7 and 8. At the same time, it appeared that 9 labeled apo 14-3-3σ more slowly than 7 and 8. To further characterize the chemical reactivity of 6–9, we assessed buffer stability in the presence and absence of glutathione (GSH). Compounds 7 and 8 were the most reactive of the four compounds (GSH t 1/2 < 5 min). Compounds 6 and 9 were comparatively more stable, but both exhibited appreciable reactivity toward GSH (GSH t 1/2 43.3 and 34.2 min respectively). This information would later prove helpful in prioritizing compounds for chemical optimization.

To provide orthogonal biophysical validation of the hits from our screening campaigns, we developed two NMR-based assays: a 19F-CPMG-1D reporter assay as well as a 1H,15N-2D-TROSY-HSQC binding assay. The ability of hit compounds to stabilize the 14-3-3σ/ERα peptide complex was first tested using the higher-throughput 19F-based reporter assay. To enable this assay, we designed and synthesized a phosphorylated (pThr594) ERα (588–595) derived peptide and introduced a CF3 group at its N-terminus and two fluorine atoms on the side chain of the C-terminal Val595 (Figure D). Upon binding of the fluoro-reporter peptide to unlabeled, full-length 14-3-3σ, the fluorine peaks lose intensity relative to the unbound form. Addition of small molecules that enhance binding of the peptide to the protein lead to an increase in 19F signal intensity, as the exchange kinetics between reporter and target are changed (moving from intermediate toward the slow exchange regime) and the fluorinated peptide is in excess. The extent by which the 19F signals are perturbed upon ligand binding is related to the stabilization potency of our hits.

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Evidence for direct binding and ternary complex stabilization from NMR spectroscopy. (A) 19F-CPMG-1D NMR spectra of fluorinated pERα peptide (25 μM) in the absence and presence of 14-3-3σ (5 μM) and FC-A 1 or compounds 6–9 (50 μM). Spectra are horizontally offset by 0.25 ppm for clarity. Asterisks denote the signal from residual TFA from the peptide synthesis. (B) Section of a 1H,15N-TROSY-HSQC spectrum of 70 μM 14-3-3σ showing the amide resonance for Gly171 in the absence (black) and presence of 150 μM nonfluorinated pERα peptide (red) and 100 μM FC-A 9(navy) or compound 7 (maroon). (C) Structure of 14-3-3σ bound to pERα peptide. Gly171 is highlighted in magenta. (D) Chemical structure of the fluorinated pERα peptide utilized in the 19F-CPMG-1D NMR reporter assay. The 19F NMR signal shown in panel A comes from the N-terminal CF3 group.

In Figure A, we show the reporter assay results for our positive control fusicoccin A (FC-A, compound 1) and compounds 6–9. Fusicoccin A led to a recovery of the broadened, due to 14-3-3σ binding, CF3 reporter signal, indicating ligand binding and stabilization of the protein-peptide complex. Negative control compound 10 did not induce any recovery of the CF3 reporter signal, as expected (see Supplementary Figure 14). Compounds 6–9 recovered the reporter 19F signal intensity to a similar degree relative to each other and to fusicoccin A, confirming that these compounds stabilize the peptide:protein complex. The small assay window was not conducive to fine-grained ranking of hits but was still useful for confirming complex stabilization prior to 2D NMR.

Hits passing the 19F-CPMG-1D reporter assay were further tested using 2D 1H,15N-TROSY-HSQC NMR. For the 2D-NMR assay, we expressed uniformly 15N, 2H labeled, full-length 14-3-3σ, complexed it with an unlabeled, phosphorylated (pThr594) peptide derived from ERα (aa 581–595) and followed small molecule-induced chemical shift perturbations of the protein 1H,15N correlated peaks. This assay allowed us to confirm with high confidence that primary hits bind to the peptide-bound target by directly observing changes in the fingerprint spectrum of 14-3-3σ. The assay was carried out by adding fusicoccin A or compounds 6–9 to labeled, pERα peptide-bound 14-3-3σ. As expected, all five molecules induced clear chemical shift perturbations of protein signals, indicating binding to the target complex (see Supplementary Figure 16). In Figure B, we highlight the spectral changes caused by the positive control ligand fusicoccin A and compound 7. Of all residues where the HN chemical shift could be confidently tracked between apo, pERα peptide-bound, and pERα peptide:compound-bound spectra of 14-3-3σ, we observed the most significant perturbation for residue Gly171. This amino acid is very close to the interface of the peptide and the ligand binding site (Figure C), as proven later by X-ray crystallography, and is thus an excellent probe to detect binding of small molecule glue compounds.

Collectively, the FP, SPR and NMR results provided confidence that compounds 6–9 were genuine stabilizers of the interaction between 14-3-3σ and the pERα peptide. To provide insight into the molecular interactions driving this activity, X-ray cocrystal structures were solved for each of the corresponding ternary complexes formed by compounds 6–9 and 14-3-3σ bound to pERα peptide (Figure A–D).

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X-ray crystal structures of compounds 6 (A), 7 (B), 8 (C) and 9 (D) bound to the complex of 14-3-3σ and pERα 15-mer peptide.

The structures confirmed that compounds 6–9 form a covalent linkage to Cys38. No electron density was observed to suggest covalent binding to Cys96, the only other cysteine residue in 14-3-3σ. In the case of compound 6, Cys38 undergoes nucleophilic attack on the nitrile carbon, which is rendered particularly electrophilic by the electron-withdrawing pyrimidine ring. Compound 7 reacts with Cys38 in a conjugate addition reaction, wherein the thiol side chain of Cys38 attacks the alkyne which is activated by the electron deficient purine ring system. The resulting product features a cis double bond. For compounds 8 and 9, Cys38 displaces the chloride substituent through an SNAr or SN2 reaction, respectively. Compounds 6–9 occupy the fusicoccin binding pocket adjacent to Val595 at the C-terminus of the pERα peptide, stabilizing the ternary complex primarily through van der Waals interactions and shape complementarity. The crystal structures did not reveal any direct or water-mediated polar interactions that make significant contributions to stabilization of the ternary complex. Compounds 6, 7, and 9 project chlorinated phenyl rings deep into the back of the pocket, reminiscent of several 14-3-3 molecular glues reported previously. ,, Compound 8, meanwhile, is unique in that it projects a benzo fused lactone ring into the same pocket. It should be noted that the X-ray diffraction data suggested that both enantiomers of 8 were bound in the complex ((R)-8 in monomer C and (S)-8 in monomer A).

With this data in hand, we were motivated to evaluate the potential for optimization of the potency and selectivity of the identified hits, all of which were less active than the natural product fusicoccin A. We chose to focus our efforts on compounds 6 and 9. Both compounds exhibited higher chemical stability than either 7 or 8 as determined by incubation with GSH in buffer (see Table ). Compound 7 was also found to be chemically unstable under moderately acidic or basic conditions, which made analogue synthesis challenging. The reversible nature of the nitrile warhead in compound 6 was appealing, as was the tendency of compound 9 to label 14-3-3σ more rapidly in the presence of pERα peptide, despite its relatively modest k inact/K I compared to benchmarks.

Beginning with compound 6, early SAR revealed that changes to the cyanopyrimidine warhead or the chlorophenyl tail led to loss of activity (data not shown). Modifications to the central linker were, however, more successful. Inspection of the cocrystal structure of compound 6 bound to the 14-3-3/pERα peptide complex indicated that the cyclopentane group was positioned adjacent to the C-terminus of the pERα peptide and offered an opportunity to design ligands that could form more extensive contacts with either the client peptide itself, or with 14-3-3 in the vicinity of the peptide binding groove. By engineering greater complementarity between the ligand and the composite pocket presented by the peptide:protein complex, we hoped to improve both stabilization and selectivity. With this goal in mind, we prepared the N-acetylpiperidine analogue 11, which yielded an improved 6.5-fold stabilization of the 14-3-3σ/pERα peptide interaction at 2 h (Figure ). After surveying a small number of acyl groups on the piperidine ring, it was found that the pyrazole ring in 12 conveyed a further gain in potency (22-fold).

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Top: FAM FP assay appK D values showing apparent affinity of the pERα peptide for 14-3-3σ in the absence (DMSO) or presence of compounds 11 and 12 at 25 μM. Fold stabilization = FP appK D (DMSO)/FP appK D (with compound). Bottom: X-ray crystal structure overlay of compounds 11 (green) and 12 (purple) bound to the complex of 14-3-3σ and pERα 15-mer peptide.

X-ray cocrystal structures of compounds 11 and 12 bound to the 14-3-3σ/pERα peptide complex confirmed that both compounds formed more extensive surface contacts with the C-terminus of the pERα peptide, and filled a greater volume of the fusicoccin pocket, than compound 6 (Figure ). Notably, compound 12 projects the acyl pyrazole side chain in such a way as to wrap around the pERα peptide terminus, potentially limiting its ability to dissociate and providing a rationalization for the enhanced stabilization relative to 11.

Despite these gains, the stabilization activity of 12 fell short of that observed for fusicoccin A, possibly due to the reversible nature of the covalent interaction with Cys38. This prompted us to explore optimization of compound 9 using a similar strategy. Initial efforts to modify the chloroacetamide warhead, cis-2,6-dimethylpiperazine core or chlorophenyl tail led to a loss of stabilization activity. The central ethylene linker, however, offered an opportunity to rigidify the structure, favor the bound conformation and form more extensive interactions with the pERα peptide. Compound 13 was an early example of an analogue that followed these principles and exhibited greater stabilization than 9 at 24 h (25-fold vs 16-fold, Table ). Like 9, stabilization by 13 was time-dependent (increasing from 2 to 24 h). Further improvement was achieved by cyclizing the gem-dimethyl group into a cyclopentane ring to give 14 (68-fold stabilization at 24 h). A dramatic increase in activity was subsequently observed upon morphing of 9 and 11 to give 15A and 15B. These compounds exhibited comparable stabilization activity as assessed by SPR (199- vs 133-fold), but a clear stereochemical preference emerged in the FP assay with a 2-h incubation time (177-fold stabilization for (R)-15A compared to 26-fold for (S)-15B). The distinction between the two compounds eroded when the incubation was extended to 24 h (136- and 105-fold stabilization for 15A and 15B respectively). These data suggest that when complete labeling of 14-3-3σ is achieved with either 15A or 15B (as is the case in the SPR experiment, and likely the 24-h FP assay) these two compounds are highly effective stabilizers of the interaction with the pERα peptide. However, 15A may label 14-3-3σ more rapidly in the presence of pERα peptide and thus exhibit more pronounced stabilization in the FP assay at the 2-h time point. Extending the N-acetyl piperidine group of 15 to incorporate a pyrazole ring (c.f. compound 12) gave 16A and 16B which exhibited a further improvement in stabilization activity. Like 15A and 15B, compounds 16A and 16B showed comparable stabilization by SPR (355-fold) and in the FP assay with 24 h incubation (184- vs 197-fold). Once again, stabilization at 2 h in the FP assay revealed a difference in activity between the stereoisomers (239-fold vs 46-fold stabilization for 16A and 16B respectively). Once again, the more active compound 16A had the (R) configuration.

2. Biochemical and SPR Data for Optimized Analogues 13–16 .

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  DMSO 13 14 15A 15B 16A 16B
FAM FP
appK D (nM, 2 h) 1650 ± 250 294 ± 155 93 ± 10 9.3 ± 2.9 64 ± 13 6.9 ± 3 36 ± 8
Fold stabilization   5.6 18 177 26 239 46
appK D (nM, 24 h) 1160 ± 133 46 ± 21 17 ± 4 8.5 ± 2.9 11 ± 3 6.3 ± 2.4 5.9 ± 2
Fold stabilization   25 68 136 105 184 197
SPR
K D (nM) 994 27.3 11.3 5.0 7.5 2.8 2.8
Fold stabilization   36 88 199 133 355 355
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a

FP assay appK D values represent the apparent affinity of the pERα peptide for 14-3-3σ in the absence (DMSO) or presence of compound at 25 μM. Fold stabilization = FP appK D (DMSO)/ FP appK D (with compound).

b

SPR K D values represent the affinity of the pERα peptide for unlabeled 14-3-3σ (DMSO) or 14-3-3σ labeled quantitatively with compounds. Fold stabilization = SPR K D (unlabeled 14-3-3σ)/SPR K D (labeled 14-3-3σ). The K D values for compounds 13, 15B and 16B are the result of a single experiment. The K D values for compounds 14, 15A and 16A are the average of three experiments (14) and two experiments (15A and 16A).

Comparison of the X-ray crystal structures of compounds 15A and 15B bound to the 14-3-3σ/pERα peptide complex revealed that the chlorophenyl and acyl piperidine substituents adopt similar positions and orientations despite an inversion of stereochemistry (Figure A, (R)-15A vs (S)-15B). In contrast, the 2,6-dimethylpiperazine core adopts a significantly different orientation in the two structures. This pattern was also observed for 16A and 16B (Figure B). Importantly, both 15A/15B and 16A/16B make increased hydrophobic and van der Waals contacts with the pERα peptide compared to the original hit compound 9. To highlight this observation, portions of the pERα peptide that reside within van der Waals contact distance of the ligands are represented by a transparent surface, while portions of the peptide outside that radius are represented by an opaque surface. Compounds 16A and 16B clearly show enhanced shape complementarity, and make more extensive contacts with the pERα peptide, than 15A or 15B. In fact, interactions between 16A/16B and the pERα peptide extend well beyond Val595 to include pThr594 and Ala593. Collectively, these observations help to rationalize how these relatively simple ligands achieve such pronounced stabilization activity.

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(A) X-ray crystal structure overlay of compounds 15A (cyan) and 15B (green) bound to 14-3-3σcomplexed with pERα 15-mer peptide. (B) X-ray crystal structure overlay of compounds 16A (magenta) and 16B (yellow) bound to 14-3-3σ complexed with pERα 15-mer peptide. Portions of the pERα peptide that reside within van der Waals contact distance of the ligands are represented by a transparent surface, while portions of the peptide outside that radius are represented by an opaque surface.

To this point, all stabilization results from the FAM-FP assay had been determined with a fixed compound concentration of 25 μM. Given the improved activity observed with compounds 15A and 16A, we proceeded to assess concentration-dependent stabilization in the FAM-FP assay with a 2-h incubation time alongside fusicoccin A (1) and hit compound 9 (Figure ). Compound 9 afforded modest stabilization at all concentrations tested. Compounds 15A and 16A, however, showed levels of stabilization that exceeded that of the natural product fusicoccin A across the concentration range. The comparison at 250 nM is especially striking, as compound 16A produces a 22-fold enhancement of the pERα peptide affinity for 14-3-3σ compared with 1.6-fold for fusicoccin A (see Supplementary Table 2 and Supplementary Figure 5).

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Concentration-dependent stabilization of the 14-3-3σ/pERα peptide complex by compounds 1, 9, 15A, 16A determined using the FAM FP assay with an incubation time of 2 h.

Members of the 14-3-3 family interact with hundreds of client proteins in human cells, thereby regulating a wide array of cellular processes. Selectivity therefore poses a significant challenge in the development of therapeutics that aim to stabilize specific 14-3-3 PPIs. ERα belongs to a relatively small group of client proteins that exhibit a mode III binding interaction with 14-3-3, where the key phosphorylated residue (Thr594) is the penultimate C-terminal amino acid of the protein. To assess selectivity of the newly developed covalent stabilizers, we tested the ability of compounds 1, 9, 15A and 16A to stabilize complexes formed between 14-3-3σ and a set of client peptides with internal binding modes (sometimes referred to as mode I/II clients) using our FAM-FP assay (Figure ). Client protein peptides assessed in this effort included B-RAF pS365, C-RAF pS233pS259, pyrin pS242, YAP pS127, Gab2 pS210pT391 and Chibby pS20. All compounds were tested at a concentration of 25 μM. As expected, fusicoccin A (1) only showed significant stabilization of the complex with pERα peptide, and little to no stabilization of any mode I/II client peptides. Compound 9, as a structurally simple hit compound, was less selective for the pERα peptide complex but did not show strong stabilization of any client in this set. Compounds 15A and 16A showed no ability to promote complex formation with YAP or B-RAF peptides, while low to moderate levels of stabilization were observed for C-RAF, pyrin, Gab2 and Chibby (<14-fold). Stabilization of the complex with pERα peptide was significantly higher, ranging from 130-fold for 15A to 160-fold for 16A under these conditions (see Supplementary Table 3 and Supplementary Figure 6). The improved selectivity of 15A and 16A for ERα compared to 9 is consistent with, and a consequence of, the design strategy described above. It should be noted that because the selectivity panel consisted only of peptides with internal binding modes (i.e., mode I/II clients), no conclusions can be drawn regarding selectivity of 9, 15A and 16A for stabilization of 14-3-3σ complexes with other mode III peptides.

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Biochemical selectivity analysis of compounds 1, 9, 15A, 16A. Binding affinity of each peptide for 14-3-3σ was determined using a fluorescence polarization assay in the absence and presence of compound at 25 μM with an incubation time of 24 h. Fold stabilization = FP appK D (DMSO)/FP appK D (with compound).

During earlier hit validation studies, the chemosensor assay was utilized to assess the rate of labeling of 14-3-3σ with compounds 7–9. Compound 9 labeled 14-3-3σ significantly faster in the presence of pERα peptide, an attractive feature given that nonspecific labeling of 14-3-3σ would be undesirable in view of the important role that the protein plays in cell cycle progression and as a tumor suppressor. Despite preferential labeling of 14-3-3σ in the presence of pERα peptide, k inact/K I values for 9 were very low (as expected for an unoptimized hit). Given the improved stabilization activity and selectivity observed in the FP assay with 15A and 16A (Figure ) we returned to the chemosensor assay to assess the reaction kinetics of these optimized compounds with 14-3-3σ in the presence and absence of pERα peptide.

Both compounds labeled 14-3-3σ significantly faster in the presence of pERα peptide (see Table and Supplementary Figure 9). The data also allowed for extraction of k inact/K I values for each compound in the presence and absence of peptide. As expected, these values were much higher in the presence of peptide and correlated with the extent of stabilization observed by FP assay and SPR with the rank order of 16A > 15A > 9. Comparing the original hit 9 and optimized compound 16A, k inact/K I was improved by a factor of 254 in the presence of pERα peptide, and by a factor of 91 with 14-3-3σ alone. This suggests that the structural features of 16A confer enhanced noncovalent affinity for both 14-3-3σ and the 14-3-3σ/pERα peptide complex (although to a greater extent with the complex). It is notable that the rate and extent of labeling of 14-3-3σ alone by compound 16A is greater compared to compound 9. This is presumably driven by enhanced interactions with the 14-3-3 protein and indicates that further optimization of this scaffold should focus on making additional interactions that are unique to the ERα/14-3-3 interface.

3. k inact/K I Values (/M/s) Determined for Compounds 9, 15A, and 16A for Labeling of 14-3-3σ in the Presence and Absence of pERα Peptide.

  Compound
  9 15A 16A
14-3-3σ 0.4 19.7 36.5
14-3-3σ + pERα peptide 12.1 1329 3075
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Throughout this effort, we were cognizant of the fact that compounds such as 15 and 16 rely on a chloroacetamide electrophile to drive stabilization activity. Such a warhead may present future challenges for optimization of selectivity, PK and safety. It has been noted that chloroacetamides are generally more reactive than acrylamides, which have been successfully utilized in the design of several marketed covalent drugs. , When incubated with buffer containing GSH, the parent compound in the lead series described here (9) exhibited a half-life of 34.2 min, a value that would need to be improved as part of any future optimization effort. Chloroacetamides derived from secondary amines offer fewer options to modulate chemical reactivity compared to acrylamides, and replacement with alternate electrophiles is not straightforward. To date, our own efforts to modify or replace the chloroacetamide group present in the lead series represented by 15 and 16 largely resulted in complete loss of stabilization activity (data not shown). Compound 12 provides an interesting counter-point to this observation by demonstrating that moderate stabilization activity can be achieved with a covalent reversible cyanopyrimidine warhead. However, significant optimization is clearly required to match the activity observed with 16A. To that end, it is encouraging that others have found some success in translating covalent ligands into noncovalent stabilizers of 14-3-3 PPIs, for example, through fragment linking to drive additional noncovalent affinity. Another consideration for optimization of 14-3-3/ERα molecular glues is the fact that 14-3-3σ is the only member of the family to feature a cysteine residue at the key location adjacent to the fusicoccin pocket. The variable expression of 14-3-3σ in metastatic and invasive breast cancer cell lines presents a potential hurdle for covalent stabilizers that rely on the presence of 14-3-3σ to inhibit ERα. − However, if drug-like small-molecule stabilizers of the 14-3-3/ERα interaction can be successfully optimized to yield development candidates, there is significant potential to address limitations in the existing arsenal of ER-targeted therapeutics.

In summary, we have deployed two distinct screening approaches to identify novel molecular glue stabilizers of the interaction between 14-3-3σ and ERα. These efforts have resulted in the discovery of four new chemotypes (6–9) that bind covalently to Cys38 of 14-3-3σ and promote the binding of an ERα phosphopeptide comprised of the 15 C-terminal amino acids of the full-length protein. A focused optimization campaign enabled the discovery of 15A and 16A which exhibit remarkable levels of stabilization as assessed by the FAM FP assay and SPR. At concentrations as low as 250 nM, compound 16A exhibited a 22-fold stabilization of the 14-3-3σ/pERα peptide interaction after 2 h compared to only 1.6-fold for fusicoccin A. Collectively, the data indicate that 16A is among the most effective stabilizers of the 14-3-3σ/ERα complex described to date. Compounds 15A and 16A also demonstrated high levels of selectivity for pERα over a panel of 14-3-3 client protein peptides. X-ray crystal structures of ternary complexes formed between 14-3-3σ, pERα peptide and compounds 15 and 16 revealed increased contact and improved complementarity between the ligands and the pERα peptide that account for improvements seen in both stabilization activity and selectivity. These findings add to the growing body of evidence that small molecule 14-3-3 PPI stabilizers can be prospectively identified and optimized using high-throughput screening approaches and structure-based ligand design.

Supplementary Material

ml6c00325_si_001.pdf (3.6MB, pdf)
ml6c00325_si_002.pdf (5.6MB, pdf)

Acknowledgments

The authors are grateful to Alice Wang, Heidi Struble and Duayne Tokushige for compound purification, chiral preparative SFC and chiral analytical SFC; to Philipp Krastel, Joanne Wong, Etienne Richard, Christian Thibaut and Tim Schuhmann for helpful discussions and access to fusicoccin A; to Damien Hubert and Guillaume Ngo for GSH stability measurements; to Roman Mariano Wille for synthesis of the fluorinated pERα NMR reporter peptide.

Glossary

Abbreviations

CPMG

Carr–Purcell–Meiboom–Gill

ERα

estrogen receptor-α

FP

fluorescence polarization

GSH

glutathione

appK D

apparent dissociation constant

K D

dissociation constant

LBD

ligand binding domain

NMR

nuclear magnetic resonance

PPI

protein–protein interaction

MS

mass spectrometry

SPR

surface plasmon resonance

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.6c00325.

  • NMR spectra (PDF)

  • Detailed methods for protein expression and purification, biochemical assays, screening, kinetic labeling experiments, intact protein MS, glutathione stability assessment, SPR, protein NMR, X-ray crystallography, and synthesis and characterization of new compounds (PDF)

All protein crystal structures have been deposited in the PDB. Accession codes: 11SD (6), 11ZU (7), 11SE (8), 11SF (9), 11SG (11), 11SH (12), 11SI (15A), 11SJ (15B), 9YC4 (16A), 9Y7E (16B).

●.

Department of Research & Early Discovery, PROTEINA Co., Ltd., Seoul, 08380, South Korea

○.

TCG Lab Soleil, South San Francisco, CA 94080, USA

□.

Antares Therapeutics, Boston, MA 02110, USA

■.

Allorion Therapeutics Inc., Natick, MA 01760, USA

▽.

Global Innovation Center, Shanghai Henlius Biotech, Inc., Shanghai 200030, P. R. China

▼.

Complement Design & Discovery, LLC, San Francisco, CA 94127, USA

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

This work was funded by Novartis.

Safety Statement: no unexpected or unusually high safety hazards were encountered.

The authors declare the following competing financial interest(s): All authors were employees of Novartis at the time this work was conducted. Authors who are current or former employees of Novartis may own Novartis stock, restricted stock units or stock options.

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Associated Data

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Supplementary Materials

ml6c00325_si_001.pdf (3.6MB, pdf)
ml6c00325_si_002.pdf (5.6MB, pdf)

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