Molecular basis and dual ligand regulation of tetrameric estrogen receptor α/14-3-3ζ protein complex.

Therapeutic strategies targeting nuclear receptors (NRs) beyond their endogenous ligand binding pocket have gained significant scientific interest driven by a need to circumvent problems associated with drug resistance and pharmacological profile. The hub protein 14-3-3 is an endogenous regulator of various NRs, providing a novel entry point for small molecule modulation of NR activity. Exemplified, 14-3-3 binding to the C-terminal F-domain of the estrogen receptor alpha (ERα), and small molecule stabilization of the ERα/14-3-3ζ protein complex by the natural product Fusicoccin A (FC-A), was demonstrated to downregulate ERα-mediated breast cancer proliferation. This presents a novel drug discovery approach to target ERα; however, structural and mechanistic insights into ERα/14-3-3 complex formation are lacking. Here, we provide an in-depth molecular understanding of the ERα/14-3-3ζ complex by isolating 14-3-3ζ in complex with an ERα protein construct comprising its ligand-binding domain (LBD) and phosphorylated F-domain. Bacterial co-expression and co-purification of the ERα/14-3-3ζ complex, followed by extensive biophysical and structural characterization, revealed a tetrameric complex between the ERα homodimer and the 14-3-3ζ homodimer. 14-3-3ζ binding to ERα, and ERα/14-3-3ζ complex stabilization by FC-A, appeared to be orthogonal to ERα endogenous agonist (E2) binding, E2-induced conformational changes, and cofactor recruitment. Similarly, the ERα antagonist 4-hydroxytamoxifen inhibited cofactor recruitment to the ERα LBD while ERα was bound to 14-3-3ζ. Furthermore, stabilization of the ERα/14-3-3ζ protein complex by FC-A was not influenced by the disease-associated and 4-hydroxytamoxifen resistant ERα-Y537S mutant. Together, these molecular and mechanistic insights provide direction for targeting ERα via the ERα/14-3-3 complex as an alternative drug discovery approach.

14-3-3 Protein-Protein Interactions: From Mechanistic Understanding to Their Small-Molecule Stabilization.

Protein-protein interactions (PPIs) are of utmost importance for maintenance of cellular homeostasis. Herein, a central role can be found for 14-3-3 proteins. These hub-proteins are known to bind hundreds of interaction partners, thereby regulating their activity, localization, and/or stabilization. Due to their ability to bind a large variety of client proteins, studies of 14-3-3 protein complexes flourished over the last decades, aiming to gain greater molecular understanding of these complexes and their role in health and disease. Because of their crucial role within the cell, 14-3-3 protein complexes are recognized as highly interesting therapeutic targets, encouraging the discovery of small molecule modulators of these PPIs. We discuss various examples of 14-3-3-mediated regulation of its binding partners on a mechanistic level, highlighting the versatile and multi-functional role of 14-3-3 within the cell. Furthermore, an overview is given on the development of stabilizers of 14-3-3 protein complexes, from initially used natural products to fragment-based approaches. These studies show the potential of 14-3-3 PPI stabilizers as novel agents in drug discovery and as tool compounds to gain greater molecular understanding of the role of 14-3-3-based protein regulation.

Discovery of 14-3-3 PPI Stabilizers by Extension of an Amidine-Substituted Thiophene Fragment.

Protein-protein interaction (PPI) modulation is a promising approach in drug discovery with the potential to expand the 'druggable' proteome and develop new therapeutic strategies. While there have been significant advancements in methodologies for developing PPI inhibitors, there is a relative scarcity of literature describing the 'bottom-up' development of PPI stabilizers (Molecular Glues). The hub protein 14-3-3 and its interactome provide an excellent platform for exploring conceptual approaches to PPI modulation, including evolution of chemical matter for Molecular Glues. In this study, we employed a fragment extension strategy to discover stabilizers for the complex of 14-3-3 protein and an Estrogen Receptor alpha-derived peptide (ERα). A focused library of analogues derived from an amidine-substituted thiophene fragment enhanced the affinity of the 14-3-3/ERα complex up to 6.2-fold. Structure-activity relationship (SAR) analysis underscored the importance of the newly added, aromatic side chain with a certain degree of rigidity. X-ray structural analysis revealed a unique intermolecular π-π stacking binding mode of the most active analogues, resulting in the simultaneous binding of two molecules to the PPI binding pocket. Notably, analogue 11 displayed selective stabilization of the 14-3-3/ERα complex.

Reversible Dual-Covalent Molecular Locking of the 14-3-3/ERRγ Protein-Protein Interaction as a Molecular Glue Drug Discovery Approach.

Molecules that stabilize protein-protein interactions (PPIs) are invaluable as tool compounds for biophysics and (structural) biology, and as starting points for molecular glue drug discovery. However, identifying initial starting points for PPI stabilizing matter is highly challenging, and chemical optimization is labor-intensive. Inspired by chemical crosslinking and reversible covalent fragment-based drug discovery, we developed an approach that we term "molecular locks" to rapidly access molecular glue-like tool compounds. These dual-covalent small molecules reversibly react with a nucleophilic amino acid on each of the partner proteins to dynamically crosslink the protein complex. The PPI between the hub protein 14-3-3 and estrogen-related receptor γ (ERRγ) was used as a pharmacologically relevant case study. Based on a focused library of dual-reactive small molecules, a molecular glue tool compound was rapidly developed. Biochemical assays and X-ray crystallographic studies validated the ternary covalent complex formation and overall PPI stabilization via dynamic covalent crosslinking. The molecular lock approach is highly selective for the specific 14-3-3/ERRγ complex, over other 14-3-3 complexes. This selectivity is driven by the interplay of molecular reactivity and molecular recognition of the composite PPI binding interface. The long lifetime of the dual-covalent locks enabled the selective stabilization of the 14-3-3/ERRγ complex even in the presence of several other competing 14-3-3 clients with higher intrinsic binding affinities. The molecular lock approach enables systematic, selective, and potent stabilization of protein complexes to support molecular glue drug discovery.

Structure-Based Optimization of Covalent, Small-Molecule Stabilizers of the 14-3-3σ/ERα Protein-Protein Interaction from Nonselective Fragments.

The stabilization of protein-protein interactions (PPIs) has emerged as a promising strategy in chemical biology and drug discovery. The identification of suitable starting points for stabilizing native PPIs and their subsequent elaboration into selective and potent molecular glues lacks structure-guided optimization strategies. We have previously identified a disulfide fragment that stabilized the hub protein 14-3-3σ bound to several of its clients, including ERα and C-RAF. Here, we show the structure-based optimization of the nonselective fragment toward selective and highly potent small-molecule stabilizers of the 14-3-3σ/ERα complex. The more elaborated molecular glues, for example, show no stabilization of 14-3-3σ/C-RAF up to 150 µM compound. Orthogonal biophysical assays, including mass spectrometry and fluorescence anisotropy, were used to establish structure-activity relationships. The binding modes of 37 compounds were elucidated with X-ray crystallography, which further assisted the concomitant structure-guided optimization. By targeting specific amino acids in the 14-3-3σ/ERα interface and locking the conformation with a spirocycle, the optimized covalent stabilizer 181 achieved potency, cooperativity, and selectivity similar to the natural product Fusicoccin-A. This case study showcases the value of addressing the structure, kinetics, and cooperativity for molecular glue development.

Crystal structure and ligandability of the 14-3-3/pyrin interface.

Overactivation of Pyrin is the cause of the inflammatory diseases Mediterranean Fever and Pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND). Binding of 14-3-3 proteins reduces the pro-inflammatory activity of Pyrin, hence small molecules that stabilize the Pyrin/14-3-3 complex could convey an anti-inflammatory effect. We have solved the atomic resolution crystal structures of phosphorylated peptides derived from PyrinpS208 and PyrinpS242 - the two principle 14-3-3 binding sites in Pyrin - in complex with 14-3-3 and analyzed the ligandability of these protein-peptide interfaces by crystal-based fragment soaking. The complex between 14-3-3 and PyrinpS242 appears to be much more amenable for small-molecule binding than that of 14-3-3/PyrinpS208. Consequently, only for the 14-3-3/PyrinpS242 complex could we find an interface-binding fragment, validating protein crystallography and fragment soaking as a method to evaluate the ligandability of protein surfaces.

From Tethered to Freestanding Stabilizers of 14-3-3 Protein-Protein Interactions through Fragment Linking.

Small-molecule stabilization of protein-protein interactions (PPIs) is a promising strategy in chemical biology and drug discovery. However, the systematic discovery of PPI stabilizers remains a largely unmet challenge. Herein we report a fragment-linking approach targeting the interface of 14-3-3 and a peptide derived from the estrogen receptor alpha (ERα) protein. Two classes of fragments-a covalent and a noncovalent fragment-were co-crystallized and subsequently linked, resulting in a noncovalent hybrid molecule in which the original fragment interactions were largely conserved. Supported by 20 crystal structures, this initial hybrid molecule was further optimized, resulting in selective, 25-fold stabilization of the 14-3-3/ERα interaction. The high-resolution structures of both the single fragments, their co-crystal structures and those of the linked fragments document a feasible strategy to develop orthosteric PPI stabilizers by linking to an initial tethered fragment.

Glucocorticoid receptor Thr524 phosphorylation by MINK1 induces interactions with 14-3-3 protein regulators.

The glucocorticoid receptor (GR) is a ligand-dependent transcription factor that plays a central role in inflammation. The GR activity is also modulated via protein-protein interactions, including binding of 14-3-3 proteins induced by GR phosphorylation. However, the specific phosphorylation sites on the GR that trigger these interactions and their functional consequences are less clear. Hence, we sought to examine this system in more detail. We used phosphorylated GR peptides, biophysical studies, and X-ray crystallography to identify key residues within the ligand-binding domain of the GR, T524 and S617, whose phosphorylation results in binding of the representative 14-3-3 protein 14-3-3ζ. A kinase screen identified misshapen-like kinase 1 (MINK1) as responsible for phosphorylating T524 and Rho-associated protein kinase 1 for phosphorylating S617; cell-based approaches confirmed the importance of both GR phosphosites and MINK1 but not Rho-associated protein kinase 1 alone in inducing GR-14-3-3 binding. Together our results provide molecular-level insight into 14-3-3-mediated regulation of the GR and highlight both MINK1 and the GR-14-3-3 axis as potential targets for future therapeutic intervention.

Indazole MRL-871 interacts with PPARγ via a binding mode that induces partial agonism.

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) plays a central role in metabolic processes. PPARγ full agonists have side effects, arguing for the discovery of PPARγ partial agonists with novel chemotypes. We report the unique binding mode of the known allosteric retinoic acid receptor-related orphan receptor gamma t (RORγt) ligand MRL-871 to PPARγ. MRL-871 binds between PPARγ helices 3, 5, 7 and 11, where it stabilizes the beta-sheet region with a hydrogen bond between its carboxylic acid moiety and PPARγ Ser370. Its unique binding mode differs from that of the benzoyl 2-methyl indoles which are well-studied, structurally similar, PPARγ ligands. MRL-871's high affinity for PPARγ induces only limited coactivator stabilization, highlighting its attractive partial agonistic characteristics. Affinity comparison of MRL-871 and related compounds towards both RORγt and PPARγ indicates the possibility for tuning of selectivity, bringing MRL-871 forward as an interesting starting point for novel PPARγ ligands.

Computational model predicts protein binding sites of a luminescent ligand equipped with guanidiniocarbonyl-pyrrole groups.

The 14-3-3 protein family, one of the first discovered phosphoserine/phosphothreonine binding proteins, has attracted interest not only because of its important role in the cell regulatory processes but also due to its enormous number of interactions with other proteins. Here, we use a computational approach to predict the binding sites of the designed hybrid compound featuring aggregation-induced emission luminophores as a potential supramolecular ligand for 14-3-3ζ in the presence and absence of C-Raf peptides. Our results suggest that the area above and below the central pore of the dimeric 14-3-3ζ protein is the most probable binding site for the ligand. Moreover, we predict that the position of the ligand is sensitive to the presence of phosphorylated C-Raf peptides. With a series of experiments, we confirmed the computational prediction of two C 2 related, dominating binding sites on 14-3-3ζ that may bind to two of the supramolecular ligand molecules.

MMP activation-associated aminopeptidase N reveals a bivalent 14-3-3 binding motif.

Aminopeptidase N (APN, CD13) is a transmembrane ectopeptidase involved in many crucial cellular functions. Besides its role as a peptidase, APN also mediates signal transduction and is involved in the activation of matrix metalloproteinases (MMPs). MMPs function in tissue remodeling within the extracellular space and are therefore involved in many human diseases, such as fibrosis, rheumatoid arthritis, tumor angiogenesis, and metastasis, as well as viral infections. However, the exact mechanism that leads to APN-driven MMP activation is unclear. It was previously shown that extracellular 14-3-3 adapter proteins bind to APN and thereby induce the transcription of MMPs. As a first step, we sought to identify potential 14-3-3-binding sites in the APN sequence. We constructed a set of phosphorylated peptides derived from APN to probe for interactions. We identified and characterized a canonical 14-3-3-binding site (site 1) within the flexible, structurally unresolved N-terminal APN region using direct binding fluorescence polarization assays and thermodynamic analysis. In addition, we identified a secondary, noncanonical binding site (site 2), which enhances the binding affinity in combination with site 1 by many orders of magnitude. Finally, we solved crystal structures of 14-3-3σ bound to mono- and bis-phosphorylated APN-derived peptides, which revealed atomic details of the binding mode of mono- and bivalent 14-3-3 interactions. Therefore, our findings shed some light on the first steps of APN-mediated MMP activation and open the field for further investigation of this important signaling pathway.

Supramolecular Enhancement of a Natural 14-3-3 Protein Ligand.

Rational design of protein-protein interaction (PPI) inhibitors is challenging. Connecting a general supramolecular protein binder with a specific peptidic ligand provides a novel conceptual approach. Thus, lysine-specific molecular tweezers were conjugated to a peptide-based 14-3-3 ligand and produced a strong PPI inhibitor with 100-fold elevated protein affinity. X-ray crystal structure elucidation of this supramolecular directed assembly provides unique molecular insight into the binding mode and fully aligns with Molecular Dynamics (MD) simulations. This new supramolecular chemical biology concept opens the path to novel chemical tools for studying PPIs.

Reversible Covalent Imine-Tethering for Selective Stabilization of 14-3-3 Hub Protein Interactions.

The stabilization of protein complexes has emerged as a promising modality, expanding the number of entry points for novel therapeutic intervention. Targeting proteins that mediate protein-protein interactions (PPIs), such as hub proteins, is equally challenging and rewarding as they offer an intervention platform for a variety of diseases, due to their large interactome. 14-3-3 hub proteins bind phosphorylated motifs of their interaction partners in a conserved binding channel. The 14-3-3 PPI interface is consequently only diversified by its different interaction partners. Therefore, it is essential to consider, additionally to the potency, also the selectivity of stabilizer molecules. Targeting a lysine residue at the interface of the composite 14-3-3 complex, which can be targeted explicitly via aldimine-forming fragments, we studied the de novo design of PPI stabilizers under consideration of potential selectivity. By applying cooperativity analysis of ternary complex formation, we developed a reversible covalent molecular glue for the 14-3-3/Pin1 interaction. This small fragment led to a more than 250-fold stabilization of the 14-3-3/Pin1 interaction by selective interfacing with a unique tryptophan in Pin1. This study illustrates how cooperative complex formation drives selective PPI stabilization. Further, it highlights how specific interactions within a hub proteins interactome can be stabilized over other interactions with a common binding motif.

Targeting the Surface of the Protein 14-3-3 by Ultrasmall (1.5†nm) Gold Nanoparticles Carrying the Specific Peptide CRaf.

The surface of ultrasmall gold nanoparticles with an average diameter of 1.55 nm was conjugated with a 14-3-3 protein-binding peptide derived from CRaf. Each particle carries 18 CRaf peptides, leading to an overall stoichiometry of Au(115)Craf(18). The binding to the protein 14-3-3 was probed by isothermal titration calorimetry (ITC) and fluorescence polarization spectroscopy (FP). The dissociation constant (KD ) was measured as 5.0 µM by ITC and 0.9 µM by FP, which was close to the affinity of dissolved CRaf to 14-3-3σ. In contrast to dissolved CRaf, which alone did not enter HeLa cells, CRAF-conjugated gold nanoparticles were well taken up by HeLa cells, opening the opportunity to target the protein inside a cell.

14-3-3 modulation of the inflammatory response.

Regulation of inflammation is a central part of the maintenance of homeostasis by the immune system. One important class of regulatory protein that has been shown to have effects on the inflammatory process are the 14-3-3 proteins. Herein we describe the roles that have been identified for 14-3-3 in regulation of the inflammatory response. These roles encompass regulation of the response that affect inflammation at the genetic, molecular and cellular levels. At a genetic level 14-3-3 is involved in the regulation of multiple transcription factors and affects the transcription of key effectors of the immune response. At a molecular level many of the constituent parts of the inflammatory process, such as pattern recognition receptors, protease activated receptors and cytokines are regulated through phosphorylation and recognition by 14-3-3 whilst disruption of the recognition processes has been observed to result in clinical syndromes. 14-3-3 is also involved in the regulation of cell proliferation and differentiation, this has been shown to affect the immune system, particularly T- and B-cells. Finally, we discuss how abnormal levels of 14-3-3 contribute to undesirable immune responses and chronic inflammatory conditions.

A biophysical and structural analysis of the interaction of BLNK with 14-3-3 proteins.

B-cell linker protein (BLNK) is an adaptor protein that orchestrates signalling downstream of B-cell receptors. It has been reported to undergo proteasomal degradation upon binding to 14-3-3 proteins. Here, we report the first biophysical and structural study of this protein-protein interaction (PPI). Specifically, we investigated the binding of mono- and di- phosphorylated BLNK peptides to 14-3-3 using fluorescent polarization (FP) and isothermal titration calorimetry assays (ITC). Our results suggest that BLNK interacts with 14-3-3 according to the gatekeeper model, where HPK1 mediated phosphorylation of Thr152 (pT152) allows BLNK anchoring to 14-3-3, and an additional phosphorylation of Ser285 (pS285) by AKT, then further improves the affinity. Finally, we have also solved a crystal structure of the BLNKpT152 peptide bound to 14-3-3σ. These findings could serve as important tool for compound discovery programs aiming to modulate this interaction with 14-3-3.

AMPK and AKT protein kinases hierarchically phosphorylate the N-terminus of the FOXO1 transcription factor, modulating interactions with 14-3-3 proteins.

Forkhead box protein O1 (FOXO1) is a transcription factor involved in various cellular processes such as glucose metabolism, development, stress resistance, and tumor suppression. FOXO1's transcriptional activity is controlled by different environmental cues through a myriad of posttranslational modifications. In response to growth factors, the serine/threonine kinase AKT phosphorylates Thr24 and Ser256 in FOXO1 to stimulate binding of 14-3-3 proteins, causing FOXO1 inactivation. In contrast, low nutrient and energy levels induce FOXO1 activity. AMP-activated protein kinase (AMPK), a master regulator of cellular energy homeostasis, partly mediates this effect through phosphorylation of Ser383 and Thr649 in FOXO1. In this study, we identified Ser22 as an additional AMPK phosphorylation site in FOXO1's N terminus, with Ser22 phosphorylation preventing binding of 14-3-3 proteins. The crystal structure of a FOXO1 peptide in complex with 14-3-3 σ at 2.3 Å resolution revealed that this is a consequence of both steric hindrance and electrostatic repulsion. Furthermore, we found that AMPK-mediated Ser22 phosphorylation impairs Thr24 phosphorylation by AKT in a hierarchical manner. Thus, numerous mechanisms maintain FOXO1 activity via AMPK signaling. AMPK-mediated Ser22 phosphorylation directly and indirectly averts binding of 14-3-3 proteins, whereas phosphorylation of Ser383 and Thr649 complementarily stimulates FOXO1 activity. Our results shed light on a mechanism that integrates inputs from both AMPK and AKT signaling pathways in a small motif to fine-tune FOXO1 transcriptional activity.

Selectivity via Cooperativity: Preferential Stabilization of the p65/14-3-3 Interaction with Semisynthetic Natural Products.

Natural compounds are an important class of potent drug molecules including some retrospectively found to act as stabilizers of protein-protein interactions (PPIs). However, the design of synthetic PPI stabilizers remains an understudied approach. To date, there are limited examples where cooperativity has been utilized to guide the optimization of a PPI stabilizer. The 14-3-3 scaffold proteins provide an excellent platform to explore PPI stabilization because these proteins mediate several hundred PPIs, and a class of natural compounds, the fusicoccanes, are known to stabilize a subset of 14-3-3 protein interactions. 14-3-3 has been reported to negatively regulate the p65 subunit of the NF-κB transcription factor, which qualifies this protein complex as a potential target for drug discovery to control cell proliferation. Here, we report the high-resolution crystal structures of two 14-3-3 binding motifs of p65 in complex with 14-3-3. A semisynthetic natural product derivative, DP-005, binds to an interface pocket of the p65/14-3-3 complex and concomitantly stabilizes it. Cooperativity analyses of this interaction, and other disease relevant 14-3-3-PPIs, demonstrated selectivity of DP-005 for the p65/14-3-3 complex. The adaptation of a cooperative binding model provided a general approach to characterize stabilization and to assay for selectivity of PPI stabilizers.

Small-molecule modulation of p53 protein-protein interactions.

Small-molecule modulation of protein-protein interactions (PPIs) is a very promising but also challenging area in drug discovery. The tumor suppressor protein p53 is one of the most frequently altered proteins in human cancers, making it an attractive target in oncology. 14-3-3 proteins have been shown to bind to and positively regulate p53 activity by protecting it from MDM2-dependent degradation or activating its DNA binding affinity. PPIs can be modulated by inhibiting or stabilizing specific interactions by small molecules. Whereas inhibition has been widely explored by the pharmaceutical industry and academia, the opposite strategy of stabilizing PPIs still remains relatively underexploited. This is rather interesting considering the number of natural compounds like rapamycin, forskolin and fusicoccin that exert their activity by stabilizing specific PPIs. In this review, we give an overview of 14-3-3 interactions with p53, explain isoform specific stabilization of the tumor suppressor protein, explore the approach of stabilizing the 14-3-3σ-p53 complex and summarize some promising small molecules inhibiting the p53-MDM2 protein-protein interaction.

Conjugated Protein Domains as Engineered Scaffold Proteins.

Assembly of proteins into higher-order complexes generates specificity and selectivity in cellular signaling. Signaling complex formation is facilitated by scaffold proteins that use modular scaffolding domains, which recruit specific pathway enzymes. Multimerization and recombination of these conjugated native domains allows the generation of libraries of engineered multidomain scaffold proteins. Analysis of these engineered proteins has provided molecular insight into the regulatory mechanism of the native scaffold proteins and the applicability of these synthetic variants. This topical review highlights the use of engineered, conjugated multidomain scaffold proteins on different length scales in the context of synthetic signaling pathways, metabolic engineering, liquid-liquid phase separation, and hydrogel formation.