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.

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.

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.

Functional mapping of the 14-3-3 hub protein as a guide to design 14-3-3 molecular glues.

Molecular glues represent an evolution in drug discovery, however, targeted stabilization of protein complexes remains challenging, owing to a paucity of drug design rules. The functional mapping of hotspots has been critical to protein-protein interaction (PPI) inhibitor research, however, the orthogonal approach to stabilize PPIs has not exploited this information. Utilizing the hub protein 14-3-3 as a case study we demonstrate that functional mapping of hotspots provides a triage map for 14-3-3 molecular glue development. Truncation and mutation studies allowed deconvoluting the energetic contributions of sidechain and backbone interactions of a 14-3-3-binding non-natural peptide. Three central 14-3-3 hotspots were identified and their thermodynamic characteristics profiled. In addition to the phospho-binding pocket; (i) Asn226, (ii) Lys122 and (iii) the hydrophobic patch formed by Leu218, Ile219 and Leu222 were critical for protein complex formation. Exploiting this hotspot information allowed a peptide-based molecular glue that elicits high cooperativity (α = 36) and selectively stabilizes the 14-3-3/ChREBP PPI to be uniquely developed.

Covalent flexible peptide docking in Rosetta.

Electrophilic peptides that form an irreversible covalent bond with their target have great potential for binding targets that have been previously considered undruggable. However, the discovery of such peptides remains a challenge. Here, we present Rosetta CovPepDock, a computational pipeline for peptide docking that incorporates covalent binding between the peptide and a receptor cysteine. We applied CovPepDock retrospectively to a dataset of 115 disulfide-bound peptides and a dataset of 54 electrophilic peptides. It produced a top-five scoring, near-native model, in 89% and 100% of the cases when docking from the native conformation, and 20% and 90% when docking from an extended peptide conformation, respectively. In addition, we developed a protocol for designing electrophilic peptide binders based on known non-covalent binders or protein-protein interfaces. We identified 7154 peptide candidates in the PDB for application of this protocol. As a proof-of-concept we validated the protocol on the non-covalent complex of 14-3-3σ and YAP1 phosphopeptide. The protocol identified seven highly potent and selective irreversible peptide binders. The predicted binding mode of one of the peptides was validated using X-ray crystallography. This case-study demonstrates the utility and impact of CovPepDock. It suggests that many new electrophilic peptide binders can be rapidly discovered, with significant potential as therapeutic molecules and chemical probes.

Structure-Activity Relationship Studies of Trisubstituted Isoxazoles as Selective Allosteric Ligands for the Retinoic-Acid-Receptor-Related Orphan Receptor γt.

The inhibition of the nuclear receptor retinoic-acid-receptor-related orphan receptor γt (RORγt) is a promising strategy in the treatment of autoimmune diseases. RORγt features an allosteric binding site within its ligand-binding domain that provides an opportunity to overcome drawbacks associated with orthosteric modulators. Recently, trisubstituted isoxazoles were identified as a novel class of allosteric RORγt inverse agonists. This chemotype offers new opportunities for optimization into selective and efficacious allosteric drug-like molecules. Here, we explore the structure-activity relationship profile of the isoxazole series utilizing a combination of structure-based design, X-ray crystallography, and biochemical assays. The initial lead isoxazole (FM26) was optimized, resulting in compounds with a ∼10-fold increase in potency (low nM), significant cellular activity, promising pharmacokinetic properties, and a good selectivity profile over the peroxisome-proliferated-activated receptor γ and the farnesoid X receptor. We envisage that this work will serve as a platform for the accelerated development of isoxazoles and other novel chemotypes for the effective allosteric targeting of RORγt.

Fluorescence Anisotropy-Based Tethering for Discovery of Protein-Protein Interaction Stabilizers.

Protein-protein interaction (PPI) networks are fundamental for cellular processes. Small-molecule PPI enhancers have been shown to be powerful tools to fundamentally study PPIs and as starting points for potential new therapeutics. Yet, systematic approaches for their discovery are not widely available, and the design prerequisites of "molecular glues" are poorly understood. Covalent fragment-based screening can identify chemical starting points for these enhancers at specific sites in PPI interfaces. We recently reported a mass spectrometry-based disulfide-trapping (tethering) approach for a cysteine residue in the hub protein 14-3-3, an important regulator of phosphorylated client proteins. Here, we invert the strategy and report the development of a functional read-out for systematic identification of PPI enhancers based on fluorescence anisotropy (FA-tethering) with the reactive handle now on a client-derived peptide. Using the DNA-binding domain of the nuclear receptor Estrogen Related Receptor gamma (ERRγ), we target a native cysteine positioned at the 14-3-3 PPI interface and identify several fragments that form a disulfide bond to ERRγ and stabilize the complex up to 5-fold. Crystallography indicates that fragments bind in a pocket comprised of 14-3-3 and the ERRγ phosphopeptide. FA-tethering presents a streamlined methodology to discover molecular glues for protein complexes.

Ultra-large chemical libraries for the discovery of high-affinity peptide binders.

High-diversity genetically-encoded combinatorial libraries (108-1013 members) are a rich source of peptide-based binding molecules, identified by affinity selection. Synthetic libraries can access broader chemical space, but typically examine only ~ 106 compounds by screening. Here we show that in-solution affinity selection can be interfaced with nano-liquid chromatography-tandem mass spectrometry peptide sequencing to identify binders from fully randomized synthetic libraries of 108 members-a 100-fold gain in diversity over standard practice. To validate this approach, we show that binders to a monoclonal antibody are identified in proportion to library diversity, as diversity is increased from 106-108. These results are then applied to the discovery of p53-like binders to MDM2, and to a family of 3-19 nM-affinity, α/ß-peptide-based binders to 14-3-3. An X-ray structure of one of these binders in complex with 14-3-3σ is determined, illustrating the role of ß-amino acids in facilitating a key binding contact.