Modulation of protein–protein interactions (PPIs) by small molecules has emerged as a valuable approach in drug discovery. Compared to direct inhibition, PPI stabilization is vastly underexplored but has strong advantages, including the ability to gain selectivity by targeting an interface formed only upon association of proteins. Here, we present the application of a site-directed screening technique based on disulfide trapping (tethering) to select for fragments that enhance the affinity between protein partners. We target the phosphorylation-dependent interaction between the hub protein 14-3-3σ and a peptide derived from Estrogen Receptor α (ERα), an important breast cancer target that is negatively regulated by 14-3-3σ. We identify orthosteric stabilizers that increase 14-3-3/ERα affinity up to 40-fold and propose the mechanism of stabilization based on X-ray crystal structures. These fragments already display partial selectivity toward ERα-like motifs over other representative 14-3-3 clients. This first of its kind study illustrates the potential of the tethering approach to overcome the hurdles in systematic PPI stabilizer discovery.
A cooperativity framework to describe and interpret small-molecule stabilization of protein–protein interactions (PPI) is presented, which allows elucidating structure–activity relationships regarding cooperativity and intrinsic affinity.
Interactions between proteins frequently involve recognition sequences based on multivalent binding events. Dimeric 14‐3‐3 adapter proteins are a prominent example and typically bind partner proteins in a phosphorylation‐dependent mono‐ or bivalent manner. Herein we describe the development of a cucurbit[8]uril (Q8)‐based supramolecular system, which in conjunction with the 14‐3‐3 protein dimer acts as a binary and bivalent protein assembly platform. We fused the phenylalanine–glycine–glycine (FGG) tripeptide motif to the N‐terminus of the 14‐3‐3‐binding epitope of the estrogen receptor α (ERα) for selective binding to Q8. Q8‐induced dimerization of the ERα epitope augmented its affinity towards 14‐3‐3 through a binary bivalent binding mode. The crystal structure of the Q8‐induced ternary complex revealed molecular insight into the multiple supramolecular interactions between the protein, the peptide, and Q8.
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.
Protein–protein interactions (PPIs) are at the core of molecular control over cellular function. Multivalency in PPI formation, such as via proteins with multiple binding sites and different valencies, requires fundamental understanding to address correlated challenges in pathologies and drug development. Thermodynamic binding models are needed to provide frameworks for describing multivalent PPIs. We established a model based on ditopic host–guest systems featuring the effective molarity, a hallmark property of multivalency, as a prime parameter governing the intramolecular binding in divalent interactions. By way of illustration, we study the interaction of the bivalent 14-3-3 protein scaffold with both the nonavalent CFTR and the hexavalent LRRK2 proteins, determining the underlying thermodynamics and providing insights into the role of individual sites in the context of the multivalent platform. Fitting of binding data reveals enthalpy–entropy correlation in both systems. Simulations of speciations for the entire phosphorylated protein domains reveal that the CFTR protein preferably binds to 14-3-3 by combinations including the strongest binding site pS768, but that other binding sites take over when this site is eliminated, leading to only a minor decrease in total affinity for 14-3-3. For LRRK2, two binding sites dominate the complex formation with 14-3-3, but the distantly located pS1444 site also plays a role in complex formation. Thermodynamic modeling of these multivalent PPIs allowed analyzing and predicting the effects of individual sites regarding their modulation via, for example, (de)phosphorylation or small-molecule targeting. The results specifically bring forward the potential of PPI stabilization, as an entry for drug discovery for multivalent PPIs.
The natural product family of fusicoccanes are stabilizers of 14-3-3 mediated protein-protein interactions (PPIs), some of which possess antitumor activity. In this study, the first use of molecular dynamics (MD) to rationally design PPI stabilizers with increased potency is presented. Synthesis of a focused library, with subsequent characterization by fluorescence polarization, mutational studies, and X-ray crystallography confirmed the power of the MD-based design approach, revealing the potential for an additional hydrogen bond with the 14-3-3 protein to lead to significantly increased potency. Additionally, these compounds exert their action in a cellular environment with increased potency. The newly found polar interaction could provide an anchoring point for new small-molecule PPI stabilizers. These results facilitate the development of fusicoccanes towards drugs or tool compounds, as well as allowing the study of the fundamental principles behind PPI stabilization.
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.
The photochemistry of a variety of 1-alkoxy-and l-(benzyloxy)-9,1O-anthraquinones in methanol has been examined. In the absence of oxygen the primary photoproducts of these reactions are 9,lO-anthrahydroquinones with MeOCH20 and ArCH(0Me)O groups at the 1 -position, respectively. Quenching studies established the multiplicity of the excited state to be triplet. Deuterium isotope effects, substituent effects, and solvent polarity studies support a mechanism in which a biradical intermediate is formed by an intramolecular &hydrogen atom transfer that occurs in one step but not a reaction pathway in which the hydrogen atom transfer is accomplished in two discrete steps via separate electron and proton transfers. Apparent rate constants for the disappearance of 1-@-XPhCH20)-2-methyl-9,lO-anthraquinone (X = H, CI, CH3, NO2, OCH3, CF3, and CN) were measured and found to vary by a factor of 3.4 from the largest (X = NOz) to the smallest (X = OCHJ. Captodative interactions account for the small differences in these rate constants.
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