Straightforward model construction and analysis of multicomponent biomolecular systems in equilibrium.

Mathematical modelling of molecular systems can be extremely helpful in elucidating complex phenomena in (bio)chemistry. However, equilibrium conditions in systems consisting of more than two components, such as for molecular glues bound to two proteins, can typically not be analytically determined without assumptions and (semi-)numerical models are not trivial to derive by the non-expert. Here we present a framework for equilibrium models, geared towards molecular glues and other contemporary multicomponent chemical biology challenges. The framework utilizes a general derivation method capable of generating custom mass-balance models for equilibrium conditions of complex molecular systems, based on the simple, reversible biomolecular reactions describing these systems. Several chemical biology concepts are revisited via the framework to demonstrate the simplicity, generality and validity of the approach. The ease of use of the framework and the ability to both analyze systems and gain additional insights in the underlying parameters driving equilibria formation strongly aids the analysis and understanding of biomolecular systems. New directions for research and analysis are brought forward based on the model formation and system and parameter analysis. This conceptual framework severely reduces the time and expertise requirements which currently impede the broad integration of such valuable equilibrium models into molecular glue development and chemical biology research.

Cooperativity as quantification and optimization paradigm for nuclear receptor modulators.

Nuclear Receptors (NRs) are highly relevant drug targets, for which small molecule modulation goes beyond a simple ligand/receptor interaction. NR-ligands modulate Protein-Protein Interactions (PPIs) with coregulator proteins. Here we bring forward a cooperativity mechanism for small molecule modulation of NR PPIs, using the Peroxisome Proliferator Activated Receptor γ (PPARγ), which describes NR-ligands as allosteric molecular glues. The cooperativity framework uses a thermodynamic model based on three-body binding events, to dissect and quantify reciprocal effects of NR-coregulator binding (K I D) and NR-ligand binding (K II D), jointly recapitulated in the cooperativity factor (α) for each specific ternary ligand·NR·coregulator complex formation. These fundamental thermodynamic parameters allow for a conceptually new way of thinking about structure-activity-relationships for NR-ligands and can steer NR modulator discovery and optimization via a completely novel approach.

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.

Light-driven release of cucurbit[8]uril from a bivalent cage.

Temporal control over supramolecular systems has great potential for the modulation of binding and assembly events, such as providing orthogonal control over protein activity. Especially light controlled triggering provides unique entries for supramolecular systems to interface in a controlled manner with enzymes. Here we report on the light-induced release of cucurbit[8]uril (CB[8]) from a bivalent cage molecule and its subsequent activation of a proteolytic enzyme, caspase-9, that itself is unresponsive to light. Central to the design is the bivalent binding of the cage with high affinity to CB[8], 100-fold stronger than the UV-inactivated products. The affinity switching occurs in the (sub-)micromolar concentration regime, matching the concentration characteristics required for dimerizing and activating caspase-9 by CB[8]. The light-responsive caged CB[8] concept presented offers a novel platform for tuning and application of switchable cucurbiturils and beyond.

Cooperativity basis for small-molecule stabilization of protein-protein interactions.

A cooperativity framework to describe and interpret small-molecule stabilization of protein-protein interactions (PPI) is presented. The stabilization of PPIs is a versatile and emerging therapeutic strategy to target specific combinations of protein partners within the protein interactome. Currently, the potency of PPI stabilizers is typically expressed by their apparent affinity or EC50. Here, we propose that the effect of a PPI stabilizer be best described involving the cooperativity factor, α, between the stabilizer and binding partners in addition to the intrinsic affinity, K D II, of the stabilizer for one of the apo-proteins. By way of illustration, we combine fluorescence polarization measurements with thermodynamic modeling to determine the α and K D II for the PPI stabilization of 14-3-3 and TASK3 by fusicoccin-A (FC-A) and validate our approach by studying other PPI-partners of 14-3-3 proteins. Finally, we characterize a library of different stabilizer compounds, and perform structure-activity relationship studies in which molecular changes could be attributed to either changes in cooperativity or intrinsic affinity. Such insights should aid in the development of more effective protein-protein stabilizer drugs.

Site-Directed Fragment-Based Screening for the Discovery of Protein-Protein Interaction Stabilizers.

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 Thermodynamic Model for Multivalency in 14-3-3 Protein-Protein Interactions.

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.

A Binary Bivalent Supramolecular Assembly Platform Based on Cucurbit[8]uril and Dimeric Adapter Protein 14-3-3.

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.