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

Bente A. Somsen, Rick J.C. Schellekens, Carlo J.A. Verhoef, Michelle R. Arkin, Christian Ottmann, Peter J. Cossar, and Luc Brunsveld

Journal of the American Chemical Society 2023

DOI: 10.1021/jacs.2c12781

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.

Quantifying KRAS G12C Covalent Drug Inhibitor Activity in Mouse Tumors Using Mass Spectrometry

John C. Tran, Thomas Hunsaker, Christina Bell, Taylur P. Ma, Emily Chan, Pablo Saenz-Lopez Larrocha, Kelsey Homyk, Liling Liu, Hank La, Jialin Mao, Cecile C. de la Cruz, Kebing Yu, Maureen Beresini, William F. Forrest, Yang Xiao, Anne Jang, Natalia Samus, Nicholas Dupuis Stesco, Marija Mentinova, Stephane Parent, Gwenael Pottiez, Michael Schirm, Hans E. Purkey, Yichin Liu, and Mark Merchant

Analytical Chemistry 2023

DOI: 10.1021/acs.analchem.2c04417

The growing opportunities recognized for covalent drug inhibitors, like KRAS G12C inhibitors, are driving the need for mass spectrometry methods that can quickly and robustly measure therapeutic drug activity in vivo for drug discovery research and development. Effective front-end sample preparation is critical for proteins extracted from tumors but is generally labor intensive and impractical for large sample numbers typical in pharmacodynamic (PD) studies. Herein, we describe an automated and integrated sample preparation method for the measurement of activity levels of KRAS G12C drug inhibitor alkylation from complex tumor samples involving high throughput detergent removal and preconcentration followed by quantitation using mass spectrometry. We introduce a robust assay with an average intra-assay coefficient of variation (CV) of 4% and an interassay CV of 6% obtained from seven studies, enabling us to understand the relationship between KRAS G12C target occupancy and the therapeutic PD effect from mouse tumor samples. Further, the data demonstrated that the drug candidate GDC-6036, a KRAS G12C covalent inhibitor, shows dose-dependent target inhibition (KRAS G12C alkylation) and MAPK pathway inhibition, which correlate with high antitumor potency in the MIA PaCa-2 pancreatic xenograft model.

Development and applications of chimera platforms for tyrosine phosphorylation

Rajaiah Pergu, Veronika M. Shoba, Santosh K. Chaudhary, Dhanushka N. P. Munkanatta Godage, Arghya Deb, Santanu Singha, Uttam Dhawa, Viktoriya Anokhina, Sameek Singh, Sachini U. Siriwardena, Amit Choudhary

bioRxiv 2023.03.05.531183; 

doi: https://doi.org/10.1101/2023.03.05.531183

Chimeric small molecules that induce post-translational modification (PTM) on a target protein by bringing it in proximity to a PTM-inducing enzyme are furnishing novel modalities to perturb protein function. Despite recent advances, such molecules are unavailable for a critical PTM, tyrosine phosphorylation. Furthermore, the contemporary design paradigm of chimeric molecules, formed by joining a non-inhibitory binder of the PTM-inducing enzyme with the binder of the target protein, prohibits the recruitment of most PTM-inducing enzymes as their non-inhibitory binders are unavailable. Here, we report two platforms to generate phosphorylation-inducing chimeric small molecules (PHICS) for tyrosine phosphorylation. We generate PHICS from both non-inhibitory binders (scantily available, platform 1) and kinase inhibitors (abundantly available, platform 2) using cysteine-based group transfer chemistry. PHICS triggered phosphorylation on tyrosine residues in diverse sequence contexts and target proteins (e.g., membrane-associated, cytosolic) and displayed multiple bioactivities, including initiation of a growth receptor signaling cascade and death of drug-resistant cancer cells. These studies provide an approach to induce biologically relevant PTM and lay the foundation for pharmacologic PTM editing (i.e., induction or removal) on target proteins using abundantly available inhibitors of PTM-inducing or erasing enzymes.

A covalent BTK ternary complex compatible with targeted protein degradation

James Schiemer, Andrew Maxwell, Reto Horst, Shenping Liu, Daniel P. Uccello, Kris Borzilleri, Nisha Rajamohan, Matthew F. Brown & Matthew F. Calabrese

Nat Commun 14, 1189, 2023

https://www.nature.com/articles/s41467-023-36738-z

Targeted protein degradation using heterobifunctional chimeras holds the potential to expand target space and grow the druggable proteome. Most acutely, this provides an opportunity to target proteins that lack enzymatic activity or have otherwise proven intractable to small molecule inhibition. Limiting this potential, however, is the remaining need to develop a ligand for the target of interest. While a number of challenging proteins have been successfully targeted by covalent ligands, unless this modification affects form or function, it may lack the ability to drive a biological response. Bridging covalent ligand discovery with chimeric degrader design has emerged as a potential mechanism to advance both fields. In this work, we employ a set of biochemical and cellular tools to deconvolute the role of covalent modification in targeted protein degradation using Bruton’s tyrosine kinase. Our results reveal that covalent target modification is fundamentally compatible with the protein degrader mechanism of action.