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.

Development of a Covalent Inhibitor of c-Jun N-Terminal Protein Kinase (JNK) 2/3 with Selectivity over JNK1

Wenchao Lu, Yao Liu, Yang Gao, Qixiang Geng, Deepak Gurbani, Lianbo Li, Scott B. Ficarro, Cynthia J. Meyer, Dhiraj Sinha, Inchul You, Jason Tse, Zhixiang He, Wenzhi Ji, Jianwei Che, Audrey Y. Kim, Tengteng Yu, Kenneth Wen, Kenneth C. Anderson, Jarrod A. Marto, Kenneth D. Westover, Tinghu Zhang, and Nathanael S. Gray

Journal of Medicinal Chemistry 2023

DOI: 10.1021/acs.jmedchem.2c01834

The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase (MAPK) family, which includes JNK1–JNK3. Interestingly, JNK1 and JNK2 show opposing functions, with JNK2 activity favoring cell survival and JNK1 stimulating apoptosis. Isoform-selective small molecule inhibitors of JNK1 or JNK2 would be useful as pharmacological probes but have been difficult to develop due to the similarity of their ATP binding pockets. Here, we describe the discovery of a covalent inhibitor YL5084, the first such inhibitor that displays selectivity for JNK2 over JNK1. We demonstrated that YL5084 forms a covalent bond with Cys116 of JNK2, exhibits a 20-fold higher Kinact/KI compared to that of JNK1, and engages JNK2 in cells. However, YL5084 exhibited JNK2-independent antiproliferative effects in multiple myeloma cells, suggesting the existence of additional targets relevant in this context. Thus, although not fully optimized, YL5084 represents a useful chemical starting point for the future development of JNK2-selective chemical probes.

Depletion of creatine phosphagen energetics with a covalent creatine kinase inhibitor

Narek Darabedian, Wenzhi Ji, Mengyang Fan, Shan Lin, Hyuk-Soo Seo, Ekaterina V. Vinogradova, Tomer M. Yaron, Evanna L. Mills, Haopeng Xiao, Kristine Senkane, Emily M. Huntsman, Jared L. Johnson, Jianwei Che, Lewis C. Cantley, Benjamin F. Cravatt, Sirano Dhe-Paganon, Kimberly Stegmaier, Tinghu Zhang, Nathanael S. Gray & Edward T. Chouchan

 Nat Chem Biol., 2023

https://www.nature.com/articles/s41589-023-01273-x

Creatine kinases (CKs) provide local ATP production in periods of elevated energetic demand, such as during rapid anabolism and growth. Thus, creatine energetics has emerged as a major metabolic liability in many rapidly proliferating cancers. Whether CKs can be targeted therapeutically is unknown because no potent or selective CK inhibitors have been developed. Here we leverage an active site cysteine present in all CK isoforms to develop a selective covalent inhibitor of creatine phosphagen energetics, CKi. Using deep chemoproteomics, we discover that CKi selectively engages the active site cysteine of CKs in cells. A co-crystal structure of CKi with creatine kinase B indicates active site inhibition that prevents bidirectional phosphotransfer. In cells, CKi and its analogs rapidly and selectively deplete creatine phosphate, and drive toxicity selectively in CK-dependent acute myeloid leukemia. Finally, we use CKi to uncover an essential role for CKs in the regulation of proinflammatory cytokine production in macrophages.

2-Ethynylbenzaldehyde-Based, Lysine-Targeting Irreversible Covalent Inhibitors for Protein Kinases and Nonkinases

Peng Chen, Guanghui Tang, Chengjun Zhu, Jie Sun, Xuan Wang, Menghua Xiang, Huisi Huang, Wei Wang, Lin Li, Zhi-Min Zhang, Liqian Gao, and Shao Q. Yao 

Journal of the American Chemical Society 2023 145 (7), 3844-3849
DOI: 10.1021/jacs.2c11595

Lysine-targeting irreversible covalent inhibitors have attracted growing interests in recent years, especially in the fields of kinase research. Despite encouraging progress, few chemistries are available to develop inhibitors that are exclusively lysine-targeting, selective, and cell-active. We report herein a 2-ethynylbenzaldehyde (EBA)-based, lysine-targeting strategy to generate potent and selective small-molecule inhibitors of ABL kinase by selectively targeting the conserved catalytic lysine in the enzyme. We showed the resulting compounds were cell-active, capable of covalently engaging endogenous ABL kinase in K562 cells with long-residence time and few off-targets. We further validated the generality of this strategy by developing EBA-based irreversible inhibitors against EGFR (a kinase) and Mcl-1 (a nonkinase) that covalently reacted with the catalytic and noncatalytic lysine within each target.