A practical method for determining the rate of covalent modification of fragments and leads

Janice Jeon, Svetlana A. Kholodar, Brian H. Tran, Kimberly E. Mallinger, Daniel A. Erlanson & Robert A. Everley 

Nat Commun 16, 11234 (2025). 

https://doi.org/10.1038/s41467-025-66924-0

The clinical success of covalent drugs such as sotorasib has renewed interest in covalency for rational drug design. The most rigorous potency metric for covalent modifiers is the second-order rate constant kinact/KI. However, existing methods for measuring kinact/KI are resource-intensive and involve complex data interpretation. We describe the diagonal dose-response time-course (dDRTC), an efficient mass spectrometry-based method for determining kinact/KI, enabling routine kinact/KI quantification earlier in programs and accelerating SAR interpretation for lead discovery. We apply dDRTC to a dozen covalent fragment and lead-like modifiers for three targets, KRASG12C and two E3 ligase complexes. Kinetic simulations comparing a range of kinact and KI values establish recommended parameters for dDRTC and reveal that the approach is particularly suited for covalent fragments and leads. Our results demonstrate accurate determination of kinact/KI values across three orders of magnitude with eight-fold increased throughput, reduced protein consumption, and simplified data analysis.

Stereoselective Degradation of Diacylglycerol Kinases Potentiate T cell Activation and Tumor Cell Cytotoxicity

Minhaj Shaikh, Surya P Mookherjee, Claire Weckerly, Adam H Libby, Aizhen Xiao, Yunge Zhao, Sagar D Vaidya, AeRyon Kim, Zhihong Li, Madeleine L Ware, Michelle Marants, Olivia Murtagh, Wesley J Wolfe, Timothy N Bullock, Benjamin W Purow, Gerald R Hammond, Ken Hsu

bioRxiv 2025.12.09.692983; 

doi: https://doi.org/10.64898/2025.12.09.692983

Stereoselective recognition is a powerful means to differentiate selective versus non-specific activity of small molecules in complex biological systems. Here, we disclose stereochemically defined, sulfonyl-triazole inhibitors of the lipid enzyme diacylglycerol kinase-alpha (DGKA), a key metabolic checkpoint for T cell effector function. Acute treatment with the covalent DGKA inhibitor AHL-7160 recruited endogenous DGKA to the plasma membrane in a stereoselective and isozyme-specific manner. The membrane translocation activity of AHL-7160 correlated with blockade of cellular phosphatidic acid production and potentiation of primary T cell-mediated killing of a glioblastoma cell line. Quantitative chemoproteomics revealed Y669 and K411 as sites of AHL-7160 modification on endogenous DGKA in cells. Extended treatments resulted in proteasome-dependent and proteome-wide selective degradation of DGKA in T cells. Collectively, these findings establish covalent DGKA ligands as potent molecular glues with translational potential in immunotherapy.

Identification of VVD-214/RO7589831, a Clinical-Stage, Covalent Allosteric Inhibitor of WRN Helicase for the Treatment of MSI-High Cancers

Shota Kikuchi, Jason C. Green, Don C. Rogness, Betty Lam, Zachary A. Owyang, Robert D. Malmstrom, Ali Tabatabaei, Aaron N. Snead, Melissa A. Hoffman, Steffen M. Bernard, Paige Ashby, Kelsey N. Lamb, Benjamin D. Horning, Kristen A. Baltgalvis, Kent T. Symons, Thomas A. Glaza, Chu-Chiao Wu, Xiaodan Song, Martha K. Pastuszka, John J. Sigler, Jonathan Pollock, Laurence Burgess, Gabriel M. Simon, Matthew P. Patricelli, and David S. Weinstein

Journal of Medicinal Chemistry 2025

DOI: 10.1021/acs.jmedchem.5c01805

Werner syndrome helicase (WRN) has emerged as a compelling therapeutic target for microsatellite instability-high (MSI-H) cancers, owing to its selective dependency on the helicase activity of WRN. Despite the inherent challenges in targeting helicases, our chemoproteomics approach enabled the identification of compounds that covalently engage C727 within an allosteric pocket of WRN, thereby inhibiting its ability to unwind DNA. Through optimization of each molecular component, particularly focusing on the vinyl sulfone warhead and C2 substitution at the pyrimidine core, an optimal balance of intrinsic reactivity, inhibitory potency, and metabolic stability was achieved, culminating in the identification of VVD-214/RO7589831. This process underscored the tunability of the vinyl sulfone warhead and its effectiveness in covalent drug discovery. VVD-214 induced tumor regression in MSI-H colorectal cancer models and is being evaluated as a promising therapeutic candidate for MSI-H cancers.

The Development of UM-203, A Reversible Covalent STING Antagonis

 Leonard Barasa, Leo DeOrsey, Maeve D. O’Reilly, Shruti Choudhary, Sara E. Cahill, Anukriti Mathur, Akumalla Allabaji, Srinivasa Rao Vidadala, Sujit Kumar Sarkar, Santoshkumar N. Patil, Harikesh Kalonia, Jeffrey Hale, Fiachra Humphries, Katherine A. Fitzgerald, and Paul R. Thompson

ACS Medicinal Chemistry Letters 2025

DOI: 10.1021/acsmedchemlett.5c00611

The cGAS-STING pathway is a critical component of the innate immune system, responsible for detecting cytosolic DNA and triggering inflammatory signaling. While essential for host defense, aberrant activation of this pathway is linked to a range of inflammatory and autoimmune disorders. Consequently, STING has emerged as a compelling therapeutic target. Herein we report the development of the first reversible covalent STING inhibitor, i.e., UM-203 which employs an alkyne-thiazole warhead. UM-203 inhibits STING-dependent signaling in both mouse and human systems. Notably, UM-203 maintains activity against the most prevalent human STING variant (R232), effectively suppresses STING signaling in primary human CD14+ monocytes, and exhibits moderate metabolic stability. Collectively, these findings highlight UM-203 as a promising scaffold for the development of therapeutics targeting STING-driven inflammatory and autoimmune diseases.

A Highly Reactive Cysteine-Targeted Acrylophenone Chemical Probe That Enables Peptide/Protein Bioconjugation and Chemoproteomics Analysis

Constantin M. Nuber, Anna V. Milton, Benedikt Nissl, Maria C. Isaza Alvarez, Benjamin R. G. Bissinger, Manjima B. Sathian, Cedric D. Pignot, Annsophie Haberhauer, Dongqing Wu, Céline Douat, Sabine Schneider, Stephan M. Hacker, Pavel Kielkowski, and David B. Konrad

J. Am. Chem. Soc. Au, 2025

 https://doi.org/10.1021/jacsau.5c00692

To date, a variety of covalent cysteine-reactive chemical probes have been reported. The most common ones include maleimide for bioconjugation and iodoacetamide alkyne (IAA) for chemoproteomics analyses. Their applicability, however, is limited due to, e.g., the hydrolytic lability of maleimide adducts and the slow reaction kinetics as well as the inability to record the entirety of the cysteinome with IAA. This is compounded by the missing potential to fine-tune their reactivity tailored to advanced applications. To generate a high-reactivity, cysteine-selective chemical probe with broad utility, we have performed an in-depth investigation into the acrylophenone scaffold. The aryl group connected to the vinyl ketone chemotype can be readily substituted, which provides the potential to fine-tune the reactivity and install a bioorthogonal handle. We took advantage of this feature by modifying acrylophenone-alkyne (APA) with two ortho chlorine groups to generate ortho-dichloroacrylophenone-alkyne (CAPA), which increased the stability of the probe and the yield of its cysteine adducts. To showcase the reactivity, we performed reaction rate analyses with model reagents. The selectivity was demonstrated by specifically labeling cysteine residues within two peptides under physiological conditions. To investigate its utility toward bioconjugation reactions, we performed the stoichiometric labeling of two proteins. Remarkably, CAPA was successfully implemented as a high-reactivity cysteine-selective chemical probe into activity-based protein profiling (ABPP) experiments using both in-gel fluorescence (in-gel ABPP) and mass spectrometry analyses. The chemoproteomics workflow, named isoDTB-ABPP, allowed us to highlight that CAPA provides a complementary approach to IAA in expanding the coverage of the cysteinome.

Preclinical characterization of EGT710, an oral non-peptidomimetic reversible covalent SARS-CoV-2 main protease inhibitor

Stephanie A. Moquin, Suresh B. Lakshminarayana, Kamal Kumar Balavenkatraman, Hilmar Schiller, Allison Claas, Barun Bhhatarai, Ioannis Loisios-Konstantinidis, Katarina Vulic, Chaitanya Kurhade, Birte K. Kalveram, John Yun-Chung Chen, Jing Zou, Xuping Xie, Laura Tandeske, Dustin Dovala, Elizabeth Ornelas, Mark S. Knapp, Daniel Fuller, Zachary Nguyen, David T. Barkan, Lidiya Bebrevska, S. Kirk Wright, Scott A. Busby, Johanne Blais, Pei-Yong Shi, Suzanne Gaudet, Renee Bergeron, Hannah Yu, Julia Zack, Christopher Sarko, Feng Gu, James E. Bradner, John A. Tallarico, Thierry T. Diagana & Julien P. N. Papillon 

npj Drug Discov. 2, 28 (2025). 

https://doi.org/10.1038/s44386-025-00030-5

EGT710 is an orally bioavailable non-peptidomimetic reversible covalent coronavirus main protease (Mpro) inhibitor with low nM cellular activity against SARS-CoV-2. Twice daily dosing of 10 mg/kg of EGT710 decreased lung viral load in a mouse model of SARS-CoV-2 infection to below the limit of detection. Resistance selection resulted in the emergence of several Mpro mutations, with recombinant viruses containing L50F + E166A substitutions showing the largest shift in potency. Development of a viral kinetics model using viremia data from clinical trials, along with a human physiologically based pharmacokinetic model, predicted efficacy in humans with once daily oral doses of >360 mg. EGT710 displays favorable pharmacokinetic properties and an acceptable in vitro and in vivo safety profile, with human exposures at the recommended clinical dose of 600 mg predicted to be below the no adverse effect level in preclinical toxicology studies. Together, EGT710 has a promising preclinical profile and has completed a Phase I study.

Fast and Site-Specific Covalent Targeting of Proteins by Arylfluorosulfate-Modified Aptamers

 Kaining Zhang, Juan Li, Yang Shi, Sining Hou, Zichen Qin, Wenhao Shi, Yiying Zhu, Jingjing Zhang, Aijun Tong, and Yu Xiang

Journal of the American Chemical Society 2025

DOI: 10.1021/jacs.5c14374

Sulfur fluoride exchange (SuFEx) is a versatile click chemistry platform with emerging applications in covalent inhibitor development, among which arylfluorosulfate (AFS) derivatives have shown great promise because of their exclusive selectivity to target proteins. Nevertheless, due to the inherently mild electrophilicity of AFS, the high target specificity often comes at the expense of target reactivity. Here, we challenge this conventional view and show that in vitro selected AFS-modified covalent aptamers (AFS-Aps) can be both highly target selective and reactive. One such AFS-Ap is able to site-specifically modify the extracellular domain (ECD) of human epidermal growth factor receptor 3 (HER3) with a “click” reaction half-life (t1/2) as short as 1 min and covalently inhibit the protein–protein interaction (PPI) of HER3/EGFRs. Another AFS-Ap targeting human vascular endothelial growth factor (VEGF) can also rapidly cross-link VEGF165 and block the PPI of VEGF/VEGFR. The inactivation rate constants (kinact) of these AFS-Aps are one to two orders of magnitude higher than many reported AFS-based covalent molecules. This unexpectedly fast and site-specific covalent targeting requires a precisely organized aptamer–protein binding interface, where the AFS structure, rather than electrophilicity, is the decisive factor. We believe the target reactivity potential of AFS derivatives may have long been underestimated in covalent inhibitor development. AFS-adapted combinatorial techniques are therefore invaluable for discovering covalent inhibitors with both high target reactivity and selectivity.