Covalent Drug Binding in Live Cells Monitored by Mid-Infrared Quantum Cascade Laser Spectroscopy: Photoactive Yellow Protein as a Model System

Srijit Mukherjee, Steven D. E. Fried, Nathalie Y. Hong, Nahal Bagheri, Jacek Kozuch, Irimpan I. Mathews, Jacob M. Kirsh, and Steven G. Boxer

Journal of the American Chemical Society 2026

DOI: 10.1021/jacs.5c14498

The detection of drug-target interactions in live cells enables analysis of therapeutic compounds in a native cellular environment. Recent advances in spectroscopy and molecular biology have facilitated the development of genetically encoded vibrational probes like nitriles that can sensitively report on molecular interactions. Nitriles are powerful tools for measuring electrostatic environments within condensed media like proteins, but such measurements in live cells have been hindered by low signal-to-noise ratios. In this study, we design a spectrometer based on a double-beam quantum cascade laser (QCL)-based transmission infrared (IR) source with balanced detection that can significantly enhance sensitivity to nitrile vibrational probes embedded in proteins within cells compared to a conventional FTIR spectrometer. Using this approach, we detect small-molecule binding in Escherichia coli, with particular focus on the interaction between para-Coumaric acid (pCA) and nitrile-incorporated photoactive yellow protein (PYP). This system effectively serves as a model for investigating covalent drug binding in a cellular environment. Notably, we observe large spectral shifts of up to 15 cm–1 for nitriles embedded in PYP between the unbound and drug-bound states directly within bacteria, in agreement with observations for purified proteins. Such large spectral shifts are ascribed to the changes in the hydrogen-bonding environment around the local environment of nitriles, accurately modeled through high-level molecular dynamics simulations using the AMOEBA force field. Our findings underscore the QCL spectrometer’s ability to enhance sensitivity for monitoring drug–protein interactions, offering new opportunities for advanced methodologies in drug development and biochemical research.

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.