Ninhydrin as a covalent warhead for chemical proteomic-enabled discovery and selective engagement of reactive arginines

Andrew Ecker, Andreas Langen, Chloe Fields, José Luis Montaňo, Minh Tran, Ian Bass Seiple, Balyn W Zaro

bioRxiv 2026.01.05.697388; 

doi: https://doi.org/10.64898/2026.01.05.697388

Covalent molecules have emerged as next-generation therapeutics and as powerful tools for perturbing fundamental biological processes. Chemical proteomic methods to screen for reactive proteinaceous amino acids have transformed small-molecule discovery pipelines, but their application remains mostly limited to sites where reactive cysteines and lysines are present. Here we report a ninhydrin-based warhead that selectively modifies arginine residues, thus expanding the repertoire of amino acids targetable by covalent molecules. Specifically, we developed alkyne-functionalized variants of ninhydrin to establish an arginine-specific chemical proteomics platform, enabling the classification of more than 6,800 unique reactive arginines. These studies uncovered potential modification sites on disease-relevant proteins, including reactive arginines within catalytic sites that are essential for function. By endowing a reversible small molecule inhibitor of cyclophilin A with a ninhydrin warhead, we achieved selective, covalent engagement, and attenuation of enzymatic activity, highlighting the potential for targeting arginines in future therapeutic development campaigns. These findings establish ninhydrin as a warhead for studying arginine reactivity and modulating protein function.

Peptide-based covalent inhibitor of tubulin detyrosination promotes mesenchymal-to-epithelial transition in lung cancer cells

Hathaichanok Impheng, Ghislain Gillard, Nuttanid Numnoi, and Krzysztof Rogowski 

PNAS 123 (1) e2514990123

https://doi.org/10.1073/pnas.2514990123

Detyrosination is a reversible posttranslational modification specific to α-tubulin, which has been implicated in cancer progression and invasiveness by promoting epithelial-to-mesenchymal transition. The members of the vasohibin family, VASH1 and VASH2, were previously identified as the first class of enzymes involved in catalyzing this modification. Here, we report the development of a covalent VASH inhibitor, which is characterized by high specificity and low toxicity. By combining the use of a new compound with molecular approaches in lung cancer cell lines, we find that tubulin detyrosination plays an important role in the maintenance of mesenchymal properties. We show that in the absence of VASH activity, collective cell migration and 3D spheroid formation are severely compromised. Moreover, we demonstrate that the observed phenotypes are caused by the accumulation of the important epithelial marker E-cadherin with simultaneous reduction in mesenchymal markers N-cadherin and vimentin. Taken together, our study establishes tubulin detyrosination as a promising target for the future development of anticancer treatment.

Discovery of a First-in-Class Covalent Allosteric SHP1 Inhibitor with Immunotherapeutic Activity

Zihan Qu,  Frederick Nguele Meke,  Zheng Zhang,  Aaron D. Krabill,  Christine S. Muli,  Brenson A. Jassim,  Jiajun Dong,  Quyen D. Nguyen,  Yunpeng Bai,  Jinyue Li,  Yiming Miao,  Bardia Asadi,  Levi M. Johnson,  Jinmin Miao,  Darci J. Trader,  W. Andy Tao,  Zhong-Yin Zhang

Angew. Chem. Int. Ed. 2025, e25126 

https://doi.org/10.1002/anie.202525126

Src homology 2 domain-containing phosphatase 1 (SHP1), encoded by PTPN6, is a key intracellular mediator of inhibitory immune signals. SHP1 is garnering attention as a potential immunotherapeutic target since SHP1 deletion elicits strong antitumor activity by boosting both innate and adaptive immunity. Unfortunately, no quality SHP1 inhibitor exists to demonstrate its translatability owing to the challenges posed by the chemistry of the phosphatase active site. Herein, we describe the discovery of a first-in-class, phenyl chloroacetamide-based covalent allosteric SHP1 inhibitor M029 through covalent fragment screening and multiparameter optimization. M029 inactivates SHP1 by covalently binding to a non-conserved and cryptic Cys480 far away from the active site, thus uncovering a novel allosteric mechanism for SHP1 inhibition. In addition, M029 is highly selective for SHP1 and exhibits robust cellular target engagement. Importantly, M029 is orally active and blocks tumor progression in a syngeneic cancer model by activating natural killer cells and cytotoxic CD8+ T cells, along with reduced T cell exhaustion. Together, this study reveals a ligandable Cys that can be exploited for allosteric inhibition of SHP1, which has been refractory to targeted pharmacologic manipulation. The work also demonstrates small-molecule SHP1 inhibition as a compelling approach for new cancer immunotherapy.

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.