Ninhydrin as a Covalent Warhead for Chemical Proteomic-Enabled Discovery and Selective Engagement of Reactive Arginines

Andrew K. Ecker, Andreas Langen, Chloe S. Fields, José L. Montaño, Minh Tran, Ian B. Seiple, Balyn W. Zaro; 

ACS Cent. Sci.  2026; 12 (10): 1610–1624. 

https://doi.org/10.1021/acscentsci.6c00922

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 from one cell line. These studies uncovered potential modification sites on disease-relevant proteins, including reactive arginines within catalytic sites that are essential for function and reactive arginines on proteins lacking reactive cysteines. By endowing a reversible small-molecule binder of cyclophilin A with a ninhydrin warhead, we achieved selective, covalent engagement in cell lysates and attenuation of enzymatic activity in vitro. These findings establish ninhydrin as a warhead for studying arginine reactivity and identifying at scale arginines amenable to covalent modification.