UCSF

Covalent molecules have emerged as powerful tools and next-generation therapeutics, promising improved specificity and sustained target engagement. Chemical proteomic methods to screen for reactive cysteine and lysine residues on proteins amenable to covalent engagement have transformed small molecule discovery pipelines. Covalent molecules have improved our understanding of fundamental biological processes and changed the way we treat human disease, but their application remains limited to sites where reactive cysteines and lysines are present. Here we report a ninhydrin-based warhead that selectively modifies arginine residues. We developed alkyne-functionalized variants of ninhydrin to establish an arginine-specific chemical proteomics platform, enabling the classification of over 6,800 arginines. These studies uncovered potential modification sites on disease-relevant proteins, including reactive arginines within catalytic sites that is essential for function. By endowing a reversible small molecule inhibitor of cyclophilin A with our ninhydrin warhead, we achieved selective, covalent engagement, highlighting the potential for targeting arginines in therapeutic development. These findings establish ninhydrin as a warhead for studying arginine reactivity and modulating protein function, enabling new avenues for covalently engaging proteins.In chapter 1, I introduce the rationale for covalently targeting arginine residues and describe the early stages of ninhydrin development as a covalent warhead. It begins with foundational concepts in activity-based protein profiling (ABPP) and discusses the unique chemical challenges posed by arginine. The chapter presents in vitro studies characterizing the kinetics and selectivity of ninhydrin reactivity toward free arginine and model proteins, establishing proof-of-concept for selective arginine modification.In chapter 2, I describe the development and implementation of the Reactive Arginine Profiling (RAP) platform to map reactive arginines across the proteome using alkyne-functionalized ninhydrin probes. It includes structural bioinformatic analyses to assess reactivity determinants and contextual features of modified residues. Mechanistic studies are presented on the inhibition of aconitase by ninhydrin and a structure-guided approach is used to design a covalent probe that selectively targets R55 in cyclophilin A. These efforts collectively demonstrate the utility of ninhydrin probes in functional proteomics and ligand development.Chapter 3, contains the extended data, detailed experimental protocols, synthetic procedures for ninhydrin probes and analogs, NMR characterization of compounds, and a comprehensive list of references. It provides the technical foundation for the studies described in Chapters 1 and 2 and serves as a resource for reproducing or extending this work.