Synthesis and characterization of covalent ligands

Division Of Basic Sciences - Nci

Investigators

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Abstract

Main activities of this project include the exploration of chemical functionalities to increase the number of electrophilic warhead options for the design of covalent modulators. Our initial efforts are focused on exploring unique and unstudied electrophilic moieties derived from structures of known natural products. 3-halo-4,5-dihydroisoxazole, which is found in natural product acivicin, reacts with cysteine residues activated by surrounding amino acid residues in the active site of a number of enzymes. To systematically evaluate 3-bromo-4,5-dihydroisoxazole (BDHI) as a molecular warhead, we have devised a synthetic strategy to rapidly install the electrophilic warhead into a library of fragments and have synthesized the first set of 300 compounds. For characterization of electrophilic compounds, we measure intrinsic chemical reactivity and their tunability for specific protein modification. We are currently employing reactivity-driven chemoproteomic methods to map the target sites of proteins. Combining competitive gel analysis and quantitative mass spectrometry-based profiling, we identify specific target engagement of a given covalent compound in T cell signaling. Our study demonstrates that 3-bromo-4,5-dihydroxazoles capably engage many cysteines in the human proteome and the selectivity landscape of cysteines liganded by BDHIs is distinct from that of traditional haloacetamide electrophiles. We validated that 3-bromo-4,5-dihydroxazoles form covalent conjugation with glutathione S-transferase Pi (GSTP1) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1). This electrophile was further exploited in Bruton's tyrosine kinase (BTK) inhibitor design using a single-step late-stage installation of the warhead onto acrylamide-containing compounds. A compound that targets multiple functionally important cysteines suppresses T cell activation and cytokine secretion without compromising cellular viability. Additionally, we identified 4-chloro-pyrazolopyridine (CPzP) scaffold as a cysteine targeting covalent warhead. The characterization of a series of analogs validated that CPzP can covalently modify non-catalytic, allosteric cysteines in prolyl endopeptidase (PREP) and heat shock protein 60 (HSP60). Coupled with functional screening, this approach provides compound-target interactions that can modulate immune responses and their associated disorders.