Covalent Inhibition by a Natural Product-Inspired Latent Electrophile

Strategies to target specific proteincysteines are critical tocovalent probe and drug discovery. 3-Bromo-4,5-dihydroisoxazole (BDHI)is a natural product-inspired, synthetically accessible electrophilicmoiety that has previously been shown to react with nucleophilic cysteinesin the active site of purified enzymes. Here, we define the globalcysteine reactivity and selectivity of a set of BDHI-functionalizedchemical fragments using competitive chemoproteomic profiling methods.Our study demonstrates that BDHIs capably engage reactive cysteineresidues in the human proteome and the selectivity landscape of cysteinesliganded by BDHI is distinct from that of haloacetamide electrophiles.Given its tempered reactivity, BDHIs showed restricted, selectiveengagement with proteins driven by interactions between a tunablebinding element and the complementary protein sites. We validate thatBDHI forms covalent conjugates with glutathione S-transferase Pi (GSTP1) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1), emerging anticancertargets. BDHI electrophile was further exploited in Bruton'styrosine kinase (BTK) inhibitor design using a single-step late-stageinstallation of the warhead onto acrylamide-containing compounds.Together, this study expands the spectrum of optimizable chemicaltools for covalent ligand discovery and highlights the utility of3-bromo-4,5-dihydroisoxazole as a cysteine-reactive electrophile.

Automated summary

This study defines the reactivity and selectivity of a set of BDHI-functionalized chemical fragments on cysteines using chemoproteomic profiling methods. The study demonstrates that BDHIs can react with reactive cysteine residues in the human proteome and their selectivity differs from that of haloacetamide electrophiles. Furthermore, BDHI forms covalent conjugates with potential anticancer targets and is used in the design of a BTK inhibitor.