Covalent Inhibition by a Natural Product-Inspired Latent Electrophile

Strategies to target specific protein
cysteines are critical to
covalent probe and drug discovery. 3-Bromo-4,5-dihydroisoxazole (BDHI)
is a natural product-inspired, synthetically accessible electrophilic
moiety that has previously been shown to react with nucleophilic cysteines
in the active site of purified enzymes. Here, we define the global
cysteine reactivity and selectivity of a set of BDHI-functionalized
chemical fragments using competitive chemoproteomic profiling methods.
Our study demonstrates that BDHIs capably engage reactive cysteine
residues in the human proteome and the selectivity landscape of cysteines
liganded by BDHI is distinct from that of haloacetamide electrophiles.
Given its tempered reactivity, BDHIs showed restricted, selective
engagement with proteins driven by interactions between a tunable
binding element and the complementary protein sites. We validate that
BDHI forms covalent conjugates with glutathione S-transferase Pi (GSTP1) and peptidyl-prolyl cis–trans isomerase NIMA-interacting 1 (PIN1), emerging anticancer
targets. BDHI 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.
Together, this study expands the spectrum of optimizable chemical
tools for covalent ligand discovery and highlights the utility of
3-bromo-4,5-dihydroisoxazole as a cysteine-reactive electrophile.