Covalent Inhibition by a Natural Product-Inspired Latent Electrophile

Abstract

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.

Solid-Phase Compatible Silane-Based Cleavable Linker Enables Custom Isobaric Quantitative Chemoproteomics

“…By enabling the discovery of covalent degraders, ,– protein–protein interaction (PPI) modulators, – novel targets with antibacterial activity, – pinpointed redox sensitive cysteines, – and even shedding light on the mode of action and off-targets of existing drugs and clinical candidates, – cysteine chemoproteomics has made considerable inroads into closing the druggability gap. Cysteine chemoproteomics has proven useful for identifying both reversible and irreversible protein modulators, including those identified through screens of cysteine-reactive electrophilic fragments, ,– fully functionalized fragments, – and latent electrophiles. – An ongoing challenge for the field of chemoproteomics is to fully establish the scope of proteins that can be targeted by cysteine-reactive chemical probes.…”

Discovery of a Tunable Heterocyclic Electrophile 4-Chloro-pyrazolopyridine That Defines a Unique Subset of Ligandable Cysteines

“…BDHI-151 showed no reactivity against other nucleophiles, such as lysine and serine (Figure S2A), suggesting that the CPzP core is more reactive with a preference for thiol nucleophiles in solution. As the BDHI warhead does not readily react with sulfhydryl groups in solution, there was no noticeable formation of Br-displaced adduct, and the desCl analogue (BDHI-151a) did not produce covalent adducts even after overnight incubation (Figure S2B).…”

Emerging and Re-emerging Warheads for Targeted Covalent Inhibitors: An Update

“…240−243 Such compounds have been subject to early reactivity/promiscuity analyses 244 which showed that the reaction proceeds through an addition−elimination mechanism, displacing the halogen at the electrophilic 3-position, which has been hypothesized to be favored in protease-like enzymes by stabilization of the tetrahedral intermediate via the oxyanion hole. 245 With respect to human target proteins, Pinto et al 246 recently showed by MS and X-ray crystallography (PDB code: 6FFM) that the BDHI 125 (Figure 39A) covalently binds to Cys151 of the KEAP1 protein, allowing the modulation of the antioxidant system NRF2/HO-1. The Conti group 247 described spirocyclic BDHIs like 126 (Figure 39A) targeting the catalytic Cys152 in human glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH).…”

“…Recently, competitive chemoproteomic profiling was used to investigate the reactivity and selectivity of BDHI-functionalized fragments on a proteome level. 245 The analysis showed the restricted reactivity of BDHI warheads to a subset of (often hyper-reactive) cysteine residues. In addition, non-covalent recognition and stabilization of the covalent reaction 39B) showed decreased yet potent activity on both BTK and BLK and a higher proteome selectivity.…”

“…3-Halo-4,5-dihydroisoxazoles (3-halo­isooxazolines) such as the natural product acivicin ( 123 ) and its more active synthetic 3-bromo-4,5-dihydro­isoxazole (BDHI) derivative 124 (Figure A) are well known for their ability to covalently inhibit cysteine-dependent enzymes like human trans­glutaminase 2 and for their activity on parasitic enzymes. − Such compounds have been subject to early reactivity/​promiscuity analyses which showed that the reaction proceeds through an addition–elimination mechanism, displacing the halogen at the electrophilic 3-position, which has been hypothesized to be favored in protease-like enzymes by stabilization of the tetrahedral intermediate via the oxyanion hole . With respect to human target proteins, Pinto et al recently showed by MS and X-ray crystallography (PDB code: 6FFM) that the BDHI 125 (Figure A) covalently binds to Cys151 of the KEAP1 protein, allowing the modulation of the antioxidant system NRF2/HO-1.…”