Weekley, C. M. | Search bioRxiv

bioRxiv2023

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-dihy-droxazole 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 3-bromo-4,5-dihydroxazole-functionalized chemical fragments using competitive chemoproteomic profiling methods. Our study demonstrates that 3-bromo-4,5-dihydroxazoles capably engage reactive cysteine residues in the human proteome and the selectivity landscape of cysteines liganded by 3-bromo-4,5-dihydroxazoles is distinct from that of haloacetamide electrophiles. Given its tem-pered reactivity, 3-bromo-4,5-dihydroxazoles showed restricted, selective engagement with proteins driven by interactions between a tunable binding element and the complementary protein sites. We further validate that 3-bromo-4,5-dihydroxazoles form covalent conjugates with glutathione S-transferase Pi (GSTP1) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1), emerging anti-cancer targets. Together, this study expands the spectrum of optimizable chemical tools for covalent ligand discovery and high-lights the utility of 3-bromo-4,5-dihydroxazole as a cysteine-reactive electrophile. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=57 SRC="FIGDIR/small/524242v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@13b987eorg.highwire.dtl.DTLVardef@1987ae1org.highwire.dtl.DTLVardef@1ab4fb9org.highwire.dtl.DTLVardef@1ff1f71_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

SARS-CoV-2 Spike receptor-binding domain with a G485R mutation in complex with human ACE2

Since SARS-CoV-2 emerged in 2019, genomic sequencing has identified mutations in the viral RNA including in the receptor-binding domain of the Spike protein. Structural characterisation of the Spike carrying point mutations aids in our understanding of how these mutations impact binding of the protein to its human receptor, ACE2, and to therapeutic antibodies. The Spike G485R mutation has been observed in multiple isolates of the virus and mutation of the adjacent residue E484 to lysine is known to contribute to antigenic escape. Here, we have crystallised the SARS-CoV-2 Spike receptor-binding domain with a G485R mutation in complex with human ACE2. The crystal structure shows that while the G485 residue does not have a direct interaction with ACE2, its mutation to arginine affects the structure of the loop made by residues 480-488 in the receptor-binding motif, disrupting the interactions of neighbouring residues with ACE2 and with potential implications for antigenic escape from vaccines, antibodies and other biologics directed against SARS-CoV-2 Spike.

biochemistry↗