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. 2024 Aug 19;19(16):e202300684.
doi: 10.1002/cmdc.202300684. Epub 2024 Jul 8.

Fluoromethylketone-Fragment Conjugates Designed as Covalent Modifiers of EcDsbA are Atypical Substrates

Bradley C Doak  1 , Rebecca L Whitehouse  1 , Kieran Rimmer  1 , Martin Williams  1 , Begoña Heras  2 , Sofia Caria  1 , Olga Ilyichova  1 , Mansha Vazirani  1 , Biswaranjan Mohanty  1   3 , Jason B Harper  4 , Martin J Scanlon  1   3 , Jamie S Simpson  1
Affiliations

Fluoromethylketone-Fragment Conjugates Designed as Covalent Modifiers of EcDsbA are Atypical Substrates

Bradley C Doak et al. ChemMedChem. .

Abstract

Disulfide bond protein A (DsbA) is an oxidoreductase enzyme that catalyzes the formation of disulfide bonds in Gram-negative bacteria. In Escherichia coli, DsbA (EcDsbA) is essential for bacterial virulence, thus inhibitors have the potential to act as antivirulence agents. A fragment-based screen was conducted against EcDsbA and herein we describe the development of a series of compounds based on a phenylthiophene hit identified from the screen. A novel thiol reactive and "clickable" ethynylfluoromethylketone was designed for reaction with azide-functionalized fragments to enable rapid and versatile attachment to a range of fragments. The resulting fluoromethylketone conjugates showed selectivity for reaction with the active site thiol of EcDsbA, however unexpectedly, turnover of the covalent adduct was observed. A mechanism for this turnover was investigated and proposed which may have wider ramifications for covalent reactions with dithiol-disulfide oxidoreducatases.

Keywords: Enzymes; Inhibitors; NMR spectroscopy; Protein Structure.

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