Bromo-Cyclobutenaminones as New Covalent UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA) Inhibitors

Abstract

Drug discovery programs against the antibacterial target UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) have already resulted in covalent inhibitors having small three- and five-membered heterocyclic rings. In the current study, the reactivity of four-membered rings was carefully modulated to obtain a novel family of covalent MurA inhibitors. Screening a small library of cyclobutenone derivatives led to the identification of bromo-cyclobutenaminones as new electrophilic warheads. The electrophilic reactivity and cysteine specificity have been determined in a glutathione (GSH) and an oligopeptide assay, respectively. Investigating the structure-activity relationship for MurA suggests a crucial role for the bromine atom in the ligand. In addition, MS/MS experiments have proven the covalent labelling of MurA at Cys115 and the observed loss of the bromine atom suggests a net nucleophilic substitution as the covalent reaction. This new set of compounds might be considered as a viable chemical starting point for the discovery of new MurA inhibitors.

Puckering the Planar Landscape of Fragments: Design and Synthesis of a 3D Cyclobutane Fragment Library

“…Our workflow produced one fragment ( 12 d , as an E/Z ‐mixture) containing an α,β‐unsaturated ketone unit rendering it an electrophilic fragment with potential to act as a Michael acceptor. This could serve as a covalent fragment, alongside other well‐known acrylamide warheads [56,57] and our previously published cyclobutenaminone fragments, which have been shown to exhibit covalent inhibition of bacterial enzymes [58] . A PMI plot was generated for the synthesised library set (Figure 3), with points labelled according to their substituent geometry ( cis =triangles; trans =squares) and functional group (amine=blue; amide=green; sulfonamide=red).…”

“…This could serve as a covalent fragment, alongside other well-known acrylamide warheads [56,57] and our previously published cyclobutenaminone fragments, which have been shown to exhibit covalent inhibition of bacterial enzymes. [58] A PMI plot was generated for the synthesised library set (Figure 3), with points labelled according to their substituent geometry (cis = triangles; trans = squares) and functional group (amine = blue; amide = green; sulfonamide = red). Interestingly, of the seven pairs of cis/trans analogues amongst the collection (10 a/11 c, 10 e/11 d, 12 a/ 13 a, 12 b/13 b, 12 e/13 d, 12 f/13 h, 12 h/13 g), there appears to be a marginally higher ΣNPR value for the trans isomer, with differences ranging from ΔΣNPR = 0.00-0.05 (mean average 0.02).…”

Advances in UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA) Covalent Inhibition

“…Nowadays, bromo-cyclobutenaminone derivatives are studied as new covalent inhibitors and electrophilic warheads. These inhibitors have an electrophilic character acting as new warheads for the covalent bonding against the Cys115 residue located in the active site of MurA E.coli (Hamilton et al, 2020). The development of a heterocyclic electrophilic fragment library revealed their potential as covalent warheads.…”

“…UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is a cytoplasmatic enzyme from the peptidoglycan pathway responsible for catalyzing the transfer of enolpyruvate from phosphoenolpyruvate to UDP-N-acetylglucosamine to form UDP-N-acetylglucosamine enolpyruvate and release inorganic phosphate ( Evangelina et al, 2021 ). Since the discovery of fosfomycin antibiotic, multiple studies have been carried out on covalent inhibitors ( Mihalovits et al, 2019 ; Hamilton et al, 2020 ). Recently, the design of new covalent inhibitors has been receiving considerable attention ( Mihalovits et al, 2021 ).…”

Special Issue “Novel Antibacterial Agents”

“…Covalent inhibitors of another bacterial enzyme for which there is no human orthologue, namely UDP-N-acetylglucosamine enolpyruvyl transferase (MurA), have been developed by G.M. Keserü et al [14], who indicated bromo-cyclobutenaminones as novel electrophilic probes by screening a small library of cyclobutenone derivatives. The bromine atom has been recognized as an essential requirement, and MS/MS experiments have led to the suggestion that Cys115 is also involved.…”