biostudies-literatureGuan XNNational Natural Science Foundation of China138-149https://www.ebi.ac.uk/biostudies/studies/S-EPMC8864484biostudies-literatureUnknown13(2)The housekeeping sortase A (SrtA), a membrane-associated cysteine transpeptidase, is responsible for anchoring surface proteins to the cell wall peptidoglycan in Gram-positive bacteria. This process is essential for the regulation of bacterial virulence and pathogenicity. Therefore, SrtA is considered to be an ideal target for antivirulence therapy. In this study, we report that ML346, a compound with a barbituric acid and cinnamaldehyde scaffold, functions as an irreversible inhibitor of <i>Staphylococcus aureus</i> SrtA (<i>Sa</i>SrtA) and <i>Streptococcus pyogenes</i> SrtA (<i>Sp</i>SrtA) <i>in vitro</i> at low micromolar concentrations. According to our X-ray crystal structure of the <i>Sp</i>SrtA_ΔN81/ML346 complex (Protein Data Bank ID: 7V6K), ML346 covalently modifies the RSC medicinal chemistryCovalent sortase A inhibitor ML346 prevents <i>Staphylococcus aureus</i> infection of <i>Galleria mellonella</i>.PMC8864484818611380462172580122107109Guan XNZhang TDong ZGan JLan LYang TYang SYang CGfalseCovalent sortase A inhibitor ML346 prevents <i>Staphylococcus aureus</i> infection of <i>Galleria mellonella</i>.The housekeeping sortase A (SrtA), a membrane-associated cysteine transpeptidase, is responsible for anchoring surface proteins to the cell wall peptidoglycan in Gram-positive bacteria. This process is essential for the regulation of bacterial virulence and pathogenicity. Therefore, SrtA is considered to be an ideal target for antivirulence therapy. In this study, we report that ML346, a compound with a barbituric acid and cinnamaldehyde scaffold, functions as an irreversible inhibitor of <i>Staphylococcus aureus</i> SrtA (<i>Sa</i>SrtA) and <i>Streptococcus pyogenes</i> SrtA (<i>Sp</i>SrtA) <i>in vitro</i> at low micromolar concentrations. According to our X-ray crystal structure of the <i>Sp</i>SrtA_ΔN81/ML346 complex (Protein Data Bank ID: 7V6K), ML346 covalently modifies the 2022-01-01T00:00:00Z2022 Feb2026-05-31T16:00:10.025Z2025-04-04T12:46:06.232ZS-EPMC88644843530803010.1039/d1md00316j