Structure-Guided Design, Synthesis, and Antivirulence Assessment of Covalent Staphylococcus aureus Sortase A Inhibitors.

Sortase A (SrtA) is a membrane-associated cysteine transpeptidase required for bacterial virulence regulation and anchors surface proteins to cell wall, thereby assisting biofilm formation. SrtA is targeted in antivirulence treatments against Gram-positive bacterial infections. However, the development of potent small-molecule SrtA inhibitors is constrained owing to the limited understanding of the mode of action of inhibitors in the SrtA binding pocket. Herein, we designed and synthesized a novel class of covalent SrtA inhibitors based on the binding mode detailed in the X-ray crystal structure of the ML346/Streptococcus pyogenes SrtA complex. ML346 analog Y40 exhibited 2-fold increased inhibitory activity on Staphylococcus aureus SrtA and showed superior inhibitory effects on biofilm formation in vitro. Y40 protected Galleria mellonella larvae fromS. aureusinfections in vivo while minimally attenuating staphylococcal growth in vitro. Our study indicates that the covalent SrtA inhibitor Y40 is an antivirulence agent that is effective againstS. aureusinfections.

Covalent sortase A inhibitor ML346 prevents Staphylococcus aureus infection of Galleria mellonella.

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 Staphylococcus aureus SrtA (SaSrtA) and Streptococcus pyogenes SrtA (SpSrtA) in vitro at low micromolar concentrations. According to our X-ray crystal structure of the SpSrtAΔN81/ML346 complex (Protein Data Bank ID: 7V6K), ML346 covalently modifies the thiol group of Cys208 in the active site of SpSrtA. Importantly, ML346 significantly attenuated the virulence phenotypes of S. aureus and exhibited inhibitory effects on Galleria mellonella larva infection caused by S. aureus. Collectively, our results indicate that ML346 has potential for development as a covalent antivirulence agent for treating S. aureus infections, including methicillin-resistant S. aureus.

Tideglusib and Its Analogues As Inhibitors of Staphylococcus aureus SrtA.

Sortase A (SrtA) anchors surface proteins to the cell wall envelope, and it has attracted increasing interesting as a potential antivirulence target. Several small-molecule inhibitors for SrtA have been developed, but target validation remains largely underexplored. Herein, we report a new class of SrtA inhibitors that supports antivirulence therapy through small-molecule targeting of SrtA. Tideglusib (TD), a drug candidate for myotonic dystrophy, was outstanding in high-throughput screening. A concise synthetic route quickly provided TD analogues, and the structure-activity relationships for SrtA inhibition have been established from those analogues. Several compounds largely retained the in vitro potency and exhibited a better solubility than TD. Additionally, TD attenuated virulence-related phenotypes in vitro and protected mice against lethal S. aureus USA300 bacteremia. Our study indicates that TD and its analogues could be new candidates as SrtA inhibitors with potential in the development of new antivirulence agents.