Study On The Inhibition Mechanism Of Inhibitors Targeting Virulence Proteins Of Staphylococcus Aureus

Staphylococcus aureus(S.aureus)is an important food-borne pathogen.Most people get S.aureus poisoning by eating contaminated food,which poses a great threat to food safety and human health.At present,due to the abuse of antibiotics and the emergence of multiple antibiotic-resistant S.aureus,it is urgent to develop new inhibitors against S.aureus.Surface protein Sortase A(SrtA)and virulence regulator Serine/threonine phosphatase(Stp1)are important virulence factors in the process of S.aureus infection.Thus,they can be used as effective inhibitoric targets against S.aureus.The new inhibitoric strategy can reduce the selection pressure of S.aureus,thereby greatly inhibit the emergence of antibiotic-resistant S.aureus.Previous literature reports indicated that the activity of SrtA can be inhibited by curcumin and its analogues,and the activity of Stp1 can be inhibited by 5,5’-methylenedisalicylic acid(MDSA)and its analogues.However,the mechanism by which these inhibitors bind to SrtA/Stp1 at the atomic level remains unclear.In order to investigate the interaction mechanism,the SrtA/Stp1-inhibitor complexes were studied by molecular dockings,molecular dynamics simulations,binding free energy decomposition calculations,steering molecular dynamics simulations and hydrogen bond analyses.Energy decomposition analyses indicated that Ala104,Pro163,Leu169,Gln172,Ile182 and Ile199 were key residues in SrtA-inhibitor complexes.The binding region of inhibitors overlapped that of LPXTG motif.The binding modes indicated that SrtA lost its catalytic activity after binding to inhibitors.Due to the absence of methoxyl groups,the binding affinity of demethoxycurcumin and bisdemethoxycurcumin were significantly weaker than that of curcumin,resulting in a decrease in their inhibitory activity.In addition,the lack of double bonds induced coiled molecular structure,which prevented tetrahydrocurcumin from binding to the catalytic pocket of SrtA,and its inhibitory activity was completely lost.Based on these results,methoxyl groups on benzene rings in conjugated molecules largely influenced the inhibitory activity of SrtA inhibitors.Energy decomposition analyses of Stp1-inhibitor complexes indicated that MDSA and hydroxymethyl MDSA could bind to the catalytic pocket of Stp1.Furthermore,Met39,Ile163,Ile164,Val167,Gly195 and Asp233 were key residues in Stp1-inhibitor complexes.Carboxyl groups and hydroxyl groups on both sides of benzene rings contributed to the interaction between inhibitors and Stp1.Comparing the binding free energy of hydroxymethyl MDSA and carboxymethyl MDSA,it was found that the contribution of carboxyl groups was greater than that of hydroxyl groups.Meanwhile,due to the lack of a double salicylate structure,salicylic acid could not bind to the active region of Stpl,resulting in complete loss of its inhibitory activity.In summary,this study revealed the binding models and the interaction mechanism of S.aureus virulence factor—SrtA/Stp1 and its inhibitors at the atomic level,providing new insights to screening and designing of SrtA/Stp1 inhibitors.