Study On The Drug Resistance Mechanism And Inhibitors Of Multidrug Resistance Protein OptrA

ABC transporter(ATP-binding cassette transporter)existed in mammals,bacteria,fungi and other cells,and associated with a variety of cell resistance.Antibiotic resistance(ARE)proteins mediated the resistance to a series of antibiotics that targeted ribosomes,and played an important role in the process of resistance.Oxazolidinones,linezolid and tedizolid,were considered to be the most potent drug for the treatment of the infection caused by VRE.However,the new found ARE protein OptrA mediating the resistance to oxazolidinones made the new drug at risk of failure in 2015.Therefore,the study on the resistance mechanism and inhibitors of OptrA was very important.In this study,the interaction between OptrA and membrane proteins and the protection of ribosomes by OptrA were determined by membrane protein crosslinking assays and translation assays in vitro respectively.And the key residues of the ATPase activity center of OptrA and the active center of OptrA mediating resistance were determined by ATP hydrolase activity assay and minimal inhibitory concentration assay.The inhibitor of OptrA was screened using fragment-based drug screening and the inhibitory effect of the inhibitor on the ATPase activity of the OptrA was demonstrated in vitro test.Although the structure of OptrA was analyzed unsuccessfully,the interaction sites of the inhibitor and OptrA were determined by molecular simulation.Results showed that OptrA was not detected on the membrane proteins of RN4220,indicating that OptrA did not interact with membrane proteins.OptrA protected the translation of DNA without antibiotic inhibition in a dose dependence manner.Replacing the glutamine at the 208 and 488 positions of full length amino acid sequence of OptrA with glutamate not only made OptrA lost its ATP hydrolase activity but also made its host cell RN4220 lost resistance to florfenicol.CP1 screened using FBS inhibited 30% of ATPase activity of OptrA.In the meantime,we failed to analysised the structure of OptrA.Results of homologous modeling and molecular simulation showed that the binding sites of CP1 and OptrA were Ile-496,Ile-292,Ile-289 and Lys-271.In summary,OptrA mediated resistance to antibiotics through protecting ribosomes.The glutamic acid at 208 and 488 positions of full length amino acid sequence of OptrA were the key residues of its ATPase activity center,and the ATPase activity center of OptrA was its active center mediating resistance.CP1 could be used as a lead compound of inhibitors of OptrA,and it laid an important experimental basis and theoretical basis for the further study of more efficient inhibitors.