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Aim:To characterize the functional and pharmacological features of a symmetrical 1-pyrrolidineacetamide,N,N’-(methylene-di-4,1-phenylene) bis-1-pyrrolidineacetamide,as a new anti-HIV compound which could competitively inhibit HIV-1 integrase (IN) binding to viral DNA.Methods:A surface plasma resonance (SPR)-based competitive assay was employed to determine the compound’s inhibitory activity,and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell assay was used to qualify the antiviral activity.The potential binding sites were predicted by molecular modeling and determined by site-directed mutagenesis and a SPR binding assay.Results:l-pyrrolidineacetamide,N,N’-(methylene-di-4,1-phenylene) bis-1-pyrrolidineacetamide could competitively inhibit IN binding to viral DNA with a 50% inhibitory concentration (IC50) value of 7.29±0.68 μmol/L as investigated by SPR-based investigation.Another antiretroviral activity assay showed that this compound exhibited inhibition against HⅣ-Ⅰ(ⅢB) replication with a 50% effective concentration (EC50) value of 40.54 μmol/L in C8166 cells,and cytotoxicity with a cytotoxic concentration value of 173.84 μmol/L in mock-infected C8166 cells.Molecular docking predicted 3 potential residues as 1-pyrrolidineacetamide,N,N’-(methylene-di-4,1-phenylene)bis-1-pyrrolidineacetamide binding sites.The importance of 3 key amino acid residues (Lys103,Lys173,and Thr174) involved in the binding was further identified by site-directed mutagenesis and a SPR binding assay.Conclusion:This present work identified a new anti-HIV compound through a new IN-binding site which is expected to supply new potential drug-binding site information for HIV-1 integrase inhibitor discovery and development.