Symmetrical 1-pyrrolidineacetamide showing anti-HIV activity through a new binding site on HIV-1 integrase

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.

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.

1-pyrrolidineacetamide, N,N'-(methylene-di-4,1-phenylene) bis-1-pyrrolidineacetamide could competitively inhibit IN binding to viral DNA with a 50% inhibitory concentration (IC(50)) value of 7.29+/-0.68 micromol/L as investigated by SPR-based investigation. Another antiretroviral activity assay showed that this compound exhibited inhibition against HIV-1(IIIB) replication with a 50% effective concentration (EC(50)) value of 40.54 micromol/L in C8166 cells, and cytotoxicity with a cytotoxic concentration value of 173.84 micromol/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.

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.