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 (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-1(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 (Lys 103, Lys 173, and Thr 174) 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.
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This work was financially supported by the National Natural Science Foundation of China (No 30525024, 20472095, and 20572023), Shanghai Pujiang Program (No 05PJ14034), Shanghai Key Basic Research Project (No 06JC14080 and 05JC14092), the State Key Program of Basic Research of China (No 2004CB58905 and 2006AA09Z447), and a grant from the Chinese Academy of Sciences (No KSCX2-YW-R-18).
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Du, L., Zhao, Yx., Yang, Lm. et al. Symmetrical 1-pyrrolidineacetamide showing anti-HIV activity through a new binding site on HIV-1 integrase. Acta Pharmacol Sin 29, 1261–1267 (2008). https://doi.org/10.1111/j.1745-7254.2008.00863.x
- HIV-1 integrase inhibitor
- surface plasma resonance
- molecular docking
- site-directed mutagenesis
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