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


CONCLUSIONS
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.
Du
Li
L
Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China.
Zhao
Ya-xue
YX
Yang
Liu-meng
LM
Zheng
Yong-tang
YT
Tang
Yun
Y
Shen
Xu
X
Jiang
Hua-liang
HL
D000081:Acetamides / Q000138:chemical synthesis* / Q000494:pharmacology*; D019380:Anti-HIV Agents / Q000138:chemical synthesis* / Q000494:pharmacology*; D001665:Binding Sites; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D019427:HIV Integrase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019428:HIV Integrase Inhibitors / Q000138:chemical synthesis* / Q000494:pharmacology*; D015497:HIV-1 / Q000187:drug effects; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D011759:Pyrrolidines / Q000138:chemical synthesis* / Q000494:pharmacology*; D013329:Structure-Activity Relationship
C540111:1-pyrrolidineacetamide; D000081:Acetamides; D019380:Anti-HIV Agents; D004279:DNA, Viral; D019428:HIV Integrase Inhibitors; D011759:Pyrrolidines; D019427:HIV Integrase