309   New Small-Molecule Inhibitors of HIV-1 Entry.

Y. Shu*¹, T. Stanchev², X. Xiao¹, P. D'Souza³, M. Nasr³, C .C. Broder², and D. S. Dimitrov¹.
¹NCI-Frederick, NIH, Frederick, MD;²Uniformed Services Univ. of the Hlth. Sci., Bethesda, MD; and³NIAID, NIH, Bethesda, MD.

Background:HIV-1 entry into cells is mediated through the formation of a complex between its envelope glycoprotein (Env), CD4 and coreceptor molecules. Disruption of the complex formation is a promising avenue for development of HIV-1 inhibitors.

Methods:The inhibitory activities of 21 compounds were measured by theb-gal reporter gene cell-cell fusion assay, a syncytium assay, and the pseudotyped luciferase-based HIV-1 infection assay. We also developed a new assay based on soluble CD4-induced fusion. The disruption of the Env-CD4-coreceptor complex was measured by an improved coimmunoprecipitation assay. Cell toxicity was measured by trypan blue exclusion.

Results:We found new small-molecule inhibitors of HIV-1 entry that affected either R5 (JRFL) or X4 (HBX) or both isolates. Some of the compounds had inhibitory efficiency (50% inhibitory concentration below 10mg/ml) comparable to or higher than that of known small-molecule (e.g. AMD3100) or peptide (e.g. AOP-RANTES) inhibitors, which were tested under the same conditions. Several of the inhibitors showed strong synergistic effects. The inhibitory effect decreased with a decrease in the concentration of the compounds, but even at 1mg/ ml some of the inhibitors showed inhibitory activity higher than 50%. The toxicity to the target cells (NIH 3T3 CD4 CCR5 (CXCR4) and U373) was minimal, even at relatively high concentrations (60mg/ml). The inhibitory efficiency of the compounds for soluble CD4-induced fusion was about 10-fold higher than in the standardb-gal reporter gene cell-cell fusion assay. Some of the inhibitors disrupted the Env-CD4-coreceptor complex formation, but others did not. We are currently investigating in more detail the mechanism of their inhibitory effects.

Conclusions:The potential importance of these new inhibitors lies in the ability of some of them to interfere with the entry of both R5 and X4 HIV-1 isolates and the potential for further improvement of their activity by rational design of novel inhibitors.