Mechanism study of novel CCR5 antagonists and their potential as anti-HIV-1 microbicides

Abstract

R5-tropic HIV-1 is predominantly transmitted during unprotected sexual contacts, rendering CCR5 antagonist as an attractive agent not only for antiretroviral therapy but also for prevention. Here, we report two 1,3,3,4-tetrasubstituted pyrrolidine embodied compounds, TD-0232 and TD-0680, as novel small molecule CCR5 antagonists and investigate their specificities, potencies and underlying mechanisms. We found that both TD-0232 and TD-0680 inhibited a diverse group of R5-tropic HIV-1 and SIV strains in both single-cycle infectivity assays and live viral PBMC assays. When compared to other CCR5 antagonists, such as TAK-779 and the only FDA-approved Maraviroc, TD-0680 displayed the highest potency with EC50 values at the subnanomolar levels (range 0.09nM-2.29nM). TD-0232 and TD-0680, but not Tenofovir, a nucleoside reverse transcriptase inhibitor, completely blocked envelope-mediated cell-cell fusion and subsequent viral transmission. Critically, TD-0680 was potent at inhibiting the replication of a TAK-779/Maraviroc-resistant HIV-1 variant in PBMCs at a subnanomolar concentration.

Interestingly, despite binding to a similar transmembrane pocket of CCR5, TD-0232 and TD-0680 functioned differently as revealed by site-directed mutagenesis and drug combination assays. Based on the sequence homology, we constructed a CCR5 molecule model using the crystallized CXCR4 as a template. By docking of CCR5 antagonists with CCR5, we identified a unique binding mode of TD-0680, which has not been described previously. TD-0680, with an exo-configuration, extended its interaction with the ECL-2 region of CCR5 in a protruding manner, thereby interrupting the interaction between the virus and its co-receptor more effectively. In an antibody recognition assay, we confirmed that TD-0680 had an enhanced inhibitory activity against the anti-ECL2 monoclonal antibodies binding.

Furthermore, we investigated the antiviral activities of TD-0232 and TD-0680 that were formulated into a thermo-reversible acidic microbicide gel. Both drugs were stable in the acidic gels and could be released rapidly for long lasting and potent antiviral activities. Although human semen could enhance the infection of HIV-1, it did not seem to affect the potencies of the TD-0232 and TD-0680 gels.

In summary, our findings suggest that TD-0232 and TD-0680 can be further developed not only as anti-HIV-1 agents for therapeutic purposes but also as potent microbicides for the prevention of sexual transmission of R5-tropic HIV-1.