18th International HIV Drug Resistance Workshop Basic Principles & Clinical Implications June 9–13 2009, Fort Myers, Florida, USA

DIFFERENT MECHANISMS OF HIV-1 INHIBITION BY CCR5 AGONISTS/ANTAGONISTS (MARAVIROC, TAK-779 AND PSC-RANTES) ARE LINKED TO DIFFERENT DRUG RESISTANCE MECHANISMS

Antivir Ther 2009; 14 Suppl 1:A14 (abstract no. 12)

M Lobritz, A Ratcliff, A Marozsan, D Dudley and E Arts
Case Western Reserve University, Cleveland, OH, USA

BACKGROUND: TAK-779 and maraviroc bind a hydrophobic cavity formed between transmembrane helices in CCR5 near the membrane surface. By contrast, PS-CRANTES interacts with the second extracellular loop of CCR5 and effectively downregulates surface CCR5 expression. However, inhibition of virus may be primarily due to competitive binding of PSC-RANTES to CCR5.

METHODS: To access differential mechanisms of drug activity and resistance to CCR5 agonists/antagonists, we employed a panel of primary HIV-1 isolates and clones with varying susceptibility to PSC-RANTES and maraviroc (100-fold variations in IC₅₀ values) in multiple and single-cycle drug susceptibility assays with varying amounts of drug (inhibitor) and virus (substrate).

RESULTS: In multiple cycle assays, primary HIV-1 isolates of subtypes A, B, C and D displayed a 100-fold variation in susceptibility to maraviroc, but only fivefold variation to PSC-RANTES inhibition (as determined by IC₅₀ values). We then selected two clones (varying in a single amino acid substitution in the V3 loop) with a 10-fold difference in IC₅₀ value to PSC-RANTES in multiple-cycle infection assays. Differential PSC-RANTES inhibition of the HIV-1 clones was mediated by competitive binding for CCR5. Increasing virus concentration could saturate the receptors on cell surface and overcome PSC-RANTES inhibition indicative of competitive inhibition. In single-cycle replication assays, the two HIV-1 clones displayed equal susceptibility to PSC-RANTES due to effective CCR5 downregulation even when the virus titres (substrate) were increased. By contrast, PSC-RANTES inhibition was evident between the two clones, even in a single-cycle assay, when the cells harbored CCR5 mutant incapable of ligand-induced downregulation. With maraviroc and TAK779, the same level of resistance was observed in both the single and multiple cycle assays.

CONCLUSIONS: In conclusion, CCR5 receptor downregulation by PSC-RANTES or RANTES provides minimal contribution to overall HIV-1 inhibition under physiological conditions. However, the other CCR5 antagonists are non-competitive, allosteric inhibitors incapable of receptor downregulation. The effect of CCR5 downregulation by PSC-RANTES appears to be short-lived as an inhibitory mechanism and as compared with subsequent competitive inhibition. Competitive inhibition, and not receptor downregulation, is the likely factor that contributed to the immediate selection of PSC-RANTES-resistant SHIV clone in vaginal microbicide model.

2009-06-09
12

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