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

HIV INTEGRASE INHIBITOR DISSOCIATION RATES CORRELATE WITH EFFICACY IN VITRO

Antivir Ther 2009; 14 Suppl 1:A27 (abstract no. 25)

JA Grobler, PM Mckenna, S Ly, KA Stillmock, CM Bahnck, RM Danovich, G Dornadula, DJ Hazuda and MD Miller
Department of Antiviral Research, Merck Research Laboratories, West Point, PA, USA

BACKGROUND: Resistance to the integrase strand transfer inhibitor (InSTI) raltegravir in the clinic and in cell culture studies is generally associated with integrase mutations at positions N155, Q148 or Y143, usually in combination with other mutations. The mechanisms by which these mutations confer resistance have not been fully elucidated.

METHODS: Time of addition and washout experiments were conducted using HIV-1 (JRFL) engineered to express green fluorescent protein. HOSCD4R5 cells were synchronously infected for 30 min using magnetic beads, washed and monitored for infection by counting the number of GFP-expressing cells 48 h later. Drug residence time on integrase/DNA complexes was measured using a scintillation proximity assay. Integrase was assembled on immobilized viral DNA ends and equilibrated with radiolabeled drug. Dissociation rates were determined by scintillation counting at regular intervals following the addition of a large excess of unlabeled compound.

RESULTS: In time-of-addition and washout studies, raltegravir effectively blocked wild-type HIV-1 replication when present during a relatively short window of time following the completion of reverse transcription. In dissociation kinetic studies, raltegravir exhibited a long residence time on the integrase/DNA complexes that is comparable to or exceeds the half-life of the pre-integration complex in the cell. The clinically important integrase mutation N155H increased the rate of raltegravir dissociation by 10-fold and allowed the inhibitory effects of the compound to be washed out in viral replication assays. The second-generation InSTI MK2048, which better inhibits raltegravir-resistant variants, exhibited a substantially longer residence time on integrase than raltegravir. While a similar increase in dissociation rate was observed with MK-2048 with integrase containing the N155H mutation, the half-life of MK2048 on the mutant is nearly that of raltegravir on the wild-type enzyme.

CONCLUSIONS: Residence times of InSTIs on integrase/DNA complexes comparable to or exceeding the half-life of the pre-integration complex in the cell results in functionally irreversible ‘one-shot kill’ of integration. Resistance conferring mutations increase the rate of inhibitor dissociation from integrase providing an opportunity for integration to occur. Improved efficacy of second- generation InSTIs will likely be driven by increases in inhibitor residence time on integrase.

2009-06-09
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