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  International HIV and Hepatitis Drug Resistance Workshop
June 8-12, 2010,
Dubrovnik Croatia
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S/GSK1349572 Demonstrates Significantly Slower Dissociation Rates than Raltegravir When Comparing Wild Type and Raltegravir Resistant Integrase Protein
  Reported by Jules Levin
International Drug Resistance Workshop, 8-12 June 2010, Dubrovnik, Croatia
Kendra E. Hightower, Ruolan Wang, Mark R. UnderwoodGlaxoSmithKline, Inc., RTP, NC, USA

The integrase inhibitor (INI) S/GSK1349572 (572) has significant activity against HIV-1 with raltegravir-associated resistance mutations. Previous evaluations demonstrated faster raltegravir dissociation with N155H, which has been suggested as a mechanism of resistance. As an initial step in characterizing the different resistance profiles for S/GSK1349572 and raltegravir, their dissociation rates were determined with wild type and resistant integrase proteins, including signature raltegravir mutations at Q148 and N155.
Methods: HIV-1 integrase BH10 wild type and site directed mutant proteins were used for inhibitor binding studies. These studies were performed by pre-incubating 40 nM 3H-labeled S/GSK1349572 or raltegravir overnight at room temperature with an integrase-donor DNA complex that was bound to SPA imaging beads. Following incubation at 37C for one hour, 40 mM cold compound was added, and dissociation at 37C was monitored for over 7 days using a ViewLux™.
Results: S/GSK1349572 dissociated slowly from wild type integrase at 37C with a half-life of approximately 100 hours (versus 10.1 hours with raltegravir). The presence of a N155H, Q148H or Q148R mutation decreased the half-life of S/GSK1349572 binding (11.6, 7.7, and 10.7 hours, respectively), but the S/GSK1349572 binding half-lives were still at least 19-times longer than raltegravir (0.6, <0.3, and 0.4 hours, respectively). Mutation at E92 had the least effect on compound dissociation (t1/2 of 23.6 and 3.9 hours for S/GSK1349572 and raltegravir, respectively) but the addition of this second mutation to N155H impacted compound dissociation (t1/2of 5.8 and <0.3 hours for S/GSK1349572 and raltegravir, respectively).
Conclusions: S/GSK1349572 had significantly slower dissociation than raltegravir from wild type integrase and all mutants examined. In fact, dissociation of S/GSK1349572 from raltegravir resistant N155H, Q148H, and Q148R mutants was comparable to dissociation of raltegravir from wild type integrase protein. The measured values for raltegravir dissociation were consistent with previously presented values. The long dissociation half-life of S/GSK1349572 may contribute to its distinct resistance profile and highlight a potential for improved activity against wild type HIV-1 and raltegravir-resistant viruses.
HIV-1 integrase catalyzes the insertion of viral DNA into the host genome through 3' end processing of the viral DNA followed by strand transfer. Since the activity of HIV-1 integrase is necessary for viral replication, integrase is a target for the development of new anti-viral therapies. Raltegravir was the first marketed integrase inhibitor (INI); some patients on raltegravir treatment have developed resistance to the drug. In general, INIs appear to dissociate slowly from wild type integrase-DNA complexes.1, 2It has been suggested that faster dissociation of raltegravir from the N155H raltegravir-resistant mutant may contribute to the mechanism of resistance.1S/GSK1349572 is an INI that demonstrates significant antiviral activity against wild type and raltegravir-resistant mutants, and is being developed for once daily dosing. We investigated dissociation of S/GSK1349572 and raltegravir from wild type and mutant integrase to obtain a better understanding of how binding could contribute to the distinctive resistance profile of S/GSK1349572.

Figure 1. INI Dissociation Experiment

·Proximity of 3H to the imaging bead when the 3H-INI is bound to the IN-DNA-bead complex resulted in emission of light from the scintillant containing beads.
·As the 3H-INI dissociated from the IN-DNA-bead complex it was replaced by cold INI which caused a signal decrease.
·Stable 3H-INI-IN-DNA-bead complexes were obtained for all proteins tested with both S/GSK1349572 and raltegravir. As shown here the 3H-572-IN-DNA-bead complex was stable for more than two weeks at 37C with wild type protein.

·For all forms of integrase used in this study, dissociation of S/GSK1349572 was significantly slower than dissociation of raltegravir.
·Relative binding represents the ratio of the background subtracted signals with and without addition of cold INI.
·Assays were performed at 37C to mimic physiological conditions.
·Dissociation went to completion for both S/GSK1349572 and raltegravir with all forms of integrase used in this study.

·S/GSK1349572 dissociated more slowly than raltegravir from raltegravir-resistant mutants.
·In vitro resistance fold change (FC) versus wild type integrase was determined in a HeLa-CD4 cell assay.3

1.Grobler JA, et al., 2009, 10thInternational Workshop on Clinical Pharmacology of HIV Therapy; Abstract O-10
2.Hluhanich et al., 2009, 49thInterscience Conference on Antimicrobial Agents and Chemotherapy; Poster H-930
3.Seki et al., 2010, 17thConference on Retroviruses and Opportunistic Infections; Poster 555
4.Yoshinaga et al., 2010, International Drug Resistance Workshop
5.DeAnda et al., 2010 International Drug Resistance Workshop