17th International HIV Drug Resistance Workshop 10-14 June 2008, Sitges, Spain

MECHANISM BY WHICH THE HIV INTEGRASE ACTIVE-SITE MUTATION N155H CONFERS RESISTANCE TO RALTEGRAVIR

Antivir Ther. 2008; 13(Suppl. 3):A41 (abstract no. 36)

JA Grobler, KA Stillmock, MD Miller and DJ Hazuda
Merck Research Laboratories, West Point, Pennsylvania, USA

BACKGROUND: Raltegravir is the first and currently the only integrase strand transfer inhibitor (InSTI) approved for the treatment of HIV infection. Raltegravir inhibits integrase by binding to and functionally sequestering essential active-site magnesium ions. The binding of the inhibitor to integrase is mediated both by interactions with the metals and via direct contact with the enzyme. The active-site mutation N155H, either alone or with additional mutations, is associated with resistance to raltegravir both in cell-culture studies and in the clinic and has been observed to confer cross-resistance to other InSTIs, including elvitegravir. The mechanism by which this mutation confers resistance has not yet been elucidated. N155 interacts with integrase active-site residues that bind the magnesium ions, and mutation of this residue may confer resistance by perturbing metal binding to the active site.

METHODS: A radioligand binding assay was employed to measure the magnesium-dependent binding of raltegravir to functional complexes of wild-type integrase (In_wt) and mutant integrase (IN_N155H) assembled with viral DNA ends.

RESULTS: Raltegravir binding affinities to In_wt and In_N155H were dependent on magnesium ion concentration. The two enzymes displayed similar binding affinities for magnesium and for raltegravir in the absence of metal. However, the binding affinity of raltegravir to integrase at saturating magnesium concentrations was lower with InN155H than with wild-type enzyme.

CONCLUSIONS: The integrase active-site mutation N155H confers resistance to raltegravir primarily by perturbing the arrangement of the active-site magnesium ions and not by affecting the affinity of the metals or by affecting direct contacts of the inhibitor with the enzyme. These studies lay a foundation for determining the roles of additional mutations in the N155H resistance pathway and for establishing the mechanisms by which other pathways confer resistance to InSTIs.

2008-06-10
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