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Preliminary Mapping of a Putative Inhibitor-Binding Pocket for Human Immunodeficiency Virus Type 1 Integrase Inhibitors

Departments of Microbiology and Molecular Genetics,¹ Medicine,² Pathology and Laboratory Medicine, University of California, Irvine, California 92697-4800³

Received 28 July 2005/ Returned for modification 6 September 2005/ Accepted 26 September 2005

Molecular modeling studies have identified a putative human immunodeficiency virus (HIV) integrase (IN) inhibitor-binding pocket for L-chicoric acid (L-CA) and other inhibitors of IN (C. A. Sotriffer, H. Ni, and A. McCammon, J. Med. Chem. 43:4109-4117, 2000). By using site-directed mutagenesis of several amino acid residues identified by modeling studies, a common inhibitor-binding pocket on IN was confirmed for L-CA and the diketo acid L-731,988. Specifically, the single mutations E92K, Q148A, K156A, K156R, G140S, and G149S, as well as the double mutations C65S-K156N and H67D-G140A were evaluated for their effects on enzymatic activity and inhibitor susceptibility. Each recombinant IN was attenuated for 3'-end processing and strand transfer activities. Most proteins were also attenuated for disintegration; the IN that contained K156R and C65S-K156N, however, displayed disintegration activity similar to that of IN from HIV_NL4-3. All mutant IN proteins demonstrated decreased susceptibility to L-CA, while all mutant proteins except E92K and K156R demonstrated resistance to L-731,988. These data validate the computer modeling data and demonstrate that L-CA and L-731,988 share an overlapping inhibitor-binding pocket that involves amino acids Q148, C65, and H67. The resistance studies confirm that L-731,988 fills one-half of the inhibitor-binding pocket and binds to Q148 but excludes E92, while L-CA fills the entire binding groove and thus interacts with E92. These results provide "wet laboratory" evidence that molecular models of the HIV IN inhibitor-binding pocket can be used for drug discovery.

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