Identification of an inhibitor-binding site to HIV-1 integrase with affinity acetylation and mass spectrometry

doi:10.1073/pnas.0400873101

Identification of an inhibitor-binding site to HIV-1 integrase with affinity acetylation and mass spectrometry

^*Ohio State University Health Sciences Center, College of Pharmacy, Center for Retrovirus Research and Comprehensive Cancer Center, Columbus, OH 43210; ^†Laboratory of Medicinal Chemistry and ^§HIV Drug Resistance Program, National Cancer Institute, Frederick, MD 21702; and ^‡National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892

Edited by John M. Coffin, Tufts University School of Medicine, Boston, MA (received for review February 6, 2004)

Abstract

We report a methodology that combines affinity acetylation with MS analysis for accurate mapping of an inhibitor-binding site to a target protein. For this purpose, we used a known HIV-1 integrase inhibitor containing aryl di-O-acetyl groups (Acetylated-Inhibitor). In addition, we designed a control compound (Acetylated-Control) that also contained an aryl di-O-acetyl group but did not inhibit HIV-1 integrase. Examination of the reactivity of these compounds with a model peptide library, which collectively contained all 20 natural amino acids, revealed that aryl di-O-acetyl compounds effectively acetylate Cys, Lys, and Tyr residues. Acetylated-Inhibitor and Acetylated-Control exhibited comparable chemical reactivity with respect to these small peptides. However, these two compounds differed markedly in their interactions with HIV-1 integrase. In particular, Acetylated-Inhibitor specifically acetylated K173 at its inhibitory concentration (3 μM) whereas this site remained unrecognized by Acetylated-Control. Our data enabled creation of a detailed model for the integrase:Acetylated-Inhibitor complex, which indicated that the inhibitor selectively binds at an architecturally critical region of the protein. The methodology reported herein has a generic application for systems involving a variety of ligand–protein interactions.

Footnotes

↵ ¶ To whom correspondence should be addressed at: Ohio State University, 500 West 12th Avenue, 238 L. M. Parks Hall, Columbus, OH 43210. E-mail: kvaratskhelia.1{at}osu.edu.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: IN, integrase; MALDI-TOF, matrix-assisted laser desorption ionization/time-of-flight; PSD, post source decay; MS/MS, tandem MS.