Destabilization of the HIV-1 Reverse Transcriptase Dimer upon Interaction with N-Acyl Hydrazone Inhibitors
- The University of Pittsburgh School of Medicine, Division of Infectious Diseases, Pittsburgh, Pennsylvania
- Michael A. Parniak, Ph.D., University of Pittsburgh School of Medicine, Division of Infectious Diseases, S818D, Scaife Hall, 3550 Terrace Street, Pittsburgh, PA 15261. E-mail: parniakm{at}msx.dept-med.pitt.edu
Abstract
N-(4-tert-butylbenzoyl)-2-hydroxy-1-naphthaldehyde hydrazone (BBNH) inhibits both the DNA polymerase and ribonuclease H (RNase H) activities of the human immunodeficiency virus type 1 reverse transcriptase. In this study, we show that BBNH binding impacts on the stability of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) heterodimer. The Gibbs free energy of dimer dissociation of HIV-1 RT is decreased in the presence of increasing concentrations of BBNH, resulting in a loss in stability of 3.8 kcal mol−1. To evaluate whether this observed phenomenon was mediated by BBNH binding to one or more sites in RT, we synthesized a variety of BBNH analogs and identified (4-t-butylbenzoyl)-2-hydroxy-1-salicylyl hydrazone (BBSH) and (4,N,N-dimethylaminobenzoyl)-2-hydroxy-1-naphthyl hydrazone as specific inhibitors of RT DNA polymerase or RT RNase H activity, respectively. Interestingly, only BBSH provided significant destabilization of the HIV-1 RT dimer. The identification of these specific inhibitors, in combination with other biochemical data, suggests a model in which two molecules of BBNH bind per RT heterodimer. In this regard, only the binding of hydrazone molecules in the DNA polymerase domain activity elicits the observed destabilization of the HIV-1 RT heterodimer.
Footnotes
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This work was supported in part by grants from the Medical Research Council of Canada and the International Research Scholars Program of the Howard Hughes Medical Institute (to M.A.P.) and a postdoctoral fellowship from the Medical Research Council of Canada (to N.S.-C.).
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This work was initiated while the authors were at the Lady Davis Institute for Medical Research and McGill University AIDS Center, Sir Mortimer B. Davis-Jewish General Hospital, Montreal, QC, Canada.
- Abbreviations:
- HIV
- human immunodeficiency virus
- BBNH
- N-(4-tert-butylbenzoyl)-2-hydroxy-1-naphthaldehyde hydrazone
- BBSH
- (4-t-butylbenzoyl)-2-hydroxy-1-salicylyl hydrazone
- CD
- circular dichroism
- DABNH
- (4,N,N-dimethylamino benzoyl)-2-hydroxy-1-naphthyl hydrazone
- HPLC
- high-performance liquid chromatography
- RDDP
- RNA-dependent DNA polymerase
- RNase H
- ribonuclease H
- RT
- reverse transcriptase
- T/P
- template-primer
- TSAOe3T
- 1-{spiro[4-amino-2,2-dioxo-1,2-oxathiole-5,3′-[2′,5′-bis-O-(tert-butyldimethylsilyl)-β-d-ribofuranosyl]]}-3-ethylthymine
- NNRTI
- nonnucleoside reverse transcriptase inhibitor
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- Received September 28, 2001.
- Accepted April 29, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
