Inhibition of HIV-1 Reverse Transcriptase-Catalyzed DNA Strand Transfer Reactions by 4-Chlorophenylhydrazone of Mesoxalic Acid
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- 作者：Wendolyn R. Davis ; John Tomsho ; Savita Nikam ; Elizabeth M. Cook ; David Somand ; and James A. Peliska
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：November 21, 2000
- 年：2000
- 卷：39
- 期：46
- 页码：14279 - 14291
- 全文大小：347K
- 年卷期：v.39,no.46(November 21, 2000)
- ISSN：1520-4995
文摘
DNA strand transfer reactions occur twice during retroviral reverse transcription catalyzed byHIV-1 reverse transcriptase. The 4-chlorophenylhydrazone of mesoxalic acid (CPHM) was found to bean inhibitor of DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase. Using a modelstrand transfer assay system described previously [Davis, W. R., et al. (1998) Biochemistry 37, 14213-14221], the mechanism of CPHM inhibition of DNA strand transfer has been characterized. CPHM wasfound to target the RNase H activity of HIV-1 reverse transcriptase. DNA polymerase activity was notsignificantly affected by CPHM; however, it did inhibit the polymerase-independent RNase H activitywith an IC50 of 2.2 
M. In the absence of DNA synthesis, CPHM appears to interfere with the translocation,or repositioning, of RT on the RNA·DNA template duplex, a step required for efficient RNA hydrolysisby RNase H. Enzyme inhibition by CPHM was found to be highly specific for HIV-1 reverse transcriptase;little or no inhibition of DNA strand transfer or DNA polymerase activity was observed with MLV orAMV reverse transcriptase, T7 DNA polymerase, or DNA polymerase I. Examination of additional4-chlorophenylhydrazones showed that the dicarboxylic acid moiety of CPHM is essential for activity,suggesting its important role for enzyme binding. Consistent with the role of the dicarboxylic acid ininhibitor function, Mg2+ was found to chelate directly to CPHM with a Kd of 2.4 mM. Together, thesestudies suggest that the inhibitor may function by binding to enzyme-bound divalent metal cofactors.
M. In the absence of DNA synthesis, CPHM appears to interfere with the translocation,or repositioning, of RT on the RNA·DNA template duplex, a step required for efficient RNA hydrolysisby RNase H. Enzyme inhibition by CPHM was found to be highly specific for HIV-1 reverse transcriptase;little or no inhibition of DNA strand transfer or DNA polymerase activity was observed with MLV orAMV reverse transcriptase, T7 DNA polymerase, or DNA polymerase I. Examination of additional4-chlorophenylhydrazones showed that the dicarboxylic acid moiety of CPHM is essential for activity,suggesting its important role for enzyme binding. Consistent with the role of the dicarboxylic acid ininhibitor function, Mg2+ was found to chelate directly to CPHM with a Kd of 2.4 mM. Together, thesestudies suggest that the inhibitor may function by binding to enzyme-bound divalent metal cofactors.