Inhibition of HIV-1 reverse transcriptase-catalyzed DNA strand transfer reactions by 4-chlorophenylhydrazone of mesoxalic acid

DNA strand transfer reactions occur twice during retroviral reverse transcr iption catalyzed by HIV-1 reverse transcriptase. The 4-chlorophenylhydrazon e of mesoxalic acid (CPHM) was found to be an inhibitor of DNA strand trans fer reactions catalyzed by HN-I reverse transcriptase. Using a model strand transfer assay system described previously [Davis, W. R., et al. (1998) Bi ochemistry 37, 14213-14221], the mechanism of CPHM inhibition of DNA strand transfer has been characterized. CPHM was found to target the RNase I-I ac tivity of HIV-I reverse transcriptase. DNA polymerase activity was not sign ificantly affected by CPHM; however, it did inhibit the polymerase-independ ent RNase H activity with an IC50 of 2.2 muM. In the absence of DNA synthes is, CPHM appears to interfere with the translocation, or repositioning, of RT on the RNA DNA template duplex, a step required for efficient RNA hydrol ysis by RNase H. Enzyme inhibition by CPHM was found to be highly specific for HIV-1 reverse transcriptase; little or no inhibition of DNA strand tran sfer or DNA polymerase activity was observed with MLV or AMV reverse transc riptase, T7 DNA polymerase, or DNA polymerase I. Examination of additional 4-chlorophenylhydrazones showed that the dicarboxylic acid moiety of CPHM i s essential for activity, suggesting its important role for enzyme binding. Consistent with the role of the dicarboxylic acid in inhibitor function, M g2+ was found to chelate directly to CPHM with a K-d Of 2.4 mM. Together, t hese studies suggest that the inhibitor may function by binding to enzyme-b ound divalent metal cofactors.