Derivatives of mesoxalic acid block translocation of HIV-1 reverse transcriptase [Microbiology]

October 29th, 2014 by Bernatchez, J. A., Paul, R., Tchesnokov, E. P., Ngure, M., Beilhartz, G. L., Berghuis, A. M., Lavoie, R., Li, L., Auger, A., Melnyk, R. A., Grobler, J. A., Miller, M. D., Hazuda, D. J., Hecht, S. M., Gotte, M.

The pyrophosphate mimic and broad spectrum antiviral phosphonoformic acid (PFA, forscarnet) was shown to freeze the pre-translocational state of the reverse transcriptase (RT) complex of the human immunodeficiency virus type 1 (HIV-1). However, PFA lacks a specificity domain, which is seen as a major reason for toxic side effects associated with the clinical use of this drug. Here we studied the mechanism of inhibition of HIV-1 RT by the 4-chlorophenylhydrazone of mesoxalic acid (CPHM), and demonstrate that this compound also blocks RT translocation. Hot spots for inhibition with PFA or CPHM occur at template positions with a bias toward pre-translocation. Mutations at active site residue D185 compromise binding of both compounds. Moreover, divalent metal ions are required for the formation of ternary complexes with either of the two compounds. However, CPHM contains both an anchor domain that likely interacts with the catalytic metal ions and a specificity domain. Thus, although the inhibitor binding sites may partly overlap, they are not identical. The K65R mutation in HIV-1 RT, which reduces affinity to PFA, increases affinity to CPHM. Details with respect to the binding sites of the two inhibitors are provided on the basis of mutagenesis studies, structure activity relationship analyses with newly designed CPHM derivatives, and in silico docking experiments. Together, these findings validate the pre-translocated complex of HIV-1 RT as a specific target for the development of novel classes of RT inhibitors.