Interaction of HIV-1 Reverse Transcriptase and T7 RNA Polymerase with Phosphonate Analogs of NTP and Inorganic Pyrophosphate

Abstract

We have examined the interaction of human immunodeficiency virus reverse transcriptase (HIV RT) and T7 RNA polymerase (T7 RNAP) with modified nucleoside triphosphates and inorganic pyrophosphate (PP_i) analogs containing nonhydrolyzable bisphosphonate groups. We have synthesized a number of derivatives of bisphosphonic acid having different aromatic and nonaromatic side substituents, as well as the NTP derivatives whose incorporation into the growing nucleotide chain during the polymerization reaction results in formation of bisphosphonates as leaving groups. The competitive character of inhibition of both enzymes has been revealed for all the compounds under study, and the inhibition constants have been estimated. One of PP_ianalogs containing a bulky aromatic substituent is characterized by similar inhibition constants for both T7 RNAP and RT. The universal character of this inhibitor can serve as evidence for a similar structure of the NPT-binding sites in the two polymerases. It has been shown that nonsubstituted methylenebisphosphate is a better leaving group than that containing additional methyl and hydroxyl groups. The NTP analogs are very weak inhibitors of T7 RNAP, whereas HIV RT is more sensitive to this type of compounds. On the basis of the X-ray crystallographic data on the T7 RNAP complex with a template and NTP, we have modeled the binding of some derivatives of bisphosphonic acid in the active center of the enzyme. The peculiarities observed in the model correlate well with the experimental data on inhibition.