Interaction of HIV-1 reverse transcriptase and T7 RNA polymerase with phosphonate analogs of NTP and inorganic pyrophosphate

We have examined the interaction of human immunodeficiency virus reverse tr anscriptase (HIV RT) and T7 RNA polymerase (T7 RNAP) with modified nucleosi de triphosphates and inorganic pyrophosphate (PPi) analogs containing nonhy drolyzable bisphosphonate groups. We have synthesized a number of derivativ es of bi-phosphonic acid having different aromatic and nonaromatic side sub stituents, as well as the NTP derivatives whose incorporation into the grow ing nucleotide chain during the polymerization reaction results in formatio n of bisphosphonates as leaving groups. The competitive character of inhibi tion of both enzymes has been revealed for all the compounds under study, a nd the inhibition constants have been estimated. One of PPi analogs contain ing a bulky aromatic substituent is characterized by similar inhibition con stants for both T7 RNAP and RT. The universal character of this inhibitor c an serve as evidence for a similar structure of the NPT-binding sites in th e two polymerases. It has been shown that nonsubstituted methylenebisphosph ate is a better leaving group than that containing additional methyl and hy droxyl 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, w e have modeled the binding of some derivatives of bisphosphonic acid in the active center of the enzyme. The peculiarities observed in the model corre late well with the experimental data on inhibition.