Mechanism of Inhibition of RNA Tumor Virus Reverse Transcriptase by Rifamycin SV Derivatives

Abstract

The rifamycin SV derivatives AF/ABDMP (2,5-dimethyl-4N-benzyl demethyl rifampicin), AF/013 (O-n-octyloxime of 3-formyl rifamycin SV) and C-27 [3-(dicyclohexyl-alkyl piperidyl)-rifamycin SV] have been tested against a variety of purified enzymes, including the a and as forms of the reverse transcriptase purified from avian myeloblastosis virus (AMV). These derivatives appear to be specific for nucleic acid polymerizing enzymes; they were found inactive when tested against non-polymerizing enzymes such as alkaline phosphatase, glutamateoxaloacetate transaminase, RNase A, DNase I, even at very high ratios of drug to enzyme molecule. The derivatives were very active against the DNA polymerase and RNase H activities of AMV. The mechanism by which the derivative AF/ABDMP inhibits the reverse transcriptase activities of AMV and murine leukemia-sarcoma virus, Moloney strain [MSV-MLV(M)] has been studied. The drug interacts with the enzyme, not with templates, and the inhibition is completely reversible. Lineweaver-Burke analysis demonstrated that the inhibition is of the non-competitive type with regard to the binding of templates and triphosphates. Inhibition occurs at an early step(s) of transcription and is the result of the cooperative binding of drug molecules to a region distinct from the active site. The kinetic data of polymerization obtained with the two different forms of the AMV enzyme, a and as were analyzed according to Hill in order to calculate the number of drug molecules that bind and inhibit the viral enzyme» A straight line with a slope of 6.5 was obtained with the a enzyme, which has one subunit. A biphasic plot was obtained with the as enzyme which has two subunits; the values obtained for the two slopes were 3 and 7, with the change in slope occurring at a drug concentration that gives approximately 65% inhibition. A slope of 1 was obtained when the inhibition of E. coli RNA polymerase by AF/ABDMP was studied in an analogous way. The evidence discussed in this paper suggests that the drug binds to a hydrophobic site on the enzyme which exerts a regulatory function on the initiation of DNA synthesis