KINETIC AND MUTATIONAL ANALYSIS OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REVERSE-TRANSCRIPTASE INHIBITION BY INOPHYLLUMS, A NOVEL CLASS OF NONNUCLEOSIDE INHIBITORS

Inophyllums are novel non-nucleoside inhibitors of human immunodeficie ncy virus (HIV) type 1 reverse transcriptase identified through an enz yme screening program and isolated from the plant Calophyllum inophyll um. The kinetics of reverse transcriptase inhibition by inophyllum B w ere characterized using recombinant purified enzyme, a heteropolymeric RNA template, and a scintillation proximity assay. Preincubation of i nhibitor with the enzyme-template-primer complex for 11 min was requir ed for maximal inhibition of reverse transcriptase to occur, suggestin g that inophyllum B had a slow on-rate and that template-primer must b ind to reverse transcriptase prior to inhibitor binding. Inhibition of reverse transcriptase by inophyllums was shown to be reversible. When thymidine triphosphate was the variable substrate, inophyllum B inhib ited reverse transcriptase noncompetitively with a K-i of 42 nM. Enzym e inhibition with respect to template-primer was uncompetitive with a K-i of 26 nM. Reverse transcriptase enzymes containing point mutations in which tyrosine 181 was changed to either cysteine or isoleucine ex hibited marginal resistance to inophyllums but were resistant to (+)-( 5S) trahydro-9-chloro-5-methyl-6-(3-methyl-2-butenyl)- imidazo[4,5,1-j ,k][1,4]benzodiazepin-2-(1H)-thione (TIBO RS2913). A mutant enzyme in which tyrosine 188 was changed to leucine was cross-resistant to both inophyllum B and TIBO RS2913, as was HIV type 2 reverse transcriptase. These studies suggest that inophyllum B and TIBO R82913 bind to disti nct but overlapping sites. Inhibition of avian myeloblastosis virus re verse transcriptase and Moloney murine leukemia virus reverse transcri ptase by inophyllum B was detectible, suggesting that these inhibitors may be more promiscuous than other previously described non-nucleosid e inhibitors. Inophyllums were active against HIV type 1 in cell cultu re with IC50 values of approximately 1.5 mu M. These studies imply tha t the inophyllums have a novel mechanism of interaction with reverse t ranscriptase and as such could conceivably play a role in combination therapy.