Kinetic and mutational analysis of human immunodeficiency virus type 1 reverse transcriptase inhibition by inophyllums, a novel class of non-nucleoside inhibitors.

P.B Taylor,J.S Culp,C Debouck,R.K Johnson,A.D Patil,D.J Woolf,I Brooks,R.P Hertzberg

doi:10.1016/s0021-9258(17)37375-1

Abstract

Inophyllums are novel non-nucleoside inhibitors of human immunodeficiency virus (HIV) type 1 reverse transcriptase identified through an enzyme screening program and isolated from the plant Calophyllum inophyllum. The kinetics of reverse transcriptase inhibition by inophyllum B were characterized using recombinant purified enzyme, a heteropolymeric RNA template, and a scintillation proximity assay. Preincubation of inhibitor with the enzyme-template-primer complex for 11 min was required for maximal inhibition of reverse transcriptase to occur, suggesting that inophyllum B had a slow on-rate and that template-primer must bind 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 inhibited reverse transcriptase noncompetitively with a Ki of 42 nM. Enzyme inhibition with respect to template-primer was uncompetitive with a Ki of 26 nM. Reverse transcriptase enzymes containing point mutations in which tyrosine 181 was changed to either cysteine or isoleucine exhibited marginal resistance to inophyllums but were resistant to (+)-(5S)-4,5,6,7-tetrahydro-9-chloro-5-methyl-6- (3-methyl-2-butenyl)-imidazo[4,5,1-j,k][1,4]benzodiazepin-2-(1H)-t hione (TIBO R82913). A mutant enzyme in which tyrosine 188 was changed to leucine was cross-resistant to both inophyllum B and TIBO R82913, as was HIV type 2 reverse transcriptase. These studies suggest that inophyllum B and TIBO R82913 bind to distinct but overlapping sites. Inhibition of avian myeloblastosis virus reverse transcriptase and Moloney murine leukemia virus reverse transcriptase by inophyllum B was detectible, suggesting that these inhibitors may be more promiscuous than other previously described non-nucleoside inhibitors. Inophyllums were active against HIV type 1 in cell culture with IC50 values of approximately 1.5 microM. These studies imply that the inophyllums have a novel mechanism of interaction with reverse transcriptase and as such could conceivably play a role in combination therapy.

Highlights

11 To whom COReSDOndenCe should be addressedPharmaceuticals,Mail Code UW-2110, Kingof Prussia, PA 19406. “el.: dimethylammoniol-I-propanesulfonicacid; Dm, dithiothreitol;PAGE, 215-270-6629;Fax: 215-270-5005
As judged from SDS-PAGE, the cleanest fractions containing the highest amounts ofRT were and active enantiomer of the naturally occurring inophyllum B pooled.Glycerol was added to a final concentration of 40% to allow was prepared.Recently another cou- storage at -20 "C without freezing.The RT mutants were purified in a marin derivative related to inophyllum B called calanolide A similar manner with the following changes
We report the kinetic characterization of HIV-1RT inhibitionby inophyllum Band TIBO using a scintillationproxflow columnA. ll other conditions werethe same

Summary

11 To whom COReSDOndenCe should be addressed

Pharmaceuticals,Mail Code UW-2110, Kingof Prussia, PA 19406. “el.: dimethylammoniol-I-propanesulfonicacid; Dm, dithiothreitol;PAGE, 215-270-6629;Fax: 215-270-5005. Inhibition of Reverse Danscriptase by Inophyllums nucleoside RT inhibitors from a mechanism-based screening S-buffer As judged from SDS-PAGE, the cleanest fractions containing the highest amounts ofRT were and active enantiomer of the naturally occurring inophyllum B pooled.Glycerol was added to a final concentration of 40% to allow was prepared (inophyllumP; see Fig. 1).Recently another cou- storage at -20 "C without freezing.The RT mutants were purified in a marin derivative related to inophyllum B called calanolide A similar manner with the following changes. Inhibitors were stored as 10-25 II~Mstock solutionsin MezSO at 4 "C From these stock solutions, 10%MezSO working solutions in assay buffer were made such that the final concentration of Me,SO in the assays was 1%.The detergents CHAPSand Triton X-100 were purchased from Sigma, and Nonidet P-40was obtained from Cal-

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