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

• Published in: The Journal of biological chemistry Vol. 269; no. 9; pp. 6325 - 6331
• Main Authors: Taylor, P B, Culp, J S, Debouck, C, Johnson, R K, Patil, A D, Woolf, D J, Brooks, I, Hertzberg, R P
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 04.03.1994
• Subjects: Antiviral Agents - pharmacology; Base Sequence; Benzodiazepines - pharmacology; Coumarins - chemistry; Coumarins - isolation & purification; Coumarins - pharmacology; HIV Reverse Transcriptase; HIV-1 - enzymology; human immunodeficiency virus 1; Imidazoles - pharmacology; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligodeoxyribonucleotides; Recombinant Proteins - antagonists & inhibitors; Recombinant Proteins - biosynthesis; Recombinant Proteins - isolation & purification; Reverse Transcriptase Inhibitors; RNA-Directed DNA Polymerase - biosynthesis; RNA-Directed DNA Polymerase - isolation & purification; Structure-Activity Relationship; Substrate Specificity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)37375-1

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.