title: Kinetic and mutational analysis of human immunodeficiency virus type 1 reverse transcriptase inhibition by inophyllums, a novel class of non-nucleoside inhibitors.
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.
authors.affiliation: Department of Biomolecular Discovery, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406.
authors.forename: P B
authors.initials: PB
authors.lastname: Taylor
authors.forename: J S
authors.initials: JS
authors.lastname: Culp
authors.forename: C
authors.initials: C
authors.lastname: Debouck
authors.forename: R K
authors.initials: RK
authors.lastname: Johnson
authors.forename: A D
authors.initials: AD
authors.lastname: Patil
authors.forename: D J
authors.initials: DJ
authors.lastname: Woolf
authors.forename: I
authors.initials: I
authors.lastname: Brooks
authors.forename: R P
authors.initials: RP
authors.lastname: Hertzberg
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D001483:Base Sequence; D001569:Benzodiazepines / Q000494:pharmacology*; D003374:Coumarins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D007093:Imidazoles / Q000494:pharmacology*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D012194:RNA-Directed DNA Polymerase / Q000096:biosynthesis / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000302:isolation & purification; D018894:Reverse Transcriptase Inhibitors; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D000998:Antiviral Agents; D003374:Coumarins; D007093:Imidazoles; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D018894:Reverse Transcriptase Inhibitors; D001569:Benzodiazepines; C086091:inophyllum B; C067413:R-82913; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase