title: The role of Thr139 in the human immunodeficiency virus type 1 reverse transcriptase sensitivity to (+)-calanolide A.
abstract: The coumarins represent a unique class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) that were isolated from tropical plants. (+)-Calanolide A, the most potent compound of this class, selects for the T139I resistance mutation in HIV-1 reverse transcriptase (RT). Seven RTs mutated at amino acid position 139 (Ala, Lys, Tyr, Asp, Ile, Ser, and Gln) were constructed by site-directed mutagenesis. The mutant T139Q enzyme retained full catalytic activity compared with wild-type RT, whereas the mutant T139I, T139S, and T139A RTs retained only 85 to 50% of the activity. Mutant T139K, T139D, and T139Y RTs had seriously impaired catalytic activities. The mutations in the T139I and T139D RTs were shown to destabilize the RT heterodimer. (+)-Calanolide A lost inhibitory activity (up to 20-fold) against the mutant T139Y, T139Q, T139K, and T139I enzymes. All of the mutant enzymes retained marked susceptibility toward the other NNRTIs, including nevirapine, delavirdine, efavirenz, thiocarboxanilide UC-781, quinoxaline GW867420X, TSAO [[2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro-5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide)] derivatives, and the nucleoside inhibitor, ddGTP. The fact that the T139I RT 1) proved to be resistant to (+)-calanolide A, 2) represents a catalytically efficient enzyme, and 3) requires only a single transition point mutation (ACA-->ATA) in codon 139 seems to explain why mutant T139I RT virus strains, but not virus strains containing other amino acid changes at this position, predominantly emerge in cell cultures under (+)-calanolide A pressure.
authors.affiliation: Rega Institute for Medical Research, Minderbroedersstraat 10, B-3000 Leuven, Belgium.
authors.forename: Joeri
authors.initials: J
authors.lastname: Auwerx
authors.forename: Fátima
authors.initials: F
authors.lastname: Rodríguez-Barrios
authors.forename: Francesca
authors.initials: F
authors.lastname: Ceccherini-Silberstein
authors.forename: Ana
authors.initials: A
authors.lastname: San-Félix
authors.forename: Sonsoles
authors.initials: S
authors.lastname: Velázquez
authors.forename: Erik
authors.initials: E
authors.lastname: De Clercq
authors.forename: María-José
authors.initials: MJ
authors.lastname: Camarasa
authors.forename: Carlo-Federico
authors.initials: CF
authors.lastname: Perno
authors.forename: Federico
authors.initials: F
authors.lastname: Gago
authors.forename: Jan
authors.initials: J
authors.lastname: Balzarini
mesh_terms: D003374:Coumarins / Q000494:pharmacology*; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D039681:Pyranocoumarins; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D013912:Threonine / Q000737:chemistry / Q000502:physiology*
chemical_list: D003374:Coumarins; D039681:Pyranocoumarins; D018894:Reverse Transcriptase Inhibitors; D013912:Threonine; D054303:HIV Reverse Transcriptase; C075959:calanolide A