The role of Thr139 in the human immunodeficiency virus type 1 reverse transcriptase sensitivity to (+)-calanolide A.
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.
Auwerx
Joeri
J
Rega Institute for Medical Research, Minderbroedersstraat 10, B-3000 Leuven, Belgium.
Rodríguez-Barrios
Fátima
F
Ceccherini-Silberstein
Francesca
F
San-Félix
Ana
A
Velázquez
Sonsoles
S
De Clercq
Erik
E
Camarasa
María-José
MJ
Perno
Carlo-Federico
CF
Gago
Federico
F
Balzarini
Jan
J
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*
D003374:Coumarins; D039681:Pyranocoumarins; D018894:Reverse Transcriptase Inhibitors; D013912:Threonine; D054303:HIV Reverse Transcriptase; C075959:calanolide A