The thiocarboxanilide nonnucleoside UC781 is a tight-binding inhibitor of HIV-1 reverse transcriptase.
The thiocarboxanilide nonnucleoside inhibitor (NNI) UC781 inhibited HIV-1 reverse transcriptase (RT) DNA polymerase activity at a 1:1 molar ratio of inhibitor to enzyme. Inhibition was linear uncompetitive with respect to template/primer (T/P) and mixed noncompetitive with respect to deoxynucleoside triphosphate (dNTP), typical of NNI. When the RT-T/P binary complex was incubated with UC781 and then separated from unbound inhibitor, recovery of enzyme activity was slow, with only about 60% activity recovered after 25 min. The inactivation of the RT-T/P complex was prevented by the presence of a large excess of UC84, another carboxanilide NNI that interacts with this RT mechanistic form. UC781 protected the RT-T/P-dNTP ternary complex from irreversible inactivation by a photoactivatable azido analog of nevirapine, implying that UC781 binds to the NNI pocket of this RT mechanistic form. UC781 did not photoprotect either the free enzyme or the RT-T/P binary complex; however, protein fluorescence quenching studies indicated that UC781 interacted with all RT mechanistic forms, with the order of affinity being RT-T/P-dNTP ternary complex > RT-T/P binary complex > free RT. Reaction progress curve analysis showed that the binding of UC781 to RT is rapid (k(on) approximately 1.7 x 10(6) M(-1) s(-1)), but that dissociation is slow (k(off) approximately 1.6 x 10(-3) s(-1)). UC781 is therefore a rapid tight-binding inhibitor of HIV-1 RT, the first NNI to demonstrate this property.
Barnard
J
J
Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, McGill AIDS Centre, McGill University, Montreal, Quebec, Canada.
Borkow
G
G
Parniak
M A
MA
D000813:Anilides / Q000378:metabolism / Q000494:pharmacology*; D001570:Benzodiazepinones / Q000494:pharmacology; D005663:Furans / Q000378:metabolism / Q000494:pharmacology*; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D007700:Kinetics; D019829:Nevirapine / Q000031:analogs & derivatives*; D011485:Protein Binding; D011725:Pyridines / Q000494:pharmacology; D018894:Reverse Transcriptase Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence; D013854:Thioamides
C092891:9-azido-5,6-dihydro-11-ethyl-6-methyl-11H-pyrido(2,3-b)(1,5)benzodiazepin-5-enone; D000813:Anilides; D001570:Benzodiazepinones; D005663:Furans; D011725:Pyridines; D018894:Reverse Transcriptase Inhibitors; D013854:Thioamides; D019829:Nevirapine; D054303:HIV Reverse Transcriptase; C100668:UC-781