The Thiocarboxanilide Nonnucleoside UC781 Is a Tight-Binding Inhibitor of HIV-1 Reverse Transcriptase

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Abstract
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 (kon ∼ 1.7 × 106 M-1 s-1), but that dissociation is slow (koff ∼ 1.6 × 10-3 s-1). UC781 is therefore a rapid tight-binding inhibitor of HIV-1 RT, the first NNI to demonstrate this property.