INHIBITION OF HIV-1 REVERSE TRANSCRIPTASE VIA A SUBSTRATE INHIBITOR COMPLEX: STRUCTURAL IMPLICATIONS. Mary L. Kopka, Mark Filipowsky and Richard E. Dickerson, Molecular Biology Institute, University of California at Los Angles, CA 90095, USA

A bis-linked distamycin drug using m-pyridyl as the linker was studied by kinetics, gel shift and nucleic acid melting point, [[Delta]]Tm, analysis as an inhibitor of HIV-1 reverse transcriptase (RT). IC50 curves run with enzyme pre-incubated with template-primer (TP) show a 4-fold increase in inhibition over conditions with TP pre-incubated with drug. This fact, coupled with increased binding of TP to RT in response to drug in gel shift assays, and weak binding of the inhibitor to the RNA/DNA TP alone from [[Delta]]Tm assays, indicates formation of a dead-end ternary complex of drug, enzyme and TP. The kinetics indicate that the inhibition occurs before processive synthesis begins. The drug-bound to the RNA/DNA template-primer is the inhibiting species, i.e. a substrate-inhibitor complex (SI). This SI complex is competitive with TP on the same site in a transition enzyme-TP complex and to a lesser extent on free enzyme. This dead-end ternary transition complex is the main inhibition intermediate.

These results will be interpreted considering the relevant conformational changes the enzyme undergoes immediately prior to and during reverse transcription.