LINKED LEXITROPSINS AND THE IN-VITRO INHIBITION OF HIV-I REVERSE-TRANSCRIPTASE RNA-DIRECTED DNA POLYMERIZATION - A NOVEL INDUCED-FIT OF 3,5-M-PYRIDYL BISDISTAMYCIN TO ENZYME-ASSOCIATED TEMPLATE-PRIMER

Five classic DNA minor groove-binding drugs and a series of bis-linked lexitropsins based on netropsin and distamycin have been screened for their effectiveness in inhibiting transcription by HIV-1 reverse tran scriptase OCT) on a poly(rA). oligo(dT) template-primer (TP). The two most effective drugs, 3,5 m-pyridyl-linked bisdistamycin (MPyr) and tr ans-vinyl-linked bisdistamycin (TVin), show (1) enhanced inhibition in reactions initiated with pre-incubated enzyme template-primer (ETP) a nd (2) reduced affinity for a ''free'' TP analog, when compared with t he parent drug distamycin. All three drugs lack the ability to inhibit processive incorporation of nucleotide, suggesting drug intervention instead at initiation or termination of processive cycles. The two bis -linked drugs exhibit different kinetic behavior with reverse transcri ptase's two substrates: template-primer and nucleotide. When primer is the variable substrate, TVin is partially noncompetitive and MPyr is dead-end competitive (K-i 6.5 mu M) with nucleotide as substrate, TVin is noncompetitive at low drug concentrations and MPyr is uncompetitiv e. Gel band mobility shift assays with MPyr indicate that the drug inh ibits via entrapment of TP on the enzyme rather than displacement of T P from the enzyme surface. The conformation of nucleic acid is most li kely altered upon MPyr binding, enhancing the induced fit of enzyme to hybrid duplex. The relevance of this novel mode of inhibition is cons idered in relation to enzyme association/dissociation with TP that occ urs prior to (-)-DNA strand transfer, and to the structural implicatio ns of an enzyme-bound hybrid RNA/DNA nucleic acid.