Linked lexitropsins and the in vitro inhibition of HIV-1 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 transcriptase (RT) on a poly(rA).oligo(dT) template-primer (TP). The two most effective drugs, 3,5 m-pyridyl-linked bisdistamycin (MPyr) and trans-vinyl-linked bisdistamycin (TVin), show (1) enhanced inhibition in reactions initiated with pre-incubated enzyme template-primer (ETP) and (2) reduced affinity for a "free" TP analog, when compared with the 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 transcriptase's two substrates: template-primer and nucleotide. When primer is the variable substrate, TVin is partially noncompetitive and MPyr is dead-end competitive (Ki = 6.5 microM). With nucleotide as substrate, TVin is noncompetitive at low drug concentrations and MPyr is uncompetitive. Gel band mobility shift assays with MPyr indicate that the drug inhibits via entrapment of TP on the enzyme rather than displacement of TP from the enzyme surface. The conformation of nucleic acid is most likely altered upon MPyr binding, enhancing the induced fit of enzyme to hybrid duplex. The relevance of this novel mode of inhibition is considered in relation to enzyme association/dissociation with TP that occurs prior to (-)-DNA strand transfer, and to the structural implications of an enzyme-bound hybrid RNA/DNA nucleic acid.
Filipowsky
M E
ME
Molecular Biology Institute, University of California at Los Angeles 90095, USA.
Kopka
M L
ML
Brazil-Zison
M
M
Lown
J W
JW
Dickerson
R E
RE
D000998:Antiviral Agents / Q000494:pharmacology*; D006690:Bisbenzimidazole / Q000494:pharmacology; D004256:DNA Polymerase I / Q000378:metabolism; D017931:DNA Primers / Q000378:metabolism; D004133:Diminazene / Q000031:analogs & derivatives / Q000494:pharmacology; D004214:Distamycins / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D007211:Indoles / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D009429:Netropsin / Q000031:analogs & derivatives* / Q000494:pharmacology; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D013698:Templates, Genetic; D013942:Thymine Nucleotides / Q000378:metabolism
D000998:Antiviral Agents; D017931:DNA Primers; D004214:Distamycins; D007211:Indoles; D013942:Thymine Nucleotides; C053654:lexitropsin; C007293:DAPI; D009429:Netropsin; C030000:stallimycin; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D004256:DNA Polymerase I; C003915:diminazene aceturate; D006690:Bisbenzimidazole; C024157:thymidine 5'-triphosphate; D004133:Diminazene