Amino acid substitutions in HIV-1 reverse transcriptase with corresponding residues from HIV-2. Effect on kinetic constants and inhibition by non-nucleoside analogs.
Nevirapine is a highly potent and specific inhibitor of human immunodeficiency virus type 1 (HIV-1) polymerase, but is inactive against HIV-2 and other polymerase. Previous studies demonstrated that residues 176-190 of HIV-1 reverse transcriptase (RT) can confer nevirapine sensitivity to HIV-2 RT. To better characterize the role of this sequence in HIV-1 RT, we have progressively substituted residues 176-190 of HIV-2 RT for those of HIV-1 RT and monitored the impact on the kinetic properties; inhibitory activity of nevirapine (11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido[2,3-b:2',3'-e] [1,4]diazepin-6-one), E-BPU (5-ethyl-1-benzyloxymethyl-6-(phenylthio)-uracil), and TIBO-R82150 ((+)-S-4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)imidazo[4,5,1-j k] [1,4]benzodiazepin-2(1H)-thione); and inhibitor-induced fluorescence changes of the mutant enzymes. The study revealed that in addition to Try-181 and Tyr-188, a new amino acid residue (Gly-190) plays an important role in determining susceptibility to nevirapine and E-BPU, but not to TIBO-R82150. These data argue that these non-nucleoside inhibitors fit differently, even though they share a common binding pocket. Nevirapine was seen to exert inhibitory activity by altering the interaction of the enzyme with the template-primer. Kinetic parameters were modulated by the template (DNA versus RNA) as well as by some of the mutations.
Bacolla
A
A
Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877-0368.
Shih
C K
CK
Rose
J M
JM
Piras
G
G
Warren
T C
TC
Grygon
C A
CA
Ingraham
R H
RH
Cousins
R C
RC
Greenwood
D J
DJ
Richman
D
D
D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D015498:HIV-2 / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019829:Nevirapine; D011725:Pyridines / Q000494:pharmacology*; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry*; D018894:Reverse Transcriptase Inhibitors; D013050:Spectrometry, Fluorescence
D000998:Antiviral Agents; D011725:Pyridines; D018894:Reverse Transcriptase Inhibitors; D019829:Nevirapine; C072416:reverse transcriptase, Human immunodeficiency virus 2; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase