Mutations M184V and Y115F in HIV-1 reverse transcriptase discriminate against "nucleotide-competing reverse transcriptase inhibitors".
Indolopyridones are potent inhibitors of reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1). Although the structure of these compounds differs from established nucleoside analogue RT inhibitors (NRTIs), previous studies suggest that the prototype compound INDOPY-1 may bind in close proximity to the polymerase active site. NRTI-associated mutations that are clustered around the active site confer decreased, e.g. M184V and Y115F, or increased, e.g. K65R, susceptibility to INDOPY-1. Here we have studied the underlying biochemical mechanism. RT enzymes containing the isolated mutations M184V and Y115F cause 2-3-fold increases in IC(50) values, while the combination of the two mutations causes a >15-fold increase. K65R can partially counteract these effects. Binding studies revealed that the M184V change reduces the affinity to INDOPY-1, while Y115F facilitates binding of the natural nucleotide substrate and the combined effects enhance the ability of the enzyme to discriminate against the inhibitor. Studies with other strategic mutations at residues Phe-61 and Ala-62, as well as the use of chemically modified templates shed further light on the putative binding site of the inhibitor and ternary complex formation. An abasic site residue at position n, i.e. opposite the 3'-end of the primer, prevents binding of INDOPY-1, while an abasic site at the adjacent position n+1 has no effect. Collectively, our findings provide strong evidence to suggest that INDOPY-1 can compete with natural deoxynucleoside triphosphates (dNTPs). We therefore propose to refer to members of this class of compounds as "nucleotide-competing RT inhibitors" (NcRTIs).
Ehteshami
Maryam
M
Department of Microbiology & Immunology, McGill University, Quebec H3A 2B4, Canada.
Scarth
Brian J
BJ
Tchesnokov
Egor P
EP
Dash
Chandravanu
C
Le Grice
Stuart F J
SF
Hallenberger
Sabine
S
Jochmans
Dirk
D
Götte
Matthias
M
D001665:Binding Sites; D020134:Catalytic Domain; D017931:DNA Primers / Q000737:chemistry; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000235:genetics*; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008968:Molecular Conformation; D009154:Mutation; D011485:Protein Binding; D018894:Reverse Transcriptase Inhibitors / Q000737:chemistry*
D017931:DNA Primers; D018894:Reverse Transcriptase Inhibitors; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase