Elucidation of HIV-1 protease resistance by characterization of interaction kinetics between inhibitors and enzyme variants.
The kinetics of the interaction between drug-resistant variants of HIV-1 protease (G48V, V82A, L90M, I84V/L90M, and G48V/V82A/I84V/L90M) and clinically used inhibitors (amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir) were determined using biosensor technology. The enzyme variants were immobilized on a biosensor chip and the association and dissociation rate constants (k(on) and k(off)) and affinities (K(D)) for interactions with inhibitors were determined. A unique interaction kinetic profile was observed for each variant/inhibitor combination. Substitution of single amino acids in the protease primarily resulted in reduced affinity through increased k(off) for the inhibitors. For inhibitors characterized by fast association rates to wild-type protease (ritonavir, amprenavir, and indinavir), additional substitutions resulted in a further reduction of affinity by a combination of decreased k(on) and increased k(off). For inhibitors characterized by slow dissociation rates to wild-type enzyme (saquinavir and nelfinavir), the decrease of affinity conferred by additional mutations was attributed to increased k(off) values. Development of resistance thus appears to be associated with a change of the distinctive kinetic parameter contributing to high affinity. Further inhibitor design should focus on improving the "weak point" of the lead compound, that being either k(on) or k(off).
Shuman
Cynthia F
CF
Department of Biochemistry, Uppsala University, BMC, Box 576, SE-751 23, Uppsala, Sweden.
Markgren
Per Olof
PO
Hämäläinen
Markku
M
Danielson
U Helena
UH
D019943:Amino Acid Substitution; D015374:Biosensing Techniques; D024882:Drug Resistance, Viral; D014644:Genetic Variation; D016333:HIV Protease / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D017320:HIV Protease Inhibitors / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D020349:Surface Plasmon Resonance
D017320:HIV Protease Inhibitors; D016333:HIV Protease