Kinetic and thermodynamic characterization of the South African subtype C HIV-1 protease : implications for drug resistance

摘要

ABSTRACTudThe magnitude of the AIDS epidemic is well documented. It has been shown thatudAfrica constitutes about 70 % of people infected with HIV worldwide. Efforts toudcontrol the AIDS epidemic have focused heavily on studies pertaining to the biology,udbiochemistry and structural biology of HIV and on the interactions between HIVudproteins and new drugs. One of the most challenging problems in AIDS therapy is thatudHIV develops drug-resistant variants rapidly. Extensive research has been dedicatedudto designing resistance-evading drugs for HIV-1 protease (predominantly subtype B),udwhich is crucial for the maturation of viral, structural and enzymatic proteins. Thereudare 10 subtypes of HIV-1 within the major group of the virus, with subtype Cudaccounting for about 95 % of infections in South Africa. Since HIV-1 antiretroviraludtreatment has been developed and tested against the B subtype, which is prevalent inudNorth America, Western Europe and Australia, an important question relates to theudeffectiveness of these drugs against the C subtype. At this point, however, little isudknown about inhibitor-resistant mutations in the subtype C. The study, therefore,udlooked at the two active site mutations (V82A and V82F/I84V) in the South AfricanudHIV-1 subtype C protease (C-SA) emerging from the viral population circulating inudpatients. These mutations are well-characterized within the framework of the subtypeudB and are known to cause cross-resistance to most of inhibitors currently in clinicaluduse. Protein engineering techniques were used to generate the V82A and theudV82F/I84V variants. Comparative studies with the wild-type HIV-1 C-SA proteaseudwere performed. The spectral properties of the V82A and the V82F/I84V variantsudindicated no changes in the secondary structure in the respective variant proteins.udTryptophan and tyrosine fluorescence indicated a major difference in the intensities atudthe emission maxima for all three proteins. The fluorescence intensity of theudV82F/I84V variant, in particular, was significantly enhanced indicating theudoccurrence of tertiary structural changes at/near the flap region. Both mutations didudnot impact significantly upon catalytic function. Both variants also had the same Kmudvalues comparable to that of the wild-type enzyme. The catalytic efficiencies and theudkinetic constants were lowered 3.6-fold for the V82A mutation and 6-fold for theudV82F/I84V mutation relative to the wild-type C-SA protease. Inhibition studies wereudperformed using four inhibitors in clinical use (saquinavir, ritonavir, indinavir andudnelfinavir). For the V82A variant, IC50 and Ki values for saquinavir and nelfinavirudivudwere not affected, whilst those for ritonavir and indinavir were 5- and 9-fold higherudthan the wild-type C-SA protease, respectively. Against the V82F/I84V variant,udhowever, the inhibition constants were drastically weaker and characterized by IC50udand Ki ratios ranging from 50 to 450. Isothermal titration calorimetry (ITC) was alsoudused to determine the binding energetics of saquinavir, ritonavir, indinavir andudnelfinavir to the wild-type C-SA, V82A and V82F/I84V HIV-1 protease. The V82Audmutation lowered the Gibbs energy of binding for the respective four clinicaludinhibitors by 0.4 kcal/mol, 1.3 kcal/mol, 1.5 kcal/mol and 0.6 kcal/mol, respectively,udrelative to the wild-type C-SA HIV-1 protease. The affinity of V82A HIV-1 proteaseudfor saquinavir, ritonavir, indinavir and nelfinavir (Kd = 1.85 nM, 2.00 nM, 12.70 nMudand 0.66 nM, respectively, at 25 °C) was in the range of 2- to 13-fold of magnitudeudweaker than that of the wild-type C-SA protein. The clinical inhibitors exhibited theudhighest binding affinity to both the wild-type and the V82A enzymes, but wereudextremely sensitive to the V82F/I84V mutation. The V82F/I84V mutant reduced theudbinding of saquinavir, ritonavir, indinavir and nelfinavir 117-, 1095-, 474- and 367-udfold, respectively. A drop in Kd values obtained for the V82F/I84V in association withudsaquinavir, ritonavir, indinavir and nelfinavir was consistent with a decrease ofudbetween 2.8 - 4.2 kcal/mol in ΔG, which is equivalent to at least 2 to 3 orders ofudmagnitude in binding affinity. Taken together, thermodynamic data indicated that theudV82A and V82F/I84V active site mutations in the C-SA subtype lower the affinity ofudthe first-generation inhibitors by making the binding entropy less positiveud(unfavorable) and making the enthalpy change slightly less favorable.