ABSTRACT
The magnitude of the AIDS epidemic is well documented. It has been shown that
Africa constitutes about 70 % of people infected with HIV worldwide. Efforts to
control the AIDS epidemic have focused heavily on studies pertaining to the biology,
biochemistry and structural biology of HIV and on the interactions between HIV
proteins and new drugs. One of the most challenging problems in AIDS therapy is that
HIV develops drug-resistant variants rapidly. Extensive research has been dedicated
to designing resistance-evading drugs for HIV-1 protease (predominantly subtype B),
which is crucial for the maturation of viral, structural and enzymatic proteins. There
are 10 subtypes of HIV-1 within the major group of the virus, with subtype C
accounting for about 95 % of infections in South Africa. Since HIV-1 antiretroviral
treatment has been developed and tested against the B subtype, which is prevalent in
North America, Western Europe and Australia, an important question relates to the
effectiveness of these drugs against the C subtype. At this point, however, little is
known about inhibitor-resistant mutations in the subtype C. The study, therefore,
looked at the two active site mutations (V82A and V82F/I84V) in the South African
HIV-1 subtype C protease (C-SA) emerging from the viral population circulating in
patients. These mutations are well-characterized within the framework of the subtype
B and are known to cause cross-resistance to most of inhibitors currently in clinical
use. Protein engineering techniques were used to generate the V82A and the
V82F/I84V variants. Comparative studies with the wild-type HIV-1 C-SA protease
were performed. The spectral properties of the V82A and the V82F/I84V variants
indicated no changes in the secondary structure in the respective variant proteins.
Tryptophan and tyrosine fluorescence indicated a major difference in the intensities at
the emission maxima for all three proteins. The fluorescence intensity of the
V82F/I84V variant, in particular, was significantly enhanced indicating the
occurrence of tertiary structural changes at/near the flap region. Both mutations did
not impact significantly upon catalytic function. Both variants also had the same Km
values comparable to that of the wild-type enzyme. The catalytic efficiencies and the
kinetic constants were lowered 3.6-fold for the V82A mutation and 6-fold for the
V82F/I84V mutation relative to the wild-type C-SA protease. Inhibition studies were
performed using four inhibitors in clinical use (saquinavir, ritonavir, indinavir and
nelfinavir). For the V82A variant, IC50 and Ki values for saquinavir and nelfinavir
iv
were not affected, whilst those for ritonavir and indinavir were 5- and 9-fold higher
than the wild-type C-SA protease, respectively. Against the V82F/I84V variant,
however, the inhibition constants were drastically weaker and characterized by IC50
and Ki ratios ranging from 50 to 450. Isothermal titration calorimetry (ITC) was also
used to determine the binding energetics of saquinavir, ritonavir, indinavir and
nelfinavir to the wild-type C-SA, V82A and V82F/I84V HIV-1 protease. The V82A
mutation lowered the Gibbs energy of binding for the respective four clinical
inhibitors by 0.4 kcal/mol, 1.3 kcal/mol, 1.5 kcal/mol and 0.6 kcal/mol, respectively,
relative to the wild-type C-SA HIV-1 protease. The affinity of V82A HIV-1 protease
for saquinavir, ritonavir, indinavir and nelfinavir (Kd = 1.85 nM, 2.00 nM, 12.70 nM
and 0.66 nM, respectively, at 25 °C) was in the range of 2- to 13-fold of magnitude
weaker than that of the wild-type C-SA protein. The clinical inhibitors exhibited the
highest binding affinity to both the wild-type and the V82A enzymes, but were
extremely sensitive to the V82F/I84V mutation. The V82F/I84V mutant reduced the
binding of saquinavir, ritonavir, indinavir and nelfinavir 117-, 1095-, 474- and 367-
fold, respectively. A drop in Kd values obtained for the V82F/I84V in association with
saquinavir, ritonavir, indinavir and nelfinavir was consistent with a decrease of
between 2.8 - 4.2 kcal/mol in ΔG, which is equivalent to at least 2 to 3 orders of
magnitude in binding affinity. Taken together, thermodynamic data indicated that the
V82A and V82F/I84V active site mutations in the C-SA subtype lower the affinity of
the first-generation inhibitors by making the binding entropy less positive
(unfavorable) and making the enthalpy change slightly less favorable.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/4712 |
| Date | 27 March 2008 |
| Creators | Mosebi, Salerwe |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 1634625 bytes, 43051 bytes, application/pdf, application/pdf, application/pdf, application/pdf |
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