Molecular analysis of human immuno-deficiency virus-1 (South African subtype C) protease drug resistance mutations emerging on Darunavir therapy.
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Date
2020
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Abstract
Human immunodeficiency virus as the causative agent of acquired immune deficiency
syndrome remains a serious infectious disease and the leading cause of deaths worldwide.
According to UNAIDS, approximately 37 million individuals are living with HIV/AIDS and
770,000 AIDS related deaths. HIV-1 subtype C strain is responsible for approximately 70 %
of individuals living with HIV. Even with this staggering statistic, not many studies have been
conducted on this subtype.
Currently, there exist no treatment that completely eradicates the virus from an infected
individual. Although, three enzymes required by the virus to undergo intracellular replication
have been targeted to delay the progression of the disease, these enzymes include; reverse
transcriptase crucial for completion of the initial stages of HIV replication, integrase essential
for the integration of pro-viral DNA into the host chromosomal DNA and finally the enzyme
for which this study will focus only is protease which is vital for the development and assembly
of infectious viral progeny. The HIV aspartyl protease plays a major role in the life cycle of
the virus and has long been a target in antiviral therapy. This advancements in the knowledge
of HIV biology, pathogenesis and pharmacology has led to unprecedented efforts to interpret
basic findings in the development of novel antiviral drug therapies.
Nonetheless, the emergence of drug resistant mutations has hampered the efficacy of HIV-1
protease inhibition therapy. These mutations reduce the binding affinity of inhibitors while
maintaining viable catalytic activity and affinity for the natural substrate. In HIV-1 protease,
mutations at the following positions V32I, I50V,154M, and I84V are associated with subtle
structural changes that confer resistance to protease inhibitors especially darunavir. These
mutations located at or adjacent to the active site cavity, compromise drug susceptibility due
to weak Van der Waals interaction and binding site distortion resulting in treatment failure. In
this study we analysed the functional effects of these mutations on the HIV-1 South African
subtype C protease. To understand how these mutations influence drug susceptibility in HIV1 CSA protease, the mutations were introduced by site directed mutagenesis and confirmed by
DNA sequencing. Over-expression and purification of wild-type and mutant protease.
Followed by enzyme kinetics, inhibition (Ki) and thermodynamics studies carried out against
six clinically approved drugs. Significant difference was not observed in the substrate affinity
of the variant protease compared to the wildtype C-SA protease with a Michaelis constant (Km)
values of 104 and 124 µM and turnover number (Kcat) of approximately 2.2 and 0.2 s-1 for
variant and wildtype protease respectively. The six clinically approved drugs used in this study
demonstrated reduced binding affinities and weaker inhibition towards the variant protease in
comparison to the wild-type HIV-1 protease. Atazanavir, amprenavir, darunavir and saquinavir
exhibited the weakest inhibition towards the variant protease with Ki ratio values of 163, 232,
465 and 247 respectively. Thermodynamic data showed less favourable Gibbs free binding
energy in selected protease inhibitors towards the variant protease, largely due to decreased
binding entropy. Vitality values for the variant protease against the selected protease inhibitors,
confirm the impact of these mutations on the HIV-1 CSA protease. In the presence of these
drug resistant mutations V32I, I50V,154M, and I84V the efficacy of the selected protease
inhibitors used in this study is significantly reduced. Future studies would involve
crystallization and structure determination. This will give an in-depth understanding on the
structural interaction of the variant protease towards the protease inhibitors.
Description
Masters Degree. University of KwaZulu-Natal, Durban.