Structural and enzymatic studies of HIV-1 subtype C protease
Date
2019
Authors
Shabangu, Bonginkosi
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Abstract
Human immunodeficiency virus (HIV) is a retrovirus that infects T-lymphocytes in the human immune system causing a condition known as Acquired Immunodeficiency Syndrome (AIDS). At the moment, there is an estimated 1.2 million people dying annually from diseases related to AIDS and ~ 36.7 million people are currently infected with HIV across the globe. There are 10 subtypes found within the major group of HIV-1 and subtype C overwhelmingly drives the South African epidemic and accounts for more than 50% of the world infections. The continuous infection and maturation of HIV-1 is yielded by the three viral enzymes; namely, reverse transcriptase, integrase and protease. HIV-1 relies on the catalytic efficacy of the protease enzyme for cleaving precursor polyproteins to yield structural and functional proteins. When HIV-1 protease is inactivated, either by an inhibitor or mutagenically, the virion will remain non-infectious. Thus, the HIV-1 protease is an opportune drug target. It is an important variant in the study of the pathogenesis, treatment and prevention of HIV-1 infections. The study intended to characterise and evaluate the catalytic activity of HIV-1 South African subtype C protease which exhibits high variability compared to subtype B protease. For characterisation properties, far-UV circular dichroism, intrinsic fluorescence spectroscopy and size exclusion high-performance liquid chromatography were used to evaluate secondary, tertiary and quaternary structure, respectively. Secondary structure results indicated a trough at 216 nm which means the protein is predominantly β-sheeted. Using intrinsic tryptophan as a probe, the tertiary structure of protease revealed that the local structural environment had not been perturbed and it was indicated by fluorescence emission intensity peak at 355 nm. The results of the size exclusion-high performance liquid chromatography study revealed that the dimeric molecular size of the wild-type protease was 22 kDa. The proteolytic efficiency of the subtype C wild-type protease was evaluated following the hydrolysis of a fluorogenic substrate resembling the CA/p2 cleavage site in the gag-pol polyprotein precursors. The KM was determined as 42.07 μM, Vmax was 0.047 μmol.min-1, specific activity was 76.46 μmol/min/mg, kcat was 24.02 s-1 and kcat/KM was 0.084 s-1μM-1. In the presence of darunavir, the Ki value for protease was determined as 3.4 nM, for saquinavir it was 6.2 nM and for ritonavir it was 9.4 nM. The protease inhibitor GRD 110D had a Ki of 27 nM, showing high susceptibility compared to the other three FDA-approved HIV protease inhibitors (PIs). A molecular docking study was carried out to analyse and compare the binding mode of the FDA-approved HIV PIs. The docking results indicated that the protease recognition site is hydrophobic due to the two-flexible glycine-dense β-sheets and that subtype C protease (PDB code: 3U71) polymorphisms result in an altered flap-hinge region, thus making it less susceptible to inhibitors as compared to subtype B protease (PDB code: 2P3B). The results of the comparative binding mode analysis of all the FDA-approved drugs could be useful for the design of new potent inhibitors of HIV-1 protease.
Description
Submitted, in fulfilment of the requirements for the degree of Master of Science
in Molecular and Cell Biology to the Faculty of Science, University of the Witwatersrand, Johannesburg July 2019
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Citation
Shabangu, Bonginkosi. (2019). Structural and enzymatic studies of HIV-1 subtype C protease. University of the Witwatersrand, https://hdl.handle.net/10539/29631