Molecular modelling and drug susceptibilty of the L38 (N) L HIV-1 sub-type C pro-tease

• Main Author: Williams, Alison
• Format: Others
• Language: en
• Published: 2018
• Online Access: https://hdl.handle.net/10539/25864

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Doctor of Philosophy May 2018 === Human Immunodeficiency Virus (HIV) is a global concern due to the 36 million people infected worldwide. HIV is genetically diverse consisting of nine subtypes. Subtype C infections predominate in sub-Saharan Africa and this subtype has been under-investigated in comparison to subtype B. Great advances have been made to combat this disease, particularly in South Africa, but drug resistance still remains a concern. HIV protease cleaves the Gag and Gag-Pol polyproteins into their functional forms, making it indispensable to the production of infectious virions. It is, as such, a major drug target. The proteolytic enzyme accumulates mutations associated with drug resistance due to the high replication rate of the virus and the error prone reverse transcriptase. These include mutations in the active site and distal regions. Insertion mutations are rarely incorporated into the hinge region and because of that the effect of these insertions are poorly characterised. The variant protease in this study (L38↑N↑L) contains a double insertion of Asparagine and Leucine at position 38, in the hinge region. For the first time L38↑N↑L protease was successfully overexpressed and purified using a thioredoxin-hexahistidine tag fusion system. Molecular dynamics simulations showed that the flap region of L38↑N↑L was less dynamic than that of a wild-type protease, suggesting a possible mechanism to evade drug binding. Induced-fit docking studies showed that the drugs lopinavir, atazanavir and darunavir do bind L38↑N↑L albeit with reduced hydrophobic contacts and hydrogen bonds. In vitro inhibition studies confirmed that these drugs do bind and inhibit L38↑N↑L. The catalytic efficiency of L38↑N↑L was diminished compared to wild-type, which resulted in reduced replication capacity of the virus. Phenotypic assays showed that L38↑N↑L had reduced susceptibility to darunavir in the presence of a Gag sequence thus confirming that this region does contribute to drug resistance. === MT 2018