In a bid to design clinical drugs to improve health outcomes for people living with a particular strain of HIV and its mutants, scientists at the University of the Witwatersrand’s Protein Structure-Function Research Unit (PSFRU) in South Africa have embarked on a clinical drug discovery journey with promising results. The aim of the research, which receives funding from the GCRF START grant, is to develop a novel inhibitor specifically designed to target the South African HIV-1 subtype C protease (C-SA PR) and its mutants.
The purpose of developing an inhibitor is to stop C-SA PR’s activity by preventing the formation of mature copies of the human immunodeficiency virus (HIV). This would improve health outcomes in line with Sustainable Development goals across South Africa by increasing drug efficacy, reducing adverse side effects and drug resistance, as well as benefitting populations infected with the HIV subtype C in other sub-Saharan countries, India, China and Brazil.
There is no permanent cure for HIV/AIDS which has claimed the lives of an estimated 32.7 million people globally since the beginning of the HIV pandemic (UNAIDS, 2020). A number of drugs have been developed and approved by the USA’s Food and Drug Association (FDA) to increase the quality and duration of life in HIV infected individuals in some parts of the world. However, these are not specific to the HIV-1 protease subtype C which is dominant in South Africa – a country with approximately 20% of the global HIV infection rate and 10.44% of the global AIDS-related deaths (UNAIDS, 2020).
To date, the scientists at the PSFRU have screened ten drugs in the FDA approved and Zinc drug databases, with seven hits that show promise for optimisation as inhibitors. They have solved one protein structure through sophisticated computational modelling, and with high-resolution X-ray crystallography data collected on beamline i03 at the UK’s national synchrotron – Diamond Light Source (Diamond) – have solved the structure of the South African HIV-1 subtype C protease (C-SA PR), the results of which were deposited in the global Protein Data Bank (6I45.pdb) in 2018 (Fig.1).
The research involves characterising the structure and function of the South African HIV-1 subtype C protease (C-SA PR) and its mutants using state-of-the-art computational and experimental methods made possible by the GCRF START grant. The scientists want to understand what role amino acid insertions and mutations the HIV-1 protease may have on clinical drug binding so they can design an effective inhibitor.
This research builds on a previous study, in which a blood sample was taken from a drug naive infant born to an HIV positive mother. To prevent the transmission of the virus to her baby, the mother had received reverse transcript inhibitor treatment (but not protease inhibitors) prior to the birth. However, when the baby was born it was found to be HIV positive and a mutation was present rendering current drug therapy ineffective.
“It is the results like this of other research on the South African HIV-1 C-SA PR, and the impact of the disease on individual lives and livelihoods, which drives our motivation. The fact that the baby had developed drug resistance mutations is very rare in mother-to-child transmission but no less concerning,” says Professor Yasien Sayed, who heads up the PSFRU and leads the research on the HIV-1 C-SA PR, “and there is evidence that some adults are also failing drug therapies. We therefore need to develop treatments which work more effectively against the HIV-1 C-SA PR and its mutants if we are going to improve clinical outcomes for the large population of HIV positive adults and children in South Africa, and further afield.”
Some of the first steps in the team’s drug discovery journey have been computational involving Molecular Dynamics Simulation of the HIV wild type C-SA PR and its mutants. After this, drugs in various databases were screened. As a result, the scientists found a promising drug from the FDA database that binds with the HIV wild type C-SA PR and its mutant with best docking scores and energies.
“We modelled the homology structures of the HIV wild type protease and its mutants using the template South African wild type HIV-1 Subtype C Protease (PDB ID: 3U71),” explains Dr Pandian who is a Post-Doctoral Research Fellow funded by the GCRF START grant specialising in the computational aspects of the research. “The structure was solved at 2.72Å using the software packages: Swiss model/modeler/I-Tasser, followed by experimental validation of the modelled structures with in-house computer software. We are excited by the preliminary results, which are better than the current FDA approved drugs, although the computational results now have to be proved through wet lab experiments, along with the best results from the screened from the Zinc database.”
To conduct these studies, the PSFRU has its own computational and wet lab facilities for Molecular Dynamics simulation, docking studies, protein expression and purification. Screening of crystals is carried out using an Oryx8Protein Crystallization Robot (Douglas Instruments, UK) and testing of crystals using a home-source X-ray diffractometer. However, synchrotron facilities are not available on the African continent, so access to the Diamond Light Source synchrotron (Beamlines i03, i04, and also i04-1 and i24) is achieved remotely from the PSFRU lab in South Africa in order to characterise the structure and function of proteins at high resolution.
“Having access through the GCRF START grant to experimental synchrotron techniques like X-Ray crystallography at Diamond to obtain crystals and to solve the structures at high resolutions has been revolutionary for us,” reports Dr Pandian. “It is ultimately the combination of computational and experimental techniques that makes it possible to see how well the drugs are binding to optimise them for the South African HIV-1 subtype C protease (C-SA PR)” .
“During the wet lab experiments,” Dr Pandian continues, “we can’t screen the whole drug library for the target protein and it’s very costly to purchase the drugs for screening. The theoretical part of the drug discovery method is therefore useful for generating three dimensional structures for any proteins when the crystal structures are not available in the PDB databases, and for sorting out the best ligand / inhibitors for the protein target before starting protein characterisation wet lab experiments.”
Scientific results are not the only progress being made by the PSFRU team in their research which is meeting the UN’s Sustainable Development Goals for health (SDG 3); great strides have also been made in the PSFRU in terms of education and capacity building in structural biology (SDG 4) with more than 30 postgraduate students involved in the collaboration with the GCRF START grant since 2019. This includes Dr Ramesh Pandian, and Ms Mpho Setshedi who is a MSc. candidate working on the wet lab studies of the HIV-1 C-SA PR and its mutants.
“The research is meaningful,” says Ms Setshedi, “I feel like we are doing a good job and doing something to solve a challenge that impacts South Africa. I hope it contributes something big – an effective HIV inhibitor. In terms of what I am learning, there are challenges in this field but once you get the hang of the techniques you just need to persevere. Getting funding is a struggle in my field generally and there aren’t a lot of women doing this work. There have also been challenges caused by the COVID-19 lockdown in 2020 – but I haven’t let anything discourage me.”
Read more about the UN’s Sustainable Development Goals here
: https://www.unaids.org/en/AIDS_SDGs accessed 3.3.2021
https://www.unaids.org/en/resources/documents/2020/UNAIDS_FactSheet accessed 3.3.2021
 https://www.avert.org/professionals/hiv-around-world/sub-saharan-africa/south-africa accessed on 4 March 2021; For latest (2019) South African country statistics see: https://www.unaids.org/en/regionscountries/countries/southafrica
 http://www.schroding-er.com ; Schrödinger, L. & DeLano, W., 2020. PyMOL, Available at: http://www.pymol.org/pymol
 https://pubmed.ncbi.nlm.nih.gov/30793914/ accessed 9.3.2021
The Molecular Dynamics (MD) simulations of validated structures were performed under physiological conditions for 100ns using GROMACS software packages. The MD simulated structures were analysed thoroughly and extracted the energy minimised structure for further analysis. Important parameters such as root mean square deviation (RMSD), root mean square fluctuation (RMSF), radiation of gyration (Rg) and hydrogen bonding analysis were carried out.
The energy minimised structures of HIV wild type and its mutants were used for the screening of drugs from different data bases such as the Zinc database and FDA approved drugs database.
The results of the binding pocket analysis of the protease complex form obtained by the docking studies with best ligand directed the scientists to modify the side chain with the combination of different R group of the drug to improve the binding affinity.