Professor Yasien Sayed
Principal investigator: Professor Y Sayed (PhD)
Academic qualifications: BSc (Wits, 1996), BSc Honours (Wits, 1997), PhD (Wits, 2001)
It is estimated that approximately 33.2 million people are infected with HIV world-wide and the vast majority of the infected individuals (about 70%) are located in sub-Saharan Africa. Thus far, the major target of antiretroviral drugs in the HI-virus has been the protease molecule. However, other drugs such as NNRTIs and RTIs have also been designed to target the reverse transcriptase molecule. To date, however, the protease molecule remains the most attractive target because very little variation exists between reverse transcriptase molecules from the various subtypes. Of significance is that all the protease inhibitors currently in clinical use have been designed to target protease molecules from subtype B viruses.
Our research seeks to address the structural and thermodynamic basis of binding of protease inhibitors to the South African HIV-1 subtype C protease. We are also interested in characterising the binding of non-nucleoside reverse transcriptase inhibitors to the reverse transcriptase from the various subtypes using a variety of techniques including X-ray crystallography and isothermal titration calorimetry (ITC).
The virus is categorised into ten subtypes (Figure 1) and the C subtype is the most prevalent subtype in sub-Saharan Africa. The C subtype appears to have originated in Southern and Central Africa and is the most frequently transmitted subtype. It has been suggested that the subtype C virus accounts for greater than 95% of infections in South Africa. The HIV-1 subtypes that are found on the African continent are not the same as those that are found in North America and Europe. In fact, the subtype B is the major cause of infection in North America and Western Europe while the A and C subtype accounts for the majority of infections in Africa (India and China as well). Within the South African subtype C framework, there appears to be significant genetic diversity amongst the subtype C strains. Coupled with this, there also appears to be no apparent genetic pattern when compared with sequences from other southern African countries [Morris et al., (2000) S. Afr. J. Sci. 96, 339-342].
The HI-virus contains THREE enzymes only:
- Reverse transcriptase (RT): Forms a heterodimer with RT/RNase H, Reverse transcribes viral RNA into DNA, Low fidelity (High mutation frequency), Viral DNA fully synthesised within 6 hours of viral entry.
- Protease (PR): The protease is an example of an aspartyl protease which functions as homodimer and is required for cleavage of Gag, Gap-Pol and Nef precursors. Protease activity is responsible for viral maturation and represents a major drug target for antiretroviral therapy.
- Integrase (IN): This enzyme is responsible for insertion of viral DNA into the genome of the host. It has exonuclease activity - removes two nucleotides from each 3 end of linear DNA AND endonuclease activity - cleaves host double-stranded DNA at the integration site. The ligase activity of this enzyme is responsible for the formation of a single covalent linkage at each end of the viral DNA.
Once viral entry into the cell has been accomplished, reverse transcription of the HIV-1 genome occurs in the cytosol of the cell via the RT enzyme. RT is, therefore, responsible for the conversion of retroviral genomic RNA into double-stranded DNA (Turner and Summers, 1999). Due to the absence of any cellular homolog, the RT enzyme has become a very attractive target for the development of HIV drugs. RT has two types of DNA polymerase activity, viz., RNA-dependant DNA polymerase activity and DNA-dependant DNA polymerase activity. In addition to this, RT also possesses ribonuclease H (RNaseH) activity (Review, Parniak and Sluis-Cremer, 2000). The above-mentioned activities are required for the proper and efficient functioning of RT in reverse transcription and, therefore, present a unique target for antiviral intervention and treatment. The biological form of RT is a heterodimer comprising two subunits of 66 kDa (p66) and 51 kDa (p51). HIV-1 protease catalyses the cleavage of 15 kDa of the C-terminal RNaseH domain from the 66 kDa subunit to generate the 51 kDa subunit. The HIV-1 heterodimer, p66/p51, is thought to be the biologically active form responsible for the reverse transcription process and also the species that is present in HIV-1 virions. This, however, has yet to be established unequivocally. The interaction between nevirapine and its target, reverse transcriptase (RT), will also be studied using isothermal titration calorimetry. This study will be amongst the first to report thermodynamic parameters for the binding of nevirapine to RT using ITC. Amino acid sequence data for the HIV-1 subtype C protease and reverse transcriptase (various subtypes) will be obtained from Professor Lynn Morris at NICD. Sequence data from both drug-treated and drug-na? individuals will be utilised to establish baseline polymorphisms and to monitor the appearance of primary and secondary resistance mutations. Sequence data will also be obtained from databases on the internet (e.g., Stanford HIV database hivdb.stanford.edu/, Los Alamos National Laboratory database www.hiv.lanl.gov/). The impact of primary and secondary resistance mutations on protease conformational stability and folding, catalytic function and vitality, energetics of substrate and antiretroviral drug binding to the protease will be determined. Determination of the three-dimensional crystal structures of the protease in the apo and drug-complexed forms will be determined. Plasmids for the over-expression of reverse transcriptase have been supplied by Dr N. Sluis-Cremer from the University of Pittsburgh, School of Medicine, USA. Studies are currently underway to over-express and purify the HIV-1 reverse transcriptase enzyme.
The PR enzyme represents the major protein target due to its role in polyprotein processing and viral maturation. There are currently 9 protease inhibitors (Indinavir, Saquinavir, Nelfinavir, Ritonavir, Amprenavir, Lopinavir, Atazanavir, Tipranavir and Darunavir) that are in clinical use for the treatment of HIV-1 infection. The HIV-1 PR molecule is described as a symmetrical, obligate homodimer. Both subunits are needed for the enzyme to be catalytically active. The high rate of viral replication together with the error prone nature of RT results in the high genetic diversity observed in the PR gene. It has been noted that genetic variability, also referred to as natural polymorphisms, can affect more that 45% of the amino acid content in the PR molecule. This is significant considering that there are only 99 amino acid residues per subunit. Therefore, it is important to assess the role of naturally occurring polymorphisms and the direct impact this will have on the efficacy of current PR inhibitors. Sequence information from drug-na? South African patients (data supplied by Professor Lynn Morris at NICD) indicates that baseline polymorphisms affect as much as 25% of amino acids in the protease molecule. The situation is worsened with the emergence of antiretroviral-induced resistance mutations. Despite the presence of natural and/or drug-induced mutations in the primary amino acid sequence, the PR remains catalytically active and conformationally stable. Mutations are described as either primary or secondary resistance mutations. Primary resistance mutations are mutations that occur at/near the active site and lower the binding affinity of the inhibitor. The presence of these mutations, although lowering the binding affinity for drugs, still allows the PR to maintain its catalytic activity. This scenario is achieved by mutating residues that interact more favourably with the drugs than with the natural substrate. Secondary resistance mutations are mutations that occur distal from the active site. Although non-active site mutations have a compensatory role, these residues have been observed in lowering inhibitor binding affinity by three orders of magnitude [Muzammil et al. (2003) Biochemistry 42, 631-638]. In a recent publication we have shown that non-B subtype polymorphisms lower the binding affinity of protease inhibitors [Velazquez-Campoy et al. (2003) AIDS Rev. 5, 165-171]. Therefore, a detailed understanding of the structural and thermodynamic consequences of drug-induced and natural polymorphisms is required if more effective drugs are to be designed. These drugs will have to be designed to target the appropriate subtype so that they bind with high affinity and a great degree of specificity. The project, therefore, focuses on a major epidemic facing South Africa as well as the other African countries. South Africa has a pivotal role in enhancing our understanding of the fundamentals of the efficacy of antiretroviral drugs in a non-B subtype context.
Post doctoral positions are available.
Current Honours student/s:
- Ms Kelly Nichols
- Ms Mpho Setshedi
Current post-graduate students:
- Tshele Mokhantso. HIV Protease MSc Candidate
- Dean Sherry. HIV Protease. PhD Candidate
- Maite Kgomokaboya. MSc Candidate
- Zaahida Sheik Ismail. HIV Protease. PhD Candidate
Post Doctoral Fellow/s
- Dr Ramesh Pandian
- Dr Allison Williams (Research Assistant)
- Dr Salerwe Mosebi
- Dr Previn Naicker
- Dr Gary Robertson
- Dr Alison Williams
- Dr Jake Zondagh
- Dr Zikhona Njengele
- Prof Heini Dirr (Wits, MCB)
- Prof Manuel Fernandes (Wits, School of Chemistry)
- Prof Lynn Morris (NICD)
- Prof Gert Kruger (University of KwaZulu-Natal)
- Prof Zenixole Tshentu (NMMU)
Research grant funding:
- University of the Witwatersrand
- NRF - Incentive funding for rated researchers
- AIDS Research Initiative Grant (Wits University)
- Carnegie Corporation of New York
- South African Medical Research Council (MRC)
- GCRF-START: Synchrotron Techniques for African Research and Technology (DIAMOND LIGHT SOURCE LIMITED)
Membership of professional bodies/associations
- South African Society of Biochemistry and Molecular Biology
- International Proteolysis Society
Publications: PubMed search results
Research supporting facilities:
- Equipment includes:
- preparative & analytical HPLC systems
- UV/VIS spectrophotometers
- fluorimeters (Hitachi 850, Perkin Elmer LS50B)
- Applied PhotoPhysics SX-18MV stopped-flow device with UV/VIS, fluorescence and CD detectors
- Jasco J-810 CD spectropolarimeter with Peltier temperature-controlled unit
- TA-Microcalorimetry ITC instrument
- MicroCal VP-isothermal titration calorimeter
- computer and molecular graphics facility for structural biology and bioinformatics
- The BioRad ExprionTM Automated Electrophoresis Statio
- The light scatter, zetasizer, nano-S system
- Single crystal X-ray diffractometer
- Wits University home page
- Wits School of Biology home page
- Joint United Nations Programme on HIV/AIDS
- HIV Drug Resistance Database
- Bioinformatics for HIV Research
- National Center for Biotechnology Information (NCBI)
- Protein Data Bank (PDB)
- ExPaSy Proteomics tools
For any enquiries, please contact
Professor Y. Sayed
27 11 717 6350 (Office)
27 11 717 6351 (Fax)