My Ongoing Research
1. HCV Enzymatic Assay A very fast and cost efficient approach to scan large number of potential inhibitors of the HCV helicase has been developed during my PhD. The collaboration continues since new compounds are being synthesised and are send for testing. Data coming out of the Assay will eventually feedback the drug design process, thus leading to even more active and potent species of the existing inhibitors.
2. Toxin from Aedes Crystallography Recently a toxin was isolated from larvae of the mosquito species Aedes that was found to play a key role to the survival of its host. The DNA coding for the protein was cloned and the protein expressed and purified. The protein is now capable of being crystallographically explored by trying different crystallisation conditions, in order to obtain a crystal viable for x-ray analysis and the determination of the three-dimensional structure of the toxin.
3. Helicases Flavi – Molecular Modelling Based on the x-ray determined structure of HCV helicase, the three dimensional structures of various other helicases that belong to the family of Flavi viruses were established by homology modelling. Eventually, new inhibitors were designed using structure based de novo drug design algorithms, in an attempt to design a universal inhibitor compound that will be active in all helicases. Various active compounds were eventually synthesised.
4. Polymerases Flavi – Molecular Modelling The three dimensional structure of the HCV and BVDV polymerases have been solved by x-ray crystallography. Based on those two structures, the three dimensional structures of the Dengue, West Nile, Yellow fever and Japanese Encephalitis Viruses, all members of the family of Flavi viruses were established by homology modelling. The protein – DNA interactions were analysed and a series of proposed inhibitors was designed.
5. Cloning / Protein Expression of HCV Helicase This project was funded and supported by ROSHE pharmaceuticals US. The gene of the HCV helicase was obtained from a cDNA library and cloned into an expression vector. The Protein was expressed by recombinant protein techniques and purified. An assay was established using immobilised double stranded DNA on biotin plates. The unwinding activity of the helicase is determined by the amount of the dislocating DNA single strand that is properly labelled. Many compounds were tested and some found quite active (manuscript in preparation).
6. Cloning / Protein Expression of Dengue Helicase and full NS3 Domain The genes of the Dengue helicase and the Dengue full NS3 domain (helicase – protease complex) were obtained from a cDNA library and cloned into an expression vector. The Protein was over expressed and purified enough to be used in the same assay setup as the HCV project above.
7. Molecular Modelling of the β-ketoacyl carrier protein synthases In bacteria, three KAS enzymes have been identified (KAS I, KAS II and KAS III), whilst in Plasmodium a KAS I/II and KAS III enzymes have been identified. This project consists of a study of the β-ketoacyl carrier protein synthases (the KAS enzymes). The ultimate aim was to study the Plasmodium enzymes as potential drug targets and to establish the structures of the KAS I/II and KAS III enzymes from their sequences. The first step was to study the active sites of enzymes from different sources (i.e. derived from different species). The main aim of the active site study was to identify the residues that comprise the active site, the ones that actually interact with the ligand and finally to identify what kind of interaction takes place when the inhibitor is docked.
8. The effect of cell immobilisation in adipocyte viability by LDH assay Fat cell immobilisation in agar beads proved to protect the adipocytes from disrupting. The viability of the fat cells was determined from their LDH release. The amount of LDH released in the medium of the fat cells is proportional to the number of dispersed cells since each cell contains a certain amount of LDH. The total LDH content of the fat cell suspension was determined by complete disruption of all the fat cells in the cell suspension, using the ice-cold homogenisation technique (Baht and Saggerson, 1988). Adipocytes immobilised in agar beads (Jones, 1992 – modified) released almost 92 percent less LDH in their medium when compared to the defatted (homogenised) cell suspension. Next erythromycin was added in the cell suspension and fat cells from this cell suspension were both homogenised and immobilised. Erythromycin, which is a wide spectrum antibiotic, was used to kill the lactic acid bacteria (lactobacci) that had contaminated the cell suspension. The results showed that the same quantity of homogenised cells with Erythromycin released almost 46 percent less LDH in their medium. That showed that 46 percent of the initial LDH, in then homogenised cell suspension, without Erythromycin was released from bacteria. Immobilisation of adipocytes in agar beads with Erythromycin showed almost 98.5 percent reduced LDH release when compared to the defatted (homogenised) cell suspension without Erythromycin. As a result 6.5 percent of the LDH that was released from the immobilised cell suspension without Erythromycin was released from bacteria.