Topoisomerases (TOPs) are essential enzymes that catalysis topological changes in DNA. TOPs are pivotal to cell survival and therefore inhibitors have been developed to target these enzymes both for antimicrobial and anti-cancer chemotherapy. The genome of the protozoan parasite Trypanosoma brucei contains 7 different genes for TOPs (TbTOPIA, TbTOPIB, TbTOPIImt, TbTOPIIα, TbTOPIIβ, TbTOPIIIα and TbTOPIIIβ). By using RNAi, it was shown that TbTOPIA, TbTOPIB, TbTOPIImt and TbTOPIIα are essential for parasite growth. These findings indicate that these four TbTOPs are good targets for anti-trypanosomal drug development. The genetic validations of the TbTOPs as suitable anti-trypanosomal drug targets are corroborated by inhibitor studies. It has been shown that different TOP inhibitors, including approved anti-cancer drugs, exhibit promising trypanocidal activities. In order to carry out screening assays to identify TOP inhibitors as new chemotherapies against T. brucei, it is necessary to produce considerable amounts (mg quantities) of active TbTOPs.
Objectives of the PhD
The aim of the project is to recombinantly expressed TbTOPs and to purify the enzymes in active form suitable for inhibitor screening assays. The individual measurable objectives are:
(i) to clone and express TbTOPs as His-tagged proteins,
(ii) to purify His-tagged TbTOPs and to determine their enzymatic activities, and
(iii) to screen compound libraries for specific inhibitors of TbTOPs.
The gene sequences of all TbTOPs are available, and these will be utilized to construct primers for the cloning of these genes in His expression vectors. His-tagged TbTOPs will be purified from cell extracts using Ni2+-NTA agarose columns. The activity of the purified TbTOPs will be determined by using topoisomerase assay kits. Recombinant TbTOPs will then be used for screening small compound libraries using a recently developed high-throughput assay. Promising compounds will be tested for their trypanocidal activity in cell culture with bloodstream forms of T. brucei.
For more information on the supervisor for this project, please go here: https://www.uea.ac.uk/medicine/people/profile/d-steverding
This is a PhD programme.
The start date of the project is 1 April 2020, 1 July 2020 or 1 October 2020.
The mode of study is full-time. The studentship length is 4 years (3 years period of study, 1 year period of registration).
Please note: Applications are processed as soon as they are received and the project may be filled before the closing date, so early application is encouraged.
This PhD project is offered on a self-funding basis. It is open to applicants with funding or those applying to funding sources. Details of tuition fees can be found at View Website.
Acceptable first degree in Biology, Biochemistry, Microbiology, Parasitology, Molecular Biology, Cell Biology, Biological Science, Biomedical Science, Medical Science, Infectious Disease, Medicinal and Biological Chemistry, Chemistry.
The standard minimum entry requirement is 2:1.
i) Deterding, A., Dungey, F.A., Thompson, K.-A. & Steverding, D. (2005) Anti-trypanosomal activities of DNA topoisomerase inhibitors. Acta Trop. 93, 311-316.
ii) Steverding, D. & Wang, X. (2009) Evaluation of anti-sleeping sickness drugs and topoisomerase inhibitors in combination on Trypanosoma brucei. J. Antimicrob. Chemother. 63, 1293-1295.
iii) Jobe, M., Anwuzia-Iwegbu, C., Banful, A., Bosier, E., Iqbal, M., Jones, K., Lecutier, S.J., Lepper, K., Redmond, M., Ross-Parker, A., Ward, E., Wernham, P., Whidden, E.M., Tyler, K.M. & Steverding, D. (2012) Differential in vitro activity of the DNA topoisomerase inhibitor idarubicin against Trypanosoma rangeli and Trypanosoma cruzi. Mem. Inst. Oswaldo Cruz 107, 946-950.
iv) Ellis, S., Sexton, D.W. & Steverding, D. (2015) Trypanotoxic activity of thiosemicarbazone iron chelators. Exp. Parasitol. 150, 7-12.