Analysis of a putative cyclooxygenase from Trypanosoma brucei and its eligibility as a possible drug target

Abstract

Human African Trypanosomiasis (HAT) is one of the deadly neglected tropical diseases affecting people living in sub-Saharan Africa. It is caused by T. brucei parasites transmitted to the human host by tsetse flies. HAT therapy still relies upon drugs which use is limited by toxicity and rigorous treatment regimes, while development of vaccines remains up highly unlikely, due to the effectiveness of antigenic variation. There is an eminent need to identify drug target and develop better drugs against HAT. Sequencing the T. brucei genome revealed approximately 50% of genes which are so far uncharacterized regarding their biochemical or cellular functions but are identified as “hypothetical proteins” with no homologs in the human host. This opens possibilities for new target identification and drug discovery. In this thesis, an annotated hypothetical protein of T. brucei genome, identified as a putative cyclooxygenase-like enzyme (pTbCOX) was analyzed for genetic validation and structure determination. The bioinformatic analyses of pTbCOX revealed no convincing homolog proteins in human or any other organism, except in kinetoplastida. No hit was found regarding specific domains, signal peptides or membrane anchors. Therefore, the pTbCOX is a prime example of a protein annotated as “hypothetical protein” in T. brucei genome which fulfilled the above criteria, i.e., no homologs in human or any other organisms to be selected as an attractive target for validation. Here we report on the cloning, expression, refolding, and purification of pTbCOX in E. coli BL21 (DE3). The pTbCOX was found most entirely in an insoluble form inside inclusion bodies. The refolded pTbCOX was purified using a Ni-NTA column on ÄKTA-FPLC system. The pTbCOX was obtained in low amount showing a high tendency to aggregate as revealed by size exclusion chromatography and DLS experiments. Next, pTbCOX was expressed in Sf9 insect and produced in vivo crystals as micro-sized needle-shaped forms 14 days after transfection using the P3 virus stock. The isolated pTbCOX crystals were identified by Western blotting and scanning electron microscopy analysis. Isolated pTbCOX crystals showed diffraction patterns using powder diffraction and a positive signal by second harmonic generation (SONICC) analysis. These experiments present a preliminary characterization of the pTbCOX crystals before XFEL beam or MicroED for data collection. DLS and CD spectroscopy of pTbCOX crystals solubilized using acetate buffer revealed optimal condition by which pTbCOX is stable and monodisperse. RNAi studies demonstrated that pTbCOX is essential for cell division and morphogenesis in bloodstream form T. brucei. The pTbCOX knock-down parasite phenotypes were analyzed by fluorescence, SEM, and TEM microscopy. Surprisingly, overexpression of pTbCOX-eGFP was lethal for bloodstream form parasites and appeared as a single dot near the kinetoplast or in between kinetoplast and nucleus. Therefore, pTbCOX seems to be a suitable drug target candidate to be added to the drug discovery pipeline for HAT therapy. However, the challenge is still ongoing to obtain native pTbCOX, solving its X-ray crystals structure for druggability and assayability studies.