Speaker
Description
Parasitic diseases, such as leishmaniasis, pose a growing global health threat, particularly in regions with high HIV/AIDS prevalence [1]. The limited efficacy and growing resistance to current treatments necessitate the discovery of novel therapeutic targets. ∆24-sterolmethyltransferase (SMT) is an attractive target as an essential enzyme in the ergosterol biosynthetic pathway of protozoa and has no mammalian homologue [2]. SMT catalyses the transfer of a methyl group from S-adenosyl-methionine to the C24 position of zymosterol or lanosterol [3]. Structural characterisation of SMTs via X-ray crystallography is crucial for structure-based drug discovery approaches. This study aims to produce and structurally characterise SMTs from Leishmania donovani and Leishmania major to identify lead compounds for antiparasitic therapies.
The ERG6 genes, encoding SMT, from L. donovani and L. major were expressed in E. coli, but resulted in insoluble protein. We then fused ERG6 to genes encoding mCherry or SUMO and successfully expressed the fusions as soluble proteins. The SMTs were cleaved from the fusion partners and purified using column chromatography methods. Furthermore, the purified SMTs have been shown to methylate lanosterol using GC-MS. Biotransformation optimisation is underway to improve conversion yields.
Crystallisation trials using the vapour-diffusion sitting-drop method are currently ongoing. Future work includes collecting X-ray diffraction data of the SMT crystals to solve their three-dimensional structures. The structural information, as well as a robust crystal system, is a prerequisite for X-ray crystallographic fragment screening, which will be used to map SMT binding pockets for inhibitor design.
References
[1] Dangarembizi, R., Wasserman, S., Hoving, J. C., et al. Parasite Immunology, (2023) e12953.
[2] Sakyi, P. O., Kwofie, S. K., Tuekpe, J. K., et al. Pharmaceuticals, (2023) 16(3), 330.
[3] Nes, W.D., Chaudhuri, M., Leaver, D.J. Biomolecules, (2024) 14:249.
