Tuberculosis (TB) remains a global health threat, especially due to the emergence and rapid evolution of resistant strains of pathogenic bacteria coupled to the lack of development in treatment. The main treatment requires infected individuals to take multiple drugs over an extended period of time. The treatment for individuals infected with strains of TB that are resistant to classic antibiotics is even heavier. The main drugs have remained the same over the past 50 years and more efficient drugs against TB are yet to be discovered. The development of new and more efficient drugs is therefore becoming increasingly urgent.
Mycobacterium tuberculosis, the causative agent of TB, is particularly difficult to kill because of the thick and highly hydrophobic cell wall. 6-O-Methylglucose lipopolysaccharides (MGLP) have a major role in the biosynthesis of fatty acids and mycolic acids. Mycolic acids are one of the main reasons for the hydrophobicity and efficiency of the cell wall in protecting the bacteria. Disrupting the synthesis of MGLP shows significant decrease in growth and bacterial activity of Mtb. Rv3030 is an S-Adenosyl-L-Methionine (SAM)-dependent methyltransferase (MTase) and is a major enzyme in the biosynthetic pathway of MGLP as it is responsible for the essential 6-O- methylation. Unfortunately, the information currently available on this protein is limited as no crystal structure has been published. Therefore, to suppress the production of MGLP through the inhibition of Rv3030, it is necessary to fully characterize Rv3030. In order to achieve this goal, we screened compounds against Rv3030 using Differential Scanning Fluorimetry (DSF) to obtain information on chemotypes that could potentially be used as a basis for the development of Rv3030 inhibitors. Additionally, we used a competitive fluorescence polarization (FP) assay to measure in vitro activity of Rv3030, which would allow for inhibitor validation studies. In parallel, Rv3030 was crystallized in an initial screen and will provide valuable information leading crystal structure determination.
The second part of this thesis focuses on the characterization of an enzyme essential for the survival of Mtb within the host. Rv1405 is an uncharacterized putative SAM- dependent MTase for which the function remains at the stage of hypothesis. This part describes the steps we have taken to obtain information on this enzyme screening multiple libraries of compounds using a DSF assay and optimizing a competitive FP assay to carry out inhibitory studies. In an attempt to gain invaluable structural knowledge of Rv1405, we obtained preliminary crystals of Rv1405 establishing a basis for further improvement of crystallization.
The last part of this thesis describes the work and experiments carried out to characterize Rv0893 and confirm that it is a SAM-dependent MTase. Knowledge about this enzyme is extremely limited, making the study of Rv0893 to be challenging. We have used DSF and competitive FP assays with the intention to identify the biological methyl acceptor of this enzyme. In parallel, we crystallized and attempted to optimize crystallization in an attempt to gain valuable structural knowledge about Rv0893.