Structural Investigation of Mycobacterium tuberculosis Drug Targets and the Evaluation of Natural Products Derived Inhibitor Candidates

Abstract

This dissertation uses two different approaches to the identification of inhibitors of Mycobacterium tuberculosis (Mtb) - a structure-based drug discovery approach and a high-throughput screening of natural product libraries based approach. In the structure-based approach the structural characterization of Mtb s-adenosylhomocysteine hydrolase (SAHH) enzyme as a drug target using x-ray crystallography is described. Crystal structure of Mtb SAHH protein was solved in complex with the substrate adenosine and the product s-adenosylhomocysteine at 1.6 Å and 2.0 Å resolutions respectively. Additionally, crystal structures of Mtb SAHH in complex with inhibitors, aristeromycine (ARI), deazaadenosine and 2-fluoroadenosine were also solved at 2.1 Å, 2.2 Å and 2.4 Å resolutions respectively. The complex structure with ARI is the first structure reported and confirms the proposed type-I mechanism based inhibition of Mtb SAHH. Differences in the active site of Mtb SAHH and human SAHH are identified and the design of lead molecules selective towards the Mtb SAHH is described using the fragment-based lead identification method. The structural characterization of a nitrogen regulatory Mtb PII protein is also described. The crystal structure of Mtb PII protein in the apo form and adenosine triphosphate bound form was solved to 1.4 Å and 2.4 Å resolutions respectively. The crystal structures suggest an alternate annotation of the protein as GlnK and also provide insights into the mechanism of action of the Mtb PII protein. The Mtb PII protein plays a versatile role in the nitrogen regulatory pathway of the microorganism and represents a potential drug target in Mtb.

Through the alternate approach to drug discovery involving the screening of natural products for whole-cell bactericidal activity a novel natural product inhibitor of Mycobacterium tuberculosis and Mycobacterium smegmatis was isolated, purified and characterized. Challenges encountered in the large scale data processing involving high-throughput screening and high performance liquid chromatography (HPLC) / mass spectrometric analysis in terms of prioritizing the crude extracts, the HPLC fractions and the masses corresponding to the compounds of interest are listed and methods for data reduction and efficient data analysis are presented. The successful identification of a novel natural product with inhibitory activity towards the human and yeast proteasome in an in-vitro enzyme assay is also described. The novel polyphenolic natural product was discovered through the screening of crude extracts in a proteasome targeted in vitro enzyme assay followed by activity based fractionation, isolation, purification and structure elucidation using analytical techniques. A technique for the chemical derivatization of a mixture of unknown secondary metabolites in crude extracts is also described, which can potentially increase the existing diversity of natural product libraries used in high-throughput screening.