Content:
As antibiotic resistance of various pathogens emerged globally, the need for new effective drugs with novel modes of action became urgent. In this thesis, we focus on infectious diseases, e.g. tuberculosis, malaria, and nosocomial infections, and the corresponding causative pathogens, Mycobacterium tuberculosis, Plasmodium falciparum, and the Gram-negative ESKAPE pathogens that underlie so many healthcare-acquired diseases. Following the same-target-other-pathogen (STOP) strategy, we attempted to comprehensively explore the properties of three promising drug targets. Signal peptidase I (SPase I), existing both in Gram-negative and Gram-positive bacteria, as well as in parasites, is vital for cell viability, due to its critical role in signal peptide cleavage, thus, protein maturation, and secreted protein transport. Three factors, comprising essentiality, a unique mode of action, and easy accessibility, make it an attractive drug target. We have established a platform, investigating the protein purification, enzymatic kinetics, and inhibition. A full-length SPase I from E. coli, including two transmembrane segments, was produced and purified in the presence of 0.5 % Triton X-100. In the in vitro biochemical assay, it exhibits proteolytic activity on antigen 85A from M. tuberculosis, with a Km of 20 µM and a kcat of 135 s-1. A series of macrocyclic oligopeptides that have been proven inhibitory to E. coli SPase I also showed potency against a panel of Gram-negative bacteria. 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is responsible for the production of methylerythritol phosphate (MEP) in the non-mevalonate pathway of isoprenoid biosynthesis, and is thus essential for cell growth. DXRs from M. tuberculosis and P. falciparum have been under investigation in our lab for years. I addressed structural and biochemical characterizations of PfDXR with analogs of 3-(N-formyl-N-hydroxyamino)propyl- phosphonate (fosmidomycin) and 3-(N-acetyl-N-hydroxyamino)propyl- phosphonate (FR-9000098), two natural products showing potency against P. falciparum. Chemical modifications, methylation at Cg, and double bond formation between Ca and Cb, were investigated to increase the pathogenicidal activity. Crystallographic complex structures of PfDXR and four novel compounds inhibitory to PfDXR in a dose-dependent manner were solved, and ligand binding will be discussed in detail. Type II NADH dehydrogenase (NDH-2) is an essential component in the respiratory chain, playing an important role in electron transfer. Biomembrane-bound NDH-2 from M. tuberculosis was over-expressed in E. coli, as well as the homolog from M. smegmatis. The purified NDH-2s were kinetically characterized, and showed a similar affinity to previously reported NDH-2s expressed M. smegmatis. A collection of novel inhibitors in the scaffold of quinolinyl pyrimidines were synthesized and tested for inhibition in a biochemical assay.
Note:
Dissertation Uppsala universitet, Strukturbiologi; Uppsala 2021
Language:
English
URN:
urn:nbn:se:uu:diva-435815
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