| Summary: | <p>The extraordinarily thick and complex cell wall of <em>Mycobacterium tuberculosis</em> (Mtb), the causative agent of tuberculosis (TB), may be considered as an Achilles’ heel of the bacillus; however, the most important class of antibiotics targeting the bacterial cell wall (the β-lactams) has not been proven to be effective as TB treatment, likely in part due to resistance mechanisms, such as hydrolysis catalysed by the β-lactamase BlaC. β-Lactams are inhibitors of a class of transpeptidases of the peptidoglycan layer of the bacterial cell wall, i.e., the penicillin binding proteins (PBPs). <em>Mtb</em> additionally applies a second class transpeptidases, the L,D-transpeptidases (Ldts), which employ a nucleophilic cysteine in catalysis, and which are reported to be resistant to inactivation by most β-lactams.</p>
<p>In <strong>Chapter 1</strong>, an introduction to TB and TB drug development is described. <strong>Chapter 2</strong> describes a literature review of the function and inhibition of the Ldts, PBPs, and BlaC of Mtb. <strong>Chapter 3</strong> describes the identification of novel inhibitors for LdtMt2, an essential Ldt of Mtb, via high-throughput screening (HTS), and detailed studies of their mechanism of inhibition. Several potent classes of inhibitors were identified, both from established inhibitor classes (e.g., β-lactams), and unexplored covalently reacting electrophilic groups (e.g., cyanamides). <strong>Chapter 4</strong> describes the mechanism of the αβ,α′β′-diepoxyketones as another class of unexplored mechanism-based inhibitors of nucleophilic cysteine enzymes identified in the HTS. <strong>Chapter 5</strong> describes studies on the mechanism of the transpeptidase activity of LdtMt2. A crystal structure of LdtMt2 in complex with the covalent intermediate of the transpeptidase reaction informs on key interactions of LdtMt2 with its substrate, and provides strategies for the design of improved inhibitors. <strong>Chapter 6</strong> describes the cellular inhibition of a selection of β-lactams and LdtMt2 inhibitors identified in the HTS with the model organism <em>Mycobacterium smegmatis</em>. The results of a fluorescent assay evaluating the inhibition of Ldt and PBP transpeptidase activity in live cells suggests multifaceted correlation of transpeptidase inhibition with inhibition of growth and suggests that inhibition of PBP transpeptidase activity alone is insufficient to inhibit growth, which was often accompanied with partial Ldt inhibition. <strong>Chapter 7</strong> describes inhibition studies of BlaC. A cephalosporin identified in the HTS manifested notable stability of the acyl-enzyme complex with BlaC, leading to inhibition similarly to that of the commonly used β-lactamase inhibitor clavulanic acid.</p>
<p>Overall, the work provides novel insights into the function and inhibition of LdtMt2. Novel potent inhibitors of LdtMt2 were identified, new insights into the protein-substrate interactions have been obtained, and understanding of the cellular role of Ldt inhibition has been advanced. The results provide guidance for the development of novel anti-TB drugs targeting the transpeptidases of <em>Mtb</em>.</p>
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