The enzymology of Streptococcus pneumoniae peptidoglycan polymerisation

Bacterial cell survival depends on intact peptidoglycan, an extensive cell wall polymer of alternating N-acetylglucosamine and N-acetylmuramic acid residues, cross-linked by short peptides. Peptidoglycan biosynthesis is a viable and validated antimicrobial target; the intracellular, membrane-bound and extracellular synthetic stages provide a multitude of enzymes for interception with inhibitors. The ultimate phase of peptidoglycan biosynthesis occurs on the extracellular face of the cytoplasmic membrane and involves the polymerisation of Lipid II (the monomeric peptidoglycan precursor) by the transglycosylase and transpeptidase activities of the Penicillin-Binding Proteins (PBPs). The work presented in this thesis primarily focused on the biochemical characterisation of the integral membrane proteins Streptococcus pneumoniae PBP1a, PBP2b and PBP2x. These enzymes are clinically relevant; they are essential targets of !-lactam antibiotics and also mediate resistance against this important antimicrobial class. Full-length and truncated forms of the PBPs were cloned, expressed and purified to high levels. Two novel spectrophotometric assays were designed and developed to study the enzymology of the individual transglycosylase and transpeptidase activities of the PBPs with their natural substrate, Lipid II. Preliminary kinetic characterisations of the bifunctional PBP1a transglycosylase activity were performed and assay conditions were optimised to recreate an in vivo environment. PBP1a active site mutants revealed that transglycosylase activity was elevated in the absence of a functional transpeptidase domain. PBP1a and PBP2x exhibited transpeptidase activity with an apparent substrate preference for glycan polymers over Lipid II. PBP2x transpeptidase activity was not detected. The recorded rates of PBP activity were insufficient to support bacterial cell integrity, highlighting a gap in the understanding of PBP requirements. Finally, the PBPs were subjected to crystallisation trials for structural characterisations. This work provides an excellent foundation for the analysis and elucidation of PBP specificities. Future information attained could contribute to the design of novel inhibitors, alleviating the global threat of antibiotic resistance

Identification of KasA as the cellular target of an anti-tubercular scaffold

Phenotypic screens for bactericidal compounds are starting to yield promising hits against tuberculosis. In this regard, whole-genome sequencing of spontaneous resistant mutants generated against an indazole sulfonamide (GSK3011724A) identifies several specific single-nucleotide polymorphisms in the essential Mycobacterium tuberculosis ketoacyl synthase (kas) A gene. Here, this genomic-based target assignment is confirmed by biochemical assays, chemical proteomics and structural resolution of a KasA-GSK3011724A complex by X-ray crystallography. Finally, M. tuberculosis GSK3011724A-resistant mutants increase the in vitro minimum inhibitory concentration and the in vivo 99% effective dose in mice, establishing in vitro and in vivo target engagement. Surprisingly, the lack of target engagement of the related-ketoacyl synthases (FabH and KasB) suggests a different mode of inhibition when compared with other Kas inhibitors of fatty acid biosynthesis in bacteria. These results clearly identify KasA as the biological target of GSK3011724A and validate this enzyme for further drug discovery efforts against tuberculosis.The research leading to these results has received funding from the European Unionś 7th framework programme (FP7- 2007-2013) under Grant Agreement No 261378, the Bill & Melinda Gates Foundation (OPP1095631), and the Medical Research Council (MR/K012118/1)

Mycobacterial drug discovery

Mycobacterium tuberculosis is the causative pathogen of the pulmonary disease tuberculosis. Despite the availability of effective treatment programs, there is a global pursuit of new anti-tubercular agents to respond to the developing threat of drug resistance, in addition to reducing the extensive duration of chemotherapy and any associated toxicity. The route to mycobacterial drug discovery can be considered from two directions: target-to-drug and drug-to-target. The former approach uses conventional methods including biochemical assays along with innovative computational screens, but is yet to yield any drug candidates to the clinic, with a high attrition rate owing to lack of whole cell activity. In the latter approach, compound libraries are screened for efficacy against the bacilli or model organisms, ensuring whole cell activity, but here subsequent target identification is the rate-limiting step. Advances in a variety of scientific fields have enabled the amalgamation of aspects of both approaches in the development of novel drug discovery tools, which are now primed to accelerate the discovery of novel hits and leads with known targets and whole cell activity. This review discusses these traditional and innovative techniques, which are widely used in the quest for new anti-tubercular compounds

Mycobacterial cell wall biosynthesis:a multifaceted antibiotic target

SUMMARYMycobacterium tuberculosis(Mtb), the etiological agent of tuberculosis (TB), is recognized as a global health emergency as promoted by the World Health Organization. Over 1 million deathsperyear, along with the emergence of multi- and extensively-drug resistant strains ofMtb, have triggered intensive research into the pathogenicity and biochemistry of this microorganism, guiding the development of anti-TB chemotherapeutic agents. The essential mycobacterial cell wall, sharing some common features with all bacteria, represents an apparent ‘Achilles heel’ that has been targeted by TB chemotherapy since the advent of TB treatment. This complex structure composed of three distinct layers, peptidoglycan, arabinogalactan and mycolic acids, is vital in supporting cell growth, virulence and providing a barrier to antibiotics. The fundamental nature of cell wall synthesis and assembly has rendered the mycobacterial cell wall as the most widely exploited target of anti-TB drugs. This review provides an overview of the biosynthesis of the prominent cell wall components, highlighting the inhibitory mechanisms of existing clinical drugs and illustrating the potential of other unexploited enzymes as future drug targets.</jats:p

DprE2 is a molecular target of the anti-tubercular nitroimidazole compounds pretomanid and delamanid

Abstract Mycobacterium tuberculosis is one of the global leading causes of death due to a single infectious agent. Pretomanid and delamanid are new antitubercular agents that have progressed through the drug discovery pipeline. These compounds are bicyclic nitroimidazoles that act as pro-drugs, requiring activation by a mycobacterial enzyme; however, the precise mechanisms of action of the active metabolite(s) are unclear. Here, we identify a molecular target of activated pretomanid and delamanid: the DprE2 subunit of decaprenylphosphoribose-2’-epimerase, an enzyme required for the synthesis of cell wall arabinogalactan. We also provide evidence for an NAD-adduct as the active metabolite of pretomanid. Our results highlight DprE2 as a potential antimycobacterial target and provide a foundation for future exploration into the active metabolites and clinical development of pretomanid and delamanid

Two-Way Regulation of MmpL3 Expression Identifies and Validates Inhibitors of MmpL3 Function in Mycobacterium tuberculosis

MmpL3, an essential mycolate transporter in the inner membrane of Mycobacterium tuberculosis (Mtb), has been identified as a target of multiple, chemically diverse antitubercular drugs. However, several of these molecules seem to have secondary targets and inhibit bacterial growth by more than one mechanism. Here, we describe a cell-based assay that utilizes two-way regulation of MmpL3 expression to readily identify MmpL3-specific inhibitors. We successfully used this assay to identify a novel guanidine-based MmpL3 inhibitor from a library of 220 compounds that inhibit growth of Mtb by largely unknown mechanisms. We furthermore identified inhibitors of cytochrome bc1-aa3 oxidase as one class of off-target hits in whole-cell screens for MmpL3 inhibitors and report a novel sulfanylacetamide as a potential QcrB inhibitor