Atica Moosa

Pyrrolo[3,4-c]pyridine-1,3(2H)-diones: A Novel Antimycobacterial Class Targeting Mycobacterial Respiration

High-throughput screening of a library of small polar molecules against Mycobacterium tuberculosis led to the identification of a phthalimide-containing ester hit compound (1), which was optimized for metabolic stability by replacing the ester moiety with a methyl oxadiazole bioisostere. A route utilizing polymer-supported reagents was designed and executed to explore structure-activity relationships with respect to the N-benzyl substituent, leading to compounds with nanomolar activity. The frontrunner compound (5h) from these studies was well tolerated in mice. A M. tuberculosis cytochrome bd oxidase deletion mutant (ΔcydKO) was hyper-susceptible to compounds from this series, and a strain carrying a single point mutation in qcrB, the gene encoding a subunit of the menaquinol cytochrome c oxidoreductase, was resistant to compounds in this series. In combination, these observations indicate that this novel class of antimycobacterial compounds inhibits the cytochrome bc1 complex, a validated drug target in M. tuberculosis.

Vitamin B12 metabolism in Mycobacterium tuberculosis

Mycobacterium tuberculosis is included among a select group of bacteria possessing the capacity for de novo biosynthesis of vitamin B12, the largest and most complex natural organometallic cofactor. The bacillus is also able to scavenge B12 and related corrinoids utilizing an ATP-binding cassette-type protein that is distinct from the only known bacterial B12-specific transporter, BtuFCD. Consistent with the inferred requirement for vitamin B12 for metabolic function, the M. tuberculosis genome encodes two B12 riboswitches and three B12-dependent enzymes. Two of these enzymes have been shown to operate in methionine biosynthesis (MetH) and propionate utilization (MutAB), while the function of the putative nrdZ-encoded ribonucleotide reductase remains unknown. Taken together, these observations suggest that M. tuberculosis has the capacity to regulate core metabolic functions according to B12 availability - whether acquired via endogenous synthesis or through uptake from the host environment - and, therefore, imply that there is a role for vitamin B12 in pathogenesis, which remains poorly understood.

Bioluminescent Reporters for Rapid Mechanism of Action Assessment in Tuberculosis Drug Discovery

ABSTRACT The tuberculosis (TB) drug discovery pipeline is fueled by compounds identified in whole-cell screens against the causative agent, Mycobacterium tuberculosis. Phenotypic screening enables the selection of molecules that inhibit essential cellular functions in live, intact bacilli grown under a chosen in vitro condition. However, deducing the mechanism of action (MOA), which is important to avoid promiscuous targets, often requires significant biological resources in a lengthy process that risks decoupling medicinal chemistry and biology efforts. Therefore, there is a need to develop methods enabling rapid MOA assessment of putative “actives” for triage decisions. Here, we describe a modified version of a bioluminescence reporter assay that allows nondestructive detection of compounds targeting either of two macromolecular processes in M. tuberculosis: cell wall biosynthesis or maintenance of DNA integrity. Coupling the luxCDABE operon from Photorhabdus luminescens to mycobacterial promoters driving expression of the iniBAC operon (PiniB-LUX) or the DNA damage-inducible genes, recA (PrecA-LUX) or radA (PradA-LUX), provided quantitative detection in real time of compounds triggering expression of any of these promoters over an extended 10- to 12-day incubation. Testing against known anti-TB agents confirmed the specificity of each reporter in registering the MOA of the applied antibiotic in M. tuberculosis, independent of bactericidal or bacteriostatic activity. Moreover, profiles obtained for experimental compounds indicated the potential to infer complex MOAs in which multiple cellular processes are disrupted. These results demonstrate the utility of the reporters for early triage of compounds based on the provisional MOA and suggest their application to investigate polypharmacology in known and experimental anti-TB agents.

Aminopyrazolo[1,5-a]pyrimidines as potential inhibitors of Mycobacterium tuberculosis: Structure activity relationships and ADME characterization

Whole-cell high-throughput screening of a diverse SoftFocus library against Mycobacterium tuberculosis (Mtb) generated a novel aminopyrazolo[1,5-a]pyrimidine hit series. The synthesis and structure activity relationship studies identified compounds with potent antimycobacterial activity. The SAR of over 140 compounds shows that the 2-pyridylmethylamine moiety at the C-7 position of the pyrazolopyrimidine scaffold was important for Mtb activity, whereas the C-3 position offered a higher degree of flexibility. The series was also profiled for in vitro cytotoxicity and microsomal metabolic stability as well as physicochemical properties. Consequently liabilities to be addressed in a future lead optimization campaign have been identified.

Susceptibility of Mycobacterium tuberculosis Cytochrome bd Oxidase Mutants to Compounds Targeting the Terminal Respiratory Oxidase, Cytochrome c

ABSTRACT We deleted subunits I (cydA) and II (cydB) of the Mycobacterium tuberculosis cytochrome bd menaquinol oxidase. The resulting ΔcydA and ΔcydAB mutants were hypersusceptible to compounds targeting the mycobacterial bc1 menaquinol-cytochrome c oxidoreductase and exhibited bioenergetic profiles indistinguishable from strains deficient in the ABC-type transporter, CydDC, predicted to be essential for cytochrome bd assembly. These results confirm CydAB and CydDC as potential targets for drugs aimed at inhibiting a terminal respiratory oxidase implicated in pathogenesis.

Real-Time Investigation of Tuberculosis Transmission: Developing the Respiratory Aerosol Sampling Chamber (RASC)

Knowledge of the airborne nature of respiratory disease transmission owes much to the pioneering experiments of Wells and Riley over half a century ago. However, the mechanical, physiological, and immunopathological processes which drive the production of infectious aerosols by a diseased host remain poorly understood. Similarly, very little is known about the specific physiological, metabolic and morphological adaptations which enable pathogens such as Mycobacterium tuberculosis (Mtb) to exit the infected host, survive exposure to the external environment during airborne carriage, and adopt a form that is able to enter the respiratory tract of a new host, avoiding innate immune and physical defenses to establish a nascent infection. As a first step towards addressing these fundamental knowledge gaps which are central to any efforts to interrupt disease transmission, we developed and characterized a small personal clean room comprising an array of sampling devices which enable isolation and representative sampling of airborne particles and organic matter from tuberculosis (TB) patients. The complete unit, termed the Respiratory Aerosol Sampling Chamber (RASC), is instrumented to provide real-time information about the particulate output of a single patient, and to capture samples via a suite of particulate impingers, impactors and filters. Applying the RASC in a clinical setting, we demonstrate that a combination of molecular and microbiological assays, as well as imaging by fluorescence and scanning electron microscopy, can be applied to investigate the identity, viability, and morphology of isolated aerosolized particles. Importantly, from a preliminary panel of active TB patients, we observed the real-time production of large numbers of airborne particles including Mtb, as confirmed by microbiological culture and polymerase chain reaction (PCR) genotyping. Moreover, direct imaging of captured samples revealed the presence of multiple rod-like Mtb organisms whose physical dimensions suggested the capacity for travel deep into the alveolar spaces of the human lung.

Novel Antitubercular 6-Dialkylaminopyrimidine Carboxamides from Phenotypic Whole-Cell High Throughput Screening of a SoftFocus Library: Structure–Activity Relationship and Target Identification Studies

A BioFocus DPI SoftFocus library of ∼35 000 compounds was screened against Mycobacterium tuberculosis (Mtb) in order to identify novel hits with antitubercular activity. The hits were evaluated in biology triage assays to exclude compounds suggested to function via frequently encountered promiscuous mechanisms of action including inhibition of the QcrB subunit of the cytochrome bc1 complex, disruption of cell–wall homeostasis, and DNA damage. Among the hits that passed this screening cascade, a 6-dialkylaminopyrimidine carboxamide series was prioritized for hit to lead optimization. Compounds from this series were active against clinical Mtb strains, while no cross-resistance to conventional antituberculosis drugs was observed. This suggested a novel mechanism of action, which was confirmed by chemoproteomic analysis leading to the identification of BCG_3193 and BCG_3827 as putative targets of the series with unknown function. Initial structure–activity relationship studies have resulted in compounds with moderate to potent antitubercular activity and improved physicochemical properties.