Dual transcriptional inhibition of glutamate and alanine racemase is synergistic in .

Synergistic interactions between chemical inhibitors, whilst informative, can be difficult to interpret, as chemical inhibitors can often have multiple targets, many of which can be unknown. Here, using multiplexed transcriptional repression, we have validated that the simultaneous repression of glutamate racemase and alanine racemase has a synergistic interaction in . This confirms prior observations from chemical interaction studies and highlights the potential of targeting multiple enzymes involved in mycobacterial cell wall synthesis.

Antibiotic resistance in alters tolerance to cell wall-targeting inhibitors.

Background: A limited ability to eliminate drug-resistant strains of is a major contributor to the morbidity of TB. Complicating this problem, little is known about how drug resistance-conferring mutations alter the ability of to tolerate antibiotic killing. Here, we investigated if drug-resistant strains of have an altered ability to tolerate killing by cell wall-targeting inhibitors.

A dual-targeting succinate dehydrogenase and FF-ATP synthase inhibitor rapidly sterilizes replicating and non-replicating Mycobacterium tuberculosis.

Mycobacterial bioenergetics is a validated target space for antitubercular drug development. Here, we identify BB2-50F, a 6-substituted 5-(N,N-hexamethylene)amiloride derivative as a potent, multi-targeting bioenergetic inhibitor of Mycobacterium tuberculosis. We show that BB2-50F rapidly sterilizes both replicating and non-replicating cultures of M.

The evolution of antibiotic resistance is associated with collateral drug phenotypes in Mycobacterium tuberculosis.

The increasing incidence of drug resistance in Mycobacterium tuberculosis has diminished the efficacy of almost all available antibiotics, complicating efforts to combat the spread of this global health burden. Alongside the development of new drugs, optimised drug combinations are needed to improve treatment success and prevent the further spread of antibiotic resistance. Typically, antibiotic resistance leads to reduced sensitivity, yet in some cases the evolution of drug resistance can lead to enhanced sensitivity to unrelated drugs.

Characterizing in vivo loss of virulence of an HN878 Mycobacterium tuberculosis isolate from a genetic duplication event.

The increase of global cases of drug resistant (DR) Mycobacterium tuberculosis (M.tb) is a serious problem for the tuberculosis research community and the goals to END TB by 2030. Due to the need for advancing and screening next generation therapeutics and vaccines, we aimed to design preclinical DR models of Beijing lineage M.

Uncovering interactions between mycobacterial respiratory complexes to target drug-resistant .

remains a leading cause of infectious disease morbidity and mortality for which new drug combination therapies are needed. Mycobacterial bioenergetics has emerged as a promising space for the development of novel therapeutics. Further to this, unique combinations of respiratory inhibitors have been shown to have synergistic or synthetic lethal interactions, suggesting that combinations of bioenergetic inhibitors could drastically shorten treatment times.

Impaired Succinate Oxidation Prevents Growth and Influences Drug Susceptibility in Mycobacterium tuberculosis.

Succinate is a major focal point in mycobacterial metabolism and respiration, serving as both an intermediate of the tricarboxylic acid (TCA) cycle and a direct electron donor for the respiratory chain. Mycobacterium tuberculosis encodes multiple enzymes predicted to be capable of catalyzing the oxidation of succinate to fumarate, including two different succinate dehydrogenases (Sdh1 and Sdh2) and a separate fumarate reductase (Frd) with possible bidirectional behavior. Previous attempts to investigate the essentiality of succinate oxidation in M.

Deciphering functional redundancy and energetics of malate oxidation in mycobacteria.

Oxidation of malate to oxaloacetate, catalyzed by either malate dehydrogenase (Mdh) or malate quinone oxidoreductase (Mqo), is a critical step of the tricarboxylic acid cycle. Both Mqo and Mdh are found in most bacterial genomes, but the level of functional redundancy between these enzymes remains unclear. A bioinformatic survey revealed that Mqo was not as widespread as Mdh in bacteria but that it was highly conserved in mycobacteria.

An amiloride derivative is active against the FF-ATP synthase and cytochrome bd oxidase of Mycobacterium tuberculosis.

Increasing antimicrobial resistance compels the search for next-generation inhibitors with differing or multiple molecular targets. In this regard, energy conservation in Mycobacterium tuberculosis has been clinically validated as a promising new drug target for combatting drug-resistant strains of M. tuberculosis.

Multiplexed transcriptional repression identifies a network of bactericidal interactions between mycobacterial respiratory complexes.

remains a leading cause of infectious disease morbidity and mortality for which new drug combination therapies are needed. Combinations of respiratory inhibitors can have synergistic or synthetic lethal interactions with sterilizing activity, suggesting that regimens with multiple bioenergetic inhibitors could shorten treatment times. However, realizing this potential requires an understanding of which combinations of respiratory complexes, when inhibited, have the strongest consequences on bacterial growth and viability.

Utilization of CRISPR interference to investigate the contribution of genes to pathogenesis in a macrophage model of Mycobacterium tuberculosis infection.

Objectives: There is an urgent need for novel drugs that target unique cellular pathways to combat infections caused by Mycobacterium tuberculosis. CRISPR interference (CRISPRi)-mediated transcriptional repression has recently been developed for use in mycobacteria as a genetic tool for identifying and validating essential genes as novel drug targets. Whilst CRISPRi has been applied to extracellular bacteria, no studies to date have determined whether CRISPRi can be used in M.

CRISPR interference identifies vulnerable cellular pathways with bactericidal phenotypes in Mycobacterium tuberculosis.

Mycobacterium tuberculosis remains a leading cause of death for which new drugs are needed. The identification of drug targets has been advanced by high-throughput and targeted genetic deletion strategies. Each though has limitations including the inability to distinguish between levels of vulnerability, lethality, and scalability as a molecular tool.

Novel Trifluoromethyl Pyrimidinone Compounds With Activity Against .

The identification and development of new anti-tubercular agents are a priority research area. We identified the trifluoromethyl pyrimidinone series of compounds in a whole-cell screen against . Fifteen primary hits had minimum inhibitory concentrations (MICs) with good potency IC is the concentration at which growth is inhibited by 90% (IC < 5 μM).

Deletion of Rv2571c confers resistance to arylamide compounds in .

Tuberculosis, caused by , is an urgent global health problem requiring new drugs, new drug targets and an increased understanding of antibiotic resistance. We have determined the mode of resistance to a series of arylamide compounds in We isolated resistant mutants to two arylamide compounds which are inhibitory to growth under host-relevant conditions (butyrate as a sole carbon source). Thirteen mutants were characterized, and all had mutations in Rv2571c; mutations included a premature stop codon and frameshifts as well as non-synonymous polymorphisms.

Multiple Mutations in Mycobacterium tuberculosis MmpL3 Increase Resistance to MmpL3 Inhibitors.

The protein MmpL3 performs an essential role in cell wall synthesis, since it effects the transport of trehalose monomycolates across the inner membrane. Numerous structurally diverse pharmacophores have been identified as inhibitors of MmpL3 largely based on the identification of resistant isolates with mutations in MmpL3. For some compounds, it is possible there are different primary or secondary targets.

Transcriptional Inhibition of the FF-Type ATP Synthase Has Bactericidal Consequences on the Viability of Mycobacteria.

Bedaquiline, an inhibitor of the mycobacterial ATP synthase, has revolutionized the treatment of infection. Although a potent inhibitor, it is characterized by poorly understood delayed time-dependent bactericidal activity. Here, we demonstrate that in contrast to bedaquiline, the transcriptional inhibition of the ATP synthase in and has rapid bactericidal activity.

Two for the price of one: Attacking the energetic-metabolic hub of mycobacteria to produce new chemotherapeutic agents.

Cellular bioenergetics is an area showing promise for the development of new antimicrobials, antimalarials and cancer therapy. Enzymes involved in central carbon metabolism and energy generation are essential mediators of bacterial physiology, persistence and pathogenicity, lending themselves natural interest for drug discovery. In particular, succinate and malate are two major focal points in both the central carbon metabolism and the respiratory chain of Mycobacterium tuberculosis.

Utilization of CRISPR Interference To Validate MmpL3 as a Drug Target in .

There is an urgent need for novel therapeutics to treat infections. Genetic strategies for validating novel targets are available, yet their time-consuming nature limits their utility. Here, using MmpL3 as a model target, we report on the application of mycobacterial CRISPR interference for the rapid validation of target essentiality and compound mode of action.

Cell wall inhibitors increase the accumulation of rifampicin in .

There is a need for new combination regimens for tuberculosis. Identifying synergistic drug combinations can avoid toxic side effects and reduce treatment times. Using a fluorescent rifampicin conjugate, we demonstrated that synergy between cell wall inhibitors and rifampicin was associated with increased accumulation of rifampicin.

Efficacy and Improved Resistance Potential of a Cofactor-Independent InhA Inhibitor of Mycobacterium tuberculosis in the C3HeB/FeJ Mouse Model.

AN12855 is a direct, cofactor-independent inhibitor of InhA in In the C3HeB/FeJ mouse model with caseous necrotic lung lesions, AN12855 proved efficacious with a significantly lower resistance frequency than isoniazid. AN12855 drug levels were better retained in necrotic lesions and caseum where the majority of hard to treat, extracellular bacilli reside. Owing to these combined attributes, AN12855 represents a promising alternative to the frontline antituberculosis agent isoniazid.