Inhibitors of Aspartate Transcarbamoylase Inhibit Mycobacterium tuberculosis Growth.

Aspartate transcarbamoylase (ATCase) plays a key role in the second step of de novo pyrimidine biosynthesis in eukaryotes and has been proposed to be a target to suppress cell proliferation in E. coli, human cells and the malarial parasite. We hypothesized that a library of ATCase inhibitors developed for malarial ATCase (PfATCase) may also contain inhibitors of the tubercular ATCase and provide a similar inhibition of cellular proliferation. Of the 70 compounds screened, 10 showed single-digit micromolar inhibition in an in vitro activity assay and were tested for their effect on M. tuberculosis cell growth in culture. The most promising compound demonstrated a MIC90 of 4 µM. A model of MtbATCase was generated using the experimental coordinates of PfATCase. In silico docking experiments showed this compound can occupy a similar allosteric pocket on MtbATCase to that seen on PfATCase, explaining the observed species selectivity seen for this compound series.

Plasma metabolome predicts trained immunity responses after antituberculosis BCG vaccination.

The antituberculosis vaccine Bacillus Calmette-Guérin (BCG) induces nonspecific protection against heterologous infections, at least partly through induction of innate immune memory (trained immunity). The amplitude of the response to BCG is variable, but the factors that influence this response are poorly understood. Metabolites, either released by cells or absorbed from the gut, are known to influence immune responses, but whether they impact BCG responses is not known. We vaccinated 325 healthy individuals with BCG, and collected blood before, 2 weeks and 3 months after vaccination, to assess the influence of circulating metabolites on the immune responses induced by BCG. Circulating metabolite concentrations after BCG vaccination were found to have a more pronounced impact on trained immunity responses, such as the increase in IL-1ß and TNF-α production upon Staphylococcus aureus stimulation, than on specific adaptive immune memory, assessed as IFN-γ production in response to Mycobacterium tuberculosis. Circulating metabolites at baseline were able to predict trained immunity responses at 3 months after vaccination and enrichment analysis based on the metabolites positively associated with trained immunity revealed enrichment of the tricarboxylic acid (TCA) cycle and glutamine metabolism, both of which were previously found to be important for trained immunity. Several new metabolic pathways that influence trained immunity were identified, among which taurine metabolism associated with BCG-induced trained immunity, a finding validated in functional experiments. In conclusion, circulating metabolites are important factors influencing BCG-induced trained immunity in humans. Modulation of metabolic pathways may be a novel strategy to improve vaccine and trained immunity responses.

An In Vitro Perspective on What Individual Antimicrobials Add to Mycobacterium avium Complex Therapies.

For Mycobacterium avium complex pulmonary disease (MAC-PD), current treatment regimens yield low cure rates. To obtain an evidence-based combination therapy, we assessed the in vitro activity of six drugs, namely, clarithromycin (CLR), rifampin (RIF), ethambutol (EMB), amikacin (AMK), clofazimine (CLO), and minocycline (MIN), alone and in combination, against Mycobacterium avium and studied the contributions of individual antibiotics to efficacy. The MICs of all antibiotics against M. avium ATCC 700898 were determined by broth microdilution. We performed kinetic time-kill assays of all single drugs and clinically relevant two-, three-, four-, and five-drug combinations against M. avium. Pharmacodynamic interactions of these combinations were assessed using area under the time-kill curve-derived effect size and Bliss independence. Adding a second drug yielded an average increase of the effect size (E) of 18.7% ± 32.9%, although antagonism was seen in some combinations. Adding a third drug showed a smaller increase in effect size (+12.2% ± 11.5%). The RIF-CLO-CLR (E of 102 log10 CFU/ml · day), RIF-AMK-CLR (E of 101 log10 CFU/ml · day), and AMK-MIN-EMB (E of 97.8 log10 CFU/ml · day) regimens proved more active than the recommended RIF-EMB-CLR regimen (E of 89.1 log10 CFU/ml · day). The addition of a fourth drug had little impact on effect size (+4.54% ± 3.08%). In vitro, several two- and three-drug regimens are as effective as the currently recommended regimen for MAC-PD. Adding a fourth drug to any regimen had little additional effect. In vitro, the most promising regimen would be RIF-AMK-macrolide or RIF-CLO-macrolide.