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Avermectins are macrocyclic lactones with anthelmintic activity. Recently, they were found to be effective against Mycobacterium tuberculosis , which accounts for one third of the worldwide deaths from antimicrobial resistance. However, their anti-mycobacterial mode of action remains to be elucidated. The activity of selamectin was determined against a panel of M. tuberculosis mutants. Two strains carrying mutations in DprE1, the decaprenylphosphoryl-β-D-ribose oxidase involved in the synthesis of mycobacterial arabinogalactan, were more susceptible to selamectin. Biochemical assays against the Mycobacterium smegmatis DprE1 protein confirmed this finding, and docking studies predicted a binding site in a loop that included Leu275. Sequence alignment revealed variants in this position among mycobacterial species, with the size and hydrophobicity of the residue correlating with their MIC values; M. smegmatis DprE1 variants carrying these point mutations validated the docking predictions. However, the correlation was not confirmed when M. smegmatis mutant strains were constructed and MIC phenotypic assays performed. Likewise, metabolic labeling of selamectin-treated M. smegmatis and M. tuberculosis cells with 14 C-labeled acetate did not reveal the expected lipid profile associated with DprE1 inhibition. Together, our results confirm the in vitro interactions of selamectin and DprE1 but suggest that selamectin could be a multi-target anti-mycobacterial compound.

The lack of effective therapies for treating tuberculosis (TB) is a global health problem. While Mycobacterium tuberculosis is notoriously resistant to most available antibiotics, we identified synthetic short cationic antimicrobial peptides that were active at low micromolar concentrations (less than 10 μM). These small peptides (averaging 10 amino acids) had remarkably broad spectra of antimicrobial activities against both bacterial and fungal pathogens and an indication of low cytotoxicity. In addition, their antimicrobial activities displayed various degrees of species specificity that were not related to taxonomy. For example, Candida albicans and Staphylococcus aureus were the best surrogates to predict peptide activity against M. tuberculosis, while Mycobacterium smegmatis was a poor surrogate. Principle component analysis of activity spectrum profiles identified unique features associated with activity against M. tuberculosis that reflect their distinctive amino acid composition; active peptides were more hydrophobic and cationic, reflecting increased tryptophan with compensating decreases in valine and other uncharged amino acids and increased lysine. These studies provide foundations for development of cationic antimicrobial peptides as potential new therapeutic agents for TB treatment.

Four butenolides, ramariolides A-D (1-4), have been isolated from the fruiting bodies of the coral mushroom Ramaria cystidiophora. Their structures were elucidated by analysis of 1D and 2D NMR data and a single-crystal X-ray diffraction analysis of 1, and their absolute configurations were established using Mosher's method. The major metabolite, ramariolide A (1), which contains an unusual spiro oxiranebutenolide moiety, showed in vitro antimicrobial activity against Mycobacterium smegmatis and Mycobacterium tuberculosis.

Therapeutic options for tuberculosis (TB) are limited and notoriously ineffective despite the wide variety of potent antibiotics available for treating other bacterial infections. We investigated an approach that enables an expansion of TB therapeutic strategies by using synergistic combinations of drugs. To achieve this, we devised a high-throughput synergy screen (HTSS) of chemical libraries having known pharmaceutical properties, including thousands that are clinically approved. Spectinomycin was used to test the concept that clinically available antibiotics with limited efficacy against Mycobacterium tuberculosis might be used for TB treatment when coadministered with a synergistic partner compound used as a sensitizer. Screens using Mycobacterium smegmatis revealed many compounds in our libraries that acted synergistically with spectinomycin. Among them, several families of antimicrobial compounds, including macrolides and azoles, were also synergistic against M. tuberculosis in vitro and in a macrophage model of M. tuberculosis infection. Strikingly, each sensitizer identified for synergy with spectinomycin uniquely enhanced the activities of other clinically used antibiotics, revealing a remarkable number of unexplored synergistic drug combinations. HTSS also revealed a novel activity for bromperidol, a butyrophenone used as an antipsychotic drug, which was discovered to be bactericidal and greatly enhanced the activities of several antibiotics and drug combinations against M. tuberculosis. Our results suggest that many compounds in the currently available pharmacopoeia could be readily mobilized for TB treatment, including disease caused by multi- and extensively drug-resistant strains for which there are no effective therapies.

Bacterial efflux pumps have traditionally been studied as low-level drug resistance determinants. Recent insights have suggested that efflux systems are often involved with fundamental cellular physiological processes, suggesting that drug extrusion may be a secondary function. In Mycobacterium tuberculosis, little is known about the physiological or drug resistance roles of efflux pumps. Using Mycobacterium bovis BCG as a model system, we showed that deletion of the Rv1410c gene encoding the P55 efflux pump made the strain more susceptible to a range of toxic compounds, including rifampin (rifampicin) and clofazimine, which are first- and second-line antituberculosis drugs. The efflux pump inhibitors carbonyl cyanide m-chlorophenylhydrazone (CCCP) and valinomycin inhibited the P55-determined drug resistance, suggesting the active export of the compounds by use of the transmembrane proton and electrochemical gradients as sources of energy. In addition, the P55 efflux pump mutant was more susceptible to redox compounds and displayed increased intracellular redox potential, suggesting an essential role of the efflux pump in detoxification processes coupled to oxidative balance within the cell. Finally, cells that lacked the p55 gene displayed smaller colony sizes and had a growth defect in liquid culture. This, together with an increased susceptibility to the cell wall-targeting compounds bacitracin and vancomycin, suggested that P55 is needed for proper cell wall assembly and normal growth in vitro. Thus, P55 plays a fundamental role in oxidative stress responses and in vitro cell growth, in addition to contributing to intrinsic antibiotic resistance. Inhibitors of the P55 efflux pump could help to improve current treatments for tuberculosis.