José Manuel Ezquerra-Aznárez’s research while affiliated with University of Zaragoza and other places

Tuberculosis remains the deadliest infectious disease of the 21st century. New antimicrobials are needed to improve treatment outcomes and enable therapy shortening. Drug repurposing is an alternative to the traditional drug discovery process. The avermectins are a family of macrocyclic lactones with anthelmintic activity active against Mycobacterium tuberculosis. However, their mode of action in mycobacteria remains unknown. In this study, we employed traditional mutant isolation approaches using Mycobacterium smegmatis, a non-pathogenic M. tuberculosis surrogate. We were only able to isolate mutants with decreased susceptibility to selamectin using the ∆nucS mutator M. smegmatis strain. This phenotype was caused by mutations in mps1 and mmpL11. Two of these mutants were used for a second experiment in which high-level selamectin-resistant mutants were isolated; however, specific mutations driving the phenotypic change to high-level resistance could not be identified. The susceptibility to selamectin in these mutants was restored to the basal level by subinhibitory concentrations of ethambutol. The selection of ethambutol resistance in a high-level selamectin-resistant mutant also resulted in multiple colonies becoming susceptible to selamectin again. These colonies carried mutations in embB, suggesting that the integrity of the cell envelope is a prerequisite for selamectin resistance. The absence of increased susceptibility to selamectin in an embB deletion strain demonstrated that the target of selamectin is not cytosolic. Our data show that the concurrence of specific multiple mutations and complete integrity of the mycobacterial envelope are necessary for selamectin resistance. Our studies provide first-time insights into the antimycobacterial mode of action of the antiparasitic avermectins. IMPORTANCE Tuberculosis is the deadliest infectious disease of the 21st century. New antibiotics are needed to improve treatment. However, developing new drugs is costly and lengthy. Drug repurposing is an alternative to the traditional drug discovery process. The avermectins are a family of drugs used to treat parasitic infections that are active against Mycobacterium tuberculosis, the bacterium that causes tuberculosis. However, their mode of action in mycobacteria remains unknown. Understanding how avermectins kill mycobacteria can facilitate its development as an anti-mycobacterial drug, including against M. tuberculosis. In this study, we used Mycobacterium smegmatis, a non-pathogenic M. tuberculosis surrogate model to understand the molecular mechanisms of how selamectin (a drug of the avermectin family selected for this study as a model) acts against mycobacteria. Our data show that the generation of resistance to selamectin is unlikely and that complete integrity of the mycobacterial envelope is necessary for selamectin resistance, providing first-time insights into the antimycobacterial mode of action of the avermectins.

The emergence of antimicrobial resistance threatens advances achieved by medicine in the last century. This situation has been aggravated by the suboptimal outcome of screening campaigns to provide novel antibiotics. In this study, we took an alternative approach to discover new chemical scaffolds with antimicrobial activity. We screened a collection of photoactive compounds originally synthesized for industrial purposes and found that 4H-pyran-4-ylidenes are active against Gram-positive bacteria. Compounds belonging to this family displayed dose-dependent bactericidal activity against wild-type Staphylococcus aureus and Methicillin Resistant S. aureus (MRSA). Importantly, they were not cytotoxic to hepatic cell lines at the concentrations at which antimicrobial activity was observed. Resistance to 4H-pyran-4-ylidenes in S. aureus was achieved via mutations in the rny locus, likely via changes in mRNA levels of genes critical for their activity. Overall, we demonstrate that chemical libraries not originally intended for drug discovery can be a fruitful source of chemical diversity for the development of novel antimicrobials.

Tuberculosis remains the deadliest infectious disease of the 21 st century. New antimicrobials are needed to improve treatment outcomes and enable therapy shortening. Drug repurposing is an alternative to the traditional drug discovery process. The avermectins are a family of macrocyclic lactones with anthelmintic activity active against Mycobacterium tuberculosis . However, their mode of action in mycobacteria remains unknown. In this study, we employed traditional mutant isolation approaches using Mycobacterium smegmatis , a non-pathogenic Mycobacterium tuberculosis surrogate. We were only able to isolate mutants with decreased susceptibility to selamectin using the Δ nucS mutator M. smegmatis strain. This phenotype was caused by mutations in mps1 and mmpL11 . Two of these mutants were used for a second experiment in which high-level selamectin-resistant mutants were isolated; however, specific mutations driving the phenotypic change to high-level resistance could not be identified. The susceptibility to selamectin in these mutants was restored to the basal level by subinhibitory concentrations of ethambutol. The selection of ethambutol resistance in a high-level selamectin-resistant mutant also resulted in multiple colonies becoming susceptible to selamectin again. These colonies carried mutations in embB , suggesting that integrity of the cell envelope is a prerequisite for selamectin resistance. The absence of increased susceptibility to selamectin in an embB deletion strain demonstrated that the target of selamectin is not cytosolic. Our data show that concurrence of specific multiple mutations and complete integrity of the mycobacterial envelope are necessary for selamectin resistance. Our studies provide first-time insights into the antimycobacterial mode of action of the antiparasitic avermectins.

Background: Tuberculosis (TB) took more than 1 million lives in 2020, being the second cause of death from a infectious disease just behind COVID-19. In the past decades, there has been an emergence of multidrug-resistant M. tuberculosis strains. Drug-resistant TB accounts for one third of the worldwide deaths attributed to antimicrobial resistant bacteria and new active compounds are urgently needed. An alternative to the traditional drug discovery process is drug-repurposing, i.e. finding new uses for clinically approved drugs. By using this approach, the anthelmintic avermectins, which were traditionally considered to be inactive against bacteria, were found to be effective against M. tuberculosis and other pathogenic mycobacteria. However, their antimycobacterial mode of action remains unknown. Materials: Mycobacterial strains were grown in Middlebrook 7H9/7H10, supplemented with 10% ADC/OADC and 0.2% glycerol. Selamectin activity was characterised using the REMA assay and time-kill kinetics. Avermectins’ IC50 against the DprE1 enzyme was measured using the Amplex Red/peroxidase coupled assay. Molecular docking between selamectin and the DprE1 structure was done using the Glide software. Mycobacterial lipid changes were analysed with 14C metabolic labelling. Results: The activity of selamectin was determined against a panel of M. tuberculosis mutant strains (Table 1). Two strains with mutations in dprE1–encoding the decaprenylphosphoryl-beta-D-ribose oxidase involved in the synthesis of mycobacterial cell-wall arabinan components–were found to be more susceptible to selamectin (Figure 1). Biochemical assays with the purified DprE1 enzyme confirmed that avermectins inhibit DprE1, being selamectin the most active with an IC50 of 2.6 µM (Table 2). Docking of selamectin to the M. tuberculosis DprE1 structure predicted a binding site including the residue Leu275 (Figure 2). Sequence alignment revealed variants in this position among mycobacterial species, with the size and hydrophobicity correlating with the selamectin MIC values. This correlation was confirmed by biochemical assays against mutants of Leu275 (Table 3). Conclusions: Selamectin inhibits the purified DprE1 enzyme. We report for the first time the role of the amino acid at positions 275 (in M. tuberculosis) in the inhibition of DprE1 activity, involved in the stabilization of selamectin interaction at the molecular level.

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 ¹⁴C-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.

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 14C-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.

Antimicrobial resistance, the so-called silent pandemic, is pushing industry and academia to find novel antimicrobial agents with new mechanisms of action in order to be active against susceptible and drug-resistant microorganisms. In the case of tuberculosis, the need of novel anti-tuberculosis drugs is specially challenging because of the intricate biology of its causative agent, Mycobacterium tuberculosis. The repurposing of medicines has arisen in recent years as a fast, low-cost, and efficient strategy to identify novel biomedical applications for already approved drugs. This review is focused on anti-parasitic drugs that have additionally demonstrated certain levels of anti-tuberculosis activity; along with this, natural products with a dual activity against parasites and against M. tuberculosis are discussed. A few clinical trials have tested antiparasitic drugs in tuberculosis patients, and have revealed effective dose and toxicity issues, which is consistent with the natural differences between tuberculosis and parasitic infections. However, through medicinal chemistry approaches, derivatives of drugs with anti-parasitic activity have become successful drugs for use in tuberculosis therapy. In summary, even when the repurposing of anti-parasitic drugs for tuberculosis treatment does not seem to be an easy job, it deserves attention as a potential contributor to fuel the anti-tuberculosis drug pipeline.