Exploring the plasticity of the InhA substrate-binding site using new diaryl ether inhibitors.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a worldwide scourge with more than 10 million people affected yearly. Among the proteins essential for the survival of Mtb, InhA has been and is still clinically validated as a therapeutic target. A new family of direct diaryl ether inhibitors, not requiring prior activation by the catalase peroxidase enzyme KatG, has been designed with the ambition of fully occupying the InhA substrate-binding site. Thus, eleven compounds, featuring three pharmacophores within the same molecule, were synthesized. One of them, 5-(((4-(2-hydroxyphenoxy)benzyl)(octyl)amino)methyl)-2-phenoxyphenol (compound 21), showed good inhibitory activity against InhA with IC50 of 0.70 µM. The crystal structure of compound 21 in complex with InhA/NAD+ showed how the molecule fills the substrate-binding site as well as the minor portal of InhA. This study represents a further step towards the design of new inhibitors of InhA.

Imidazoquinoline Derivatives as Potential Inhibitors of InhA Enzyme and Mycobacterium tuberculosis.

Tuberculosis is a serious public health problem worldwide. The search for new antibiotics has become a priority, especially with the emergence of resistant strains. A new family of imidazoquinoline derivatives, structurally analogous to triazolophthalazines, which had previously shown good antituberculosis activity, were designed to inhibit InhA, an essential enzyme for Mycobacterium tuberculosis survival. Over twenty molecules were synthesized and the results showed modest inhibitory efficacy against the protein. Docking experiments were carried out to show how these molecules could interact with the protein's substrate binding site. Disappointingly, unlike triazolophthlazines, these imidazoquinoline derivatives showed an absence of inhibition on mycobacterial growth.

Mycobacterium abscessus Infections in Cystic Fibrosis Individuals: A Review on Therapeutic Options.

Mycobacterium abscessus is an opportunistic pathogen that mainly colonizes and infects cystic fibrosis patients' lungs. M. abscessus is naturally resistant to many antibiotics such as rifamycin, tetracyclines and ß-lactams. The current therapeutic regimens are not very effective and are mostly based on repurposed drugs used against Mycobacterium tuberculosis infections. Thus, new approaches and novel strategies are urgently needed. This review aims to provide an overview of the latest ongoing findings to fight M. abscessus infections by analyzing emerging and alternative treatments, novel drug delivery strategies, and innovative molecules.

New Drugs and Novel Cellular Targets against Tuberculosis.

Mycobacterium tuberculosis (Mtb) is the etiological agent of tuberculosis (TB), one of the most life-threatening communicable diseases, which causes 10 million new cases each year and results in an estimated 1 [...].

The Veterinary Anti-Parasitic Selamectin Is a Novel Inhibitor of the Mycobacterium tuberculosis DprE1 Enzyme.

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-ß-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.

Gut Microbial Signatures in Sporadic and Hereditary Colorectal Cancer.

Colorectal cancer (CRC) is the fourth most common cause of cancer-related death and the third most common cancer in the world. Depending on the origin of the mutation, colorectal carcinomas are classified as sporadic or hereditary. Cancers derived from mutations appearing during life, affecting individual cells and their descendants, are called sporadic and account for almost 95% of the CRCs. Less than 5% of CRC cases result from constitutional mutations conferring a very high risk of developing cancer. Screening for hereditary-related cancers is offered to individuals at risk for hereditary CRC, who have either not undergone genetic evaluation or have uncertain genetic test results. In this review, we briefly summarize the main findings on the correlation between sporadic CRC and the gut microbiota, and we specifically focus on the few evidences about the role that gut microorganisms have on the development of CRC hereditary syndromes. The characterization of a gut microbiota associated with an increased risk of developing CRC could have a profound impact for prevention purposes. We also discuss the potential role of the gut microbiota as therapeutic treatment.

The Antimalarial Mefloquine Shows Activity against Mycobacterium abscessus, Inhibiting Mycolic Acid Metabolism.

Some nontuberculous mycobacteria (NTM) are considered opportunistic pathogens. Nevertheless, NTM infections are increasing worldwide, becoming a major public health threat. Furthermore, there is no current specific drugs to treat these infections, and the recommended regimens generally lack efficacy, emphasizing the need for novel antibacterial compounds. In this paper, we focused on the essential mycolic acids transporter MmpL3, which is a well-characterized target of several antimycobacterial agents, to identify new compounds active against Mycobacterium abscessus (Mab). From the crystal structure of MmpL3 in complex with known inhibitors, through an in silico approach, we developed a pharmacophore that was used as a three-dimensional filter to identify new putative MmpL3 ligands within databases of known drugs. Among the prioritized compounds, mefloquine showed appreciable activity against Mab (MIC = 16 µg/mL). The compound was confirmed to interfere with mycolic acids biosynthesis, and proved to also be active against other NTMs, including drug-resistant clinical isolates. Importantly, mefloquine is a well-known antimalarial agent, opening the possibility of repurposing an already approved drug, which is a useful strategy to reduce the time and cost of disclosing novel drug candidates.

First triclosan-based macrocyclic inhibitors of InhA enzyme.

Two macrocyclic derivatives based on the triclosan frame were designed and synthesized as inhibitors of Mycobacterium tuberculosis InhA enzyme. One of the two molecules M02 displayed promising inhibitory activity against InhA enzyme with an IC50 of 4.7 µM. Molecular docking studies of these two compounds were performed and confirmed that M02 was more efficient as inhibitor of InhA activity. These molecules are the first macrocyclic direct inhibitors of InhA enzyme able to bind into the substrate pocket. Furthermore, these biaryl ether compounds exhibited antitubercular activities comparable to that of triclosan against M. tuberculosis H37Rv strain.

Mycobacterium abscessus, an Emerging and Worrisome Pathogen among Cystic Fibrosis Patients.

Nontuberculous mycobacteria (NTM) have recently emerged as important pathogens among cystic fibrosis (CF) patients worldwide. Mycobacterium abscessus is becoming the most worrisome NTM in this cohort of patients and recent findings clarified why this pathogen is so prone to this disease. M. abscessus drug therapy takes up to 2 years and its failure causes an accelerated lung function decline. The M. abscessus colonization of lung alveoli begins with smooth strains producing glycopeptidolipids and biofilm, whilst in the invasive infection, "rough" mutants are responsible for the production of trehalose dimycolate, and consequently, cording formation. Human-to-human M. abscessus transmission was demonstrated among geographically separated CF patients by whole-genome sequencing of clinical isolates worldwide. Using a M. abscessus infected CF zebrafish model, it was demonstrated that CFTR (cystic fibrosis transmembrane conductance regulator) dysfunction seems to have a specific role in the immune control of M. abscessus infections only. This pathogen is also intrinsically resistant to many drugs, thanks to its physiology and to the acquisition of new mechanisms of drug resistance. Few new compounds or drug formulations active against M. abscessus are present in preclinical and clinical development, but recently alternative strategies have been investigated, such as phage therapy and the use of ß-lactamase inhibitors.

Mechanochemical Synthesis and Biological Evaluation of Novel Isoniazid Derivatives with Potent Antitubercular Activity.

A series of isoniazid derivatives bearing a phenolic or heteroaromatic coupled frame were obtained by mechanochemical means. Their pH stability and their structural (conformer/isomer) analysis were checked. The activity of prepared derivatives against Mycobacterium tuberculosis cell growth was evaluated. Some compounds such as phenolic hydrazine 1a and almost all heteroaromatic ones, especially 2, 5 and 7, are more active than isoniazid, and their activity against some M. tuberculosis MDR clinical isolates was determined. Compounds 1a and 7 present a selectivity index >1400 evaluated on MRC5 human fibroblast cells. The mechanism of action of selected hydrazones was demonstrated to block mycolic acid synthesis due to InhA inhibition inside the mycobacterial cell.

The Redox State Regulates the Conformation of Rv2466c to Activate the Antitubercular Prodrug TP053.

Rv2466c is a key oxidoreductase that mediates the reductive activation of TP053, a thienopyrimidine derivative that kills replicating and non-replicating Mycobacterium tuberculosis, but whose mode of action remains enigmatic. Rv2466c is a homodimer in which each subunit displays a modular architecture comprising a canonical thioredoxin-fold with a Cys(19)-Pro(20)-Trp(21)-Cys(22) motif, and an insertion consisting of a four α-helical bundle and a short α-helical hairpin. Strong evidence is provided for dramatic conformational changes during the Rv2466c redox cycle, which are essential for TP053 activity. Strikingly, a new crystal structure of the reduced form of Rv2466c revealed the binding of a C-terminal extension in α-helical conformation to a pocket next to the active site cysteine pair at the interface between the thioredoxin domain and the helical insertion domain. The ab initio low-resolution envelopes obtained from small angle x-ray scattering showed that the fully reduced form of Rv2466c adopts a "closed" compact conformation in solution, similar to that observed in the crystal structure. In contrast, the oxidized form of Rv2466c displays an "open" conformation, where tertiary structural changes in the α-helical subdomain suffice to account for the observed conformational transitions. Altogether our structural, biochemical, and biophysical data strongly support a model in which the formation of the catalytic disulfide bond upon TP053 reduction triggers local structural changes that open the substrate binding site of Rv2466c allowing the release of the activated, reduced form of TP053. Our studies suggest that similar structural changes might have a functional role in other members of the thioredoxin-fold superfamily.

Differential roles of RND efflux pumps in antimicrobial drug resistance of sessile and planktonic Burkholderia cenocepacia cells.

Burkholderia cenocepacia is notorious for causing respiratory tract infections in people with cystic fibrosis. Infections with this organism are particularly difficult to treat due to its high level of intrinsic resistance to most antibiotics. Multidrug resistance in B. cenocepacia can be ascribed to different mechanisms, including the activity of efflux pumps and biofilm formation. In the present study, the effects of deletion of the 16 operons encoding resistance-nodulation-cell division (RND)-type efflux pumps in B. cenocepacia strain J2315 were investigated by determining the MICs of various antibiotics and by investigating the antibiofilm effect of these antibiotics. Finally, the expression levels of selected RND genes in treated and untreated cultures were investigated using reverse transcriptase quantitative PCR (RT-qPCR). Our data indicate that the RND-3 and RND-4 efflux pumps are important for resistance to various antimicrobial drugs (including tobramycin and ciprofloxacin) in planktonic B. cenocepacia J2315 populations, while the RND-3, RND-8, and RND-9 efflux systems protect biofilm-grown cells against tobramycin. The RND-8 and RND-9 efflux pumps are not involved in ciprofloxacin resistance. Results from the RT-qPCR experiments on the wild-type strain B. cenocepacia J2315 suggest that there is little regulation at the level of mRNA expression for these efflux pumps under the conditions tested.

The novel drug candidate VOMG kills Mycobacterium abscessus and other pathogens by inhibiting cell division.

AIMS: The incidence of lung infections is increasing worldwide in individuals suffering from cystic fibrosis and chronic obstructive pulmonary disease. Mycobacterium abscessus is associated with chronic lung deterioration in these populations. The intrinsic resistance of M. abscessus to most conventional antibiotics jeopardizes treatment success rates. To date, no single drug has been developed targeting M. abscessus specifically. The objective of this study was to characterize VOMG, a pyrithione-core drug-like small molecule, as a new compound active against M. abscessus and other pathogens. METHODS: A multi-disciplinary approach including microbiological, chemical, biochemical and transcriptomics procedures was used to validate VOMG as a promising anti-M. abscessus drug candidate. RESULTS: To the authors' knowledge, this is the first study to report the in-vitro and in-vivo bactericidal activity of VOMG against M. abscessus and other pathogens. Besides being active against M. abscessus biofilm, the compound showed a favourable pharmacological (ADME-Tox) profile. Frequency of resistance studies were unable to isolate resistant mutants. VOMG inhibits cell division, particularly the FtsZ enzyme. CONCLUSIONS: VOMG is a new drug-like molecule active against M. abscessus, inhibiting cell division with broad-spectrum activity against other microbial pathogens.

The DprE1 enzyme, one of the most vulnerable targets of Mycobacterium tuberculosis.

The re-emergence of tuberculosis in recent years led the World Health Organization (WHO) to launch the Stop TB Strategy program. Beside repurposing the existing drugs and exploring novel molecular combinations, an essential step to face the burden of tuberculosis will be to develop new drugs by identifying vulnerable bacterial targets. Recent studies have focused on decaprenylphosphoryl-D-ribose oxidase (DprE1) of Mycobacterium tuberculosis, an essential enzyme involved in cell wall metabolism, for which new promising molecules have proved efficacy as antitubercular agents. This review summarizes the state of the art concerning DprE1 in terms of structure, enzymatic activity and inhibitors. This enzyme is emerging as one of the most vulnerable target in M. tuberculosis.

Expanding the knowledge around antitubercular 5-(2-aminothiazol-4-yl)isoxazole-3-carboxamides: Hit-to-lead optimization and release of a novel antitubercular chemotype via scaffold derivatization.

Tuberculosis is one of the deadliest infectious diseases in the world, and the increased number of multidrug-resistant and extensively drug-resistant strains is a reason for concern. We have previously reported a series of substituted 5-(2-aminothiazol-4-yl)isoxazole-3-carboxamides with growth inhibitory activity against Mycobacterium tuberculosis strains and low propensity to be substrate of efflux pumps. Encouraged by these preliminary results, we have undertaken a medicinal chemistry campaign to determine the metabolic fate of these compounds and to delineate a reliable body of Structure-Activity Relationships. Keeping intact the (thiazol-4-yl)isoxazole-3-carboxamide core, as it is deemed to be the pharmacophore of the molecule, we have extensively explored the structural modifications able to confer good activity and avoid rapid clearance. Also, a small set of analogues based on isostere manipulation of the 2-aminothiazole were prepared and tested, with the aim to disclose novel antitubercular chemotypes. These studies, combined, were instrumental in designing improved compounds such as 42g and 42l, escaping metabolic degradation by human liver microsomes and, at the same time, maintaining good antitubercular activity against both drug-susceptible and drug-resistant strains.

CanB, a Druggable Cellular Target in Mycobacterium tuberculosis.

Treatment against tuberculosis can lead to the selection of drug-resistant Mycobacterium tuberculosis strains. To tackle this serious threat, new targets from M. tuberculosis are needed to develop novel effective drugs. In this work, we aimed to provide a possible workflow to validate new targets and inhibitors by combining genetic, in silico, and enzymological approaches. CanB is one of the three M. tuberculosis ß-carbonic anhydrases that catalyze the reversible reaction of CO2 hydration to form HCO3- and H+. To this end, we precisely demonstrated that CanB is essential for the survival of the pathogen in vitro by constructing conditional mutants. In addition, to search for CanB inhibitors, conditional canB mutants were also constructed using the Pip-ON system. By molecular docking and minimum inhibitory concentration assays, we selected three molecules that inhibit the growth in vitro of M. tuberculosis wild-type strain and canB conditional mutants, thus implementing a target-to-drug approach. The lead compound also showed a bactericidal activity by the time-killing assay. We further studied the interactions of these molecules with CanB using enzymatic assays and differential scanning fluorimetry thermal shift analysis. In conclusion, the compounds identified by the in silico screening proved to have a high affinity as CanB ligands endowed with antitubercular activity.

Implementing best practices on data generation and reporting of Mycobacterium tuberculosis in vitro assays within the ERA4TB consortium.

Tuberculosis (TB) is the historical leading cause of death by a single infectious agent. The European Regimen Accelerator for Tuberculosis (ERA4TB) is a public-private partnership of 30+ institutions with the objective to progress new anti-TB regimens into the clinic. Thus, robust and replicable results across independent laboratories are essential for reliable interpretation of treatment efficacy. A standardization workgroup unified in vitro protocols and data reporting templates. Time-kill assays provide essential input data for pharmacometric model-informed translation of single agents and regimens activity from in vitro to in vivo and the clinic. Five conditions were assessed by time-kill assays in six independent laboratories using four bacterial plating methods. Baseline bacterial burden varied between laboratories but variability was limited in net drug effect, confirming 2.5 µL equally robust as 100 µL plating. This exercise establishes the foundations of collaborative data generation, reporting, and integration within the overarching Antimicrobial Resistance Accelerator program.

MmpL3 is the cellular target of the antitubercular pyrrole derivative BM212.

The 1,5-diarylpyrrole derivative BM212 was previously shown to be active against multidrug-resistant clinical isolates and Mycobacterium tuberculosis residing within macrophages as well as against Mycobacterium avium and other atypical mycobacteria. To determine its mechanism of action, we identified the cellular target. Spontaneous Mycobacterium smegmatis, Mycobacterium bovis BCG, and M. tuberculosis H37Rv mutants that were resistant to BM212 were isolated. By the screening of genomic libraries and by whole-genome sequencing, we found that all the characterized mutants showed mutations in the mmpL3 gene, allowing us to conclude that resistance to BM212 maps to the MmpL3 protein, a member of the MmpL (mycobacterial membrane protein, large) family. Susceptibility was unaffected by the efflux pump inhibitors reserpine, carbonylcyanide m-chlorophenylhydrazone, and verapamil. Uptake/efflux experiments with [(14)C]BM212 demonstrated that resistance is not driven by the efflux of BM212. Together, these data strongly suggest that the MmpL3 protein is the cellular target of BM212.

Antituberculars which target decaprenylphosphoryl-ß-D-ribofuranose 2'-oxidase DprE1: state of art.

Multidrug resistance is a major barrier in the battle against tuberculosis and still a leading cause of death worldwide. In order to fight this pathogen, two routes are practicable: vaccination or drug treatment. Vaccination against Mycobacterium tuberculosis with the current vaccine Mycobacterium bovis Bacillus Calmette-Guerin is partially successful, being its efficacy variable. A few new tuberculosis vaccines are now in various phases of clinical trials. The emergence of multidrug-resistant strains of M. tuberculosis gave the impulse to discover new effective antitubercular drugs, a few of which are in clinical development. Here we focus on three different classes of very promising antitubercular drugs recently discovered (benzothiazinones, dinitrobenzamides, and benzoquinoxalines) that share the same cellular target: a subunit of the heteromeric decaprenylphosphoryl-ß-D: -ribose 2'-epimerase, encoded by the dprE1 (or Rv3790) gene. This enzyme is involved in the biosynthesis of D: -arabinose which is crucial for the synthesis of the mycobacterial cell wall and essential for the pathogen's survival.

A New Benzothiazolthiazolidine Derivative, 11726172, Is Active In Vitro, In Vivo, and against Nonreplicating Cells of Mycobacterium tuberculosis.

Tuberculosis (TB) still poses a global menace as one of the deadliest infectious diseases. A quarter of the human population is indeed latently infected with Mycobacterium tuberculosis. People with latent infection have a 5 to 10% lifetime risk of becoming ill with TB, representing a reservoir for TB active infection. This is a worrisome problem to overcome in the case of relapse; unfortunately, few drugs are effective against nonreplicating M. tuberculosis cells. Novel strategies to combat TB, including its latent form, are urgently needed. In response to the lack of new effective drugs and after screening about 500 original chemical molecules, we selected a compound, 11726172, that is endowed with potent antitubercular activity against M. tuberculosis both in vitro and in vivo and importantly also against dormant nonculturable bacilli. We also investigated the mechanism of action of 11726172 by applying a multidisciplinary approach, including transcriptomic, labeled metabolomic, biochemical, and microbiological procedures. Our results represent an important step forward in the development of a new antitubercular compound with a novel mechanism of action active against latent bacilli. IMPORTANCE The discontinuation of TB services due to COVID-19 causes concern about a future resurgence of TB, also considering that latent infection affects a high number of people worldwide. To combat this situation, the identification of antitubercular compounds targeting Mycobacterium tuberculosis through novel mechanisms of action is necessary. These compounds should be active against not only replicating bacteria cells but also nonreplicating cells to limit the reservoir of latently infected people on which the bacterium can rely to spread after reactivation.