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.

Distinct Effects of Moxifloxacin and Bedaquiline on Growing and 'Non-Culturable' Mycobacterium abscessus.

Mycobacterium abscessus has recently emerged as the cause of an increasing number of human infections worldwide. Unfortunately, it is highly resistant to existing drugs, and new specific agents to combat M. abscessus have not yet been found. The discovery of antibiotics that are effective not only against replicating but also against dormant and often recalcitrant cells is a daunting challenge. In this study, we developed a model of non-replicating M. abscessus, which represents a valuable screening tool for antibacterial agents. Thus, we demonstrated that, under a deficiency of potassium ions in the growth media and prolonged incubation, M. abscessus entered a 'non-culturable' state with a significant loss of colony-forming ability, but it retained viability, as confirmed using the most-probable-number (MPN) assay. The 'non-culturable' mycobacteria possessed decelerated cellular metabolism and noticeable differences in cell morphology from actively growing mycobacteria. 'Non-culturable' cells were used in a comprehensive screening of the efficacy of antibiotics, along with actively growing cells. Both CFU and MPN tests confirmed the prominent bactericidal effect of moxifloxacin on actively growing and 'non-culturable' M. abscessus, as proven by less than 0.01% of cells surviving after antibiotic treatment and prolonged storage. Bedaquiline exhibited a comparable bactericidal effect only on metabolically inactive non-culturable cells aged for 44 days. There were reductions ranging from 1000 to 10,000-fold in CFU and MPN, but it was not so efficient with respect to active cells, resulting in a bacteriostatic effect. The demonstrated specificity of bedaquiline in relation to inert non-replicating M. abscessus offers a new and unexpected result. Based on the findings of this research, moxifloxacin and bedaquiline can be regarded as potential treatments for infections caused by M. abscessus. In addition, a key outcome is the proposal to include the combination of viability assays for comprehensive testing of drug candidates. Relying on CFU-based assays alone resulted in overestimates of antibacterial efficacy, as demonstrated in our experiments.

Diaphragm Fatigue in SMNΔ7 Mice and Its Molecular Determinants: An Underestimated Issue.

Spinal muscular atrophy (SMA) is a genetic disorder characterized by the loss of spinal motor neurons leading to muscle weakness and respiratory failure. Mitochondrial dysfunctions are found in the skeletal muscle of patients with SMA. For obvious ethical reasons, the diaphragm muscle is poorly studied, notwithstanding the very important role that respiratory involvement plays in SMA mortality. The main goal of this study was to investigate diaphragm functionality and the underlying molecular adaptations in SMNΔ7 mice, a mouse model that exhibits symptoms similar to that of patients with intermediate type II SMA. Functional, biochemical, and molecular analyses on isolated diaphragm were performed. The obtained results suggest the presence of an intrinsic energetic imbalance associated with mitochondrial dysfunction and a significant accumulation of reactive oxygen species (ROS). In turn, ROS accumulation can affect muscle fatigue, cause diaphragm wasting, and, in the long run, respiratory failure in SMNΔ7 mice. Exposure to the antioxidant molecule ergothioneine leads to the functional recovery of the diaphragm, confirming the presence of mitochondrial impairment and redox imbalance. These findings suggest the possibility of carrying out a dietary supplementation in SMNΔ7 mice to preserve their diaphragm function and increase their lifespan.

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.

Mechanochemical Studies on Coupling of Hydrazines and Hydrazine Amides with Phenolic and Furanyl Aldehydes-Hydrazones with Antileishmanial and Antibacterial Activities.

Hydrazone compounds represent an important area of research that includes, among others, synthetic approaches and biological studies. A series of 17 hydrazones have been synthesized by mechanochemical means. The fragments chosen were phenolic and furanyl aldehydes coupled with 12 heterocyclic hydrazines or hydrazinamides. All compounds can be obtained quantitatively when operating on a planetary ball mill and a maximum reaction time of 180 min (6 cycles of 30 min each). Complete spectroscopic analyses of hydrazones revealed eight compounds (3-5, 8-11, 16) present in one geometric form, six compounds (1, 2, 13-15) present in two isomeric forms, and three compounds (6, 7, 12) where one rotation is restricted giving rise to two different forms. The single crystal X-ray structure of one of the hydrazones bearing the isoniazid fragment (8) indicates a crystal lattice consisting of two symmetry-independent molecules with different geometries. All compounds obtained were tested for anti-infectious and antibacterial activities. Four compounds (1, 3, 5 and 8) showed good activity against Mycobacterium tuberculosis, and one (7) was very potent against Staphylococcus aureus. Most interesting, this series of compounds displayed very promising antileishmanial activity. Among all, compound 9 exhibited an IC50 value of 0.3 µM on the Leishmania donovani intramacrophage amastigote in vitro model and a good selectivity index, better than miltefosine, making it worth evaluating in vivo.

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.

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.

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.

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.

Age-related decline in murine heart and skeletal muscle performance is attenuated by reduced Ahnak1 expression.

BACKGROUND: Aging is associated with a progressive reduction in cellular function leading to poor health and loss of physical performance. Mitochondrial dysfunction is one of the hallmarks of aging; hence, interventions targeting mitochondrial dysfunction have the potential to provide preventive and therapeutic benefits to elderly individuals. Meta-analyses of age-related gene expression profiles showed that the expression of Ahnak1, a protein regulating several signal-transduction pathways including metabolic homeostasis, is increased with age, which is associated with low VO2MAX and poor muscle fitness. However, the role of Ahnak1 in the aging process remained unknown. Here, we investigated the age-related role of Ahnak1 in murine exercise capacity, mitochondrial function, and contractile function of cardiac and skeletal muscles. METHODS: We employed 15- to 16-month-old female and male Ahnak1-knockout (Ahnak1-KO) and wild-type (WT) mice and performed morphometric, biochemical, and bioenergetics assays to evaluate the effects of Ahnak1 on exercise capacity and mitochondrial morphology and function in cardiomyocytes and tibialis anterior (TA) muscle. A human left ventricular (LV) cardiomyocyte cell line (AC16) was used to investigate the direct role of Ahnak1 in cardiomyocytes. RESULTS: We found that the level of Ahnak1 protein is significantly up-regulated with age in the murine LV (1.9-fold) and TA (1.8-fold) tissues. The suppression of Ahnak1 was associated with improved exercise tolerance, as all aged adult Ahnak1-KO mice (100%) successfully completed the running programme, whereas approximately 31% male and 8% female WT mice could maintain the required running speed and distance. Transmission electron microscopic studies showed that LV and TA tissue specimens of aged adult Ahnak1-KO of both sexes have significantly more enlarged/elongated mitochondria and less small mitochondria compared with WT littermates (P < 0.01 and P < 0.001, respectively) at basal level. Further, we observed a shift in mitochondrial fission/fusion balance towards fusion in cardiomyocytes and TA muscle from aged adult Ahnak1-KO mice. The maximal and reserve respiratory capacities were significantly higher in cardiomyocytes from aged adult Ahnak1-KO mice compared with the WT counterparts (P < 0.05 and P < 0.01, respectively). Cardiomyocyte contractility and fatigue resistance of TA muscles were significantly increased in Ahnak1-KO mice of both sexes, compared with the WT groups. In vitro studies using AC16 cells have confirmed that the alteration of mitochondrial function is indeed a direct effect of Ahnak1. Finally, we presented Ahnak1 as a novel cardiac mitochondrial membrane-associated protein. CONCLUSIONS: Our data suggest that Ahnak1 is involved in age-related cardiac and skeletal muscle dysfunction and could therefore serve as a promising therapeutical target.

Rebalancing expression of HMGB1 redox isoforms to counteract muscular dystrophy.

Muscular dystrophies (MDs) are a group of genetic diseases characterized by progressive muscle wasting associated to oxidative stress and persistent inflammation. It is essential to deepen our knowledge on the mechanism connecting these two processes because current treatments for MDs have limited efficacy and/or are associated with side effects. Here, we identified the alarmin high-mobility group box 1 (HMGB1) as a functional link between oxidative stress and inflammation in MDs. The oxidation of HMGB1 cysteines switches its extracellular activities from the orchestration of tissue regeneration to the exacerbation of inflammation. Extracellular HMGB1 is present at high amount and undergoes oxidation in patients with MDs and in mouse models of Duchenne muscular dystrophy (DMD) and limb-girdle muscular dystrophy 3 (LGMDR3) compared to controls. Genetic ablation of HMGB1 in muscles of DMD mice leads to an amelioration of the dystrophic phenotype as evidenced by the reduced inflammation and muscle degeneration, indicating that HMGB1 oxidation is a detrimental process in MDs. Pharmacological treatment with an engineered nonoxidizable variant of HMGB1, called 3S, improves functional performance, muscle regeneration, and satellite cell engraftment in dystrophic mice while reducing inflammation and fibrosis. Overall, our data demonstrate that the balance between HMGB1 redox isoforms dictates whether skeletal muscle is in an inflamed or regenerating state, and that the nonoxidizable form of HMGB1 is a possible therapeutic approach to counteract the progression of the dystrophic phenotype. Rebalancing the HMGB1 redox isoforms may also be a therapeutic strategy for other disorders characterized by chronic oxidative stress and inflammation.

Reduction of Movement in Neurological Diseases: Effects on Neural Stem Cells Characteristics.

Both astronauts and patients affected by chronic movement-limiting pathologies face impairment in muscle and/or brain performance. Increased patient survival expectations and the expected longer stays in space by astronauts may result in prolonged motor deprivation and consequent pathological effects. Severe movement limitation can influence not only the motor and metabolic systems but also the nervous system, altering neurogenesis and the interaction between motoneurons and muscle cells. Little information is yet available about the effect of prolonged muscle disuse on neural stem cells characteristics. Our in vitro study aims to fill this gap by focusing on the biological and molecular properties of neural stem cells (NSCs). Our analysis shows that NSCs derived from the SVZ of HU mice had shown a reduced proliferation capability and an altered cell cycle. Furthermore, NSCs obtained from HU animals present an incomplete differentiation/maturation. The overall results support the existence of a link between reduction of exercise and muscle disuse and metabolism in the brain and thus represent valuable new information that could clarify how circumstances such as the absence of load and the lack of movement that occurs in people with some neurological diseases, may affect the properties of NSCs and contribute to the negative manifestations of these conditions.