Cyclic AMP-Mediated Inhibition of Cholesterol Catabolism in Mycobacterium tuberculosis by the Novel Drug Candidate GSK2556286.

Despite the deployment of combination tuberculosis (TB) chemotherapy, efforts to identify shorter, nonrelapsing treatments have resulted in limited success. Recent evidence indicates that GSK2556286 (GSK286), which acts via Rv1625c, a membrane-bound adenylyl cyclase in Mycobacterium tuberculosis, shortens treatment in rodents relative to standard of care drugs. Moreover, GSK286 can replace linezolid in the three-drug, Nix-TB regimen. Given its therapeutic potential, we sought to better understand the mechanism of action of GSK286. The compound blocked growth of M. tuberculosis in cholesterol media and increased intracellular cAMP levels ~50-fold. GSK286 did not inhibit growth of an rv1625c transposon mutant in cholesterol media and did not induce cyclic AMP (cAMP) production in this mutant, suggesting that the compound acts on this adenylyl cyclase. GSK286 also induced cAMP production in Rhodococcus jostii RHA1, a cholesterol-catabolizing actinobacterium, when Rv1625c was heterologously expressed. However, these elevated levels of cAMP did not inhibit growth of R. jostii RHA1 in cholesterol medium. Mutations in rv1625c conferred cross-resistance to GSK286 and the known Rv1625c agonist, mCLB073. Metabolic profiling of M. tuberculosis cells revealed that elevated cAMP levels, induced using either an agonist or a genetic tool, did not significantly affect pools of steroid metabolites in cholesterol-incubated cells. Finally, the inhibitory effect of agonists was not dependent on the N-acetyltransferase MtPat. Together, these data establish that GSK286 is an Rv1625c agonist and sheds light on how cAMP signaling can be manipulated as a novel antibiotic strategy to shorten TB treatments. Nevertheless, the detailed mechanism of action of these compounds remains to be elucidated.

GSK2556286 Is a Novel Antitubercular Drug Candidate Effective In Vivo with the Potential To Shorten Tuberculosis Treatment.

As a result of a high-throughput compound screening campaign using Mycobacterium tuberculosis-infected macrophages, a new drug candidate for the treatment of tuberculosis has been identified. GSK2556286 inhibits growth within human macrophages (50% inhibitory concentration [IC50] = 0.07 µM), is active against extracellular bacteria in cholesterol-containing culture medium, and exhibits no cross-resistance with known antitubercular drugs. In addition, it has shown efficacy in different mouse models of tuberculosis (TB) and has an adequate safety profile in two preclinical species. These features indicate a compound with a novel mode of action, although still not fully defined, that is effective against both multidrug-resistant (MDR) or extensively drug-resistant (XDR) and drug-sensitive (DS) M. tuberculosis with the potential to shorten the duration of treatment in novel combination drug regimens. (This study has been registered at ClinicalTrials.gov under identifier NCT04472897).

Discovery of Novel Oral Protein Synthesis Inhibitors of Mycobacterium tuberculosis That Target Leucyl-tRNA Synthetase.

The recent development and spread of extensively drug-resistant and totally drug-resistant resistant (TDR) strains of Mycobacterium tuberculosis highlight the need for new antitubercular drugs. Protein synthesis inhibitors have played an important role in the treatment of tuberculosis (TB) starting with the inclusion of streptomycin in the first combination therapies. Although parenteral aminoglycosides are a key component of therapy for multidrug-resistant TB, the oxazolidinone linezolid is the only orally available protein synthesis inhibitor that is effective against TB. Here, we show that small-molecule inhibitors of aminoacyl-tRNA synthetases (AARSs), which are known to be excellent antibacterial protein synthesis targets, are orally bioavailable and effective against M. tuberculosis in TB mouse infection models. We applied the oxaborole tRNA-trapping (OBORT) mechanism, which was first developed to target fungal cytoplasmic leucyl-tRNA synthetase (LeuRS), to M. tuberculosis LeuRS. X-ray crystallography was used to guide the design of LeuRS inhibitors that have good biochemical potency and excellent whole-cell activity against M. tuberculosis Importantly, their good oral bioavailability translates into in vivo efficacy in both the acute and chronic mouse models of TB with potency comparable to that of the frontline drug isoniazid.

Rapid cytolysis of Mycobacterium tuberculosis by faropenem, an orally bioavailable ß-lactam antibiotic.

Recent clinical studies indicate that meropenem, a ß-lactam antibiotic, is a promising candidate for therapy of drug-resistant tuberculosis. However, meropenem is chemically unstable, requires frequent intravenous injection, and must be combined with a ß-lactamase inhibitor (clavulanate) for optimal activity. Here, we report that faropenem, a stable and orally bioavailable ß-lactam, efficiently kills Mycobacterium tuberculosis even in the absence of clavulanate. The target enzymes, L,D-transpeptidases, were inactivated 6- to 22-fold more efficiently by faropenem than by meropenem. Using a real-time assay based on quantitative time-lapse microscopy and microfluidics, we demonstrate the superiority of faropenem to the frontline antituberculosis drug isoniazid in its ability to induce the rapid cytolysis of single cells. Faropenem also showed superior activity against a cryptic subpopulation of nongrowing but metabolically active cells, which may correspond to the viable but nonculturable forms believed to be responsible for relapses following prolonged chemotherapy. These results identify faropenem to be a potential candidate for alternative therapy of drug-resistant tuberculosis.

Testing tuberculosis drug efficacy in a zebrafish high-throughput translational medicine screen.

The translational value of zebrafish high-throughput screens can be improved when more knowledge is available on uptake characteristics of potential drugs. We investigated reference antibiotics and 15 preclinical compounds in a translational zebrafish-rodent screening system for tuberculosis. As a major advance, we have developed a new tool for testing drug uptake in the zebrafish model. This is important, because despite the many applications of assessing drug efficacy in zebrafish research, the current methods for measuring uptake using mass spectrometry do not take into account the possible adherence of drugs to the larval surface. Our approach combines nanoliter sampling from the yolk using a microneedle, followed by mass spectrometric analysis. To date, no single physicochemical property has been identified to accurately predict compound uptake; our method offers a great possibility to monitor how any novel compound behaves within the system. We have correlated the uptake data with high-throughput drug-screening data from Mycobacterium marinum-infected zebrafish larvae. As a result, we present an improved zebrafish larva drug-screening platform which offers new insights into drug efficacy and identifies potential false negatives and drugs that are effective in zebrafish and rodents. We demonstrate that this improved zebrafish drug-screening platform can complement conventional models of in vivo Mycobacterium tuberculosis-infected rodent assays. The detailed comparison of two vertebrate systems, fish and rodent, may give more predictive value for efficacy of drugs in humans.

Hydrolysis of clavulanate by Mycobacterium tuberculosis ß-lactamase BlaC harboring a canonical SDN motif.

Combinations of ß-lactams with clavulanate are currently being investigated for tuberculosis treatment. Since Mycobacterium tuberculosis produces a broad spectrum ß-lactamase, BlaC, the success of this approach could be compromised by the emergence of clavulanate-resistant variants, as observed for inhibitor-resistant TEM variants in enterobacteria. Previous analyses based on site-directed mutagenesis of BlaC have led to the conclusion that this risk was limited. Here, we used a different approach based on determination of the crystal structure of ß-lactamase BlaMAb of Mycobacterium abscessus, which efficiently hydrolyzes clavulanate. Comparison of BlaMAb and BlaC allowed for structure-assisted site-directed mutagenesis of BlaC and identification of the G(132)N substitution that was sufficient to switch the interaction of BlaC with clavulanate from irreversible inactivation to efficient hydrolysis. The substitution, which restored the canonical SDN motif (SDG→SDN), allowed for efficient hydrolysis of clavulanate, with a more than 10(4)-fold increase in k cat (0.41 s(-1)), without affecting the hydrolysis of other ß-lactams. Mass spectrometry revealed that acylation of BlaC and of its G(132)N variant by clavulanate follows similar paths, involving sequential formation of two acylenzymes. Decarboxylation of the first acylenzyme results in a stable secondary acylenzyme in BlaC, whereas hydrolysis occurs in the G(132)N variant. The SDN/SDG polymorphism defines two mycobacterial lineages comprising rapidly and slowly growing species, respectively. Together, these results suggest that the efficacy of ß-lactam-clavulanate combinations may be limited by the emergence of resistance. ß-Lactams active without clavulanate, such as faropenem, should be prioritized for the development of new therapies.

Mycobacterium tuberculosis gyrase inhibitors as a new class of antitubercular drugs.

One way to speed up the TB drug discovery process is to search for antitubercular activity among compound series that already possess some of the key properties needed in anti-infective drug discovery, such as whole-cell activity and oral absorption. Here, we present MGIs, a new series of Mycobacterium tuberculosis gyrase inhibitors, which stem from the long-term efforts GSK has dedicated to the discovery and development of novel bacterial topoisomerase inhibitors (NBTIs). The compounds identified were found to be devoid of fluoroquinolone (FQ) cross-resistance and seem to operate through a mechanism similar to that of the previously described NBTI GSK antibacterial drug candidate. The remarkable in vitro and in vivo antitubercular profiles showed by the hits has prompted us to further advance the MGI project to full lead optimization.

Determinants of psychotherapists' attitudes to online psychotherapy.

Introduction: Online psychotherapy is a form of work that is becoming more and more popular. Public health problems, such as COVID-19, forced mental health professionals and patients to incorporate new methodologies such as the use of electronic media and internet to provide follow-up, treatment and also supervision. The aim of this study was to investigate which factors shape the therapists' attitudes toward online psychotherapy during a pandemic taking into account: (1) attitudes toward the COVID-19 pandemic (fear of contagion, pandemic fatigue, etc.), (2) personal characteristics of the psychotherapists (age, gender, feeling of efficacy, anxiety, depression, etc.), and (3) characteristics of the psychotherapeutic practice (guideline procedure, client age group, professional experience, etc). Materials and methods: Study participants were 177 psychotherapists from four European countries: Poland (n = 48), Germany (n = 44), Sweden (n = 49), and Portugal (n = 36). Data were collected by means of an individual online survey through the original questionnaire and the standardized scales: a modified version of the Attitudes toward Psychological Online Interventions Scale (APOI), Fear of Contagion by COVID-19 Scale (FCS COVID-19), Pandemic Fatigue Scale (PFS), Hospital Anxiety and Depression Scale (HADS), Social Support Questionnaire (F-SozU K-14), and the Sense of Efficiency Test (SET). Results: Determinants that impacted psychotherapists' attitudes toward online therapy were: COVID-19 belief in prevention-keeping distance and hand disinfection, pandemic behavioral fatigue, previous online therapy experience (including voice call), working with youth and adults. Our study showed that belief in the sense of prevention in the form of taking care of hand disinfection before the session, pandemic behavioral fatigue and experience in working with adults were significant predictors of negative attitudes of therapists toward online psychological interventions. On the other hand, belief in the sense of prevention in the form of keeping distance during the session had a positive effect on general attitudes toward therapy conducted via the internet. Discussion: The online therapy boom during the COVID-19 pandemic has spawned a powerful tool for psychotherapists. More research in this area and training of psychotherapists are needed for online psychological interventions to become an effective therapy format that is accepted by patients and therapists alike.

Redescription and DNA barcoding of diurnal moth Athroolopha latimargoRothschild, 1914 bona sp., stat. rev. from the southern Iberian Peninsula (Lepidoptera: Geometridae: Ennominae).

The rare and diurnal geometrid moth Athroolopha latimargo Rothschild, 1914 bona sp., stat. rev. is re-discovered and redescribed from the furthest point of the south of the Iberian Peninsula, for the first time since its original description as a subspecies of Athroolopha chrysitaria (Hübner, 1813) from North Africa. The range of this taxon is questioned. A mitochondrial COI barcode sequence was generated for the specimens and compared with Iberian and Sicilian Athroolopha species.

Obstructive sleep apnea and dyslipidemia: from animal models to clinical evidence.

Lipid metabolism deregulation constitutes the pathogenic basis for the development of atherosclerosis and justifies a high incidence of cardiovascular-related morbidity and mortality. Some data suggest that dyslipidemia may be associated with sleep-disordered breathing, mainly obstructive sleep apnea (OSA), due to alterations in fundamental biochemical processes, such as intermittent hypoxia (IH). The aim of this systematic review was to identify and critically evaluate the current evidence supporting the existence of a possible relationship between OSA and alterations in lipid metabolism. Much evidence shows that, during the fasting state, OSA and IH increase lipid delivery from the adipose tissue to the liver through an up-regulation of the sterol regulatory element-binding protein-1 and stearoyl-CoA desaturase-1, increasing the synthesis of cholesterol esters and triglycerides. In the postprandial state, lipoprotein clearance is delayed due to lower lipoprotein lipase activity, probably secondary to IH-up-regulation of angiopoietin-like protein 4 and decreased activity of the peroxisome proliferator-activated receptor alpha. Moreover, oxidative stress can generate dysfunctional oxidized lipids and reduce the capacity of high-density lipoproteins (HDL) to prevent low-density lipoprotein (LDL) oxidation. In the clinical field, several observational studies and a meta-regression analysis support the existence of a link between OSA and dyslipidemia. Although there is evidence of improved lipid profile after apnea-hypopnea suppression with continuous positive airway pressure (CPAP), the majority of the data come from observational studies. In contrast, randomized controlled trials evaluating the effects of CPAP on lipid metabolism present inconclusive results and two meta-analyses provide contradictory evidence.

Bio-behavioral survey of syphilis in homeless men in Central Brazil: a cross-sectional study.

The objective of this study was to investigate the prevalence and factors associated with syphilis in homeless men in Central Brazil. It is a cross-sectional study conducted with 481 individuals attending four therapeutic communities between October and December 2015. A structured interview was conducted to collect sociodemographic data and risk factors for syphilis. Rapid/point-of-care and VDRL tests were performed to determine exposure to syphilis and the presence of active syphilis, respectively. Poisson regression analysis was used to verify the risk factors associated with the outcomes investigated. Of the study participants, 10.2% were reactive to the rapid test, and 5.4% had active syphilis. At the multiple regression analysis, schooling (adjusted prevalence ratio - APR: 0.89; p = 0.005), history of genital ulcer (APR: 2.59; p = 0.002), STI history (APR: 1.97; p = 0.042), and sexual intercourse under drug effects (APR: 1.60; p = 0.022) were independent factors associated with lifetime syphilis. Also, history of genital ulcer (APR: 2.19; p = 0.019), STI history (APR: 1.74; p = 0.033) and number of sexual partners in the last year (APR: 1.02; p = 0.044) were associated with active syphilis. The prevalence of syphilis among homeless men was rather high, confirming the vulnerability of this group to this infection. These results emphasize the need for educational intervention, improvement of risk reduction programs, availability of diagnostic tests, especially the rapid test, and treatment.

HIV infection, high-risk behaviors and substance use in homeless men sheltered in therapeutic communities in Central Brazil.

Homeless men present high vulnerability to HIV infection, mainly due to sexual risk behaviors and substance use. The objective was to estimate the prevalence of HIV infection, risk behaviors and substance use in homeless men. A cross-sectional study was conducted in 481 homeless men recruited in four therapeutic communities in the Goiás State, Central Brazil. All were interviewed about sociodemographic characteristics, substance use, and risk behaviors. Furthermore, all were tested for HIV. Poisson regression was used to verify factors associated with HIV infection. HIV prevalence was 1.24% (95.0% CI: 0.57 to 2.69%). Previous HIV testing (adjusted prevalence ratio [APR]: 10.0; 95.0% CI: 1.86-55.8) and years of education (APR: 0.76; 95.0% CI: 0.60-0.97) were factors associated with HIV infection. Participants had high rates of hazardous alcohol use and illicit drug use. The prevalence of HIV infection among homeless men was higher than that found in the Brazilian male population and we identified a high rate of risk behaviors for HIV among the homeless men investigated. Thus, it is necessary to expand HIV prevention measures in Brazil, such as health education, condom availability, regular HIV testing and increased testing coverage in this population, and treatment for alcohol and/or illicit drug dependence/abuse.

Identification and Profiling of Hydantoins-A Novel Class of Potent Antimycobacterial DprE1 Inhibitors.

Tuberculosis is the leading cause of death worldwide from infectious diseases. With the development of drug-resistant strains of Mycobacterium tuberculosis, there is an acute need for new medicines with novel modes of action. Herein, we report the discovery and profiling of a novel hydantoin-based family of antimycobacterial inhibitors of the decaprenylphospho-ß-d-ribofuranose 2-oxidase (DprE1). In this study, we have prepared a library of more than a 100 compounds and evaluated them for their biological and physicochemical properties. The series is characterized by high enzymatic and whole-cell activity, low cytotoxicity, and a good overall physicochemical profile. In addition, we show that the series acts via reversible inhibition of the DprE1 enzyme. Overall, the novel compound family forms an attractive base for progression to further stages of optimization and may provide a promising drug candidate in the future.

Screening of a Novel Fragment Library with Functional Complexity against Mycobacterium tuberculosis InhA.

Our findings reported herein provide support for the benefits of including functional group complexity (FGC) within fragments when screening against protein targets such as Mycobacterium tuberculosis InhA. We show that InhA fragment actives with FGC maintained their binding pose during elaboration. Furthermore, weak fragment hits with functional group handles also allowed for facile fragment elaboration to afford novel and potent InhA inhibitors with good ligand efficiency metrics for optimization.

Target Identification of Mycobacterium tuberculosis Phenotypic Hits Using a Concerted Chemogenomic, Biophysical, and Structural Approach.

Mycobacterium phenotypic hits are a good reservoir for new chemotypes for the treatment of tuberculosis. However, the absence of defined molecular targets and modes of action could lead to failure in drug development. Therefore, a combination of ligand-based and structure-based chemogenomic approaches followed by biophysical and biochemical validation have been used to identify targets for Mycobacterium tuberculosis phenotypic hits. Our approach identified EthR and InhA as targets for several hits, with some showing dual activity against these proteins. From the 35 predicted EthR inhibitors, eight exhibited an IC50 below 50 µM against M. tuberculosis EthR and three were confirmed to be also simultaneously active against InhA. Further hit validation was performed using X-ray crystallography yielding eight new crystal structures of EthR inhibitors. Although the EthR inhibitors attain their activity against M. tuberculosis by hitting yet undefined targets, these results provide new lead compounds that could be further developed to be used to potentiate the effect of EthA activated pro-drugs, such as ethionamide, thus enhancing their bactericidal effect.

Inhibiting mycobacterial tryptophan synthase by targeting the inter-subunit interface.

Drug discovery efforts against the pathogen Mycobacterium tuberculosis (Mtb) have been advanced through phenotypic screens of extensive compound libraries. Such a screen revealed sulfolane 1 and indoline-5-sulfonamides 2 and 3 as potent inhibitors of mycobacterial growth. Optimization in the sulfolane series led to compound 4, which has proven activity in an in vivo murine model of Mtb infection. Here we identify the target and mode of inhibition of these compounds based on whole genome sequencing of spontaneous resistant mutants, which identified mutations locating to the essential α- and ß-subunits of tryptophan synthase. Over-expression studies confirmed tryptophan synthase as the biological target. Biochemical techniques probed the mechanism of inhibition, revealing the mutant enzyme complex incurs a fitness cost but does not prevent inhibitor binding. Mapping of the resistance conferring mutations onto a low-resolution crystal structure of Mtb tryptophan synthase showed they locate to the interface between the α- and ß-subunits. The discovery of anti-tubercular agents inhibiting tryptophan synthase highlights the therapeutic potential of this enzyme and draws attention to the prospect of other amino acid biosynthetic pathways as future Mtb drug targets.

A Phenotypic Based Target Screening Approach Delivers New Antitubercular CTP Synthetase Inhibitors.

Despite its great potential, the target-based approach has been mostly unsuccessful in tuberculosis drug discovery, while whole cell phenotypic screening has delivered several active compounds. However, for many of these hits, the cellular target has not yet been identified, thus preventing further target-based optimization of the compounds. In this context, the newly validated drug target CTP synthetase PyrG was exploited to assess a target-based approach of already known, but untargeted, antimycobacterial compounds. To this purpose the publically available GlaxoSmithKline antimycobacterial compound set was assayed, uncovering a series of 4-(pyridin-2-yl)thiazole derivatives which efficiently inhibit the Mycobacterium tuberculosis PyrG enzyme activity, one of them showing low activity against the human CTP synthetase. The three best compounds were ATP binding site competitive inhibitors, with Ki values ranging from 3 to 20 µM, but did not show any activity against a small panel of different prokaryotic and eukaryotic kinases, thus demonstrating specificity for the CTP synthetases. Metabolic labeling experiments demonstrated that the compounds directly interfere not only with CTP biosynthesis, but also with other CTP dependent biochemical pathways, such as lipid biosynthesis. Moreover, using a M. tuberculosis pyrG conditional knock-down strain, it was shown that the activity of two compounds is dependent on the intracellular concentration of the CTP synthetase. All these results strongly suggest a role of PyrG as a target of these compounds, thus strengthening the value of this kind of approach for the identification of new scaffolds for drug development.

Discovery of a Potent and Specific M. tuberculosis Leucyl-tRNA Synthetase Inhibitor: (S)-3-(Aminomethyl)-4-chloro-7-(2-hydroxyethoxy)benzo[c][1,2]oxaborol-1(3H)-ol (GSK656).

There is an urgent need to develop new and safer antitubercular agents that possess a novel mode of action. We synthesized and evaluated a novel series of 3-aminomethyl 4-halogen benzoxaboroles as Mycobacterium tuberculosis (Mtb) leucyl-tRNA synthetase (LeuRS) inhibitors. A number of Mtb LeuRS inhibitors were identified that demonstrated good antitubercular activity with high selectivity over human mitochondrial and cytoplasmic LeuRS. Further evaluation of these Mtb LeuRS inhibitors by in vivo pharmacokinetics (PK) and murine tuberculosis (TB) efficacy models led to the discovery of GSK3036656 (abbreviated as GSK656). This molecule shows potent inhibition of Mtb LeuRS (IC50 = 0.20 µM) and in vitro antitubercular activity (Mtb H37Rv MIC = 0.08 µM). Additionally, it is highly selective for the Mtb LeuRS enzyme with IC50 of >300 µM and 132 µM for human mitochondrial LeuRS and human cytoplasmic LeuRS, respectively. In addition, it exhibits remarkable PK profiles and efficacy against Mtb in mouse TB infection models with superior tolerability over initial leads. This compound has been progressed to clinical development for the treatment of tuberculosis.

Repurposing clinically approved cephalosporins for tuberculosis therapy.

While modern cephalosporins developed for broad spectrum antibacterial activities have never been pursued for tuberculosis (TB) therapy, we identified first generation cephalosporins having clinically relevant inhibitory concentrations, both alone and in synergistic drug combinations. Common chemical patterns required for activity against Mycobacterium tuberculosis were identified using structure-activity relationships (SAR) studies. Numerous cephalosporins were synergistic with rifampicin, the cornerstone drug for TB therapy, and ethambutol, a first-line anti-TB drug. Synergy was observed even under intracellular growth conditions where beta-lactams typically have limited activities. Cephalosporins and rifampicin were 4- to 64-fold more active in combination than either drug alone; however, limited synergy was observed with rifapentine or rifabutin. Clavulanate was a key synergistic partner in triple combinations. Cephalosporins (and other beta-lactams) together with clavulanate rescued the activity of rifampicin against a rifampicin resistant strain. Synergy was not due exclusively to increased rifampicin accumulation within the mycobacterial cells. Cephalosporins were also synergistic with new anti-TB drugs such as bedaquiline and delamanid. Studies will be needed to validate their in vivo activities. However, the fact that cephalosporins are orally bioavailable with good safety profiles, together with their anti-mycobacterial activities reported here, suggest that they could be repurposed within new combinatorial TB therapies.