Design, synthesis and evaluation of biological activities of some novel anti-TB agents with bio-reducible functional group

Introduction: With regard to the anti-mycobacterial activity of 2-pyrazinoic acid esters (POEs), recent studies have shown that both pyrazine core and alkyl part of POE interact with the fatty acid synthase type (I) (FAS (I)) precluding a complex formation between NADPH and FAS (I). Methods: Considering this interaction at the reductase site of FAS (I) responsible for reduction of β-ketoacyl-CoA to β-hydroxyacyl-CoA, we hypothesized that POE containing a bioreducible center in its alkyl part might show an increased anti-tubercular activity due to the involvement of FAS (I) in extra bio-reduction reaction. Thus, we synthesized novel POEs, confirmed their structures by spectral data, and subsequently evaluated their anti-mycobacterial activity against Mycobacterium tuberculosis (Mtb) (H37Rv) strain at 10 μg/mL concentration. Results: Compounds 3c, 3j, and 3m showed higher activity with regard to the inhibition of Mtb growth by 45.4, 45.7, and 51.2% respectively. Unexpectedly, the maltol derived POE 3l having the lowest log p value among the POEs indicated the highest anti-mycobacterial growth activity with 56% prevention. Compounds 3c and 3l showed no remarkable cytotoxicity on human macrophages at 10 μg/mL concentration as analyzed by xCELLigence real-time cell analysis. In further experiments, some of the tested POEs, unlike pyrazinamide (PZA), exhibited significant antibacterial and also anti-fungal activities. POEs showed an enhanced bactericidal activity on gram-positive bacteria as shown for Staphylococcus aureus, e.g. compound 3b with a MIC value of 125 μg/mL but not E. coli as a gram-negative bacteria, except for maltol derived POE (3l) that showed an inverse activity in the susceptibility test. In the anticancer activity test against the human leukemia K562 cell lines using MTT assay, compounds 3e and 3j showed the highest cytotoxic effect with IC50 values of 25±8.0 μΜ and 25±5.0 μΜ, respectively. Conclusion: It was found that the majority of POEs containing a bioreducible center showed higher inhibitory activities on Mtb growth when compared to the similar compounds without a bio-reducible functional group

Inhibitors of Aspartate Transcarbamoylase Inhibit Mycobacterium tuberculosis Growth

Aspartate transcarbamoylase (ATCase) plays a key role in the second step of de novo pyrimidine biosynthesis in eukaryotes and has been proposed to be a target to suppress cell proliferation in E. coli, human cells and the malarial parasite. We hypothesized that a library of ATCase inhibitors developed for malarial ATCase (PfATCase) may also contain inhibitors of the tubercular ATCase and provide a similar inhibition of cellular proliferation. Of the 70 compounds screened, 10 showed single-digit micromolar inhibition in an in vitro activity assay and were tested for their effect on M. tuberculosis cell growth in culture. The most promising compound demonstrated a MIC 90 of 4 μM. A model of MtbATCase was generated using the experimental coordinates of PfATCase. In silico docking experiments showed this compound can occupy a similar allosteric pocket on MtbATCase to that seen on PfATCase, explaining the observed species selectivity seen for this compound series. </p

Not Every Hit-Identification Technique Works on 1-Deoxy-D-Xylulose 5-Phosphate Synthase (DXPS):Making the Most of a Virtual Screening Campaign

In this work, we demonstrate how important it is to investigate not only on-target activity but to keep antibiotic activity against critical pathogens in mind. Since antimicrobial resistance is spreading in bacteria such as Mycobacterium tuberculosis, investigations into new targets are urgently needed. One promising new target is 1-deoxy-D-xylulose 5-phosphate synthase (DXPS) of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. We have recently solved the crystal structure of truncated M. tuberculosis DXPS and used it to perform a virtual screening in collaboration with Atomwise Inc. using their deep convolutional neural network-based AtomNet® platform. Of 94 virtual hit compounds only one showed interesting results in binding and activity studies. We synthesized 30 close derivatives using a straightforward synthetic route that allowed for easy derivatization. However, no improvement in activity was observed for any of the derivatives. Therefore, we tested them against a variety of pathogens and found them to be good inhibitors against Escherichia coli.</p

Potential role for IL-2 ELISpot in differentiating recent and remote infection in tuberculosis contact tracing

Interferon (IFN)-γ release assays (IGRA) have improved tuberculosis contact tracing, but discrimination of recent from remote Mycobacterium tuberculosis contacts is not possible by IGRA alone. We present results of a tuberculosis contact investigation with a new early-secretory-antigenic-target (ESAT)-6 and culture-filtrate-protein (CFP)-10 specific interleukin (IL)-2 ELISpot in addition to ESAT-6 and CFP-10 specific IFN-γ ELISpot and tuberculin skin testing (TST). Results of the TST, IFN-γ ELISpot and IL-2 ELISpot were positive in 6/172 (3.4%), 7/167 (4.2%) and 6/196 (3.1%) of contacts, respectively. Close contact (≥100 hours) to the index case increased the risk of positive results in the IFN-γ ELISpot, TST, and IL-2 ELISpot by 40.8, 19.3, and 2.5 times, respectively. Individuals with a positive IFN-γ ELISpot/negative IL-2 ELISpot result had a median (IQR) duration of index case exposure of 568 hours (133_1000) compared to individuals with a positive IFN-γ ELISpot/positive IL-2 ELISpot result (median=24 hours; 20_130; p-value=0.047). Combination of a M. tuberculosis specific IFN-γ ELISpot with a M. tuberculosis specific IL-2 ELISpot significantly improved the identification of individuals with the highest risk of recent M. tuberculosis infection and is a promising method that should be explored to target tuberculosis preventive chemotherapy

Tuberculostearic Acid-Containing Phosphatidylinositols as Markers of Bacterial Burden in Tuberculosis

One-fourth of the global human population is estimated to be infected with strains of the Mycobacterium tuberculosis complex (MTBC), the causative agent of tuberculosis (TB). Using lipidomic approaches, we show that tuberculostearic acid (TSA)-containing phosphatidylinositols (PIs) are molecular markers for infection with clinically relevant MTBC strains and signify bacterial burden. For the most abundant lipid marker, detection limits of ∼10$^{2}$ colony forming units (CFUs) and ∼10$^{3}$ CFUs for bacterial and cell culture systems were determined, respectively. We developed a targeted lipid assay, which can be performed within a day including sample preparation─roughly 30-fold faster than in conventional methods based on bacterial culture. This indirect and culture-free detection approach allowed us to determine pathogen loads in infected murine macrophages, human neutrophils, and murine lung tissue. These marker lipids inferred from mycobacterial PIs were found in higher levels in peripheral blood mononuclear cells of TB patients compared to healthy individuals. Moreover, in a small cohort of drug-susceptible TB patients, elevated levels of these molecular markers were detected at the start of therapy and declined upon successful anti-TB treatment. Thus, the concentration of TSA-containing PIs can be used as a correlate for the mycobacterial burden in experimental models and in vitro systems and may prospectively also provide a clinically relevant tool to monitor TB severity

Discovery of novel drug-like antitubercular hits targeting the MEP pathway enzyme DXPS by strategic application of ligand-based virtual screening.

In the present manuscript, we describe how we successfully used ligand-based virtual screening (LBVS) to identify two small-molecule, drug-like hit classes with excellent ADMET profiles against the difficult to address microbial enzyme 1-deoxy-d-xylulose-5-phosphate synthase (DXPS). In the fight against antimicrobial resistance (AMR), it has become increasingly important to address novel targets such as DXPS, the first enzyme of the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway, which affords the universal isoprenoid precursors. This pathway is absent in humans but essential for pathogens such as Mycobacterium tuberculosis, making it a rich source of drug targets for the development of novel anti-infectives. Standard computer-aided drug-design tools, frequently applied in other areas of drug development, often fail for targets with large, hydrophilic binding sites such as DXPS. Therefore, we introduce the concept of pseudo-inhibitors, combining the benefits of pseudo-ligands (defining a pharmacophore) and pseudo-receptors (defining anchor points in the binding site), for providing the basis to perform a LBVS against M. tuberculosis DXPS. Starting from a diverse set of reference ligands showing weak inhibition of the orthologue from Deinococcus radiodurans DXPS, we identified three structurally unrelated classes with promising in vitro (against M. tuberculosis DXPS) and whole-cell activity including extensively drug-resistant strains of M. tuberculosis. The hits were validated to be specific inhibitors of DXPS and to have a unique mechanism of inhibition. Furthermore, two of the hits have a balanced profile in terms of metabolic and plasma stability and display a low frequency of resistance development, making them ideal starting points for hit-to-lead optimization of antibiotics with an unprecedented mode of action

Discovery of novel drug-like antitubercular hits targeting the MEP pathway enzyme DXPS by strategic application of ligand-based virtual screening

In the present manuscript, we describe how we successfully used ligand-based virtual screening (LBVS) to identify two small-molecule, drug-like hit classes with excellent ADMET profiles against the difficult to address microbial enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXPS). In the fight against antimicrobial resistance (AMR), it has become increasingly important to address novel targets such as DXPS, the first enzyme of the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, which affords the universal isoprenoid precursors. This pathway is absent in humans but essential for pathogens such as Mycobacterium tuberculosis, making it a rich source of drug targets for the development of novel anti-infectives. Standard computer-aided drug-design tools, frequently applied in other areas of drug development, often fail for targets with large, hydrophilic binding sites such as DXPS. Therefore, we introduce the concept of pseudo-inhibitors, combining the benefits of pseudo-ligands (defining a pharmacophore) and pseudo-receptors (defining anchor points in the binding site), for providing the basis to perform a LBVS against M. tuberculosis DXPS. Starting from a diverse set of reference ligands showing weak inhibition of the orthologue from Deinococcus radiodurans DXPS, we identified three structurally unrelated classes with promising in vitro (against M. tuberculosis DXPS) and whole-cell activity including extensively drug-resistant strains of M. tuberculosis. The hits were validated to be specific inhibitors of DXPS and to have a unique mechanism of inhibition. Furthermore, two of the hits have a balanced profile in terms of metabolic and plasma stability and display a low frequency of resistance development, making them ideal starting points for hit-to-lead optimization of antibiotics with an unprecedented mode of action

Allele Summation of Diabetes Risk Genes Predicts Impaired Glucose Tolerance in Female and Obese Individuals

INTRODUCTION: Single nucleotide polymorphisms (SNPs) in approximately 40 genes have been associated with an increased risk for type 2 diabetes (T2D) in genome-wide association studies. It is not known whether a similar genetic impact on the risk of prediabetes (impaired glucose tolerance [IGT] or impaired fasting glycemia [IFG]) exists. METHODS: In our cohort of 1442 non-diabetic subjects of European origin (normal glucose tolerance [NGT] n = 1046, isolated IFG n = 142, isolated IGT n = 140, IFG+IGT n = 114), an impact on glucose homeostasis has been shown for 9 SNPs in previous studies in this specific cohort. We analyzed these SNPs (within or in the vicinity of the genes TCF7L2, KCNJ11, HHEX, SLC30A8, WFS1, KCNQ1, MTNR1B, FTO, PPARG) for association with prediabetes. RESULTS: The genetic risk load was significantly associated with the risk for IGT (p = 0.0006) in a model including gender, age, BMI and insulin sensitivity. To further evaluate potential confounding effects, we stratified the population on gender, BMI and insulin sensitivity. The association of the risk score with IGT was present in female participants (p = 0.008), but not in male participants. The risk score was significantly associated with IGT (p = 0.008) in subjects with a body mass index higher than 30 kg/m(2) but not in non-obese individuals. Furthermore, only in insulin resistant subjects a significant association between the genetic load and the risk for IGT (p = 0.01) was found. DISCUSSION: We found that T2D genetic risk alleles cause an increased risk for IGT. This effect was not present in male, lean and insulin sensitive subjects, suggesting a protective role of beneficial environmental factors on the genetic risk

Identification of Candida glabrata genes involved in pH modulation and modification of the phagosomal environment in macrophages

notes: PMCID: PMC4006850types: Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov'tCandida glabrata currently ranks as the second most frequent cause of invasive candidiasis. Our previous work has shown that C. glabrata is adapted to intracellular survival in macrophages and replicates within non-acidified late endosomal-stage phagosomes. In contrast, heat killed yeasts are found in acidified matured phagosomes. In the present study, we aimed at elucidating the processes leading to inhibition of phagosome acidification and maturation. We show that phagosomes containing viable C. glabrata cells do not fuse with pre-labeled lysosomes and possess low phagosomal hydrolase activity. Inhibition of acidification occurs independent of macrophage type (human/murine), differentiation (M1-/M2-type) or activation status (vitamin D3 stimulation). We observed no differential activation of macrophage MAPK or NFκB signaling cascades downstream of pattern recognition receptors after internalization of viable compared to heat killed yeasts, but Syk activation decayed faster in macrophages containing viable yeasts. Thus, delivery of viable yeasts to non-matured phagosomes is likely not triggered by initial recognition events via MAPK or NFκB signaling, but Syk activation may be involved. Although V-ATPase is abundant in C. glabrata phagosomes, the influence of this proton pump on intracellular survival is low since blocking V-ATPase activity with bafilomycin A1 has no influence on fungal viability. Active pH modulation is one possible fungal strategy to change phagosome pH. In fact, C. glabrata is able to alkalinize its extracellular environment, when growing on amino acids as the sole carbon source in vitro. By screening a C. glabrata mutant library we identified genes important for environmental alkalinization that were further tested for their impact on phagosome pH. We found that the lack of fungal mannosyltransferases resulted in severely reduced alkalinization in vitro and in the delivery of C. glabrata to acidified phagosomes. Therefore, protein mannosylation may play a key role in alterations of phagosomal properties caused by C. glabrata.Deutsche ForschungsgemeinschaftNational Institutes for HealthWellcome TrustBBSR