A novel class of antimicrobial drugs selectively targets a Mycobacterium tuberculosis PE-PGRS protein.

The continued spread of drug-resistant tuberculosis is one of the most pressing and complex challenges facing tuberculosis management worldwide. Therefore, developing a new class of drugs is necessary and urgently needed to cope with the increasing threat of drug-resistant tuberculosis. This study aims to discover a potential new class of tuberculosis drug candidates different from existing tuberculosis drugs. By screening a library of compounds, methyl (S)-1-((3-alkoxy-6,7-dimethoxyphenanthren-9-yl)methyl)-5-oxopyrrolidine-2-carboxylate (PP) derivatives with antitubercular activity were discovered. MIC ranges for PP1S, PP2S, and PP3S against clinically isolated drug-resistant Mycobacterium tuberculosis strains were 0.78 to 3.13, 0.19 to 1.56, and 0.78 to 6.25 µg/ml, respectively. PPs demonstrated antitubercular activities in macrophage and tuberculosis mouse models, showing no detectable toxicity in all assays tested. PPs specifically inhibited M. tuberculosis without significantly changing the intestinal microbiome in mice. Mutants selected in vitro suggest that the drug targets the PE-PGRS57, which has been found only in the genomes of the M. tuberculosis complex, highlighting the specificity and safety potency of this compound. As PPs show an excellent safety profile and highly selective toxicity specific to M. tuberculosis, PPs are considered a promising new candidate for the treatment of drug-resistant tuberculosis while maintaining microbiome homeostasis.

Synthesis and activity of BNF15 against drug-resistant Mycobacterium tuberculosis.

Aim: Tuberculosis is the leading cause of mortality among infectious diseases worldwide. Finding a new competent anti tubercular therapy is essential. Materials & methods: We screened thousands of compounds and evaluated their efficacy against Mycobacterium tuberculosis. Results: Initially, 2-nitronaphtho[2,3-b]benzofuran-6,11-dione was active against M. tuberculosis. Next, among 15 newly synthesized derivatives, BNF15 showed promising effect against all drug-sensitive and drug-resistant M. tuberculosis (MIC: 0.02-0.78 µg/ml). BNF15 effectively killed intracellular M. tuberculosis and nontuberculous mycobacteria. BNF15 exhibited a prolonged post antibiotic effect superior to isoniazid, streptomycin, and ethambutol and synergistic interaction with rifampicin. In acute oral toxicity test, BNF15 did not show toxic effect at a concentration up to 2000 mg/kg. Conclusion: These results highlight the perspective of BNF15 to treat drug-resistant M. tuberculosis.

In vitro activity of DNF-3 against drug-resistant Mycobacterium tuberculosis.

Due to the emergence of multidrug-resistant and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis, new antituberculosis drugs are urgently required to improve the efficacy of current tuberculosis (TB) treatment. To achieve this goal, ca. 1000 chemical compounds were screened for potential antimycobacterial activity, among which methyl 5-(2-diethylaminoethoxy)-7,12-dioxo-7,12 dihydrodinaphtho[1,2-b;2',3'-d]furan-6-carboxylate (DNF-3) showed strong activity against all of the tested drug-susceptible and -resistant M. tuberculosis strains, with 50% minimum inhibitory concentrations (MIC50 values) of 0.02-0.39 µg/mL both in culture broth and within murine RAW 264.7 macrophage cells. When DNF-3 was used in combination with rifampicin or streptomycin, it exhibited direct synergy against XDR-TB and an additive effect against M. tuberculosis H37Rv. DNF-3 displayed a long post-antibiotic effect (PAE) that was comparable with rifampicin but was superior to isoniazid, streptomycin and ethambutol. Importantly, DNF-3 showed no cytotoxicity to any cell line tested, with a selectivity index (SI) of >32. DNF-3 was also active against 27 nontuberculous mycobacteria (NTM) strains, Staphylococcus spp. and Streptococcus spp. Taken together, these results indicate that DNF-3 is a promising new candidate drug for treating TB. Further studies are warranted to establish the in vivo effect and therapeutic potential of DNF-3.

In vitro activity of collinin isolated from the leaves of Zanthoxylum schinifolium against multidrug- and extensively drug-resistant Mycobacterium tuberculosis.

BACKGROUND: Tuberculosis is a very serious infectious disease that threatens humanity, and the emergence of multidrug-resistant (MDR), extensively drug-resistant (XDR) strains resistant to drugs suggests that new drug development is urgent. In order to develop new tuberculosis drug, we have conducted in vitro anti-tubercular tests on thousands of plant-derived substances and finally found collinin extracted from the leaves of Zanthoxylum schinifolium, which has an excellent anti-tuberculosis effect. PURPOSE: To isolate an anti-tubercular bioactive compound from the leaves of Z. schinifolium and evaluate whether this agent demonstrates any potential in vitro characteristics suitable for the development of future anti-tubercular drugs to treat MDR and XDR Mycobacterium tuberculosis. METHODS: The methanolic extracts of the leaves of Z. schinifolium were subjected to bioassay-guided fractionation against M. tuberculosis using a microbial cell viability assay. In addition, following cell cytotoxicity assay, an intracellular anti-mycobacterial activity of the most active anti-tubercular compound was investigated after it was purified. RESULTS: The active compound with anti-tubercular activity isolated from leaves of Z. schinifolium was identified as a collinin. The extracted collinin showed anti-tubercular activity against both drug-susceptible and -resistant strains of M. tuberculosis at 50% minimum inhibitory concentrations (MIC50s) of 3.13-6.25 µg/ml in culture broth and MIC50s of 6.25-12.50 µg/ml inside Raw264.7 and A549 cells. Collinin had no cytotoxicity against human lung pneumocytes up to a concentration of 100 µg/ml (selectivity index > 16-32). CONCLUSIONS: Collinin extracted from the leaves of Z. schinifolium significantly inhibits the growth of MDR and XDR M. tuberculosis in the culture broth. In addition, it also inhibits the growth of intracellular drug-susceptible and drug-resistant tuberculosis in Raw264.7 and A549 cells. To our knowledge, this is the first report on the in vitro anti-tubercular activity of collinin, and our data suggest collinin as a potential drug to treat drug-resistant tuberculosis. Further studies are warranted to assess the in vivo efficacy and therapeutic potential of collinin.

In Vitro Effect of DFC-2 on Mycolic Acid Biosynthesis in Mycobacterium tuberculosis.

DFC-2, a methyl 5-[2-(dimethylamino)ethoxy]-7,12-dioxo-7,12-dihydrodinaphtho[1,2-b:2',3'-d]furan-6-carboxylate, is reported to have antitubercular effects against Mycobacterium tuberculosis. At concentrations ranging from 0.19 to 0.39 µg/ml, DFC-2 inhibited both drug-susceptible and -resistant strains of M. tuberculosis. Microarray analyses were employed to gain insights into the molecular mechanisms of DFC-2's action in M. tuberculosis. The most affected functional gene category was "lipid biosynthesis," which is involved in mycolic acid synthesis. The decrease in transcription of genes related to mycolic acid synthesis was confirmed by RT-PCR. Furthermore, we found that DFC-2 triggered a reduction in mycolic acid levels, showing a similar pattern to that of mycolic acid synthesis inhibitor isoniazid. These results may explain how this compound kills mycobacteria efficiently by inhibiting mycolic acid synthesis.

In vitro Antitubercular Activity of 3-Deoxysappanchalcone Isolated From the Heartwood of Caesalpinia sappan Linn.

Responsible for nearly 1.5 million deaths every year, the infectious disease tuberculosis remains one of the most serious challenges to global health. The emergence of multidrug-resistant tuberculosis and, more recently, extensively drug-resistant tuberculosis poses a significant threat in our effort to control this epidemic. New drugs are urgently needed to combat the growing threat of antimicrobial resistance. To achieve this goal, we screened approximately 500 species of medicinal plant methanol extracts and their solvent partitioned fractions for potential inhibitors of Mycobacterium tuberculosis growth. Using microdilution screening, the ethyl acetate solvent partitioned fraction from the heartwood of Caesalpinia sappan exhibited strong antitubercular activity. We isolated the active compound and identified it as 3-deoxysappanchalcone. The extracted 3-deoxysappanchalcone possessed activity against both drug-susceptible and drug-resistant strains of M. tuberculosis at MIC50 s of 3.125-12.5 µg/mL in culture broth and MIC50 s of 6.25-12.5 µg/mL inside macrophages and pneumocytes. 3-Deoxysappanchalcone was also found to act in partial synergy with streptomycin/ethambutol against M. tuberculosis H37Rv. 3-Deoxysappanchalcone had no cytotoxicity against the A549 cell line up to a concentration of 100 µg/mL (selectivity index > 8-32). Further studies are warranted to establish the in vivo effect and therapeutic potential of 3-deoxysappanchalcone. Copyright © 2017 John Wiley & Sons, Ltd.

Naphthofuroquinone derivatives show strong antimycobacterial activities against drug-resistant Mycobacteria.

Tuberculosis, one of the world's major health problems, has become more serious due to the emergence of multi-drug resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis (MTB). In this study, we performed three anti-MTB assays to evaluate the anti-mycobacterial activity of naphthofuroquinone derivatives against drug-resistant MTB. Among them, methyl 5-[2-(dimethylamino)ethoxy]-7,12-dioxo-7,12-dihydrodinaphtho[1,2-b:2',3'-d]furan-6-carboxylate (DFC2) exhibited strong anti-mycobacterial activity against MTB H37Ra, H37Rv and four drug-resistant MTB strains. The MIC of DFC2 ranged from 0.19-0.39 µg/ml to 0.78-1.56 µg/ml against all tested MTB strains. Moreover, DFC2 showed low cytotoxicity against fibroblast cells (L929) at concentrations 10-40-fold higher than their MICs. The IC50 value of DFC2 against L929 cells was 15.218 µg/ml. In addition, DFC2 reduced the number of intracellular M. tuberculosis in macrophages in a dose-dependent manner. Taken together, our results indicate DFC2 to be promising new candidate agents for the treatment of tuberculosis.

In vitro activity of (-)-deoxypergularinine, on its own and in combination with anti-tubercular drugs, against resistant strains of Mycobacterium tuberculosis.

BACKGROUND: The increasing incidence of multidrug-resistant tuberculosis (MDR-TB) infections has created a need for new effective drugs that also target extensively drug-resistant tuberculosis (XDR-TB) and/or augment the activities of existing drugs against tuberculosis. AIM: This study searched natural products for a new lead compound that targets MDR/XDR-TB. METHODS: An active compound was purified from the roots of Cynanchum atratum Bunge (Asclepiadaceae) after screening 1640 plant extracts, and its inhibitory effects against MDR/XDR strains and synergistic effects with existing anti-TB drugs were assessed using the resazurin, MGIT, and checkboard assays. RESULTS: (-)-Deoxypergularinine, purified from the roots of C. atratum, inhibited not only M. tuberculosis but also MDR/XDR strains. The minimum inhibitory concentrations (MICs) of (-)-deoxypergularinine for H37Ra, H37Rv, MDR, and XDR strains were all about 12.5 µg/ml. Moreover, combinations of (-)-deoxypergularinine with the first-line standard drugs rifampicin or isoniazid afforded six- and eight-fold reductions in drug MIC values, respectively, against strain H37Ra. CONCLUSIONS: (-)-Deoxypergularinine exerts anti-tubercular activities not only against normal tuberculosis strains but also MDR/XDR strains, and synergic effects with rifampicin and isoniazid for the H37Ra strain. The alkaloid may be valuable for targeting M/XDR M. tuberculosis.

Antimycobacterial activity of methanolic plant extract of Artemisia capillaris containing ursolic acid and hydroquinone against Mycobacterium tuberculosis.

OBJECTIVE: In order to protect against Mycobacterium tuberculosis (MTB) infection, novel drugs and new targets should be screened from the vast source of plants. We investigated the potentiality of the herbal plant of Artemisia capillaris extract (AC) against Mycobacterium tuberculosis. DESIGN: In this study, we isolated ursolic acid and hydroquinone by bio-activity guided fractionation from the methanol extracts of AC, and tested the inhibitory effects against several strains of MTB. Anti-mycobacterial evaluation of these compounds was carried out using the MGIT™ 960 and resazurin assay. Mycobacterial morphological changes due to the treatment of these compounds were further evaluated by Transmission electron microscopy (TEM). RESULTS: Ursolic acid (UA) and hydroquinone (HQ) inhibited the growth of both susceptible and resistant strains of M. tuberculosis. The MIC (minimum inhibitory concentration) values of both UA and HQ were 12.5 µg/ml against the susceptible strains of M. tuberculosis. Also both UA and HQ showed 12.5-25 µg/ml of MIC values against MDR/XDR MTB strains. However, against clinical strains of MTB, UA was found sensitive against those strains that are sensitive against both INH and RFP but resistant against those strains that are resistant to INH. On the other hand HQ was sensitive against all clinical strains. TEM image-analysis of the strain H37Ra after treatment with UA revealed cell wall lysis, whereas HQ-treated cells showed deformed cytoplasmic morphology. CONCLUSION: All these results indicate that AC extracts containing UA and HQ possess promising chemotherapeutic potency against MTB for future use.

Anti-Mycobacterial Activity of Tamoxifen Against Drug-Resistant and Intra-Macrophage Mycobacterium tuberculosis.

Recently, it has become a struggle to treat tuberculosis with the current commercial antituberculosis drugs because of the increasing emergence of multidrug-resistant (MDR) tuberculosis and extensively drug-resistant (XDR) tuberculosis. We evaluated here the antimycobacterial activity of tamoxifen, known as a synthetic anti-estrogen, against eight drug-sensitive or resistant strains of Mycobacterium tuberculosis (TB), and the active intracellular killing of tamoxifen on TB in macrophages. The results showed that tamoxifen had antituberculosis activity against drug-sensitive strains (MIC, 3.125-6.25 µg/ml) as well as drug-resistant strains (MIC, 6.25 to 12.5 µg/ml). In addition, tamoxifen profoundly decreased the number of intracellular TB in macrophages in a dose-dependent manner.

Dioscorea batatas extract attenuates high-fat diet-induced obesity in mice by decreasing expression of inflammatory cytokines.

BACKGROUND: The objective of the present study was to determine whether Dioscorea batatas (DB) extract reduces visceral fat accumulation and obesity-related biomarkers in mice fed a high-fat diet (HFD) and whether genes associated with adipogenesis and inflammation could be modulated by a diet containing DB extract. MATERIAL AND METHODS: Male C57BL/6J mice were divided into 4 groups (n=10 per group): normal diet (ND), HFD, 100 mg/kg DB extract-gavage with HFD, and 200 mg/kg DB extract-gavage with HFD. The mice were fed the experimental diets for 14 weeks. At 12 weeks, micro-computed X-ray tomography (micro-CT) was performed. RESULTS: Supplementation of the diet with DB extract for 14 weeks significantly prevented HFD-induced increases in body weight, visceral adipose tissue, plasma lipid levels, and leptins. The area of visceral fat was reduced by DB extract supplementation when examined by micro-CT. Supplementation with DB extract resulted in the downregulation of the adipogenic transcription factor (C/ERBa) and its target gene (CD36) in epididymal adipose tissue, compared to HFD alone. DB extract decreased the expression of proinflammatory cytokines (TNF-α, MCP-1, and IL-6) in epididymal adipose tissue. CONCLUSIONS: Our results suggest that DB extract may prevent HFD-induced obesity by downregulating the expression of genes related to adipogenesis and inflammation in visceral adipose tissue.

Ursolic Acid Activates Intracellular Killing Effect of Macrophages During Mycobacterium tuberculosis Infection.

Tuberculosis is one of the most threatening infectious diseases to public health all over the world, for which Mycobacterium tuberculosis (MTB) is the etiological agent of pathogenesis. Ursolic acid (UA) has immunomodulatory function and exhibits antimycobacterial activity. However, the intracellular killing effect of UA has yet to be elucidated. The aim of this study was to evaluate the intracellular killing effect of UA during mycobacterial infection. The intracellular killing activity of UA was evaluated in the macrophage cell line THP-1 by the MGIT 960 system as well as by CFU count. The production of reactive oxygen species (ROS) and the level of nitric oxide (NO) were measured using DCF-DA and Griess reagent, respectively. Phagocytosis was observed by a fluorescence-based staining method, and the colony forming units were enumerated on 7H11 agar medium following infection. In addition, MRP8 mRNA expression was measured by qRT-PCR. UA significantly decreased the number of intracellular Mycobacterium through generation of ROS and NO. In addition, it profoundly activated the phagocytosis process of THP-1 cells during MTB-infection. Furthermore, our data demonstrated that UA activated the phagocytosis process in human monocyte cells through MRP8 induction. These data suggest that UA firmly contributes to the intracellular killing effect of macrophages during mycobacterial infection.

Determination of platycodin D and platycodin D3 in rat plasma using liquid chromatography-tandem mass spectrometry.

Platycodon grandiflorum has long been used as a traditional oriental medicine for respiratory disorder. Platycodin D (PD) is known as the main component isolated from the root of PG. A simple and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the quantitation of PD in rat plasma. Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization and multiple reaction monitoring in positive ion mode. The total chromatographic run time was 4.0 min, and the calibration curves of PD were linear over the concentration range of 50-10,000 ng/mL in rat plasma. The coefficient of variation and relative error at five QC levels were 1.0 to 8.8% and 0.7 to 8.7%, respectively. After a single oral administration of 500 mg/kg and a single intravenous administration of 25 mg/kg of 3% PD extract (a PG extract including 3% of PD), platycodin D and platycodin D3 were detected and pharmacokinetic parameters were estimated. The oral bioavailability of platycodin D and platycodin D3 was 0.29% and 1.35% in rats at 500 mg/kg of 3% PD extract of PG, respectively. The present method can be applied to pharmacokinetic analysis of platycodins and platycosides of the PG.

Effects of mushroom, Pleurotus eryngii, extracts on bone metabolism.

We performed the present study to investigate whether Pleurotus eryngii extracts (PEX) play a role in bone metabolism. PEX treatment showed increase in the alkaline phosphatase activity of the osteoblasts and in the osteocalcin mRNA expression from primary osteoblasts. PEX also increased the expression of the Runx2 gene, and the secretion of osteoprotegerin from the osteoblasts showed marked increases after treatment with PEX. In addition, PEX treatment decreased the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and resorption areas. In vivo studies, using rats with ovariectomy-induced osteoporosis revealed that PEX alleviated the decrease in the trabecular bond mineral density.

Suppressive effect of AIF, a water extract from three herbs, on collagen-induced arthritis in mice.

AIF has been formulated using three herbs known to have anti-inflammatory and anti-osteolytic effects. In this study, the potential therapeutic effects of AIF for rheumatoid arthritis were assessed in vitro and in vivo. The effects of AIF on the inflammation (TNF-alpha, IL-1, iNO), cartilage protection (MMP-13), and selective killing of activated T cells were examined, in vitro. In addition, the therapeutic effect of AIF was evaluated using a collagen-induced arthritis (CIA) mouse model. DBA/1 mice were immunized with type II collagen. Following booster immunization, mice were treated with the oral administration of 276 mg/kg/d AIF once a day for 18 days, then, the severity of CIA was evaluated by macroscopic scoring and histopathological assessment. AIF significantly inhibited the production of TNF-alpha, IL-1, iNO, and MMP-13 in a dose dependent manner in vitro. Also, AIF killed activated T cells selectively, conserving naïve T cells. The oral administration of AIF in CIA mice suppressed the progression of CIA significantly and decreased synovial hyperplasia, cartilage destruction, and bone erosion. AIF showed potent anti-inflammatory effects in vitro and substantial protective effect for the progression of CIA in vivo. These results suggest that AIF contains effective compound(s) which may modify the progression of rheumatoid arthritis.