New Terpenoids from the Corticioid Fungus Punctularia atropurpurascens and their Antimycobacterial Evaluation.

Chemical investigation of Punctularia atropurpurascens strain HM1 (Punctulariaceae), a corticioid isolated from a decorticated piece of Quercus bark collected in Bosque de Tlalpan, Mexico City, led to the isolation of a new drimane, 1-α-hydroxy-isodrimenine (1: ) and a new tetrahydroxy kauranol, 16-hydroxy-phlebia-nor-kauranol (2: ), together with the known N-phenylacetamide (3: ). Structures of all compounds were elucidated by spectroscopic and spectrometric methods, and the absolute configuration of 1: and 2: was confirmed via single-crystal X-ray crystallography. The isolated compounds showed modest antimycobacterial activity.

Antitubercular polyhalogenated phenothiazines and phenoselenazine with reduced binding to CNS receptors.

Targeting energy metabolism in Mycobacterium tuberculosis (Mtb) is a new paradigm in the search for innovative anti-TB drugs. NADH:menaquinone oxidoreductase is a non-proton translocating type II NADH dehydrogenase (NDH-2) that is an essential enzyme in the respiratory chain of Mtb and is not found in mammalian mitochondria. Phenothiazines (PTZs) represent one of the most known class of NDH-2 inhibitors, but their use as anti-TB drugs is currently limited by the wide range of potentially serious off-target effects. In this work, we designed and synthesized a series of new PTZs by decorating the scaffold in an unconventional way, introducing various halogen atoms. By replacing the sulfur atom with selenium, a dibromophenoselenazine 20 was also synthesized. Among the synthesized poly-halogenated PTZs (HPTZs), dibromo and tetrachloro derivatives 9 and 11, along with the phenoselenazine 20, emerged with a better anti-TB profile than the therapeutic thioridazine (TZ). They targeted non-replicating Mtb, were bactericidal, and synergized with rifampin and bedaquiline. Moreover, their anti-TB activity was found to be related to the NDH-2 inhibition. Most important, they showed a markedly reduced affinity to dopaminergic and serotonergic receptors respect to the TZ. From this work emerged, for the first time, as the poly-halogenation of the PTZ core, while permitting to maintain good anti-TB profile could conceivably lead to fewer CNS side-effects risk, making more tangible the use of PTZs for this alternative therapeutic application.

Strategies in anti-Mycobacterium tuberculosis drug discovery based on phenotypic screening.

The rise of multi- and extensively drug-resistant Mycobacterium tuberculosis (M. tb) strains and co-infection with human immunodeficiency virus has escalated the need for new anti-M. tb drugs. Numerous challenges associated with the M. tb, in particular slow growth and pathogenicity level 3, discouraged use of this organism in past primary screening efforts. From current knowledge of the physiology and drug susceptibility of mycobacteria in general and M. tb specifically, it can be assumed that many potentially useful drug leads were missed by failing to screen directly against this pathogen. This review discusses recent high-throughput phenotypic screening strategies for anti-M. tb drug discovery. Emphasis is placed on prioritization of hits, including their extensive biological and chemical profiling, as well as the development status of promising drug candidates discovered with phenotypic screening.

Small Molecules Targeting Mycobacterium tuberculosis Type II NADH Dehydrogenase Exhibit Antimycobacterial Activity.

The generation of ATP through oxidative phosphorylation is an essential metabolic function for Mycobaterium tuberculosis (Mtb), regardless of the growth environment. The type II NADH dehydrogenase (Ndh-2) is the conduit for electrons into the pathway, and is absent in the mammalian genome, thus making it a potential drug target. Herein, we report the identification of two types of small molecules as selective inhibitors for Ndh-2 through a multicomponent high-throughput screen. Both compounds block ATP synthesis, lead to effects consistent with loss of NADH turnover, and importantly, exert bactericidal activity against Mtb. Extensive medicinal chemistry optimization afforded the best analogue with an MIC of 90 nm against Mtb. Moreover, the two scaffolds have differential inhibitory activities against the two homologous Ndh-2 enzymes in Mtb, which will allow precise control over Ndh-2 function in Mtb to facilitate the assessment of this anti-TB drug target.

Development of (6 R)-2-Nitro-6-[4-(trifluoromethoxy)phenoxy]-6,7-dihydro-5 H-imidazo[2,1- b][1,3]oxazine (DNDI-8219): A New Lead for Visceral Leishmaniasis.

Discovery of the potent antileishmanial effects of antitubercular 6-nitro-2,3-dihydroimidazo[2,1- b][1,3]oxazoles and 7-substituted 2-nitro-5,6-dihydroimidazo[2,1- b][1,3]oxazines stimulated the examination of further scaffolds (e.g., 2-nitro-5,6,7,8-tetrahydroimidazo[2,1- b][1,3]oxazepines), but the results for these seemed less attractive. Following the screening of a 900-compound pretomanid analogue library, several hits with more suitable potency, solubility, and microsomal stability were identified, and the superior efficacy of newly synthesized 6 R enantiomers with phenylpyridine-based side chains was established through head-to-head assessments in a Leishmania donovani mouse model. Two such leads ( R-84 and R-89) displayed promising activity in the more stringent Leishmania infantum hamster model but were unexpectedly found to be potent inhibitors of hERG. An extensive structure-activity relationship investigation pinpointed two compounds ( R-6 and pyridine R-136) with better solubility and pharmacokinetic properties that also provided excellent oral efficacy in the same hamster model (>97% parasite clearance at 25 mg/kg, twice daily) and exhibited minimal hERG inhibition. Additional profiling earmarked R-6 as the favored backup development candidate.

In Vitro Activities of Enantiopure and Racemic 1'-Acetoxychavicol Acetate against Clinical Isolates of Mycobacterium tuberculosis.

In the process of evaluating the effect of several plant extracts against Mycobacterium tuberculosis using the Microplate Alamar Blue Assay (MABA), an extract of Thai herb Alpinia galanga rhizome and its major component, 1'-acetoxychavicol acetate (ACA), exhibited marked anti-tuberculosis activity. The minimal inhibition concentrations (MICs) of the S-enantiomer of ACA (S-ACA) against M. tuberculosis H37Ra ATCC 25177 and H37Rv ATCC 27294 strains were 0.2 µg/mL and 0.7 µg/mL, respectively. More than 95% of 100 drug-sensitive and 50 drug-resistant mycobacterial clinical isolates were inhibited by extracted S-ACA at 1.0 µg/mL. All of the remaining isolates were inhibited at 2.0 µg/mL. In contrast to the S-enantiomer, synthetic racemic 1'-R,S-ACA (rac-ACA) showed MICs of 0.5 µg/mL and 2.7 µg/mL for M. tuberculosis H37Ra ATCC 25177 and H37Rv ATCC 27294, respectively, suggesting that the anti-tuberculosis effect might be primarily due to the S-form. These observations were in line with the MICs of rac-ACA against 98% of 93 drug-resistant clinical isolates, which showed the effective inhibitory dose at 2.0 µg/mL. After exposure to 2.7 µg/mL of rac-ACA for at least 3 h, the tubercle bacilli were completely killed. These demonstrated that ACA had potent anti-TB activity.

Fluorescence-based assay for polyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA) and identification of novel antimycobacterial WecA inhibitors.

Polyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA) is an essential enzyme for the growth of Mycobacterium tuberculosis (Mtb) and some other bacteria. Mtb WecA catalyzes the transformation from UDP-GlcNAc to decaprenyl-P-P-GlcNAc, the first membrane-anchored glycophospholipid that is responsible for the biosynthesis of mycolylarabinogalactan in Mtb. Inhibition of WecA will block the entire biosynthesis of essential cell wall components of Mtb in both replicating and non-replicating states, making this enzyme a target for development of novel drugs. Here, we report a fluorescence-based method for the assay of WecA using a modified UDP-GlcNAc, UDP-Glucosamine-C6-FITC (1), a membrane fraction prepared from an M. smegmatis strain, and the E. coli B21WecA. Under the optimized conditions, UDP-Glucosamine-C6-FITC (1) can be converted to the corresponding decaprenyl-P-P-Glucosamine-C6-FITC (3) in 61.5% yield. Decaprenyl-P-P-Glucosamine-C6-FITC is readily extracted with n-butanol and can be quantified by ultraviolet-visible (UV-vis) spectrometry. Screening of the compound libraries designed for bacterial phosphotransferases resulted in the discovery of a selective WecA inhibitor, UT-01320 (12) that kills both replicating and non-replicating Mtb at low concentration. UT-01320 (12) also kills the intracellular Mtb in macrophages. We conclude that the WecA assay reported here is amenable to medium- and high-throughput screening, thus facilitating the discovery of novel WecA inhibitors.

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.

Imidazo[1,2-a]Pyridine-3-Carboxamides Are Active Antimicrobial Agents against Mycobacterium avium Infection In Vivo.

A panel of six imidazo[1,2-a]pyridine-3-carboxamides (IAPs) were shown to have low-micromolar activity against Mycobacterium avium strains. Compound ND-10885 (compound 2) showed significant activity in the lung, spleen, and liver in a mouse M. avium infection model. A combined regimen consisting of ND-10885 (compound 2) and rifampin was additive in its anti-M. avium activity in the lung. Our data indicate that IAPs represent a new class of antibiotics that are active against M. avium and could potentially serve as an effective addition to a combined treatment regimen.

Bioassay-Guided Isolation and Structural Modification of the Anti-TB Resorcinols from Ardisia gigantifolia.

Tuberculosis (TB) is a highly contagious disease mainly caused by Mycobacterium tuberculosis H37 RV . Antitubercular (anti-TB) bioassay-guided isolation of the CHCl3 extract of the leaves and stems of the medicinal plant Ardisia gigantifolia led to the isolation of two anti-TB 5-alkylresorcinols, 5-(8Z-heptadecenyl) resorcinol (1) and 5-(8Z-pentadecenyl) resorcinol (2). We further synthesized 15 derivatives based on these two natural products. These compounds (natural and synthetic) were evaluated for their anti-TB activity against Mycobacterium tuberculosis H37 RV . Resorcinols 1 and 2 exhibited anti-TB activity with MIC values at 34.4 and 79.2 µm in MABA assay, respectively, and 91.7 and 168.3 µm in LORA assay, respectively. Among these derivatives, compound 8 was found to show improved anti-TB activity than its synthetic precursor (2) with MIC values at 42.0 µm in MABA assay and 100.2 µm in LORA assay. The active compounds should be regarded as new hits for further study as a novel class of anti-TB agents. The distinct structure-activity correlations of the parent compound were elucidated based on these derivatives.

The cyclic peptide ecumicin targeting ClpC1 is active against Mycobacterium tuberculosis in vivo.

Drug-resistant tuberculosis (TB) has lent urgency to finding new drug leads with novel modes of action. A high-throughput screening campaign of >65,000 actinomycete extracts for inhibition of Mycobacterium tuberculosis viability identified ecumicin, a macrocyclic tridecapeptide that exerts potent, selective bactericidal activity against M. tuberculosis in vitro, including nonreplicating cells. Ecumicin retains activity against isolated multiple-drug-resistant (MDR) and extensively drug-resistant (XDR) strains of M. tuberculosis. The subcutaneous administration to mice of ecumicin in a micellar formulation at 20 mg/kg body weight resulted in plasma and lung exposures exceeding the MIC. Complete inhibition of M. tuberculosis growth in the lungs of mice was achieved following 12 doses at 20 or 32 mg/kg. Genome mining of lab-generated, spontaneous ecumicin-resistant M. tuberculosis strains identified the ClpC1 ATPase complex as the putative target, and this was confirmed by a drug affinity response test. ClpC1 functions in protein breakdown with the ClpP1P2 protease complex. Ecumicin markedly enhanced the ATPase activity of wild-type (WT) ClpC1 but prevented activation of proteolysis by ClpC1. Less stimulation was observed with ClpC1 from ecumicin-resistant mutants. Thus, ClpC1 is a valid drug target against M. tuberculosis, and ecumicin may serve as a lead compound for anti-TB drug development.

Antitubercular constituents from Premna odorata Blanco.

ETHNOPHARMACOLOGICAL RELEVANCE: Premna odorata Blanco (Lamiaceae) is a medicinal plant traditionally used in Albay Province, in southeastern Luzon, Philippines to treat tuberculosis. This study aimed to determine the antitubercular property of the crude extract and sub-extracts of the leaves, and to isolate the bioactive principles from the active fractions. MATERIALS AND METHODS: Through extraction, solvent polarity-based fractionation and silica gel chromatography purification of the DCM sub-extract, compound mixtures from the bioactive fractions were isolated and screened for their in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv using the colorimetric Microplate Alamar Blue assay (MABA). RESULTS: The crude methanolic extract and sub-extracts showed poor inhibitory activity against Mycobacterium tuberculosis H37Rv (MIC≥128µg/mL). However, increased inhibitory potency was observed for fractions eluted from the DCM sub-extract (MIC=54 to 120µg/mL). Further purification of the most active fraction (MIC=54µg/mL) led to the isolation of a 1-heneicosyl formate (1), 4:1 mixture of ß-sitosterol (2), stigmasterol (3) and diosmetin (4), which were identified through GC-MS analysis (with dereplication) and NMR experiments. The MIC of compound 1 was 8µg/mL. CONCLUSIONS: The results of this study provide scientific basis for the traditional use of Premna odorata as treatment for tuberculosis.

Airborne antituberculosis activity of Eucalyptus citriodora essential oil.

The rapid emergence of multi- and extensively drug-resistant tuberculosis (MDR/XDR-TB) has created a pressing public health problem, which mostly affects regions with HIV/AIDS prevalence and represents a new constraint in the already challenging disease management of tuberculosis (TB). The present work responds to the need to reduce the number of contagious MDR/XRD-TB patients, protect their immediate environment, and interrupt the rapid spread by laying the groundwork for an inhalation therapy based on anti-TB-active constituents of the essential oil (EO) of Eucalyptus citriodora. In order to address the metabolomic complexity of EO constituents and active principles in botanicals, this study applied biochemometrics, a 3-D analytical approach that involves high-resolution CCC fractionation, GC-MS analysis, bioactivity measurements, and chemometric analysis. Thus, 32 airborne anti-TB-active compounds were identified in E. citriodora EO: the monoterpenes citronellol (1), linalool (3), isopulegol (5), and α-terpineol (7) and the sesquiterpenoids spathulenol (11), ß-eudesmol (23), and τ-cadinol (25). The impact of the interaction of multiple components in EOs was studied using various artificial mixtures (AMxs) of the active monoterpenes 1, 2, and 5 and the inactive eucalyptol (33). Both neat 1 and the AMx containing 1, 2, and 33 showed airborne TB inhibition of >90%, while the major E. citriodora EO component, 2, was only weakly active, at 18% inhibition.

New finding of an anti-TB compound in the genus Marsypopetalum (Annonaceae) from a traditional herbal remedy of Laos.

ETHNOPHARMACOLOGICAL RELEVANCE: There is widespread use of traditional herbal remedies in the Lao PDR (Laos). It is common practice to treat many diseases with local plants. This research project documented and analysed some of these traditional remedies used to treat symptoms of tuberculosis (TB). MATERIALS AND METHODS: This research was executed by interviewing healers about plants used traditionally to treat the symptoms of TB. Samples of some of the plants were collected, and extracts of 77 species were submitted to various in vitro assays in order to determine the amount of growth inhibition of virulent Mycobacterium tuberculosis H37Rv (Mtb), as opposed to other microbes and mammalian Vero cells. RESULTS: Interviews took place with 58 contemporary healers in 5 different provinces about plants currently used, giving a list of 341 plants. Bioassay-guided fractionation was performed on Marsypopetalum modestum (Pierre) B. Xue and R.M.K. Saunders (Annonaceae), leading to the isolation of dipyrithione, an anti-mycobacterial compound isolated for the first time from the genus Marsypopetalum through this research. CONCLUSIONS: This research has helped to increase awareness of Laos' rich diversity of medicinal plants and will hopefully provide incentive to preserve the undeveloped forested areas that remain, which still hold a wealth of medical information for future discoveries.

Quantitative purity-activity relationships of natural products: the case of anti-tuberculosis active triterpenes from Oplopanax horridus.

The present study provides an extension of the previously developed concept of purity-activity relationships (PARs) and enables the quantitative evaluation of the effects of multiple minor components on the bioactivity of residually complex natural products. The anti-tuberculosis active triterpenes from the Alaskan ethnobotanical Oplopanax horridus were selected as a case for the development of the quantitative PAR (QPAR) concept. The residual complexity of the purified triterpenes was initially evaluated by 1D- and 2D-NMR and identified as a combination of structurally related and unrelated impurities. Using a biochemometric approach, the qHNMR purity and anti-TB activity of successive chromatographic fractions of O. horridus triterpenes were correlated by linear regression analysis to generate a mathematical QPAR model. The results demonstrate that impurities, such as widely occurring monoglycerides, can have a profound impact on the observed antimycobacterial activity of triterpene-enriched fractions. The QPAR concept is shown to be capable of providing a quantitative assessment in situations where residually complex constitution contributes toward the biological activity of natural products.

Current prospects of synthetic curcumin analogs and chalcone derivatives against mycobacterium tuberculosis.

Tuberculosis, caused by Mycobacterium tuberculosis, is amongst the foremost infectious diseases. Treatment of tuberculosis is a complex process due to various factors including a patient's inability to persevere with a combined treatment regimen, the difficulty in eradicating the infection in immune-suppressed patients, and multidrug resistance (MDR). Extensive research circumscribing molecules to counteract this disease has led to the identification of many inhibitory small molecules. Among these are chalcone derivatives along with curcumin analogs. In this review article, we summarize the reported literature regarding anti tubercular activity of chalcone derivatives and synthetic curcumin analogs. Our goal is to provide an analysis of research to date in order to facilitate the synthesis of superior antitubercular chalcone derivatives and curcumin analogs.

Antitubercular activity of the semi-polar extractives of Uvaria rufa.

OBJECTIVE: To investigate the inhibitory activity of the chloroform extract, petroleum ether and chloroform sub-extracts, lead-acetate treated chloroform extract, fractions and secondary metabolites of Uvaria rufa (U. rufa) against Mycobacterium tuberculosis (M. tuberculosis) H(37)Rv. METHODS: The antituberculosis susceptibility assay was carried out using the colorimetric Microplate Alamar blue assay (MABA). In addition, the cytotoxicity of the most active fraction was evaluated using the VERO cell toxicity assay. RESULTS: The in vitro inhibitory activity against M. tuberculosis H(37)Rv increased as purification progressed to fractionation (MIC up to 23 µg/mL). The chloroform extract and its sub-extracts showed moderate toxicity while the most active fraction from chloroform sub-extract exhibited no cytotoxicity against VERO cells. Meanwhile, the lead acetate-treated crude chloroform extract and its fractions showed complete inhibitions (100%) with MIC values up to 8 µg/mL. Phytochemical screening of the most active fraction showed, in general, the presence of terpenoids, steroids and phenolic compounds. Evaluation of the antimycobacterial activity of known secondary metabolites isolated showed no promising inhibitory activity against the test organism. CONCLUSIONS: The present results demonstrate the potential of U. rufa as a phytomedicinal source of compounds that may exhibit promising antituberculosis activity. In addition, elimination of polar pigments revealed enhanced inhibition against M. tuberculosis H(37)Rv. While several compounds known for this plant did not show antimycobacterial activity, the obtained results are considered sufficient reason for further study to isolate the metabolites from U. rufa responsible for the antitubercular activity.

Comprehensive analysis of methods used for the evaluation of compounds against Mycobacterium tuberculosis.

In drug development, there are typically a series of preclinical studies that must be completed with new compounds or regimens before use in humans. A sequence of in vitro assays followed by in vivo testing in validated animal models to assess the activity against Mycobacterium tuberculosis, pharmacology and toxicity is generally used for advancing compounds against tuberculosis in a preclinical stage. A plethora of different assay systems and conditions are used to study the effect of drug candidates on the growth of M. tuberculosis, making it difficult to compare data from one laboratory to another. The Bill and Melinda Gates Foundation recognized the scientific gap to delineate the spectrum of variables in experimental protocols, identify which of these are biologically significant, and converge towards a rationally derived standard set of optimized assays for evaluating compounds. The goals of this document are to recommend protocols and hence accelerate the process of TB drug discovery and testing. Data gathered from preclinical in vitro and in vivo assays during personal visits to laboratories and an electronic survey of methodologies sent to investigators is reported. Comments, opinions, experiences as well as final recommendations from those currently engaged in such preclinical studies for TB drug testing are being presented. Certain in vitro assays and mouse efficacy models were re-evaluated in the laboratory as head-to-head experiments and a summary is provided on the results obtained. It is our hope that this information will be a valuable resource for investigators in the field to move forward in an efficient way and that key variables of assays are included to ensure accuracy of results which can then be used for designing human clinical trials. This document then concludes with remaining questions and critical gaps that are in need of further validation and experimentation.

Unbiased evaluation of bioactive secondary metabolites in complex matrices.

The majority of bioactive principles in a complex matrix such as natural products and botanical medicines are secondary rather than primary metabolites. In addition to being chemically diverse, the bioactivity of an ethnobotanical can comprise from one to several bioactive compounds, present in a complex mixture. Conventional discovery efforts utilize bioassay-guided fractionation (BGF) to isolate individual active compounds. When applied to complex natural products, BGF is often challenged by an apparent loss of activity during fractionation, resulting in weakly active isolated compounds. Metabolomic analysis can potentially complement existing the BGF paradigm by capturing the chemical complexity of the metabolites. The proposed biochemometric approach establishes a link between the chemistry of a secondary metabolome and a deserved health impact, using a high-throughput, high-resolution capable biological endpoint. The proof of principle is demonstrated for the anti-tuberculosis (TB) activity of the Alaskan ethnobotanical, Oplopanax horridus. Biochemometric analysis identified the 100 most active constituents from thousands of metabolites in the active extract by means of 2D orthogonal chromatography using countercurrent and GC-MS methods. Previously isolated O. horridus phytoconstituents were used as reference markers of known structure and bio (in)activity. Positive correlations allowed distinction of anti-TB actives from inactive compounds. A total of 29 bioactives from 3 main structural classes were assigned based on MS data. Biochemometric analysis is a new tool for the standardization of herbal medicines and ethnobotanicals, as well as for drug discovery from nature. The method can assign multiple active compounds in complex mixtures without their prior isolation or structure elucidation, while still providing an interface to structural information.

Photoactivated [3+2] addition of 6,7-seco-angustilobine B to fullerene [C60].

A synthesis of a new alkaloid-fullerene conjugate (1) is reported. The reaction was carried out by photoinduced [3+2] cycloaddition of the Alstonia indole alkaloid, 6,7-seco-angustilobine B (2), to fullerene[C60] (3) under aerobic conditions. The major monoaddition photoadduct (1) was characterized unambiguously by UV, IR, MALDI-TOFMS and NMR experiments. A mechanism highlighted by sequential photoinduced electron transfer andradical recombination pathways is also proposed. No significant enhancement in inhibition against M. tuberculosis H37Rv was observed for 1 compared with its parent compounds 2 and 3.