Large-scale chemical-genetics yields new M. tuberculosis inhibitor classes.

New antibiotics are needed to combat rising levels of resistance, with new Mycobacterium tuberculosis (Mtb) drugs having the highest priority. However, conventional whole-cell and biochemical antibiotic screens have failed. Here we develop a strategy termed PROSPECT (primary screening of strains to prioritize expanded chemistry and targets), in which we screen compounds against pools of strains depleted of essential bacterial targets. We engineered strains that target 474 essential Mtb genes and screened pools of 100-150 strains against activity-enriched and unbiased compound libraries, probing more than 8.5 million chemical-genetic interactions. Primary screens identified over tenfold more hits than screening wild-type Mtb alone, with chemical-genetic interactions providing immediate, direct target insights. We identified over 40 compounds that target DNA gyrase, the cell wall, tryptophan, folate biosynthesis and RNA polymerase, as well as inhibitors that target EfpA. Chemical optimization yielded EfpA inhibitors with potent wild-type activity, thus demonstrating the ability of PROSPECT to yield inhibitors against targets that would have eluded conventional drug discovery.

Identification of Axinellamines A and B as Anti-Tubercular Agents.

Tuberculosis remains a significant global health pandemic. There is an urgent need for new anti-tubercular agents to combat the rising incidence of drug resistance and to offer effective and additive therapeutic options. High-throughput screening of a subset of the NatureBank marine fraction library (n = 2000) identified a sample derived from an Australian marine sponge belonging to the order Haplosclerida that displayed promising anti-mycobacterial activity. Bioassay-guided fractionation of the organic extract from this Haplosclerida sponge led to the purification of previously identified antimicrobial pyrrole alkaloids, axinellamines A (1) and B (2). The axinellamine compounds were found to have a 90% minimum inhibitory concentration (MIC90) of 18 µM and 15 µM, respectively. The removal of protein and complex carbon sources reduced the MIC90 of 1 and 2 to 0.6 and 0.8 µM, respectively. The axinellamines were not toxic to mammalian cells at 25 µM and significantly reduced the intracellular bacterial load by >5-fold. These data demonstrate that axinellamines A and B are effective anti-tubercular agents and promising targets for future medicinal chemistry efforts.

Chrysomycins, Anti-Tuberculosis C-Glycoside Polyketides from Streptomyces sp. MS751.

A new dimeric C-glycoside polyketide chrysomycin F (1), along with four new monomeric compounds, chrysomycins G (2), H (3), I (4), J (5), as well as three known analogues, chrysomycins A (6), B (7), and C (8), were isolated and characterised from a strain of Streptomyces sp. obtained from a sediment sample collected from the South China Sea. Their structures were determined by detailed spectroscopic analysis. Chrysomycin F contains two diastereomers, whose structures were further elucidated by a biomimetic [2 + 2] photodimerisation of chrysomycin A. Chrysomycins B and C showed potent anti-tuberculosis activity against both wild-type Mycobacterium tuberculosis and a number of clinically isolated MDR M. tuberculosis strains.

Kallopterolides A-I, a New Subclass of seco-Diterpenes Isolated from the Southwestern Caribbean Sea Plume Antillogorgia kallos.

Kallopterolides A-I (1-9), a family of nine diterpenoids possessing either a cleaved pseudopterane or a severed cembrane skeleton, along with several known compounds were isolated from the Caribbean Sea plume Antillogorgia kallos. The structures and relative configurations of 1-9 were characterized by analysis of HR-MS, IR, UV, and NMR spectroscopic data in addition to computational methods and side-by-side comparisons with published NMR data of related congeners. An investigation was conducted as to the potential of the kallopterolides as plausible in vitro anti-inflammatory, antiprotozoal, and antituberculosis agents.

Anti-mycobacterial activity of heat and pH stable high molecular weight protein(s) secreted by a bacterial laboratory contaminant.

BACKGROUND: Tuberculosis currently stands as the second leading cause of deaths worldwide due to single  infectious agent after Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The current challenges of drug resistance in tuberculosis highlight an urgent need to develop newer anti-mycobacterial compounds. In the present study, we report the serendipitous discovery of a bacterial laboratory contaminant (LC-1) exhibiting a zone of growth inhibition on an agar plate seeded with Mycobacterium tuberculosis. RESULTS: We utilized microbiological, biochemical and biophysical approaches to characterize LC-1 and anti-mycobacterial compound(s) in its secretome. Based on 16S rRNA sequencing and BIOLOG analysis, LC-1 was identified as Staphylococcus hominis, a human bacterial commensal. Anti-mycobacterial activity was initially found in 30 kDa retentate that was obtained by ultrafiltration of culture filtrate (CF). SDS-PAGE analysis of peak fractions obtained by size exclusion chromatography of 30 kDa retentate confirmed the presence of high molecular weight (≥ 30 kDa) proteins. Peak fraction-1 (F-1) exhibited inhibitory activity against M. bovis BCG, but not against M. smegmatis, E. coli and S. aureus. The active fraction F-1 was inactivated by treatment with Proteinase K and α-chymotrypsin. However, it retained its anti-mycobacterial activity over a wide range of heat and pH treatment. The anti-mycobacterial activity of F-1 was found to be maintained even after a long storage (~12 months) at - 20 °C. Mass spectrometry analysis revealed that the identified peptide masses do not match with any previously known bacteriocins. CONCLUSIONS: The present study highlights the anti-mycobacterial activity of high molecular weight protein(s) present in culture filtrate of LC-1, which may be tested further to target M. tuberculosis. The heat and pH stability of these proteins add to their characteristics as therapeutic proteins and may contribute to their long shelf life. LC-1 being a human commensal can be tested in future for its potential as a probiotic to treat tuberculosis.

Study of the Structure-Activity Relationship of an Anti-Dormant Mycobacterial Substance 3-(Phenethylamino)Demethyl(oxy)aaptamine to Create a Probe Molecule for Detecting Its Target Protein.

The current tuberculosis treatment regimen is long and complex, and its failure leads to relapse and emergence of drug resistance. One of the major reasons underlying the extended chemotherapeutic regimen is the ability of Mycobacterium tuberculosis to attain a dormant state. Therefore, the identification of new lead compounds with chemical structures different from those of conventional anti-tuberculosis drugs is essential. The compound 3-(phenethylamino)demethyl(oxy)aaptamine (PDOA, 1), isolated from marine sponge of Aaptos sp., is known as an anti-dormant mycobacterial substance, and has been reported to be effective against the drug resistant strains of M. tuberculosis. However, its target protein still remains unclear. This study aims to clarify the structure-activity relationship of 1 using 15 synthetic analogues, in order to prepare a probe molecule for detecting the target protein of 1. We succeeded in creating the compound 15 with a photoaffinity group that retained antimicrobial activity, which proved to be a suitable probe molecule for identifying the target protein of 1.

Bioactive Dimeric Tetrahydroxanthones with 2,2'- and 4,4'-Axial Linkages from the Entomopathogenic Fungus Aschersonia confluens.

Thirteen tetrahydroxanthone dimers, atrop-ascherxanthone A (1), ascherxanthones C-G (2-6), and confluxanthones A-G (7-13), were isolated from the entomopathogenic fungus Aschersonia confluens BCC53152. The chemical structures were determined based on analysis of NMR spectroscopic and mass spectrometric data. The absolute configurations of compounds 1 and 7 were confirmed by single-crystal X-ray diffraction experiments, while the configurations of other compounds were assigned based upon evidence from NOESY and NOEDIFF experiments, modified Mosher's method, and ECD spectroscopic data together with biogenetic considerations. Compounds 1, 3-5, 7-11, and 13 showed antimalarial activity against Plasmodium falciparum (K1, multidrug-resistant strain) (IC50 0.6-6.1 µM), antitubercular activity against Mycobacterium tuberculosis H37Ra (MIC 6.3-25.0 µg/mL), and cytotoxicity against NCI-H187 (IC50 0.5-3.5 µM) and Vero (IC50 0.9-6.1 µM) cells. All tested compounds except for compound 9 exhibited cytotoxicity against KB cells (IC50 1.3-9.7 µM).

Identification of fusarielin M as a novel inhibitor of Mycobacterium tuberculosis protein tyrosine phosphatase B (MptpB).

The secreted Mycobacterium tuberculosis (Mtb) protein tyrosine phosphatase B (MptpB) is an essential virulence factor required for the intracellular survival of Mtb within host macrophages. MptpB has become a promising target for the development of novel anti-tuberculosis (TB) drugs. In this study, two new fusarielins, fusarielins M (1) and N (2), and a biogenetically related known compound, fusarielin G (3) were isolated from the marine-derived fungus Fusarium graminearum SYSU-MS5127. Their inhibitory effects on MptpB were evaluated. Among these compounds, fusarielin M substantially inhibited MptpB with a half-maximal inhibitory concentration (IC50) of 1.05 ± 0.08 µM, and an inhibition constant (Ki) of 1.03 ± 0.39 µM. Surface plasmon resonance analysis was used to characterize the interaction between fusarielin M and MptpB in vitro. Fusarielin M also exhibited cellular activity in blocking MptpB-mediated Erk1/2 and p38 inactivation in macrophages. Importantly, fusarielin M (20 µM) substantially reduced intracellular mycobacterial growth within macrophages, causing a 62% reduction in the bacterial burden. The binding mode of fusarielin M was further explored via molecular docking which suggested that fusarielin M binds to the active site of MptpB, forming a hydrogen bond with the side chain of Asp165; this is unique in the P-loop of MptpB compared to conventional human PTPs. The contact between fusarielin M and Asp165 in the catalytic loop provides a potential basis for inhibitor selectivity. Therefore, fusarielin M shows great potential as an anti-TB drug candidate.

Marine Pharmacology in 2016-2017: Marine Compounds with Antibacterial, Antidiabetic, Antifungal, Anti-Inflammatory, Antiprotozoal, Antituberculosis and Antiviral Activities; Affecting the Immune and Nervous Systems, and Other Miscellaneous Mechanisms of Action.

The review of the 2016-2017 marine pharmacology literature was prepared in a manner similar as the 10 prior reviews of this series. Preclinical marine pharmacology research during 2016-2017 assessed 313 marine compounds with novel pharmacology reported by a growing number of investigators from 54 countries. The peer-reviewed literature reported antibacterial, antifungal, antiprotozoal, antituberculosis, and antiviral activities for 123 marine natural products, 111 marine compounds with antidiabetic and anti-inflammatory activities as well as affecting the immune and nervous system, while in contrast 79 marine compounds displayed miscellaneous mechanisms of action which upon further investigation may contribute to several pharmacological classes. Therefore, in 2016-2017, the preclinical marine natural product pharmacology pipeline generated both novel pharmacology as well as potentially new lead compounds for the growing clinical marine pharmaceutical pipeline, and thus sustained with its contributions the global research for novel and effective therapeutic strategies for multiple disease categories.

Blutaparon portulacoides ethanolic extract reduced IL-1ß and inflammatory parameters induced by the Mycobacterium complex and carrageenan in mice.

Information on the health benefits of ethanolic extracts obtained from Blutaparon portulacoides stem (EEBP) hasn´t been consistently described in the literature until the present moment. This study investigated the antimycobacterial, anti-inflammatory and toxicological effects of EEBP in models of inflammation/infection, as well as its chemical composition. Chemical analysis of EEBP by electrospray ionization-mass spectrometry/HPLC-MS/MS identified 3,5,3'-Trihydroxy-4'-methoxy-6,7-methylenedioxy-flavone, gomphrenol, ferulic, vanillic, and caffeic acids. The minimum inhibitory concentration of EEBP and isoniazid in the presence of Mycobacterium tuberculosis was 123.4 and 0.030 µg/ml, respectively. EEBP oral administration (p.o.) (300-1000 mg/kg) or dexamethasone subcutaneous injection (s.c.) (1 mg/kg) significantly inhibited leukocytes and proteins resulting from carrageenan-induced pleurisy in Swiss mice. In the BCG-induced pleurisy model, the oral treatments performed once a day for 7 days, with EEBP (30 and 100 mg/kg) and isoniazid (25 mg/kg), inhibited the increase in plasmatic IL-1ß levels and in pleural exudate from C57BL-6 mice, and reduced M. tuberculosis growth in organs (colony forming units assays). EEBP (30-300 mg/kg, p.o.) and dexamethasone (1 mg/s.c.) significantly prevented carrageenan-induced oedema and mechanical hyperalgesia in Swiss mice. The treatments (once a day for 22 days) with EEBP (30 mg/kg, p.o.) and dexamethasone (1 mg/s.c.) substantially inhibited oedema and mechanical- and cold-hyperalgesia at 11, 16 and 22 days after the administration of Freund's Complete Adjuvant in C57bL6 mice. No evidence of physio-pathologic was observed in Wistar rats acutely treated with EEBP (2000 mg/kg, p.o.). This study confirms the anti-inflammatory and antibiotic properties of EEBP, opening possibilities for the development of safe new drugs with dual anti-inflammatory/antimycobacterial activities which could be favorable from a pharmacoeconomic perspective.

Nonpeptidal compounds from the insect Polyphaga plancyi and their biological evaluation.

Five new nitrogen-containing compounds (1-3, 5, and 6), two compounds which was firstly isolated from natural origin (7 and 10), along with six known ones, were isolated from the ethanol extract of the whole bodies of Polyphaga plancyi. The structures of the new compounds including their absolute configurations at stereogenic centers were assigned on the basis of spectroscopic analyses and computational methods. Racemic 10 was separated by chiral HPLC. Biological activities of these isolates against extracellular matrix components in rat renal proximal tubular cells, EV71, COX-2, ROCK2, JAK3, and tuberculosis were evaluated. Importantly, 8 was found to be a selective Smad3 phosphorylation inhibitor.

Bioactive Metabolites from the Deep-Sea-Derived Fungus Diaporthe longicolla FS429.

The chemical investigation of a methanol extract of the deep-sea-derived fungus Diaporthe longicolla FS429 led to the isolation of two novel diterpenoids longidiacids A and B (1 and 2), two new polyketides (3 and 4), two new cytochalasin analogues longichalasins A and B (6 and 8) and three known analogues 5, 7, 9. Their structures were elucidated through comprehensive spectroscopic analysis, while the absolute configurations were established by the comparison of the experimental and quantum chemical calculated ECD spectra. The structure of compound 7 was confirmed through X-ray diffraction for the first time. In the bioassays compound 8 exhibited antiproliferative effects against SF-268, with an IC50 value of 16.44 µM. Moreover, compounds 1 and 8 were detected to inhibit 35.4% and 53.5% of enzyme activity of Mycobacterium tuberculosis protein tyrosine phosphatase B (MptpB) at a concentration of 50 µM.

Flavanones from the Twigs and Barks of Artocarpus lakoocha Having Antiplasmodial and Anti-TB Activities.

Chromatographic separation of the acetone extracts from the twigs and barks of Artocarpus lakoocha led to the isolation of the one new flavanone, lakoochanone (1), together with eleven known compounds (2-12). Lakoochanone (1) and moracin C (4) exhibited weak antiplasmodial activity against Plasmodium falciparum Dd2 with IC50 values of 36.7 and 33.9 µM, respectively. Moreover, moracin C (4) and sanggenofuran B (5) showed cytotoxic activity against A2780 cell line with the respective IC50 values of 15.0 and 57.1 µM. In addition, cyclocommunin (7) displayed strong antimycobacterial activity against Mycobacterium tuberculosis H37Ra with the minimum inhibitory concentration (MIC) value of 12.3 µM.

5-Alkylamino-N-phenylpyrazine-2-carboxamides: Design, Preparation, and Antimycobacterial Evaluation.

According to the World Health Organization, tuberculosis is still in the top ten causes of death from a single infectious agent, killing more than 1.7 million people worldwide each year. The rising resistance developed by Mycobacterium tuberculosis against currently used antituberculars is an imperative to develop new compounds with potential antimycobacterial activity. As a part of our continuous research on structural derivatives of the first-line antitubercular pyrazinamide, we have designed, prepared, and assessed the in vitro whole cell growth inhibition activity of forty-two novel 5-alkylamino-N-phenylpyrazine-2-carboxamides with various length of the alkylamino chain (propylamino to octylamino) and various simple substituents on the benzene ring. Final compounds were tested against Mycobacterium tuberculosis H37Ra and four other mycobacterial strains (M. aurum, M. smegmatis, M. kansasii, M. avium) in a modified Microplate Alamar Blue Assay. We identified several candidate molecules with micromolar MIC against M. tuberculosis H37Ra and low in vitro cytotoxicity in HepG2 cell line, for example, N-(4-hydroxyphenyl)-5-(pentylamino)pyrazine-2-carboxamide (3c, MIC = 3.91 µg/mL or 13.02 µM, SI > 38) and 5-(heptylamino)-N-(p-tolyl)pyrazine-2-carboxamide (4e, MIC = 0.78 µg/mL or 2.39 µM, SI > 20). In a complementary screening, we evaluated the in vitro activity against bacterial and fungal strains of clinical importance. We observed no antibacterial activity and sporadic antifungal activity against the Candida genus.

Identification and Characterization of pantocin wh-1, a Novel Cyclic Polypeptide Produced by Pantoea dispersa W18.

Pantoea dispersa W18, isolated from contaminated soil, was found to exert antimicrobial activity against Mycobacterium species, including Mycobacterium tuberculosis, an important human pathogen. Here, the anti-mycobacterial compound produced by Pantoea dispersa W18 was purified by a combination of hydrophobic interaction chromatography, cation exchange chromatography, and reverse phase HPLC. Active compounds from Pantoea dispersa W18 were identified as a natural peptide named pantocin wh-1 with a 1927 Da molecular weight. The primary structure of this compound was detected by N-terminal amino acid sequencing. The amino acid sequence of pantocin wh-1 consisted of 16 amino acid residues with a cyclic structure. The pantocin wh-1 could be inactivated by protease K, but was heat stable and unaffected by pH (2-12). However, the activity was not completely inactivated by trypsin and pepsin. This is the first report of a cyclic polypeptide purified from a strain of Pantoea dispersa.

Bioassay-guided isolation of a Mycobacterium tuberculosis bioflim inhibitor from Arisaema sinii Krause.

Mycobacterium tuberculosis biofilms harbour drug-tolerant bacteria. Identification of drugs that inhibit biofilm formation could enable the dramatic shortening of tuberculosis treatments using standard antibiotics. Arisaema sinii Krause is used to treat pulmonary and lymphatic tuberculosis by Dong People of China. Current study was aimed to purify the active components against M. tuberculosis biofilms from Arisaema sinii extract by using bioassay-guided isolation. (E)-2-(methyl (phenyl) amino) ethyl 2-(2-hydroxyundecanamido)-7, 11-dimethyl-3-oxotetradec-4-enoate, compound 1, was identified as the active component. It could inhibit mycobacterial biofilm formation, disperse the preformed biofilms, and disrupt the mature biofilms at concentration of 4, 8, and 32 µg/ml, respectively. At the dose of 32 µg/ml, it could potentiate the bactericidal activity of isoniazid against M. tuberculosis in mature biofilms. The results of this study indicate that compound 1 might be a novel lead compound against mycobacterial biofilm formation.

Isolation of Tryptanthrin and Reassessment of Evidence for Its Isobaric Isostere Wrightiadione in Plants of the Wrightia Genus.

A series of Wrightia hanleyi extracts was screened for activity against Mycobacterium tuberculosis H37Rv. One active fraction contained a compound that initially appeared to be either the isoflavonoid wrightiadione or the alkaloid tryptanthrin, both of which have been previously reported in other Wrightia species. Characterization by NMR and MS, as well as evaluation of the literature describing these compounds, led to the conclusion that wrightiadione (1) was misidentified in the first report of its isolation from W. tomentosa in 1992 and again in 2015 when reported in W. pubescens and W. religiosa. Instead, the molecule described in these reports and in the present work is almost certainly the isobaric (same nominal mass) and isosteric (same number of atoms, valency, and shape) tryptanthrin (2), a well-known quinazolinone alkaloid found in a variety of plants including Wrightia species. Tryptanthrin (2) is also accessible synthetically via several routes and has been thoroughly characterized. Wrightiadione (1) has been synthesized and characterized and may have useful biological activity; however, this compound can no longer be said to be known to exist in Nature. To our knowledge, this misidentification of wrightiadione (1) has heretofore been unrecognized.

Lassomycin and lariatin lasso peptides as suitable antibiotics for combating mycobacterial infections: current state of biosynthesis and perspectives for production.

Lasso peptides are ribosomally synthesized and post-translationally modified natural products with a characteristic slipknot-like structure, which confers these peptides remarkable stability and diverse pharmacologically relevant bioactivities. Among all the reported lasso peptides, lassomycin and lariatins are unique lasso peptides that exhibit noticeable anti-tuberculosis (TB) activity. Due to the unique threaded structure and the unusual bactericidal mechanism toward Mycobacterium tuberculosis, these peptides have drawn considerable interest, not only in the field of total synthesis but also in several other fields including biosynthesis, bioengineering, and structure-activity studies. During the past few years, significant progress has been made in understanding the biosynthetic mechanism of these intriguing compounds, which has provided a solid foundation for future work. This review highlights recent achievements in the discovery, structure elucidation, biological activity, and the unique anti-TB mechanism of lasso peptides. Moreover, the discovery of their biosynthetic pathway has laid the foundation for combinatorial biosynthesis of their analogs, which provides new perspectives for the production of novel anti-TB lasso peptides.

Diterpenoids from the roots of Euphorbia ebracteolata and their anti-tuberculosis effects.

Euphorbia ebracteolata was a natural medicine for the treatment of tuberculosis. The present work has performed the investigation of bioactive chemical substances from the roots of E. ebracteolata. Using various chromatographic techniques, 15 compounds were obtained from the roots of E. ebracteolata. On the basis of widely spectroscopic data analyses, the isolated compounds were determined to be diterpenoids, including rosane derivatives (1-12), isopimarane (13), abietane (14), and lathyrane (15), among which compounds 1-4, and 9 were undescribed previously. The inhibitory effects of isolated diterpenoids against Mycobacterium tuberculosis were evaluated using an Alamar blue cell viability assay. And two rosane-type diterpenoids 3 and 8 displayed moderate inhibitory effects on with the MIC values of 18 µg/mL and 25 µg/mL, respectively. For the potential inhibitor 3, the inhibitory effect against the target enzyme GlmU was evaluated, which displayed a moderate inhibitory effect with the IC50 12.5 µg/mL. Therefore, the diterpenoids from the roots of E. ebracteolata displayed anti-tuberculosis effects, which would be pay more attentions for the anti-tuberculosis agents.

Antitubercular evaluation of root extract and isolated phytochemicals from Lophira lanceolata against two resistant strains of Mycobacterium tuberculosis.

CONTEXT: The roots of Lophira lanceolata Van Tiegh. Ex Keay (Ochnaceae) have numerous medicinal values in the Central African region. Even though the MeOH extract of the roots has shown antimycobacterial activities, the constituents responsible for this inhibitory activity remain unknown. OBJECTIVE: Phytochemical investigation of the MeOH root extract of L. lanceolata and determination of the antimycobacterial activities of that extract and constituents against the growth of Mycobacterium tuberculosis. MATERIALS AND METHODS: Column chromatography was used to provide bioactive phytoconstituents. Those compounds were elucidated using MS and NMR spectroscopic data. Antimycobacterial screening of the extract (4.882-5000 µg/mL in DMSO during 24 h at 37 °C) and isolated compounds (0.244-250 µg/mL in DMSO during 24 h at 37 °C) was performed by microplate alamar blue assay (MABA) against two mycobacterial strains. RESULTS: The investigation of L. lanceolata MeOH roots extract provided of mixture of unseparated biflavonoids with a newly described one, dihydrolophirone A (1a) associated to lophirone A (1b). The bioactive compounds that effectively inhibited the growth of M. tuberculosis AC45 were found to be compounds 1 and 2. They exhibited MIC values of 31.25 and 15.75 µg/mL, respectively, and their MIC was found to be 62.5 µg/mL against resistant strain AC83. DISCUSSION AND CONCLUSIONS: It is clearly evident from the results obtained that the mycobacterial activity of L. lanceolata could be related mainly to its steroid and flavonoid contents. Therefore, this study suggests the potential of the above-mentioned classes of compounds as promising candidate agents for developing new anti-tuberculosis drugs.