Mavintramycin A is a promising antibiotic for treating Mycobacterium avium complex infectious disease.

Mycobacterium avium complex (MAC) is a serious disease that is mainly caused by infection with the non-tuberculous mycobacteria (NTM), Mycobacterium avium and Mycobacterium intracellulare. Seven new compounds, designated mavintramycins A-G (1-7), were isolated along with structurally related compounds, including amicetin (9) and plicacetin (10), from the culture broth of Streptomyces sp. OPMA40551 as anti-MAC compounds that were active against M. avium and M. intracellulare. Among them, mavintramycin A showed the most potent and selective inhibition of M. avium and M. intracellulare. Furthermore, mavintramycin A was active against more than 40 clinically isolated M. avium, including multidrug-resistant strains, and inhibited the growth of M. avium in a persistent infection cell model using THP-1 macrophages. Mavintramycin A also exhibited in vivo efficacy in silkworm and mouse infection assays with NTM. An experiment to elucidate its mechanism of action revealed that mavintramycin A inhibits protein synthesis by binding to 23S ribosomal RNA in NTM. Mavintramycin A, with a different chemical structure from those of clinically used agents, is a promising drug candidate for the treatment of MAC infectious disease.

Evaluation of Anti-Mycobacterial Compounds in a Silkworm Infection Model with Mycobacteroides abscessus.

Among four mycobacteria, Mycobacterium avium, M. intracellulare, M. bovis BCG and Mycobacteroides (My.) abscessus, we established a silkworm infection assay with My. abscessus. When silkworms (fifth-instar larvae, n = 5) were infected through the hemolymph with My. abscessus (7.5 × 107 CFU/larva) and bred at 37 °C, they all died around 40 h after injection. Under the conditions, clarithromycin and amikacin, clinically used antimicrobial agents, exhibited therapeutic effects in a dose-dependent manner. Furthermore, five kinds of microbial compounds, lariatin A, nosiheptide, ohmyungsamycins A and B, quinomycin and steffimycin, screened in an in vitro assay to observe anti-My. abscessus activity from 400 microbial products were evaluated in this silkworm infection assay. Lariatin A and nosiheptide exhibited therapeutic efficacy. The silkworm infection model with My. abscessus is useful to screen for therapeutically effective anti-My. abscessus antibiotics.

Discovery of Nosiheptide, Griseoviridin, and Etamycin as Potent Anti-Mycobacterial Agents against Mycobacterium avium Complex.

Mycobacterium avium complex (MAC) is a serious disease mainly caused by M. avium and M. intracellulare. Although the incidence of MAC infection is increasing worldwide, only a few agents are clinically used, and their therapeutic effects are limited. Therefore, new anti-MAC agents are needed. Approximately 6600 microbial samples were screened for new anti-mycobacterial agents that inhibit the growth of both M. avium and M. intracellulare, and two culture broths derived from marine actinomycete strains OPMA1245 and OPMA1730 had strong activity. Nosiheptide (1) was isolated from the culture broth of OPMA1245, and griseoviridin (2) and etamycin (viridogrisein) (3) were isolated from the culture broth of OPMA1730. They had potent anti-mycobacterial activity against M. avium and M. intracellulare with minimum inhibitory concentrations (MICs) between 0.024 and 1.56 µg/mL. In addition, a combination of 2 and 3 markedly enhanced the anti-mycobacterial activity against both M. avium and M. intracellulare. Furthermore, a combination 2 and 3 had a therapeutic effect comparable to that of ethambutol in a silkworm infection assay with M. smegmatis.

Identification and characterization of natural microbial products that alter the free d-aspartate content of mammalian cells.

Mammalian cells possess the molecular apparatus necessary to take up, degrade, synthesize, and release free d-aspartate, which plays an important role in physiological functions within the body. Here, biologically active microbial compounds and pre-existing drugs were screened for their ability to alter the intracellular d-aspartate level in mammalian cells, and several candidate compounds were identified. Detailed analytical studies suggested that two of these compounds, mithramycin A and geldanamycin, suppress the biosynthesis of d-aspartate in cells. Further studies suggested that these compounds act at distinct sites within the cell. These compounds may advance our current understanding of biosynthesis of d-aspartate in mammals, a whole picture of which remains to be disclosed.

Mass spectral similarity for untargeted metabolomics data analysis of complex mixtures.

While in nucleotide sequencing, the analysis of DNA from complex mixtures of organisms is common, this is not yet true for mass spectrometric data analysis of complex mixtures. The comparative analyses of mass spectrometry data of microbial communities at the molecular level is difficult to perform, especially in the context of a host. The challenge does not lie in generating the mass spectrometry data, rather much of the difficulty falls in the realm of how to derive relevant information from this data. The informatics based techniques to visualize and organize datasets are well established for metagenome sequencing; however, due to the scarcity of informatics strategies in mass spectrometry, it is currently difficult to cross correlate two very different mass spectrometry data sets from microbial communities and their hosts. We highlight that molecular networking can be used as an organizational tool of tandem mass spectrometry data, automated database search for rapid identification of metabolites, and as a workflow to manage and compare mass spectrometry data from complex mixtures of organisms. To demonstrate this platform, we show data analysis from hard corals and a human lung associated with cystic fibrosis.

The antiviral drug acyclovir is a slow-binding inhibitor of (D)-amino acid oxidase.

d-Amino acid oxidase (DAO) is a degradative enzyme that is stereospecific for d-amino acids, including d-serine and d-alanine, which are believed to be coagonists of the N-methyl-d-aspartate (NMDA) receptor. To identify a new class of DAO inhibitor(s) that can be used to elucidate the molecular details of the active site environment of DAO, manifold biologically active compounds of microbial origin and pre-existing drugs were screened for their ability to inhibit DAO activity, and several compounds were identified as candidates. One of these compounds, acyclovir (ACV), a well-known antiviral drug used for the treatment of herpesvirus infections, was characterized and evaluated as a novel DAO inhibitor in vitro. Analysis showed that ACV acts on DAO as a reversible slow-binding inhibitor, and interestingly, the time required to achieve equilibrium between DAO, ACV, and the DAO/ACV complex was highly dependent on temperature. The binding mechanism of ACV to DAO was investigated in detail by several approaches, including kinetic analysis, structural modeling of DAO complexed with ACV, and site-specific mutagenesis of an active site residue postulated to be involved in the binding of ACV. The results confirm that ACV is a novel, active site-directed inhibitor of DAO that can be a valuable tool for investigating the structure-function relationships of DAO, including the molecular details of the active site environment of DAO. In particular, it appears that ACV can serve as an active site probe to study the structural basis of temperature-induced conformational changes of DAO.

Discovery of nosokophic acid, a predicted intermediate of moenomycins, from nosokomycin-producing Streptomyces sp. K04-0144.

A new actinomycete metabolite designated nosokophic acid was isolated from the culture broth of nosokomycin-producing Streptomyces sp. K04-0144, and the structure was elucidated by various NMR experiments. Nosokophic acid was found to be 3-phosphoglycosyl-2-sesquiterpenyl dihydroxypropionic acid, a predicted biosynthetic intermediate of nosokomycin-related moenomycins. The compound showed no activity against MRSA, but potentiated imipenem activity against MRSA by 512-fold.

Synthesis and antimycobacterial activity of calpinactam derivatives.

Synthesis of calpinactam 1, a fungal antimycobacterial metabolite, utilizing solid-phase peptide synthesis is described. To explore the structure-activity relationships of 1, its derivatives with different amino acids were also synthesized on the basis of the same synthetic strategy. These derivatives were examined for antimycobacterial activity against Mycobacterium smegmatis. Among them, only peptide 6d having d-Ala in place of d-Glu showed moderate activity.

Search method for inhibitors of Staphyloxanthin production by methicillin-resistant Staphylococcus aureus.

Staphyloxanthin, a yellow pigment produced by methicillin-resistant Staphylococcus aureus (MRSA), is a virulent factor escaping from the host immune system. A new screening method for inhibitors of staphyloxanthin production by MRSA was established using paper disks. By this screening method, inhibitors of staphyloxanthin production were selected from the natural product library (ca. 300) and from actinomycete culture broths (ca. 1000). From the natural product library, four known inhibitors of lipid metabolism, cerulenin, dihydrobisvertinol, xanthohumol and zaragozic acid, were found to inhibit staphyloxanthin production; however, typical antibiotics used clinically, including vancomycin, had no effect on staphyloxanthin production. From actinomycete culture broths, two known anthraquinones, 6-deoxy-8-O-methylrabelomycin and tetrangomycin, were found to inhibit staphyloxanthin production by MRSA in the paper disk assay. These results suggested that this screening method is useful and effective to find compounds targeting staphyloxanthin production, leading to a new type of chemotherapeutics against MRSA infection.

Anti-infectious agents against MRSA.

Clinically useful antibiotics, ß-lactams and vancomycin, are known to inhibit bacterial cell wall peptidoglycan synthesis. Methicillin-resistant Staphylococcus aureus (MRSA) has a unique cell wall structure consisting of peptidoglycan and wall teichoic acid. In recent years, new anti-infectious agents (spirohexaline, tripropeptin C, DMPI, CDFI, cyslabdan, 1835F03, and BPH-652) targeting MRSA cell wall biosynthesis have been discovered using unique screening methods. These agents were found to inhibit important enzymes involved in cell wall biosynthesis such as undecaprenyl pyrophosphate (UPP) synthase, FemA, flippase, or UPP phosphatase. In this review, the discovery, the mechanism of action, and the future of these anti-infectious agents are described.

Kimidinomycin, a new antibiotic against Mycobacterium avium complex, produced by Streptomyces sp. KKTA-0263.

During our screening for antibiotics against Mycobacterium avium complex (MAC) with a mass spectrometry network-based indexing approach, a new compound named kimidinomycin was isolated from the culture broth of Streptomyces sp. KKTA-0263 by solvent extraction, HP20 column chromatography, and preparative HPLC. From the structural elucidation, the compound possesses a 38-membered macrolide structure with an N-methylguanidyl group at the terminal side chain. The compound exhibited antimycobacterial activity against M. avium, M. intracellulare, M. smegmatis, and M. bovis BCG with respective MIC values of 12.5, 0.78, 12.5, and 25.0 µg ml-1.

Quantitative analysis of selective glycosylation of saccharides with aromatic amines.

Glycosylation, a part of the Maillard reaction, occurs non-enzymatically in food and biological processes. The selectivity of N-glycosylation was analyzed based on the reactivity of monosaccharides with aromatic amines, including aromatic amino acids, and the degree of molecular interaction (MI) measured using liquid chromatography. Furthermore, the chemical structures of reaction products were determined using X-ray crystallography and/or NMR. The possible reaction products were estimated in silico using the optimized energy values of different conformations. The MI energy values of amino groups and saccharides were calculated using in silico analysis using a model phase. Saccharides having larger MI values easily produced stable crystals of N-glycosides. The reaction rate of glucose (an energy saccharide) was slow, and it easily produced the Amadori compounds. The study of the reactivity of aromatic amines with saccharides, the measurement of the retention of monosaccharides on amino phase in chromatography, and the synthesis of N-glycosides for the determination of their structures will provide useful information about selective glycosylation for the modification of drug candidates to improve their water solubility.

Steffimycin E, a new anti-mycobacterial agent against Mycobacterium avium complex, produced by Streptomyces sp. OPMA02852.

The marine actinomycete strain OPMA02852, identified as the genus Streptomyces, was found to produce anti-mycobacterial compounds against Mycobacterium avium complex (MAC). One new compound, designated as steffimycin E (1), was isolated together with three known steffimycins (steffimycin (2), 10-dihydrosteffimycin (3), and 8-demethoxysteffimycin (4)) from the culture broth of this producing microorganism by solvent extraction, ODS column chromatography, and preparative HPLC. Compound 1 has a tetracyclic quinone structure with a sugar moiety. Compound 1 exhibited anti-mycobacterial activity against M. intracellulare, M. bovis BCG, and M. smegmatis.

A new diketopiperazine-like inhibitor of bone morphogenetic protein-induced osteoblastic differentiation produced by marine-derived Aspergillus sp. BFM-0085.

A new diketopiperazine-like compound, designated protuboxepin K (1), was isolated together with the known structurally related protuboxepin A (2) from culture broth of the marine-derived fungal strain Aspergillus sp. BFM-0085 isolated from a sediment sample of Tokyo Bay. The structure of protuboxepin K was elucidated by spectroscopic data, including 1D and 2D NMR. Compounds 1 and 2 inhibited bone morphogenetic protein (BMP)-induced alkaline phosphatase activity with IC50 values of 4.7 and 25.2 µM, respectively, in mutant BMP receptor-carrying C2C12(R206H) cells.

Inhibition of lipid droplet accumulation in mouse macrophages by stemphone derivatives.

From a study on the biological activity of fungal stemphones and their derivatives, five derivatives having an O-alkyl moiety at C-11 of stemphone C were found to inhibit lipid droplet accumulation in macrophages without any cytotoxic effect. Among the derivatives, those having O-isopropyl and O-isobutyl were the most potent inhibitors by blocking the synthesis of both cholesteryl ester (CE) and triacylglycerol (TG), the main constituents of lipid droplets in macrophages.

Structure-activity relationships of stemphones, potentiators of imipenem activity against methicillin-resistant Staphylococcus aureus.

From a further purification study, four new stemphones D to G were isolated along with previously reported stemphones B and C from the culture broth of Aspergillus sp. FKI-2136. Twenty-one derivatives were semisynthetically prepared from stemphones C, E and G. Potentiation of imipenem activity against methicillin-resistant Staphylococcus aureus (MRSA) by all the stemphones including natural and semisynthetic ones was compared to study the structure-activity relationships. Derivatives with a free hydroxy or an O-acyl residue having a C2 to C5 carbon length at C-4 held the potentiating activity, but those with a longer acyl residue lost the activity. The presence of an oxo or a free hydroxy residue at C-10 is important for the potentiating activity because introduction of an alkyl or acyl residue at this position resulted in a loss of activity. Among them, stemphone E exhibited the most potent potentiation of imipenem activity against MRSA and the lowest cytotoxic activity against Jurkat cells.

Anti-Rhizopus activity of tanzawaic acids produced by the hot spring-derived fungus Penicillium sp. BF-0005.

A silkworm infection assay with the pathogenic fungus Rhizopus oryzae was established. Microbial culture broths were screened for anti-Rhizopus antibiotics using this assay. A new compound, tanzawaic acid R was isolated along with known and structurally related tanzawaic acids and arohynapene A from the culture broth of the hot spring-derived fungus Penicillium sp. BF-0005. The structure of tanzawaic acid R was elucidated by various spectroscopic data including 1D and 2D nuclear magnetic resonance spectroscopy. Tanzawaic acids A, B, C, and R and arohynapene A exhibited antifungal activity against R. oryzae. Tanzawaic acids A and B dose-dependently exerted therapeutic effects in the silkworm infection assay with R. oryzae.

Fungal isobisvertinol, a new inhibitor of lipid droplet accumulation in mouse macrophages.

[structure: see text] New isobisvertinol and known bisvertinol were isolated from the culture broth of Aspergillus sp. FKI-1746. Isobisvertinol with the two alkenyl side chains extending in the same direction inhibited lipid droplet accumulation in macrophages, whereas bisvertinol with those extending in the reverse direction had almost no effect on the accumulation.

Structure-Activity Analysis of Gram-positive Bacterium-producing Lasso Peptides with Anti-mycobacterial Activity.

Lariatin A, an 18-residue lasso peptide encoded by the five-gene cluster larABCDE, displays potent and selective anti-mycobacterial activity. The structural feature is an N-terminal macrolactam ring, through which the C-terminal passed to form the rigid lariat-protoknot structure. In the present study, we established a convergent expression system by the strategy in which larA mutant gene-carrying plasmids were transformed into larA-deficient Rhodococcus jostii, and generated 36 lariatin variants of the precursor protein LarA to investigate the biosynthesis and the structure-activity relationships. The mutational analysis revealed that four amino acid residues (Gly1, Arg7, Glu8, and Trp9) in lariatin A are essential for the maturation and production in the biosynthetic machinery. Furthermore, the study on structure-activity relationships demonstrated that Tyr6, Gly11, and Asn14 are responsible for the anti-mycobacterial activity, and the residues at positions 15, 16 and 18 in lariatin A are critical for enhancing the activity. This study will not only provide a useful platform for genetically engineering Gram-positive bacterium-producing lasso peptides, but also an important foundation to rationally design more promising drug candidates for combatting tuberculosis.

A metabolomics guided exploration of marine natural product chemical space.

INTRODUCTION: Natural products from culture collections have enormous impact in advancing discovery programs for metabolites of biotechnological importance. These discovery efforts rely on the metabolomic characterization of strain collections. OBJECTIVE: Many emerging approaches compare metabolomic profiles of such collections, but few enable the analysis and prioritization of thousands of samples from diverse organisms while delivering chemistry specific read outs. METHOD: In this work we utilize untargeted LC-MS/MS based metabolomics together with molecular networking to. RESULT: This approach annotated 76 molecular families (a spectral match rate of 28 %), including clinically and biotechnologically important molecules such as valinomycin, actinomycin D, and desferrioxamine E. Targeting a molecular family produced primarily by one microorganism led to the isolation and structure elucidation of two new molecules designated maridric acids A and B. CONCLUSION: Molecular networking guided exploration of large culture collections allows for rapid dereplication of know molecules and can highlight producers of uniques metabolites. These methods, together with large culture collections and growing databases, allow for data driven strain prioritization with a focus on novel chemistries.