Sattahipmycin, a Hexacyclic Xanthone Produced by a Marine-Derived Streptomyces.

Sattahipmycin was isolated from the mycelium of marine-derived Streptomyces sp. GKU 257-1 by following the antibiofilm activity against E. coli NBRC 3972 throughout the purification steps. The structure of sattahipmycin was determined to be a new polycyclic xanthone related to xantholipin but lacking a dioxymethylene and a chlorinated carbon. This compound showed activity toward Gram-positive bacteria and Plasmodium falciparum, antibiofilm formation of Escherichia coli, and cytotoxicity to human cancer cell lines. Using genome sequence data, a biosynthetic pathway leading to sattahipmycin has been proposed involving an uncharacterized type II polyketide synthase biosynthetic gene cluster.

New Antimicrobial Phenyl Alkenoic Acids Isolated from an Oil Palm Rhizosphere-Associated Actinomycete, Streptomyces palmae CMU-AB204T.

Basal stem rot (BSR), or Ganoderma rot disease, is the most serious disease associated with the oil palm plant of Southeast Asian countries. A basidiomycetous fungus, Ganoderma boninense, is the causative microbe of this disease. To control BSR in oil palm plantations, biological control agents are gaining attention as a major alternative to chemical fungicides. In the course of searching for effective actinomycetes as potential biological control agents for BSR, Streptomyces palmae CMU-AB204T was isolated from oil palm rhizosphere soil collected on the campus of Chiang Mai University. The culture broth of this strain showed significant antimicrobial activities against several bacteria and phytopathogenic fungi including G. boninense. Antifungal and antibacterial compounds were isolated by antimicrobial activity-guided purification using chromatographic methods. Their structures were elucidated by spectroscopic techniques, including Nuclear Magnetic Resonance (NMR), Mass Spectrometry (MS), Ultraviolet (UV), and Infrared (IR) analyses. The current study isolated new phenyl alkenoic acids 1-6 and three known compounds, anguinomycin A (7), leptomycin A (8), and actinopyrone A (9) as antimicrobial agents. Compounds 1 and 2 displayed broad antifungal activity, though they did not show antibacterial activity. Compounds 3 and 4 revealed a strong antibacterial activity against both Gram-positive and Gram-negative bacteria including the phytopathogenic strain Xanthomonas campestris pv. oryzae. Compounds 7-9 displayed antifungal activity against Ganoderma. Thus, the antifungal compounds obtained in this study may play a role in protecting oil palm plants from Ganoderma infection with the strain S. palmae CMU-AB204T.

Discovery of Antiamebic Compounds That Inhibit Cysteine Synthase From the Enteric Parasitic Protist Entamoeba histolytica by Screening of Microbial Secondary Metabolites.

Amebiasis is caused by infection with the protozoan parasite Entamoeba histolytica. Although metronidazole has been a drug of choice against amebiasis for decades, it shows side effects and low efficacy against asymptomatic cyst carriers. In addition, metronidazole resistance has been documented for bacteria and protozoa that share its targets, anaerobic energy metabolism. Therefore, drugs with new mode of action or targets are urgently needed. L-cysteine is the major thiol and an essential amino acid for proliferation and anti-oxidative defense of E. histolytica trophozoites. E. histolytica possesses the de novo L-cysteine biosynthetic pathway, consisting of two reactions catalyzed by serine acetyltransferase and cysteine synthase (CS, O-acetylserine sulfhydrylase). As the pathway is missing in humans, it is considered to be a rational drug target against amebiasis. In this study, we established a protocol to screen both a library of structurally known compounds and microbial culture extracts to discover compounds that target de novo cysteine biosynthesis of E. histolytica. The new screening system allowed us to identify the compounds that differentially affect the growth of the trophozoites in the cysteine-deprived medium compared to the cysteine-containing medium. A total of 431 structurally defined compounds of the Kitasato Natural Products Library and 6,900 microbial culture broth extracts were screened on the system described above. Five compounds, aspochalasin B, chaetoglobosin A, prochaetoglobosin III, cerulenin, and deoxyfrenolicin, from the Kitasato Natural Products Library, showed differential antiamebic activities in the cysteine-deprived medium when compared to the growth in the cysteine-containing medium. The selectivity of three cytochalasans apparently depends on their structural instability. Eleven microbial extracts showed selective antiamebic activities, and one fungal secondary metabolite, pencolide, was isolated. Pencolide showed cysteine deprivation-dependent antiamebic activity (7.6 times lower IC50 in the absence of cysteine than that in the presence of cysteine), although the IC50 value in the cysteine-deprived medium was rather high (283 µM). Pencolide also showed inhibitory activity against both CS1 and CS3 isoenzymes with comparable IC50 values (233 and 217 µM, respectively). These results indicated that antiamebic activity of pencolide is attributable to inhibition of CS. Cytotoxicity of pencolide was 6.7 times weaker against mammalian MRC-5 cell line than E. histotytica. Pencolide has the maleimide structure, which is easily attacked by Michael donors including the thiol moiety of cysteine. The cysteine-adducts of pencolide were detected by mass spectrometric analysis as predicted. As CS inhibition by the pencolide adducts was weak and their IC50 values to CS was comparable to that to the parasite in the cysteine-containing medium, the cysteine-adducts of pencolide likely contribute to toxicity of pencolide to the parasite in the cysteine-rich conditions. However, we cannot exclude a possibility that pencolide inactivates a variety of targets other than CSs in the absence of cysteine. Taken together, pencolide is the first compound that inhibits CS and amebic cell growth in a cysteine-dependent manner with relatively low mammalian cytotoxicity.

Pestynol, an Antifungal Compound Discovered Using a Saccharomyces cerevisiae 12geneΔ0HSR-iERG6-Based Assay.

The multidrug-sensitive budding yeast, Saccharomyces cerevisiae 12geneΔ0HSR-iERG6, is very useful in antifungal screens. A novel compound, named pestynol (1), was discovered from a culture of the fungus Pestalotiopsis humus FKI-7473 using the multidrug-sensitive yeast. The structure of 1 was elucidated by NMR studies and modified Mosher's method as (1 R,2 R,3 R,4 R)-( E)-5-(7,11-dimethyl-3-methylenedodeca-6,10-dien-1-yn-1-yl)cyclohex-5-ene-1,2,3,4-tetraol. Compound 1 showed antimicrobial activity against the Gram-positive bacteria, Klebsiella pneumoniae, and S. cerevisiae 12geneΔ0HSR-iERG6 and Mucor racemosus, but displayed only weak cytotoxicity against various human cancer cell lines. Compound 1 displayed antifungal activities against S. cerevisiae 12geneΔ0HSR-iERG6 and Mucor racemosus at 10 µg/disc.

Tolyprolinol, a new dipeptide from Tolypocladium sp. FKI-7981.

A new dipeptide, named tolyprolinol, was isolated from the static culture of a fungus, Tolypocladium sp. FKI-7981. The structure of tolyprolinol was elucidated as N-acetyl-L-phenylalanyl-L-prolinol. It showed moderate antimalarial activity but did not show cytotoxicity or any other antimicrobial property.

Characterization and validation of Entamoeba histolytica pantothenate kinase as a novel anti-amebic drug target.

The Coenzyme A (CoA), as a cofactor involved in >100 metabolic reactions, is essential to the basic biochemistry of life. Here, we investigated the CoA biosynthetic pathway of Entamoeba histolytica (E. histolytica), an enteric protozoan parasite responsible for human amebiasis. We identified four key enzymes involved in the CoA pathway: pantothenate kinase (PanK, EC 2.7.1.33), bifunctional phosphopantothenate-cysteine ligase/decarboxylase (PPCS-PPCDC), phosphopantetheine adenylyltransferase (PPAT) and dephospho-CoA kinase (DPCK). Cytosolic enzyme PanK, was selected for further biochemical, genetic, and phylogenetic characterization. Since E. histolytica PanK (EhPanK) is physiologically important and sufficiently divergent from its human orthologs, this enzyme represents an attractive target for the development of novel anti-amebic chemotherapies. Epigenetic gene silencing of PanK resulted in a significant reduction of PanK activity, intracellular CoA concentrations, and growth retardation in vitro, reinforcing the importance of this gene in E. histolytica. Furthermore, we screened the Kitasato Natural Products Library for inhibitors of recombinant EhPanK, and identified 14 such compounds. One compound demonstrated moderate inhibition of PanK activity and cell growth at a low concentration, as well as differential toxicity towards E. histolytica and human cells.

Identification and Characterization of a Novel NADPH Oxidase 1 (Nox1) Inhibitor That Suppresses Proliferation of Colon and Stomach Cancer Cells.

Reactive oxygen species (ROS) generated by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox)1 mediate cellular signalings involved in normal physiological processes, and aberrant control of Nox1 has been implicated in the pathogenesis of various diseases. Therefore, Nox1 could have great potential as a therapeutic target. Here, we identified a novel Nox1 inhibitor, NOS31 secreted from Stretomyces sp. and analyzed its chemical structure. Furthermore, NOS31 was found to selectively inhibit Nox1-mediated ROS generation, with only a marginal effect on other Nox isoforms (Nox2-5) and no ROS scavenging activity. This compound blocked both Nox organizer 1 (NOXO1)/Nox activator 1 (NOXA1)-dependent and phorbol 12-myristate 13-acetate-stimulated Nox1-mediated ROS production in colon cancer cells. NOS31 inhibited the proliferation of several colon carcinoma and gastric cancer cell lines that upregulate the Nox1 system, whereas it had no appreciable effect on normal cells with low levels of Nox1. The finding suggests that NOS31 is a unique, potent Nox1 inhibitor of microbial origin and raises its possibility as a therapeutic agent for inhibiting gastrointestinal cancer cell growth.

Marcanine G, a new cytotoxic 1-azaanthraquinone from the stem bark of Goniothalamus marcanii Craib.

The ethanolic extract from the stem bark of Goniothalamus marcanii Craib was shown in preliminary brine shrimp lethality data having good cytotoxic activity. Further bioassay guided isolation was done by means of solvent partition, chromatography and precipitation to provide four isolated compounds: a novel compound 1 with the core structure of 1-azaanthraquinone moiety referred as marcanine G; as well as compounds 2-4 with known aristolactam structures namely, piperolactam C, cepharanone B and taliscanine. These compounds were characterised by spectroscopic techniques. The assessment of cytotoxicity was established on an SRB assay using doxorubicin as a positive control. Marcanine G (1) was considered the most active compound indicating the IC50 values of 14.87 and 15.18 µM against human lung cancer cells (A549) and human breast cancer cells (MCF7), respectively. However, 2 showed mild activity with the IC50 values of 83.72 and 82.32 µM against A549 and MCF7 cells, respectively.

1-Methoxypyrrole-2-carboxamide-A new pyrrole compound isolated from Streptomyces griseocarneus SWW368.

A new pyrrole compound, 1-methoxypyrrole-2-carboxamide, was obtained from a culture broth of Streptomyces griseocarneus SWW368, which was isolated from the rhizospheric soil under a Para rubber tree (Hevea brasiliensis). The chemical structure was elucidated by 1D NMR, 2D NMR, and MS, as a pyrrole ring with a N-methoxy group and a primary amide group. It exhibited antibacterial properties against Kocuria rhizophila, Staphylococcus aureus and Xanthomonas campestris pv. oryzae; however, cytotoxicity of the compound at 714 µM against several mammalian tumor cell lines, i.e. A549, PANC1, HT29, HT1299 and HeLa S3, were not detected.

Decatamariic acid, a new mitochondrial respiration inhibitor discovered by pesticidal screening using drug-sensitive Saccharomyces cerevisiae.

A new decalin, decatamariic acid, was isolated from a cultured broth of the fungus Aspergillus tamarii FKI-6817. Its absolute configuration was elucidated by NMR and electronic circular dichroism. Decatamariic acid (10 µM) elicited ~50% inhibition of the ATP production in mitochondria isolated from wild-type Saccharomyces cerevisiae without affecting the activities of respiratory enzymes. The action manner of this compound may be interesting as a possible seed for new pesticides.

Iminimycin A, the new iminium metabolite produced by Streptomyces griseus OS-3601.

A new natural product, designated iminimycin A, was isolated from the cultured broth of a streptomycin-producing microbial strain, Streptomyces griseus OS-3601, via a physicochemical screening method using HP-20, silica gel and ODS column chromatographies and subsequent preparative HPLC. Iminimycin A is an indolizidine alkaloid, containing of an unusual iminium group and a cyclopropane ring with a triene side chain. The absolute configuration of iminimycin A was elucidated by NMR studies and electronic circular dichroism analysis. Iminimycin A shows anti-bacterial activity against Bacillus subtilis, Kocuria rhizophila and Xanthomonas campestris pv. orizae, and cytotoxic activity against HeLa S3 and Jurkat cells with IC50 values of 43 and 36 µM, respectively.

Actinoplanes lichenis sp. nov., isolated from lichen.

A novel species of the genus Actinoplanes, strain LDG1-22T, for which we propose the name Actinoplanes lichenis sp. nov., was isolated from a lichen sample collected from tree bark in Thailand. The taxonomic position of the species has been described based on a polyphasic approach. Strain LDG1-22T produced irregular sporangia on agar media. It contained meso-diaminopimelic acid in the cell-wall peptidoglycan. The major menaquinone was MK-9(H4); the polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannosides and phosphatidylglycerol. Whole-cell hydrolysates contained ribose, glucose, mannose and small amounts of arabinose and xylose. The major cellular fatty acids were anteiso-C15 : 0 (31.2 %) and iso-C16 : 0 (14.2 %). Mycolic acids were absent. The G+C content was 73.6 %. 16S rRNA gene sequence analysis of strain LDG1-22T showed highest similarity (98.8 %) to Actinoplanes friuliensis DSM 45797T and it clustered with Actinoplanes nipponensis JCM 3264T and Actinoplanes missouriensis JCM 3121T in phylogenetic tree analysis. On the basis of the phenotypic characteristics and DNA-DNA relatedness, strain LDG1-22T could be distinguished from related species of the genus Actinoplanes and so represents a novel species of this genus. The type strain of Actinoplanes lichenis sp. nov. is LDG1-22T ( = JCM 30485T = TISTR 2343T = PCU 344T).

Identification of natural inhibitors of Entamoeba histolytica cysteine synthase from microbial secondary metabolites.

Amebiasis is a common worldwide diarrheal disease, caused by the protozoan parasite, Entamoeba histolytica. Metronidazole has been a drug of choice against amebiasis for decades despite its known side effects and low efficacy against asymptomatic cyst carriers. E. histolytica is also capable of surviving sub-therapeutic levels of metronidazole in vitro. Novel drugs with different mode of action are therefore urgently needed. The sulfur assimilatory de novo L-cysteine biosynthetic pathway is essential for various cellular activities, including the proliferation and anti-oxidative defense of E. histolytica. Since the pathway, consisting of two reactions catalyzed by serine acetyltransferase (SAT) and cysteine synthase (CS, O-acetylserine sulfhydrylase), does not exist in humans, it is a rational drug target against amebiasis. To discover inhibitors against the CS of E. histolytica (EhCS), the compounds of Kitasato Natural Products Library were screened against two recombinant CS isozymes: EhCS1 and EhCS3. Nine compounds inhibited EhCS1 and EhCS3 with IC50 values of 0.31-490 µM. Of those, seven compounds share a naphthoquinone moiety, indicating the structural importance of the moiety for binding to the active site of EhCS1 and EhCS3. We further screened >9,000 microbial broths for CS inhibition and purified two compounds, xanthofulvin and exophillic acid from fungal broths. Xanthofulvin inhibited EhCS1 and EhCS3. Exophillic acid showed high selectivity against EhCS1, but exhibited no inhibition against EhCS3. In vitro anti-amebic activity of the 11 EhCS inhibitors was also examined. Deacetylkinamycin C and nanaomycin A showed more potent amebicidal activity with IC50 values of 18 and 0.8 µM, respectively, in the cysteine deprived conditions. The differential sensitivity of trophozoites against deacetylkinamycin C in the presence or absence of L-cysteine in the medium and the IC50 values against EhCS suggest the amebicidal effect of deacetylkinamycin C is due to CS inhibition.

Micromonospora fluostatini sp. nov., isolated from marine sediment.

The novel actinomycete strain PWB-003T, which produced fluostatins B and C antibiotics, was isolated from nearshore sediment collected from Panwa Cape, Phuket Province, Thailand. Data from the present polyphasic study indicated that strain PWB-003T represented a member of the genus Micromonospora. It produced single spores on substrate mycelia and contained meso-diaminopimelic acid in the cell-wall peptidoglycan. Whole-cell hydrolysate contained ribose, xylose, arabinose, mannose and glucose. The predominant menaquinone was MK-10 (H4). Cellular fatty acids comprised C18 : 1ω9c, iso-C16 : 0, anteiso-C17 : 0, iso-C15 : 0 and iso-C17 : 0. On the basis of 16S rRNA gene sequence similarity analysis, the novel strain was closely related to Micromonospora eburnea LK2-10T (99.38 %), Micromonospora chaiyaphumensis MC5-1T (99.16 %), Micromonospora yangpuensis FXJ6.011T (98.97 %), Micromonospora echinaurantiaca DSM 43904T (98.97 %), Micromonospora pallida DSM 43817T (98.97 %), Micromonospora sagamiensis DSM 43912T and Micromonospora auratinigra JCM 12357T (both 98.97 %). The G+C content of the DNA was 74.5 mol%. DNA-DNA relatedness values among strain PWB-003T and related type strains ranged from 11.3 ± 1.3 to 38.8 ± 1.1 %. On the basis of these observations, strain PWB-003T could be distinguished from its closely related type strains and is considered to represent a novel species of the genus Micromonospora, for which the name Micromonospora fluostatini sp. nov. (type strain PWB-003T = JCM 30529T = PCU 341T = TISTR 2345T) is proposed.

Trichopolyn VI: a new peptaibol insecticidal compound discovered using a recombinant Saccharomyces cerevisiae screening system.

In the course of searching for insecticides from soil microorganisms, we found that a fermentation broth of the fungus, Trichoderma brevicompactum FKI-6324, produced Trichopolyn VI, a new peptaibol, which possessed significant insecticidal potential. Spectroscopic analysis showed the compound to be a new trichopolyn I derivative. This paper describes the isolation, structure elucidation and biological activity of trichopolyn VI.

Streptomyces siamensis sp. nov., and Streptomyces similanensis sp. nov., isolated from Thai soils.

Three actinomycete strains, KC-038(T), KC-031 and KC-106(T), were isolated from soil samples collected in the southern Thailand. The morphological and chemotaxonomic properties of strains KC-038(T), KC-031 and KC-106(T) were consistent with the characteristics of members of the genus Streptomyces, that is, the formation of aerial mycelia bearing spiral spore chains; the presence of LL-diaminopimelic acid in the cell wall, MK-9 (H6), MK-9 (H4) and MK-9 (H8) as the predominant menaquinones; and C16:0, iso-C16:0 and anteiso-C15:0 as the major cellular fatty acids. 16S rRNA gene sequence analyses indicated that strains KC-038(T) and KC-031 were highly similar (99.9%), and they were closely related to S. olivochromogenes NBRC 3178(T) (98.1%) and S. psammoticus NBRC 13971(T) (98.1%). Strain KC-106(T) was closely related to S. seoulensis NBRC 16668(T) (98.9%), S. recifensis NBRC 12813(T) (98.9%), S. chartreusis NBRC 12753(T) (98.7%) and S. griseoluteus NBRC 13375(T) (98.4%). The values of DNA-DNA relatedness between the isolates and the type strains of the related species were below 70%. On the basis of the polyphasic evidence, the isolates should be classified as two novel species, namely Streptomyces siamensis sp. nov. (type strain, KC-038(T) = NBRC 108799(T) = PCU 328(T) = TISTR 2107(T)) and Streptomyces similanensis sp. nov. (type strain, KC-106(T) = NBRC 108798(T) = PCU 329(T) = TISTR 2104(T)).

Nonomuraea thailandensis sp. nov. isolated from Thai soil.

A novel actinomycete, strain KC-061(T) was isolated from a soil sample collected from Angthong Islands National Park, Surat Thani province, Thailand. Strain KC-061(T) was identified as a member of the genus Nonomuraea through the use of a polyphasic approach. Phylogenetic analyses based on 16S rRNA gene sequencing, strain KC-061(T) belongs to the genus Nonomuraea and was closely related to 'N. monospora' PT708(T) (99.3%), N. rhizophila YIM 67092(T) (98.6%), N. dietziae DSM 44320(T) (98.5%) and N. rosea GW 12687(T) (98.3%). On the basis of phenotypic characteristics, DNA-DNA relatedness and phylogenetic distinctiveness, the novel isolate was identified as representing a novel species of the genus Nonomuraea, for which the name Nonomuraea thailandensis sp. nov. (type strain KC-061(T) =JCM 18408(T)=KCTC 29074(T) =PCU 327(T)) is proposed.

Sinefungin VA and dehydrosinefungin V, new antitrypanosomal antibiotics produced by Streptomyces sp. K05-0178.

Two new nucleotide antibiotics, named sinefungin VA and dehydrosinefungin V, were separated by cation exchange column chromatography and purified by HPLC from the culture broth of Streptomyces sp. K05-0178, together with the known antibiotics, sinefungin, dehydrosinefungin and KSA-9342. The structures of the two novel sinefungin analogs were elucidated by spectroscopic studies, including various NMR and advanced peptide chemical methods. Sinefungin VA consists of adenosine and ornithylvalylalanine, whereas dehydrosinefungin V consists of 4',5'-dehydroadenosine and ornithylvaline. Sinefungin VA showed potent antitrypanosomal activity with an IC(50) value of 0.0026 µg ml(-1) in vitro without cytotoxicity against MRC-5 cells. Dehydrosinefungin V showed moderate antitrypanosomal activity (IC(50)=0.15 µg ml(-1)).