New liposidomycin congeners produced by Streptomyces sp. TMPU-20A065, anti-Mycobacterium avium complex agents with therapeutic efficacy in a silkworm infection model.

Three new liposidomycin congeners (1, 2, and 4), together with 14 known liposidomycins (3 and 5-17), were isolated from the culture broth of Streptomyces sp. TMPU-20A065 as anti-Mycobacterium avium complex agents. The structures of liposidomycins were elucidated by spectroscopic analyses, including NMR and MS. Compounds 1, 2, and 4 belong to type-I liposidomycin-containing sulfate groups and methylglutaric acid, each with a different acyl side chain in the structure. Compounds 1-17 exhibited in vitro anti-M. avium and M. intracellulare activities with MIC values ranging between 2.0 and 64 µg ml-1. Furthermore, 1-17 exerted potent therapeutic effects in an in vivo-mimic silkworm infection model with ED50 values ranging between 0.12 and 3.7 µg larva-1 g-1.

Synthesis and biological evaluation of nectriatide derivatives, potentiators of amphotericin B activity.

Nectriatide 1a, a naturally occurring cyclic tetrapeptide, has been reported to a potentiator of amphotericin B (AmB) activity. In order to elucidate its structure-activity relationships, we synthesized nectriatide derivatives with different amino acids in solution-phase synthesis and evaluated AmB-potentiating activity against Candida albicans. Among them, C-and N-terminal protected linear peptides were found to show the most potent AmB-potentiating activity.

New potentiators of amphotericin B activity, shodoamides A to C produced by Pseudophialophora sp. BF-0158.

During our screening program for new potentiators of amphotericin B activity against Candida albicans, shodoamides A to C (1-3) were isolated from a culture broth of the fungus Pseudophialophora sp. BF-0158 fermented under shaking conditions. A known congener named shodoamide D (4) in this paper was obtained from a culture broth of the BF-0158 strain fermented under static conditions. The structures of 1-4 were assigned based on spectroscopic analyses, including NMR and MS, and were found to have a common N-(2´,3´,4´-trihydroxybutyl)-6-methyl-2,4-tetradecadienamide structure. Compounds 1-3 exhibited no antifungal activity, but they induced up to 32-fold increases in amphotericin B activity against C. albicans by a microdilution method.

New piericidin rhamnosides as potentiators of amphotericin B activity against Candida albicans produced by actinomycete strain TMPU-A0287.

Four new piericidin rhamnosides (2, 4-6) together with three known piericidins (1, 3, 7) were isolated from the culture broth of the unidentified actinomycete strain TMPU-A0287 as potentiators of antifungal amphotericin B (AmB) activity. The structures of piericidins were elucidated by spectroscopic analyses, including NMR and MS. Compounds 2 and 4-6 possessed a ketone at C-10 and one or two methoxy groups on the rhamnose in their structures. Compounds 1-7 did not exhibit antifungal activity against Candida albicans and all potentiated AmB activity. The MIC values of AmB against C. albicans combined with 1-7 (4.0 µg ml-1) decreased from 0.50 to 0.063 or 0.031 µg ml-1, yielding an 8- or 16-fold increase in AmB activity.

Discovery of prescopranone, a key intermediate in scopranone biosynthesis.

A key intermediate in scopranone biosynthesis, prescopranone, accumulated in the mycelium of Streptomyces avermitilis SUKA carrying the biosynthetic gene cluster for scopranone lacking the sprT encoding the monooxygenase. The structure of prescopranone was elucidated by NMR and other spectral data. Prescopranone consists of a 2-pyranone ring with two atypical scoop-like moieties (1-ethyl-1-propenyl and 2-ethylbutyl groups), which was deduced as a product of the modular polyketide syntheses encoded by sprA, sprB, and sprC. Prescopranone inhibited bone morphogenetic protein (BMP)-induced alkaline phosphatase activity in a BMP receptor mutant cell line.

Synthesis and Evaluation of Habiterpenol Analogs.

Habiterpenol is a G2 checkpoint inhibitor isolated from the culture broth of Phytohabitans sp. 3787_5. Here, we report the synthesis of new habiterpenol analogs through the total synthesis process of habiterpenol and evaluating the analogs for G2 checkpoint inhibitory activity. We investigated two different synthetic approaches for total synthesis, with intramolecular conjugate addition and Ti(III)-mediated radical cyclization as key reactions. Although the former was unsuccessful, the latter reaction facilitated stereoselective total synthesis and determination of the absolute configuration of habiterpenol. The extension of these chemistries to a structure-activity relationship (SAR) study gave new habiterpenol analogs, which could not be derived from natural habiterpenol and only be synthesized by applying the total synthesis. Therefore, this study provides important insights into SAR studies of habiterpenol.

Germacrane sesquiterpenes from leaves of Eupatorium chinense inhibit protein tyrosine phosphatase.

Three new germacrane-type sesquiterpene lactones (1-3) were isolated alongside seven known related congeners (4-10) from the leaves of Eupatorium chinense L. (Compositae). The planar structures of 1-3 were elucidated by their spectroscopic data, including 1D and 2D NMR spectra. The relative and absolute configurations of 1-3 were determined using NOESY experiments and electronic circular dichroism analyses. Compounds 1, 4, 5, and 7 inhibited protein tyrosine phosphatase (PTP) 1B activity with IC50 values of 25, 11, 28, and 24 µM, respectively. Among these, compound 4 exhibited an inhibitory effect on T-cell PTP (TCPTP) with an IC50 value of 25 µM. To our knowledge, this is the first study demonstrating the PTP inhibitory activity of the germacrane sesquiterpenes. The results show that compound 4 acts as an inhibitor of both PTP1B and TCPTP.

Inhibitory effects of sesquiterpene lactones from the Indonesian marine sponge Lamellodysidea cf. herbacea on bone morphogenetic protein-induced osteoblastic differentiation.

A new unique sesquiterpene lactone, bicyclolamellolactone A (1), was isolated together with two known monocyclofarnesol-type sesquiterpenes, lamellolactones A (2) and B (3), from the Indonesian marine sponge Lamellodysidea sp. (cf. L. herbacea). The planar structure of 1 was assigned based on its spectroscopic data (1D and 2D NMR, HRESIMS, UV, and IR spectra). The relative and absolute configuration of 1 was determined by comparison of its calculated and experimental electronic circular dichroism spectra in combination with NOESY correlations. Compounds 1-3 inhibited bone morphogenic protein (BMP)-induced alkaline phosphatase activity in mutant BMP receptor-carrying C2C12 cells with IC50 values of 51, 4.6, and 20 µM, respectively.

Development of an in vivo-mimic silkworm infection model with Mycobacterium avium complex.

In vivo-mimic silkworm infection models with Mycobacterium avium and Mycobacterium intracellulare were newly established to evaluate the therapeutic effects of anti-M. avium complex (MAC) antibiotics. Silkworms raised at 37°C died within 72 hours of an injection of M. avium or M.intracellulare (2.5 × 107 colony-forming unit (CFU)/larva·g) into the hemolymph. Clinical anti-mycobacterial (tuberculosis) antibiotics were evaluated under these conditions. Clarithromycin, kanamycin, streptomycin, amikacin, and ciprofloxacin exerted therapeutic effects in a dose-dependent manner, which was consistent with those in the mouse model. Furthermore, three effective actinomycete culture broths were selected in the screening program of our microbial broth library using the silkworm model, and four active metabolites, ohmyungsamycins A and B (1 and 2), chartreusin (3), and griseoviridin (4), were identified. Among these compounds, 1 showed the lowest 50% effective dose (ED50) value (8.5 µg/larva·g), while 3 had the best ED50/minimum inhibitory concentration (MIC) ratio (7.4). These results indicate that silkworm models are a useful tool for identifying anti-MAC antibiotics candidates with veritable therapeutic effects.

Screening for Small Molecule Inhibitors of BMP-Induced Osteoblastic Differentiation from Indonesian Marine Invertebrates.

Fibrodysplasia ossificans progressiva (FOP) is a rare congenital disorder with heterotopic ossification (HO) in soft tissues. The abnormal activation of bone morphogenetic protein (BMP) signaling by a mutant activin receptor-like kinase-2 (ALK2) leads to the development of HO in FOP patients, and, thus, BMP signaling inhibitors are promising therapeutic applications for FOP. In the present study, we screened extracts of 188 Indonesian marine invertebrates for small molecular inhibitors of BMP-induced alkaline phosphatase (ALP) activity, a marker of osteoblastic differentiation in a C2C12 cell line stably expressing ALK2(R206H) (C2C12(R206H) cells), and identified five marine sponges with potent ALP inhibitory activities. The activity-guided purification of an EtOH extract of marine sponge Dysidea sp. (No. 256) resulted in the isolation of dysidenin (1), herbasterol (2), and stellettasterol (3) as active components. Compounds 1-3 inhibited ALP activity in C2C12(R206H) cells with IC50 values of 2.3, 4.3, and 4.2 µM, respectively, without any cytotoxicity, even at 18.4-21.4 µM. The direct effects of BMP signaling examined using the Id1WT4F-luciferase reporter assay showed that compounds 1-3 did not decrease the reporter activity, suggesting that they inhibit the downstream of the Smad transcriptional step in BMP signaling.

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.

Screening of Indonesian Edible Plants for Bioactive Constituents and a New Protein Tyrosine Phosphatase 1B Inhibitory Acylbenzene Derivative from Leaves of Indonesian Syzygium polyanthum.

Bioassay screening using Indonesian plants, such as traditional foods (vegetables, spices, and tea) and folk medicinal herbs, identified eight protein tyrosine phosphatase (PTP) 1B inhibitory and two antibacterial plants. The leaves of Syzygium polyanthum (Wight) Walp. were examined in more detail to define PTP1B inhibitory components, resulting in the isolation of a new active acylbenzene (1) along with four related congeners of 1 (2-5) and four oleanane triterpenes (6-9). The structure of 1 was elucidated as 12-oxo-12-(2,3,5-trihydroxy-4-methylphenyl)dodecanoic acid based on its spectroscopic data. The acylbenzenes 1 and 3-5 inhibited PTP1B activity with IC50 values ranging between 9.5 and 14 µM, whereas the triterpenes 7-9 also suppressed this activity with IC50 values of 3.3-5.7 µM.

Antifungal trichothecene sesquiterpenes obtained from the culture broth of marine-derived Trichoderma cf. brevicompactum and their structure-activity relationship.

Two new trichothecene sesquiterpenes, trichobreols D (1) and E (2), were isolated from the culture broth of marine-derived Trichoderma cf. brevicompactum together with trichobreol A (3). The structures of 1 and 2 were assigned on the basis of their spectroscopic data. Compound 1 inhibited the growth of two yeast-like fungi, Candida albicans and Cryptococcus neoformans, with equivalent MIC values (6.3 µg/mL), while 2 gave MIC values of 12.5 and 25 µg/mL, respectively. The antifungal activities of five semisynthetic derivatives (4-8) prepared from 3 were evaluated and compared to investigate the preliminary structure-activity relationship.

Synthesis and Absolute Configuration of Habiterpenol.

The synthesis of habiterpenol, a G2 checkpoint inhibitor, was achieved through the stereoselective Ti(III)-mediated radical cyclization of a ß-epoxide as the key reaction. Moreover, the absolute configuration of habiterpenol was determined.

Epipolythiodiketopiperazine and trichothecene derivatives from the NaI-containing fermentation of marine-derived Trichoderma cf. brevicompactum.

The marine-derived fungus Trichoderma sp. TPU199 (cf. Trichoderma brevicompactum) produces pretrichodermamide A (1) and gliovirin (2), which possess a rare type of epipolythiodiketopiperazine (ETP) structure with a disulfide bridge between the α- and ß-positions of two amino acid residues. We previously reported that this strain gave the halogenated ETPs, DC1149B (4), DC1149R (6), and iododithiobrevamide (7), when fermented with sodium halides (NaCl, NaBr, and NaI). Further analyses of the metabolites obtained under NaI-containing culture conditions resulted in the isolation of two new ETP derivatives (11 and 12) and three new trichothecene sesquiterpenes (13-15). The structures of 11 and 12, including their absolute configurations, were elucidated based on spectroscopic data for 11 and 12 and comparisons with those for 1 and related compounds, revealing that 11 was an epimer of 1 at the C-5 position and 12 was a trithio-derivative of 11. The structures of 13-15 were established by analyzing their 1D and 2D NMR data. The absolute configurations of 13-15 were assigned by comparing their experimental electronic circular dichroism (ECD) spectra with the calculated ECD spectrum of 13.

Voluhemins, new inhibitors of sterol O-acyltransferase, produced by Volutella citrinella BF-0440.

New compounds, designated voluhemins A (1) and B (2), are isolated from the culture broth of the fungal strain Volutella citrinella BF-0440 along with structurally related known NK12838 (3). Spectroscopic data, including 1D and 2D NMR, elucidated their structures. Compounds 1-3 have a common indoline-diterpene core and two additional isoprenyl moieties. Compounds 1 and 3 contain a hemiaminal unit, while 2 is O-methylated 1. Their inhibitory activities toward sterol O-acyltransferase (SOAT) 1 and 2 isozymes in SOAT1- and SOAT2-expressing Chinese hamster ovary (CHO) cells show that 2 selectively inhibits the SOAT2 isozyme.

Polyketide glycosides phialotides A to H, new potentiators of amphotericin B activity, produced by Pseudophialophora sp. BF-0158.

Eight new potentiators of antifungal amphotericin B (AmB) activity, phialotides A to H, were isolated from the fermentation broths of the rare fungus Pseudophialophora sp. BF-0158. The structures of phialotides were elucidated by spectroscopic analyses, including NMR and MS, and degradation studies. Phialotides were novel polyketide glycosides consisting of a 1,3-dimethylbut-1-ene (C6-unit) repeating substructure and one to three hexopyranoses. None of the phialotides exhibited antifungal activity, whereas all potentiated AmB activity against several fungi. Phialotide F was the most effective potentiator of AmB activity against Candida albicans, with a decrease in the MIC from 0.50 to 0.016 µg ml-1 being observed in combination with phialotide F at 1.0 µg ml-1.

An Unusual Extender Unit Is Incorporated into the Modular Polyketide Synthase of Scopranones Biosynthesis.

Scopranones, produced by Streptomyces sp. BYK-11038, are the novel bone morphogenetic protein inhibitors characterized by atypical two scoop-like moieties and a 3-furanone moiety. Two scoop-like moieties connected to a 3-furanone have not previously been reported in natural products, and their biosynthesis must occur via a unique pathway. Feeding experiments using 13C-labeled precursors indicated that scopranones were synthesized from three acetates and three butyrates in polyketide-type biosynthesis. Genome mining of Streptomyces sp. BYK-11038 revealed that the candidate biosynthetic gene cluster contains 21 open reading frames (ORFs), including three modular polyketide synthases (PKSs; SprA, SprB, and SprC), which were composed of 4 modules with one loading module and 18 additional ORFs (SprD to SprU) spanning a distance of 55 kbp. The characterization of in-frame deletion mutants and feeding experiments with the predicted extender units indicated that two genes, sprP and sprR, encoding discrete 3-oxoacyl-ACP synthases, and a gene, sprO, encoding crotonyl-CoA reductase, were involved in assembling an unusual C8 branched extender unit, 2-(2-ethylbutyl)malonyl-CoA. Additionally, three ORFs, sprM, sprN, and sprT, encoding cytochrome P450s and a monooxygenase, are important tailoring enzymes in post-PKS modification. SprT is an essential enzyme for decarboxylative ring contraction via oxidation, which converts the 2-pyranone to a 3-furanone.