Binding Mode-Based Physicochemical Screening Method Using d-Ala-d-Ala Silica Gel and Chemical Modification Approach to Facilitate Discovery of New Macrolactams, Banglactams A and B, from Nonomuraea bangladeshensis K18-0086.

Utilizing a binding mode-based physicochemical screening method using d-Ala-d-Ala silica gel, two new macrolactams, named banglactams A (1) and B (2), were discovered from the culture broth of Nonomuraea bangladeshensis K18-0086. In the course of our investigation, we found that d-Ala-d-Ala silica gel precisely differentiated the chemical structures of banglactams and separated them. However, we were not able to obtain enough of 1 to elucidate the structure due to its instability and insolubility. To overcome this challenge, we chemically modified 1 to improve solubility, enabling us to obtain a sufficient material supply for the indirect determination of the structure. Antibacterial activity evaluation of banglactams revealed that 1 binding to d-Ala-d-Ala silica gel exhibited antibacterial activity against Staphylococcus aureus; however, this was not the case with 2. This research indicates the utility of our original binding mode-based PC screening method, and the combination strategy of PC and chemical modifications led us to discover novel antibacterial compounds.

Virgaricins C and D, new pramanicin analogs produced by Apiospora sp. FKI-8058.

Two new pramanicin analogs, named virgaricins C (1) and D (2), were discovered by physicochemical screening from a static cultured material of Apiospora sp. FKI-8058. Their structures were elucidated by MS and NMR analyses and chemical derivatization. Compounds 1 and 2 showed moderate antimalarial activity and cytotoxicity.

Design and Synthesis of d-Ala-d-Ala Silica Gel for a Binding Mode-Based Physicochemical Screening Method.

Vancomycin is a potent and broad-spectrum antibiotic that binds to the d-Ala-d-Ala moiety of the growing bacterial cell wall and kills bacteria. This fascinating binding model prompted us to design and synthesize d-Ala-d-Ala silica gels for the establishment of a new physicochemical (PC) screening method. In this report, we confirmed that vancomycin binds to d-Ala-d-Ala silica gel and can be eluted with MeOH containing 50 mM TFA. Finally, d-Ala-d-Ala silica gel enables to purify vancomycin from the culture broth of a vancomycin-producing strain, Amycolatopsis orientalis.

Growth and Migration Blocking Effect of Nanaomycin K, a Compound Produced by Streptomyces sp., on Prostate Cancer Cell Lines In Vitro and In Vivo.

Since castration-resistant prostate cancer (CRPC) acquires resistance to molecularly targeted drugs, discovering a class of drugs with different mechanisms of action is needed for more efficient treatment. In this study, we investigated the anti-tumor effects of nanaomycin K, derived from "Streptomyces rosa subsp. notoensis" OS-3966. The cell lines used were LNCaP (non-CRPC), PC-3 (CRPC), and TRAMP-C2 (CRPC). Experiments included cell proliferation analysis, wound healing analysis, and Western blotting. In addition, nanaomycin K was administered intratumorally to TRAMP-C2 carcinoma-bearing mice to assess effects on tumor growth. Furthermore, immuno-histochemistry staining was performed on excised tissues. Nanaomycin K suppressed cell proliferation in all cell lines (p < 0.001) and suppressed wound healing in TRAMP-C2 (p = 0.008). Nanaomycin K suppressed or showed a tendency to suppress the expression of N-cadherin, Vimentin, Slug, and Ras in all cell lines, and suppressed the phosphorylation of p38, SAPK/JNK, and Erk1/2 in LNCaP and TRAMP-C2. In vivo, nanaomycin K safely inhibited tumor growth (p = 0.001). In addition, suppression of phospho-Erk1/2 and increased expression of E-cadherin and cleaved-Caspase3 were observed in excised tumors. Nanaomycin K inhibits tumor growth and suppresses migration by inhibiting epithelial-mesenchymal transition in prostate cancer. Its mechanism of action is related to the inhibition of phosphorylation of the MAPK signaling pathway.

Mycelial differentiation linked avermectin production in Streptomyces avermitilis studied with Raman imaging.

Streptomyces avermitilis is a gram-positive bacterium that undergoes complex physiological and morphological differentiation during its life cycle, which has implications in secondary metabolite production. Avermectin, produced by S. avermitilis, is widely used as an anthelmintic and insecticidal agent. In this study, we have applied Raman microspectroscopic imaging to elucidate the correlation between production of avermectin and the morphological differentiation in S. avermitilis. We demonstrate distinctive variations in the localization of secondary metabolites at various stages of morphological differentiation. Under solid culture, avermectin was detected in the mycelia formed at the later stages of morphological differentiation (e.g., spore-bearing mycelium and spiral spore chains), but not in the early-stage substrate mycelium. On the contrary, under liquid culture condition, avermectin was found concentrated in the mycelial pellet formed at the early MII stage of differentiation. Furthermore, the chemical profiles of the mycelia were substantially different depending on the culture condition. Raman spectra corresponding to proteins, lipids, and cytochrome were observed in the mycelia irrespective of the stage of morphological differentiation, however, carotenoid was observed under solid culture condition particularly in spore-bearing mycelium and spiral spore chains. KEY POINTS: • Avermectin production is regulated during mycelial differentiation • Liquid and solid culture conditions affects mycelial differentiation • Raman microspectroscopic analysis reveals localization profiles of avermectin.

New nitrogen-compounds, penicidones E and F, produced by the fungal strain Oidiodendron sp. FKI-7498.

The nitrogen rule in mass spectrometry was used to search for new nitrogen-compounds from microbial metabolites. During this program, two new nitrogen-containing compounds, penicidones E and F, were discovered from the filamentous fungal strain FKI-7498, which was isolated from soil collected in Tokushima, Japan, and identified as Oidiodendron sp. by sequence analysis of the internal transcribed spacer region, including 5.8S ribosomal RNA. The structures of penicidones E and F were determined by mass spectrometry, nuclear magnetic resonance spectroscopy, and chemical modification analyses. These analyses revealed that penicidones E and F have a core structure of 3,5-dihydroxy-2-(4-pyridone-3-carbonyl)benzoic acid. Penicidone E exhibited hydroxyl radical scavenging activity.

Detection and characterization of new mangromicin analogs by tandem mass spectrometry.

Many useful natural products are usually screened based on their biological activities. On the other hand, various natural products can be detected based on their physicochemical properties. We have already reported the isolation and characterization of mangromicins from a cultural broth of Lechevalieria aerocolonigenes K10-0216 using physicochemical screening. In this report, we have conducted the mass spectrometry-based screening of new mangromicin analogs based on the neutral loss pattern originated from the unique cyclopentadecane skeleton of mangromicins. Two novel analogs were detected showing characteristic neutral loss pattern found in eight known mangromicin analogs. We propose the structures of the newly-found analogs based on the mass spectrometric as well as genomic and metabolic pathway data.

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.

Raman Microspectroscopy Imaging Analysis of Extracellular Vesicles Biogenesis by Filamentous Fungus Penicilium chrysogenum.

The mechanism of production of extracellular vesicles (EVs) and their molecular contents are of great interest due to their diverse roles in biological systems and are far from being completely understood. Even though cellular cargo releases mediated by EVs have been demonstrated in several cases, their role in secondary metabolite production and release remains elusive. In this study, this aspect is investigated in detail using Raman microspectroscopic imaging. Considerable evidence is provided to suggest that the release of antibiotic penicillin by the filamentous fungus Penicillium chrysogenum involves EVs. Further, the study also reveals morphological modifications of the fungal body during biogenesis, changes in cell composition at the locus of biogenesis, and major molecular contents of the released EVs. The results suggest a possible general role of EVs in the release of antibiotics from the producing organisms.

Trehangelins ameliorate inflammation-induced skin senescence by suppressing the epidermal YAP-CCN1 axis.

Trehangelins (THG) are newly identified trehalose compounds derived from broth cultures of an endophytic actinomycete, Polymorphospora rubra. THG are known to suppress Cellular Communication Network factor 1 (CCN1), which regulates collagen homeostasis in the dermis. Although the physical properties of THG suggest a high penetration of the stratum corneum, the effect of THG on the epidermis has not been reported. Here we describe a possible mechanism involved in skin aging focusing on the effect of THG on epidermal CCN1. This study shows that: (1) THG suppress epidermal CCN1 expression by inhibiting the translocation of Yes-Associated Protein (YAP) to nuclei. (2) Epidermal CCN1, localized at the basement membrane, regulates the balance between the growth and differentiation of keratinocytes. (3) Keratinocytes secrete more CCN1 than fibroblasts, which leads to disruption of the basement membrane and extracellular matrix components. (4) The secretion of CCN1 from keratinocytes is increased by ultraviolet B exposure, especially in aged keratinocytes, and deteriorates the elastic fiber structures in the underlying dermis. (5) Topical application of THG ameliorates the structure of the basement membrane in ex vivo human skin explants. Taken together, THG might be a promising treatment for aged skin by suppressing the aberrant YAP-CCN1 axis.

Identification and Analysis of the Biosynthetic Gene Cluster for the Indolizidine Alkaloid Iminimycin in Streptomyces griseus.

Indolizidine alkaloids, which have versatile bioactivities, are produced by various organisms. Although the biosynthesis of some indolizidine alkaloids has been studied, the enzymatic machinery for their biosynthesis in Streptomyces remains elusive. Here, we report the identification and analysis of the biosynthetic gene cluster for iminimycin, an indolizidine alkaloid with a 6-5-3 tricyclic system containing an iminium cation from Streptomyces griseus. The gene cluster has 22 genes, including four genes encoding polyketide synthases (PKSs), which consist of eight modules in total. In vitro analysis of the first module revealed that its acyltransferase domain selects malonyl-CoA, although predicted to select methylmalonyl-CoA. Inactivation of seven tailoring enzyme-encoding genes and structural elucidation of four compounds accumulated in mutants provided important insights into iminimycin biosynthesis, although some of these compounds appeared to be shunt products. This study expands our knowledge of the biosynthetic machinery of indolizidine alkaloids and the enzymatic chemistry of PKS.

Actinomadura violacea sp. nov., a madurastatin A1-producing strain isolated from lichen in Thailand.

An actinomycete strain, LCR2-06T, isolated from a lichen sample on rock collected from Chiang Rai Province (Pong Phra Bat Waterfall), Thailand, was characterized using a polyphasic approach. The strain grew at 25-45 °C, pH 6-11 and on International Streptomyces Project 2 agar plate with 5 % (w/v) NaCl. It contained meso-diaminopimelic acid as the diamino acid in whole-cell hydrolysates. Rhamnose, ribose, xylose, madurose, glucose and galactose were detected as whole-cell sugar hydrolysates. Mycolic acids were absent. The N-acyl type of muramic acid was acetyl. The strain contained C16 : 0, TBSA 10-methyl C18 : 0 and 2-hydroxy C16 : 0 as the predominant fatty acids and MK-9(H6), MK-9(H4) and MK-9(H8) as the major menaquinones. The major polar lipids were diphosphatidylglycerol, phosphatidylinositol and unidentified phospholipid. The draft genome of strain LCR2-06T was closely related to Actinomadura barringtoniae TBRC 7225T (99.2 %), Actinomadura nitritigenes NBRC 15918T (98.8 %), Actinomadura montaniterrae TISTR 2400T (98.5 %) and Actinomadura physcomitrii JCM 33455T (97.9 %). The draft genome of LCR2-06T was 11.1 Mb with 10 588 coding sequences with an average G+C content of 72.7 mol%. Results of genomic analysis revealed that the ANIb and ANIm values between strain LCR2-06T and A. montaniterrae TISTR 2400T were 90.0 and 92.0 %, respectively. The digital DNA-DNA hybridization value was 43.9 % in comparison with the draft genome of A. montaniterrae TISTR 2400T. The strain produced an antibacterial compound active against Bacillus subtilis ATCC 6633 and Kocuria rhizophila ATCC 9341. The results of taxonomic analysis suggested that strain LCR2-06T represented a novel species of the genus Actinomadura for which the name Actinomadura violacea sp. nov. is proposed. The type strain is LCR2-06T (=JCM 33065T=KCTC 49547T=NBRC 114810T=LMG 32136T=TISTR 2935T).

Nanaomycin K inhibited epithelial mesenchymal transition and tumor growth in bladder cancer cells in vitro and in vivo.

Nanaomycin K, derived from Streptomyces rosa subsp. notoensis OS-3966T, has been discovered to have inhibitory bioactivity on epithelial-mesenchymal transition (EMT), an important mechanism of cancer cell invasion and migration. In this study, we examined the anti-EMT and anti-tumor effect of nanaomycin K in bladder cancer, where EMT has important roles in progression. We treated two bladder cancer lines, non-muscle-invasive KK47 and muscle-invasive T24, with nanaomycin K to determine the effects on cell proliferation, apoptosis and expression of EMT markers in vitro. Wound-healing assays were performed to assess cell invasion and migration. We conducted an in vivo xenograft study in which mice were inoculated with bladder cancer cells and treated with intratumoral administration of nanaomycin K to investigate its anti-tumor and EMT inhibition effects. As the results, nanaomycin K (50 µg/mL) significantly inhibited cell proliferation in KK47 (p < 0.01) and T24 (p < 0.01) in the presence of TGF-ß, which is an EMT-inducer. Nanaomycin K (50 µg/mL) also significantly inhibited cell migration in KK47 (p < 0.01) and T24 (p < 0.01), and induced apoptosis in both cell lines in the presence of TGF-ß (p < 0.01). Nanaomycin K increased the expression of E-cadherin and inhibited the expression of N-cadherin and vimentin in both cell lines. Nanaomycin K also decreased expression of Snail, Slug, phospho-p38 and phospho-SAPK/JNK especially in T24. Intratumoral administration of nanaomycin K significantly inhibited tumor growth in both KK47 and T24 cells at high dose (1.0 mg/body) (p = 0.009 and p = 0.003, respectively) with no obvious adverse events. In addition, nanaomycin K reversed EMT and significantly inhibited the expression of Ki-67 especially in T24. In conclusion, we demonstrated that nanaomycin K had significant anti-EMT and anti-tumor effects in bladder cancer cells, suggesting that nanaomycin K may be a therapeutic candidate for bladder cancer treatment.

Aldsulfin, a novel unusual anti-mannheimiosis epithiodiketopiperazine antibiotic produced by Lasiodiplodia pseudotheobromae FKI-4499.

An anti-mannheimiosis agent, aldsulfin, was isolated from a culture broth of the fungus Lasiodiplodia pseudotheobromae FKI-4499, together with a known compound, lasiodipline C, using bioassay-guided fractionation. Spectroscopic analysis of aldsulfin, using NMR, mass spectrometry, and CD analyses revealed it to be an epithiodiketopiperazine with an unstable and unusual hemithioaminal moiety. Aldsulfin showed antibacterial activity against Mannheimia haemolytica and Pasteurella multocida.

On Selecting a Suitable Spectral Matching Method for Automated Analytical Applications of Raman Spectroscopy.

Raman spectra are molecular structure-specific and hence are employed in applications requiring chemical identification. The advent of efficient handheld and smartphone-based Raman instruments is promoting widespread applications of the technique, which often involve less trained end users. Software modules that enable spectral library searches based on spectral pattern matching is an essential part of such applications. The Raman spectrum recorded by end users will naturally have varying levels of signal to noise (SN), baseline fluctuations, etc., depending on the sample environment. Further, in biological, forensic, food, pharmaceuticals, etc., fields where a vast amount of Raman spectral data is generated, careful removal of background is often impossible. In other words, a 100% match between the library spectrum and user input cannot be often guaranteed or expected. Often, such influences are discounted upon developing mathematical methods for general applications. In this manuscript, we carefully examine how such effects would determine the results of spectral similarity-based library search. We show that several popular mathematical spectral matching approaches give incorrect results under the influence of small changes in the baseline and/or the noise. We also discuss the points to be carefully considered while generating a spectral library. We believe our results will be a guiding note for developing applications of Raman spectroscopy that uses a standard spectral library and mathematical spectral matching.

Detection of Penicillin G Produced by Penicillium chrysogenum with Raman Microspectroscopy and Multivariate Curve Resolution-Alternating Least-Squares Methods.

Raman microspectroscopy is a minimally invasive technique that can identify molecules without labeling. In this study, we demonstrate the detection of penicillin G inside Penicillium chrysogenum KF425 fungal cells. Raman spectra acquired from the fungal cells had highly overlapped spectroscopic signatures and hence were analyzed with multivariate curve resolution by alternating least-squares (MCR-ALS) to extract the spectra of individual molecular constituents. In addition to detecting spatial distribution of multiple constituents such as proteins and lipids inside the fungal body, we could also observe the subcellular localization of penicillin G. This methodology has the potential to be employed in screening the production of bioactive compounds by microorganisms.

Hatsusamides A and B: Two New Metabolites Produced by the Deep-Sea-Derived Fungal Strain Penicillium steckii FKJ-0213.

Two new nitrogen-containing metabolites, designated hatsusamide A (1) and B (2), were isolated from a culture broth of Penicilliumsteckii FKJ-0213 together with the known compounds tanzawaic acid B (3) and trichodermamide C (4) by physicochemical (PC) screening. The structures of 1 and 2 were determined as a tanzawaic acid B-trichodermamide C hybrid structure and a new analog of aspergillazines, respectively. The absolute configuration of 1 was determined by comparing the values of tanzawaic acid B and trichodermamide C in the literatures, such as 1H-nuclear magnetic resonance (1H-NMR) data and optical rotation, after hydrolysis of 1. Compounds 1-4 were evaluated for cytotoxicity and anti-malarial activities. Compounds 1 and 3 exhibited weak anti-malarial activity at half-maximal inhibitory concentration (IC50) values of 27.2 and 78.5 µM against the K1 strain, and 27.9 and 79.2 µM against the FCR3 strain of Plasmodium falciparum, respectively. Furthermore, 1 exhibited cytotoxicity against HeLa S3, A549, Panc1, HT29 and H1299 cells, with IC50 values of 15.0, 13.7, 12.9, 6.8, and 18.7 µM, respectively.

Oral administration of trehangelin-A alleviates metabolic disorders caused by a high-fat diet through improvement of lipid metabolism and restored beneficial microbiota.

The present study investigated whether or not the oral administration of trehangelin-A (THG-A) is effective for metabolic disorders caused by a high-fat diet, as we previously showed that the intraperitoneal administration of THG-A improved metabolic disorders caused by a high-fat diet. Mice received a control diet or high-fat diet for eight weeks. Concurrently, mice were orally administered 0.2 ml/mouse phosphate-buffered saline (PBS) or 1 or 10 mg/0.2 ml/mouse of THG-A once daily during the experiment. The weight gain caused by a high-fat diet was significantly suppressed by oral THG-A compared to a high-fat diet without THG-A. In addition, at eight weeks after starting the diet, the increased plasma total-cholesterol (T-CHO) and low-density lipoprotein-cholesterol (LDL-C) levels caused by a high-fat diet were significantly reduced by 10 mg/mouse THG-A and tended to attenuated by 1 mg/mouse THG-A. The LDL receptor and CYP7A1 mRNA expression in liver associated with lipid metabolism for reducing plasma LDL-C levels was significantly enhanced by oral THG-A. In contrast, oral THG-A exerted no marked effects on mice fed the control diet. The dysbiosis of a high-fat diet fed mice, which is in the form of an increased Firmicutes-to-Bacteroidetes ratio, also recovered, and the high-fat diet induced decreased levels of Bacteroides and Akkermansia genera, which are beneficial microbiota against metabolic disorders, were also restored by oral THG-A. These results indicate that oral THG-A administration acts on metabolic disorders by improving the lipid metabolism and restoring beneficial microbiota to resolve high-fat diet induced dysbiosis.

Thioporidiols A and B: Two New Sulfur Compounds Discovered by Molybdenum-Catalyzed Oxidation Screening from Trichoderma polypori FKI-7382.

Two new sulfur compounds, designated thioporidiol A (1) and B (2), were discovered by the MoS-screening program from a culture broth of Trichoderma polypori FKI-7382. The structures of 1 and 2 were determined as C13 lipid structures with an N-acetylcysteine moiety. The relative configuration at the C-5 and C-6 position of 1 was determined by the derivatives of -methoxy--phenylacetic acid diesters, and the absolute configuration of the N-acetylcysteine moiety was determined by advanced Marfey's analysis. Compounds 1 and 2 were evaluated for anti-microbial, cytotoxic and anti-malarial activities. Compound 2 exhibited anti-microbial activity against Candida albicans ATCC 64548.

Screening for Sulfur Compounds by Molybdenum-Catalyzed Oxidation Combined with Liquid Chromatography-Mass Spectrometry.

The molybdenum (Mo)-catalyzed oxidation of sulfide under neutral conditions yields sulfone. This reaction proceeds more smoothly than olefin epoxidation and primary or secondary alcohol oxidation. In this study, Mo-catalyzed oxidation was used to screen for sulfur compounds (named "MoS-screening") in microbial broths by liquid chromatography-mass spectrometry (LC/MS). To demonstrate proof-of-concept, known sulfur microbial compounds were successfully identified from a mixture of non-sulfur microbial compounds as sulfinyl or sulfonyl products of Mo-catalyzed oxidation. Then our MoS-screening method was used to screen 300 samples of microbial broth for sulfur compounds. One of the identified compounds was a kitasetaline-containing N-acetyl cysteine moiety produced by an actinomycete strain. These results demonstrate the potential of MoS-screening in the search for new sulfur compounds from microbial sources.