New Analogues of Amides and Quinolinones Produced by Streptomyces sp. YIM S01983.

Streptomide (1), a new amide analogue, streptomynone (2), a new quinolinone, and ten known compounds including three aliphatic acids (3-5), two amides (6-7), four cyclic dipeptides (8-11), and an adenosine (12) were isolated from the fermentation broth of Streptomyces sp. YIM S01983 isolated from a sediment sample collected in Bendong Village, Huadong Town, Chuxiong, China. Their structures were determined by analysis of the 1D/2D-NMR and HR-ESI-MS spectra. Compound 12 presented weak antimicrobial activities against Candida albicans and Aligenes faecalis (MIC=64 µg/mL). Compounds 7 and 12 showed weak cytotoxic activity against MHCC97H.

Jiangchuanmycin, a New Pyrrolizidine Analog from Streptomyces sp. YIM S01863.

Jiangchuanmycin (1), a new indole containing pyrrolizidine, and six known peptides (2-7) were obtained from the fermentation broth of a Streptomyces isolate collected from a sediment sample of Xingyun Lake, Jiangchuan, China. Their structures were elucidated on the detailed analysis of the HR-ESI-MS, 1D and 2D NMR, ECD, and X-ray crystallographic data. Jiangchuanmycin (1) presented weak inhibitory effects on cell lines of H1299, MHCC97H, HCT116 with the IC50 values of 97.6 µM, 98.6 µM and 40.6 µM, respectively.

Steroidal alkaloid with unprecedented triheterocyclic architecture.

Veratrazine A (1), a steroidal alkaloid with a unique 6/5/5 triheterocyclic scaffold as the side chain, was isolated from Veratrum stenophyllum, and its structure was established via spectroscopic analyses and X-ray diffraction. A plausible biosynthetic pathway for 1 is proposed. Bioassy exhibits moderate anti-inflammatory activities in vitro and in vivo.

Discovery of potent immune-modulating molecule taccaoside A against cancers from structures-active relationships of natural steroidal saponins.

BACKGROUND: In recent years, the T-cell therapy and immune checkpoint inhibitors toward CTLA-4 and PD-1/PD-L1 axis antibody therapy have acquired encouraging success. However, most of patients were still not benefited with lots of troubles, such as low penetration of tissues/cells, strong immunogenicity and cytokine release syndrome, and long manufacturing process and expensive costs. By contrast, the immune-modulating small molecules possessed natural advantages to overcome these obstacles and might achieve greater success. PURPOSE: Exploring the potent immune-modulating natural small molecules and revealing what kinds of molecules or structures with the immunomodulatory activity against cancers. METHODS: A novel non-cytotoxic T-cell immunomodulating screening model was used to identify the cytotoxic/selective/immunomodulatory bioactivity for 148 natural steroidal saponins. The structure-activity relationships (SARs) research was used to reveal the key groups for immunomodulation/cytotoxicity/selectivity. The negative selection was used to isolate and purify the T-cell. The cell viability assay was used to measure the anti-cancer effect in vitro. The ELISA assay was used to detect the cytokines for IL-1ß, IL-6, TNF-α, IFN-γ, IL-12, perforin and granzyme B (GZMB). The western blotting assay was used to research the immunomodulatory mechanism. The siRNA knockdown was used to generate the IFN-γ resistant melanoma cells. The NOG immune-deficient mice were used to evaluate the anti-tumor efficacy in vivo. The peripheral blood samples from 10 cancer patients were used to detect the broad population anti-tumor efficacy. RESULTS: It was reported that the correlation among structures and immunomodulation/ cytotoxicity/selectivity, in which opening ring-F with 26-O-glucopyranosyl, disaccharide and trisaccharide chains at C-3, steric hindrance and polarity of C-22 were key immunomodulatory groups. Moreover, taccaoside A was identified as the most potent candidate against cancer cells, including non-small cell lung cancer, triple negative breast cancer, and the IFN-γ resistant melanoma, partly through enhancing T lymphocyte mTORC1-Blimp-1 signal to secrete GZMB. Besides, 10 patients derived T-cell also would be modulated against cancer cells in vitro. Moreover, the overall survival was great extended (>140 days vs 93 days) with nearly 100% tumor burden disappearance (0 mm3vs 1006 ± 79.5 mm3) in mice. CONCLUSION: This work demonstrated one possibility for this concerned purpose, and identified a potent immune-modulating natural molecule taccaoside A, which might contribute to cancer immunotherapy in future.

New steroidal alkaloids with anti-inflammatory and analgesic effects from Veratrum grandiflorum.

ETHNOPHARMACOLOGICAL RELEVANCE: "Li-Lu", the roots and rhizomes of Veratrum grandiflorum (Melianthiaceae), has been historically used as a traditional folk medicine for the treatment of wrist pain, fractures, sores, and inflammation in Yunnan Province, China. However, the anti-inflammatory and analgesic studies of this plant have seldom reported. AIM OF THE STUDY: To evaluate the anti-inflammatory and analgesic properties related to the traditional usage of V. grandiflorum both in vitro and in vivo, and further explore the accurate bioactive compounds from the medicinal plant. MATERIALS AND METHODS: Phytochemical investigation was carried out by chromatographic methods and their structures were established based on extensive spectra and comparison with corresponding data in the reported literatures. Anti-inflammatory activities were assessed by the suppression of lipopolysaccharide-activated inflammatory mediators in RAW 264.7 macrophage cells in vitro. Furthermore, anti-inflammatory and analgesic effects were evaluated based on carrageenan-induced paw edema and acetic acid-stimulated writhing in mice. RESULTS: The methanol extract (ME) of V. grandiflorum significantly alleviated the paw edema caused by carrageenan and the writhing numbers induced by acetic acid. Subsequent phytochemical investigation led to isolated of 21 steroidal alkaloids, including seven new compounds, veragranines C-I (1-7). Anti-inflammatory test indicated that steroidal alkaloids could decrease the expression of cyclooxygenase-2 (COX-2), interleukin-1ß (IL-1ß), and tumor necrosis factor α (TNF-α) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells at a concentration of 5.0 µg/ml in vitro, comparable to DXM. Moreover, five new steroidal alkaloids (2, 4, 5, 6, and 7) and two major steroidal alkaloids (9 and 13) significantly decreased the numbers of writhing in mice at the doses of 0.5 and/or 1.0 mg/kg (p < 0.01/0.05), roughly comparable to Dolantin™ at 10.0 mg/kg. CONCLUSIONS: The investigation supported the traditional use of V. grandiflorum and provided new steroidal alkaloids as potent analgesic agents.

Neothalfine, a potent natural anti-tumor agent against metastatic colorectal cancer and its primary mechanism.

Neothalfine is a natural bisbenzylisoquinoline alkaloid with the abundant resource in medicinal plants and has not been reported its anti-tumor efficacy. In the present study, the anti-tumor efficacy was investigated and it showed broad-spectrum activity against several cancer cell lines, especially metastatic colorectal cancer (HCT116, SW620, T84) with the IC50 values of 7.2, 5.9, 8.2 nM, respectively, roughly equal to well-known anti-tumor agent docetaxel (4.0, 4.7, 2.7 nM) and nearly 1000 folds than CPT-11 (4.4, 5.1, 6.9 µM). Furthermore, neothalfine inhibited colorectal cell proliferation by resulting in cell cycle arrest at the G2/M phase and induced apoptosis through the dysfunction of mitochondria to trigger intrinsic apoptotic pathway by untargeted metabolomic method, mitochondrial membrane potential, and caspase-3/7 activity assay. Moreover, neothalfine damaged colorectal cancer clonal spheres expansion significantly at the concentration of 3.5 nM with nearly 1000 folds efficacy than CPT-11 (3.0 µM). The results supported that neothalfine might be an anti-tumor lead for further investigation.

Structures/cytotoxicity/selectivity relationship of natural steroidal saponins against GSCs and primary mechanism of tribulosaponin A.

Glioblastoma multiform (GBM) is the highly aggressive brain tumor with poor prognosis. Glioma stem cells (GSCs), small population of cancer cells that exist in GBM tissues, resistant to chemotherapy and radiotherapy and usually driving GBM recurrence, have been developed as effective therapeutic target. Steroidal saponins are one of important resources for anti-tumor agent and may be benefited to selectively clear GSCs. In this report, total of 97 natural steroidal saponins were investigated the relationship among structures/cytotoxicity/selectivity against GSCs, glioma cell lines and human untransformed cells, and revealed that tribulosaponin A was the most potent compound. Further investigation suggested that tribulosaponin A up-regulated the expression of NCF1 and NOX1 to accumulate ROS for triggering apoptosis in GSCs, but not in untransformed cells, and it was further supported by the assay that N-acetyl-l-cysteine (NAC) clearing ROS delayed GSCs apoptosis. Besides, tribulosaponin A damaged GSCs recapturing tumor spheres formation.

Cytotoxic androstane derivatives from Sarcococca ruscifolia.

Sarcorusones A-D (1-4), four new androstane (C19-steroid) derivatives were characterized from Sarcococca ruscifolia along with five known compounds. Their structures were elucidated on the basis of extensive MS and NMR spectroscopic analysis. All the new structures share common 14-hydroxyl and 17-ketone functional groups, and compounds 2-4 feature a seneciamide group connecting to C-3 position. The inhibitory activities of all the isolates against melanoma cell B16F10 and lung cancer cell H1299 were evaluated, and compounds 2, 3, 5, and 6 exhibited moderate cytotoxic activities against B16F10 and H1299 cell lines with IC50 values 2.7-8.0 µM.

Herbicidins from Streptomyces sp. CB01388 Showing Anti- Cryptosporidium Activity.

A high-content imaging assay was used to screen the fraction collection of the Natural Product Library at The Scripps Research Institute for inhibitors of Cryptosporidium parvum. A chemical investigation of one strain, Streptomyces sp. CB01388, resulted in the isolation of six herbicidins (1-6), one of which is new (herbicidin L, 1). Five of the six herbicidins (1-3, 5, 6) showed moderate inhibitory activity against C. parvum, with 1 and 6 comparable to the FDA-approved drug nitazoxanide, and 2-6 showed no toxicity to the host HCT-8 cells and human HEK293T and HepG2 cells. These findings highlight the herbicidin scaffold for anti- Cryptosporidium drug development.

Discovery of Alternative Producers of the Enediyne Antitumor Antibiotic C-1027 with High Titers.

The potent cytotoxicity and unique mode of action make the enediyne antitumor antibiotic C-1027 an exquisite drug candidate for anticancer chemotherapy. However, clinical development of C-1027 has been hampered by its low titer from the original producer Streptomyces globisporus C-1027. Here we report three new C-1027 alternative producers, Streptomyces sp. CB00657, CB02329, and CB03608, from The Scripps Research Institute actinomycetes strain collection. Together with the previously disclosed Streptomyces sp. CB02366 strain, four C-1027 alternative producers with C-1027 titers of up to 11-fold higher than the original producer have been discovered. The five C-1027 producers, isolated from distant geographic locations, are distinct Streptomyces strains based on morphology and taxonomy. Pulsed-field gel electrophoresis and Southern analysis of the five C-1027 producers reveal that their C-1027 biosynthetic gene clusters (BGCs) are all located on giant plasmids of varying sizes. The high nucleotide sequence similarity among the five C-1027 BGCs implies that they most likely have evolved from a common ancestor.

Lovastatin analogues and other metabolites from soil-derived Aspergillus terreus YIM PH30711.

Eight previously undescribed metabolites including of lovastatin analogues, a pair of diastereoisomers, a cyclopentenone dimer, and three polyketides were isolated from the culture of Aspergillus terreus YIM PH30711. Two types of unprecedented skeletons, benzene-cyclopentanone complex and linear polyketide, and an unusual dimer structure were determined by spectral analysis. Compound, 3α-hydroxy-3,5-dihydromonacolin L showed moderate activity against HMG-CoA reductase, with an inhibition ratio of 34% at the concentration of 50 µM, while lovastatin and dihydromonacolin K ethyl ester presented much stronger activity against HMGR with inhibition rates of 85% and 90% at the concentration of 50 µM, respectively. Aspereusin A was active against AChE with a ratio of 62% at the concentration of 50 µM, while its stereomers did not showed obvious inhibition (<10%). The configuration at C-4 of these three diastereoisomers was crucial in the inhibition against AChE, and the ß-orientation of substituted methoxyl acrylic acid should be beneficial to the combining with AChE.

Discovery of the leinamycin family of natural products by mining actinobacterial genomes.

Nature's ability to generate diverse natural products from simple building blocks has inspired combinatorial biosynthesis. The knowledge-based approach to combinatorial biosynthesis has allowed the production of designer analogs by rational metabolic pathway engineering. While successful, structural alterations are limited, with designer analogs often produced in compromised titers. The discovery-based approach to combinatorial biosynthesis complements the knowledge-based approach by exploring the vast combinatorial biosynthesis repertoire found in Nature. Here we showcase the discovery-based approach to combinatorial biosynthesis by targeting the domain of unknown function and cysteine lyase domain (DUF-SH) didomain, specific for sulfur incorporation from the leinamycin (LNM) biosynthetic machinery, to discover the LNM family of natural products. By mining bacterial genomes from public databases and the actinomycetes strain collection at The Scripps Research Institute, we discovered 49 potential producers that could be grouped into 18 distinct clades based on phylogenetic analysis of the DUF-SH didomains. Further analysis of the representative genomes from each of the clades identified 28 lnm-type gene clusters. Structural diversities encoded by the LNM-type biosynthetic machineries were predicted based on bioinformatics and confirmed by in vitro characterization of selected adenylation proteins and isolation and structural elucidation of the guangnanmycins and weishanmycins. These findings demonstrate the power of the discovery-based approach to combinatorial biosynthesis for natural product discovery and structural diversity and highlight Nature's rich biosynthetic repertoire. Comparative analysis of the LNM-type biosynthetic machineries provides outstanding opportunities to dissect Nature's biosynthetic strategies and apply these findings to combinatorial biosynthesis for natural product discovery and structural diversity.

Phytotoxic, antibacterial, and antioxidant activities of mycotoxins and other metabolites from Trichoderma sp.

A new natural mycotoxin was isolated from the fermentation broth of Trichoderma sp. Jing-8 and the structure was determined as alternariol 1'-hydroxy-9-methyl ether (1), together with twelve known compounds. The structures were elucidated on the basis of their 1D, 2D NMR spectra and mass spectrometric data. Compounds 1, 8 and 9 indicated inhibitions against germination of the seeds of cabbage with MICs < 3 µg/mL. The compound 1 showed the antibacterial activity against Bacillus subtilis and Staphylococcus aureus with MICs at 64 µg/mL. Compound 1 and 3 showed significant DPPH radical-scavenging activities with IC50 at 12 µg/mL, respectively. The OH at C-1' in compound 1 decreased the cytotoxicity of these mycotoxins. A primary structure-activity relationship about the alternariol derivatives was discussed. Compounds 2-7 and 8 were the first time to be isolated from the Trichoderma.

Strain Prioritization and Genome Mining for Enediyne Natural Products.

The enediyne family of natural products has had a profound impact on modern chemistry, biology, and medicine, and yet only 11 enediynes have been structurally characterized to date. Here we report a genome survey of 3,400 actinomycetes, identifying 81 strains that harbor genes encoding the enediyne polyketide synthase cassettes that could be grouped into 28 distinct clades based on phylogenetic analysis. Genome sequencing of 31 representative strains confirmed that each clade harbors a distinct enediyne biosynthetic gene cluster. A genome neighborhood network allows prediction of new structural features and biosynthetic insights that could be exploited for enediyne discovery. We confirmed one clade as new C-1027 producers, with a significantly higher C-1027 titer than the original producer, and discovered a new family of enediyne natural products, the tiancimycins (TNMs), that exhibit potent cytotoxicity against a broad spectrum of cancer cell lines. Our results demonstrate the feasibility of rapid discovery of new enediynes from a large strain collection. IMPORTANCE: Recent advances in microbial genomics clearly revealed that the biosynthetic potential of soil actinomycetes to produce enediynes is underappreciated. A great challenge is to develop innovative methods to discover new enediynes and produce them in sufficient quantities for chemical, biological, and clinical investigations. This work demonstrated the feasibility of rapid discovery of new enediynes from a large strain collection. The new C-1027 producers, with a significantly higher C-1027 titer than the original producer, will impact the practical supply of this important drug lead. The TNMs, with their extremely potent cytotoxicity against various cancer cells and their rapid and complete cancer cell killing characteristics, in comparison with the payloads used in FDA-approved antibody-drug conjugates (ADCs), are poised to be exploited as payload candidates for the next generation of anticancer ADCs. Follow-up studies on the other identified hits promise the discovery of new enediynes, radically expanding the chemical space for the enediyne family.

Angucyclines and Angucyclinones from Streptomyces sp. CB01913 Featuring C-Ring Cleavage and Expansion.

Angucyclines and angucyclinones are aromatic polyketides with a tetracyclic benz[a]anthracene skeleton. The benz[a]anthracene scaffold is biosynthesized by type II polyketide synthases that catalyze the decarboxylative condensation of a short acyl-CoA starter and nine extender units. Angucyclines and angucyclinones, the largest group of polycyclic aromatic polyketides, achieve structural diversity via subsequent oxidation, ring cleavage, amino acid incorporation, and glycosylation. We here report the discovery of 14 angucyclinones and two angucyclines (1-16) from Streptomyces sp. CB01913, identifying 12 new compounds featuring various oxidations on rings A and C (1, 2, and 4), different sugar moieties attached to rings A and B (3 and 6), and C-ring cleavage (5 and 10-14) and expansion (8). These new structural features, highlighted by C-ring cleavage and expansion, enrich the structural diversity of angucyclines and angucyclinones. All compounds were tested for cytotoxicity and antibacterial activities, with 1, 5, 15, and 16 showing moderate activities against selected cancer cell lines or bacterial strains.

Stackebrandtia endophytica sp. nov., an actinobacterium isolated from Tripterygium wilfordii.

A novel endophytic actinobacterium, designated strain YIM 64602T, was isolated from healthy stems of Tripterygium wilfordii. It grew at 15-40 °C, pH 6.0-9.0 and in the presence of 0-3 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain YIM 64602T belongs to the genus Stackebrandtia. Whole-cell hydrolysates of strain YIM 64602T contained the amino acid meso-diaminopimelic acid with the sugars mannose, rhamnose and glucose, and a trace of ribose. The major polar lipids were diphosphatidylglycerol, phosphatidylmethylethanolamine and phosphatidylethanolamine. MK-10(H6), MK-10(H4) and MK-11(H4) were the predominant components in the quinone system. The fatty-acid pattern was mainly composed of the saturated branched-chain acids iso-C16 : 0, anteiso-C17 : 0, iso-C15 : 0 and iso-C17 : 0. The DNA G+C content was 72.4 mol%. 16S rRNA gene sequence analysis showed the highest pairwise sequence identity (96.0-98.5 %) with the members of the genus Stackebrandtia. Strain YIM 64602T displayed a DNA-DNA relatedness of 43.9±0.4 % with the type strain Stackebrandtia albiflava YIM 45751T. Based on evidence from this polyphasic study, strain YIM 64602T ( = BCRC 16954T = DSM 45928T) is considered to represent a novel species of the genus Stackebrandtia, for which the name Stackebrandtia endophytica is proposed.

New duclauxamide from Penicillium manginii YIM PH30375 and structure revision of the duclauxin family.

Duclauxamide A1 (1), a new polyketide-derived heptacyclic oligophenalenone dimer with a N-2-hydroxyethyl moiety, was isolated from Penicillium manginii YIM PH30375. Spectroscopic analysis, X-ray single crystal diffraction, and (13)C NMR DFT calculations confirmed that compound 1 and other duclauxin analogues possess the unified S configuration at C-9', which corrects a long-standing misrepresentation of duclauxins as C-9'R epimers. A plausible biosynthetic pathway for duclauxins is proposed on the basis of previous acetate labeling results for duclauxin and sclerodin.

Blastococcus endophyticus sp. nov., an actinobacterium isolated from Camptotheca acuminata.

A novel endophytic actinobacterium, designated strain YIM 68236(T), was isolated from healthy leaves of Camptotheca acuminata. and characterized by using a polyphasic approach. Cells of this strain occurred singly, in pairs or in tetrads. It grew at 10-45 °C, at pH 5.0-8.0 (optimum pH 7.0) and in the presence of 0-3% (w/v) NaCl. The DNA G+C content was 71.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM 68236(T) belongs to the genus Blastococcus. However, it differed from its closest relatives, Blastococcus aggregatus DSM 4725(T), Blastococcus saxobsidens DSM 44509(T) and Blastococcus jejuensis DSM 19597(T) in many phenotypic characteristics. Moreover, the DNA-DNA relatedness values between the novel isolate and the three above-mentioned type strains were 49.0 ± 1.6%, 46.1 ± 3.2% and 39.8 ± 1.5%, respectively. Based on comparative analysis of physiological and chemotaxonomic data, strain YIM 68236(T) represents a novel species of the genus Blastococcus, for which the name Blastococcus endophyticus sp. nov. is proposed. The type strain is YIM 68236(T) ( =CCTCC AA 209045(T) =DSM 45413(T) =KCTC 19998(T)).

Isolation and characterization of new p-Terphenyls with antifungal, antibacterial, and antioxidant activities from halophilic actinomycete Nocardiopsis gilva YIM 90087.

A new p-terphenyl 1 and a novel p-terphenyl derivative 3 bearing a benzothiazole moiety were isolated from halophilic actinomycete Nocardiopsis gilva YIM 90087, along with known p-terphenyl 2, antibiotic novobiocin 4, cyclodipeptides 5-13, and aromatic acids 14 and 15. Their structures were elucidated on the basis of the interpretation of spectral data and by comparison of the corresponding data with those reported previously. The p-terphenyl 1 showed antifungal activity against the three pathogenic fungi, including Fusarium avenaceum, Fusarium graminearum, and Fusarium culmorum, that caused Fusarium head blight with minimal inhibitory concentrations (MICs) of 8, 16, and 128 µg/mL, respectively. Compound 1 showed antifungal activity against Candida albicans with a MIC of 32 µg/mL and antibacterial activity against Bacillus subtilis with a MIC of 64 µg/mL. Novobiocin 4 showed antifungal activity against Pyricularia oryzae with a MIC of 16 µg/mL and antibacterial activity against B. subtilis with a MIC of 16 µg/mL and Staphylococcus aureus with a MIC of 64 µg/mL. The 1,1-diphenyl-2-picryl-hydrazyl assay suggested that 1, 3, and 4 exhibited 54.9% (2 mg/mL), 14.3% (4 mg/mL), and 47.7% (2 mg/mL) free radical scavenging activity, respectively. The positively charged 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radical (ABTS(+•)) scavenging assay indicated that 1, 3, 4, and 8 exhibited 68.6% (1 mg/mL), 28.4% (2 mg/mL), 78.2% (0.5 mg/mL), and 54.6% (2 mg/mL) ABTS(+•) scavenging capacity, respectively. The superoxide anion radical scavenging assay suggested that 4 exhibited 77.9% superoxide anion radical scavenging capacity at 2 mg/mL. N. gilva YIM 90087 is a new resource for novobiocin 4.

New megastigmane glycoside and alkaloids from Streptomyces sp. YIM 63342.

New sesquiterpene glycoside, cyclodipeptide and piperidine derivative were isolated from Streptomyces sp. YIM 63342. On the basis of spectral data, their structures were determined as 3R, 5R, 6S, 7E, 9R-megastigman-7-en-3,5,6,9-tetrol-9-O-ß-D-apiofuranosyl-(1→ 2)-ß-D-glucopyranoside (1), cyclo (L-Pro-L-OMet) (2) and (R)-(E, E)-2-(l,3-pentadienyl) piperidine (3), together with three known compounds as N-acetyltyramine (4), lycoperodine-1 (5), cyclo(L-Pro-L-Tyr)(6).