Peniapyrones A-I, Cytotoxic Tricyclic-Fused α-Pyrone Derivatives from an Endophytic Penicillium brefeldianum F4a.

Five cyclopenta[d]pyrano[4,3-b]pyran-1,7(6H)-dione 6/6/5-fused tricyclic ring system containing metabolites peniapyrones A-E (1-5), and four previously undescribed cyclopenta[4,5]furo[3,2-c]pyran-1-one 6/5/5-fused tricyclic ring system containing compounds peniapyrones F-I (6-9), were isolated from the endophytic Penicillium brefeldianum F4a. Their structures, including absolute configurations, were determined through spectroscopic analysis and quantum chemical calculations. Peniapyrones D (4) and E (5) were a pair of diastereoisomers. Compounds 1, 3, and 5-9 showed cytotoxic activity against AsPC-1, CRL-2234, and MCF-7 cancer cell lines. Compounds 1, 3, 6, 8, and 9 inhibited the Kirsten rat sarcoma viral oncogene homologue (KRAS) mutant AsPC-1 cell line.

5-(4-Hydroxyphenyl)-3H-1,2-dithiole-3-thione derivatives of brefeldin A: Design, synthesis and cytotoxicity in MDA-MB-231 human breast cancer cells.

27 novel 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione derivatives of brefeldin A were designed and synthesized to make them more conducive to the cancer treatment. The antiproliferative activity of all the target compounds was tested against six human cancer cell lines and one human normal cell line. Compound 10d exhibited nearly the most potent cytotoxicity with IC50 values of 0.58, 0.69, 1.82, 0.85, 0.75, 0.33 and 1.75 µM against A549, DU-145, A375, HeLa, HepG2, MDA-MB-231 and L-02 cell lines. Moreover, 10d inhibited metastasis and induced apoptosis of MDA-MB-231 cells in a dose-dependent manner. The potent anticancer effects of 10d were prompted based on the aforementioned results, the therapeutic potential of 10d for breast cancer was worth further exploration.

Design and Synthesis of Brefeldin A-Isothiocyanate Derivatives with Selectivity and Their Potential for Cervical Cancer Therapy.

Brefeldin A has a wide range of anticancer activity against a variety of tumor cells. Its poor pharmacokinetic properties and significant toxicity seriously hinder its further development. In this manuscript, 25 brefeldin A-isothiocyanate derivatives were designed and synthesized. Most derivatives showed good selectivity between HeLa cells and L-02 cells. In particular, 6 exhibited potent antiproliferative activity against HeLa cells (IC50 = 1.84 µM) with no obvious cytotoxic activity to L-02 (IC50 > 80 µM). Further cellular mechanism tests indicated that 6 induced HeLa cell cycle arrest at G1 phase. Cell nucleus fragmentation and decreased mitochondrial membrane potential suggested 6 could induce apoptosis in HeLa cells through the mitochondrial-dependent pathway.

Genome Mining and Molecular Networking Guided Isolation of Antimycin Analogs with Antifeedant Activities from the Deep-Sea-Derived Streptomyces sp. NA13.

Polyphagous insects could affect agricultural production, which leads to serious economic losses. Due to the negative effects of synthesized insecticides, finding eco-friendly and new biopesticides is emergent. To develop natural origin insecticides, an integrative approach combining antifeedant activity screening, genome mining, and molecular networking has been applied to discover antifeedant secondary metabolites from Streptomyces sp. NA13, which leads to the isolation of a novel antimycin Q (1) and six known antimycin analogs (antimycins A1a, A2a, A3a, A4a, A7a, and N-formylantimycic acid methyl ester, 2-7). Their structures were identified by high-resolution mass spectrometry (HR-MS) and nuclear magnetic resonance (NMR) spectroscopic. The absolute configuration of 1 was elucidated by the comparison of coupling constant, electronic circular dichroism (ECD) analysis, and NMR calculations. 1-6 exhibited different levels of antifeedant activities against Helicoverpa armigera, especially 1-4. At the same time, the antifeedant activity of antimycin was reported firstly.

Penipentenone A and brefeldin A derivatives potently inhibit KRAS mutant cancer cells from an endophytic fungus Penicillium brefeldianum F4a.

Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation is one of the most important carcinogenic factors in many solid tumors, which leads to the poor prognosis and therapy resistance of cancer. In order to develop direct or indirect KRAS inhibitors, one unique asymmetric dicyclopentenone penipentenone A, three undescribed brefeldin A (BFA) derivatives, and five known BFA derivatives were discovered from the endophytic fungus Penicillium brefeldianum guided by LC-MS/MS and cytotoxic activities. Their structures were elucidated by optical rotation, mass spectrometry, and NMR spectroscopic data. The absolute configurations of four undescribed compounds were elucidated by comparison of the experimental and calculated ECD spectra. The antiproliferative activities of obtained compounds against three KRAS mutant tumor cell lines and two BFA derivative-sensitive cell lines were evaluated. Besides 4-epi-15-epi-brefeldin A, the other compounds showed significant inhibitory activities against those tumor cell lines with IC50 values ranging from 0.82 to 18.87 µM. Intriguingly, penipentenone A selectively inhibited KRAS mutant cancer cells SW620 (KRASG12V) and ASPC-1 (KRASG12D). BFA and four derivatives showed potent cytotoxic activities against all selected tumor cell lines H358 (KRASG12C), SW620 (KRASG12V), ASPC-1 (KRASG12D), PC-3, and HepG-2. These findings will provide undescribed lead compounds for developing drugs that target KRAS mutations.

Nigirpexin E, a new azaphilone derivative with anti-tobacco mosaic virus activity from soil-derived fungus Trichoderma afroharzianum LTR-2.

A new compound classified as one new azaphilone derivative, nigirpexin E (1), was obtained from the soil-derived fungus Trichoderma afroharzianum LTR-2, together with seven known compounds (2-8). The structures of 1-8 were determined by their HRESIMS, optical rotation, and NMR spectroscopic data. The absolute configuration of nigirpexin E (1) was determined on the basis of comparisons of experimental and theoretically calculated ECD spectra. Compound 3 was firstly isolated from Trichoderma. Bioactivities of the isolated compounds were assayed their anti-tobacco mosaic virus (anti-TMV) activities. The results showed that compound 1 exhibited significant inactivation effect against TMV with an inhibition rate of 67.25% (0.5 mg ml-1), which was higher than that of positive control ribavirin (56.74%). This is the first report of the anti-TMV activity of azaphilone derivatives.

Novel Antioxidants and α-Glycosidase and Protein Tyrosine Phosphatase 1B Inhibitors from an Endophytic Fungus Penicillium brefeldianum F4a.

Oxidative stress plays a very important role in the progression of diabetes and its complications. A therapeutic agent that is both antidiabetic and antioxidant would be the preferred choice for the treatment of diabetes. The crude extract of the endophytic fungus Penicillium brefeldianum F4a has significant antioxidant and α-glycosidase and protein tyrosine phosphatase 1B (PTP1B) inhibition activities. Chemical investigation of P. brefeldianum F4a using an activity-guided isolation led to the discovery of three new compounds called peniorcinols A-C (1-3) along with six known compounds: penialidins A (4), penialidin F (5), myxotrichin C (6), riboflavin (7), indole-3-acetic acid (8), and 2-(4-hydroxy-2-methoxy-6-methylphenyl) acetic acid (9). Their chemical structures were established by their NMR and HRESIMS. The absolute configurations of 1 and 3 were determined by experimental and calculated electronic circular dichroism (ECD). Their antioxidant activities were evaluated by DPPH• and ABTS•+ scavenging assays. Compounds 1-6 and 8-9 showed moderate to strong free radical scavenging activities. Significantly, 4-6 exhibited more potent ABTS•+ scavenging activity than that of the positive control. Their α-glycosidase and PTP1B inhibition activities were tested. Among them, compound 3 showed α-glucosidase inhibition activity, and compounds 7 and 8 showed PTP1B inhibitory activity for the first time. It is worth noting that 3 and 8 displayed both antioxidant and α-glycosidase or PTP1B inhibition activities. These finding suggest that compounds 3 and 8 could be used as lead compounds to generate new potent drugs for the treatment of oxidative stress-related diabetes.

Structure-based molecular networking for the target discovery of novel germicidin derivatives from the sponge-associated streptomyces sp. 18A01.

Four new α-pyrone derivatives, named germicidins P-S (1-4) along with nine known analogues (5-13) were discovered from the sponge-associated Streptomyces sp. 18A01 guided by Global Natural Products Social (GNPS) molecular networking, the LC-DAD-MS profile, and hexokinase II (HK2) inhibitory activity. The structures of 1-13 were elucidated by analysis of their HRMS, optical rotation, and NMR spectroscopic data. The absolute configurations of germicidin P (1) and germicidin Q (2) were determined on the basis of comparisons of experimental and theoretically calculated ECD spectra. Bioactivities of the isolated compounds were assayed against human HK2. The bioassay results showed that compounds 1-4 and 11-13 exhibited significant inhibitory activities against HK2, with IC50 values ranging from 5.1 to 11.0 µM. A molecular docking simulation demonstrated that these germicidins were docked in the inner active site tunnel of HK2. Interestingly, the amino residue Arg91 has a better binding affinity and efficacy than the amino residue Asn89 in the process of HK2 binding to the ligands, resulting in better hexokinase inhibitory activity. This result provided a valuable perspective for better understanding their HK2 inhibition activity.

Cytotoxic and antimicrobial activities of secondary metabolites isolated from the deep-sea-derived Actinoalloteichus cyanogriseus 12A22.

A new deep-sea-derived actinomycete 12A22 was isolated from the sediment of the South China Sea which showed potential cytotoxic and antimicrobial activities. The actinomycete was identified as Actinoalloteichus cyanogriseus by investigating morphological characteristics and phylogenetic analyses based on its 16S rRNA gene sequence. Two compounds, cyclo-(L-Pro-D-Pro-L-Tyr-L-Tyr) (1) and 2-hydroxyethyl-3-methyl-1,4-naphthoquinone (2), were isolated and characterized from the fermentation broth of the strain 12A22. Compound 2 exhibited significant inhibitory activities against a variety of phytopathogenic fungi (Fusarium oxysporum f. sp. cucumerinum, Setosphaeria turcica, and Botrytis cinerea) and Gram-positive bacterium (Bacillus subtilis). In particular, this compound showed better antifungal activity against Botrytis cinerea than positive control amphotericin B. Besides, compound 2 showed moderate cytotoxic activity against human breast cancer MDA-MB-435 cells with IC50 10.59 µM, weaker than the positive control diaminedichloroplatinum with 5.91 µM. Our results suggested that this naphthoquinone could be used as a potential antimicrobial and antitumor agent. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02846-0.

Genome mining and metabolic profiling illuminate the chemistry driving diverse biological activities of Bacillus siamensis SCSIO 05746.

Bacillus spp. are important producers of bioactive natural products with potential applications in medicine and agriculture. Bacillus sp. SCSIO 05476 from a deep-sea sediment exhibits broad-spectrum antimicrobial activities and strong cytotoxic activity. Here, an integrative approach combining genome mining and metabolic profiling has been applied to decipher the chemical origins of this strain's varied and significant biological activities. First, genome mining revealed 19 candidate gene clusters encoding the biosynthesis of diverse secondary metabolites. Then, a series of bacillibactins, fengycins, bacillomycins, surfactins, bacillaenes, macrolactins, and related species were found by LC-DAD-MS. Finally, three new linear bacillibactins, linbacillibactins A-C (1-3), along with 11 known secondary metabolites, bacillibactin (4), normal-C13 Val7 surfactin (5), anteiso-C13 Leu7 surfactin (6), iso-C14 Leu7 surfactin (7), normal-C14 Leu7 surfactin (8), anteiso-C14 Leu7 surfactin (9), macrolactin D (10), normal-C14 bacillomycin D (11), iso-C16 bacillomycin D (12), normal-C17 bacillomycin D (13), and iso-C17 bacillomycin D (14), were obtained and elucidated by bioactivity and structure-guided isolation from the fermentation of strain SCSIO 05746. Among them, new compounds 1-3 show significant siderophore activities comparable to that of bacillibactin (4), compounds 13 and 14 exhibit strong cytotoxic activity. At the same time, the strain classification status was confirmed by genomic analyses, and the complete genome sequence of Bacillus siamensis was presented firstly. This study provides a foundation for understanding the mechanisms driving SCSIO 05746's multiple bioactivities and demonstrates a successful way of discovering bioactive metabolites using a combination of genome mining and metabolic profiling methods.

Characterization and abolishment of the cyclopiazonic acids produced by Aspergillus oryzae HMP-F28.

Extracellular alkalinization and H2O2 production are important early events during induced resistance establishment in plants. In a screen for metabolites as plant resistance activators from 98 fungal isolates associated with marine sponge Hymeniacidon perleve, the cyclopiazonic acids (CPAs) produced by Aspergillus oryzae HMP-F28 induced significant extracellular alkalinization coupled with augmented H2O2 production in tobacco cell suspensions. Bioassay-guided fractionation led to the isolation and structural elucidation of a new CPA congener (4, 3-hydroxysperadine A) and three known ones (1-3). To construct a mutasynthetic strain to generate unnatural CPA analogues, a hybrid pks-nrps gene (cpaS) was disrupted to abolish the production of the critical precursor of cyclo-acetoacetyl-L-tryptophan (cAATrp) and all the downstream CPA products. Elimination of cAATrp will allow cAATrp mimics being processed by the CPA biosynthetic machinery to produce CPA derivatives with designed structural features.

Novel hybrids of brefeldin A and nitrogen mustards with improved antiproliferative selectivity: Design, synthesis and antitumor biological evaluation.

A series of novel conjugates of brefeldin A (11a-c, 12a-c and 13a-c) were obtained by introducing a variety of nitrogen mustards at 4-OH or 7-OH position to explore more efficacious and less toxic antitumor agents. The antiproliferative activities were tested against three cancer cell lines (HL-60, PC-3 and Bel-7402) and one multidrug resistant cell line Bel-7402/5-FU. Among them, compound 11a was the strongest derivative with IC50 values of 4.48, 9.37, 0.2 and 0.84 µM, respectively, and more potent than nitrogen mustards. Though the antiproliferative potency was weaker than the lead compound brefeldin A, 11a displayed lower toxicity than brefeldin A (IC50 < 0.001 µM) with an IC50 of 9.74 µM against normal human liver L-O2 cells, showing good selectivity between normal and malignant liver cells. The mechanism studies confirmed that 11a could induce apoptosis, arrest cell cycle at the G1 phase and lead to mitochondrial dysfunction in Bel-7402 cells at submicromolar concentrations. Furthermore, 11a induced the intrinsic apoptotic mitochondrial pathway in Bel-7402 cells, evidenced by the enhanced expression of the pro-apoptotic protein Bax, cyto-c and p53, and the reduced expression of the anti-apoptotic protein Bcl-2. The caspase-9 and -3 levels were also up-regulated.

Nitric oxide-releasing derivatives of brefeldin A as potent and highly selective anticancer agents.

A series of NO-donating mono- or diester derivatives of brefeldin A were designed, synthesized and biologically evaluated. Some derivatives exhibited potent antiproliferative activity with low IC50 values. The most potent NO-donating hybrid 13b exhibited stronger cytotoxicity against human prostate cancer PC-3 cells, human colon carcinoma HT-29 cells and human liver cancer HepG-2 cells than BFA with IC50 values of 25 nM, 160 nM and 180 nM, respectively. More importantly, compound 13b showed good selectivity between human normal and tumor liver cells with selectivity index of 33. Additionally, 13b released higher levels of NO in HepG-2 cells than L-02 cells. Further mechanism concerning cellular apoptosis showed that 13b induced apoptosis and S phase cell cycle arrest in HepG-2 cells. Incubation with 13b increased the number of HepG-2 cells with collapsed mitochondrial membrane at low concentrations in dose-dependent manner. In addition, by using the Human Apoptosis Protein Array kit, several apoptosis-related proteins, including HO-1, HO-2 and survivin, were found to be markedly downregulated by 13b in HepG-2 cells. Furthermore, in western blot assay, 13b increased the expression of Bax, Cyt c and caspase 3, and reduced the relative levels of Bcl-2, Bcl-xl and pro-caspase 3 in HepG-2 cells.

The complete genome sequence of Bacillus velezensis 9912D reveals its biocontrol mechanism as a novel commercial biological fungicide agent.

A Bacillus sp. 9912 mutant, 9912D, was approved as a new biological fungicide agent by the Ministry of Agriculture of the People's Republic of China in 2016 owing to its excellent inhibitory effect on various plant pathogens and being environment-friendly. Here, we present the genome of 9912D with a circular chromosome having 4436 coding DNA sequences (CDSs), and a circular plasmid encoding 59 CDSs. This strain was finally designated as Bacillus velezensis based on phylogenomic analyses. Genome analysis revealed a total of 19 candidate gene clusters involved in secondary metabolite biosynthesis, including potential new type II lantibiotics. The absence of fengycin biosynthetic gene cluster is noteworthy. Our data offer insights into the genetic, biological and physiological characteristics of this strain and aid in deeper understanding of its biocontrol mechanism.

Paenibacillin A, a new 2(1H)-pyrazinone ring-containing natural product from the endophytic bacterium Paenibacillus sp. Xy-2.

A new 2(1H)-pyrazinone ring-containing natural product, paenibacillin A (1), together with five known diketopiperazine derivatives 2-6 and two known isoflavones 7-8, was isolated from the culture of an endophytic bacterium Paenibacillus sp. Xy-2. The structure of compound 1 was elucidated by extensive spectral methods, including UV, IR, HR-ESI-MS, 1D and 2D NMR and ECD experiments. Compound 1 exhibited moderate cytotoxicity against HL-60 cell line with IC50 value of 50.48 µM.

Nocardiopsis oceani sp. nov. and Nocardiopsis nanhaiensis sp. nov., actinomycetes isolated from marine sediment of the South China Sea.

Two actinomycete strains, designated 10A08AT and 10A08BT, were isolated from marine sediment samples of the South China Sea and their taxonomic positions were determined by a polyphasic approach. The two Gram-stain-positive, aerobic strains produced branched substrate mycelium and aerial hyphae, and no diffusible pigment was produced in the media tested. At maturity, spore chains were formed on aerial hyphae and all mycelium fragmented with age. Whole-cell hydrolysates of both strains contained meso-diaminopimelic acid and no diagnostic sugars. Their predominant menaquinones (>10 %) were MK-9(H4), MK-9(H6) and MK-10(H6) for strain 10A08AT and MK-9(H4), MK-9(H6), MK-10(H4) and MK-10(H6) for strain 10A08BT. The polar lipids detected from the two strains were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine and unknown phosphoglycolipids and phospholipids. The major fatty acids (>10 %) of both strains were iso-C16 : 0 and summed feature 4 (iso-C17 : 1 I and/or anteiso-C17 : 1 B). The genomic DNA G+C contents of strains 10A08AT and 10A08BT were 70.9 and 71.6 mol%, respectively. On the basis of 16S rRNA gene sequence similarities, the two strains were shown to be most closely related to species of the genus Nocardiopsis. DNA­DNA hybridization relatedness values of < 70 % between these two isolates and their closest neighbour, Nocardiopsis terrae YIM 90022T, and between the two strains supported the conclusion that they represent two novel species. Based on phylogenetic analysis and phenotypic and genotypic data, it is concluded that the two isolates belong to the genus Nocardiopsis, and the names Nocardiopsis oceani sp. nov. (type strain 10A08AT = DSM 45931T = BCRC 16951T) and Nocardiopsis nanhaiensis sp. nov. (type strain 10A08BT = CGMCC 47227T = BCRC 16952T) are proposed.

Draft genome sequence of the novel strain Pseudomonas sp. 10B238 with potential ability to produce antibiotics from deep-sea sediment.

Pseudomonas sp. 10B238 was a putatively novel species of Pseudomonas, isolated from a deep-sea sediment of the South China Sea, which had the genetic potential to produce secondary metabolites related to nonribosomal peptides (NRPs), as well as showed moderate antimicrobial activities. Here we report a high quality draft genome of Pseudomonas sp. 10B238, which comprises 4,933,052bp with the G+C content of 60.23%. A total of 11 potential secondary metabolite biosynthetic gene clusters were predicted, including a NRP for new peptide siderophore. And many anaerobic respiratory terminal enzymes were found for life in deep-sea environments. Our results may provide insights into biosynthetic pathway for antimicrobial bioactive compounds and be helpful to understand the physiological characteristic of this species.

Two new amides from a halotolerant fungus, Myrothecium sp. GS-17.

Two new amides, named N-acetyl-2,4,10,17-tetrahydroxyheptadecylamine (1) and N-acetyl-3,5,11,18-tetrahydroxyoctadecyl-2-amine (2), were isolated from a halotolerant fungus, Myrothecium sp. GS-17. Their structures were identified on the basis of spectroscopic characteristics. The cancer cell cytotoxicities of two compounds were evaluated, and compound 2 exhibited weak cytotoxicity in HL-60 cell line.

Streptomyces bohaiensis sp. nov., a novel actinomycete isolated from Scomberomorus niphonius in the Bohai Sea.

A novel actinomycete strain, designated 11A07(T), was isolated from young Scomberomorus niphonius in the Bohai Sea. Basic local alignment search tool analyses showed that this isolate had the highest 16S rRNA gene sequence similarity of 97.41% with Streptomyces rimosus subsp. paromomycinus DSM 41429(T). Phylogenetic tree revealed that strain 11A07(T) formed a distinct lineage clustered with Streptomyces panacagri Gsoil 519(T), Streptomyces sodiiphilus YIM 80305(T) and Streptomyces albus subsp. albus NRRL B-2365(T) having similarities of 97.30%, 97.10% and 96.83%, respectively. Multilocus sequence analysis further demonstrated that the new isolate was different from the selected representatives of Streptomyces as a separate phylogenetic line. Strain 11A07(T) produced straight or rectiflexibile spore chains with smooth surface, white aerial mycelia and brown diffusible pigments on international streptomyces project 2 medium. Maximum tolerated NaCl concentration for growth was 11.0%. Whole-cell sugars were mannose, ribose, glucose, galactose and xylose. The predominant menaquinones were MK-9(H2), MK-9(H4) and MK-9 (H6). The fatty-acid profile contained iso-C16:0, C18:0 10-methyl (tuberculostearic acid) and anteiso-C17:0 as the major compositions. The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and an unknown phospholipid. The G+C content of the genomic DNA was 71.4 mol%. These morphological, phenotypic and chemotaxonomic properties showed that strain 11A07(T) could be readily distinguished from the most closely related members of the genus Streptomyces. Thus, based on the polyphasic taxonomic data, strain 11A07(T) (=JCM 19630(T)=CCTCC AA 2013020(T)=KCTC 29263(T)) represents a novel species within the genus Streptomyces, for which the name Streptomyces bohaiensis sp. nov. is proposed.

Identification and characterization of the antifungal substances of a novel Streptomyces cavourensis NA4.

A new actinomycete strain NA4 was isolated from a deep-sea sediment collected from the South China Sea and showed promising antifungal activities against soilborne fungal pathogens. It was identified as Streptomyces cavourensis by morphological, physiological, and phylogenetic analyses based on its 16S rRNA gene sequence. The main antifungal components were isolated and identified from the fermentation culture as bafilomycins B1 and C1. These compounds exhibited significant antifungal activities and a broad antifungal spectrum. The results suggest that the Streptomyces cavourensis NA4 and bafilomycins B1 and C1 could be used as potential biocontrol agents for soilborne fungal diseases of plants.