A facile strategy for the construction of a phage display cyclic peptide library for the selection of functional macrocycles.

Cyclic peptides represent invaluable scaffolds in biological affinity, providing diverse collections for discovering functional molecules targeting challenging biological entities and protein-protein interactions. The field increasingly focuses on developing cyclization strategies and chemically modified combinatorial libraries in conjunction with M13 phage display, to identify macrocyclic peptide inhibitors for traditionally challenging targets. Here, we introduce a cyclization strategy utilizing ortho-phthalaldehyde (OPA) for the discovery of active macrocycles characterized by asymmetric scaffolds with side-chain cyclization. Through this approach, aldehyde groups attached to free molecules sequentially attack the ε-amine of lysine and the thiol of cysteine, facilitating the rapid cyclization of genetically encoded linear precursor libraries displayed on phage particles. The construction of a 109-member library and subsequent screening successfully identified cyclic peptide binders targeting three therapeutically relevant proteins: PTP1B, NEK7, and hKeap1. The results confirm the efficacy in rapidly obtaining active ligands with micromolar potency. This work provides a fast and efficient operable high-throughput platform for screening functional peptide macrocycles, which hold promise for broad application in therapeutics, chemically biological probes, and disease diagnosis.

Harzianic acids and oxazolidinone from the endophytic fungus Ilyonectria sp. and their cytotoxicity activity.

Four undescribed compounds including three harzianic acids (1, 3 and 4) and one oxazolidinone (2), along with three known ones (5-7) were isolated from the solid fermented product of endophytic fungus Ilyonectria sp., their structures were elucidated as 1-amino-harzianic acid (1), ilyonectria-oxazolidinone (2),10'-nor- isoharzianic acid (3), isohomoharzianic acid (4), harzianic acid (5), isoharzianic acid (6), homoharzianic acid (7) by means of detailed chemical evidences and spectroscopic data analysis. All the compounds were evaluated for cytotoxicity against SMMC-7721 human cancer cell lines by MTS assay. Among the seven tested compounds, 1-amino-harzianic acid (1) demonstrated well cytotoxic activity against SMMC-7721 with IC50 value of 26.84 µM. The results of molecular docking indicated that compound exhibited moderate anti-tumor activity may through binding to apoptosis related proteins.

Chromones from the endophytic fungus Bipolaris eleusines.

Eight previously undescribed chromones eleusineketones A-H (1-8), as well as eight known compounds (9-16), were isolated from the endophytic fungus Bipolaris eleusines. These planar structures were created using an in-depth analysis of their spectral data, which included 1D, 2D, and HRESIMS data. Furthermore, the absolute configurations of compounds 1, 2, and 6 were determined by spectroscopic analysis and quantum chemical computational approaches, and compound 5 was determined by single-crystal X-ray diffraction analysis. The cytotoxic activity assay revealed that compounds 1 and 5 both inhibited MDA-MB-231 cells with IC50 values of 14.48 µM and 17.99 µM, respectively.

The antifungal properties of terpenoids from the endophytic fungus Bipolaris eleusines.

A series of terpenoids (1-17), comprising six new compounds designated bipolariterpenes A-F (1-6) and eleven recognized compounds (7-17), were isolated from the wheat culture of the potato endophytic fungus Bipolaris eleusines. Their structures and stereochemistry were clarified by HRESIMS, NMR, DP4 + probability analyses, and computations for electronic circular dichroism (ECD). All compounds are made up of six meroterpenoids, four sesterterpenes and seven sesquiterpenes. Among them, four sesterterpenes (4, 5, 10, 11) were investigated for their antifungal, antibacterial and cytotoxic properties, and six meroterpenoids (1-3, 7-9) were evaluated for their antifungal properties. The compounds 7, 9, and 10 had substantial antifungal activity against Epidermophyton floccosum at a concentration of 100 µM. No antibacterial and cytotoxic activities were observed.

Boerelasins A-D, Four Unprecedented Cytochalasins from the Endophytic Fungus Boeremia Exigua.

Four undescribed cytochalasins (1-4) were isolated from the endophytic fungus Boeremia exigua. Structurally, boerelasin A (1) represents the first example of a cytochalasin with a rare 5/5 bicyclic carbon core. Boerelasin B (2) possesses an unprecedented 5/6/5/6/8 pentacyclic ring system. Boerelasin C (3), a derivative from the common biosynthetic intermediate to 1, is a macrocyclic ring-opening cytochalasin, and boerelasin D (4) contains an uncommon six-carbon alkyl acid side chain. The structures were elucidated based on spectroscopic methods, electronic circular dichroism, spin-spin coupling constants, and calculated nuclear magnetic resonance with DP4+ analysis. These compounds exhibited significant cytotoxicity against the tumor cells.

Six Unprecedented Cytochalasin Derivatives from the Potato Endophytic Fungus Xylaria curta E10 and Their Cytotoxicity.

Six previously undescribed cytochalasins, Curtachalasins X1-X6 (1-6), together with six known compounds (7-12) were isolated from the endophytic fungus Xylaria curta E10 harbored in the plant Solanum tuberosum. The structures were elucidated by the interpretation of HRESIMS, UV, and NMR data. The absolute configurations of Curtachalasins X1-X6 were determined by comparison of their experimental and calculated electronic circular dichroism (ECD) spectra. In bioassays, Curtachalasin X1 (1) and X5 (5) showed cytotoxic activity against the MCF-7 cell line with IC50 values of 2.03 µM and 0.85 µM, respectively.

Pimarane Diterpenes from Fungi.

Pimarane diterpenes are a kind of tricyclic diterpene, generally isolated from plant and fungi. In nature, fungi distribute widely and there are nearly two to three million species. They provide many secondary metabolites, including pimarane diterpenes, with novel skeletons and bioactivities. These natural products from fungi have the potential to be developed into clinical medicines. Herein, the structures and bioactivities of 197 pimarane diterpenes are summarized and the biosynthesis and pharmacological researches of pimarane diterpenes are introduced. This review may be useful improving the understanding of pimarane diterpenes from fungi.

Paecilins F-P, new dimeric chromanones isolated from the endophytic fungus Xylaria curta E10, and structural revision of paecilin A.

A total of eleven new dimeric chromanones, paecilins F-P (2-12), were isolated from the endophytic fungus Xylaria curta E10, along with four known analogs (1, 13-15). Their structures and absolute configurations were determined by extensive experimental spectroscopic methods, single-crystal X-ray diffraction, and equivalent circulating density (ECD) calculations. In addition, the structure of paecilin A, which was reported to be a symmetric C8-C8' dimeric pattern, was revised by analysis of the nuclear magnetic resonance (NMR) data, and single-crystal X-ray diffraction. Compound 1 showed antifungal activity against the human pathogenic fungus Candida albicans with a minimum inhibitory concentration of 16 µg/mL, and Compounds 8 and 10 showed antibacterial activity against the gram-negative bacterium Escherichia coli with the same minimum inhibitory concentration of 16 µg/mL.

Four New Highly Oxygenated Eremophilane Sesquiterpenes from an Endophytic Fungus Boeremia exigua Isolated from Fritillaria hupehensis.

Four new eremophilane-type sesquiterpenes, boeremialanes A-D (1-4) were obtained from solid substrate cultures of Boeremia exigua (Didymellaceae), an endophytic fungus isolated from Fritillaria hupehensis (Liliaceae). Boeremialanes A-C (1-3) are highly oxygenated eremophilanes with a benzoate unit attached at the C-13 position and are rarely found in nature. Their structures and absolute configurations were determined by extensive spectroscopic methods, electronic circular dichroism (ECD), and nuclear magnetic resonance (NMR) calculations with DP4+ analysis. Boeremialane D (4) potently inhibited nitric oxide production in lipopolysaccharide-treated RAW264.7 macrophages with an IC50 of 8.62 µM and was more potent than the positive control, pyrrolidinedithiocarbamate (IC50 = 23.1 µM).

Three new pyrrole alkaloids from the endophytic fungus Albifimbria viridis.

Three new pyrrole alkaloids albifipyrrols A-C (1-3), were isolated from the endophytic fungus Albifimbria viridis collected from the Chinese medicinal plant. Their structures were elucidated by extensive NMR and HRESIMS spectrometric analyses. All compounds were evaluated for immunosuppressive activity. Fortunately, compound 2 exhibits certain inhibition specifically against the LPS-induced proliferation of B lymphocyte cells with IC50 value 16.16 µM.

Oblongolides from endophytic fungus Phoma bellidis Neerg. harbored in Tricyrtis maculata (D. Don) J.F.Macbr.

Five previously undescribed oblongolides, namely phomaones A-E, along with four known compounds, were isolated from the endophytic fungus Phoma bellidis Neerg.. Their structures and absolute configurations were determined by extensive experimental spectroscopic methods as well as single crystal X-ray diffractions, ECD calculations and GIAO 13C NMR calculations. Phomaone A represent the first example of oblongolides with glycol directly linked by two C-C bonds, and its biosynthetic pathway were proposed. The cytotoxicity of obtained compounds was evaluated against human cancer cell lines MCF-7, DU145, and SW480. All compounds except phomaone A showed the cytotoxicity against MCF-7 with IC50 value ranging from 12.45 to 49.84 µM.

Ophiorrhines F and G, Key Biogenetic Intermediates of Ophiorrhine Alkaloids from Ophiorrhiza japonica and Their Immunosuppressant Activities.

Continued interest in bioactive alkaloids led to the isolation of two undescribed alkaloids, ophiorrhines F (1) and G (2), from the aerial parts of Ophiorrhiza japonica. Their structures were elucidated based on spectroscopic methods, electronic circular dichroism, and calculated NMR with DP4+ analysis. These two alkaloids represent key biological genetic intermediates in the formation of ring C in the ophiorrhines. Compound 1 exhibited good inhibition on LPS-induced B cell proliferation with an IC50 value of 0.38 µM and showed significant selective inhibitory activity on a B cell proliferation response with a selective index of 548.42. A preliminary study indicated that 1 may have a new mechanism of immunosuppression.

Structure and cytotoxicity of trichothecenes produced by the potato-associated fungus Trichothecium crotocinigenum.

Seven previously undescribed trichothecenes, named trichothecrotocins M-S (1-7), along with five known compounds, were isolated from rice cultures of the potato-associated fungus Trichothecium crotocinigenum. Their structures and absolute configurations were determined through spectroscopic methods, single-crystal X-ray diffraction, and quantum chemistry calculations on ECD. Compound 1 possesses a rare 6,11-epoxy moiety in the trichothecene family. Compound 6 exhibited strong cytotoxic activity against MCF-7 cancer cell lines with an IC50 value of 2.34 ± 0.45 µM. It promoted apoptosis induction in MCF-7 cells. Moreover, cell cycle analysis showed cell cycle arrest caused by compound 6 at the G2/M phase which resulted to cell proliferation inhibition and pro-apoptotic activity. Further quantitative real-time PCR (qRT-PCR) analysis confirmed that the G2/M arrest was accompanied by upregulation of p21 and down regulation of cyclins B1 in 6-treated MCF-7 cells.

Identification and Bioactivities of Secondary Metabolites Derived from Endophytic Fungi Isolated from Ethnomedicinal Plants of Tujia in Hubei Province: A Review.

Tujia is a national minority, inhabiting in the mountainous Wuling area in China. Since 1978, Tujia medicine has been studied, summarized and developed, leading to numerous achievements by Chinese researchers, such as the publishing of approximately 30 monographs of Tujia medicine. These publications are focused on summarizing and improving the theory of Tujia medicine and developing clinical therapies from this system of medicine. The shortage of natural medicinal plants used in Tujia medicine has created the need to discover new resources to replace them and protect endangered natural plant species. Endophytic fungi are one of the conservation options, are considered a source of new bioactive natural products, and are a renewable and inexhaustible source of new drugs and agrochemicals. This review summarizes 260 compounds from endophytic fungi that have been previously isolated from the medicinal plants of Tujia. These compounds include steroids, terpenoids, meroterpenoids, polyketides, alkaloids, peptides, aliphatic compounds, aromatic compounds, and heterocyclic compounds.

Immunosuppressive Isopimarane Diterpenes From Cultures of the Endophytic Fungus Ilyonectria robusta.

Five new isopimarane diterpenes, robustaditerpene A-E (1-5), which include 19-nor-isopimarane skeleton and isopimarane skeleton, were isolated from the liquid fermentation of the endophytic fungus Ilyonectria robusta collected from Bletilla striata. The structure elucidation and relative configuration assignments of all compounds were accomplished by interpretation of NMR and HRESIMS spectrometric analyses and 13C NMR calculation. And the absolute configuration of 1-5 were identified by single-crystal X-ray diffraction and ECD calculation. Compound 3 inhibited lipopolysaccharide-induced B lymphocytes cell proliferation with an IC50 value at 17.42 ± 1.57 µM while compound 5 inhibited concanavalin A-induced T lymphocytes cell proliferation with an IC50 value at 75.22 ± 6.10 µM. These data suggested that compounds 3 and 5 may possess potential immunosuppressive prospect.

Characterization of the complete chloroplast genome of a well-known Chinese medicinal herb, Scrophularia ningpoensis.

Scrophularia ningpoensis has been used as a famous traditional medicinal herb in Asian countries to treat jaundice, dysentery, and the pain of rheumatism. In this paper, the complete chloroplast (cp) genome sequence of S. ningpoensis was reported and characterized. The cp genome is 153,175 bp in length, composed of a pair of 25,490 bp inverted repeat (IR) regions separated by a large single copy (LSC) region of 84,257 bp and a small single copy (SSC) region of 17,938 bp. There were 130 predicted genes (85 protein-coding genes, 37 tRNA genes, and 8 rRNA genes) in the genome, and the overall GC content of the genome is 38%. Phylogenetic analysis based on the cp genome data showed that S. ningpoensis was sister to S. buergeriana.

Comparative transcriptome combined with metabolome analyses revealed key factors involved in nitric oxide (NO)-regulated cadmium stress adaptation in tall fescue.

BACKGROUND: It has been reported that nitric oxide (NO) could ameliorate cadmium (Cd) toxicity in tall fescue; however, the underlying mechanisms of NO mediated Cd detoxification are largely unknown. In this study, we investigated the possible molecular mechanisms of Cd detoxification process by comparative transcriptomic and metabolomic approaches. RESULTS: The application of Sodium nitroprusside (SNP) as NO donor decreased the Cd content of tall fescue by 11% under Cd stress (T1 treatment), but the Cd content was increased by 24% when treated with Carboxy-PTIO (c-PTIO) together with Nitro-L-arginine methyl ester (L-NAME) (T2 treatment). RNA-seq analysis revealed that 904 (414 up- and 490 down-regulated) and 118 (74 up- and 44 down-regulated) DEGs were identified in the T1 vs Cd (only Cd treatment) and T2 vs Cd comparisons, respectively. Moreover, metabolite profile analysis showed that 99 (65 up- and 34-down- regulated) and 131 (45 up- and 86 down-regulated) metabolites were altered in the T1 vs Cd and T2 vs Cd comparisons, respectively. The integrated analyses of transcriptomic and metabolic data showed that 81 DEGs and 15 differentially expressed metabolites were involved in 20 NO-induced pathways. The dominant pathways were antioxidant activities such as glutathione metabolism, arginine and proline metabolism, secondary metabolites such as flavone and flavonol biosynthesis and phenylpropanoid biosynthesis, ABC transporters, and nitrogen metabolism. CONCLUSIONS: In general, the results revealed that there are three major mechanisms involved in NO-mediated Cd detoxification in tall fescue, including (a) antioxidant capacity enhancement; (b) accumulation of secondary metabolites related to cadmium chelation and sequestration; and (c) regulation of cadmium ion transportation, such as ABC transporter activation. In conclusion, this study provides new insights into the NO-mediated cadmium stress response.

Trichothecrotocins D-L, Antifungal Agents from a Potato-Associated Trichothecium crotocinigenum.

Seven new merosesquiterpenoids, trichothecrotocins D-J (1-7), two new trichothecene sesquiterpenoids, trichothecrotocins K (12) and L (13), and six known compounds (8-11, 14, and 15), were isolated from a potato-associated fungus, Trichothecium crotocinigenum. Compounds 5 and 6 were racemates which were further separated as pure enantiomers. Structures together with absolute configurations were established by extensive spectroscopic analysis, as well as quantum chemistry calculations on ECD and optical rotations. Compounds 1-4 are rare meroterpenoids featuring a seco-phenyl group, while 1 and 2 possessed a novel 6-6/5 fused ring system. Compounds 1-4, 8, 11, and 12 showed antifungal activity against four plant pathogens with MIC values of 8-128 µg/mL. It is suggested that the meroterpenoids produced by T. crotocinigenum may play an important role in the antifungal property of the fungus, thereby protecting the host plant, i.e., potato.

Depsidones and diaryl ethers from potato endophytic fungus Boeremia exigua.

Three depsidones boremexins A-C (1-3), two diaryl ethers boremexins D (4) and E (5), together with four known compounds were obtained from cultures of potato endophytic fungus Boeremia exigua. Their structures with absolute configurations were established by extensive spectroscopic methods and electronic circular dichroism (ECD) calculations. Compounds 1-4, 6, and 9 displayed anti-inflammatory properties on nitric oxide production in LPS-induced RAW264.7 macrophages with an IC50 range of 19.4-34.4 µM. Compounds 2 and 5 exhibited cytotoxicities to human breast cancer cell line (MCF-7) with IC50 values of 33.1 and 4.0 µM, respectively.

Novel Metabolites from the Endophytic Fungus Chaetomium subaffine L01.

One natural p-terphenyl glycoside, gliocladinin C, and two furano-polyene derivatives, chaetominins A and B, were isolated from potato endophytic fungus Chaetomium subaffine. The absolute configurations of these compounds were elucidated by HR-ESI-MS, NMR, the DP4+ probabilities and electronic circular dichroism (ECD) spectra. Furthermore, gliocladinin C and chaetominin A showed cytotoxic activity against two selected human tumor cell lines (Hep-2 and HepG-2).