Divergent Biosynthesis of Bridged Polycyclic Sesquiterpenoids by a Minimal Fungal Biosynthetic Gene Cluster.

The bridged polycyclic sesquiterpenoids derived from sativene, isosativene, and longifolene have unique structures, and many chemical synthesis approaches with at least 10 steps have been reported. However, their biosynthetic pathway remains undescribed. A minimal biosynthetic gene cluster (BGC), named bip, encoding a sesquiterpene cyclase (BipA) and a cytochrome P450 (BipB) is characterized to produce such complex sesquiterpenoids with multiple carbon skeletons based on enzymatic assays, heterologous expression, and precursor experiments. BipA is demonstrated as a versatile cyclase with (-)-sativene as the dominant product and (-)-isosativene and (-)-longifolene as minor ones. BipB is capable of hydroxylating different enantiomeric sesquiterpenes, such as (-)-longifolene and (+)-longifolene, at C-15 and C-14 in turn. The C-15- or both C-15- and C-14-hydroxylated products are then further oxidized by unclustered oxidases, resulting in a structurally diverse array of sesquiterpenoids. Bioinformatic analysis reveals the BipB homologues as a discrete clade of fungal sesquiterpene P450s. These findings elucidate the concise and divergent biosynthesis of such intricate bridged polycyclic sesquiterpenoids, offer valuable biocatalysts for biotransformation, and highlight the distinct biosynthetic strategy employed by nature compared to chemical synthesis.

Genome Mining of Linaridins Provides Insights into the Widely Distributed LinC Oxidoreductases.

Linaridins are a family of underexplored ribosomally synthesized and post-translationally modified peptides despite the prevalence of their biosynthetic gene clusters (BGCs) in microbial genomes, as shown by bioinformatic studies. Our genome mining efforts reveal that 96 putative oxidoreductase genes, namely, LinC, are encoded in linaridin BGCs. We heterologously expressed two such LinC-containing linaridin BGCs, yan and ydn, from Streptomyces yunnanensis and obtained three new linaridins, named yunnanaridins A-C (1-3). Their structures are characterized by Z-configurations of the dehydrobutyrines and the presence of a variety of epimerized amino acid residues. Yunnanaridin A (1) is the sixth member of the family of type-B linaridins, whereas yunnanaridins B (2) and C (3) represent the first examples of expressed type-C linaridins. Interestingly, heterologous expression of the same BGCs with LinC in-frame knockouts produced the same compounds. This work expands the structural diversity of linaridins and provides evidence for the notion that the widespread LinCs may not be involved in linaridin biosynthesis.

Jatrophane diterpenoids with cytotoxic activity from the whole plant of Euphorbia heliosocpia L.

Eight undescribed jatrophane diterpenoids, euphohelinoids A-H, along with 11 known analogues were isolated from the whole plant of Euphorbia heliosocpia L. Among them, euphohelinoids A and B contain a rare type of jatrophane diterpenoid skeleton with a 7,8-seco scaffold. To the best of our knowledge, only two such jatrophane diterpenoids have been reported. In addition, euphohelinoids G and H belong to a rare class of jatrophane diterpene possessing a ß-hydroxy group at C-11. Structure elucidation of these undescribed diterpenoids was performed by spectroscopic analysis, including NMR, HRESIMS, IR, electronic circular dichroism (ECD) and DP4+ analysis. The cytotoxicity of 17 abundant jatrophane diterpenes was evaluated against HepG2, HeLa, HL-60, and SMMC-7721 cell lines. Seven compounds presented potent cytotoxicity against the four tested cell lines with IC50 values from 8.1 to 29.7 µM. Moreover, preliminary structure-activity relationships for these jatrophane diterpenoids were discussed.

Cardiac glycosides from Digitalis lanata and their cytotoxic activities.

Cardiac glycosides (CGs) are good candidates as drug leads in the treatment of cancer because of their structural diversities and potent biological activities. In this study, fifteen CGs including three new ones (1-3) were isolated from Digitalis lanata Ehrh. Their structures were elucidated by HRESIMS, NMR spectroscopic methods, including homonuclear and heteronuclear coupling constant analysis, and acid-catalyzed hydrolysis and derivatization analysis of the sugar chain. The cytotoxic activities of these CGs were evaluated against three human cancer cell lines (A549, HeLa and MCF-7 cell lines), and all of them showed strong activities at nanomolar scale. The flow cytometric analysis indicated that compound 1 induced cell cycle arrest in the G2/M phase. Transcriptome analysis revealed a panel of possible targets for compound 1. RT-PCR and western blot experiments showed that 1 significantly inhibited the expression of vasohibin-2 (VASH2). Moreover, compound 1 restrained angiogenesis in a concentration-dependent manner in the chick embryo chorioallantoic membrane (CAM) model.

Generation of Fused Seven-Membered Polycyclic Systems via Ring Expansion and Application to the Total Synthesis of Sesquiterpenoids.

Seven-membered polycyclic architectures, widely present in natural products and molecular drugs, are challenging synthetic targets. However, methods for synthesizing fused medium-sized bicyclo[m.n.0] ring systems, including the benzo-cycloheptane systems, are still urgent. Herein we describe a base-induced ring expansion as a general strategy to construct a wide range of fused seven-membered ring systems. The application of this method was demonstrated by the efficient total syntheses of two sesquiterpenoids, plecarpenene and plecarpenone, both bearing a fused bicyclo[5.3.0]decane skeleton.

Total Synthesis of Complex Peptidyl Nucleoside Antibiotics: Asymmetric De Novo Syntheses of Miharamycin†B and Its Biosynthetic Precursor.

Miharamycins belong to a class of peptidyl nucleoside antibiotics with a unique nine-carbon pyranosyl amino acid core and a rare 2-aminopurine moiety. Herein, we report the de novo total synthesis of miharamycin B and its biosynthetic precursor from 3-bromofuran and Garner's aldehyde through a modified Achmatowicz reaction. Many challenges were resolved toward the de novo synthesis of miharamycin B, including the introduction of a dense array of functional groups, the stereoselective construction of consecutive stereocenters, dealing with the variability of the anomeric positions, and promoting site-selectivity in the cyclization to form the tetrahydrofuran ring. This de novo synthesis strategy enables efficient preparation of 3'-substituted saccharides, allowing the study of their structure-activity relationships and mode of action, and meets the growing demand for the development of novel antibiotics inspired by miharamycin natural products.

Lanthipeptides from the Same Core Sequence: Characterization of a Class II Lanthipeptide Synthetase from Microcystis aeruginosa NIES-88.

Class II lanthipeptide synthetases (LanMs) are relatively promiscuous to core peptide variations. Previous studies have shown that different LanMs catalyze identical reactions on the same core sequence fused to their respective cognate leaders. We characterized a new LanM enzyme from Microcystis aeruginosa NIES-88, MalM, and demonstrated that MalM and ProcM exhibited disparate dehydration and cyclization patterns on identical core peptides. Our study provided new insights into the regioselectivity of LanMs and showcased an appropriate strategy for lanthipeptide structural diversity engineering.

Biochemical Reconstitution Reveals the Biosynthetic Timing and Substrate Specificity for Thioamitides.

Thioamitides are apoptosis-inducing ribosomally synthesized and post-translationally modified peptides (RiPPs) with substantial post-translational modifications (PTMs), whose biosynthetic details remain elusive. We reconstituted their key PTMs through in vitro enzymatic reactions and gene coexpressions in E. coli and rigorously demonstrated the order of those modifications. Notably, thioamitide biosynthesis involves N- to C-terminal thioamidations and employs both leader-dependent and leader-independent reactions followed by leader removal by successive degradation. Our study provides a comprehensive overview of thioamitide biosynthesis and lays the foundation for thioamitide engineering in E. coli.

Recent Advances in the Discovery and Biosynthetic Study of Eukaryotic RiPP Natural Products.

Natural products have played indispensable roles in drug development and biomedical research. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a group of fast-expanding natural products attribute to genome mining efforts in recent years. Most RiPP natural products were discovered from bacteria, yet many eukaryotic cyclic peptides turned out to be of RiPP origin. This review article presents recent advances in the discovery of eukaryotic RiPP natural products, the elucidation of their biosynthetic pathways, and the molecular basis for their biosynthetic enzyme catalysis.

Which neoadjuvant chemotherapy regimen should be recommended for patients with advanced nasopharyngeal carcinoma?: A network meta-analysis.

BACKGROUND: The clinical application has widespread disagreement on the different regimens of neoadjuvant chemotherapy (NCT) in the treatment of locoregionally advanced nasopharyngeal carcinoma (NPC). We conducted a network meta-analysis (NMA) to evaluate the efficacy of the different NCT regimens in the treatment of NPC. METHODS: A systematic literature search was performed using PubMed, Embase, and Cochran Library. Totally, 31 randomized controlled trials (RCTs) (n = 4062) met study selection criteria and were incorporated in this NMA study. RESULTS: Our study showed that certain NCT regimens improved the prognosis of patients, and found out the relative best solution for each endpoint, such as paclitaxel, carboplatin, and gemcitabine for 1-year overall survival (OS) rate, cisplatin, calcium folinate, and 5-fluorouracil for 2-year OS rate, vinorelbine and cisplatin (NP) for 3-year OS rate, cyclophosphamide, cisplatin, and 5-fluorouracil for 5-year OS rate, NP for complete remission rate, cisplatin and gemcitabine for overall remission rate of the primary tumor. In addition, for certain grade 3 and above toxicity, the results of the NMA reflected certain NCT regimens can reduce toxicity of chemoradiotherapy (CRT) to a minimum, such as NP for anemia, mucositis, and thrombocytopenia, paclitaxel, epirubicin, and cisplatin for neutropenia and skin toxicity. CONCLUSION: Our NMA showed that certain cisplatin-based NCT regimens improved the prognosis of patients with NPC and reduced the toxicity of CRT. However, in view of survival rate and response rate, the best NCT regimen is not entirely consistent. Therefore, which NCT regimen will benefit most patients will need further explored.

CRISPR-Cas9 strategy for activation of silent Streptomyces biosynthetic gene clusters.

Here we report an efficient CRISPR-Cas9 knock-in strategy to activate silent biosynthetic gene clusters (BGCs) in streptomycetes. We applied this one-step strategy to activate multiple BGCs of different classes in five Streptomyces species and triggered the production of unique metabolites, including a novel pentangular type II polyketide in Streptomyces viridochromogenes. This potentially scalable strategy complements existing activation approaches and facilitates discovery efforts to uncover new compounds with interesting bioactivities.

Microseiramide from the Freshwater Cyanobacterium Microseira sp. UIC 10445.

Microseiramide (1), a cyclic heptapeptide, was isolated from a sample of the freshwater cyanobacterium Microseira sp. UIC 10445 collected in a shallow lake in Northern Indiana. Taxonomic identification of UIC 10445 was performed by a combination of morphological and phylogenetic characterization. Phylogenetic analysis revealed that UIC 10445 was a member of the recently described genus Microseira, which is phylogenetically distinct from the morphologically similar genera. Moorea and Lyngbya. The planar structure of microseiramide (1) was determined by extensive 1D and 2D NMR experiments as well as HRESIMS analysis. The absolute configurations of amino acid residues were determined using acid hydrolysis followed by the advanced Marfey's analysis. microseiramide (1) is the first cyclic peptide reported from a Microseira sp., and the structure of microseiramide (1) is distinct from the previously known metabolites from cyanobacteria of the genera Moorea and Lyngbya.

Trichormamides C and D, antiproliferative cyclic lipopeptides from the cultured freshwater cyanobacterium cf. Oscillatoria sp. UIC 10045.

Extract from the cultured freshwater cf. Oscillatoria sp. UIC 10045 showed antiproliferative activity against HT-29 cell line. Bioassay-guided fractionation led to the isolation of two new cyclic lipopeptides, named trichormamides C (1) and D (2). The planar structures were determined by combined analyses of HRESIMS, Q-TOF ESIMS/MS, and 1D and 2D NMR spectra. The absolute configurations of the amino acid residues were assigned by advanced Marfey's analysis after partial and complete acid hydrolysis. Trichormamides C (1) is a cyclic undecapeptide and D (2) is a cyclic dodecapeptide, both containing a lipophilic ß-aminodecanoic acid residue. Trichormamide C (1) displayed antiproliferative activities against HT-29 and MDA-MB-435 cancer cell lines with IC50 values of 1.7 and 1.0µM, respectively, as well as anti-Mycobacterium tuberculosis activity with MIC value of 23.8µg/mL (17.3µM). Trichormamide D (2) was found to be less potent against both HT-29 and MDA-MB-435 cancer cell lines with IC50 values of 11.5 and 11.7µM, respectively.

A novel GC-MS method for determination of chrysophanol in rat plasma and tissues: Application to the pharmacokinetics, tissue distribution and plasma protein binding studies.

The purpose of this study was to explore the plasma protein binding (PPB), pharmacokinetics profiles and tissue distribution of chrysophanol in rats. Biological samples were extracted by ethyl ether, separated and determined by a novel, sensitive and specific GC-MS method under selected ion monitoring (SIM) mode for the first time. This method was validated for the quantification of chrysophanol, in the range of 0.005-1.50µgmL-1 in rat plasma and 0.04-12µgmL-1 in tissue homogenates, respectively. The limit of detection (LOD) was 0.4ngmL-1 or 5.0ngg-1, which obtained excellent sensitivity for the pharmacokinetics analysis in rats. The intra- and inter-day assay of precisions in plasma and tissues were less than 10% and the intra- and inter-day accuracies were 82.1-92.9%. The method recoveries of all samples were more than 90%, except for liver samples (>80%), which indicated that chrysophanol may be partly metabolized in liver homogenates. The PPB rates in rat plasma, human plasma and bovine serum albumin were 83±2, 88±3 and 58±3%, respectively. The main pharmacokinetic parameters were the time of peak concentration (Tmax)=(0.665±0.034) h, the peak concentration (Cmax)=(929.8±102.7)ngmL-1, the area under the curve (AUC)=(1197.6±258.5)nghmL-1 and the clearance (CL)=(0.013±0.0028)mLmg-1h-1. The tissue distribution of chrysophanol in rats after the oral administration showed a decreasing tendency in different tissues (heart>kidney>spleen>liver>lung>cerebrum).

Carbamidocyclophanes F and G with Anti-Mycobacterium tuberculosis Activity from the Cultured Freshwater Cyanobacterium Nostoc sp.

Two new (1 and 2) and three known (3-5) carbamidocyclophanes were isolated from a cultured freshwater cyanobacterium Nostoc sp. (UIC 10274) obtained from a sample collected at Des Plaines, Illinois. Their planar structures and stereoconfigurations were determined by extensive spectroscopic analysis including 1D/2D NMR experiments, HRESIMS as well as CD spectroscopy. Carbamidocyclophane F (1) showed potent anti-Mycobacterium tuberculosis activity in the microplate Alamar blue assay and low-oxygen-recovery assay with MIC values of 0.8 and 5.4 µM, respectively. Carbamidocyclophane F (1) also displayed antimicrobial activities against the gram positive bacteria Staphylococcus aureus and Enterococcus faecalis with MIC values of 0.1 and 0.2 µM, respectively. Carbamidocyclophane F (1) and Carbamidocyclophane G (2) both showed antiproliferative activity against MDA-MB-435 and HT-29 human cancer cell lines with IC50 values in the range from 0.5 to 0.7 µM.

Trichormamides A and B with Antiproliferative Activity from the Cultured Freshwater Cyanobacterium Trichormus sp. UIC 10339.

Two new cyclic lipopeptides, trichormamides A (1) and B (2), were isolated from the cultured freshwater cyanobacterium Trichormus sp. UIC 10339. The strain was obtained from a sample collected in Raven Lake in Northern Wisconsin. The planar structures of trichormamides A (1) and B (2) were determined using a combination of spectroscopic analyses including HRESIMS and 1D and 2D NMR experiments. The absolute configurations of the amino acid residues were assigned by the advanced Marfey's method after acid hydrolysis. Trichormamide A (1) is a cyclic undecapeptide containing two D-amino acid residues (D-Tyr and D-Leu) and one ß-amino acid residue (ß-aminodecanoic acid). Trichormamide B (2) is a cyclic dodecapeptide characterized by the presence of four nonstandard α-amino acid residues (homoserine, N-methylisoleucine, and two 3-hydroxyleucines) and one ß-amino acid residue (ß-aminodecanoic acid). Trichormamide B (2) was cytotoxic against MDA-MB-435 and HT-29 cancer cell lines with IC50 values of 0.8 and 1.5 µM, respectively.

Development of a liquid chromatography-mass spectrometry method for the determination of ursolic acid in rat plasma and tissue: application to the pharmacokinetic and tissue distribution study.

A fast and sensitive liquid chromatography-mass spectrometry method was developed for the determination of ursolic acid (UA) in rat plasma and tissues. Glycyrrhetinic acid was used as the internal standard (IS). Chromatographic separation was performed on a 3.5 µm Zorbax SB-C18 column (30 mm × 2.1 mm) with a mobile phase consisting of methanol and aqueous 10 mM ammonium acetate using gradient elution. Quantification was performed by selected ion monitoring with (m/z)(-) 455 for UA and (m/z)(-) 469 for the IS. The method was validated in the concentration range of 2.5 - 1470 ng mL(-1) for plasma samples and 20 - 11760 ng g(-1) for tissue homogenates. The intra- and inter-day assay of precision in plasma and tissues ranged from 1.6% to 7.1% and 3.7% to 9.0%, respectively, and the intra- and inter-day assay accuracy was 84.2 - 106.9% and 82.1 - 108.1%, respectively. Recoveries in plasma and tissues ranged from 83.2% to 106.2%. The limits of detections were 0.5 ng mL(-1) or 4.0 ng g(-1). The recoveries for all samples were >90%, except for liver, which indicated that ursolic acid may metabolize in liver. The main pharmacokinetic parameters obtained were T(max) = 0.42 ± 0.11 h, C(max) = 1.10 ± 0.31 µg mL(-1), AUC = 1.45 ± 0.21 µg h mL(-1) and K(a) = 5.64 ± 1.89 h(-1). The concentrations of UA in rat lung, spleen, liver, heart, and cerebellum were studied for the first time. This method is validated and could be applicable to the investigation of the pharmacokinetics and tissue distribution of UA in rats.