Sorbremnoids A and B: NLRP3 Inflammasome Inhibitors Discovered from Spatially Restricted Crosstalk of Biosynthetic Pathways.

Crosstalk-oriented chemical evolution of natural products (NPs) is an efficacious strategy for generating novel skeletons through coupling reactions between NP fragments. In this study, two NOD-like receptor protein 3 (NLRP3) inflammasome inhibitors, sorbremnoids A and B (1 and 2), with unprecedented chemical architectures were identified from a fungus Penicillium citrinum. Compounds 1 and 2 exemplify rare instances of hybrid NPs formed via a major facilitator superfamily (MFS)-like enzyme by coupling reactive intermediates from two separate biosynthetic gene clusters (BGCs), pcisor and pci56. Both sorbremnoids A and B are NLRP3 inflammasome inhibitors. Sorbremnoid A demonstrated strong inhibition of IL-1ß by directly binding to the NLRP3 protein, inhibiting the assembly and activation of the NLRP3 inflammasome in vitro, with potential application in diabetic refractory wound healing through the suppression of excessive inflammatory responses. This research will inspire the development of anti-NLRP3 inflammasome agents as lead treatments and enhance knowledge pertaining to NPs derived from biosynthetic crosstalk.

Cyclo-diphenylalanine production in Aspergillus nidulans through stepwise metabolic engineering.

Cyclo-diphenylalanine (cFF) is a symmetrical aromatic diketopiperazine (DKP) found wide-spread in microbes, plants, and resulting food products. As different bioactivities continue being discovered and relevant food and pharmaceutical applications gradually emerge for cFF, there is a growing need for establishing convenient and efficient methods to access this type of compound. Here, we present a robust cFF production system which entailed stepwise engineering of the filamentous fungal strain Aspergillus nidulans A1145 as a heterologous expression host. We first established a preliminary cFF producing strain by introducing the heterologous nonribosomal peptide synthetase (NRPS) gene penP1 to A. nidulans A1145. Key metabolic pathways involving shikimate and aromatic amino acid biosynthetic support were then engineered through a combination of gene deletions of competitive pathway steps, over-expressing feedback-insensitive enzymes in phenylalanine biosynthesis, and introducing a phosphoketolase-based pathway, which diverted glycolytic flux toward the formation of erythrose 4-phosphate (E4P). Through the stepwise engineering of A. nidulans A1145 outlined above, involving both heterologous pathway addition and native pathway metabolic engineering, we were able to produce cFF with titers reaching 611 mg/L in shake flask culture and 2.5 g/L in bench-scale fed-batch bioreactor culture. Our study establishes a production platform for cFF biosynthesis and successfully demonstrates engineering of phenylalanine derived diketopiperazines in a filamentous fungal host.

Evolution-Based Discovery of Polyketide Acylated Valine from a Cytochalasin-Like Gene Cluster in Simplicillium lamelliciola HDN13430.

Utilizing a gene evolution-oriented approach for gene cluster mining, a cryptic cytochalasin-like gene cluster (sla) in Antarctic-derived Simplicillium lamelliciola HDN13430 was identified. Compared with the canonical cytochalasin biosynthetic gene clusters (BGCs), the sla gene cluster lacks the key α,ß-hydrolase gene. Heterologous expression of the sla gene cluster led to the discovery of a new compound, slamysin (1), characterized by an N-acylated amino acid structure and demonstrating weak anti-Bacillus cereus activity. These findings underscore the potential of genetic evolution in uncovering novel compounds and indicating specific adaptive evolution within specialized habitats.

Phenylhydrazone Alkaloids from the Deep-Sea Cold Seep Derived Fungus Talaromyces amestolkiae HDN21-0307.

Alkaloids with a phenylhydrazone architecture are rarely found in nature. Four unusual phenylhydrazone alkaloids named talarohydrazones A-D (1-4) were isolated from the deep-sea cold seep derived fungus Talaromyces amestolkiae HDN21-0307 using the one strain-many compounds (OSMAC) approach and MS/MS-based molecular networking (MN) combined with network annotation propagation (NAP) and the unsupervised substructure annotation method MS2LDA. Their structures were elucidated by spectroscopic data analysis, single-crystal X-ray diffraction, and quantum chemical calculations. Talarohydrazone A (1) possessed an unusual skeleton combining 2,4-pyridinedione and phenylhydrazone. Talarohydrazone B (2) represents the first natural phenylhydrazone-bearing azadophilone. Bioactivity evaluation revealed that compound 1 exhibited cytotoxic activity against NCI-H446 cells with an IC50 value of 4.1 µM. In addition, compound 1 displayed weak antibacterial activity toward Staphylococcus aureus with an MIC value of 32 µg/mL.

Polyketide Derivatives from the Mangrove-Derived Fungus Penicillium sp. HDN15-312.

Four new polyketides, namely furantides A-B (1-2), talamin E (3) and arugosinacid A (4), and two known polyketides were obtained from the mangrove-derived fungus Penicillium sp. HDN15-312 using the One Strain Many Compounds (OSMAC) strategy. Their chemical structures, including configurations, were elucidated by detailed analysis of extensive NMR spectra, HRESIMS and ECD. The DPPH radicals scavenging activity of 3, with an IC50 value of 6.79 µM, was better than vitamin C.

Impacts of irrigation with treated livestock wastewater on the accumulation characteristic of ARGs in the farmland soil: a case study in Hohhot, China.

Irrigation with treated livestock wastewater (TWW) is a promising strategy for reusing resources. However, TWW irrigation might introduce antibiotic resistant genes (ARGs) into the soil, posing environmental risks associated with antibiotic resistance. This study focuses on investigating the influence of irrigation amounts and duration on the fate of ARGs and identifies key factors driving their changes. The results showed that there were 13 ARGs in TWW, while only 5 ARGs were detected in irrigated soil. That is some introduced ARGs from TWW could not persistently exist in the soil. After 1-year irrigation, an increase in irrigation amount from 0.016 t/m2 to 0.048 t/m2 significantly enhanced the abundance of tetC by 29.81%, while ermB and sul2 decreased by 45.37% and 76.47%, respectively (p < 0.01). After 2-year irrigation, the abundance of tetC, ermB, ermF, dfrA1, and total ARGs significantly increased (p < 0.05) when the irrigation amount increased. The abundances of ARGs after 2-year irrigation were found to be 2.5-34.4 times higher than 1 year. Obviously, the irrigation years intensified the positive correlation between ARGs abundance and irrigation amount. TetC and ermF were the dominant genes resulting in the accumulation of ARGs. TWW irrigation increased the content of organic matter and total nitrogen in the soil, which affected microbial community structure. The changes of the potential host were the determining factors driving the ARGs abundance. Our study demonstrated that continuous TWW irrigation for 2 years led to a substantial accumulation of ARGs in soil.

Discovery, Total Synthesis, and Anti-Inflammatory Evaluation of Naturally Occurring Naphthopyrone-Macrolide Hybrids as Potent NLRP3 Inflammasome Inhibitors.

Numerous clinical disorders have been linked to the etiology of dysregulated NLRP3 (NACHT, LRR, and PYD domain-containing protein 3) inflammasome activation. Despite its potential as a pharmacological target, modulation of NLRP3 activity remains challenging. Only a sparse number of compounds have been reported that can modulate NLRP3 and none of them have been developed into a commercially available drug. In this research, we identified three potent NLRP3 inflammasome inhibitors, gymnoasins A-C (1-3), with unprecedented pentacyclic scaffolds, from an Antarctic fungus Pseudogymnoascus sp. HDN17-895, which represent the first naturally occurring naphthopyrone-macrolide hybrids. Additionally, biomimetic synthesis of gymnoasin A (1) was also achieved validating the chemical structure and affording ample amounts of material for exhaustive bioactivity assessments. Biological assays indicated that 1 could significantly inhibited in vitro NLRP3 inflammasome activation and in vivo pro-inflammatory cytokine IL-1ß release, representing a valuable new lead compound for the development of novel therapeutics with the potential to inhibit the NLRP3 inflammasome.

Microbial Dimerization and Chlorination of Isoflavones by a Takla Makan Desert-Derived Streptomyces sp. HDN154127.

Sixteen new biisoflavones, bisoflavolins A-N (1-16), were discovered from cultures of the Takla Makan desert-derived strain Streptomyces sp. HDN154127. The chemical structures, including axial chirality, were elucidated by NMR, MS, and ECD analyses. Antibacterial activity of dimerized compounds was tested against seven different bacteria. The dimerized compounds showed better activity (MIC from 0.8 to 50.0 µM) than the corresponding monomers (daidzein and genistein, MIC > 50.0 µM). The rare dimeric and chlorinated structures in 1-16 were proved to be biotransformation products obtained from soy isoflavones and sodium chloride, which constituted the culture medium. This is the first report of an actinomycete that promotes both dimerization and chlorination utilizing natural isoflavones as skeletons sources.

Extending the Structural Diversity of Labdane Diterpenoids from Marine-Derived Fungus Talaromyces sp. HDN151403 Using Heterologous Expression.

Heterologous biosynthesis has become an effective means to activate fungal silent biosynthetic gene clusters (BGCs) and efficiently utilize fungal genetic resources. Herein, thirteen labdane diterpene derivatives, including five undescribed ones named talarobicins A-E (3-7), were discovered via heterologous expression of a silent BGC (labd) in Aspergillus nidulans. Their structures with absolute configurations were elucidated using extensive MS and NMR spectroscopic methods, as well as electronic circular dichroism (ECD) calculations. These labdanes belong to four skeleton types, and talarobicin B (4) is the first 3,18-dinor-2,3:4,18-diseco-labdane diterpene with the cleavage of the C2-C3 bond in ring A and the decarboxylation at C-3 and C-18. Talarobicin B (4) represents the key intermediate in the biosynthesis of penioxalicin and compound 13. The combinatorial heterologous expression and feeding experiments revealed that the cytochrome P450 enzymes LabdC, LabdE, and LabdF were responsible for catalyzing various chemical reactions, such as oxidation, decarboxylation, and methylation. All of the compounds are noncytotoxic, and compounds 2 and 8 displayed inhibitory effects against methicillin-resistant coagulase-negative staphylococci (MRCNS) and Bacillus cereus.

Overexpression of Global Regulator SCrp Leads to the Discovery of New Angucyclines in Streptomyces sp. XS-16.

Six angucyclines including three unreported compounds (1-3) were isolated from Streptomyces sp. XS-16 by overexpressing the native global regulator of SCrp (cyclic AMP receptor). The structures were characterized based on nuclear magnetic resonance (NMR) and spectrometry analysis and assisted by electronic circular dichroism (ECD) calculations. All compounds were tested for their antitumor and antimicrobial activities, and compound 1 showed different inhibitory activities against various tumor cell lines with IC50 values ranging from 0.32 to 5.33 µM.

Activation of a Silent Polyketide Synthase SlPKS4 Encoding the C7-Methylated Isocoumarin in a Marine-Derived Fungus Simplicillium lamellicola HDN13-430.

Coumarins, isocoumarins and their derivatives are polyketides abundant in fungal metabolites. Although they were first discovered over 50 years ago, the biosynthetic process is still not entirely understood. Herein, we report the activation of a silent nonreducing polyketide synthase that encodes a C7-methylated isocoumarin, similanpyrone B (1), in a marine-derived fungus Simplicillium lamellicola HDN13-430 by heterologous expression. Feeding studies revealed the host enzymes can change 1 into its hydroxylated derivatives pestapyrone A (2). Compounds 1 and 2 showed moderate radical scavenging activities with ED50 values of 67.4 µM and 104.2 µM. Our discovery fills the gap in the enzymatic elucidation of naturally occurring C7-methylated isocoumarin derivatives.

Dimeric Tetracenomycin Derivatives from a Taklamakan Desert-Derived Streptomyces sp. HDN154193.

Bitetracenomycin A (1) and its diastereomers [(±)-bitetracenomycin B, (±)-2] were discovered from the cultures of Streptomyces sp. HDN154193. Compounds 1 and (±)-2 were the first tetracenomycin dimers obtained from a natural source with sp3 methine protons at the bridge positions (C-12/12'), which also exhibited broad-spectrum antibacterial activity. The racemate (±)-2 was semisynthesized and separated into enantiomers (+)-2 and (-)-2, and the absolute configurations were determined by specific rotation and ECD data. These metabolites exhibited potent antibacterial activity especially against drug-resistant strains (MRSA and MRCNS) with MIC values ranging from 1.0 to 1.9 µg/mL.

Cytotoxic Nitrobenzoyl Sesquiterpenoids from an Antarctica Sponge-Derived Aspergillus insulicola.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive neoplastic diseases of the pancreas with fatal proliferation and metastasis and no medicine available for treatment. From an Antarctica sponge-derived fungus, Aspergillus insulicola HDN151418, four new nitrobenzoyl sesquiterpenoids, namely, insulicolides D-G (1-4), were isolated. Compounds 3 and 4 exhibited selective inhibition against human PDAC cell lines. Further studies indicated that compound 4 could significantly suppress cell proliferation to induce apoptosis and blocked migration and invasion of PDAC cells. Compound 4 could also avoid resistance and improved the therapeutic effect of the chemotherapy drug gemcitabine. A preliminary mechanism study showed that compound 4 can significantly inhibit the expression of EGFR and XIAP in PDAC cells. Altogether, 4 is a potential lead compound for anti-PDAC drug research.

Citreobenzofuran D-F and Phomenone A-B: Five Novel Sesquiterpenoids from the Mangrove-Derived Fungus Penicillium sp. HDN13-494.

Five new sesquiterpenoids, citreobenzofuran D-F (1-3) and phomenone A-B (4-5), along with one known compound, xylarenone A (6), were isolated from the culture of the mangrove-derived fungus Penicillium sp. HDN13-494. Their structures were deduced from extensive spectroscopic data, high-resolution electrospray ionization mass spectrometry (HRESIMS), and electronic circular dichroism (ECD) calculations. Furthermore, the absolute structures of 1 were determined by single-crystal X-ray diffraction analysis. Citreobenzofuran E-F (2-3) are eremophilane-type sesquiterpenoids with rare benzofuran frameworks, while phomenone A (4) contains a rare thiomethyl group, which is the first report of this kind of sesquiterpene with sulfur elements in the skeleton. All the compounds were tested for their antimicrobial and antitumor activity, and phomenone B (5) showed moderate activity against Bacillus subtilis, with an MIC value of 6.25 µM.

Brasilterpenes A-E, Bergamotane Sesquiterpenoid Derivatives with Hypoglycemic Activity from the Deep Sea-Derived Fungus Paraconiothyrium brasiliense HDN15-135.

Five bergamotane sesquiterpenoid derivatives, brasilterpenes A-E (1-5), bearing an unreported spiral 6/4/5 tricyclic ring system, were isolated from the deep sea-derived ascomycete fungus Paraconiothyrium brasiliense HDN15-135. Their structures, including absolute configurations, were established by extensive spectroscopic methods complemented by single-crystal X-ray diffraction analyses, electronic circular dichroism (ECD), and density-functional theory (DFT) calculations of nuclear magnetic resonance (NMR) data including DP4+ analysis. The hypoglycemic activity of these compounds was assessed using a diabetic zebrafish model. Brasilterpenes A (1) and C (3) significantly reduced free blood glucose in hyperglycemic zebrafish in vivo by improving insulin sensitivity and suppressing gluconeogenesis. Moreover, the hypoglycemic activity of compound 3 was comparable to the positive control, anti-diabetes drug rosiglitazone. These results suggested brasilterpene C (3) had promising anti-diabetes potential.

Unusual Tetrahydropyridoindole-Containing Tetrapeptides with Human Nicotinic Acetylcholine Receptors Targeting Activity Discovered from Antarctica-Derived Psychrophilic Pseudogymnoascus sp. HDN17-933.

Chemical investigation of the psychrophilic fungus Pseudogymnoascus sp. HDN17-933 derived from Antarctica led to the discovery of six new tetrapeptides psegymamides A-F (1-6), whose planar structures were elucidated by extensive NMR and MS spectrometric analyses. Structurally, psegymamides D-F (4-6) possess unique backbones bearing a tetrahydropyridoindoles unit, which make them the first examples discovered in naturally occurring peptides. The absolute configurations of structures were unambiguously determined using solid-phase total synthesis assisted by Marfey's method, and all compounds were evaluated for their inhibition of human (h) nicotinic acetylcholine receptor subtypes. Compound 2 showed significant inhibitory activity. A preliminary structure-activity relationship investigation revealed that the tryptophan residue and the C-terminal with methoxy group were important to the inhibitory activity. Further, the high binding affinity of compound 2 to hα4ß2 was explained by molecular docking studies.

Clinical efficacy of norepinephrine combined with cimetidine in treatment of neonatal upper gastrointestinal hemorrhage and its adverse reactions.

Objectives: To investigate the clinical efficacy of norepinephrine combined with cimetidine in the treatment of neonatal upper gastrointestinal hemorrhage and its adverse reactions. Methods: A total of 68 cases of neonatal upper gastrointestinal hemorrhage in Huangshi Maternal and Child Health Care Hospital from please mention dates October 2018 to February 2020 were selected and randomly divided into treatment group and control group by coin tossing, with 34 infants in each group. The control group received conventional therapy, and the treatment group was additionally treated with norepinephrine combined with cimetidine. The efficacy and safety were compared between the two groups. Results: The time when the bleeding stops, the time of fecal occult blood turning negative and hospital stay of the treatment group were shorter than those of the control group (P < 0.05). Superoxide dismutase (SOD) level increased while malondialdehyde (MDA) level decreased in both groups after treatment compared with those before treatment (P < 0.05). After treatment, the SOD level was higher while the MDA level was lower in the treatment group than those in the control group (P < 0.05). The effective rate of the treatment group was higher than that of the control group (P < 0.05). However, no significance was found in adverse reactions between the two groups (P > 0.05). Conclusion: Norepinephrine combined with cimetidine in the treatment of neonatal upper gastrointestinal hemorrhage can shorten the recovery time of symptoms, improve efficacy and reduce stress reaction. It is safe, effective and worthy of use in clinical practice.

Effective Generation of Glucosylpiericidins with Selective Cytotoxicities and Insights into Their Biosynthesis.

Exploring unknown glycosyltransferases (GTs) is important for compound structural glycodiversification during the search for drug candidates. Piericidin glycosides have been reported to have potent bioactivities; however, the GT responsible for piericidin glucosylation remains unknown. Herein, BmmGT1, a macrolide GT with broad substrate selectivity and isolated from Bacillus methylotrophicus B-9987, was found to be able to glucosylate piericidin A1 in vitro. Next, the codon-optimized GT gene sbmGT1, which was designed based on BmmGT1, was heterologously expressed in the piericidin producer Streptomyces youssoufiensis OUC6819. Piericidin glycosides thus significantly accumulated, leading to the identification of four new glucopiericidins (compounds 3, 4, 6, and 7). Furthermore, using BmmGT1 as the probe, GT1507 was identified in the genome of S. youssoufiensis OUC6819 and demonstrated to be associated with piericidin glucosylation; the overexpression of this gene led to the identification of another new piericidin glycoside, N-acetylglucosamine-piericidin (compound 8). Compounds 4, 7, and 8 displayed cytotoxic selectivity toward A549, A375, HCT-116, and HT-29 solid cancer cell lines compared to the THP-1 lymphoma cell line. Moreover, database mining of GT1507 homologs revealed their wide distribution in bacteria, mainly in those belonging to the high-GC Gram-positive and Firmicutes clades, thus representing the potential for identification of novel tool enzymes for compound glycodiversification. IMPORTANCE Numerous bioactive natural products are appended with sugar moieties and are often critical for their bioactivities. Glycosyltransferases (GTs) are powerful tools for the glycodiversification of natural products. Although piericidin glycosides display potent bioactivities, the GT involved in glucosylation is unclear. In this study, five new piericidin glycosides (compounds 3, 4, 6, 7, and 8) were generated following the overexpression of GT-coding genes in a piericidin producer. Three of them (compounds 4, 7, and 8) displayed cytotoxic selectivity. Notably, GT1507 was demonstrated to be related to piericidin glucosylation in vivo. Furthermore, mining of GT1507 homologs from the GenBank database revealed their wide distribution across numerous bacteria. Our findings would greatly facilitate the exploration of GTs to glycodiversify small molecules in the search for drug candidates.

HDN-1 induces cell differentiation toward apoptosis in promyelocytic leukemia cells depending on its selective effect on client proteins of Hsp90.

Heat Shock Protein 90 (Hsp90) is frequently upregulated in many cancers, and its inhibition simultaneously blocks multiple signaling pathways, resulting in cell differentiation or apoptosis. However, the complexity of Hsp90 in differentiation and its relation with apoptosis have remained unsettled. In this study, we demonstrated that HDN-1, a C-terminal inhibitor of Hsp90, induced the differentiation of HL-60 cells toward apoptosis. HDN-1 induced the differentiation of cells containing mutant AML1-ETO into mature granulocytes, which was related to its selective effect on client proteins of Hsp90. HDN-1 destabilized AML1-ETO and preserved C/EBPß at the same time, thereby induced a total increase in C/EBPß levels because of AML1-ETO negative regulation to C/EBPß expression. Neither HDN-1 nor 17-AAG (an N-terminal inhibitor of Hsp90) led to the differentiation of NB4 cells because mutant PML-RARα was not affected as a client protein of Hsp90; thus, no additional expression of C/EBPß was induced. 17-AAG did not affect the differentiation of HL-60 cells due to decreased AML1-ETO and C/EBPß levels. These results indicate that HDN-1 drives cell differentiation toward apoptosis depending on its selective influence on client proteins of Hsp90, establishing the relationship between differentiation and apoptosis and uncovering the mechanism of HDN-1 in promyelocytic leukemia cell differentiation. Moreover, HDN-1 is very promising for the development of anticancer agents with the induction of differentiation.

Ascandinines A-D, Indole Diterpenoids, from the Sponge-Derived Fungus Aspergillus candidus HDN15-152.

Four new indole diterpenoids, ascandinines A-D (1-4), were isolated from an Antarctic sponge-derived fungus Aspergillus candidus HDN15-152. Their structures, including absolute configurations, were established based on NMR data, computational calculations, and biosynthetic considerations. Ascandinine A (1) possesses an unprecedented 2-oxabicyclo[2.2.2]octan-3-ol motif embedded in a pentacyclic ring system, while compounds 2-4 represent a rare type of indole diterpenoid featuring the 6/5/5/6/6/6/6-fused ring system. Compound 3 displayed anti-influenza virus A (H1N1) activity with an IC50 value of 26 µM, while compound 4 showed cytotoxicity against HL-60 cells with an IC50 value of 7.8 µM.