Synthesis and biological evaluation of novel penindolone derivatives as potential antiproliferative agents against SCLC in vitro.

Small cell lung cancer (SCLC) keeps on the leading cause of cancer mortality world widely, while there is lack of efficient therapeutic drugs especially for the resistant ones. In this work, a compound named penindolone (PND) with new skeleton was found to show weak inhibitory effect (IC50 = 42.5 µM) on H69AR cells (SCLC, adriamycin-resistant) proliferation by screening our in-house compound library. With the aim of improving its low potency, a series of PND derivatives were synthesized and biologically evaluated by the Sulforhodamine B (SRB) assay. Among all tested derivatives, compound 5h possessed higher antiproliferation potency (IC50 = 1.6 µM). Furthermore, preliminary mechanism investigation revealed that 5h was able to induce apoptosis and arrest the cell cycle at G0/G1 phase. These findings suggest that this novel skeleton has expanded the anti-SCLC compound reservoir and provided a new drug lead.

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.

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.

Combining the Elicitor Up-Regulated Production of Unusual Linear Diterpene-Derived Variants for an In-Depth Assessment of the Application Value and Risk of the Medicinal and Edible Basidiomycete Schizophyllum commune.

To better assess the practical value and avoid potential risks of the traditionally medicinal and edible basidiomycete Schizophyllum commune, which may arise from undescribed metabolites, a combination of elicitors was introduced for the first time to discover products from cryptic and low-expressed gene clusters under laboratory cultivation. Treating S. commune NJFU21 with the combination of five elicitors led to the upregulated production of a class of unusual linear diterpene-derived variants, including eleven new ones (1-11), along with three known ones (12-14). The structures and stereochemistry were determined by 1D and 2D NMR, HRESIMS, ECD, OR and VCD calculations. Notably, the elongation terminus of all the diterpenes was decorated by an unusual butenedioic acid moiety. Compound 1 was a rare monocyclic diterpene, while 2-6 possessed a tetrahydrofuran moiety. The truncated metabolites 4, 5 and 13 belong to the trinorditerpenes. All the diterpenes displayed approximately 70% scavenging of hydroxyl radicals at 50 µM and null cytotoxic activity at 10 µM. In addition, compound 1 exhibited potent antifungal activity against the plant pathogenic fungi Colletotrichum camelliae, with MIC values of 8 µg/mL. Our findings indicated that this class of diterpenes could provide valuable protectants for cosmetic ingredients and the lead compounds for agricultural fungicide development.

Competing Domino Knoevenagel-Cyclization Sequences with N-Arylcinnamylamines.

Domino Knoevenagel-cyclization reactions of N-arylcinnamylamines were carried out with active methylene reagents, which took place with five competing cyclization mechanisms: intramolecular hetero Diels-Alder reaction, stepwise polar [2 + 2] cycloaddition, styryl or aza-Diels-Alder reactions followed by rearomatization, and [1,5]-hydride shift-6-endo cyclization. In the stepwise aza-Diels-Alder reaction, the N-vinylpyridinium moiety acted as an azadiene, producing a condensed heterocycle with tetrahydroquinolizinium and tetrahydroquiniline subunits. Antiproliferative activity with low micromolar IC50 values was identified for some of the novel scaffolds.

The Discovery of Acremochlorins O-R from an Acremonium sp. through Integrated Genomic and Molecular Networking.

The fermentation of a soil-derived fungus Acremonium sp. led to the isolation of thirteen ascochlorin congeners through integrated genomic and Global Natural Product Social (GNPS) molecular networking. Among the isolated compounds, we identified two unusual bicyclic types, acremochlorins O (1) and P (2), as well as two linear types, acremochlorin Q (3) and R (4). Compounds 1 and 2 contain an unusual benzopyran moiety and are diastereoisomers of each other, the first reported for the ascochlorins. Additionally, we elucidated the structure of 5, a 4-chloro-5-methylbenzene-1,3-diol with a linear farnesyl side chain, and confirmed the presence of eight known ascochlorin analogs (6-13). The structures were determined by the detailed interpretation of 1D and 2D NMR spectroscopy, MS, and ECD calculations. Compounds 3 and 9 showed potent antibacterial activity against Staphylococcus aureus and Bacillus cereus, with MIC values ranging from 2 to 16 µg/mL.

Genome Mining Reveals a UbiA-Type Prenyltransferase Access to Farnesylation of Diketopiperazines.

UbiA-type prenyltransferases (PTases) are significant enzymes that lead to structurally diverse meroterpenoids. Herein, we report the identification and characterization of an undescribed UbiA-type PTase, FtaB, that is responsible for the farnesylation of indole-containing diketopiperazines (DKPs) through genome mining. Heterologous expression of the fta gene cluster and non-native pathways result in the production of a series of new C2-farnesylated DKPs. This study broadens the reaction scope of UbiA-type PTases and expands the chemical diversity of meroterpenoids.

Exploring the Diverse Landscape of Fungal Cytochrome P450-Catalyzed Regio- and Stereoselective Dimerization of Diketopiperazines.

Dimeric indole-containing diketopiperazines (di-DKPs) are a diverse group of natural products produced through cytochrome P450-catalyzed C-C or C-N coupling reactions. The regio- and stereoselectivity of these reactions plays a significant role in the structural diversity of di-DKPs. Despite their pivotal role, the mechanisms governing the selectivity in fungi are not fully understood. Employing bioinformatics analysis and heterologous expression experiments, five undescribed P450 enzymes (AmiP450, AcrP450, AtP450, AcP450, and AtuP450) responsible for the regio- and stereoselective dimerization of diketopiperazines (DKPs) in fungi are identified. The function of these P450s is consistent with phylogenetic analysis, highlighting their dominant role in controlling the dimerization modes. Combinatorial biosynthesis-based pathway reconstitution of non-native gene clusters expands the chemical space of fungal di-DKPs and reveals that the regioselectivity is influenced by the substrate. Furthermore, multiple sequence alignment and molecular docking of these enzymes demonstrate a C-terminal variable region near the substrate tunnel entrance in AtuP450 that is crucial for its regioselectivity. These findings not only reveal the secret of fungal di-DKPs diversity but also deepen understanding of the mechanisms and catalytic specificity involved in P450-catalyzed dimerization reactions.

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.

Rapid FRET Assay for the Early Detection of Alpha-Synuclein Aggregation in Parkinson's Disease.

Alpha-synuclein (α-Syn) is a key protein of Parkinson's disease (PD). Oligomers formed by misfolding and aggregation of α-Syn can cause many pathological phenomena and aggravate the development of PD. Therefore, sensitive and accurate detection of oligomers is essential to understanding the pathology of PD and beneficial to screening and developing new drugs against PD. Here, we demonstrated a simple and sensitive method to detect the early aggregation of α-Syn via Förster resonance energy transfer (FRET) technology. We performed systematic investigations of the FRET sensitizations, efficiencies, and donor-to-acceptor distances during α-Syn aggregation, which was proved to be more sensitive to reflect small distance changes in the early stage of α-Syn aggregation, especially for α-Syn oligomers. The FRET assays were also applied to study the influence of Ser129 phosphorylation (pS129) on the aggregation rate of α-Syn. Our results showed that pS129 modification promotes α-Syn aggregation and enhances the ability of preformed fibrils to induce monomer aggregation. pS129 also increased the cytotoxicity of α-Syn. These results are of great significance for a better understanding of the pathological mechanisms of PD and future PD drug development.

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.

Antiviral and cytotoxic indole diterpenoids from the antarctic sponge-derived fungus Aspergillus candidus HDN15-152.

One new indole diterpenoid, ascandinine T (1), and three known analogues (2-4) were isolated from an Antarctic sponge-derived fungus Aspergillus candidus HDN15-152. The structures, including absolute configurations, were established based on NMR, HRESIMS, and electronic circular dichroism (ECD) calculations. All isolated compounds were tested for antiviral and anticancer activity. Compound 4 displayed antiviral activity against influenza A virus (IAV) of A/PR/8/34(H1N1) strain with an IC50 value of 39.2 µM, while compound 2 showed cytotoxicity against NCI-H446, NCI-H446/EP and L-02 cells with IC50 values ranging from 9.77 to 13.91 µM.

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.

AS1041, a novel derivative of marine natural compound Aspergiolide A, induces senescence of leukemia cells via oxidative stress-induced DNA damage and BCR-ABL degradation.

Chronic myelogenous leukemia (CML) is characterized by the constitutive activation of BCR-ABL tyrosine kinase. Imatinib was approved for CML therapy, however, BCR-ABL-dependent drug resistance, especially BCR-ABL-T315I mutation, restricts its clinical application. In this study, we reported anthraquinone lactone AS1041, a synthesized derivative of marine natural compound Aspergiolide A, showed anti-leukemia effect in vitro and in vivo by promoting cell senescence. Mechanistic study revealed the pro-senescence effect of AS1041 was dependent on oxidative stress-induced DNA damage, and the resultant activation of P53/P21 and P16INK4a/Rb. Also, AS1041 promoted ubiquitin proteasome system (UPS)-mediated BCR-ABL degradation, which also contributed to AS1041-induced senescence. In vivo, AS1041-induced senescence promoted tumor growth inhibition. In summary, the in vitro and in vivo antitumor effect of AS1041 suggests it can serve as a pro-senescence agent for alternative antileukemia therapy and imatinib-resistant cancer therapy by enhancing cellular oxidative stress and BCR-ABL degradation.

Antibacterial p-terphenyl and α­pyrone derivates isolated from the marine-derived actinomycete Nocardiopsis sp. HDN154086.

Assisted by OSMAC strategy, one new p-terphenyl and two new α­pyrone derivates, namely nocarterphenyl I (1) and nocardiopyrone D-E (2-3), were obtained and characterized from the marine sediment-derived actinomycete Nocardiopsis sp. HDN154086. The structures of these compounds were determined on the basis of MS, NMR spectroscopic data and single-crystal X-ray diffraction. Compound 1 with a rare 2,2'-bithiazole structure among natural products showed promising activity against five bacteria with MIC values ranging from 0.8 to 1.6 µM and 3 exhibited notable antibacterial activity against MRSA compared the positive control ciprofloxacin.

Organocatalyzed Enantioselective [2 + 2] Cycloaddition of C,N-Cyclic Ketimines and Allenoates.

We report a novel enantioselective and regioselective [2 + 2] cycloaddition of allenoate and C,N-cyclic ketimine catalyzed by a quinidine derivative. The methodology enables the synthesis of fused tricyclic azetidines with a quaternary stereogenic center exhibiting high enantioselectivities. The broad range of substrates demonstrates the generality of the protocol, and the resulting functional products can be easily converted to a variety of valuable synthons. To elucidate the plausible reaction mechanism and how the catalyst affects absolute stereocontrol over the products, we conducted the corresponding density functional theory (DFT) calculations.

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.

Plastoquinone synthesis inhibition by tetrabromo biphenyldiol as a widespread algicidal mechanism of marine bacteria.

Algae and bacteria have complex and intimate interactions in the ocean. Besides mutualism, bacteria have evolved a variety of molecular-based anti-algal strategies. However, limited by the unknown mechanism of synthesis and action of these molecules, these strategies and their global prevalence remain unknown. Here we identify a novel strategy through which a marine representative of the Gammaproteobacteria produced 3,3',5,5'-tetrabromo-2,2'-biphenyldiol (4-BP), that kills or inhibits diverse phytoplankton by inhibiting plastoquinone synthesis and its effect cascades to many other key metabolic processes of the algae. Through comparative genomic analysis between the 4-BP-producing bacterium and its algicidally inactive mutant, combined with gene function verification, we identified the gene cluster responsible for 4-BP synthesis, which contains genes encoding chorismate lyase, flavin-dependent halogenase and cytochrome P450. We demonstrated that in near in situ simulated algal blooming seawater, even low concentrations of 4-BP can cause changes in overall phytoplankton community structure with a decline in dinoflagellates and diatoms. Further analyses of the gene sequences from the Tara Oceans expeditions and 2750 whole genome sequences confirmed the ubiquitous presence of 4-BP synthetic genes in diverse bacterial members in the global ocean, suggesting that it is a bacterial tool potentially widely used in global oceans to mediate bacteria-algae antagonistic relationships.

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.