Diverse Combinatorial Biosynthesis Strategies for C-H Functionalization of Anthracyclinones.

Streptomyces spp. are "nature's antibiotic factories" that produce valuable bioactive metabolites, such as the cytotoxic anthracycline polyketides. While the anthracyclines have hundreds of natural and chemically synthesized analogues, much of the chemical diversity stems from enzymatic modifications to the saccharide chains and, to a lesser extent, from alterations to the core scaffold. Previous work has resulted in the generation of a BioBricks synthetic biology toolbox in Streptomyces coelicolor M1152ΔmatAB that could produce aklavinone, 9-epi-aklavinone, auramycinone, and nogalamycinone. In this work, we extended the platform to generate oxidatively modified analogues via two crucial strategies. (i) We swapped the ketoreductase and first-ring cyclase enzymes for the aromatase cyclase from the mithramycin biosynthetic pathway in our polyketide synthase (PKS) cassettes to generate 2-hydroxylated analogues. (ii) Next, we engineered several multioxygenase cassettes to catalyze 11-hydroxylation, 1-hydroxylation, 10-hydroxylation, 10-decarboxylation, and 4-hydroxyl regioisomerization. We also developed improved plasmid vectors and S. coelicolor M1152ΔmatAB expression hosts to produce anthracyclinones. This work sets the stage for the combinatorial biosynthesis of bespoke anthracyclines using recombinant Streptomyces spp. hosts.

Biochemical and Structural Studies of the Carminomycin 4-O-Methyltransferase DnrK.

Structural and functional studies of the carminomycin 4-O-methyltransferase DnrK are described, with an emphasis on interrogating the acceptor substrate scope of DnrK. Specifically, the evaluation of 100 structurally and functionally diverse natural products and natural product mimetics revealed an array of pharmacophores as productive DnrK substrates. Representative newly identified DnrK substrates from this study included anthracyclines, angucyclines, anthraquinone-fused enediynes, flavonoids, pyranonaphthoquinones, and polyketides. The ligand-bound structure of DnrK bound to a non-native fluorescent hydroxycoumarin acceptor, 4-methylumbelliferone, along with corresponding DnrK kinetic parameters for 4-methylumbelliferone and native acceptor carminomycin are also reported for the first time. The demonstrated unique permissivity of DnrK highlights the potential for DnrK as a new tool in future biocatalytic and/or strain engineering applications. In addition, the comparative bioactivity assessment (cancer cell line cytotoxicity, 4E-BP1 phosphorylation, and axolotl embryo tail regeneration) of a select set of DnrK substrates/products highlights the ability of anthracycline 4-O-methylation to dictate diverse functional outcomes.

Bioactivity and Metabolomics Profiling of Endophytic Actinobacteria Isolated from Roots of the Medicinal Plants Dominant in South Asian Region.

Background: Plant-derived endophytic actinobacteria are the center of attention due to their capacity to produce diverse antimicrobial and anticancer compounds and their metabolites influence plant growth. Methods: In this study, 40 endophytic actinobacteria strains were isolated from the roots of eight medicinal plants used as folk medicine in South Asian region. The isolates were characterized morphologically, biochemically and physiologically and the genus level identification of the selected strains was done by 16SrRNA gene sequencing. In small scale cultivation (50ml broth), the isolates were grown in A-medium to prepare the crude extracts. These crude extracts were subsequently evaluated for their antimicrobial, anticancer and antioxidant activity and the metabolomics profile of each of the extract was determined by TLC and HPLC-UV/MS. Results: The taxonomic studies showed that the isolates belong to the group actinobacteria based on their morphological and physiological characteristics and the 16SrRNA gene sequencing of the selected strains identified the genera including Streptomyces, Micromonospora and Nocardia. Cumulatively,53% of extracts exhibited anti-Gram-(+) activity,47% exhibited anti-Gram-(-) activity,32% exhibited antifungal activity and 30% were cytotoxic to PC3 and A549 cancer cell lines and most of the extracts have shown antioxidant activity greater than 50%. The metabolomics analysis predicted the presence of an array of low molecular weight metabolites and indicated the promising isolates in collection for further studies for novel bioactive metabolite isolation and structure elucidation. Conclusion: Overall the study provides an overview of the endophytic actinobacteria residing in the roots of the selected medicinal plants prevalent in south Asian region and their potential to produce the medicinally and biotechnologically useful compounds.

Engineering BioBricks for Deoxysugar Biosynthesis and Generation of New Tetracenomycins.

Tetracenomycins and elloramycins are polyketide natural products produced by several actinomycetes that exhibit antibacterial and anticancer activities. They inhibit ribosomal translation by binding in the polypeptide exit channel of the large ribosomal subunit. The tetracenomycins and elloramycins are typified by a shared oxidatively modified linear decaketide core, yet they are distinguished by the extent of O-methylation and the presence of a 2',3',4'-tri-O-methyl-α-l-rhamnose appended at the 8-position of elloramycin. The transfer of the TDP-l-rhamnose donor to the 8-demethyl-tetracenomycin C aglycone acceptor is catalyzed by the promiscuous glycosyltransferase ElmGT. ElmGT exhibits remarkable flexibility toward transfer of many TDP-deoxysugar substrates to 8-demethyltetracenomycin C, including TDP-2,6-dideoxysugars, TDP-2,3,6-trideoxysugars, and methyl-branched deoxysugars in both d- and l-configurations. Previously, we developed an improved host, Streptomyces coelicolor M1146::cos16F4iE, which is a stable integrant harboring the required genes for 8-demethyltetracenomycin C biosynthesis and expression of ElmGT. In this work, we developed BioBricks gene cassettes for the metabolic engineering of deoxysugar biosynthesis in Streptomyces spp. As a proof of concept, we used the BioBricks expression platform to engineer biosynthesis for d-configured TDP-deoxysugars, including known compounds 8-O-d-glucosyl-tetracenomycin C, 8-O-d-olivosyl-tetracenomycin C, 8-O-d-mycarosyl-tetracenomycin C, and 8-O-d-digitoxosyl-tetracenomycin C. In addition, we generated four new tetracenomycins including one modified with a ketosugar, 8-O-4'-keto-d-digitoxosyl-tetracenomycin C, and three modified with 6-deoxysugars, including 8-O-d-fucosyl-tetracenomycin C, 8-O-d-allosyl-tetracenomycin C, and 8-O-d-quinovosyl-tetracenomycin C. Our work demonstrates the feasibility of BioBricks cloning, with the ability to recycle intermediate constructs, for the rapid assembly of diverse carbohydrate pathways and glycodiversification of a variety of natural products.

Paecilins Q and R: Antifungal Chromanones Produced by the Endophytic Fungus Pseudofusicoccum stromaticum CMRP4328.

Chemical investigation of the endophyte Pseudofusicoccum stromaticum CMRP4328 isolated from the medicinal plant Stryphnodendron adstringens yielded ten compounds, including two new dihydrochromones, paecilins Q (1: ) and R (2: ). The antifungal activity of the isolated metabolites was assessed against an important citrus pathogen, Phyllosticta citricarpa. Cytochalasin H (6: ) (78.3%), phomoxanthone A (3: ) (70.2%), phomoxanthone B (4: ) (63.1%), and paecilin Q (1: ) (50.5%) decreased in vitro the number of pycnidia produced by P. citricarpa, which are responsible for the disease dissemination in orchards. In addition, compounds 3: and 6: inhibited the development of citrus black spot symptoms in citrus fruits. Cytochalasin H (6: ) and one of the new compounds, paecilin Q (1: ), appear particularly promising, as they showed strong activity against this citrus pathogen, and low or no cytotoxic activity. The strain CMRP4328 of P. stromaticum and its metabolites deserve further investigation for the control of citrus black spot disease.

Bioprospecting of desert actinobacteria with special emphases on griseoviridin, mitomycin C and a new bacterial metabolite producing Streptomyces sp. PU-KB10-4.

BACKGROUND: Bioprospecting of actinobacteria isolated from Kubuqi desert, China for antibacterial, antifungal and cytotoxic metabolites production and their structure elucidation. RESULTS: A total of 100 actinobacteria strains were selectively isolated from Kubuqi desert, Inner Mongolia, China. The taxonomic characterization revealed Streptomyces as the predominant genus comprising 37 different species, along with the rare actinobacterial genus Lentzea. The methanolic extracts of 60.8% of strains exhibited potent antimicrobial activities against Staphylococcus aureus, Micrococcus luteus, Bacillus subtilis, Escherichia coli, Salmonella enterica, Saccharomyces cerevisiae and high to mild in vitro cytotoxicity against PC3 (prostate cancer) and A549 (lung carcinoma) cell lines. The metabolomics analysis by TLC, HPLC-UV/vis, HPLC-MS and NMR showed the presence of compounds with molecular weights ranging from 100 to 1000 Da. The scale-up fermentation of the prioritized anti-Gram-negative strain PU-KB10-4 (Streptomyces griseoviridis), yielded three pure compounds including; griseoviridin (1; 42.0 mgL- 1) with 20 fold increased production as compared to previous reports and its crystal structure as monohydrate form is herein reported for the first time, mitomycin C (2; 0.3 mgL- 1) and a new bacterial metabolite 4-hydroxycinnamide (3; 0.59 mgL- 1). CONCLUSIONS: This is the first report of the bioprospecting and exploration of actinobacteria from Kubuqi desert and the metabolite 4-hydroxycinnamide (3) is first time isolated from a bacterial source. This study demonstrated that actinobacteria from Kubuqi desert are a potential source of novel bioactive natural products. Underexplored harsh environments like the Kubuqi desert may harbor a wider diversity of actinobacteria, particularly Streptomyces, which produce unique metabolites and are an intriguing source to develop medicinally valuable natural products.

A BioBricks Metabolic Engineering Platform for the Biosynthesis of Anthracyclinones in Streptomyces coelicolor.

Actinomycetes produce a variety of clinically indispensable molecules, such as antineoplastic anthracyclines. However, the actinomycetes are hindered in their further development as genetically engineered hosts for the synthesis of new anthracycline analogues due to their slow growth kinetics associated with their mycelial life cycle and the lack of a comprehensive genetic toolbox for combinatorial biosynthesis. In this report, we tackled both issues via the development of the BIOPOLYMER (BIOBricks POLYketide Metabolic EngineeRing) toolbox: a comprehensive synthetic biology toolbox consisting of engineered strains, promoters, vectors, and biosynthetic genes for the synthesis of anthracyclinones. An improved derivative of the production host Streptomyces coelicolor M1152 was created by deleting the matAB gene cluster that specifies extracellular poly-ß-1,6-N-acetylglucosamine (PNAG). This resulted in a loss of mycelial aggregation, with improved biomass accumulation and anthracyclinone production. We then leveraged BIOPOLYMER to engineer four distinct anthracyclinone pathways, identifying optimal combinations of promoters, genes, and vectors to produce aklavinone, 9-epi-aklavinone, auramycinone, and nogalamycinone at titers between 15-20 mg/L. Optimization of nogalamycinone production strains resulted in titers of 103 mg/L. We structurally characterized six anthracyclinone products from fermentations, including new compounds 9,10-seco-7-deoxy-nogalamycinone and 4-O-ß-d-glucosyl-nogalamycinone. Lastly, we tested the antiproliferative activity of the anthracyclinones in a mammalian cancer cell viability assay, in which nogalamycinone, auramycinone, and aklavinone exhibited moderate cytotoxicity against several cancer cell lines. We envision that BIOPOLYMER will serve as a foundational platform technology for the synthesis of designer anthracycline analogues.

Diverse polyketides from the marine endophytic Alternaria sp. LV52: Structure determination and cytotoxic activities.

We report the isolation and characterization of five polyketides [alternariol (1), alternariol-9-methyl ether (2), altertoxin I (3), altertoxin II (4) and tenuazonic acid (5)] from the marine endophytic Alternaria sp. LV52 derived from Cystoseira tamariscifolia, collected from the Red Sea at Nabq-Bay, Egypt. The chemical structures of compounds 1-5 were identified by extensive 1D, 2D NMR, and HR mass measurements. Isolation and phenotypic and genotypic characterization of the producing fungus is reported. The antimicrobial activity of the produced extract and derived compounds was examined against a panel of test organisms. In addition, an in vitro cytotoxic activity of 1-5 was performed against diverse cancer cell lines: HEPG2, HELA, A549 and PC3, revealing that compounds 2 and 4 are potentially cytotoxic against A549 and PC3 with EC50 of 0.73 µg/ml (2.69 µM) and 0.17 µg/ml (0.64 µM) for 2, and 0.40 µg/ml (1.15 µM) and 0.12 µg/ml (0.33 µM) for 4, respectively.

Himalaquinones A-G, Angucyclinone-Derived Metabolites Produced by the Himalayan Isolate Streptomyces sp. PU-MM59.

Himalaquinones A-G, seven new anthraquinone-derived metabolites, were obtained from the Himalayan-based Streptomyces sp. PU-MM59. The chemical structures of the new compounds were identified based on cumulative analyses of HRESIMS and NMR spectra. Himalaquinones A-F were determined to be unique anthraquinones that contained unusual C-4a 3-methylbut-3-enoic acid aromatic substitutions, while himalaquinone G was identified as a new 5,6-dihydrodiol-bearing angucyclinone. Comparative bioactivity assessment (antimicrobial, cancer cell line cytotoxicity, impact on 4E-BP1 phosphorylation, and effect on axolotl embryo tail regeneration) revealed cytotoxic landomycin and saquayamycin analogues to inhibit 4E-BP1p and inhibit regeneration. In contrast, himalaquinone G, while also cytotoxic and a regeneration inhibitor, did not affect 4E-BP1p status at the doses tested. As such, this work implicates a unique mechanism for himalaquinone G and possibly other 5,6-dihydrodiol-bearing angucyclinones.

Taxonomic and Metabolomics Profiling of Actinobacteria Strains from Himalayan Collection Sites in Pakistan.

Actinobacteria have proven themselves as the major producers of bioactive compounds with wide applications. In this study, 35 actinobacteria strains were isolated from soil samples collected from the Himalayan mountains region in Pakistan. The isolated strains were identified by polyphasic taxonomy and were prioritized based on biological and chemical screening to identify the strains with ability to produce inimitable metabolites. The biological screening included antimicrobial activity against Staphylococcus aureus, Micrococcus luteus, Salmonella enterica, Escherichia coli, Mycobacterium aurum, and Bacillus subtilis and anticancer activity using human cancer cell lines PC3 and A549. For chemical screening, methanolic extracts were investigated using TLC, HPLC-UV/MS. The actinobacteria strain PU-MM93 was selected for scale-up fermentation based on its unique chemical profile and cytotoxicity (50-60% growth inhibition) against PC3 and A549 cell lines. The scale-up fermentation of PU-MM93, followed by purification and structure elucidation of compounds revealed this strain as a promising producer of the cytotoxic anthracycline aranciamycin and aglycone SM-173-B along with the potent neuroprotective carboxamide oxachelin C. Other interesting metabolites produced include taurocholic acid as first report herein from microbial origin, pactamycate and cyclo(L-Pro-L-Leu). The study suggested exploring more bioactive microorganisms from the untapped Himalayan region in Pakistan, which can produce commercially significant compounds.

Chemical genetics of regeneration: Contrasting temporal effects of CoCl2 on axolotl tail regeneration.

BACKGROUND: Histone deacetylases (HDACs) regulate transcriptional responses to injury stimuli that are critical for successful tissue regeneration. Previously we showed that HDAC inhibitor romidepsin potently inhibits axolotl tail regeneration when applied for only 1-minute postamputation (MPA). RESULTS: Here we tested CoCl2, a chemical that induces hypoxia and cellular stress, for potential to reverse romidepsin inhibition of tail regeneration. Partial rescue of regeneration was observed among embryos co-treated with romidepsin and CoCl2 for 1 MPA, however, extending the CoCl2 dosage window either inhibited regeneration (CoCl2 :0 to 30 MPA) or was lethal (CoCl2 :0 to 24 hours postamputation; HPA). CoCl2 :0 to 30 MPA caused tissue damage, tissue loss, and cell death at the distal tail tip, while CoCl2 treatment of non-amputated embryos or CoCl2 :60 to 90 MPA treatment after re-epithelialization did not inhibit tail regeneration. CoCl2 -romidepsin:1 MPA treatment partially restored expression of transcription factors that are typical of appendage regeneration, while CoCl2 :0 to 30 MPA significantly increased expression of genes associated with cell stress and inflammation. Additional experiments showed that CoCl2 :0 to 1 MPA and CoCl2 :0 to 30 MPA significantly increased levels of glutathione and reactive oxygen species, respectively. CONCLUSION: Our study identifies a temporal window from tail amputation to re-epithelialization, within which injury activated cells are highly sensitive to CoCl2 perturbation of redox homeostasis.

Metal-free domino amination-Knoevenagel condensation approach to access new coumarins as potent nanomolar inhibitors of VEGFR-2 and EGFR.

A metal-free, atom-economy and simple work-up domino amination-Knoevenagel condensation approach to construct new coumarin analogous (4a-f and 8a-e) was described. Further, new formyl (5a,d-f) and nitro (9a,d-f) coumarin derivatives were synthesized via C-N coupling reaction of various cyclic secondary amines and 4-chloro-3-(formyl-/nitro)coumarins (1a,c), respectively. The confirmed compounds were screened for their in vitro anti-proliferative activity against KB-3-1, A549 and PC3 human cancer cell lines using resazurin cellular-based assay. Among them, coumarin derivatives 4e and 8e displayed the best anti-cervical cancer potency (KB-3-1) with IC50 values of 15.5 ± 3.54 and 21 ± 4.24 µM, respectively. Also, 4e showed the most promising cytotoxicity toward A549 with IC50 value of 12.94 ± 1.51 µM. As well, 9d presented a more significant impact of potency against PC3 with IC50 7.31 ± 0.48 µM. Moreover, 8d manifested selectivity against PC3 (IC50 = 20.16 ± 0.07 µM), while 8e was selective toward KB-3-1 cell line (IC50 = 21 ± 4.24 µM). Matching with docking profile, the enzymatic assay divulged that 8e is a dual potent single-digit nanomolar inhibitor of VEGFR-2 and EGFR with IC50 values of 24.67 nM and 31.6 nM that were almost equipotent to sorafenib (31.08 nM) and erlotinib (26.79 nM), respectively.

HDAC Inhibitor Titration of Transcription and Axolotl Tail Regeneration.

New patterns of gene expression are enacted and regulated during tissue regeneration. Histone deacetylases (HDACs) regulate gene expression by removing acetylated lysine residues from histones and proteins that function directly or indirectly in transcriptional regulation. Previously we showed that romidepsin, an FDA-approved HDAC inhibitor, potently blocks axolotl embryo tail regeneration by altering initial transcriptional responses to injury. Here, we report on the concentration-dependent effect of romidepsin on transcription and regeneration outcome, introducing an experimental and conceptual framework for investigating small molecule mechanisms of action. A range of romidepsin concentrations (0-10 µM) were administered from 0 to 6 or 0 to 12 h post amputation (HPA) and distal tail tip tissue was collected for gene expression analysis. Above a threshold concentration, romidepsin potently inhibited regeneration. Sigmoidal and biphasic transcription response curve modeling identified genes with inflection points aligning to the threshold concentration defining regenerative failure verses success. Regeneration inhibitory concentrations of romidepsin increased and decreased the expression of key genes. Genes that associate with oxidative stress, negative regulation of cell signaling, negative regulation of cell cycle progression, and cellular differentiation were increased, while genes that are typically up-regulated during appendage regeneration were decreased, including genes expressed by fibroblast-like progenitor cells. Using single-nuclei RNA-Seq at 6 HPA, we found that key genes were altered by romidepin in the same direction across multiple cell types. Our results implicate HDAC activity as a transcriptional mechanism that operates across cell types to regulate the alternative expression of genes that associate with regenerative success versus failure outcomes.

RF-3192C and other polyketides from the marine endophytic Aspergillus niger ASSB4: structure assignment and bioactivity investigation.

Chemical investigation of the methanolic extract of endophytic Aspergillus niger SB4, isolated from the marine alga Laurencia obtuse, afforded the pentacyclic polyketide, RF-3192C (1), the dimeric coumarin orlandin (2), fonsecin B (3), TMC-256A1 (4), cyclo-(Leu-Ala) (5), and cerebroside A (6).The chemical structure of RF-3192C (1) is assigned herein for the first time using 1D/2D NMR and HRESI-MS. Additionally, the revision of the NMR assignments of orlandin (2) was reported herein as well. Investigation of the antimicrobial activities of isolated compounds revealed the high activity of RF-3192C (1) against Pseudomonas aeruginosa and Bacillus subtilis, and moderate activity against yeast. Moreover, an in vitro cytotoxic activity against liver (HEPG2), cervical (HELA), lung (A549), prostate (PC3), and breast (MCF7) cancer cell lines of the isolated compounds was evaluated. The isolation and taxonomical characterization of the producing fungus was reported as well.

Mithramycin 2'-Oximes with Improved Selectivity, Pharmacokinetics, and Ewing Sarcoma Antitumor Efficacy.

Mithramycin A (MTM) inhibits the oncogenic transcription factor EWS-FLI1 in Ewing sarcoma, but poor pharmacokinetics (PK) and toxicity limit its clinical use. To address this limitation, we report an efficient MTM 2'-oxime (MTMox) conjugation strategy for rapid MTM diversification. Comparative cytotoxicity assays of 41 MTMox analogues using E-twenty-six (ETS) fusion-dependent and ETS fusion-independent cancer cell lines revealed improved ETS fusion-independent/dependent selectivity indices for select 2'-conjugated analogues as compared to MTM. Luciferase-based reporter assays demonstrated target engagement at low nM concentrations, and molecular assays revealed that analogues inhibit the transcriptional activity of EWS-FLI1. These in vitro screens identified MTMox32E (a Phe-Trp dipeptide-based 2'-conjugate) for in vivo testing. Relative to MTM, MTMox32E displayed an 11-fold increase in plasma exposure and improved efficacy in an Ewing sarcoma xenograft. Importantly, these studies are the first to point to simple C3 aliphatic side-chain modification of MTM as an effective strategy to improve PK.

Sugar-Pirating as an Enabling Platform for the Synthesis of 4,6-Dideoxyhexoses.

An efficient divergent synthetic strategy that leverages the natural product spectinomycin to access uniquely functionalized monosaccharides is described. Stereoselective 2'- and 3'-reduction of key spectinomycin-derived intermediates enabled facile access to all eight possible 2,3-stereoisomers of 4,6-dideoxyhexoses as well as representative 3,4,6-trideoxysugars and 3,4,6-trideoxy-3-aminohexoses. In addition, the method was applied to the synthesis of two functionalized sugars commonly associated with macrolide antibiotics-the 3-O-alkyl-4,6-dideoxysugar d-chalcose and the 3-N-alkyl-3,4,6-trideoxysugar d-desosamine.

OleD Loki as a Catalyst for Hydroxamate Glycosylation.

Herein we describe the ability of the permissive glycosyltransferase (GT) OleD Loki to convert a diverse set of >15 histone deacetylase (HDAC) inhibitors (HDACis) into their corresponding hydroxamate glycosyl esters. Representative glycosyl esters were subsequently evaluated in assays for cancer cell line cytotoxicity, chemical and enzymatic stability, and axolotl embryo tail regeneration. Computational substrate docking models were predictive of enzyme-catalyzed turnover and suggest certain HDACis may form unproductive, potentially inhibitory, complexes with GTs.

Dihydroisocoumarins produced by Diaporthe cf. heveae LGMF1631 inhibiting citrus pathogens.

Citrus black spot (CBS) and post-bloom fruit drop (PFD), caused by Phyllosticta citricarpa and Colletotrichum abscissum, respectively, are two important citrus diseases worldwide. CBS depreciates the market value and prevents exportation of citrus fruits to Europe. PFD under favorable climatic conditions can cause the abscission of flowers, thereby reducing citrus production by 80%. An ecofriendly alternative to control plant diseases is the use of endophytic microorganisms, or secondary metabolites produced by them. Strain LGMF1631, close related to Diaporthe cf. heveae 1, was isolated from the medicinal plant Stryphnodendron adstringens and showed significant antimicrobial activity, in a previous study. In view of the potential presented by strain LGMF1631, and the absence of chemical data for secondary metabolites produced by D. cf. heveae, we decided to characterize the compounds produced by strain LGMF1631. Based on ITS, TEF1, and TUB phylogenetic analysis, strain LGMF1631 was confirmed to belong to D. cf. heveae 1. Chemical assessment of the fungal strain LGMF1631 revealed one new seco-dihydroisocoumarin [cladosporin B (1)] along with six other related, already known dihydroisocoumarin derivatives and one monoterpene [(-)-(1S,2R,3S,4R)-p-menthane-1,2,3-triol (8)]. Among the isolated metabolites, compound 5 drastically reduced the growth of both phytopathogens in vitro and completely inhibited the development of CBS and PFD in citrus fruits and flowers. In addition, compound 5 did not show toxicity against human cancer cell lines or citrus leaves, at concentrations higher than used for the inhibition of the phytopathogens, suggesting the potential use of (-)-(3R,4R)-cis-4-hydroxy-5-methylmellein (5) to control citrus diseases.

Structure Determination, Functional Characterization, and Biosynthetic Implications of Nybomycin Metabolites from a Mining Reclamation Site-Associated Streptomyces.

We report the isolation and characterization of three new nybomycins (nybomycins B-D, 1-3) and six known compounds (nybomycin, 4; deoxynyboquinone, 5; α-rubromycin, 6; ß-rubromycin, 7; γ-rubromycin, 8; and [2α(1E,3E),4ß]-2-(1,3-pentadienyl)-4-piperidinol, 9) from the Rock Creek (McCreary County, KY) underground coal mine acid reclamation site isolate Streptomyces sp. AD-3-6. Nybomycin D (3) and deoxynyboquinone (5) displayed moderate (3) to potent (5) cancer cell line cytotoxicity and displayed weak to moderate anti-Gram-(+) bacterial activity, whereas rubromycins 6-8 displayed little to no cancer cell line cytotoxicity but moderate to potent anti-Gram-(+) bacterial and antifungal activity. Assessment of the impact of 3 or 5 cancer cell line treatment on 4E-BP1 phosphorylation, a predictive marker of ROS-mediated control of cap-dependent translation, also revealed deoxynyboquinone (5)-mediated downstream inhibition of 4E-BP1p. Evaluation of 1-9 in a recently established axolotl embryo tail regeneration assay also highlighted the prototypical telomerase inhibitor γ-rubromycin (8) as a new inhibitor of tail regeneration. Cumulatively, this work highlights an alternative nybomycin production strain, a small set of new nybomycin metabolites, and previously unknown functions of rubromycins (antifungal activity and inhibition of tail regeneration) and also provides a basis for revision of the previously proposed nybomycin biosynthetic pathway.

Total synthesis of griseusins and elucidation of the griseusin mechanism of action.

A divergent modular strategy for the enantioselective total synthesis of 12 naturally-occurring griseusin type pyranonaphthoquinones and 8 structurally-similar analogues is described. Key synthetic highlights include Cu-catalyzed enantioselective boration-hydroxylation and hydroxyl-directed C-H olefination to afford the central pharmacophore followed by epoxidation-cyclization and maturation via diastereoselective reduction and regioselective acetylation. Structural revision of griseusin D and absolute structural assignment of 2a,8a-epoxy-epi-4'-deacetyl griseusin B are also reported. Subsequent mechanistic studies establish, for the first time, griseusins as potent inhibitors of peroxiredoxin 1 (Prx1) and glutaredoxin 3 (Grx3). Biological evaluation, including comparative cancer cell line cytotoxicity and axolotl embryo tail inhibition studies, highlights the potential of griseusins as potent molecular probes and/or early stage leads in cancer and regenerative biology.