A series of new coumarins was synthesized via an efficient protocol-triethylamine-promoted metal-free regioselective divergent C-N bond formation and/or cascade heterocyclization reactions of 4-chloro-3-formylcoumarin, coumarin-derived phenacyl bromide, and 4-chloro-3-nitrocoumarin with primary amines. These reactions proceeded under mild conditions, and fused pyrido-/pyrimidino-/pyrrolocoumarins, aminocoumarins, and bis-coumarins were isolated in high yields without column chromatography work-up. The main SNAr reaction involves an addition-elimination mechanism, followed by a cyclization process. Molecular structural elucidation was achieved through NMR and ESI-MS analyses. The synthesized derivatives were evaluated for their in vitro cytotoxicity against four human cancer cell lines. 4-[(5-Bromopyrimidin-2-yl)amino]-2-oxo-2Hchromene-3-carbaldehyde (7) and N-{3-[(3-nitro-2-oxo-2H-chromen-4-yl)amino]-phenyl}acetamide (10a) emerged as suitable lead molecules. Compound 7 displayed pronounced cytotoxicity against Caco-2 colon cancer cells (IC50 = 8.62 +/- 0.36 mu M) and was found to induce a significant G2/M phase cell cycle arrest (24.7%) and apoptosis (37.95%) compared to the untreated control (13.3% and 0.67%, respectively). Coumarin 10a exhibited a dual mechanism of action. It demonstrated good antiangiogenic activity by inhibiting the VEGFR-2 enzyme with an IC50 of 175 +/- 9.0 nM, which correlates with its molecular docking against 4ASD, showing a binding affinity of -8.3 kcal/mol, forming a significant hydrogen bond between the NHCOCH3 fragment and the key residue Asp1046 in the VEGFR-2 active site. Additionally, compound 10a significantly suppressed the migratory ability of Caco-2 cells in a wound healing assay. These findings, together with its favorable physicochemical properties and DFT-based reactivity descriptors (high electrophilicity and polarizability), highlight coumarin 10a as a suitable lead for further optimization in antiangiogenic and anticancer drug development.

Purpose:To develop a translational tumor growth inhibition and time-to-event (TGI-TTE) modeling framework linking drug exposure, tumor dynamics, and survival. Modeling used preclinical efficacy studies with MTMSA-Trp, a preclinical-stage anti-tumor agent for Ewing sarcoma. Methods:Tumor volume and survival data from Ewing sarcoma mouse xenografts treated with MTMSA-Trp (0.3-2.85 mg/kg) were analyzed. A Simeoni TGI model was used to estimate individual exponential and linear tumor growth rates. A parametric log-logistic TTE model was used to describe mouse survival across treatment groups by incorporating post hoc TGI metrics and regimen-specific average concentrations derived from the PK model as covariates predictive of survival. Models were evaluated using the precision of parameter estimates, goodness-of-fit plots, and bootstraps. Results:The TGI model accurately described individual tumor growth dynamics, yielding precise parameter estimates (RSEs < 15%). The final TTE model successfully captured the observed Kaplan-Meier curves across treatment groups (RSEs < 20%). Higher regimen-specific average concentration was significantly associated with longer survival (coefficient = 0.63). Conversely, higher exponential and linear tumor growth rates were significantly associated with shorter survival (coefficients = -0.75 and -0.46, respectively). Conclusion:This framework quantitatively links tumor dynamics, drug exposure, and survival and may support the design, analysis, and simulation-based evaluation of dose regimens in preclinical oncology studies.

Purpose:To develop a pharmacokinetic model for a novel mithramycin analogue, MTMSA-Trp, in mice and characterize dose-dependent disposition to support future pharmacokinetic-pharmacodynamic (PK/PD) and exposure-efficacy analyses. Methods:Non-linear mixed-effects modeling was used to develop a population pharmacokinetic (popPK) model in MonolixSuite 2024R1 using 121 plasma concentrations from 70 female athymic nude mice after single IV bolus doses of 0.3, 1, 3, 5, and 10 mg/kg. Model selection was guided by the objective function value (OFV), parameter precision, and diagnostic plots. The final model was evaluated using bootstrap resampling (1000 replicates) and visual predictive checks (VPC; 1000 simulated datasets). Results:A one-compartment model with first-order elimination and an empirical power relationship between dose and clearance best described the data. Including dose as a covariate in the clearance model significantly improved model fit relative to the linear base model (ΔOFV = - 26.19). Typical clearance and volume of distribution were 39.18 mL/h/kg (at 3 mg/kg) and 53.06 mL/kg, respectively, and the dose-clearance exponent was β = -0.30, indicating decreasing clearance with increasing dose. Fixed-effect parameters were estimated with high precision (RSE ≤ 11%). Shrinkage was high for clearance (81%) and moderate for volume of distribution (39.1%). Bootstrap and VPC results supported model robustness and predictive performance. Conclusion:A robust popPK model describing dose-dependent MTMSA-Trp disposition in mice was developed and is suitable for simulation to support subsequent PK/PD and exposure-efficacy analyses.

Purpose To develop and verify a physiologically based pharmacokinetic (PBPK) modeling strategy for mithramycin (MTM) and its analog, MTMSA-Trp, with the aim of projecting first-in-human plasma pharmacokinetics and supporting the translational development of MTMSA-Trp for Ewing sarcoma treatment. Methods PBPK models were created in GastroPlus® using a middle-out approach, incorporating preclinical pharmacokinetic data from mice, rats, and cynomolgus monkeys. Human clearance was estimated through three methods: an additional clearance approach, allometric scaling, and single-species scaling from monkeys. The model was evaluated using clinical MTM plasma PK data and then employed to project human MTMSA-Trp plasma PK, with tissue predictions considered exploratory. Results The additional clearance approach provided the most accurate prediction of human MTM plasma PK. Across all clearance prediction methods, MTMSA-Trp was predicted to achieve 8- to 15-fold higher human plasma exposure than MTM at the same dose. Model-derived liver exposures were 2- to 4-fold higher, with a lower predicted liver partition for MTMSA-Trp; however, these tissue predictions remained sensitive to distribution assumptions. Parameter sensitivity analysis identified the blood-to-plasma ratio as the most influential parameter among those examined. Conclusion PBPK modeling supports the projection that MTMSA-Trp will achieve substantially higher plasma exposure than MTM in humans. This empirically developed workflow may inform translational efforts for the first-in-human development of MTMSA-Trp.

Ewing Sarcoma (EWS) is characterized by aberrant activity of the oncogenic EWS-FLI1 transcription. Mithramycin (MTM) has been identified to be a specific inhibitor of transcription mediated by the EWS-FLI1. A phase I/II trial and PK study in Ewing sarcoma patients demonstrated that MTM had poor PK and dose-limiting hepatic and hematologic toxicities at subtherapeutic concentrations. Here, we are presenting a novel MTM analogue with significantly improved PK and a wider therapeutic window for targeted inhibition of. and Discussion: MTMSA-Trp was prepared by chemical conversion of MTM to the MTMSA analogue and the subsequent conjugation of a tryptophan amino acid. This substitution appears to shift the ionization properties of the two hydroxy groups on the tricyclic core and to increase protein binding, which significantly improves PK. The cytotoxicity of MTMSA-Trp is in the order of low nM across several cell lines expressing EWS-ETS fusions, and the compound disrupts the expression of positively regulated proteins (NR0B1, ID2) and induces expression of negatively regulated ones (PHLDA1, LOX). MTMSA-Trp mediates apoptosis during which upstream caspases proteolytically processes for Caspase-7 activation. This effector caspase drives the cleavage of downstream substrates PARP and high secondary DNA damage (gH2A.X). Luciferase reporter assay demonstrates a dose-dependent effect of MTMSA-Trp on the attenuation of EWS-FLI1 transcription and DNA binding, and based on CETSA evidence, there is physical interaction between MTMSA-Trp and EWS-FLI1. MTMSA-Trp was found to modify the prototypical activity of EWS-FLI1 by disrupting R-loops. Evaluation of MTMSA-Trp effect on EWS-FLI1 mediated transcriptional activity by RNA Seq highlighted 287 genes among overlapping DEGs. ATAC seq analysis revealed that TEAD, ETS, CEBP, and NFKB TF families have divergent TF binding profiles between Ewing and non-Ewing cells treated with MTMSA-Trp. EWS-FLI1 modulation persists longer when cells are exposed to GI90 concentration for a short time compared to the GI50 for longer times, suggesting that daily dosing or continuous exposure is not required. MTMSA-Trp has improved PK in athymic nu/nu mice with a greater than 10-fold reduction in clearance and significantly reduced partition in the liver compared to MTM. Initial efficacy studies in TC32 EWS cells demonstrated tumor regression and improved survival at the maximum tolerated dose (MTD) and at of the MTD on a daily x 5 intravenous bolus injection. Significantly, a more protracted dosing schedule, which allowed higher doses every 3 days for 6 doses, led to an impressive reduction of tumors whose initial size was 1200-1600 mm3. MTMSA-Trp demonstrate that improving the pharmacologic properties of mithramycin may lead to the development of an EWS-FLI1 inhibitor. Rajesh Yetirajam, Srijan Acharya, Kumar Kuldeep Niloy, Yasuda Kazuto, Jamie Horn, Thomas Prisinzano, Jon S. Thorson, Oleg Tsodikov, Jurgen Rohr, Markos Leggas. Development of a novel mithramycin analogue with improved pharmacokinetics and therapeutic window for targeted inhibition of EWS-FLI1 in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4360.

Background: Mithramycin (MTM) is a polyketide anti-cancer natural product previously identified as an EWS-FLI1 inhibitor. This oncogenic transcription factor is a canonical target for drug development in Ewing sarcoma. However, poor pharmacokinetics have been identified as a critical liability of MTM, preventing its further development. Through semisynthetic chemical modifications, we identified mithramycin SA-Trp (MTMSA-Trp) as being a pharmacologically superior congener. To explore their pharmacokinetic (PK) differences, this study examined the plasma PKs and plasma protein binding (PPB) of MTM and MTMSA-Trp in mice, rats, and monkeys. Methods: Protein binding was investigated by rapid equilibrium dialysis in plasma from mice, rats, monkeys, and humans. The pharmacokinetics were investigated at milligram- and microgram-level doses in mice and rats. The pharmacokinetics in monkeys were investigated using the cassette dosing approach at two microgram-level doses. The MTMSA-Trp pharmacokinetic linearity was evaluated in mice at 0.3, 1, 3, and 10 mg/kg doses. All samples were analyzed using LC-MS/MS. Results: Plasma protein binding was higher for MTMSA-Trp (1-4% unbound) than for MTM (10-30% unbound) across species, except in athymic nude mice (1-4% unbound and <1% for mithramycin and MTMSA-Trp, respectively). In mice and rats, MTMSA-Trp had significantly lower clearance than MTM at both milligram and microgram doses; however, the difference in plasma exposure was more pronounced at milligram doses. Consistent with the rodent PK results, cassette microdosing in monkeys showed that the clearance of MTMSA-Trp was lower than that of MTM, but the differences were less pronounced. In the dose proportionality study, MTMSA-Trp showed linear pharmacokinetics at 1, 3, and 10 mg/kg doses. Conclusions: MTMSA-Trp has significantly lower clearance than MTM in rodent models. This is a significant improvement compared to the parent drug, MTM, and warrants further evaluation of PKs in non-rodent models to enable the prediction of MTMSA-Trp PK in humans.

Enediynes are among the most potent antitumor and antibacterial natural products. Studies on their biosynthetic pathways have identified a shared, linear polyene precursor generated from an iterative type I polyketide synthase (PKSE) as the source of the enediyne warhead. A key step is the release of this polyene from the PKSE by a discrete thioesterase (TE). Here, we used X-ray crystallography, site-directed mutagenesis, and heterologous coexpression of PKSEs and TEs to elucidate how enediyne TEs mediate the production of the polyene. We solved the structure of wild-type EspE7 from esperamicin producer Actinomodura verrucosospora. The substrate binding pocket was also defined upon serendipitous cocrystallization of an EspE7 mutant with a fatty acyl-CoA ligand. Structural data and in vitro activity assays with EspE7 mutants provide strong evidence that Glu68 in EspE7 and the analogous Glu residue in other enediyne TEs functions as a key catalytic residue, thus supporting a hydrolysis mechanism for enediyne TEs that aligns with that of Pseudomonas sp. 4-HB-CoA TE. Furthermore, combinations of 9- and 10-membered enediyne PKSEs and TEs produced 1,3,5,7,9,11,13-pentadecaheptaene (1) as the major product. Thus, the data further support previous conclusions that 1 serves as the sole precursor for the biosynthesis of all enediyne cores.

Tetracenomycins are anticancer polyketides that arrest cancer cell proliferation via binding to the large mammalian ribosomal subunit near the polypeptide exit channel. The tetracenomycins are natural products that many members of the actinomycete family produce. The first goal of this study was to improve the biosynthesis of tetracenomycin analogs via metabolic engineering. The second goal was to probe more deeply into the antiproliferative activity of tetracenomycin aglycones. The tetracenomycins were assessed via several assays, including cell viability assays, clonogenic assays, and flow cytometry apoptosis assays. The data suggest that tetracenomycins C and X inhibit cell proliferation and arrest cell growth, supporting their cytostatic action mechanism. In addition, tetracenomycins C and X induced degeneration of 3D spheroid cultures and exhibited concentration-dependent inhibition of cell survival and colony formation in clonogenic assays. This work demonstrates that tetracenomycins act mainly as cytostatic rather than apoptotic agents.

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.

An investigation of the soils developed on ultramafic rocks in the State Line Serpentinite Belt in southeastern Pennsylvania demonstrated that the mineral assemblages are dominated by quartz, with lesser amounts of the serpentine group minerals lizardite and antigorite, and clinochlore, among other minerals. The samples have up to 39.91% MgO, 22,500 mu g/g Cr, and 3300 mu g/g Ni (ash basis). The light rare earth elements have a significant correlation to MgO/(MgO + SiO2) while the distribution of Cr is random. The organic matter in the soil bears a strong similarity to lignite and subbituminous macerals. Wood-derived macerals showed variations from relatively pristine wood to degraded and attrital forms with evidence of fungal and faunal activity. Macrinite ranged from coprolitic forms to amorphous masses with little or no recognizable structure. The pollen assemblages were dominated by Pinus sp., Quercus sp., and Ribes sp. Analysis of the fungal assemblages and guild structures suggests that the Goat Hill Barrens assemblage differed from the New Texas Barrens and Nottingham Barrens assemblages and that the guild structures encountered are both similar to those encountered in peatlands while also very different, especially in the proportion of arbuscular mycorrhizal fungal remains.

IntroductionThe exploration of new bioactive compounds for agricultural applications is critical for sustainable development. Endophytic fungi, particularly those from underexplored biomes in Brazil, represent a promising source of natural compounds. This study focused on isolation and bioprospecting endophytic fungi from the medicinal plant Vochysia divergens (Pohl), grown in Serra do Amolar (Brazilian Pantanal Biome), with an additional emphasis on conserving microbial biodiversity.Methods and resultsLeaves and petioles were collected from 18 V. divergens specimens, from which 293 endophytes were isolated and grouped by morphological characteristics into 91 phenotypes. One representative of each phenotype was selected for secondary metabolite extraction and taxonomic identification. Fungi belonging to 27 families and 32 different genera were identified, with Diaporthe, Phyllosticta, and Pseudofusicoccum as the most predominant. We also introduce and describe a new endophytic species, Diaporthe amolarensis. Multiple extracts inhibited mycelial growth of the phytopathogenic fungus Colletotrichum abscissum, with a superior effect compared to the fungicide control. These extracts were produced by Diaporthe amolarensis, Xylaria arbuscula, and Nemania primolutea. Additionally, the extract from one X. arbuscula isolate displayed moderate activity against the phytopathogen Phyllosticta citricarpa. HPLC-UV and HPLC-MS analyses of these most inhibitory extracts revealed natural products with beneficial potential that need characterization and to have their modes of action elucidated.DiscussionFinally, a very important contribution of this study was the ex situ conservation of the biodiversity of the Serra do Amolar, allowing future studies and biotechnological applications involving endophytes from this region.

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.

Tetracenomycins andelloramycins are polyketide natural productsproduced by several actinomycetes that exhibit antibacterial and anticanceractivities. They inhibit ribosomal translation by binding in the polypeptideexit channel of the large ribosomal subunit. The tetracenomycins andelloramycins are typified by a shared oxidatively modified lineardecaketide core, yet they are distinguished by the extent of O-methylationand the presence of a 2 ',3 ',4 '-tri-O-methyl-alpha-l-rhamnose appended at the 8-position ofelloramycin. The transfer of the TDP-l-rhamnose donor tothe 8-demethyl-tetracenomycin C aglycone acceptor is catalyzed bythe promiscuous glycosyltransferase ElmGT. ElmGT exhibits remarkableflexibility toward transfer of many TDP-deoxysugar substrates to 8-demethyltetracenomycinC, including TDP-2,6-dideoxysugars, TDP-2,3,6-trideoxysugars, andmethyl-branched deoxysugars in both d- and l-configurations.Previously, we developed an improved host, Streptomycescoelicolor M1146::cos16F4iE, which is a stable integrantharboring the required genes for 8-demethyltetracenomycin C biosynthesisand expression of ElmGT. In this work, we developed BioBricks genecassettes for the metabolic engineering of deoxysugar biosynthesisin Streptomyces spp. As a proof of concept, we usedthe 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-tetracenomycinC. In addition, we generated four new tetracenomycins including onemodified 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 demonstratesthe feasibility of BioBricks cloning, with the ability to recycleintermediate constructs, for the rapid assembly of diverse carbohydratepathways and glycodiversification of a variety of natural products.

AbstractBackgroundBioprospecting of actinobacteria isolated from Kubuqi desert, China for antibacterial, antifungal and cytotoxic metabolites production and their structure elucidation.ResultsA total of 100 actinobacteria strains were selectively isolated from Kubuqi desert, Inner Mongolia, China. The taxonomic characterization revealedStreptomycesas the predominant genus comprising 37 different species, along with the rare actinobacterial genusLentzea. The methanolic extracts of 60.8% of strains exhibited potent antimicrobial activities againstStaphylococcus aureus,Micrococcus luteus,Bacillus subtilis,Escherichia coli,Salmonella enterica,Saccharomyces cerevisiaeand 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).ConclusionsThis 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, particularlyStreptomyces, which produce unique metabolites and are an intriguing source to develop medicinally valuable natural products.

Abstract Ewing Sarcoma, a devastating malignancy affecting mainly pediatric and young adult populations, is characterized by the aberrant activity of the oncogenic EWS-FLI1 transcription. However, the development of treatments against EWS-FLI1 is lacking. Mithramycin analogues, exhibiting specificity towards EWS-FLI1, have been posited as a groundbreaking approach in drug discovery for the treatment of Ewing Sarcoma. A phase I/II trial and pharmacokinetic (PK) study in Ewing sarcoma patients demonstrated that mithramycin (MTM) had poor PK and dose-limiting hepatic and hematologic toxicities at subtherapeutic concentrations. Here we present a novel MTM analogue with significantly improved PK and a wider therapeutic window. MTMSA-Trp was prepared by chemical conversion of MTM to the MTMSA analogue and the subsequent conjugation of a tryptophan amino acid. The bulky amino acid substitution on the 3-side chain of the molecule appears to shift the ionization properties of the two hydroxy groups on the tricyclic core and to increase protein binding, both of which appear to lead to significantly improved pharmacokinetics. Further, based on crystallographic evaluation, the bulky hydrophobic substitution on the 3-side chain protrudes outside the DNA helix and interacts with bound FLI1. Luciferase reporter and FRET assays demonstrate a dose-dependent effect of MTMSA-Trp on the attenuation of EWS-FLI1 transcription and DNA binding, and based on CETSA evidence, there is physical interaction between MTMSA-Trp and EWS-FLI1. The cytotoxicity of MTMSA-Trp is in the order of low nM across several cell lines expressing EWS-ETS fusions, and the compound disrupts the expression of positively regulated proteins (NR0B1, ID2) and induces expression of negatively regulated ones (CD44, LOX). EWS-FLI1 modulation persists longer when cells are exposed to the GI90 concentration for a short time compared to the GI50 for longer times, suggesting that daily dosing or continuous exposure is not required. MTMSA-Trp has improved PK in athymic nu/nu mice with a greater than 10-fold reduction in clearance and significantly reduced partition in the liver as compared to MTM. Initial efficacy studies in TC32 Ewing Sarcoma cells demonstrated tumor regression and improved survival at the maximum tolerated dose (MTD) and at 2/3 of the MTD on a daily x 5 intravenous bolus injection. Significantly, a more protracted dosing schedule which allowed for higher doses every third day for six doses led to an impressive reduction of tumors whose initial size was above 15 mm in one diameter (i.e., 1200-1600 mm3). Our studies with MTMSA-Trp demonstrate that improving the pharmacologic properties of mithramycin may lead to the development of an EWS-FLI1 inhibitor. Ongoing studies are evaluating the efficacy of MTMSA-Trp in additional Ewing models, and future work will focus on pre-IND studies. Citation Format: Markos Leggas, Kumar K Niloy, Rajesh Yetijaram, Jamie Horn, Yasuda Kazuto, Thomas Prisinzano, Jon S Thorson, Oleg Tsoikov, Jurgen Rohr. Targeting EWS-FLI1 with mithramycin analogues for Ewing sarcoma treatment [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B150.

The enediynes are structurally characterized by a 1,5-diyne-3-ene motif within a 9- or 10-membered enediyne core. The anthraquinone-fused enediynes (AFEs) are a subclass of 10-membered enediynes that contain an anthraquinone moiety fused to the enediyne core as exemplified by dynemicins and tiancimycins. A conserved iterative type I polyketide synthase (PKSE) is known to initiate the biosynthesis of all enediyne cores, and evidence has recently been reported to suggest that the anthraquinone moiety also originates from the PKSE product. However, the identity of the PKSE product that is converted to the enediyne core or anthraquinone moiety has not been established. Here, we report the utilization of recombinant E. coli coexpressing various combinations of genes that encode a PKSE and a thioesterase (TE) from either 9- or 10-membered enediyne biosynthetic gene clusters to chemically complement Δ PKSE mutant strains of the producers of dynemicins and tiancimycins. Additionally, 13 C-labeling experiments were performed to track the fate of the PKSE/TE product in the Δ PKSE mutants. These studies reveal that 1,3,5,7,9,11,13-pentadecaheptaene is the nascent, discrete product of the PKSE/TE that is converted to the enediyne core. Furthermore, a second molecule of 1,3,5,7,9,11,13-pentadecaheptaene is demonstrated to serve as the precursor of the anthraquinone moiety. The results establish a unified biosynthetic paradigm for AFEs, solidify an unprecedented biosynthetic logic for aromatic polyketides, and have implications for the biosynthesis of not only AFEs but all enediynes.

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.

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.

Dynemicin is an enediyne natural product from Micromonospora chersina ATCC53710. Access to the biosynthetic gene cluster of dynemicin has enabled the in vitro study of gene products within the cluster to decipher their roles in assembling this unique molecule. This paper reports the crystal structure of DynF, the gene product of one of the genes within the biosynthetic gene cluster of dynemicin. DynF is revealed to be a dimeric eight-stranded β-barrel structure with palmitic acid bound within a cavity. The presence of palmitic acid suggests that DynF may be involved in binding the precursor polyene heptaene, which is central to the synthesis of the ten-membered ring of the enediyne core.