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.

Abstract Background: Ewing sarcoma is an aggressive pediatric cancer driven by the EWS-FLI1 fusion oncoprotein, which assembles into nuclear transcriptional condensates essential for oncogenic gene regulation. MTMSA-Trp is a synthetic mithramycin (MTM) analogue with improved pharmacokinetics and in vivo efficacy in Ewing sarcoma. MTMSA-Trp binds to the minor groove of DNA and interacts with the major groove-bound EWS-FLI1, but the mechanistic details of these interactions are not well understood. Methods and Results: Using luciferase reporter assays, we show that MTMSA-Trp selectively inhibits EWS-FLI1-dependent transcription at nanomolar potency, with weaker effects on Sp1-driven transcription. MTMSA-Trp activity is attenuated in EWS-FLI1 knockdown cells, indicating functional dependence. Western blotting confirmed MTMSA-Trp-mediated suppression of EWS-FLI1-regulated targets in multiple Ewing sarcoma cell lines, while non-Ewing lines exhibited minimal response. qRT-PCR analyses revealed that MTMSA-Trp downregulates EWS-FLI1 mRNA, while paradoxically stabilizing its protein and shifting downstream transcriptional programs consistent with EWS-FLI1 antagonism. Biophysical assays demonstrated increased thermal and proteolytic stability of EWS-FLI1 upon MTMSA-Trp treatment, suggesting drug-induced stabilization of the EWS-FLI1 complex. Protein stability assays further showed that MTMSA-Trp prolongs the half-life of EWS-FLI1 in an EWS-FLI1-dependent manner, whereas ETV6, though functionally linked, was not stabilized but essentially evicted from the nucleus. Subcellular fractionation revealed that MTMSA-Trp increases nuclear retention of EWS-FLI1 while dynamically redistributing ETV6 in an EWS-FLI1-dependent manner. These effects extended to chromatin-associated partners of EWS-FLI1 condensates, including BAF155, BAF60a, and ARID1a. Immunofluorescence confirmed that MTMSA-Trp preserved ARID1a-containing nuclear condensates, protecting them from degradation by cycloheximide, and did so only in the presence of EWS-FLI1. At the transcriptional level, MTMSA-Trp downregulated CDK7 and hyperphosphorylated RNA Pol II CTD accompanied by accelerated RPB1 degradation. These effects were abolished when EWS-FLI1 was silenced. Conclusions: Our results show that MTMSA-Trp binds to and stabilizes the EWS-FLI1 transcriptional complex, maintains its associated condensates, and alters gene expression by disrupting RNA Pol II activity. These findings reveal a previously overlooked mechanism of action for MTM analogues in their interaction with the transcriptional complex, ETV6, and phase-separated oncogenic complexes. They further support the potential of these compounds as a therapeutic approach for Ewing sarcoma. Citation Format: Srijan Acharya, Rajesh Yetirajam, Yasuda Kazuto, Suhas Bhosale, Jurgen Rohr, Markos Leggas. Mithramycin analogues trap the EWS-FLI1 transcriptional complex, evict ETV6, and disable oncogenic condensate function in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3504.

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.

Mithramycin (MTM) is a polyketide anticancer natural product, which functions by noncovalent binding to DNA in the minor groove without intercalation, resulting in inhibiting transcription at G/C-rich promoters. MTM is a potent inhibitor of cancer cells, such as Ewing sarcoma, driven by abnormal fusions involving E26 transformation-specific (ETS) family transcription factors friend leukemia integration 1 (FLI1) and ETS-related gene (ERG). However, MTM is rather toxic and nonselective; therefore, safer, selective analogues of MTM are required for use in the clinic as anticancer drugs. Herein, by using a combination of X-ray crystallographic, biophysical, and cell and molecular biological techniques, the structural and functional consequences of 3-side chain methylation at positions 5, 6, and 7 of the indole ring of the potent analogue MTM SA-Trp are explored. The conformation of the analogues in complexes with DNA, their DNA binding function, cytotoxicity, selectivity, and potency as transcription antagonists depended on the position of the methylation. MTM SA-5-methyl-Trp emerged as the most selective analogue, presumably due to the right balance of the DNA binding and the solvent exposure of the 3-side chain. This study demonstrates that minor chemical changes can have strong effects in analogue development and paves the way to further development of next-generation MTM analogues.

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.

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.

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.

DNA coordinating platinum (Pt) containing compounds cisplatin and carboplatin have been used for the treatment of ovarian cancer therapy for four decades. However, recurrent Pt-resistant cancers are a major cause of mortality. To combat Pt-resistant ovarian cancers, we designed and synthesized a conjugate of an anticancer drug mithramycin with a reactive Pt(II) bearing moiety, which we termed mithplatin. The conjugates displayed both the Mg2+-dependent noncovalent DNA binding characteristic of mithramycin and the covalent crosslinking to DNA of the Pt. The conjugate was three times as potent as cisplatin against ovarian cancer cells. The DNA lesions caused by the conjugate led to the generation of DNA double-strand breaks, as also observed with cisplatin. Nevertheless, the conjugate was highly active against both Pt-sensitive and Pt-resistant ovarian cancer cells. This study paves the way to developing mithplatins to combat Pt-resistant ovarian cancers.

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.

Background: Recent studies demonstrated that the aberrant oncogenic activity of ETS transcription factor fusions such as EWS-FLI1 and EWS-ERG in Ewing sarcoma can be targeted with the natural product mithramycin (MTM), which is known to have poor pharmacology. We hypothesized that the pharmacological properties of MTM can be ameliorated by rational semisynthetic modifications that improve its pharmacokinetic (PK) liabilities and expand its efficacy toxicity window. Further, we reasoned that the MTM core of each analog can facilitate DNA docking while the substitutions may interact uniquely with oncogenic ETS transcriptional complexes in different cancers. Here we present the pharmacologic properties of two analogs and their unique transcriptome profiles in TC32 Ewing sarcoma and VCaP prostate cancer cells, both of which depend on the aberrant oncogenic activity of an ETS gene fusion. Methods: MTM analogs were generated by semisynthetic conjugation to the 3-side chain of MTM. Cytotoxicity (GI50) was assessed using the resazurin assay following 72 hr incubation with test compounds (0.3nM-10uM). PK properties were assessed in athymic nu/nu mice after a bolus tail-vein injection and concentrations were assessed in plasma using LC/MS/MS methods. The maximum tolerated dose (MTD) was estimated based on weight loss and blood hematology. In vivo efficacy was assessed in subcutaneous xenografts established, which were dosed SIDx5 at the respective MTD or fractions of the MTD. Transcriptomic profiles were assessed in cells exposed to their respective 72 hr GI50 for 24 hr and in livers of mice that were dosed SIDx5. Results: Here we demonstrate that select modifications displayed unique cytotoxicity and efficacy Ewing sarcoma (EWS-FLI1) and VCaP prostate (TMPRSS2-ERG) cancer cell lines. Analogues with bulky amino acid substitutions in the 3-side chain of MTM had significantly improved PK in mice, as compared to MTM. Select analogs displayed selective efficacy in xenograft models of Ewing sarcoma at doses ranging as low as 1/3 of the MTD. Significantly, analogues of MTM displayed significantly different transcriptomic profiles in each cell line, which suggests that these molecules have unique properties despite sharing a core DNA-binding structure with MTM. Conclusions: MTM analogues with bulky hydrophobic amino acid substitutions on the 3-side chain showed the greatest improvement in PK properties and efficacy. Presumably PK are affected due to increased protein binding and an increase in the pKa, which leads to a lower ionized fraction and decreased uptake in the liver by organic anion transporters. This increase in plasma exposure may lead to increased drug partition in the tumor, as compared to MTM, and, therefore, improved efficacy. Transcriptomic data suggest that the analogs have unique transcriptional effects and therefore unlikely to manifest the same toxicity profile as MTM. Citation Format: Markos Leggas, Ambika Dudhate, Jurgen Rohr. Selective targeting of aberrant ETS transcription factors using uniquely modified mithramycin analogs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1851.

Objectives To screen the efficacy of potential chemotherapeutics against platinum-sensitive & resistant ovarian cancer cell lines. Methods We performed in-vitro screening on mithramycin, an antineoplastic antibiotic; telaglenastat, a glutaminase inhibitor; savolitinib, a c-met tyrosine kinase inhibitor; and AMG-232, an MDM2 inhibitor. We tested all agents against a platinum-resistant cell line (OVCAR3) and a platinum-sensitive line (CAOV3). Additionally, we tested mithramycin against UWB1.289, a BRCA mutant, and an induced platinum-resistant UWB1.289 line. DMSO and cisplatin were the negative and positive controls, respectively, and we performed all experiments in triplicate. Cell viability was determined by measuring cellular ATP content. Results The IC50 values of mithramycin ranged from 42.4 to 65.5 nM. Cisplatin IC50 values ranged from a median of 2067 to 7267nM. The IC50 values of telaglenastat, savolitinib, and AMG-232 did not reach a level of 10µm in any of the tested cell lines. Table 1. IC50 values of agents sorted by cell lines. N/A indicating value not achieved at threshold of 10um. Conclusions Platinum-resistant ovarian cancer has a poor prognosis highlighting the need for new therapeutic modalities. Neither telaglenastat, savolitinib, nor AMG-232 exhibit any appreciable cytotoxicity; however, mithramycin demonstrates cytotoxicity in the low nanomolar range in several representative ovarian cancer cell lines, including two platinum-resistant lines. The potential therapeutic benefit of mithramycin warrants further preclinical evaluation.

ETS family transcription factors of ERG and FLI1 play a key role in oncogenesis of prostate cancer and Ewing sarcoma by binding regulatory DNA sites and interfering with function of other factors. Mithramycin (MTM) is an anti-cancer, DNA binding natural product that functions as a potent antagonist of ERG and FLI1 by an unknown mechanism. We present a series of crystal structures of the DNA binding domain (DBD) of ERG/FLI1 culminating in a structure of a high-order complex of the ERG/FLI1 DBD, transcription factor Runx2, core-binding factor beta (Cbfβ), and MTM on a DNA enhancer site, along with supporting DNA binding studies using MTM and its analogues. Taken together, these data provide insight into allosteric mechanisms underlying ERG and FLI1 transactions and their disruption by MTM analogues.

Plant diseases caused by phytopathogens are responsible for significant crop losses worldwide. Resistance induction and biological control have been exploited in agriculture due to their enormous potential. In this study, we investigated the antimicrobial potential of endophytic fungi of leaves and petioles of medicinal plants Vochysia divergens and Stryphnodendron adstringens located in two regions of high diversity in Brazil, Pantanal, and Cerrado, respectively. We recovered 1,304 fungal isolates and based on the characteristics of the culture, were assigned to 159 phenotypes. One isolate was selected as representative of each phenotype and studied for antimicrobial activity against phytopathogens. Isolates with better biological activities were identified based on DNA sequences and phylogenetic analyzes. Among the 159 representative isolates, extracts from 12 endophytes that inhibited the mycelial growth (IG) of Colletotrichum abscissum (≥40%) were selected to expand the antimicrobial analysis. The minimum inhibitory concentrations (MIC) of the extracts were determined against citrus pathogens, C. abscissum, Phyllosticta citricarpa and Xanthomonas citri subsp. citri and the maize pathogen Fusarium graminearum. The highest activity against C. abscissum were from extracts of Pseudofusicoccum stromaticum CMRP4328 (IG: 83% and MIC: 40 μg/mL) and Diaporthe vochysiae CMRP4322 (IG: 75% and MIC: 1 μg/mL), both extracts also inhibited the development of post-bloom fruit drop symptoms in citrus flowers. The extracts were promising in inhibiting the mycelial growth of P. citricarpa and reducing the production of pycnidia in citrus leaves. Among the isolates that showed activity, the genus Diaporthe was the most common, including the new species D. cerradensis described in this study. In addition, high performance liquid chromatography, UV detection, and mass spectrometry and thin layer chromatography analyzes of extracts produced by endophytes that showed high activity, indicated D. vochysiae CMRP4322 and P. stromaticum CMRP4328 as promising strains that produce new bioactive natural products. We report here the capacity of endophytic fungi of medicinal plants to produce secondary metabolites with biological activities against phytopathogenic fungi and bacteria. The description of the new species D. cerradensis, reinforces the ability of medicinal plants found in Brazil to host a diverse group of fungi with biotechnological potential.

Ovarian cancer is a highly deadly malignancy in which recurrence is considered incurable. Resistance to platinum-based chemotherapy bodes a particularly abysmal prognosis, underscoring the need for novel therapeutic agents and strategies. The use of mithramycin, an antineoplastic antibiotic, has been previously limited by its narrow therapeutic window. Recent advances in semisynthetic methods have led to mithramycin analogs with improved pharmacological profiles. Mithramycin inhibits the activity of the transcription factor Sp1, which is closely linked with ovarian tumorigenesis and platinum-resistance. This article summarizes recent clinical developments related to mithramycin and postulates a role for the use of mithramycin, or its analog, in the treatment of platinum-resistant ovarian cancer.

Landomycins are angucyclines with promising antineoplastic activity produced by Streptomyces bacteria. The aglycone landomycinone is the distinctive core, while the oligosaccharide chain differs within derivatives. Herein, we report that landomycins spontaneously form Michael adducts with biothiols, including reduced cysteine and glutathione, both cell-free or intracellularly involving the benz[a]anthraquinone moiety of landomycinone. While landomycins generally do not display emissive properties, the respective Michael adducts exerted intense blue fluorescence in a glycosidic chain-dependent manner. This allowed label-free tracking of the short-lived nature of the mono-SH-adduct followed by oxygen-dependent evolution with addition of another SH-group. Accordingly, hypoxia distinctly stabilized the fluorescent mono-adduct. While extracellular adduct formation completely blocked the cytotoxic activity of landomycins, intracellularly it led to massively decreased reduced glutathione levels. Accordingly, landomycin E strongly synergized with glutathione-depleting agents like menadione but exerted reduced activity under hypoxia. Summarizing, landomycins represent natural glutathione-depleting agents and fluorescence probes for intracellular anthraquinone-based angucycline metabolism.

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.

AbstractMtmOIV and MtmW catalyze the final two reactions in the mithramycin (MTM) biosynthetic pathway, the Baeyer–Villiger opening of the fourth ring of premithramycin B (PMB), creating the C3 pentyl side chain, strictly followed by reduction of the distal keto group on the new side chain. Unexpectedly this results in a C2 stereoisomer of mithramycin, iso‐mithramycin (iso‐MTM). Iso‐MTM undergoes a non‐enzymatic isomerization to MTM catalyzed by Mg2+ ions. Crystal structures of MtmW and its complexes with co‐substrate NADPH and PEG, suggest a catalytic mechanism of MtmW. The structures also show that a tetrameric assembly of this enzyme strikingly resembles the ring‐shaped β subunit of a vertebrate ion channel. We show that MtmW and MtmOIV form a complex in the presence of PMB and NADPH, presumably to hand over the unstable MtmOIV product to MtmW, yielding iso‐MTM, as a potential self‐resistance mechanism against MTM toxicity.