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Heterogeneous nuclear ribonucleoprotein L‐like (HNRNPLL), a suppressor of colorectal cancer (CRC) metastasis, is transcriptionally downregulated when CRC cells undergo epithelial‐mesenchymal transition (EMT). Here we show that decrease of MYB mediates the downregulation of HNRNPLL during EMT. The promoter activity was attributed to a region from −273 to −10 base pairs upstream of the transcription start site identified by 5'‐RACE analysis, and the region contained potential binding sites for MYB and SP1. Luciferase reporter gene assays and knockdown or knockout experiments for genes encoding the MYB family proteins, MYB, MYBL1, and MYBL2, revealed that MYB was responsible for approximately half of the promoter activity. On the other hand, treatment with mithramycin A, an inhibitor for SP1 and SP3, suppressed the promoter activity and their additive contribution was confirmed by knockout experiments. The expression level of MYB was reduced on EMT while that of SP1 and SP3 was unchanged, suggesting that the downregulation of HNRNPLL during EMT was mediated by the decrease of MYB expression while SP1 and SP3 determine the basal transcription level of HNRNPLL. Histopathological analysis confirmed the accumulation of MYB‐downregulated cancer cells at the invasion front of clinical CRC tissues. These results provide an insight into the molecular mechanism underlying CRC progression., Heterogeneous nuclear ribonucleoprotein L‐like (HNRNPLL), a suppressor of colorectal cancer (CRC) metastasis, is transcriptionally downregulated when CRC cells undergo epithelial‐mesenchymal transition (EMT). Here we show that decrease of MYB mediates the downregulation of HNRNPLL during EMT.

Recent studies have shown that tumor-derived exosomes participate in the communication between tumor cells and their microenvironment and mediate malignant biological behaviors including immune escape. In this study, we found that gastric cancer (GC) cell-derived exosomes could be effectively uptaken by Vγ9Vδ2 T cells, decrease the cell viability of Vγ9Vδ2 T cells, induce apoptosis, and reduce the production of cytotoxic cytokines IFN-γ and TNF-α. Furthermore, we demonstrated that exosomal miR-135b-5p was delivered into Vγ9Vδ2 T cells. Exosomal miR-135b-5p impaired the function of Vγ9Vδ2 T cells by targeting specificity protein 1 (SP1). More importantly, blocking the SP1 function by Plicamycin, an SP1 inhibitor, abolished the effect of stable miR-135b-5p knockdown GC cell-derived exosomes on Vγ9Vδ2 T cell function. Collectively, our results suggest that GC cell-derived exosomes impair the function of Vγ9Vδ2 T cells via miR-135b-5p/SP1 pathway, and targeting exosomal miR-135b-5p/SP1 axis may improve the efficiency of GC immunotherapy based on Vγ9Vδ2 T cells.

Specific protein 1 (SP1) might act as a critical transcription regulator in myocardial infarction (MI), but little evidence about its function in regulating cardiac apoptosis, a major cause of MI development, has been revealed. This study tried to investigate the role of SP1 in MI and its interaction with poly-ADP-ribose polymerase (PARP)-1 by using SP1 inhibitor, mithramycin A (mithA). Primary mouse cardiomyocytes and commercial mouse cardiomyocytes were subjected to mithA treatment under hypoxia conditions, while cell viability, Nix promoter activity, and its expression were detected correspondingly. PARP overexpression and knockdown were conducted, respectively, in mithA-treated and SP1-overexpressing cells. Co-immunoprecipitation was used to verify the interaction between PARP and SP1. For in vivo experiments, mithA administration was performed after the injections of adenovirus for PARP overexpression, and then, MI introduction was carried out. Infarct size and lactate dehydrogenase level were measured to assess MI injury. SP1 inhibitor mithA attenuated hypoxia-induced decrease of cell viability and Nix transcriptional activation, which could be inhibited by PARP overexpression. Knockdown of PARP prevented SP1-induced transcription of Nix and cell viability change, and PARP showed direct interaction with SP1. Furthermore, mithA administration reduced MI injuries, while PARP overexpression could suppress the improvement. The cardioprotective role of SP1 inhibitor mithA was demonstrated here expanding the role of SP1 in MI development involving hypoxia-induced cardiac apoptosis. Moreover, PARP acted as a transcriptional coactivator in Nix transcription involving its interaction with SP1.

DNA damage triggers cell death mechanisms contributing to neuronal loss and cognitive decline in neurological disorders, including traumatic brain injury (TBI), and as a side effect of chemotherapy. Mithramycin, which competitively targets chromatin-binding sites of specificity protein 1 (Sp1), was used to examine previously unexplored neuronal cell death regulatory mechanisms via rat primary neurons in vitro and after TBI in mice (males). In primary neurons exposed to DNA-damage-inducing chemotherapy drugs in vitro we showed that DNA breaks sequentially initiate DNA-damage responses, including phosphorylation of ATM, H2AX and tumor protein 53 (p53), transcriptional activation of pro-apoptotic BH3-only proteins, and mitochondrial outer membrane permeabilization (MOMP), activating caspase-dependent and caspase-independent intrinsic apoptosis. Mithramycin was highly neuroprotective in DNA-damage-dependent neuronal cell death, inhibiting chemotherapeutic-induced cell death cascades downstream of ATM and p53 phosphorylation/activation but upstream of p53-induced expression of pro-apoptotic molecules. Mithramycin reduced neuronal upregulation of BH3-only proteins and mitochondrial dysfunction, attenuated caspase-3/7 activation and caspase substrates’ cleavage, and limited c-Jun activation. Chromatin immunoprecipitation indicated that mithramycin attenuates Sp1 binding to pro-apoptotic gene promoters without altering p53 binding suggesting it acts by removing cofactors required for p53 transactivation. In contrast, the DNA-damage-independent neuronal death models displayed caspase initiation in the absence of p53/BH3 activation and were not protected even when mithramycin reduced caspase activation. Interestingly, experimental TBI triggers a multiplicity of neuronal death mechanisms. Although markers of DNA-damage/p53-dependent intrinsic apoptosis are detected acutely in the injured cortex and are attenuated by mithramycin, these processes may play a reduced role in early neuronal death after TBI, as caspase-dependent mechanisms are repressed in mature neurons while other, mithramycin-resistant mechanisms are active. Our data suggest that Sp1 is required for p53-mediated transactivation of neuronal pro-apoptotic molecules and that mithramycin may attenuate neuronal cell death in conditions predominantly involving DNA-damage-induced p53-dependent intrinsic apoptosis.

There is a need to develop novel approaches to improve the balance between efficacy and toxicity for transcription factor–targeted therapies. In this study, we exploit context-dependent differences in RNA polymerase II processivity as an approach to improve the activity and limit the toxicity of the EWS-FLI1–targeted small molecule, mithramycin, for Ewing sarcoma. The clinical activity of mithramycin for Ewing sarcoma is limited by off-target liver toxicity that restricts the serum concentration to levels insufficient to inhibit EWS-FLI1. In this study, we perform an siRNA screen of the druggable genome followed by a matrix drug screen to identify mithramycin potentiators and a synergistic “class” effect with cyclin-dependent kinase 9 (CDK9) inhibitors. These CDK9 inhibitors enhanced the mithramycin-mediated suppression of the EWS-FLI1 transcriptional program leading to a shift in the IC50 and striking regressions of Ewing sarcoma xenografts. To determine whether these compounds may also be liver protective, we performed a qPCR screen of all known liver toxicity genes in HepG2 cells to identify mithramycin-driven transcriptional changes that contribute to the liver toxicity. Mithramycin induces expression of the BTG2 gene in HepG2 but not Ewing sarcoma cells, which leads to a liver-specific accumulation of reactive oxygen species (ROS). siRNA silencing of BTG2 rescues the induction of ROS and the cytotoxicity of mithramycin in these cells. Furthermore, CDK9 inhibition blocked the induction of BTG2 to limit cytotoxicity in HepG2, but not Ewing sarcoma cells. These studies provide the basis for a synergistic and less toxic EWS-FLI1–targeted combination therapy for Ewing sarcoma.

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.

The free-living amebae Naegleria, Acanthamoeba, and Balamuthia cause rare but life-threatening infections. All three parasites can cause meningoencephalitis. Acanthamoeba can also cause chronic keratitis and both Balamuthia and Acanthamoeba can cause skin and systemic infections. There are minimal drug development pipelines for these pathogens despite a lack of available treatment regimens and high fatality rates. To identify anti-amebic drugs, we screened 159 compounds from a high-value repurposed library against trophozoites of the three amebae. Our efforts identified 38 compounds with activity against at least one ameba. Multiple drugs that bind the ATP-binding pocket of mTOR and PI3K are active, highlighting these compounds as important inhibitors of these parasites. Importantly, 24 active compounds have progressed at least to phase II clinical studies and overall 15 compounds were active against all three amebae. Based on central nervous system (CNS) penetration or exceptional potency against one amebic species, we identified sixteen priority compounds for the treatment of meningoencephalitis caused by these pathogens. The top five compounds are (i) plicamycin, active against all three free-living amebae and previously U.S. Food and Drug Administration (FDA) approved, (ii) TG02, active against all three amebae, (iii and iv) FDA-approved panobinostat and FDA orphan drug lestaurtinib, both highly potent against Naegleria, and (v) GDC-0084, a CNS penetrant mTOR inhibitor, active against at least two of the three amebae. These results set the stage for further investigation of these clinically advanced compounds for treatment of infections caused by the free-living amebae, including treatment of the highly fatal meningoencephalitis., Graphical abstract Image 1, Highlights • Focused screen of ReFRAME library (159 compounds) on three free-living amebae. • 16 priority compounds selected based on potency, advanced clinical studies and CNS penetration. • Identified compounds with fast killing kinetics against Naegleria and Balamuthia. • Identified compounds capable of delaying Balamuthia trophozoite recrudescence. • 5 compounds of high interest for treatment of CNS infections with free-living amebae.

Background Sarcomas comprise a group of aggressive malignancies with very little treatment options beyond standard chemotherapy. Reposition of approved drugs represents an attractive approach to identify effective therapeutic compounds. One example is mithramycin (MTM), a natural antibiotic which has demonstrated a strong antitumour activity in several tumour types, including sarcomas. However, its widespread use in the clinic was limited by its poor toxicity profile. Results In order to improve the therapeutic index of MTM, we have loaded MTM into newly developed nanocarrier formulations. First, polylactide (PLA) polymeric nanoparticles (NPs) were generated by nanoprecipitation. Also, liposomes (LIP) were prepared by ethanol injection and evaporation solvent method. Finally, MTM-loaded hydrogels (HG) were obtained by passive loading using a urea derivative non-peptidic hydrogelator. MTM-loaded NPs and LIP display optimal hydrodynamic radii between 80 and 105 nm with a very low polydispersity index (PdI) and encapsulation efficiencies (EE) of 92 and 30%, respectively. All formulations show a high stability and different release rates ranging from a fast release in HG (100% after 30 min) to more sustained release from NPs (100% after 24 h) and LIP (40% after 48 h). In vitro assays confirmed that all assayed MTM formulations retain the cytotoxic, anti-invasive and anti-stemness potential of free MTM in models of myxoid liposarcoma, undifferentiated pleomorphic sarcoma and chondrosarcoma. In addition, whole genome transcriptomic analysis evidenced the ability of MTM, both free and encapsulated, to act as a multi-repressor of several tumour-promoting pathways at once. Importantly, the treatment of mice bearing sarcoma xenografts showed that encapsulated MTM exhibited enhanced therapeutic effects and was better tolerated than free MTM. Conclusions Overall, these novel formulations may represent an efficient and safer MTM-delivering alternative for sarcoma treatment. Graphical abstract

A large number of deaths have been caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) worldwide, turning it into a serious and momentous threat to public health. This study tends to contribute to the development of effective treatment strategies through a computational approach, investigating the mechanisms in relation to the binding and subsequent inhibition of SARS-CoV-2 ribonucleic acid (RNA)-dependent RNA polymerase (RdRp). Molecular docking was performed to screen six naturally occurring molecules with antineoplastic properties (Ellipticine, Ecteinascidin, Homoharringtonine, Dolastatin 10, Halichondrin, and Plicamycin). Absorption, distribution, metabolism, and excretion (ADME) investigation was also conducted to analyze the drug-like properties of these compounds. The docked results have clearly shown binding of ligands to the SARS-CoV-2 RdRp protein. Interestingly, all ligands were found to obey Lipinski’s rule of five. These results provide a basis for repurposing and using molecules, derived from plants and animals, as a potential treatment for the coronavirus disease 2019 (COVID-19) infection as they could be effective therapeutics for the same., Graphical abstract Image 1

Cancer stands in the frontline among leading killers worldwide and the annual mortality rate is expected to reach 16.4 million by 2040. Humans suffer from about 200 different types of cancers and many of them have a small number of approved therapeutic agents. Moreover, several types of major cancers are diagnosed at advanced stages as a result of which the existing therapies have limited efficacy against them and contribute to a dismal prognosis. Therefore, it is essential to develop novel potent anticancer agents to counteract cancer-driven lethality. Natural sources such as bacteria, plants, fungi, and marine microorganisms have been serving as an inexhaustible source of anticancer agents. Notably, over 13,000 natural compounds endowed with different pharmacological properties have been isolated from different bacterial sources. In the present article, we have discussed about the importance of natural products, with special emphasis on bacterial metabolites for cancer therapy. Subsequently, we have comprehensively discussed the various sources, mechanisms of action, toxicity issues, and off-target effects of clinically used anticancer drugs (such as actinomycin D, bleomycin, carfilzomib, doxorubicin, ixabepilone, mitomycin C, pentostatin, rapalogs, and romidepsin) that have been derived from different bacteria. Furthermore, we have also discussed some of the major secondary metabolites (antimycins, chartreusin, elsamicins, geldanamycin, monensin, plicamycin, prodigiosin, rebeccamycin, salinomycin, and salinosporamide) that are currently in the clinical trials or which have demonstrated potent anticancer activity in preclinical models. Besides, we have elaborated on the application of metagenomics in drug discovery and briefly described about anticancer agents (bryostatin 1 and ET-743) identified through the metagenomics approach.

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 (MTM(ox)) conjugation strategy for rapid MTM diversification. Comparative cytotoxicity assays of 41 MTM(ox) 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 MTM(ox)32E (a Phe–Trp dipeptide-based 2′-conjugate) for in vivo testing. Relative to MTM, MTM(ox)32E 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.

The discovery of the isocitrate dehydrogenase (IDH) mutation in glioma led to a paradigm shift on how we see glioma biology. Difficulties in cultivating IDH mutant glioma stem cells (IDHmut GSCs) resulted in a paucity of preclinical models in IDHmut glioma, limiting the discovery of new effective chemotherapeutic agents. To fill this gap, we used six recently developed patient-derived IDHmut GSC lines and performed a large-scale drug screening with 147 Food and Drug Administration (FDA)-approved anticancer drugs. GSCs were subjected to the test compounds for 72 h in concentrations ranging from 0.0001 to 1 &micro, M. Cell viability was assessed by CellTiterGlo and the induction of apoptosis by flow cytometry with Annexin V/propidium iodide staining. The initial screen was performed with two IDHmut GSC lines and identified seven drugs (bortezomib, carfilzomib, daunorubicin, doxorubicin, epirubicin, omacetaxine, plicamycin) with a substantial antiproliferative activity, as reflected by half maximal inhibitory concentrations (IC50) below 1 &micro, M and maximum inhibitory effects (Emax) below 25%. These findings were validated in an additional four IDHmut GSC lines. The candidate drugs, of which plicamycin and omacetaxine are known to cross the blood brain barrier, were used for subsequent cell death analyses. A significant induction of apoptosis was observed at IC50 values of the respective drugs. In summary, we were able to identify seven FDA-approved drugs that should be further taken into clinical investigations for the treatment of IDHmut gliomas.

Numerous studies have focused on the deteriorate role of amyloid-β (Aβ) on retina, implying the potential pathogenic mechanism underlying age-related macular degeneration (AMD). However, the mechanism underlying the Aβ deposition in AMD patients remains unknown. Beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1), rate-limiting enzyme for Aβ production, plays an important role in Aβ deposition in the brain. In the current study, we aimed to clarify the regulation mechanism of BACE1 and explore potential drug targets using a lipofuscinfluorophore A2E-mediated photo-oxidation model. In this model, Aβ1-40 and Aβ1-42 levels increased simultaneously with the enhanced BACE1 expression. These changes were associated with the hypomethylation of specific loci within the BACE1 gene promoter and the decreased levels of DNA methyltransferase 1 (DNMT1). Furthermore, we noticed overlapping regions of differentially methylated CpG islands and specificity protein (Sp1) binding sites within the BACE1 promoter. We employed chromatin immunoprecipitation (ChIP) assay to verify that the decreased BACE1 promoter methylation by DNMT1 enabled increased binding between Sp1 and the BACE1 promoter, which further enhanced BACE1 transcription. The inhibition of Sp1 with mithramycin A (MTM) could down-regulate the expression of BACE1 as well as alleviate the RPE barrier morphology and function impairment. Our results for the first time show the competitive regulation of BACE1 by transcription factor Sp1 and DNMT1 after photo-oxidation and confirm the potential novel protective role of MTM on RPE cells.

We previously developed an efficient deletion system for streptomycetes based on the positive selection of double-crossover events using bpsA, a gene for producing the blue pigment indigoidine. Using this system, we removed interfering secondary metabolite clusters from Streptomyces lividans TK24, resulting in RedStrep strains with dramatically increased heterologous production of mithramycin A (up to 3-g/l culture). This system, however, required a time-consuming step to remove the resistance marker genes. In order to simplify markerless deletions, we prepared a new system based on the plasmid pAMR18A. This plasmid contains a large polylinker with many unique restriction sites flanked by apramycin and kanamycin resistance genes and the bpsA gene for selecting a double-crossover event. The utility of this new markerless deletion system was demonstrated by its deletion of a 21-kb actinorhodin gene cluster from Streptomyces lividans TK24 with 30% efficiency. We used this system to efficiently remove the matA and matB genes in selected RedStrep strains, resulting in biotechnologically improved strains with a highly dispersed growth phenotype involving non-pelleting small and open mycelia. No further increase in mithramycin A production was observed in these new RedStrep strains, however. We also used this system for the markerless insertion of a heterologous mCherry gene, an improved variant of the monomeric red fluorescent protein, under the control of the strong secretory signal sequence of the subtilisin inhibitor protein, into the chromosome of S. lividans TK24. The resulting recombinant strains efficiently secreted mCherry into the growth medium in a yield of 30 mg/l.

Mithramycin A (1) was identified as the top potential inhibitor of the aberrant ETS transcription factor EWS-FLI1, which causes Ewing sarcoma. Unfortunately, 1 has a narrow therapeutic window, compelling us to seek less toxic and more selective analogues. Here, we used MTMSA (2) to generate analogues via peptide coupling and fragment-based drug development strategies. Cytotoxicity assays in ETS and non-ETS dependent cell lines identified two dipeptide analogues, 60 and 61, with 19.1- and 15.6-fold selectivity, respectively, compared to 1.5-fold for 1. Importantly, the cytotoxicity of 60 and 61 is

The progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) is one of the hallmarks of Parkinson's disease (PD). Neuroinflammation has been proposed to contributes to the progressive nature of the disease. Early growth response-1 (Egr-1), a zinc finger transcription factor, has been shown to have a crucial role in both neuronal death and the inflammatory response. However, whether and how Egr-1 is involved in the pathogenesis of PD has not been investigated. Using the subacute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD, we identified early peak induction of Egr-1 in the SNpc but not in the striatum. In situ immunofluorescent analysis showed that Egr-1 predominantly locates in the nuclei of nigral AldoC (+) astrocytes upon MPTP treatment. Genetic ablation of Egr-1 or inhibition of its transcriptional activity by Mithramycin A significantly suppresses the activation of both astrocytes and microglia, decreases proinflammatory cytokine expression, and protects dopaminergic cell bodies from degeneration in the SNpc. Taken together, these findings demonstrate that the induction of Egr-1 promotes neuroinflammation and dopaminergic cell body loss in the SNpc of MPTP-induced mouse model, suggesting an important role of astrocytic Egr-1 in neuroinflammation in PD.

Xu-Jie Liu, Liang Li, Xiu-Jun Liu, Yi Li, Chun-Yan Zhao, Rui-Qi Wang, Yong-Su Zhen Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People’s Republic of China Abstract: Previous studies have shown that mithramycin A (MIT) is a promising candidate for the treatment of pancreatic carcinoma through inhibiting transcription factor Sp1. However, systemic toxicities may limit its clinical application. Here, we report a rationally designed formulation of MIT-loaded nanoparticles (MIT-NPs) with a small size and sustained release for improved passive targeting and enhanced therapeutic efficacy. Nearly spherical MIT-NPs with a mean particle size of 25.0±4.6nm were prepared by encapsulating MIT into methoxy poly(ethylene glycol)-block-poly(D,L-lactic-co-glycolic acid) (mPEG-PLGA) nanoparticles (NPs) with drug loading of 2.11%±0.51%. The in vitro release of the MIT-NPs lasted for >48h with a sustained-release pattern. The cytotoxicity of MIT-NPs to human pancreatic cancer BxPC-3 and MIA Paca-2 cells was comparable to that of free MIT. Determined by flow cytometry and confocal microscopy, the NPs internalized into the cells quickly and efficiently, reaching the peak level at 1–2h. In vivo fluorescence imaging showed that the prepared NPs were gradually accumulated in BxPC-3 and MIA Paca-2 xenografts and retained for 168h. The fluorescence intensity in both BxPC-3 and MIA Paca-2 tumors was much stronger than that of various tested organs. Therapeutic efficacy was evaluated with the poorly permeable BxPC-3 pancreatic carcinoma xenograft model. At a well-tolerated dose of 2mg/kg, MIT-NPs suppressed BxPC-3 tumor growth by 96%. Compared at an equivalent dose, MIT-NPs exerted significantly higher therapeutic effect than free MIT (86% versus 51%, P

After sudden traumatic brain injuries, secondary injuries may occur during the following days or weeks, which leads to the accumulation of reactive oxygen species (ROS). Since ROS exacerbate brain damage, it is important to protect neurons against their activity. Zinc finger protein 179 (Znf179) was shown to act as a neuroprotective factor, but the regulation of gene expression under oxidative stress remains unknown. In this study, we demonstrated an increase in Znf179 protein levels in both in vitro model of hydrogen peroxide (H2O2)-induced ROS accumulation and animal models of traumatic brain injury. Additionally, we examined the sub-cellular localization of Znf179, and demonstrated that oxidative stress increases Znf179 nuclear shuttling and its interaction with specificity protein 1 (Sp1). Subsequently, the positive autoregulation of Znf179 expression, which is Sp1-dependent, was further demonstrated using luciferase reporter assay and green fluorescent protein (GFP)-Znf179-expressing cells and transgenic mice. The upregulation of Sp1 transcriptional activity induced by the treatment with nerve growth factor (NGF) led to an increase in Znf179 levels, which further protected cells against H2O2-induced damage. However, Sp1 inhibitor, mithramycin A, was shown to inhibit NGF effects, leading to a decrease in Znf179 expression and lower cellular protection. In conclusion, the results obtained in this study show that Znf179 autoregulation through Sp1-dependent mechanism plays an important role in neuroprotection, and NGF-induced Sp1 signaling may help attenuate more extensive (ROS-induced) damage following brain injury., Graphical abstract fx1, Highlights • Znf179 levels increase in vitro after hydrogen peroxide treatment. • Znf179 levels increase in vivo in traumatic brain injury mouse model. • Oxidative stress increases Znf179 translocation to nucleus. • Znf179 autoregulates its expression through Sp1-dependent mechanism. • Sp1-Znf179 pathway plays an important role in neuroprotection.

Spanish Ministry of Economy and Competitiveness [BIO2008-00269, BIO2011-25398]; grant "Apoyo a grupos de excelencia", Principado de Asturias-FEDER [FC-15-GRUPIN14-014]; Spanish Ministry of Economy and Competiveness, This work was supported by grants to C.M. from the Spanish Ministry of Economy and Competitiveness (Grants BIO2008-00269 and BIO2011-25398) and by the grant “Apoyo a grupos de excelencia”, Principado de Asturias-FEDER (FC-15-GRUPIN14-014). D.Z. was recipient of a predoctoral fellowship from the Spanish Ministry of Economy and Competiveness.

Since the end of 2019, the coronavirus SARS-CoV-2 has caused more than 1000000 deaths all over the world and still lacks a medical treatment despite the attention of the whole scientific community. Human angiotensin-converting enzyme 2 (ACE2) was recently recognized as the transmembrane protein that serves as the point of entry of SARS-CoV-2 into cells, thus constituting the first biomolecular event leading to COVID-19 disease. Here, by means of a state-of-the-art computational approach, we propose a rational evaluation of the molecular mechanisms behind the formation of the protein complex. Moreover, the free energy of binding between ACE2 and the active receptor binding domain of the SARS-CoV-2 spike protein is evaluated quantitatively, providing for the first time the thermodynamics of virus-receptor recognition. Furthermore, the action of different ACE2 ligands is also examined in particular in their capacity to disrupt SARS-CoV-2 recognition, also providing via a free energy profile the quantification of the ligand-induced decreased affinity. These results improve our knowledge on molecular grounds of the SARS-CoV-2 infection and allow us to suggest rationales that could be useful for the subsequent wise molecular design for the treatment of COVID-19 cases.

The dependency of prostate cancer (PCa) growth on androgen receptor (AR) signaling has been harnessed to develop first-line therapies for high-risk localized and metastatic PCa treatment. However, the occurrence of aberrant expression, mutated or splice variants of AR confers resistance to androgen ablation therapy (ADT), radiotherapy or chemotherapy in AR-positive PCa. Therapeutic strategies that effectively inhibit the expression and/or transcriptional activity of full-length AR, mutated AR and AR splice variants have remained elusive. In this study, we report that mithramycin (MTM), an antineoplastic antibiotic, suppresses cell proliferation and exhibits dual inhibitory effects on expression and transcriptional activity of AR and AR splice variants. MTM blocks AR recruitment to its genomic targets by occupying AR enhancers and causes downregulation of AR target genes, which includes key DNA repair factors in DNA damage repair (DDR). We show that MTM significantly impairs DDR and enhances the effectiveness of ionizing radiation or the radiomimetic agent Bleomycin in PCa. Thus, the combination of MTM treatment with RT or radiomimetic agents, such as bleomycin, may present a novel effective therapeutic strategy for patients with high-risk, clinically localized PCa.

The restricted expression of Wilms tumor 1 (WT1) and cyclin A1 (CCNA1) in normal tissues, as opposed to their abnormal expression in leukemia demonstrates the applicability of WT1 and CCNA1 as cancer antigens for immunotherapy, and as markers for prognosis and relapse. In this study, the WT1 and CCNA1 mRNA levels were found to be elevated in bone marrow samples from pediatric acute promyelocytic leukemia (APL or AML‑M3) patients, and to be quite varied in pediatric acute lymphocytic leukemia (ALL) patients, compared to non‑leukemic bone marrow controls. Consistent with the observed upregulation of both WT1 and CCNA1 in APL, WT1 overexpression elevated the CCNA1 mRNA levels in K562 leukemia cells. Treatment with curcumin decreased the WT1 levels in K562 cells, and also decreased CCNA1 protein expression. The examination of the CCNA1 promoter identified potential canonical WT1 binding sites within the 3‑kb region upstream of the transcription start site. Chromatin immunoprecipitation and luciferase reporter assays confirmed WT1 binding and the activation of the CCNA1 promoter. Furthermore, the GC‑rich core CCNA1 promoter region provided additional non‑canonical WT1 activation sites, as revealed by promoter assays. The importance of the GC‑rich core region of the CCNA1 promoter was confirmed by treating the K562 cells with mithramycin A, which blocks the binding of zinc finger transcription factors to GC‑rich sequences. Mithramycin A subsequently suppressed both CCNA1 promoter activity and protein expression in the K562 cells. Taken together, the data from the WT1 overexpression, and curcumin and mithramycin A treatment experiments, as well as those from chromatin binding assays, along with inferences from patient RNA analyses, establish a plausible link between WT1 and CCNA1, and support the functional significance of an elevated WT1 expression in leukemia, which may also affect CCNA1 expression.

MtmOIV 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 Mg(2+) 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 of the ring-shaped β subunit of a vertebrate ion channel. We show that MtmW and MmOIV 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.

Mithramycin (MTM) has potent anticancer activity, but severe toxicities restrict its clinical use. Semi-synthetic approaches have yielded novel MTM analogs with potentially lower toxicity and similar efficacy. In an effort to transition these analogs into in vivo models, a bioanalytical method was developed for their quantification in mouse plasma. Here we present the validation of the method for the quantitation of mithramycin SA-tryptophan (MTMSA-Trp) as well as the applicability of the methodology for assaying additional analogs, including MTM, mithramycin SK (MTMSK) and mithramycin SA-phenylalanine (MTMSA-Phe) with run times of 6 min. Assay linearity ranged from 5 to 100 ng/mL. Accuracies of calibration standards and quality control samples were within 15% of nominal with precision variability of

Triptolide (TP), a primary bioactive ingredient isolated from the traditional Chinese herbal medicine Tripterygium wilfordii Hook. F. (TWHF), has attracted great interest for its therapeutic biological activities in inflammation and autoimmune disease. However, its clinical use is limited by severe testicular toxicity, and the underlying mechanism has not been elucidated. Our preliminary evidence demonstrated that TP disrupted glucose metabolism and caused testicular toxicity. During spermatogenesis, Sertoli cells (SCs) provide lactate as an energy source to germ cells by glycolysis. The transcription factors GATA-binding protein 4 (GATA4) and specificity protein 1 (Sp1) can regulate glycolysis. Based on this evidence, we speculate that TP causes abnormal glycolysis in SCs by influencing the expression of the transcription factors GATA4 and Sp1. The mechanism of TP-induced testicular toxicity was investigated in vitro and in vivo. The data indicated that TP decreased glucose consumption, lactate production, and the mRNA levels of glycolysis-related transporters and enzymes. TP also downregulated the protein expression of the transcription factors GATA4 and Sp1, as well as the glycolytic enzyme phosphofructokinase platelet (PFKP). Phosphorylated GATA4 and nuclear GATA4 protein levels were reduced in a dose- and time-dependent manner after TP incubation. Similar effects were observed in shGata4-treated TM4 cells and BALB/c mice administered 0.4 mg/kg TP for 28 days, and glycolysis was also inhibited. Gata4 knockdown downregulated Sp1 and PFKP expression. Furthermore, the Sp1 inhibitor plicamycin inhibited PFKP protein levels in TM4 cells. In conclusion, TP inhibited GATA4-mediated glycolysis by suppressing Sp1-dependent PFKP expression in SCs and caused testicular toxicity.

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.

Transcription factors have been considered undruggable, but this paradigm has been recently challenged. DNA binding natural product mithramycin (MTM) is a potent antagonist of oncogenic transcription factor EWS-FLI1. Structural details of MTM recognition of DNA, including the FLI1 binding sequence GGA(A/T), are needed to understand how MTM interferes with EWS-FLI1. We report a crystal structure of an MTM analogue MTM SA-Trp bound to a DNA oligomer containing a site GGCC, and two structures of a novel analogue MTM SA-Phe in complex with DNA. MTM SA-Phe is bound to sites AGGG and GGGT on one DNA, and to AGGG and GGGA(T) (a FLI1 binding site) on the other, revealing how MTM recognizes different DNA sequences. Unexpectedly, at sub-micromolar concentrations MTMs stabilize FLI1-DNA complex on GGAA repeats, which are critical for the oncogenic function of EWS-FLI1. We also directly demonstrate by nuclear magnetic resonance formation of a ternary FLI1-DNA-MTM complex on a single GGAA FLI1/MTM binding site. These biochemical and structural data and a new FLI1-DNA structure suggest that MTM binds the minor groove and perturbs FLI1 bound nearby in the major groove. This ternary complex model may lead to development of novel MTM analogues that selectively target EWS-FLI1 or other oncogenic transcription factors, as anti-cancer therapeutics.

Objective Melatonin, an endogenous neurohormone secreted predominately by the pineal gland, has a variety of physiological functions. However, its protective role in atherosclerosis is not clear. In this study, we sought to investigate the potential effects of melatonin in modulating atherosclerotic plaque stability in apolipoprotein E knockout (ApoE) mice. Method and results Smooth muscle cells were treated with melatonin, which significantly increased mRNA and protein levels of a key intracellular enzyme essential for collagen maturation and secretion, prolyl-4-hydroxylase α1 (P4Hα1). Mechanistically, melatonin increased Akt phosphorylation and transcriptional activation of specificity protein 1 (Sp1), which bound with the P4Hα1 promoter and then induced P4Hα1 expression. Pretreatment with either Akt inhibitor LY294002 or Sp1 inhibitor mithramycin A (MTM) could inhibit melatonin-induced P4Hα1 expression. Finally, atherosclerotic lesions were induced by placing a perivascular collar on the right common carotid artery of ApoE mice, which were received with or without different doses of melatonin or MTM. High-dose melatonin enhanced atherosclerotic plaque stability in ApoE mice in vivo by inducing the expression of P4Hα1, which was reversed by MTM. Conclusion We propose that melatonin supplementation may provide a novel and promising approach to atherosclerosis treatment.

Mithramycin demonstrates preclinical anticancer activity, but its therapeutic dose is limited by the development of hepatotoxicity that remains poorly characterized. A pharmacogenomics characterization of mithramycin-induced transaminitis revealed that hepatotoxicity is associated with germline variants in genes involved in bile disposition: ABCB4 (multidrug resistance 3) rs2302387 and ABCB11 [bile salt export pump (BSEP)] rs4668115 reduce transporter expression (P < 0.05) and were associated with ≥grade 3 transaminitis developing 24 hours after the third infusion of mithramycin (25 mcg/kg, 6 hours/infusion, every day ×7, every 28 days; P < 0.0040). A similar relationship was observed in a pediatric cohort. We therefore undertook to characterize the mechanism of mithramycin-induced acute transaminitis. As mithramycin affects cellular response to bile acid treatment by altering the expression of multiple bile transporters (e.g., ABCB4, ABCB11, sodium/taurocholate cotransporting polypeptide, organic solute transporter α/β) in several cell lines [Huh7, HepaRG, HepaRG BSEP (-/-)] and primary human hepatocytes, we hypothesized that mithramycin inhibited bile-mediated activation of the farnesoid X receptor (FXR). FXR was downregulated in all hepatocyte cell lines and primary human hepatocytes (P < 0.0001), and mithramycin inhibited chenodeoxycholic acid- and GW4046-induced FXR-galactose-induced gene 4 luciferase reporter activity (P < 0.001). Mithramycin promoted glycochenodeoxycholic acid-induced cytotoxicity in ABCB11 (-/-) cells and increased the overall intracellular concentration of bile acids in primary human hepatocytes grown in sandwich culture (P < 0.01). Mithramycin is a FXR expression and FXR transactivation inhibitor that inhibits bile flow and potentiates bile-induced cellular toxicity, particularly in cells with low ABCB11 function. These results suggest that mithramycin causes hepatotoxicity through derangement of bile acid disposition; results also suggest that pharmacogenomic markers may be useful to identify patients who may tolerate higher mithramycin doses. SIGNIFICANCE STATEMENT: The present study characterizes a novel mechanism of drug-induced hepatotoxicity in which mithramycin not only alters farnesoid X receptor (FXR) and small heterodimer partner gene expression but also inhibits bile acid binding to FXR, resulting in deregulation of cellular bile homeostasis. Two novel single-nucleotide polymorphisms in bile flow transporters are associated with mithramycin-induced liver function test elevations, and the present results are the rationale for a genotype-directed clinical trial using mithramycin in patients with thoracic malignancies.

Drugs that target insulin secretion are useful to understand β cell function and the pathogenesis of diabetes. Kalwat et al. investigate an aureolic acid that inhibits insulin secretion and reveal that it disrupts Wnt signaling, interferes with gene expression, and suppresses Ca2+ influx in β cells., Modulators of insulin secretion could be used to treat diabetes and as tools to investigate β cell regulatory pathways in order to increase our understanding of pancreatic islet function. Toward this goal, we previously used an insulin-linked luciferase that is cosecreted with insulin in MIN6 β cells to perform a high-throughput screen of natural products for chronic effects on glucose-stimulated insulin secretion. In this study, using multiple phenotypic analyses, we found that one of the top natural product hits, chromomycin A2 (CMA2), potently inhibited insulin secretion by at least three potential mechanisms: disruption of Wnt signaling, interference of β cell gene expression, and partial suppression of Ca2+ influx. Chronic treatment with CMA2 largely ablated glucose-stimulated insulin secretion even after washout, but it did not inhibit glucose-stimulated generation of ATP or Ca2+ influx. However, by using the KATP channel opener diazoxide, we uncovered defects in depolarization-induced Ca2+ influx that may contribute to the suppressed secretory response. Glucose-responsive ERK1/2 and S6 phosphorylation were also disrupted by chronic CMA2 treatment. By querying the FUSION bioinformatic database, we revealed that the phenotypic effects of CMA2 cluster with a number of Wnt–GSK3 pathway-related genes. Furthermore, CMA2 consistently decreased GSK3β phosphorylation and suppressed activation of a β-catenin activity reporter. CMA2 and a related compound, mithramycin, are known to have DNA interaction properties, possibly abrogating transcription factor binding to critical β cell gene promoters. We observed that CMA2 but not mithramycin suppressed expression of PDX1 and UCN3. However, neither expression of INSI/II nor insulin content was affected by chronic CMA2. The mechanisms of CMA2-induced insulin secretion defects may involve components both proximal and distal to Ca2+ influx. Therefore, CMA2 is an example of a chemical that can simultaneously disrupt β cell function through both noncytotoxic and cytotoxic mechanisms. Future therapeutic applications of CMA2 and similar aureolic acid analogues should consider their potential effects on pancreatic islet function.

Osteoarthritis (OA) is the most common and increasing joint disease worldwide. Current treatment for OA is limited to control of symptoms. The purpose of this study was to determine the effect of specificity protein 1 (SP1) inhibitor Mithramycin A (MitA) on chondrocyte catabolism and OA pathogenesis and to explore the underlying molecular mechanisms involving SP1 and other key factors that are critical for OA. Here, we show that MitA markedly inhibited expressions of matrix-degrading enzymes induced by pro-inflammatory cytokine interleukin-1β (IL-1β) in mouse primary chondrocytes. Intra-articular injection of MitA into mouse knee joint alleviated OA cartilage destruction induced by surgical destabilization of the medial meniscus (DMM). However, modulation of SP1 level in chondrocyte and mouse cartilage did not alter catabolic gene expression or cartilage integrity, respectively. Instead, MitA significantly impaired the expression of HIF-2α known to be critical for OA pathogenesis. Such reduction in expression of HIF-2α by MitA was caused by inhibition of NF-κB activation, at least in part. These results suggest that MitA can alleviate OA pathogenesis by suppressing NF-κB-HIF-2α pathway, thus providing insight into therapeutic strategy for OA.

The antineoplastic and antibiotic natural product mithramycin (MTM) is used against cancer-related hypercalcemia and, experimentally, against Ewing sarcoma and lung cancers. MTM exerts its cytotoxic effect by binding DNA as a divalent metal ion (Me2+)-coordinated dimer and disrupting the function of transcription factors. A precise molecular mechanism of action of MTM, needed to develop MTM analogues selective against desired transcription factors, is lacking. Although it is known that MTM binds G/C-rich DNA, the exact DNA recognition rules that would allow one to map MTM binding sites remain incompletely understood. Towards this goal, we quantitatively investigated dimerization of MTM and several of its analogues, MTM SDK (for Short side chain, DiKeto), MTM SA-Trp (for Short side chain and Acid), MTM SA-Ala, and a biosynthetic precursor premithramycin B (PreMTM B), and measured the binding affinities of these molecules to DNA oligomers of different sequences and structural forms at physiological salt concentrations. We show that MTM and its analogues form stable dimers even in the absence of DNA. All molecules, except for PreMTM B, can bind DNA with the following rank order of affinities (strong to weak): MTM = MTM SDK > MTM SA-Trp > MTM SA-Ala. An X(G/C)(G/C)X motif, where X is any base, is necessary and sufficient for MTM binding to DNA, without a strong dependence on DNA conformation. These recognition rules will aid in mapping MTM sites across different promoters towards development of MTM analogues as useful anticancer agents.

Mammalian cells possess the molecular apparatus necessary to take up, degrade, synthesize, and release free d-aspartate, which plays an important role in physiological functions within the body. Here, biologically active microbial compounds and pre-existing drugs were screened for their ability to alter the intracellular d-aspartate level in mammalian cells, and several candidate compounds were identified. Detailed analytical studies suggested that two of these compounds, mithramycin A and geldanamycin, suppress the biosynthesis of d-aspartate in cells. Further studies suggested that these compounds act at distinct sites within the cell. These compounds may advance our current understanding of biosynthesis of d-aspartate in mammals, a whole picture of which remains to be disclosed.

More and more post-PKS tailoring enzymes are recognized to be multifunctional and co-dependent on other tailoring enzymes. One of the recently discovered intriguing examples is MtmC, a bifunctional TDP-4-keto-d-olivose ketoreductase-methyltransferase, which – in co-dependence with glycosyltransferase MtmGIV – is a key contributor to the biosynthesis of the critical trisaccharide chain of the antitumor antibiotic mithramycin (MTM), produced by Streptomyces argillaceus. We report crystal structures of three binary complexes of MtmC with its methylation co-substrate SAM, its co-product SAH, and a nucleotide TDP as well as crystal structures of two ternary complexes, MtmC-SAH-TDP-4-keto-d-olivose and MtmC-SAM-TDP, in the range of 2.2-2.7 Å in resolution. The structures reveal general and sugar-specific recognition and catalytic structural features of MtmC. Depending on the catalytic function that is carried out by MtmC, it must bind either NADPH or SAM in the same co-factor binding pocket. A tyrosine residue (Tyr79) appears as a lid covering the sugar moiety of the substrate during the methyl transfer reaction. This residue swings out of the active site by about 180° in the absence of the substrate. This unique conformational change likely serves to release the methylated product and, possibly, to open up the active site for binding the bulkier co-substrate NADPH prior to the reduction reaction.

Bone marrow microenvironment mediated downregulation of BCL6 is critical for maintaining cell quiescence and modulating therapeutic response in B-cell acute lymphoblastic leukemia (ALL). In the present study, we have performed a high throughput cell death assay using BCL6 knockdown REH ALL cell line to screen a library of FDA-approved oncology drugs. In the process, we have identified a microtubule inhibitor, cabazitaxel (CAB), and a RNA synthesis inhibitor, plicamycin (PLI) as potential anti-leukemic agents. CAB and PLI inhibited cell proliferation in not only the BCL6 knockdown REH cell line, but also six other ALL cell lines. Furthermore, combination of CAB and PLI had a synergistic effect in inhibiting proliferation in a cytarabine-resistant (REH/Ara-C) ALL cell line. Use of nanoparticles for delivery of CAB and PLI demonstrated that the combination was very effective when tested in a co-culture model that mimics the in vivo bone marrow microenvironment that typically supports ALL cell survival and migration into protective niches. Furthermore, exposure to PLI inhibited SOX2 transcription and exposure to CAB inhibited not only Mcl-1 expression but also chemotaxis in ALL cells. Taken together, our study demonstrates the utility of concomitantly targeting different critical regulatory pathways to induce cell death in drug resistant ALL cells.

Aberrant activation of AKT disturbs the proliferation, survival and metabolic homeostasis of various human cancers. Thus, it is critical to understand the upstream signalling pathways governing AKT activation. Here, we report that AKT undergoes SETDB1-mediated lysine methylation to promote its activation, which is antagonized by the Jumonji-family demethylase KDM4B. Notably, compared with wild-type mice, mice harbouring non-methylated mutant Akt1 not only exhibited reduced body size but were also less prone to carcinogen-induced skin tumours, in part due to reduced AKT activation. Mechanistically, the interaction of phosphatidylinositol (3,4,5)-trisphosphate with AKT facilitates its interaction with SETDB1 for subsequent AKT methylation, which in turn sustains AKT phosphorylation. Pathologically, genetic alterations, including SETDB1 amplification, aberrantly promote AKT methylation to facilitate its activation and oncogenic functions. Thus, AKT methylation is an important step, synergizing with PI3K signalling to control AKT activation. This suggests that targeting SETDB1 signalling could be a potential therapeutic strategy for combatting hyperactive AKT-driven cancers. Guo et al. identify SETDB1 and KDM4B as the methyltransferase and demethylase, respectively, for AKT. AKT methylation promotes its kinase activity and the subsequent tumorigenesis.

Mithramycin A is an antitumor compound used for treatment of several types of cancer including chronic and acute myeloid leukemia, testicular carcinoma, hypercalcemia and Paget's disease. Selective modifications of this molecule by combinatorial biosynthesis and biocatalysis opened the possibility to produce mithramycin analogues with improved properties that are currently under preclinical development. The mithramycin A biosynthetic gene cluster from Streptomyces argillaceus ATCC12956 was cloned by transformation assisted recombination in Saccharomyces cerevisiae and heterologous expression in Streptomyces lividans TK24 was evaluated. Mithramycin A was efficiently produced by S. lividans TK24 under standard fermentation conditions. To improve the yield of heterologously produced mithramycin A, a collection of derivative strains of S. lividans TK24 were constructed by sequential deletion of known potentially interfering secondary metabolite gene clusters using a protocol based on the positive selection of double crossover events with blue pigment indigoidine-producing gene. Mithramycin A production was evaluated in these S. lividans strains and substantially improved mithramycin A production was observed depending on the deleted gene clusters. A collection of S. lividans strains suitable for heterologous expression of actinomycetes secondary metabolites were generated and efficient production of mithramycin A with yields close to 3 g/L, under the tested fermentation conditions was achieved using these optimized collection of strains.

Gliostatin (GLS) has angiogenic and arthritogenic activities and enzymatic activity as thymidine phosphorylase. Aberrant GLS production has been observed in the synovial membranes of patients with rheumatoid arthritis (RA). Matrix metalloproteinases (MMPs) are involved in joint destruction. Promoters of GLS and some MMP genes contain Sp1 binding sites. We examined the inhibitory effect of the Sp1 inhibitor mithramycin on GLS-induced GLS and MMP expression in cultured fibroblast-like synoviocytes (FLSs).Synovial tissue samples were obtained from patients with RA. FLSs pretreated with mithramycin were cultured with GLS. The mRNA expression levels of GLS and MMP-1, MMP-2, MMP-3, MMP-9, and MMP-13 were determined using reverse transcription polymerase chain reactions. Protein levels were measured using enzyme immunoassay and gelatin zymography.GLS upregulated the expression of GLS itself and of MMP-1, MMP-3, MMP-9, and MMP-13, an effect significantly reduced by treatment with mithramycin. GLS and mithramycin had no effect on MMP-2 expression.Mithramycin downregulated the increased expression of GLS and MMP-1, MMP-3, MMP-9, and MMP-13 in FLSs treated with GLS. Because GLS plays a pathological role in RA, blocking GLS stimulation using an agent such as mithramycin may be a novel approach to antirheumatic therapy.

Mithramycin exhibits certain anticancer effects in glioma, metastatic cerebral carcinoma, malignant lymphoma, chorionic carcinoma and breast cancer. However, its effects on salivary adenoid cystic carcinoma remain unclear. Here, we report that mithramycin significantly inhibited epithelial-to-mesenchymal transition and invasion in human salivary adenoid cystic carcinoma cell lines. The underlying mechanism for this activity was further demonstrated to involve decreasing the expression of the transcription factors specificity protein 1 and SNAI1. Specificity protein 1 is a pro-tumourigenic transcription factor that is overexpressed in SACC-LM and SACC-83 cells, and its expression is inhibited by mithramycin. Moreover, chromatin immunoprecipitation assays showed that specificity protein 1 induced SNAI1 transcription through direct binding to the SNAI1 promoter. In summary, this study uncovered the mechanism through which mithramycin inhibits epithelial-to-mesenchymal transition and invasion in salivary adenoid cystic carcinoma cell lines, namely, via downregulating specificity protein 1 and SNAI1 expression, which suggests mithramycin may be a promising therapeutic option for salivary adenoid cystic carcinoma.

The present study highlights the biological effects of chromomycin A2 toward metastatic melanoma cells in culture. Besides chromomycin A2, chromomycin A3 and demethylchromomycin A2 were also identified from the extract derived from Streptomyces sp., recovered from Paracuru Beach, located in the northeast region of Brazil. The cytotoxic activity of chromomycin A2 was evaluated across a panel of human tumor cell lines, which found IC50 values in the nM-range for exposures of 48 and 72 h. MALME-3M, a metastatic melanoma cell line, showed the highest sensitivity to chromomycin A2 after 48h incubation, and was chosen as a model to investigate this potent cytotoxic effect. Treatment with chromomycin A2 at 30 nM reduced cell proliferation, but had no significant effect upon cell viability. Additionally, chromomycin A2 induced accumulation of cells in G0/G1 phase of the cell cycle, with consequent reduction of S and G2/M and unbalanced expression of cyclins. Chromomycin A2 treated cells depicted several cellular fragments resembling autophagosomes and increased expression of proteins LC3-A and LC3-B. Moreover, exposure to chromomycin A2 also induced the appearance of acidic vacuolar organelles in treated cells. These features combined are suggestive of the induction of autophagy promoted by chromomycin A2, a feature not previously described for chromomycins.

Mithramycin is a neoplastic antibiotic synthesized by various Streptomyces bacteria. It is under investigation as a chemotherapeutic treatment for a wide variety of cancers. Ongoing and forthcoming clinical trials will require pharmacokinetic analysis of mithramycin in humans, both to see if target concentrations are achieved and to optimize dosing and correlate outcomes (response/toxicity) with pharmacokinetics. Two published methods for mithramycin quantitation exist, but both are immunoassays that lack current bioanalytical standards of selectivity and sensitivity. To provide an upgraded and more widely applicable assay, a UPLC-MS/MS method for quantitation of mithramycin in human plasma was developed. Solid phase extraction allowed for excellent recoveries (>90%) necessary for high throughput analyses on sensitive instrumentation. However, a ~55% reduction in analyte signal was observed as a result of plasma matrix effects. Mithramycin and the internal standard chromomycin were separated on a Waters Acquity BEH C18 column (2.1x50mm, 1.7um) and detected using electrospray ionization operated in the negative mode at mass transitions m/z 1083.5→268.9 and 1181.5→269.0, respectively, on an AB Sciex QTrap 5500. The assay range was 0.5–500 ng/mL and proved to be linear (r2>0.996), accurate (≤10% deviation), and precise (CV

In cultured human colonic epithelial cells and mouse colonic tissue, exposure to the common food additive carrageenan leads to inflammation, activation of Wnt signaling, increased Wnt9A expression, and decline in the activity of the enzyme arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase). In this study, the novel transcriptional mechanism by which carrageenan and decline in ARSB increase Wnt9A expression in NCM460 and HT-29 human colonic epithelial cells and in mouse colon is presented. Increased expression of Wnt9A has been associated with multiple malignancies, including colon carcinoma, and with ectodermal and mesoendodermal morphogenesis. When ARSB activity was reduced by siRNA or by exposure to carrageenan (1 μg/ml for 24 h), degradation of chondroitin 4-sulfate (C4S) was inhibited, leading to accumulation of more highly sulfated C4S, which binds less galectin-3, a β-galactoside-binding protein. Nuclear galectin-3 increased and mediated increased binding of Sp1 to the Sp1 consensus sequence in the Wnt9A promoter, shown by oligonucleotide-binding assay and by chromatin immunoprecipitation assay. When galectin-3 was silenced, the increases in Sp1 binding to the Wnt9A promoter and in Wnt9A expression, which followed carrageenan or ARSB silencing, were inhibited. Mithramycin A, a specific inhibitor of Sp1 oligonucleotide binding, and Sp1 siRNA blocked the carrageenan- and ARSB siRNA-induced increases in Wnt9A expression. These studies reveal how carrageenan exposure can lead to transcriptional events in colonic epithelial cells through decline in arylsulfatase B activity, with subsequent impact on C4S, galectin-3, Sp1, and Wnt9A and can exert significant effects on Wnt-initiated signaling and related vital cell processes.

Prenyl diphosphate synthase subunit 2 (PDSS2) is the first key enzyme in the CoQ10 biosynthesis pathway, and contributes to various metabolic and nephritic diseases. It has been reported that PDSS2 is downregulated in several types of tumors and acts as a potential tumor suppressor gene to inhibit the proliferation and migration of cancer cells. However, the regulatory mechanism of PDSS2 expression remains elusive. In the present study, we first identified and characterized the PDSS2 promoter region. We established four different luciferase reporter constructs which mainly cover the 2 kb region upstream of the PDSS2 gene transcription initiation site. Series luciferase reporter assay demonstrated that all four constructs have prominent promoter activity, and the core promoter of PDSS2 is mainly located within the 202 bp region near its transcription initiation site. Transcription factor binding site analysis revealed that the PDSS2 promoter contains binding sites for canonical transcription factors such as Sp1 and GATA-1. Overexpression of Sp1 significantly inhibited PDSS2 promoter activity, as well as its endogenous expression, at both mRNA and protein levels in lung cancer cells. Site-directed mutagenesis assay further confirmed that the Sp1 binding sites are essential for proximal prompter activity of PDSS2. Consistently, a selective Sp1 inhibitor, mithramycin A, treatment repressed the PDSS2 promoter activity, as well as its endogenous expression. Chromatin immunoprecipitation (ChIP) assay revealed that Sp1 binds to the PDSS2 promoter in vivo. Of note, the expression of Sp1 and PDSS2 are negatively correlated, and higher Sp1 expression with low PDSS2 expression is significantly associated with poor prognosis in lung cancer. Taken together, our results strongly suggest the essential role of Sp1 in maintaining the basic constitutive expression of PDSS2, and the pathogenic implication of Sp1-mediated PDSS2 transcriptional repression in lung cancer cells.

Mithramycin (MTM) is a polyketide antitumor compound produced by Streptomyces argillaceus constituted by a tricyclic aglycone with two aliphatic side chains, a trisaccharide and a disaccharide chain. The biosynthesis of the polyketide aglycone is initiated by the condensation of ten malonyl-CoA units to render a carbon chain that is modified to a tetracyclic intermediate and sequentially glycosylated by five deoxysugars originated from glucose-1-phosphate. Further oxidation and reduction render the final compound. We aimed to increase the precursor supply of malonyl-CoA and/or glucose-1-phosphate in S. argillaceus to enhance MTM production. We have shown that by overexpressing either the S. coelicolor phosphoglucomutase gene pgm or the acetyl-CoA carboxylase ovmGIH genes from the oviedomycin biosynthesis gene cluster in S. argillaceus, we were able to increase the intracellular pool of glucose-1-phosphate and malonyl-CoA, respectively. Moreover, we have cloned the S. argillaceus ADP-glucose pyrophosphorylase gene glgCa and the acyl-CoA:diacylglycerol acyltransferase gene aftAa, and we showed that by inactivating them, an increase of the intracellular concentration of glucose-1-phosphate/glucose-6-phosphate and malonyl-CoA/acetyl-CoA was observed, respectively. Each individual modification resulted in an enhancement of MTM production but the highest production level was obtained by combining all strategies together. In addition, some of these strategies were successfully applied to increase production of four MTM derivatives with improved pharmacological properties: demycarosyl-mithramycin, demycarosyl-3D-β-D-digitoxosyl-mithramycin, mithramycin SK and mithramycin SDK.

Background It is currently accepted that complex behavior and mental disorder results from a combination of biological susceptibility and exposure to environmental stimuli. Most of the gene–environment interaction models focus on the interaction between the stimuli and a single candidate gene. We suggest that an alternative approach is interference with the expression of multiple genes followed by exposure to environmental insults. Methods Early interference with gene transcription was performed by treatment of 7 days old Wistar male rats for 4 days with the Sp1/DNA binding inhibitor, mithramycin. Environmental insult was mimicked by exposing these rats during adulthood (34 days) to sub-chronic (12 days, n = 30) or chronic stress (28 days, n = 48). The effects of mithramycin and stress treatment on the behavioral response and serum corticosterone concentration were assessed. Results Exposure of mithramycin treated rats to sub-chronic stress led to anxious behavior in the open field test, high startle response, low sucrose preference, indifference to novel objects and high serum corticosterone concentration. However, exposure to chronic stress resulted in normal sucrose preference, startle response and serum corticosterone, novelty seeking behavior and reduced anxiety. In saline treated rats the extension of stress duration led to behavioral and hormonal adaptation to stress. Conclusion Our study suggests that postnatal temporal interference with multiple gene expression can lead to hyper-responsiveness to environmental stimuli, the features of which affects the phenotypic outcomes. Such a paradigm may be used to model gene–environmental interaction in the etiology of behavioral disorders.

Endothelins (ETs), a family of vasoconstrictor peptides, are up-regulated in several pathological conditions in the brain, and induce astrocytic proliferation. We previously observed that ET-1 increased the expression of cyclin D1 protein. Thus, we confirmed the intracellular up-regulation of cyclin D1 by ET-1 in rat cultured astrocytes. Real-time PCR analysis indicated that ET-1 (100 nM) and Ala 1,3,11,15 -ET-1 (100 nM), a selective agonist of the ET B receptor, induced a time-dependent and transient increase in cyclin D1 mRNA. The effect of ET-1 was diminished by an ET B antagonist (1 μM BQ788) or inhibitors of Sp1 (500 nM mithramycin), ERK (50 μM PD98059), p38 (20 μM SB203580) and JNK (1 μM SP600125), but not inhibitors of NF-κB (10 μM SN50 and 100 μM pyrrolidine dithiocarbamate). The binding assay for Sp1 indicated that ET-1 increased the binding activity of Sp1 to consensus sequences, and two oligonucleotides of the cyclin D1 promoter including the Sp1-binding sites diminished the effect of ET-1. Western blot analysis showed that ET-1 induced time-dependent and transient phosphorylation of Sp1 on Thr453 and Thr739 via the ET B receptor. ET-1-induced phosphorylation of Sp1 was attenuated by PD98059 and SP600125. Additionally, ET-1 increased the incorporation of bromodeoxyuridine (BrdU) in cultured astrocytes and the number of BrdU-positive cells decreased in the presence of PD98059, SP600125 and mithramycin. These results suggest that ET-1 increases the expression of cyclin D1 via activation of Sp1 and induces astrocytic proliferation.

Mithramycin (MTM) is a potent anti-cancer agent that has recently garnered renewed attention. This manuscript describes the design and development of mithramycin derivatives through a combinational approach of biosynthetic analogue generation followed by synthetic manipulation for further derivatization. Mithramycin SA is a previously discovered analogue produced by the M7W1 mutant strain alongside the improved mithramycin analogues mithramycin SK and mithramycin SDK. Mithramycin SA shows decreased anti-cancer activity compared to mithramycin and has a shorter, two carbon aglycon side chain that is terminated in a carboxylic acid. The aglycon side chain is responsible for an interaction with the DNA-phosphate backbone as mithramycin interacts with its target DNA. It was therefore decided to further functionalize this side chain through reactions with the terminal carboxylic acid in an effort to enhance the interaction with the DNA phosphate backbone and improve the anti-cancer activity. This side chain was modified with a variety of molecules increasing the anti-cancer activity to a comparable level to mithramycin SK. This work shows the ability to transform the previously useless mithramycin SA into a valuable molecule and opens the door to further functionalization and semi-synthetic modification for the development of molecules with increased specificity and/or drug formulation.

Linker for activation of T cells (LAT) is a raft-associated, transmembrane adapter protein critical for T-cell development and function. LAT expression is transiently upregulated upon T-cell receptor (TCR) engagement, but molecular mechanisms conveying TCR signaling to enhanced LAT transcription are not fully understood. Here we found that a Jurkat subline J.CaM2, initially characterized as LAT deficient, conditionally re-expressed LAT upon the treatment with a protein kinase C activator, phorbol 12-myristate 13-acetate (PMA). We took advantage of the above observation for studying cis-elements and trans-acting factors contributing to the activation-induced expression of LAT. We identified a LAT gene region spanning nucleotide position -14 to +357 relative to the ATG start codon as containing novel cis-regulatory elements that were able to promote PMA-induced reporter transcription in the absence of the core LAT promoter. Interestingly, a point mutation in LAT intron 1, identified in J.CaM2 cells, downmodulated LAT promoter activity by 50%. Mithramycin A, a selective Sp1 DNA-binding inhibitor, abolished LAT expression upon PMA treatment as did calcium ionophore ionomycin (Iono) and valproic acid (VPA), widely used as an anti-epileptic drug. Our data introduce J.CaM2 cells as a model for dissecting drivers and blockers of activation induced expression of LAT.

Cooling induces several responses that are modulated by molecular inhibitors and activators and receptor signaling. Information regarding potential targets involved in cold response mechanisms is still insufficient. We examined levels of the receptor-signaling mediator β-arrestin-1 and phospho-Ser-412 β-arrestin-1 in 3T3-L1 adipocytes exposed to 4-37 °C or treated with some molecular agents at 37°C. We also cooled cells with or without modification and signal-modulating agents. These conditions did not decrease cell viability, and western blot analysis revealed that exposure to 4 °C for 1.5h and to 28 and 32 °C for 24 and 48 h increased phospho-β-arrestin-1 and β-arrestin-1 levels and that exposure to 4 and 18 °C for 3 and 4.5h increased β-arrestin-1 level. Serum removal and rewarming abolished β-arrestin-1 alterations induced by cooling. Mithramycin A (a transcription inhibitor) treatment for 4 and 24h increased the level of β-arrestin-1 but not that of phospho-β-arrestin-1. The level of phospho-β-arrestin-1 was increased by okadaic acid (a phosphatase inhibitor), decreased by epinephrine and aluminum fluoride (receptor-signaling modulators), and unaffected by N-ethylmaleimide (an alkylating agent) at 37 °C. N-Ethylmaleimide and the receptor-signaling modulators did not alter β-arrestin-1 expression at 37 °C but impaired the induction of phospho-β-arrestin-1 at 28 and 32 °C without affecting the induction of β-arrestin-1. We show that cold-induced β-arrestin-1 alterations are partially mimicked by molecular agents and that the responsive machinery for β-arrestin-1 requires serum factors and N-ethylmaleimide-sensitive sites and is linked to rewarming- and receptor signaling-responsive machinery. Our findings provide helpful information for clarifying the cold-responsive machinery for β-arrestin-1 and elucidating low-temperature responses.