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The Journal of Biological Chemistry Oct 2016Rifampicin monooxygenase (RIFMO) catalyzes the N-hydroxylation of the natural product antibiotic rifampicin (RIF) to 2'-N-hydroxy-4-oxo-rifampicin, a metabolite with...
Rifampicin monooxygenase (RIFMO) catalyzes the N-hydroxylation of the natural product antibiotic rifampicin (RIF) to 2'-N-hydroxy-4-oxo-rifampicin, a metabolite with much lower antimicrobial activity. RIFMO shares moderate sequence similarity with well characterized flavoprotein monooxygenases, but the protein has not been isolated and characterized at the molecular level. Herein, we report crystal structures of RIFMO from Nocardia farcinica, the determination of the oligomeric state in solution with small angle x-ray scattering, and the spectrophotometric characterization of substrate binding. The structure identifies RIFMO as a class A flavoprotein monooxygenase and is similar in fold and quaternary structure to MtmOIV and OxyS, which are enzymes in the mithramycin and oxytetracycline biosynthetic pathways, respectively. RIFMO is distinguished from other class A flavoprotein monooxygenases by its unique middle domain, which is involved in binding RIF. Small angle x-ray scattering analysis shows that RIFMO dimerizes via the FAD-binding domain to form a bell-shaped homodimer in solution with a maximal dimension of 110 Å. RIF binding was monitored using absorbance at 525 nm to determine a dissociation constant of 13 μm Steady-state oxygen consumption assays show that NADPH efficiently reduces the FAD only when RIF is present, implying that RIF binds before NADPH in the catalytic scheme. The 1.8 Å resolution structure of RIFMO complexed with RIF represents the precatalytic conformation that occurs before formation of the ternary E-RIF-NADPH complex. The RIF naphthoquinone blocks access to the FAD N5 atom, implying that large conformational changes are required for NADPH to reduce the FAD. A model for these conformational changes is proposed.
Topics: Bacterial Proteins; Flavin-Adenine Dinucleotide; Flavoproteins; Hydroxylation; Mixed Function Oxygenases; NADP; Nocardia; Protein Domains; Protein Multimerization; Rifampin
PubMed: 27557658
DOI: 10.1074/jbc.M116.745315 -
Blood Aug 2003We report in this paper that the DNA-binding drug mithramycin is a potent inducer of gamma-globin mRNA accumulation and fetal hemoglobin (HbF) production in erythroid...
We report in this paper that the DNA-binding drug mithramycin is a potent inducer of gamma-globin mRNA accumulation and fetal hemoglobin (HbF) production in erythroid cells from healthy human subjects and beta-thalassemia patients. Erythroid precursors derived from peripheral blood were grown in 2-phase liquid culture. In this procedure, early erythroid progenitors proliferate and differentiate during phase 1 (in the absence of erythropoietin) into late progenitors. In phase 2, in the presence of erythropoietin, the latter cells continue their proliferation and mature into Hb-containing orthochromatic normoblasts. Compounds were added on days 4 to 5 of phase 2 (when cells started to synthesize Hb), and cells were harvested on day 12. Accumulation of mRNAs for gamma-globin, beta-globin, alpha-globin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-actin were measured by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR); induction of HbF was analyzed by high-performance liquid chromatography (HPLC) and, at cellular level, by flow cytometry. We demonstrated that mithramycin was able to up-regulate preferentially gamma-globin mRNA production and to increase HbF accumulation, the percentage of HbF-containing cells, and their HbF content. Mithramycin was more effective than hydroxyurea, being, in addition, not cytotoxic. This was shown by the lack of cytotoxicity on erythroid and myeloid in vitro primary cell cultures treated with mithramycin at concentrations effective for HbF induction. These results are of potential clinical significance because an increase of HbF alleviates the symptoms underlying beta-thalassemia and sickle cell anemia. The results of this report suggest that mithramycin and its analogs warrant further evaluation as potential therapeutic drugs.
Topics: Actins; Cell Culture Techniques; Cell Differentiation; Cell Division; Erythroid Precursor Cells; Erythropoietin; Fetal Hemoglobin; Flow Cytometry; Globins; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; Hydroxyurea; K562 Cells; Plicamycin; RNA, Messenger; Reverse Transcriptase Polymerase Chain Reaction; Thalassemia; Up-Regulation
PubMed: 12738678
DOI: 10.1182/blood-2002-10-3096 -
International Journal of Nanomedicine 2017Previous studies have shown that mithramycin A (MIT) is a promising candidate for the treatment of pancreatic carcinoma through inhibiting transcription factor Sp1....
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±4.6 nm were prepared by encapsulating MIT into methoxy poly(ethylene glycol)-block-poly(d,l-lactic--glycolic acid) (mPEG-PLGA) nanoparticles (NPs) with drug loading of 2.11%±0.51%. The in vitro release of the MIT-NPs lasted for >48 h 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 1-2 h. In vivo fluorescence imaging showed that the prepared NPs were gradually accumulated in BxPC-3 and MIA Paca-2 xenografts and retained for 168 h. 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 2 mg/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%, <0.01). Moreover, the treatment of MIT and MIT-NPs reduced the expression level of oncogene regulated by Sp1, and notably, both of them decreased the protein level of CD47. In summary, the novel formulation of MIT-NPs shows highly therapeutic efficacy against pancreatic carcinoma xenograft. In addition, MIT-NPs can downregulate CD47 expression, implying that it might play a positive role in cancer immunotherapy.
Topics: Animals; Antibiotics, Antineoplastic; Cell Line, Tumor; Drug Carriers; Drug Liberation; Female; Humans; Mice, Inbred BALB C; Microscopy, Confocal; Nanoparticles; Pancreatic Neoplasms; Particle Size; Plicamycin; Polyesters; Polyethylene Glycols; Tissue Distribution; Xenograft Model Antitumor Assays
PubMed: 28769562
DOI: 10.2147/IJN.S139507 -
Laboratory Investigation; a Journal of... May 2019Nasal natural killer/T-cell lymphoma (NNKTL) is closely associated with Epstein-Barr virus (EBV) and is characterized by poor prognosis, resulting from rapid progression...
Nasal natural killer/T-cell lymphoma (NNKTL) is closely associated with Epstein-Barr virus (EBV) and is characterized by poor prognosis, resulting from rapid progression of lesions in the affected organs. Recent data have shown that NNKTL is associated with the aberrant expression of cyclin-dependent kinase 1 (CDK1) and its downstream target survivin, but little is known about the functional roles of CDK1 and survivin in NNKTL. In the current study, we show that knockdown of the EBV-encoded oncoprotein latent membrane protein 1 (LMP1) induces downregulation of CDK1 and survivin in NNKTL cells. Immunohistochemistry detected CDK1 and survivin expression in LMP1-positive cells of NNKTL biopsy specimens. Inhibition of CDK1 and survivin in NNKTL cells with several inhibitors led to a dose-dependent decrease in cell proliferation. In addition, the Sp1 inhibitor mithramycin, which can downregulate both CDK1 and survivin, significantly suppressed the growth of established NNKTL in a murine xenograft model. Our results suggest that LMP1 upregulation of CDK1 and survivin may be essential for NNKTL progression. Furthermore, targeting CDK1 and survivin with Sp1 inhibitors such as mithramycin may be an effective approach to treat NNKTL, which is considered to be a treatment-refractory lymphoma.
Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; CDC2 Protein Kinase; Cell Line, Tumor; Female; Humans; Killer Cells, Natural; Lymphoma, T-Cell; Male; Mice, Inbred NOD; Mice, Knockout; Mice, SCID; Middle Aged; Nose Neoplasms; Plicamycin; RNA Interference; Survivin; Viral Matrix Proteins; Xenograft Model Antitumor Assays
PubMed: 30664711
DOI: 10.1038/s41374-018-0182-9 -
Arthritis Research & Therapy Apr 2012Gliostatin/thymidine phosphorylase (GLS/TP) has angiogenic and arthritogenic activities, and aberrant GLS production has been observed in the active synovial membranes...
INTRODUCTION
Gliostatin/thymidine phosphorylase (GLS/TP) has angiogenic and arthritogenic activities, and aberrant GLS production has been observed in the active synovial membranes of rheumatoid arthritis (RA) patients. The human GLS gene promoter contains at least seven consensus binding sites for the DNA binding protein Sp1. Here we examined whether Sp1 is necessary for GLS production in RA. We also studied the effects of the Sp1 inhibitor mithramycin on GLS production in RA fibroblast-like synoviocytes (FLSs).
METHODS
FLSs from RA patients were treated with specific inhibitors. The gene and protein expression of GLS were studied using the quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and an enzyme immunoassay. Intracellular signalling pathway activation was determined by western blotting analysis, a luciferase assay, a chromatin immunoprecipitation (ChIP) assay and a small interfering RNA (siRNA) transfection.
RESULTS
The luciferase and ChIP assays showed that Sp1 binding sites in the GLS promoter were essential for GLS messenger RNA (mRNA) expression. GLS production was suppressed in FLSs by siRNA against Sp1 transfection. Mithramycin decreased GLS promoter activity, mRNA and protein expression in FLSs. Tumour necrosis factor-α (TNF-α) significantly increased GLS expression in RA FLSs; this effect was reduced by pre-treatment with cycloheximide and mithramycin.
CONCLUSIONS
Pretreatment of mithramycin and Sp1 silencing resulted in a significant suppression of GLS production in TNF-α-stimulated FLSs compared to controls. GLS gene expression enhanced by TNF-α was partly mediated through Sp1. As physiological concentrations of mithramycin can regulate GLS production in RA, mithramycin is a promising candidate for anti-rheumatic therapy.
Topics: Aged; Arthritis, Rheumatoid; Cells, Cultured; Female; Fibroblasts; Gene Expression Regulation; Humans; Male; Middle Aged; Promoter Regions, Genetic; Sp1 Transcription Factor; Synovial Fluid; Thymidine Phosphorylase
PubMed: 22534375
DOI: 10.1186/ar3811 -
Genes Oct 2023The anticancer drug mithramycin (MTH), has been proposed for drug repurposing after the finding that it is a potent inducer of fetal hemoglobin (HbF) production in...
The anticancer drug mithramycin (MTH), has been proposed for drug repurposing after the finding that it is a potent inducer of fetal hemoglobin (HbF) production in erythroid precursor cells (ErPCs) from β-thalassemia patients. In this respect, previously published studies indicate that MTH is very active in inducing increased expression of γ-globin genes in erythroid cells. This is clinically relevant, as it is firmly established that HbF induction is a valuable approach for the therapy of β-thalassemia and for ameliorating the clinical parameters of sickle-cell disease (SCD). Therefore, the identification of MTH biochemical/molecular targets is of great interest. This study is inspired by recent robust evidence indicating that the expression of γ-globin genes is controlled in adult erythroid cells by different transcriptional repressors, including Oct4, MYB, BCL11A, Sp1, KLF3 and others. Among these, BCL11A is very important. In the present paper we report evidence indicating that alterations of BCL11A gene expression and biological functions occur during MTH-mediated erythroid differentiation. Our study demonstrates that one of the mechanisms of action of MTH is a down-regulation of the transcription of the BCL11A gene, while a second mechanism of action is the inhibition of the molecular interactions between the BCL11A complex and specific sequences of the γ-globin gene promoter.
Topics: Humans; gamma-Globins; beta-Thalassemia; Plicamycin; Repressor Proteins; Transcription Factors; Fetal Hemoglobin; Gene Expression; Kruppel-Like Transcription Factors
PubMed: 37895276
DOI: 10.3390/genes14101927 -
ACS Synthetic Biology May 2024spp. are "nature's antibiotic factories" that produce valuable bioactive metabolites, such as the cytotoxic anthracycline polyketides. While the anthracyclines have...
spp. are "nature's antibiotic factories" that produce valuable bioactive metabolites, such as the cytotoxic anthracycline polyketides. While the anthracyclines have hundreds of natural and chemically synthesized analogues, much of the chemical diversity stems from enzymatic modifications to the saccharide chains and, to a lesser extent, from alterations to the core scaffold. Previous work has resulted in the generation of a BioBricks synthetic biology toolbox in M1152Δ that could produce aklavinone, 9--aklavinone, auramycinone, and nogalamycinone. In this work, we extended the platform to generate oxidatively modified analogues two crucial strategies. (i) We swapped the ketoreductase and first-ring cyclase enzymes for the aromatase cyclase from the mithramycin biosynthetic pathway in our polyketide synthase (PKS) cassettes to generate 2-hydroxylated analogues. (ii) Next, we engineered several multioxygenase cassettes to catalyze 11-hydroxylation, 1-hydroxylation, 10-hydroxylation, 10-decarboxylation, and 4-hydroxyl regioisomerization. We also developed improved plasmid vectors and M1152Δ expression hosts to produce anthracyclinones. This work sets the stage for the combinatorial biosynthesis of bespoke anthracyclines using recombinant hosts.
Topics: Polyketide Synthases; Anthracyclines; Streptomyces coelicolor; Streptomyces; Biosynthetic Pathways; Hydroxylation; Anti-Bacterial Agents
PubMed: 38662967
DOI: 10.1021/acssynbio.4c00043 -
Identification of potential therapeutic drugs for huntington's disease using Caenorhabditis elegans.PloS One Jun 2007The prolonged time course of Huntington's disease (HD) neurodegeneration increases both the time and cost of testing potential therapeutic compounds in mammalian models....
BACKGROUND
The prolonged time course of Huntington's disease (HD) neurodegeneration increases both the time and cost of testing potential therapeutic compounds in mammalian models. An alternative is to initially assess the efficacy of compounds in invertebrate models, reducing time of testing from months to days.
METHODOLOGY/PRINCIPAL FINDINGS
We screened candidate therapeutic compounds that were identified previously in cell culture/animal studies in a C. elegans HD model and found that two FDA approved drugs, lithium chloride and mithramycin, independently and in combination suppressed HD neurotoxicity. Aging is a critical contributor to late onset neurodegenerative diseases. Using a genetic strategy and a novel assay, we demonstrate that lithium chloride and mithramycin remain neuroprotective independent of activity of the forkhead transcription factor DAF-16, which mediates the effects of the insulin-like signaling pathway on aging.
CONCLUSIONS/SIGNIFICANCE
These results suggest that pathways involved in polyglutamine-induced degeneration are distinct from specific aging pathways. The assays presented here will be useful for rapid and inexpensive testing of other potential HD drugs and elucidating pathways of drug action. Additionally, the neuroprotection conferred by lithium chloride and mithramycin suggests that these drugs may be useful for polyglutamine disease therapy.
Topics: Aging; Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Disease Models, Animal; Drug Combinations; Forkhead Transcription Factors; Huntington Disease; Lithium Chloride; Longevity; Neuroprotective Agents; Peptides; Plicamycin; Transcription Factors
PubMed: 17551584
DOI: 10.1371/journal.pone.0000504 -
Microbial Biotechnology Dec 2022Coelimycin P1 and argimycins P are two types of polyketide alkaloids produced by Streptomyces coelicolor and Streptomyces argillaceus, respectively. Their biosynthesis...
Coelimycin P1 and argimycins P are two types of polyketide alkaloids produced by Streptomyces coelicolor and Streptomyces argillaceus, respectively. Their biosynthesis pathways share some early steps that render very similar aminated polyketide chains, diverging the pathways afterwards. By expressing the putative isomerase cpkE and/or the putative epoxidase/dehydrogenase cpkD from the coelimycin P1 gene cluster into S. argillaceus wild type and in argimycin mutant strains, five novel hybrid argimycins were generated. Chemical characterization of those compounds revealed that four of them show unprecedented scaffolds (quinolizidine and pyranopyridine) never found before in the argimycin family of compounds. One of these compounds (argimycin DM104) shows improved antibiotic activity. Noticeable, biosynthesis of these quinolizidine argimycins results from a hybrid pathway created by combining enzymes from two different pathways, which utilizes an aminated polyketide chain as precursor instead of lysine as it occurs for other quinolizidines.
Topics: Plicamycin; Streptomyces; Multigene Family; Anti-Bacterial Agents
PubMed: 36346129
DOI: 10.1111/1751-7915.14167 -
Scientific Reports Oct 2019The pivotal role of cancer initiating stem cells (CSCs) in tumor initiation, growth, metastasis and drug resistance has led to the postulation of a 'total cancer...
The pivotal role of cancer initiating stem cells (CSCs) in tumor initiation, growth, metastasis and drug resistance has led to the postulation of a 'total cancer therapy' paradigm, which involves targeting both cancer cells and CSCs for effective therapy. However, the progress in identifying drugs for total cancer therapy has been limited. Herein, we show for the first time that mithramycin A (Mit-A) can successfully inhibit CSC proliferation, in addition to inhibiting bulk cancer cells in a model of colorectal cancer (CRC), the second leading cause of death among men and women in the United States. To this end, a polymeric nanofiber scaffold culture system was established to develop 3D tumor organoids (tumoroids) from CRC cell lines such as HT29, HCT116, KM12, CT26 and MC38 as well as ex vivo mouse tumors. These tumoroids possessed increased expression of CSC markers and transcription factors, expanded the number of CSCs in culture and increased CSC functional properties measured by aldehyde dehydrogenase activity. Screening of an NCI library of FDA approved drugs led to the identification of Mit-A as a potential total cancer therapy drug. In both sphere and tumoroid culture, Mit-A inhibits cancer growth by reducing the expression of cancer stemness markers. In addition, Mit-A inhibits the expression of SP1, a previously known target in CRCs. Moreover, Mit-A significantly reduces growth of tumoroids in ex vivo cultures and CRC tumor growth in vivo. Finally, a dose-dependent treatment on CRC cells indicate that Mit-A significantly induces the cell death and PARP-cleavage of both CSC and non-CSC cells. Taken together the results of these in vitro, ex vivo and in vivo studies lead to the inference that Mit-A is a promising drug candidate for total cancer therapy of CRCs.
Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; HCT116 Cells; HT29 Cells; Humans; Mice, Inbred C57BL; Neoplastic Stem Cells; Plicamycin
PubMed: 31645574
DOI: 10.1038/s41598-019-50917-3