RESUMO
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.
Assuntos
Plicamicina , Streptomyces , Plicamicina/química , Plicamicina/metabolismo , Família Multigênica , Antibacterianos/metabolismoRESUMO
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 (Cbfß), 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.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Plicamicina/farmacologia , Proteína Proto-Oncogênica c-fli-1/química , Regulação Alostérica/efeitos dos fármacos , Antibióticos Antineoplásicos/química , Sítios de Ligação , Subunidade alfa 1 de Fator de Ligação ao Core/química , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade beta de Fator de Ligação ao Core/química , Subunidade beta de Fator de Ligação ao Core/metabolismo , Humanos , Simulação de Acoplamento Molecular , Plicamicina/química , Ligação Proteica , Proteína Proto-Oncogênica c-fli-1/metabolismo , Regulador Transcricional ERG/química , Regulador Transcricional ERG/metabolismoRESUMO
Mithramycin A (MTM) inhibits the oncogenic transcription factor EWS-FLI1 in Ewing sarcoma, but poor pharmacokinetics (PK) and toxicity limit its clinical use. To address this limitation, we report an efficient MTM 2'-oxime (MTMox) conjugation strategy for rapid MTM diversification. Comparative cytotoxicity assays of 41 MTMox analogues using E-twenty-six (ETS) fusion-dependent and ETS fusion-independent cancer cell lines revealed improved ETS fusion-independent/dependent selectivity indices for select 2'-conjugated analogues as compared to MTM. Luciferase-based reporter assays demonstrated target engagement at low nM concentrations, and molecular assays revealed that analogues inhibit the transcriptional activity of EWS-FLI1. These in vitro screens identified MTMox32E (a Phe-Trp dipeptide-based 2'-conjugate) for in vivo testing. Relative to MTM, MTMox32E displayed an 11-fold increase in plasma exposure and improved efficacy in an Ewing sarcoma xenograft. Importantly, these studies are the first to point to simple C3 aliphatic side-chain modification of MTM as an effective strategy to improve PK.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Antibióticos Antineoplásicos/farmacocinética , Neoplasias Ósseas/tratamento farmacológico , Oximas/química , Plicamicina/química , Sarcoma de Ewing/tratamento farmacológico , Animais , Antibióticos Antineoplásicos/química , Apoptose , Neoplasias Ósseas/patologia , Proliferação de Células , Feminino , Humanos , Camundongos , Camundongos SCID , Sarcoma de Ewing/patologia , Distribuição Tecidual , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Betacoronavirus/metabolismo , Ligantes , Peptidil Dipeptidase A/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Enzima de Conversão de Angiotensina 2 , Sítios de Ligação , COVID-19 , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Diosmina/química , Diosmina/metabolismo , Humanos , Simulação de Dinâmica Molecular , Pandemias , Peptidil Dipeptidase A/química , Plicamicina/química , Plicamicina/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Ligação Proteica , Domínios Proteicos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/química , TermodinâmicaRESUMO
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 <20%. MTMSA-Trp was stable for 30 days at -80°C and for at least three freeze-thaw cycles. Methanol (-80°C) extraction afforded 92% of MTMSA-Trp from plasma. Calibration curves for MTM and analogs were also linear from ≤5 to 100 ng/mL. This versatile method was used to quantitate MTM analogs in plasma samples collected during preclinical pharmacokinetic studies.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Plicamicina/análogos & derivados , Plicamicina/sangue , Animais , Antibióticos Antineoplásicos , Estabilidade de Medicamentos , Feminino , Limite de Detecção , Modelos Lineares , Camundongos , Plicamicina/química , Plicamicina/farmacocinética , Reprodutibilidade dos TestesRESUMO
The experimental phase determination of crystal structures of nucleic acids and nucleic acid-ligand complexes would benefit from a facile method. Even for double-stranded DNA, software-generated models are generally insufficiently accurate to serve as molecular replacement search models, necessitating experimental phasing. Here, it is demonstrated that Zn2+ ions coordinated to the N7 atom of guanine bases generate sufficient anomalous signal for single-wavelength anomalous diffraction (SAD) phasing of DNA crystal structures. Using zinc SAD, three crystal structures of double-stranded DNA oligomers, 5'-AGGGATCCCT-3', 5'-GGGATCCC-3' and 5'-GAGGCCTC-3', were determined. By determining the crystal structure of one of these oligomers, GAGGCCTC, in the presence of Mg2+ instead of Zn2+, it was demonstrated that Zn2+ is not structurally perturbing. These structures allowed the analysis of structural changes in the DNA on the binding of analogues of the natural product mithramycin to two of these oligomers, AGGGATCCCT and GAGGCCTC. Zinc SAD may become a routine approach for determining the crystal structures of nucleic acids and their complexes with small molecules.
Assuntos
Cristalografia por Raios X/métodos , DNA/química , Guanina/química , Oligonucleotídeos/química , Zinco/química , Íons , Plicamicina/químicaRESUMO
A new antibiotic complex of six aureolic acids was isolated from the marine sediment-associated strain Streptomyces sp. KMM 9048. Four of the compounds (3-6) were found to be similar but not identical to the known chromomycins A2, A3, demethyl chromomycin A3 and A4. The two remaining.compounds; A2â1 (1) and A3â1 (2), were established as novel chromomycin analogs, which did not contain sugar B. Spectroscopic methods including ID and 2D NMR, and HRMS and MS/MS were applied for structure elucidation. Compounds 1-5 showed strong antimicrobial activity against Gram-positive indicatory bacteria Enterococcusfaecium, Staphylococcus aureus, S. epidernzidis, and Bacillus subtilis. Antitumor assay indicated that all tested compounds, in different manners, inhibited colony formation of RPMI-7951 and SK-Mel-28 cancer cells. This is the first study reporting the inhibitory effects of chromomycin analogs 1-5 on the colony formation of the investigated cancer cell lines. Compound 3, in a concentration of 5 nM, inhibited colony formation of RPMI-7951 and SK-Mel-28 cells by 82 % and 72 %, respectively. Our finding indicated that, of the compounds tested, 3 and 4 are promising anticancer and antimicrobial agents.
Assuntos
Antibacterianos/farmacologia , Antineoplásicos/farmacocinética , Sedimentos Geológicos/microbiologia , Plicamicina/farmacologia , Streptomyces/química , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Linhagem Celular Tumoral , Cromomicinas/química , Cromomicinas/isolamento & purificação , Cromomicinas/farmacologia , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/crescimento & desenvolvimento , Humanos , Testes de Sensibilidade Microbiana , Plicamicina/química , Plicamicina/isolamento & purificação , Streptomyces/genética , Streptomyces/isolamento & purificação , Streptomyces/metabolismo , Espectrometria de Massas em TandemRESUMO
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.
Assuntos
DNA/química , Plicamicina/química , Proteína Proto-Oncogênica c-fli-1/química , Sequência de Bases , DNA/metabolismo , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/metabolismo , Plicamicina/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteína Proto-Oncogênica c-fli-1/metabolismo , Relação Estrutura-AtividadeRESUMO
Targeting autophagic pathways might play a critical role in designing novel chemotherapeutic approaches in the treatment of human cancers, and the prevention of tumor-derived chemoresistance. Marine compounds were found to decrease tumor cell growth in vitro and in vivo. Some of them were shown to induce autophagic flux in tumor cells. In this study, we observed that the selected marine life-derived compounds (Chromomycin A2, Psammaplin A, and Ilimaquinone) induce expression of several autophagic signaling intermediates in human squamous cell carcinoma, glioblastoma, and colorectal carcinoma cells in vitro through a transcriptional regulation by tumor protein (TP)-p53 family members. These conclusions were supported by specific qPCR expression analysis, luciferase reporter promoter assay, and chromatin immunoprecipitation of promoter sequences bound to the TP53 family proteins, and silencing of the TP53 members in tumor cells.
Assuntos
Antineoplásicos/farmacologia , Organismos Aquáticos/química , Autofagia/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Imunoprecipitação da Cromatina , Dissulfetos/química , Dissulfetos/isolamento & purificação , Dissulfetos/farmacologia , Humanos , Plicamicina/análogos & derivados , Plicamicina/química , Plicamicina/isolamento & purificação , Plicamicina/farmacologia , Quinonas/química , Quinonas/isolamento & purificação , Quinonas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Sesquiterpenos/química , Sesquiterpenos/isolamento & purificação , Sesquiterpenos/farmacologia , Proteína Supressora de Tumor p53/genética , Tirosina/análogos & derivados , Tirosina/química , Tirosina/isolamento & purificação , Tirosina/farmacologiaRESUMO
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 (Me(2+))-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.
Assuntos
Antibióticos Antineoplásicos/química , DNA/química , Plicamicina/química , DimerizaçãoRESUMO
Demycarosyl-3D-ß-D-digitoxosyl-mithramycin SK (DIG-MSK) is a recently isolated analogue of mithramycin A (MTA) that showed differences with MTA in the DNA binding strength and selectivity. These differences correlated with a better therapeutic index and less toxicity in animal studies. Herein, we show that DIG-MSK displays a potent anti-tumor activity against different types of cancer cell lines, ovarian tumor cells being particularly sensitive to this drug. Of relevance, DIG-MSK exerts low toxicity on fibroblasts and peripheral blood mononuclear cells, this toxicity being significantly lower than that of MTA. In correlation with its antitumor activity, DIG-MSK strongly inhibited Sp1-mediated transcription and endogenous Sp1 mRNA expression, which correlated with the inhibition of the expression of key Sp1-regulated genes involved in tumorigenesis, including VEGFA, BCL2L1 (Bcl-XL), hTERT, BRCA2, MYC and SRC in several ovarian cells. Significantly, DIG-MSK was a stronger inhibitor of VEGFA expression than MTA. Accordingly, DIG-MSK also exhibited potent anti-angiogenic activity on microvascular endothelial cells. Likewise, it significantly inhibited the gene expression of VEGFR1, VEGFR2, FGFR, PDGFB and PDGFRA and, additionally, it induced the expression of the anti-angiogenic factors angiostatin and tunstatin. These effects correlated with a pro-apoptotic effect on proliferating microvascular endothelial cells and the inhibition of the formation of endothelial capillary structures. Overall, the pleiotropic activity of DIG-MSK in inhibiting key oncogenic and angiogenic pathways, together with its low toxicity profile, highlight the therapeutic potential of this new drug.
Assuntos
Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Plicamicina/análogos & derivados , Indutores da Angiogênese/metabolismo , Antineoplásicos/química , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Plicamicina/química , Plicamicina/toxicidade , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Mithramycin is an antitumor compound of the aureolic acid family produced by Streptomyces argillaceus. It has been used to treat several types of cancer including testicular carcinoma, chronic and acute myeloid leukemia as well as hypercalcemias and Paget's disease. Although the use of mithramycin in humans has been limited because its side effects, in recent years a renewed interest has arisen since new uses and activities have been ascribed to it. Chemically, mithramycin is characterized by a tricyclic aglycone bearing two aliphatic side chains attached at C3 and C7, and disaccharide and trisaccharide units attached at positions 2 and 6, respectively. The mithramycin gene cluster has been characterized. This has allowed for the development of several mithramycin analogs ("mithralogs") by combinatorial biosynthesis and/or biocatalysis. The combinatorial biosynthesis strategies include gene inactivation and/or the use of sugar biosynthesis plasmids for sugar modification. In addition, lipase-based biocatalysis enabled selective modifications of the hydroxyl groups, providing further mithramycin analogs. As a result, new mithramycin analogs with higher antitumor activity and/or less toxicity have been generated. One, demycarosyl-3D-ß-D-digitoxosyl-mithramycin SK (EC-8042), is being tested in regulatory preclinical assays, representing an opportunity to open the therapeutic window of this promising molecular scaffold.
Assuntos
Antibióticos Antineoplásicos/química , Plicamicina/análogos & derivados , Animais , Antibióticos Antineoplásicos/uso terapêutico , Biocatálise , Técnicas de Química Combinatória , Humanos , Plicamicina/química , Plicamicina/uso terapêutico , Streptomyces/químicaRESUMO
Mithramycin (MTR), an aureolic acid group of antitumor antibiotic is used for the treatment of several types of tumors. We have reported here the association of MTR with an essential micronutrient, manganese (Mn(2+)). Spectroscopic methods have been used to characterize and understand the kinetics and mechanism of complex formation between them. MTR forms a single type of complex with Mn(2+) in the mole ratio of 2:1 [MTR: Mn(2+)] via a two step kinetic process. Circular dichroism (CD) spectroscopic study indicates that the complex [(MTR)2 Mn(2+)] has a right-handed twist conformation similar in structure with the complexes reported for Mg(2+) and Zn(2+). This conformation allows binding via minor groove of DNA with (G, C) base preference during the interaction with double-stranded B-DNA. Using absorbance, fluorescence, and CD spectroscopy we have shown that [(MTR)2 Mn(2+)] complex binds to double-stranded DNA with an apparent dissociation constant of 32 µM and binding site size of 0.2 (drug/nucleotide). It binds to chicken liver chromatin with apparent dissociation constant value 298 µM. Presence of histone proteins in chromatin inhibits the accessibility of the complex for chromosomal DNA. We have also shown that MTR binds to Mn(2+) containing metalloenzyme manganese superoxide dismutase from Escherichia coli.
Assuntos
Antibióticos Antineoplásicos/química , Manganês/química , Plicamicina/química , Animais , Galinhas , Cromatina/química , DNA/química , Escherichia coli , Proteínas de Escherichia coli/química , Cinética , Superóxido Dismutase/química , TermodinâmicaRESUMO
Pathogenic bacteria that are resistant to ß-lactam antibiotics mostly utilize serine ß-lactamases to degrade the antibiotics. Current studies have shown that different subclasses of metallo ß-lactamases (E[MBL]) are involved in the defense mechanism of drug resistant bacteria. Here we report that the Zn(2+) containing subclass B1 E[MBL] from Bacillus cereus binds to a naturally occurring anti-cancer drug mithramycin (MTR). Spectroscopic (CD and fluorescence) and isothermal titration calorimetry studies show that MTR forms a high affinity complex with the Zn(2+) ion containing E[MBL]. Abolished interaction of MTR with apo E[MBL] suggests that the formation of this high affinity complex occurs due to the potential of MTR to bind bivalent metal ions like Zn(2+). Furthermore, CD spectroscopy, dynamic light scattering and differential scanning calorimetry studies indicate that the strong association with sub-micromolar dissociation constant leads to an alteration in the enzyme conformation at both secondary and tertiary structural levels. The enzyme activity decreases as a consequence to this conformational disruption arising from the formation of a ternary complex involving MTR, catalytic Zn(2+) and the enzyme. Our results suggest that the naturally occurring antibiotic MTR, a generic drug, has the potential as an E[MBL] inhibitor.
Assuntos
Antibióticos Antineoplásicos/química , Bacillus cereus/enzimologia , Proteínas de Bactérias/química , Plicamicina/química , Zinco/química , beta-Lactamases/químicaRESUMO
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 â¼55% 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 (2.1×50 mm, 1.7 µm) and detected using electrospray ionization operated in the negative mode at mass transitions m/z 1083.5â268.9 and 1181.5â269.0, respectively, on an AB Sciex QTrap 5500. The assay range was 0.5-500 ng/mL and proved to be linear (r(2)>0.996), accurate (≤10% deviation), and precise (CV<15%). Mithramycin was stable in plasma at room temperature for 24 h, as well as through three freeze-thaw cycles. This method was subsequently used to quantitate mithramycin plasma concentrations from patients enrolled on two clinical trials at the NCI.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Plicamicina/sangue , Espectrometria de Massas em Tandem/métodos , Proteínas Sanguíneas/metabolismo , Humanos , Modelos Lineares , Plicamicina/química , Plicamicina/farmacocinética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Extração em Fase SólidaRESUMO
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.
Assuntos
Antibióticos Antineoplásicos , DNA de Neoplasias/química , Neoplasias/tratamento farmacológico , Plicamicina , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacologia , Linhagem Celular Tumoral , DNA de Neoplasias/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Humanos , Neoplasias/química , Neoplasias/metabolismo , Plicamicina/análogos & derivados , Plicamicina/química , Plicamicina/farmacologiaRESUMO
Mithramycin (Mith) forms a drug-metal complex with a 2:1 stoichiometry by chelation with a Ni(II) ion, which was determined using circular dichroism spectroscopy. Mith exhibits an increased affinity (~55 fold) for Ni(II) in the presence of DNA compared to the absence of DNA, suggesting that DNA acts as an effective template to facilitate chelation. Also, we characterized the DNA-acting properties of a Ni(II) derivative of Mith. Kinetic analysis using surface plasmon resonance and UV melting studies revealed that Ni(II)(Mith)(2) binds to duplex DNA with a higher affinity compared to Mg(II)(Mith)(2). The thermodynamic parameters revealed a higher free energy of formation for duplex DNA in the presence of Ni(II)(Mith)(2) compared to duplex DNA in the presence of Mg(II)(Mith)(2). The results of a DNA-break assay indicated that Ni(II)(Mith)(2) is capable of promoting one-strand cleavage of plasmid DNA in the presence of hydrogen peroxide; the DNA cleavage rate of Ni(II)(Mith)(2) was calculated to be 4.1 × 10(-4) s(-1). In cell-based experiments, Ni(II)(Mith)(2) exhibited a more efficient reduction of c-myc and increased cytotoxicity compared to Mith alone because of its increased DNA-binding and cleavage activity. The evidence obtained in this study suggests that the biological effects of Ni(II)(Mith)(2) require further investigation in the future.
Assuntos
Antibióticos Antineoplásicos/química , Quelantes/química , Complexos de Coordenação/química , Níquel/química , Plicamicina/química , Tiazolidinedionas/química , Antibióticos Antineoplásicos/farmacologia , Quelantes/farmacologia , Complexos de Coordenação/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Expressão Gênica/efeitos dos fármacos , Células HeLa , Células Hep G2 , Humanos , Concentração Inibidora 50 , Sequências Repetidas Invertidas , Ferro/química , Plasmídeos/química , Plicamicina/farmacologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ressonância de Plasmônio de Superfície , Temperatura de TransiçãoRESUMO
A marine-derived actinomycete (Streptomyces sp. WBF16) exhibiting antitumor activities was investigated. The strain was identified using morphological, biochemical and genetic techniques. 16S rDNA sequence of the isolate indicated that it was most closely related to Streptomyces coelicolor A3 (2). Furthermore, a new aureolic acid (Chromomycin B, 1), along with Chromomycin A(2) (2) and Chromomycin A(3) (3) were isolated from its secondary metabolites. Their structures were determined by chemical and spectroscopic methods including 1D, 2D NMR and HRMS. Compounds 1-3 showed strong cytotoxicity against SGC7901, HepG2, A549, HCT116 and COC1 and HUVEC.
Assuntos
Cromomicinas/química , Cromomicinas/farmacologia , Plicamicina/química , Plicamicina/farmacologia , Streptomyces/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Cromomicinas/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Células HCT116 , Células Hep G2 , Células Endoteliais da Veia Umbilical Humana , Humanos , Espectroscopia de Ressonância Magnética/métodos , Biologia Marinha , Plicamicina/metabolismo , Streptomyces/química , Streptomyces/classificaçãoRESUMO
This report shows that the DNA-binding drug, mithramycin, can be efficiently encapsulated in polymeric micelles (PM-MTH), based on Pluronic(®) block copolymers, by a new microfluidic approach. The effect of different production parameters has been investigated for their effect on PM-MTH characteristics. The compared analysis of PM-MTH produced by microfluidic and conventional bulk mixing procedures revealed that microfluidics provides a useful platform for the production of PM-MTH with improved controllability, reproducibility, smaller size, and polydispersity. Finally, an investigation of the effects of PM-MTH, produced by microfluidic and conventional bulk mixing procedures, on the erythroid differentiation of both human erythroleukemia and human erythroid precursor cells is reported. It is demonstrated that PM-MTH exhibited a slightly lower toxicity and more pronounced differentiative activity when compared to the free drug. In addition, PM-MTH were able to upregulate preferentially γ-globin messenger ribonucleic acid production and to increase fetal hemoglobin (HbF) accumulation, the percentage of HbF-containing cells, and their HbF content without stimulating α-globin gene expression, which is responsible for the clinical symptoms of ß-thalassemia. These results represent an important first step toward a potential clinical application, since an increase in HbF could alleviate the symptoms underlying ß-thalassemia and sickle cell anemia. In conclusion, this report suggests that PM-MTH produced by microfluidic approach warrants further evaluation as a potential therapeutic protocol for ß-thalassemia.
Assuntos
Química Farmacêutica/métodos , Micelas , Microfluídica , Plicamicina/análogos & derivados , Polímeros , Talassemia beta/tratamento farmacológico , Análise de Variância , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Eritrócitos/efeitos dos fármacos , Eritrócitos/patologia , Células Precursoras Eritroides , Humanos , Células K562/efeitos dos fármacos , Dispositivos Lab-On-A-Chip , Plicamicina/administração & dosagem , Plicamicina/química , Reprodutibilidade dos TestesRESUMO
Here we have examined the association of an aureolic acid antibiotic, chromomycin A3 (CHR), with Cu(2+). CHR forms a high affinity 2:1 (CHR:Cu(2+)) complex with dissociation constant of 0.08 × 10(-10) M(2) at 25°C, pH 8.0. The affinity of CHR for Cu(2+) is higher than those for Mg(2+) and Zn(2+) reported earlier from our laboratory. CHR binds preferentially to Cu(2+) in presence of equimolar amount of Zn(2+). Complex formation between CHR and Cu(2+) is an entropy driven endothermic process. Difference between calorimetric and van't Hoff enthalpies indicate the presence of multiple equilibria, supported from biphasic nature of the kinetics of association. Circular dichroism spectroscopy show that [(CHR)(2):Cu(2+)] complex assumes a structure different from either of the Mg(2+) and Zn(2+) complex reported earlier. Both [(CHR)(2):Mg(2+)] and [(CHR)(2):Zn(2+)] complexes are known to bind DNA. In contrast, [(CHR)(2):Cu(2+)] complex does not interact with double helical DNA, verified by means of Isothermal Titration Calorimetry of its association with calf thymus DNA and the double stranded decamer (5'-CCGGCGCCGG-3'). In order to interact with double helical DNA, the (antibiotic)(2) : metal (Mg(2+) and Zn(2+)) complexes require a isohelical conformation. Nuclear Magnetic Resonance spectroscopy shows that the Cu(2+) complex adopts a distorted octahedral structure, which cannot assume the required conformation to bind to the DNA. This report demonstrates the negative effect of a bivalent metal upon the DNA binding property of CHR, which otherwise binds to DNA in presence of metals like Mg(2+) and Zn(2+). The results also indicate that CHR has a potential for chelation therapy in Cu(2+) accumulation diseases. However cytotoxicity of the antibiotic might restrict the use.