Quinoxaline Antibiotics Research Articles

Europium Nanoparticle-Based Lateral Flow Strip Biosensors for the Detection of Quinoxaline Antibiotics and Their Main Metabolites in Fish Feeds and Tissues.

Olaquindox (OLA) and quinocetone (QCT) have been prohibited in aquatic products due to their significant toxicity and side effects. In this study, rapid and visual europium nanoparticle (EuNP)-based lateral flow strip biosensors (LFSBs) were developed for the simultaneous quantitative detection of OLA, QCT, and 3-methyl-quinoxaline-2-carboxylic acid (MQCA) in fish feed and tissue. The EuNP-LFSBs enabled sensitive detection for OLA, QCT, and MQCA with a limit of detection of 0.067, 0.017, and 0.099 ng/mL (R2 ≥ 0.9776) within 10 min. The average recovery of the EuNP-LFSBs was 95.13%, and relative standard deviations were below 9.38%. The method was verified by high-performance liquid chromatography (HPLC), and the test results were consistent. Therefore, the proposed LFSBs serve as a powerful tool to monitor quinoxalines in fish feeds and their residues in fish tissues.

Multi-class, multi-residue determination of 132 veterinary drugs in milk by magnetic solid-phase extraction based on magnetic hypercrosslinked polystyrene prior to their determination by high-performance liquid chromatography – tandem mass spectrometry

A quantitative multi-class multi-residue analytical method was developed for the determination of veterinary drugs in milk by high-performance liquid chromatography – tandem mass spectrometry (HPLC-MS/MS). A total of 132 veterinary drugs investigated belonged to almost 15 classes including sulfonamides, β-lactams, tetracyclines, quinolones, macrolides, nitrofurans, nitroimidazoles, phenicols, lincosamides, pleuromutilins, macrocyclic lactones, quinoxaline antibiotics, benzimidazoles, anthelmintics, coccidiostats and some others. A magnetic solid-phase extraction procedure was developed using magnetic hypercrosslinked polystyrene (HCP/Fe3O4) for the sample preparation prior to HPLC-MS/MS without deproteinization step. The results indicated recoveries of 85–107% for 14 sulfonamides, 85–120% for 13 β-lactams, 89–115% for 4 tetracyclines, 82–119% for 14 quinolones, 82–115% for 8 macrolides, 97–109% for 4 nitrofurans, 84–115% for 10 nitroimidazoles, 89–114% for 3 phenicols, 86–111% for 3 lincosamides, 97–102% for 2 pleuromutilins, 72–88% for 4 macrocyclic lactones, 87–104% for 4 quinoxaline antibiotics, 76–119% for 21 benzimidazoles, 79–115% for 12 anthelmintics, 81–118% for 12 coccidiostats and 75–119 % for 5 unclassified drugs, with relative standard deviations (RSDs) of less than 20%, and the LOQs ranged from 0.05 to 1 μg kg−1. This methodology was then applied to field-collected real milk samples and trace levels of some veterinary drugs were detected.

Encapsulation of echinomycin in cyclodextrin inclusion complexes into liposomes: in vitro anti-proliferative and anti-invasive activity in glioblastoma.

Echinomycin, a DNA bis-intercalator peptide, belongs to the family of quinoxaline antibiotics. Echinomycin exhibits potent antitumor and antimicrobial activity. However, it is highly water insoluble and suffers from low bioavailability and unwanted side effects. Therefore, developing new formulations and delivery systems that can enhance echinomycin solubility and therapeutic potency is needed for further clinical application. In this study, echinomycin has been complexed into the hydrophobic cavity of γ-cyclodextrin (γCD) then encapsulated into PEGylated liposomes. The anti-proliferative and anti-invasive effect has been evaluated against U-87 MG glioblastoma cells. Echinomycin-in-γCD inclusion complexes have been characterized by phase solubility assay, TLC, and 1H-NMR. The echinomycin-in-γCD inclusion complexes have been loaded into liposomes using a thin film hydration method to end up with echinomycin-in-γCD-in-liposomes. Drug-loaded liposomes were able to inhibit cell proliferation with IC50 of 1.0 nM. Moreover, echinomycin-in-γCD-in-liposomes were found to inhibit the invasion of U-87 MG cells using the spheroid gel invasion assay. In conclusion, the current work describes for the first time γCD-echinomycin complexes and their encapsulation into PEGylated liposomes.

Abstract 1727: Quinomycin A inhibits pancreatic cancer growth and affects stem cell viability by inhibiting oncogenic YAP1 function

Abstract Background: Pancreatic cancer (PCa) remains a leading cause of death in the United States. Cancer stem cells (CSC) are responsible for tumor behavior, and therapeutic resistance. Quinomycin (Qui) is an orally administered quinoxaline antibiotic that bifunctionally intercalates with double stranded DNA. As a first step for repurposing this drug, we determined whether Qui affects PCa growth, and if so whether this is by suppressing stem cells. Method: Growth and apoptosis of PCa lines (MiaPaCa-2, PanC-1, BxPC-3) and normal ductal epithelial cells (HPNE) was measured by hexosaminidase and clonogenicity, and caspase 3/7 assays, respectively. Pancosphere formation and FACS sorting were used to identify effects on stem cells. For in vivo, MiaPaCa-2 xenografts were developed in the flanks of nude mice. Immunohistochemistry was performed for stem cell markers and Hippo signaling proteins. Results: Qui demonstrated a dose- and time-dependent inhibition of proliferation and colony formation in all three PCa lines but not HPNE cells. Qui induced PCa cells to undergo apoptosis. It also significantly reduced the number and size of pancospheres, and flow cytometry and western blot analyses confirmed that Qui suppressed PCa stem cell marker proteins DCLK1, CD44, CD24 and EPCAM. We next determined whether Hippo signaling pathway is affected. For this, we tested the effect of Qui on Hippo signaling pathway, which is an active pathway in CSCs including in PCa. The key effector protein, YAP1 has been shown to be oncogenic in many cancer types. In the canonical Hippo signaling pathway, YAP1 function is inhibited. When YAP1 is phosphorylated at Ser127 by the action of upstream Mst1/2 and Lats1/2 kinases, it is sequestered in the cytoplasm, and therefore no induction of downstream gene expression. Qui significantly decreased the phosphorylation oncogenic YAP1. Furthermore, Qui inhibited the expression of YAP interacting proteins TEAD1, TEAD2, and TEAD4. On the other hand, ectopic expression of the TEAD1 partially rescued the cells from Qui-mediated growth suppression. To determine the effect of Qui on tumor growth in vivo, mice carrying MiaPaCa-2 tumor xenografts were administered the compound intraperitoneally (20 μg/kg bw) every day for 21 days. Qui treatment significantly suppressed tumor xenograft growth, with notably lower tumor volume and weight. Western blot and immunohistochemistry analyses demonstrated significant inhibition in the expression of CSC marker proteins, oncogenic YAP1 phosphorylation and YAP1 interacting proteins TEAD1 in the Qui-treated xenograft tissues. Conclusion: Together, these data suggest that Qui suppresses PCa growth that targets that targets stem cells by inhibiting oncogenic YAP1 in Hippo signaling pathway. Citation Format: Dharmalingam Subramaniam, Sivapriya Ponnurangam, Gaurav Fnu, Prabhu Ramamoorthy, Animesh Dhar, Ossama W. Tawfik, Shahid Umar, Scott J. Weir, Roy A. Jensen, Arun Balakrishnan, Shrikant Anant. Quinomycin A inhibits pancreatic cancer growth and affects stem cell viability by inhibiting oncogenic YAP1 function. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1727.

The Benzyl Moiety in a Quinoxaline-Based Scaffold Acts as a DNA Intercalation Switch.

Quinoxaline antibiotics intercalate dsDNA and exhibit antitumor properties. However, they are difficult to synthesize and their structural complexity impedes a clear mechanistic understanding of DNA binding. Therefore design and synthesis of minimal-intercalators, using only part of the antibiotic scaffold so as to retain the key DNA-binding property, is extremely important. Reported is a unique example of a monomeric quinoxaline derivative of a 6-nitroquinoxaline-2,3-diamine scaffold which binds dsDNA by two different modes. While benzyl derivatives bound DNA in a sequential fashion, with intercalation as the second event, nonbenzyl derivatives showed only the first binding event. The benzyl intercalation switch provides important insights about molecular architecture which control specific DNA binding modes and would be useful in designing functionally important monomeric quinoxaline DNA binders and benchmarking molecular simulations.

Abstract 4214: Quinomycin A affects pancreatic cancer stem cells in part through suppression of notch signaling pathway

Abstract Background: Pancreatic cancer (PCa) remains a leading cause of death in the United States. Cancer stem cells (CSC) are responsible for tumor behavior, and therapeutic resistance. Quinomycin (Qui) is an orally administered quinoxaline antibiotic that bifunctionally intercalates with double stranded DNA. As a first step for repurposing this drug, we determined whether Qui affects PCa growth, and if so whether this is by suppressing stem cells. Method: Growth and apoptosis of PCa lines (MiaPaCa-2, PanC-1, BxPC-3) and normal ductal epithelial cells (HPNE) was measured by hexosaminidase and clonogenicity, and caspase 3/7 assays, respectively. Pancosphere formation and FACS sorting were used for identifying effects on stem cells. For in vivo effects, MiaPaCa-2 xenografts were developed in the flanks of nude mice. Immunohistochemistry was performed for stem cell markers and Notch signaling proteins. Results: Qui treatment resulted in a dose- and time-dependent inhibition of proliferation and colony formation in all three PCa lines but not HPNE cells. Qui also induced PCa cells to undergo apoptosis. Qui also significantly reduced the number and size of pancospheres, suggesting effects on stem cells. In addition, flow cytometry and western blot analyses showed that Qui suppressed PCa stem cell marker proteins Doublecortin Calmodulin-like kinase 1 (DCLK1), CD44 and CD24. We next determined whether Qui affects the Notch signaling pathway, a pathway that is important in maintaining CSC population. Notch receptor and its ligands are up-regulated in human PCa tissues. Qui treatment significantly downregulated Notch-1, 2 and 3 expression and that of its ligand Jagged-1. Notch activation requires cleavage by the γ-secretase complex. Qui inhibits the expression of members of the complex Presenilin 1 and Nicastrin. Moreover, ectopic expression of the Notch Intracellular domain (NICD) rescued the cells from Qui -mediated growth suppression. These data demonstrate that Qui mediated effects of PCa stem cells is in part through downregulating Notch1 activation. To determine the effect of Qui on tumor growth in vivo, mice carrying MiaPaCa-2 tumor xenografts were administered the compound intraperitoneally (20 μg/kg bw) every day for 21 days. Qui treatment significantly suppressed tumor xenograft growth, with notably lower tumor volume and weight. Western blot and immunohistochemistry analyses demonstrated significant inhibition of CSC marker proteins DCLK1, CD44 and CD24 and also the Notch signaling related proteins in the Qui-treated xenograft tissues. Conclusion: Together, these data suggest that Qui suppresses PCa growth by inhibiting the Notch signaling pathway. Qui may therefore be a novel compound to target pancreatic cancers. Citation Format: Dharmalingam Subramaniam, Sivapriya Ponnurangam, Afreen Sayed, Animesh Dhar, Dan A. Dixon, Ossama Tawfik, Rajashri R. Parab, Prabhu Dutt Mishra, Prafull Ranadive, Rajiv Sharma, Girish Mahajan, Aravind Sugumar, Scott J. Weir, Roy A. Jensen, Arun Balakrishnan, Shrikant Anant. Quinomycin A affects pancreatic cancer stem cells in part through suppression of notch signaling pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4214. doi:10.1158/1538-7445.AM2015-4214

Determination of quinoxaline antibiotics in fish feed by enzyme-linked immunosorbent assay using a monoclonal antibody

Olaquindox (OLA), mequindox (MEQ), and quincetone (QCT) are widely used synthetic antibiotics of the quinoxaline-1,4-dioxide family.

A sensitive and specific ELISA for determining a residue marker of three quinoxaline antibiotics in swine liver

Methyl-3-quinoxaline-2-carboxylic acid (MQCA) is a possible residue marker for three quinoxaline veterinary medicines (olaquindox, mequindox, and quinocetone). The wide application of mequindox/quinocetone or the illegal use of olaquindox leads to MQCA residue in animal's original food, thereby threatening the safety of human food. The indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) with a specific coating antigen and monoclonal antibody (MAB) was established and optimized for detecting MQCA in swine liver. Samples were acidified with 2 mol l(-1) hydrochloric acid, extracted with ethyl acetate-hexane-isopropanol (8 + 1 + 1, v/v/v) and then detected by IC-ELISA. The logarithm correlation of standards to OD values ranged from 0.2 to 200 μg l(-1), with IC(50) of 6.46 μg l(-1). Negligible cross-reactivity happened to five quinoxaline antibiotics (olaquindox, mequindox, quinocetone, carbadox, and cyadox) and the metabolite of carbadox and cyadox (quinoxaline-2-carboxylic acid). When spiked with 1 to 100 μg kg(-1) of MQCA, the recoveries ranged from 85.44 to 100.02 %, with the intra-assay coefficient of variation (CV) of 6.64-10.57 % and inter-assay CV of 7.29-10.88 %. The limit of detection for MQCA was 1.0 μg kg(-1) in swine liver. Furthermore, incurred samples were detected by the IC-ELISA and then conformed by a reported LC/MS/MS method, it shown that there was good correlation between the two methods. All these results indicated that the IC-ELISA method is appropriate for surveillance MQCA residue in animal tissues.

Segregation of the nucleolar materials produced by quinoxaline antibiotics in JTC-13 cells. Comparison of effects among 4-nitro-quinoline 1-oxide, actinomycin-B, and quinoxaline antibiotics.

Segregation of the nucleolar materials produced by quinoxaline antibiotics in JTC-13 cells. Comparison of effects among 4-nitro-quinoline 1-oxide, actinomycin-B, and quinoxaline antibiotics.

Enzymatic Macrolactonization in the Presence of DNA Leading to Triostin A Analogs

Excised thioesterase domains are versatile catalysts for macrocyclization. However, thioesterase-catalyzed cyclization is often precluded due to the occurrence of hydrolysis and product inhibition. To circumvent these obstacles, we devised an unprecedented strategy: coincubation with DNA to capture the cyclic products possessing DNA-binding properties. In experiments involving echinomycin thioesterase-catalyzed macrolactonization leading to the cyclic triostin A analog TANDEM, we found that the addition of DNA drastically improved the yield of TANDEM (19% --> 67%), with a complete reversal of the cyclization:hydrolysis ratio (1:2 --> 18:1). Furthermore, the applicability of this protocol was demonstrated for a variety of substrates. The results described herein provide insight into the mechanism of echinomycin thioesterase-catalyzed conversions and also pave the way for chemoenzymatic synthesis of the quinoxaline antibiotics and their analogs.

Solid-Phase Synthesis of Oxathiocoraline by a Key Intermolecular Disulfide Dimer

Oxathiocoraline, a member of the quinoxaline antibiotic family, has been synthesized on solid-phase. The depsipeptide exhibits high synthetic complexity owing to the presence of consecutive NMe amino acids, two ester moieties, a disulfide bridge, and two SMe Cys residues. Because of internal diketopiperazine formation, standard stepwise or convergent approaches failed to deliver the linear octadepsipeptide precursor. Therefore, an alternative methodology where an intermolecular disulfide dimer is formed on solid-phase was developed. Cleavage of the dimer from the solid-phase and subsequent bismacrolactamization followed by incorporation of the heterocyclic unit afforded the target compound. Oxathiocoraline showed antitumoral activity in three tumor cell lines.

Functional Cross-talk between Fatty Acid Synthesis and Nonribosomal Peptide Synthesis in Quinoxaline Antibiotic-producing Streptomycetes

Quinoxaline antibiotics are chromopeptide lactones embracing the two families of triostins and quinomycins, each having characteristic sulfur-containing cross-bridges. Interest in these compounds stems from their antineoplastic activities and their specific binding to DNA via bifunctional intercalation of the twin chromophores represented by quinoxaline-2-carboxylic acid (QA). Enzymatic analysis of triostin A-producing Streptomyces triostinicus and quinomycin A-producing Streptomyces echinatus revealed four nonribosomal peptide synthetase modules for the assembly of the quinoxalinoyl tetrapeptide backbone of the quinoxaline antibiotics. The modules were contained in three protein fractions, referred to as triostin synthetases (TrsII, III, and IV). TrsII is a 245-kDa bimodular nonribosomal peptide synthetase activating as thioesters for both serine and alanine, the first two amino acids of the quinoxalinoyl tetrapeptide chain. TrsIII, represented by a protein of 250 kDa, activates cysteine as a thioester. TrsIV, an unstable protein of apparent Mr about 280,000, was identified by its ability to activate and N-methylate valine, the last amino acid. QA, the chromophore, was shown to be recruited by a free-standing adenylation domain, TrsI, in conjunction with a QA-binding protein, AcpPSE. Cloning of the gene for the QA-binding protein revealed that it is the fatty acyl carrier protein, AcpPSE, of the fatty acid synthase of S. echinatus and S. triostinicus. Analysis of the acylation reaction of AcpPSE by TrsI along with other A-domains and the aroyl carrier protein AcmACP from actinomycin biosynthesis revealed a specific requirement for AcpPSE in the activation and also in the condensation of QA with serine in the initiation step of QA tetrapeptide assembly on TrsII. These data show for the first time a functional interaction between nonribosomal peptide synthesis and fatty acid synthesis.

Synthesis and Biological Activity of New Quinoxaline Antibiotics of Echinomycin Analogues.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Echinomycin inhibits chromosomal DNA replication and embryonic development in vertebrates.

Echinomycin, a member of the quinoxaline family of antibiotics, is known to be a strong inhibitor of RNA synthesis which has been attributed to its ability to bind to double-helical DNA. Here we study the effect of echinomycin upon DNA replication using egg extracts and embryos from Xenopus laevis as well as cultured human cells. Evidence is presented that echinomycin interferes with chromatin decondensation, nuclear assembly and DNA replication. In the absence of transcription and translation, the drug specifically blocks DNA replication in both Xenopus sperm chromatin and HeLa cell nuclei in vitro. By contrast, replication of single-stranded DNA is not inhibited indicating that echinomycin acts by interacting with the DNA and not the replication elongation proteins of chromatin. The addition of the antibiotic to HeLa cells and X.laevis embryos results in anaphase bridges and cell death. Importantly, in X.laevis embryos injected with echinomycin at the two-cell stage the drug specifically inhibits the cell cycle prior to the onset of transcription, suggesting that quinoxaline antibiotics could exert anti- proliferative effects by inhibition of chromosomal DNA replication.

Synthesis and biological activity of new quinoxaline antibiotics of echinomycin analogues

Novel quinoxaline antibiotics having the methylenedithioether bridge as an analogue of echinomycin have been synthesized by insertion of methylene moiety between –S–S– bond. The compound 1a shows remarkable cytotoxicities against human tumor various cell lines, and is active VRE (vancomycin-resistant enterococci) within MIC range 0.5–8 μg/mL. According to the eukaryotic or prokaryotic data, 1a might be a first analogue to replace echinomycin.

Kakadumycins, novel antibiotics from Streptomyces sp. NRRL 30566, an endophyte of Grevillea pteridifolia

An endophytic streptomycete (NRRL 30566) is described and partially characterized from a fern-leaved grevillea ( Grevillea pteridifolia) tree growing in the Northern Territory of Australia. This endophytic streptomycete produces, in culture, novel antibiotics – the kakadumycins. Methods are outlined for the production and chemical characterization of kakadumycin A and related compounds. This antibiotic is structurally related to a quinoxaline antibiotic, echinomycin. Each contains, by virtue of their amino acid compositions, alanine, serine and an unknown amino acid. Other biological, spectral and chromatographic differences between these two compounds occur and are given. Kakadumycin A has wide spectrum antibiotic activity, especially against Gram-positive bacteria, and it generally displays better bioactivity than echinomycin. For instance, against Bacillus anthracis strains, kakadumycin A has minimum inhibitory concentrations of 0.2–0.3 μg ml −1 in contrast to echinomycin at 1.0–1.2 μg ml −1. Both echinomycin and kakadumycin A have impressive activity against the malarial parasite Plasmodium falciparum with LD 50s in the range of 7–10 ng ml −1. In macromolecular synthesis assays both kakadumycin A and echinomycin have similar effects on the inhibition of RNA synthesis. It appears that the endophytic Streptomyces sp. offer some promise for the discovery of novel antibiotics with pharmacological potential.

Molecular design, chemical synthesis and biological evaluation of quinoxaline–carbohydrate hybrids as novel and selective photo-induced DNA cleaving and cytotoxic agents

The quinoxaline moiety in antitumor quinoxaline antibiotics cleaved double stranded DNA at the 5′ side guanine of the 5′-GG-3′ site upon irradiation with UV light with a long wavelength and without any additive. The quinoxaline–carbohydrate hybrid system was very effective for the DNA cleavage. Furthermore, the quinoxaline–carbohydrate hybrids exhibited strong and selective cytotoxicity against cancer cells with photoirradiation.

Molecular design and evaluation of quinoxaline-carbohydrate hybrids as novel and efficient photo-induced GG-selective DNA cleaving agents.

Quinoxaline, found in antitumor quinoxaline antibiotics, was found to cleave double stranded DNA at the 5' side guanine of 5'-GG-3' site on irradiation with long wavelength UV light without any additive; furthermore, a bis(quinoxaline-carbohydrate) hybrid system was very effective for DNA cleavage.

Preferred Binding Sites for [N-MeCys3,N-MeCys7]TANDEM Determined Using a Universal Footprinting Substrate

We have prepared a novel footprinting substrate which contains all 136 tetranucleotide sequences and have used this to determine the preferred binding sites for the synthetic quinoxaline antibiotic [N-MeCys3,N-MeCys7]TANDEM. We find that, although the ligand binds to all TpA steps, it binds best to the tetranucleotide sequence ATAT and shows only weak interaction with TTAA and GTAC. The best binding sites contain the sequences ATAX and XTAT.

DNA Recognition by Quinoline Antibiotics: Use of Base-Modified DNA Molecules to Investigate Determinants of Sequence-Specific Binding of Luzopeptin

The luzopeptin antibiotics contain a cyclic decadepsipeptide to which are attached two quinoline chromophores that bisintercalate into DNA. Although they bind DNA less tightly than the structurally related quinoxaline antibiotics echinomycin and triostin A, the molecular basis of their interaction remains unclear. We have used the PCR in conjunction with novel nucleotides to create specifically modified DNA for footprinting experiments. In order to study the influence that removal, addition or relocation of the guanine 2-amino group, which normally identifies G. C base pairs from the minor groove, has on the interaction of luzopeptin antibiotics with DNA. The presence of a purine 2-amino group is not strictly required for binding of luzopeptin to DNA, but the exact location of this group can alter the position of preferred drug binding sites. It is, however, not the sole determinant of nucleotide sequence recognition in luzopeptin-DNA interaction. Nor can the selectivity of luzopeptin be attributed to the quinoline chromophores, suggesting that an analogue mode of DNA recognition may be operative. This is in contrast to the digital readout that seems to predominate with the quinoxaline antibiotics.