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Stephen C. Ekker - One of the best experts on this subject based on the ideXlab platform.
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a primer for Morpholino use in zebrafish
Zebrafish, 2009Co-Authors: Brent R Bill, Stephen C. Ekker, Andrew M Petzold, Karl J Clark, Lisa A SchimmentiAbstract:Abstract Morpholino oligonucleotides are the most common anti-sense “knockdown” technique used in zebrafish (Danio rerio). This review discusses common practices for the design, preparation, and deployment of Morpholinos in this vertebrate model system. Off-targeting effects of Morpholinos are discussed as well as method to minimize this potentially confounding variable via co-injection of a tP53-targeting Morpholino. Finally, new uses of Morpholinos are summarized and contextualized with respect to the complementary, DNA-based knockout technologies recently developed for zebrafish.
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amod a Morpholino oligonucleotide selection tool
Nucleic Acids Research, 2005Co-Authors: Eric W Klee, Stephen C. Ekker, Kyong Jin Shim, Michael A Pickart, Lynda B M EllisAbstract:AMOD is a web-based program that aids in the functional evaluation of nucleotide sequences through sequence characterization and antisense Morpholino oligonucleotide (target site) selection. Submitted sequences are analyzed by translation initiation site prediction algorithms and sequence-to-sequence comparisons; results are used to characterize sequence features required for Morpholino design. Within a defined subsequence, base composition and homodimerization values are computed for all putative Morpholino oligonucleotides. Using these properties, Morpholino candidates are selected and compared with genomic and transcriptome databases with the goal to identify target-specific enriched Morpholinos. AMOD has been used at the University of Minnesota to design approximately 200 Morpholinos for a functional genomics screen in zebrafish. The AMOD web server and a tutorial are freely available to both academic and commercial users at http://www.secretomes.umn.edu/AMOD/.
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xenopus frizzled 7 morphant displays defects in dorsoventral patterning and convergent extension movements during gastrulation
Genesis, 2001Co-Authors: Saulius Sumanas, Stephen C. EkkerAbstract:Wnt signaling has been implicated in the process of gastrulation in Xenopus laevis. Overexpression of Wnt molecules, such as a putative dominant-negative form of Wnt8 (Hoppler et al., 1996), Wnt11 (Tada and Smith, 2000), as well as wild type Wnt5A and Wnt11 proteins (Du et al., 1995; Gradl et al., 1999; Moon et al., 1993) interferes with convergent extension movements during gastrulation. Overexpression of some other Wnt signaling pathway members such as full-length and truncated forms of Xenopus frizzled-7 (Djiane et al., 2000; Sumanas et al., 2000), a truncated form of Xenopus disheveled (Sokol, 1996) also causes similar defects. However, evidence for the requirement of a specific Wnt pathway protein in the gastrulation process is still missing. Here we investigated the function of Xenopus frizzled-7 (Xfz7) utilizing the Morpholino phosphorodiamidate antisense oligonucleotides approach (Heasman et al., 2000; Summerton, 1999). A mixture of two Morpholinos designed against two different orthologous copies of Xfz7 was injected into unfertilized oocytes. The resulting embryos (Xfz7 morphants) failed to properly close the blastopore, whereas the more severely affected embryos exogastrulated. The gastrulation defect was caused by inhibition of convergent extension movements as demonstrated by an animal cap cell motility assay (Smith et al., 1990). Adding synthetic Xfz7 RNA alleviated the observed gastrulation defect arguing for the specificity of the Morpholino-based targeting. At tadpole stages, Xfz7 morphants displayed loss or reduction of anterior structures. Molecular analysis showed that approximately half of Xfz7 morphants had reduced expression of a dorsal marker Xnr3 at the gastrula stage. Xnr3 is known to be the direct target of the maternal Wnt axis induction pathway (Smith et al., 1995; McKendry et al., 1997) and has previously been shown to be under maternal Xfz7 control (Sumanas et al., 2000). The results presented here argue for two different Xfz7 functions. They confirm our previous findings that Xfz7 is required for the dorsoventral Wnt/b-catenin axis induction pathway (Sumanas et al., 2000) and demonstrate a Xfz7 requirement in a zygotic Wnt pathway that regulates morphogenetic movements during gastrulation. This is the first evidence for a vertebrate Frizzled protein to act in two distinct Wnt-signaling pathways, as known to be the case for Drosophila Frizzled-1 (Adler, 1992; Bhanot et al., 1999; Chen and Struhl, 1999). To study Xfz7 function during development, we designed two different Morpholinos against the 5’UTR of two distinct orthologous copies of the Xfz7 gene present due to the allotetraploid nature of Xenopus (Kobel and Du Pasquier, 1986). One ortholog has been reported by Djiane et al. (2000)., Medina et al. (2000), and Sumanas et al. (2000), and the other ortholog of Xfz7 was reported by Wheeler and Hoppler (1999). These two Xfz7 orthologs are 97% identical in amino acid sequences, but contain more significant differences in the UTR sequences (data not shown). Both orthologs have similar expression patterns at all stages reported. A mixture of the two different Morpholinos (MO-1 and MO-2) was injected into unfertilized oocytes, and the oocytes were subsequently fertilized using the host–transfer method (Heasman et al., 1994). Injection of individual Morpholinos up to 20 ng resulted in no significant developmental defects, whether delivered into early embryos or into oocytes followed by host transfer fertilization (data not shown). We used doses of 5 ng (2.5ng each MO) and 10 ng (5ng each MO) of the Morpholino mixture to simultaneously knockdown both orthologous copies of Xfz7. Xfz7 morphants developed seemingly normally with only a slight developmental delay until the start of gastrulation. During gastrulation, Xfz7 morphants formed greatly enlarged blastopores, which subsequently failed to involute properly, resulting in exogastrulating embryos at higher doses of Morpholino used (Fig. 1a–c, i). Microinjecting synthetic Xfz7 RNA into oocytes significantly reduced the severity of the phenotype, resulting in a greater percentage of normal and weakly affected embryos (Fig. 1d, i). At tailbud and tadpole stages, Xfz7 morphants displayed defects in dorsoanterior structures. They commonly had a reduced or missing head and cement gland
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Zebrafish frizzled-2 morphant displays defects in body axis elongation.
genesis, 2001Co-Authors: Saulius Sumanas, Hyon Kim, Spencer Hermanson, Stephen C. EkkerAbstract:Wnt signaling has been implicated in many patterning processes in a vertebrate embryo including morphogenetic cell rearrangements during gastrulation. Pipetail (wnt5) and silberblick (wnt11) zebrafish mutants undergo abnormal gastrulation with an undulated notochord ( pipetail) and cyclopic embryos ( silberblick) at later stages of development (Hammerschmidt et al., 1996; Heisenberg et al., 1996; Heisenberg et al., 2000; Rauch et al., 1997). Wnt proteins are known to transmit signals via the Frizzled seven-pass transmembrane receptor family (Bhanot et al., 1996). We have isolated cDNA encoding a new Frizzled family protein, zebrafish Frizzled-2 (Fz2). Zebrafish frizzled-2 is a likely ortholog of human frizzled-2 by the sequence homology alignments and mapping data analysis. Fz2 is expressed zygotically within the notochord, somitic and posterior paraxial mesoderm. For protein knockdown studies, we utilized Morpholino phosphorodiamidate antisense oligonucleotides (Morpholinos, MOs). Injection of two different Morpholinos into early zebrafish embryos targeted to fz2 caused similar developmental defects. Fz2-Morpholino injected embryos (morphants) are shorter than controls and display an undulating or kinked notochord, neural tube, and hypochord. Similar, although weaker, undulations of axial structures have been observed in pipetail mutant embryos, raising possibility that fz2 may function as a receptor in the wnt5 pathway regulating gastrulation movements. We isolated a novel zebrafish frizzled homolog from gastrula stage cDNA library by the polymerase chain reaction (PCR). The isolated gene encodes a putative Frizzled protein with highest homology to the frizzled-2 subfamily as evident from BLAST and CLUSTAL alignments (Fig. 1a, b). Radiation hybrid mapping using panel LN54 (Hukriede et al., 1999) places fz2 on linkage group 3, linked to the marker Z22516 (distance 0.00cR, LOD score 22.2). To confirm that we isolated true frizzled-2 ortholog from zebrafish, we performed synteny analysis between corresponding zebrafish and human chromosome regions. Zebrafish fz2 maps to the marker Z22516, which is located between the hoxb9a and dlx8 genes on linkage group 3 in the LN54 panel (Hukriede et al., 1999; marker list at http://zfin.org/ZFIN/). This region of zebrafish linkage group 3 is syntenic to the human chromosome 17 region. Human fz2 maps by FISH to 17q21.1 (Zhao et al., 1995), human hoxB9 maps to 17q21-q22 (Acampora et al., 1989; Apiou et al., 1996; HUGO database), and human Dlx4, an ortholog of zebrafish Dlx8, maps to 17q21.33 (Nakamura et al., 1996; Stock et al., 1996). Thus, human frizzled-2 is likely to be the true ortholog of the zebrafish frizzled-2 gene. Using Northern blotting, we detected a single, zygotically expressed transcript of fz2, with maximal expression level approximately at the five-somite stage (Fig. 1c). In situ hybridization revealed weak and ubiquitous expression starting from the shield stage (data not shown). During early somitogenesis, fz2 RNA is detected within the forming somites as well as in two longitudinal stripes within the paraxial posterior mesoderm (Fig. 1, d–g). There is also weak expression within notochordal cells anterior to fz2 expression in the forming somites. At the 20-somite stage, fz2 expression is detected in the posterior part of the notochord and in the posterior somitic mesoderm (Fig. 1h, i). At 26 hpf, fz2 is localized within the tailbud mesoderm (Fig. 1k). Injecting 100–500 pg fz2 RNA into fish embryos at the 1–2 cell stage caused severe hyperdorsalization in .90% of experimental embryos (data not shown), as noted previously for other Frizzled proteins (Nasevicius et al., 1998). A number of fz2-overexpressing embryos displayed secondary axes (data not shown). This illustrates that fz2 is capable of activating dorsoventral axis induction pathway when overexpressed in zebrafish embryos. To study fz2 function, we utilized an antisense Morpholino oligonucleotide approach (Nasevicius and Ekker, 2000; Summerton, 1999). Two nonoverlapping Morpholinos were designed against the fz2 sequence. Injecting either Morpholino against fz2 produced similar phenotypes, indicating a likely high specificity of targeting to fz2 transcripts. The resulting embryos (fz2 morphants) display characteristic defects not commonly seen with other unrelated Morpholinos. At 70% epiboly, fz2 morphants have an oblong shape compared with
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effective targeted gene knockdown in zebrafish
Nature Genetics, 2000Co-Authors: Aidas Nasevicius, Stephen C. EkkerAbstract:The sequencing of the zebrafish genome should be completed by the end of 2002. Direct assignment of function on the basis of this information would be facilitated by the development of a rapid, targeted ‘knockdown’ technology in this model vertebrate. We show here that antisense, Morpholino-modified oligonucleotides1 (Morpholinos) are effective and specific translational inhibitors in zebrafish. We generated phenocopies of mutations of the genes no tail (ref. 2), chordin (ref. 3), one-eyed-pinhead (ref. 4), nacre (ref. 5) and sparse (ref. 6), removing gene function from maternal through post-segmentation and organogenesis developmental stages. We blocked expression from a ubiquitous green fluorescent protein (GFP) transgene, showing that, unlike tissue-restricted limitations found with RNA-based interference in the nematode7, all zebrafish cells readily respond to this technique. We also developed also Morpholino-based zebrafish models of human disease. Morpholinos targeted to the uroporphyrinogen decarboxylase gene8 result in embryos with hepatoerythropoietic porphyria. We also used Morpholinos for the determination of new gene functions. We showed that embryos with reduced sonic hedgehog (ref. 9) signalling and reduced tiggy-winkle hedgehog (ref. 10) function exhibit partial cyclopia and other specific midline abnormalities, providing a zebrafish genetic model for the common human disorder holoprosencephaly. Conserved vertebrate processes and diseases are now amenable to a systematic, in vivo, reverse-genetic paradigm using zebrafish embryos.
Paul A Morcos - One of the best experts on this subject based on the ideXlab platform.
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Achieving targeted and quantifiable alteration of mRNA splicing with Morpholino oligos. Biochemical and Biophysical Research Communications
2020Co-Authors: Paul A MorcosAbstract:Abstract This work represents the first guide for using steric-block antisense oligos as tools for effective and targeted modification of RNA splicing. Comparison of several steric-block oligo types shows the properties of Morpholinos provide significant advantages over other potential splice-blocking oligos. The procedures and complications of designing effective splice-blocking Morpholino oligos are described. The design process requires complete pre-mRNA sequence for defining suitable targets, which usually generate specific predictable messengers. To validate the targeting procedure, the level and nature of transcript alteration is characterized by RT-PCR analysis of splice modification in a b-globin splice model system. An oligo-walking study reveals that while U1 and U2 small nuclear RiboNucleoProtein (snRNP) binding sites are the most effective targets for blocking splicing, inclusion of these sites is not required to achieve effective splice modifications. The most effective targeting strategy employs simultaneously blocking snRNP binding sites and splice-junctions. The work presented here continues to be the basis for most of the successful Morpholino oligos designed for the worldwide research community to block RNA splicing
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octa guanidine Morpholino restores dystrophin expression in cardiac and skeletal muscles and ameliorates pathology in dystrophic mdx mice
Molecular Therapy, 2009Co-Authors: Paul A Morcos, Timothy DoranAbstract:Steric-block antisense oligonucleotides (AONs) are able to target RNAs for destruction and splicing alteration. Reading frame restoration of the dystrophin transcript can be achieved by AON-mediated exon skipping in the dystrophic mdx mouse model. However, simple, unmodified AONs exhibit inefficient delivery systemically, leading to dystrophin induction with high variability in skeletal muscles and barely detectable in cardiac muscle. Here, we examined a Morpholino oligomer conjugated with a dendrimeric octaguanidine (Vivo-Morpholino) and demonstrated that the delivery moiety significantly improved dystrophin production in both skeletal and cardiac muscles in mdx mice in vivo. Single intravenous (IV) injections of 6 mg/kg Vivo-MorpholinoE23 (Vivo-ME23) generated dystrophin expression in skeletal muscles at the levels higher than the injection of 300 mg/kg unmodified ME23. Repeated injections at biweekly intervals achieved near 100% of fibers expressing dystrophin in skeletal muscles bodywide without eliciting a detectable immune response. Dystrophin protein was restored to ~50 and 10% of normal levels in skeletal and cardiac muscles, respectively. Vivo-Morpholinos showed no signs of toxicity with the effective dosages and regime, thus offering realistic prospects for the treatment of a majority of Duchenne muscular dystrophy (DMD) patients and many other diseases by targeting RNAs.
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achieving efficient delivery of Morpholino oligos with nucleofection
2008Co-Authors: Shannon T Knuth, Jon D Moulton, Paul A MorcosAbstract:Morpholino oligos (MOs), also known as phosphodiamidate Morpholino oligos, are designed to bind to complementary RNA sequence and sterically inhibit proteins from binding the RNA. Morpholinos are commonly used to knock down gene expression and alter mRNA splicing in a variety of organisms, tissues or cells (for a searchable data base of references see http://www.genetools. com/Publications/). For gene knock downs the MOs are designed to complement mRNA sequence in the 5’ untranslated region and/or the first 25 bases of mRNA coding sequence, sterically inhibiting the movement of the translation initiation complex toward the start codon. This prevents the large subunit of the ribosome from assembling and, as a consequence, causing reduced gene expression. Morpholinos are also used to modify pre-mRNA splicing by tar geting the oligo to a splice junction or regulatory site, sterically blocking binding of snRNPs or other splice factors. Splice-blocking Morpholinos have great promise for therapeutic use correcting and/or reducing the impact of the mutations that lead to b-Thalessemia , Duchenne muscular dystrophy and other diseases caused by Achieving Efficient Delivery of Morpholino Oligos with Nucleofection
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achieving targeted and quantifiable alteration of mrna splicing with Morpholino oligos
Biochemical and Biophysical Research Communications, 2007Co-Authors: Paul A MorcosAbstract:This work represents the first guide for using steric-block antisense oligos as tools for effective and targeted modification of RNA splicing. Comparison of several steric-block oligo types shows the properties of Morpholinos provide significant advantages over other potential splice-blocking oligos. The procedures and complications of designing effective splice-blocking Morpholino oligos are described. The design process requires complete pre-mRNA sequence for defining suitable targets, which usually generate specific predictable messengers. To validate the targeting procedure, the level and nature of transcript alteration is characterized by RT-PCR analysis of splice modification in a beta-globin splice model system. An oligo-walking study reveals that while U1 and U2 small nuclear RiboNucleoProtein (snRNP) binding sites are the most effective targets for blocking splicing, inclusion of these sites is not required to achieve effective splice modifications. The most effective targeting strategy employs simultaneously blocking snRNP binding sites and splice-junctions. The work presented here continues to be the basis for most of the successful Morpholino oligos designed for the worldwide research community to block RNA splicing.
Andrew M Petzold - One of the best experts on this subject based on the ideXlab platform.
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a primer for Morpholino use in zebrafish
Zebrafish, 2009Co-Authors: Brent R Bill, Stephen C. Ekker, Andrew M Petzold, Karl J Clark, Lisa A SchimmentiAbstract:Abstract Morpholino oligonucleotides are the most common anti-sense “knockdown” technique used in zebrafish (Danio rerio). This review discusses common practices for the design, preparation, and deployment of Morpholinos in this vertebrate model system. Off-targeting effects of Morpholinos are discussed as well as method to minimize this potentially confounding variable via co-injection of a tP53-targeting Morpholino. Finally, new uses of Morpholinos are summarized and contextualized with respect to the complementary, DNA-based knockout technologies recently developed for zebrafish.
Xiaoling Fan Cockcroft - One of the best experts on this subject based on the ideXlab platform.
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dna dependent protein kinase dna pk inhibitors synthesis and biological activity of quinolin 4 one and pyridopyrimidin 4 one surrogates for the chromen 4 one chemotype
Journal of Medicinal Chemistry, 2010Co-Authors: Celine Cano, Xiaoling Fan Cockcroft, Nicola J Curtin, Mark Frigerio, Bernard T Golding, Ian R Hardcastle, Olivier Barbeau, Christine Bailey, Heather Mary Ellen Duggan, Marc Geoffrey HummersoneAbstract:Following the discovery of dibenzo[b,d]thiophen-4-yl)-2-Morpholino-4H-chromen-4-one (NU7441) (Leahy, J. J. J.; Golding, B. T.; Griffin, R. J.; Hardcastle, I. R.; Richardson, C.; Rigoreau, L.; Smith, G. C. M. Bioorg. Med. Chem. Lett. 2004, 14, 6083−6087) as a potent inhibitor (IC50 = 30 nM) of DNA-dependent protein kinase (DNA-PK), we have investigated analogues in which the chromen-4-one core template has been replaced by aza-heterocyclic systems: 9-substituted 2-morpholin-4-ylpyrido[1,2-a]pyrimidin-4-ones and 8-substituted 2-morpholin-4-yl-1H-quinolin-4-ones. The 8- and 9-substituents were either dibenzothiophen-4-yl or dibenzofuran-4-yl, which were each further substituted at the 1-position with water-solubilizing groups [NHCO(CH2)nNR1R2, where n = 1 or 2 and the moiety R1R2N was derived from a library of primary and secondary amines (e.g., morpholine)]. The inhibitors were synthesized by employing a multiple-parallel approach in which the two heterocyclic components were assembled by Suzuki−Miyaura cro...
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pyranone thiopyranone and pyridone inhibitors of phosphatidylinositol 3 kinase related kinases structure activity relationships for dna dependent protein kinase inhibition and identification of the first potent and selective inhibitor of the ataxia t
Journal of Medicinal Chemistry, 2007Co-Authors: Jonathan J Hollick, Laurent Rigoreau, Celine Canosoumillac, Xiaoling Fan Cockcroft, Nicola J Curtin, Mark Frigerio, Bernard T Golding, Sophie Guiard, Ian R Hardcastle, Ian HicksonAbstract:Structure−activity relationships have been investigated for inhibition of DNA-dependent protein kinase (DNA-PK) and ATM kinase by a series of pyran-2-ones, pyran-4-ones, thiopyran-4-ones, and pyridin-4-ones. A wide range of IC50 values were observed for pyranones and thiopyranones substituted at the 6-position, with the 3- and 5-positions proving intolerant to substitution. Related pyran-2-ones, pyran-4-ones, and thiopyran-4-ones showed similar IC50 values against DNA-PK, whereas the pyridin-4-one system proved, in general, ineffective at inhibiting DNA-PK. Extended libraries exploring the 6-position of 2-Morpholino-pyran-4-ones and 2-Morpholino-thiopyrano-4-ones identified the first highly potent and selective ATM inhibitor 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one (151C; ATM; IC50 = 13 nM) and revealed constrained SARs for ATM inhibition compared with DNA-PK. One of the most potent DNA-PK inhibitors identified, 2-(4-methoxyphenyl)-6-(morpholin-4-yl)pyran-4-one (16; DNA-PK; IC50 = 220 nM) effectivel...
Ian R Hardcastle - One of the best experts on this subject based on the ideXlab platform.
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dna dependent protein kinase dna pk inhibitors synthesis and biological activity of quinolin 4 one and pyridopyrimidin 4 one surrogates for the chromen 4 one chemotype
Journal of Medicinal Chemistry, 2010Co-Authors: Celine Cano, Xiaoling Fan Cockcroft, Nicola J Curtin, Mark Frigerio, Bernard T Golding, Ian R Hardcastle, Olivier Barbeau, Christine Bailey, Heather Mary Ellen Duggan, Marc Geoffrey HummersoneAbstract:Following the discovery of dibenzo[b,d]thiophen-4-yl)-2-Morpholino-4H-chromen-4-one (NU7441) (Leahy, J. J. J.; Golding, B. T.; Griffin, R. J.; Hardcastle, I. R.; Richardson, C.; Rigoreau, L.; Smith, G. C. M. Bioorg. Med. Chem. Lett. 2004, 14, 6083−6087) as a potent inhibitor (IC50 = 30 nM) of DNA-dependent protein kinase (DNA-PK), we have investigated analogues in which the chromen-4-one core template has been replaced by aza-heterocyclic systems: 9-substituted 2-morpholin-4-ylpyrido[1,2-a]pyrimidin-4-ones and 8-substituted 2-morpholin-4-yl-1H-quinolin-4-ones. The 8- and 9-substituents were either dibenzothiophen-4-yl or dibenzofuran-4-yl, which were each further substituted at the 1-position with water-solubilizing groups [NHCO(CH2)nNR1R2, where n = 1 or 2 and the moiety R1R2N was derived from a library of primary and secondary amines (e.g., morpholine)]. The inhibitors were synthesized by employing a multiple-parallel approach in which the two heterocyclic components were assembled by Suzuki−Miyaura cro...
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pyranone thiopyranone and pyridone inhibitors of phosphatidylinositol 3 kinase related kinases structure activity relationships for dna dependent protein kinase inhibition and identification of the first potent and selective inhibitor of the ataxia t
Journal of Medicinal Chemistry, 2007Co-Authors: Jonathan J Hollick, Laurent Rigoreau, Celine Canosoumillac, Xiaoling Fan Cockcroft, Nicola J Curtin, Mark Frigerio, Bernard T Golding, Sophie Guiard, Ian R Hardcastle, Ian HicksonAbstract:Structure−activity relationships have been investigated for inhibition of DNA-dependent protein kinase (DNA-PK) and ATM kinase by a series of pyran-2-ones, pyran-4-ones, thiopyran-4-ones, and pyridin-4-ones. A wide range of IC50 values were observed for pyranones and thiopyranones substituted at the 6-position, with the 3- and 5-positions proving intolerant to substitution. Related pyran-2-ones, pyran-4-ones, and thiopyran-4-ones showed similar IC50 values against DNA-PK, whereas the pyridin-4-one system proved, in general, ineffective at inhibiting DNA-PK. Extended libraries exploring the 6-position of 2-Morpholino-pyran-4-ones and 2-Morpholino-thiopyrano-4-ones identified the first highly potent and selective ATM inhibitor 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one (151C; ATM; IC50 = 13 nM) and revealed constrained SARs for ATM inhibition compared with DNA-PK. One of the most potent DNA-PK inhibitors identified, 2-(4-methoxyphenyl)-6-(morpholin-4-yl)pyran-4-one (16; DNA-PK; IC50 = 220 nM) effectivel...