Identification of a potent and selective chemical probe for exploring the role of Mediator complex-associated protein kinases CDK8 and CDK19 in human disease
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ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Robert Poele
PROVIDER: E-GEOD-67849 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
A selective chemical probe for exploring the role of CDK8 and CDK19 in human disease.
Dale Trevor T Clarke Paul A PA Esdar Christina C Waalboer Dennis D Adeniji-Popoola Olajumoke O Ortiz-Ruiz Maria-Jesus MJ Mallinger Aurélie A Samant Rahul S RS Czodrowski Paul P Musil Djordje D Schwarz Daniel D Schneider Klaus K Stubbs Mark M Ewan Ken K Fraser Elizabeth E TePoele Robert R Court Will W Box Gary G Valenti Melanie M de Haven Brandon Alexis A Gowan Sharon S Rohdich Felix F Raynaud Florence F Schneider Richard R Poeschke Oliver O Blaukat Andree A Workman Paul P Schiemann Kai K Eccles Suzanne A SA Wienke Dirk D Blagg Julian J
Nature chemical biology 20151026 12
There is unmet need for chemical tools to explore the role of the Mediator complex in human pathologies ranging from cancer to cardiovascular disease. Here we determine that CCT251545, a small-molecule inhibitor of the WNT pathway discovered through cell-based screening, is a potent and selective chemical probe for the human Mediator complex-associated protein kinases CDK8 and CDK19 with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates a type 1 binding mode involv ...[more]
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Project description:There is an unmet need for chemical tools to explore the role of the Mediator complex in human pathologies ranging from cancer to cardiovascular disease. Here we determine that CCT251545 a WNT-pathway inhibitor discovered by cell-based screening is a potent and selective chemical probe for the Mediator complex-associated protein kinases CDK8 and CDK19. CCT251545 is an ATP competitive inhibitor with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates Type 1 binding involving insertion of the CDK8 C-terminus into the ligand binding site. In contrast to Type II inhibitors of CDK8/19 CCT251545 displays potent cell-based activity. We show that CCT251545 and close analogues not only alter WNT-pathway regulated gene expression but also other CDK8/19 targets including STAT1-regulated gene expression. Consistent with this we find that phospho-STAT1SER727 is a biomarker of CDK8 kinase activity in vitro and in vivo. Finally we demonstrate in vivo activity of CCT251545 in WNT-dependent tumours. Compound 1,2, 6 and 9 are close analogues of a 3,4,5-trisubstituted pyridine series identified from a high-throughput cell-based reporter assay of WNT signalling. They were shown to be potent and selective inhibitors of the Mediator complex-associated protein kinases CDK8 and CDK19. They are ATP competitive inhibitors with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates Type 1 binding involving insertion of the CDK8 C-terminus into the ligand-binding site. A 3D culture model of murine intestinal-derived organoids expressing a transgenic doxycycline-inducible mutant beta-catenin expression following removal of doxycycline results in reduced WNT signalling and was compared to treatment for 24 hours with coumpound 2, 6 and 9. Compound 2 (n=3), compound 6 (n=4), compound 9 (n=3), doxycycline removal (n=4), doxycycline treatment (n=7). Samples were hybridized agianst mouse reference. Compounds 2, 6 and 9 are close analogues of a 3,4,5-trisubstituted pyridine series identified from a high-throughput cell-based reporter assay of WNT signalling. They were shown to be potent and selective inhibitors of the Mediator complex-associated protein kinases CDK8 and CDK19. They are ATP competitive inhibitors with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates Type 1 binding involving insertion of the CDK8 C-terminus into the ligand-binding site.
2015-10-26 | E-GEOD-67847 | biostudies-arrayexpress
Project description:There is an unmet need for chemical tools to explore the role of the Mediator complex in human pathologies ranging from cancer to cardiovascular disease. Here we determine that CCT251545 a WNT-pathway inhibitor discovered by cell-based screening is a potent and selective chemical probe for the Mediator complex-associated protein kinases CDK8 and CDK19. CCT251545 is an ATP competitive inhibitor with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates Type 1 binding involving insertion of the CDK8 C-terminus into the ligand binding site. In contrast to Type II inhibitors of CDK8/19 CCT251545 displays potent cell-based activity. We show that CCT251545 and close analogues not only alter WNT-pathway regulated gene expression but also other CDK8/19 targets including STAT1-regulated gene expression. Consistent with this we find that phospho-STAT1SER727 is a biomarker of CDK8 kinase activity in vitro and in vivo. Finally we demonstrate in vivo activity of CCT251545 in WNT-dependent tumours. LS-174-T colon cancer cells were treated with 715 nM of Compound 6 (IC90 of TCF dependent luciferase) for 0, 4 or 24h. Three samples with three biological repeats each. Sample were hybridized against human reference. Compound 6 is a close analogue of a 3,4,5-trisubstituted pyridine series identified from a high-throughput cell-based reporter assay of WNT signalling. It was shown to be potent and selective inhibitor of the Mediator complex-associated protein kinases CDK8 and CDK19. Its is ATP competitive inhibitor with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates Type 1 binding involving insertion of the CDK8 C-terminus into the ligand-binding site.
2015-10-26 | E-GEOD-67846 | biostudies-arrayexpress
Project description:There is an unmet need for chemical tools to explore the role of the Mediator complex in human pathologies ranging from cancer to cardiovascular disease. Here we determine that CCT251545 a WNT-pathway inhibitor discovered by cell-based screening is a potent and selective chemical probe for the Mediator complex-associated protein kinases CDK8 and CDK19. CCT251545 is an ATP competitive inhibitor with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates Type 1 binding involving insertion of the CDK8 C-terminus into the ligand binding site. In contrast to Type II inhibitors of CDK8/19 CCT251545 displays potent cell-based activity. We show that CCT251545 and close analogues not only alter WNT-pathway regulated gene expression but also other CDK8/19 targets including STAT1-regulated gene expression. Consistent with this we find that phospho-STAT1SER727 is a biomarker of CDK8 kinase activity in vitro and in vivo. Finally we demonstrate in vivo activity of CCT251545 in WNT-dependent tumours. Colo205 colon cancer cells were treated with 350 nM of Compound 1 or with vehicle (DMSO) for 2 or 6h. Four samples with four biological repeats each. Samples were hybridized against human reference. Compound 1 is a close analogue of a 3,4,5-trisubstituted pyridine series identified from a high-throughput cell-based reporter assay of WNT signalling. It was shown to be potent and selective inhibitor of the Mediator complex-associated protein kinases CDK8 and CDK19. Its is ATP competitive inhibitor with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates Type 1 binding involving insertion of the CDK8 C-terminus into the ligand-binding site.
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2015-02-03 | E-MTAB-3023 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2013-06-07 | E-GEOD-46814 | biostudies-arrayexpress
Project description:The potential for xenoestrogens and other environmental chemicals to alter female reproductive function calls for the study of their affects on endogenous hormone-regulated gene expression pathways during uterine tissue remodeling which occurs as part of the menstrual/estrus cycle. However, our knowledge of these pathways is limited. Here, we characterize changes in the CD-1 mouse uterine luminal epithelial cell transcriptome during proestrus and estrus, which are regulated by estrogen and progesterone in preparation of the uterus for pregnancy. Mice were staged beginning at 6 wk of age and uterine horns were harvested at estrus and proestrus. RNA was extracted from lumenal epithelium of uterine horns. Microarray analysis identified 2,251 genes differentially expressed in estrus compared to proestrus in lumenal epithelial cells. These complement other studies where RNA was extracted from whole uterine horns of mice in estrus and proestrus (GSE43064). Beginning on postnatal day 41, female CD-1 mice were monitored for reproductive stage cycle and total RNA was isolated from lumenal epithelium of uterine horns at estrus or proestrus. Tissue was collected on postnatal days 41-61. Samples were pooled for microarray analysis as follows. Seven litters were used to generate 2 tissue pools for estrus and 2 tissue pools for proestrus. Each pool was comprised of uterine tissue RNA from n=4-5 individual mice and n=3-4 individual litters. Fluorescent labeling of RNA and hybridization of the Alexa 555-labeled (green) and Alexa 647-labeled (red) RNA samples to Agilent-026655 Whole Mouse Genome Microarray 4x44K v2 (Agilent Technology, Palo Alto, CA; catalog # G4846A) were carried out, with dye swapping for each estrus cycle stage comparison.
2013-06-07 | E-GEOD-46813 | biostudies-arrayexpress
Project description:The potential for xenoestrogens and other environmental chemicals to alter female reproductive function calls for the study of their affects on endogenous hormone-regulated gene expression pathways during uterine tissue remodeling which occurs as part of the menstrual/estrus cycle. However, our knowledge of these pathways is limited. Here, we characterize changes in the CD-1 mouse uterine transcriptome during proestrus and estrus, which are regulated by estrogen and progesterone in preparation of the uterus for pregnancy. Mice were staged beginning at 6 wk of age and uterine horns were harvested after monitoring two estrus cycles. Microarray analysis identified 2,429 genes differentially expressed in estrus compared to proestrus, indicating that the mouse uterus undergoes remarkable remodeling during the estrus cycle, affecting ~10% of all protein-coding genes. Changes in gene expression associated with structural alteration of the uterus include remodeling of the extracellular matrix, changes in cell keratins and adhesion molecules, activation of mitosis, MHC class II presentation, complement and coagulation cascades, and cytochrome P450 expression. Signaling pathways regulated during the estrus cycle, involving ligand-gated channels, Wnt and hedgehog signaling, and several transcription factors with poorly understood roles in reproductive tissues, include several genes and gene networks that have been implicated in pathological states. The information presented here builds a background for understanding of mechanisms involved in uterine tissue response to endocrine disruptors and the development of reproductive tract diseases. Beginning on postnatal day 42, female CD-1 mice were monitored for reproductive stage cycle for 2 consecutive cycles and then total RNA was isolated from uterine horns at estrus or proestrus. Samples were pooled for microarray as follows. Six litters were used to generate 2 tissue pools for estrus and 2 tissue pools for proestrus, with estrus pool 1 and proestrus pool 1 comprised of mice from the same 3 litters, and estrus pool 2 and proestrus pool 2 comprised of mice from the other 3 litters. Each pool was comprised of uterine tissue RNA from n= 4-6 individual mice. Fluorescent labeling of RNA and hybridization of the Alexa 555-labeled (green) and Alexa 647-labeled (red) RNA samples to Agilent-026655 Whole Mouse Genome Microarray 4x44K v2 (Agilent Technology, Palo Alto, CA; catalog # G4846A) were carried out, with dye swapping for each estrus cycle stage comparison.
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Project description:Mycobacterium abscessus [M. abscessus (sensu lato) or M. abscessus group] comprises three closely related taxa with taxonomic status under revision: M. abscessus sensu stricto, M. bolletii and M. massiliense. We describe here a simple, robust and cost effective PCR-based method for distinguishing among M. abscessus, M. massiliense and bolletii. Based on the M. abscessus ATCC 19977T genome, discriminatory regions were identified between M. abscessus and M. massiliense from array-based comparative genomic hybridization. A typing scheme using PCR primers designed for four of these locations was applied to 46 well-characterized clinical isolates comprising 29 M. abscessus, 15 M. massiliense and 2 M. bolletii previously identified by multi-target sequencing. Interestingly, 2 isolates unequivocally identified as M. massiliense were shown to have a full length erm(41) instead of the expected gene deletion and showed inducible clarithromycin resistance after 14 days. We propose using this PCR-based typing scheme combined with erm(41) PCR for a straightforward identification of M. abscessus, M. massiliense and M. bolletii and assessment of inducible clarithromycin resistance. This method can be easily implemented into a routine workflow providing subspecies level identification within 24 hours of isolation of M. abscessus group. Two-color CGH with 4 independent Mycobacterium clinical isolates and the M massiliense type strain (CCUG 48898) labeled with Cy3 were cohybridized with the M abscessus type strain (ATCC 19977) labeled with Cy5 on a tiling array designed against the M abscessus type strain
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