Journal: Scientific Reports

Article Title: Development of an orally available inhibitor of CLK1 for skipping a mutated dystrophin exon in Duchenne muscular dystrophy

doi: 10.1038/srep46126

Figure Lengend Snippet: ( a ) TG693 and TG003 chemical structures. ( b ) Pharmacokinetic profile of TG693 after a single 30 mg kg −1 dose administered by subcutaneous injection in imprinting control region (ICR) mice. Data indicate the mean ± SEM (n = 3). ( c ) Recombinant CLK1 was incubated with the substrate peptide in the presence of the indicated concentrations of small molecules. Data represent the means ± SD (n = 3). Representative dose-response curves with Hill slopes are shown. ( d ) TG693 competitive ATP inhibition is shown in Michaelis-Menten (left) and Hanes-Woolf (right) plots. CLK1 kinase activity was measured at the indicated concentrations of TG693 and ATP. Velocity was plotted versus [ATP] and [ATP]/velocity was plotted versus [ATP]. ( e ) Map of the inhibitory activities of TG693 on a kinase dendrogram. Percent inhibition by 1 μM TG693 was measured for a panel of 313 kinases. Red circles indicate the inhibited kinases and are sized according to percent inhibition. The illustration was reproduced courtesy of Cell Signaling Technology, Inc. ( www.cellsignal.com ).

Article Snippet: The reaction mixture containing serially diluted inhibitors, 10 mM MOPS-KOH (pH 6.5), 10 mM magnesium chloride, 200 μM EDTA, 1 μM ATP, 0.167 μCi of [γ- 32 P] ATP, 0.417 μg of synthetic RS peptide, and recombinant GST-tagged human CLK1 (Cat #04–126, Carna Biosciences, Kobe, Japan) was prepared in a final volume of 25 μL.

Techniques: Injection, Recombinant, Incubation, Inhibition, Activity Assay

Journal: Scientific Reports

Article Title: Development of an orally available inhibitor of CLK1 for skipping a mutated dystrophin exon in Duchenne muscular dystrophy

doi: 10.1038/srep46126

Figure Lengend Snippet: ( a ) SR protein phosphorylation was assessed in HeLa cells treated with TG693 and TG003 for 1 h. Lamin B served as a loading control. Uncropped images have been provided in . ( b , c ) Effect of TG693 on exon 31 skipping with the reporter plasmid. Transfected HeLa cells were incubated in the presence of TG693, TG003, or DMSO vehicle for 24 h. Reporter and endogenous CLK1 splicing was then analyzed by RT-PCR. GAPDH served as a control. The Splicing ratios were quantified by intensity analysis and normalized to GAPDH expression. Uncropped images have been provided in and , respectively. Data represent the means ± SD (n = 3).

Article Snippet: The reaction mixture containing serially diluted inhibitors, 10 mM MOPS-KOH (pH 6.5), 10 mM magnesium chloride, 200 μM EDTA, 1 μM ATP, 0.167 μCi of [γ- 32 P] ATP, 0.417 μg of synthetic RS peptide, and recombinant GST-tagged human CLK1 (Cat #04–126, Carna Biosciences, Kobe, Japan) was prepared in a final volume of 25 μL.

Techniques: Plasmid Preparation, Transfection, Incubation, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Scientific Reports

Article Title: Development of an orally available inhibitor of CLK1 for skipping a mutated dystrophin exon in Duchenne muscular dystrophy

doi: 10.1038/srep46126

Figure Lengend Snippet: ( a ) TG693 bioavailability in the tibialis anterior (TA) muscle of ICR mice after oral administration of a single 30 mg kg −1 dose. Data represent the mean ± SEM (n = 3). ( b ) SR protein phosphorylation status in the TA muscle of ICR mice after oral administration. Lamin B served as a loading control. SRSF4 phosphorylation was quantified by densitometry. Uncropped images are provided in . Data represent means ± SD (n = 5). * p < 0.05. ( c,d ) Clk1 expression in the TA muscle, heart and diaphragm were analyzed by RT-PCR with a GAPDH internal control. Uncropped images are provided in and in , respectively. Data represent the means ± SD (n = 3). * p < 0.05.

Article Snippet: The reaction mixture containing serially diluted inhibitors, 10 mM MOPS-KOH (pH 6.5), 10 mM magnesium chloride, 200 μM EDTA, 1 μM ATP, 0.167 μCi of [γ- 32 P] ATP, 0.417 μg of synthetic RS peptide, and recombinant GST-tagged human CLK1 (Cat #04–126, Carna Biosciences, Kobe, Japan) was prepared in a final volume of 25 μL.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: eLife

Article Title: Selective inhibition reveals the regulatory function of DYRK2 in protein synthesis and calcium entry

doi: 10.7554/eLife.77696

Figure Lengend Snippet: ( A ) Chemical structure of C17. ( B ) IC 50 values of C17 against DYRK1A, DYRKIB, DYRK3, Haspin and MARK3. ( C ) Kinome profiling of C17 at 500 nM was carried out using 468 human kinases ( https://www.discoverx.com/ ). ( D ) C17 inhibits Rpt3-Thr25 phosphorylation. HEK293T cells stably expressing FLAG-DYRK2 were treated with the indicated concentrations of C17 for 1 hr. The cells were lysed, and immunoblotting was carried out with the indicated antibodies. Figure 2—source data 1. Raw data of C17 Kinome profiling list for . Figure 2—source data 2. Raw data of western blot for .

Article Snippet: peptide, recombinant protein , GST-MARK3 protein , Carna Biosciences , Cat#: 02–122 , .

Techniques: Stable Transfection, Expressing, Western Blot

Journal: eLife

Article Title: Selective inhibition reveals the regulatory function of DYRK2 in protein synthesis and calcium entry

doi: 10.7554/eLife.77696

Figure Lengend Snippet: ( A–E ) IC 50 of C17 on DYRK2, DYRK1A, DYRK1B, DYRK3, Haspin and MARK3. The IC 50 graph was plotted using GraphPad Prism 8.4.0 software. The results are presented as the percentage of kinase activity relative to the DMSO-treated control. Results are means ± SD for triplicate reactions with similar results obtained in at least one other experiment.

Article Snippet: peptide, recombinant protein , GST-MARK3 protein , Carna Biosciences , Cat#: 02–122 , .

Techniques: Software, Activity Assay

Journal: eLife

Article Title: Selective inhibition reveals the regulatory function of DYRK2 in protein synthesis and calcium entry

doi: 10.7554/eLife.77696

Figure Lengend Snippet:

Article Snippet: peptide, recombinant protein , GST-MARK3 protein , Carna Biosciences , Cat#: 02–122 , .

Techniques: Recombinant, Plasmid Preparation, Generated, Modification, Transfection, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Kinase Assay, Software

Journal: bioRxiv

Article Title: GHB confers neuroprotection by stabilizing the CaMKIIα hub domain

doi: 10.1101/2020.09.28.310474

Figure Lengend Snippet: (A) Approach for target identification using a combination of competitive photoaffinity labeling (PAL) and affinity purification in rat hippocampal homogenate followed by quantitative proteomics. (B) Representative anti-biotin Western blot of hippocampal homogenate after PAL and competition with 2 . (C) Identification of CaMKIIα from LC-MS/MS data as the best hit from non-linear regression analysis for all proteins, and (D) concentration-dependent competition of individual proteins by 2 during PAL. (E) Target validation by [ 3 H]- 1 autoradiography using brain slices from Camk2a and Camk2b wildtype (+/+) and knockout (-/-) mice (cresyl violet staining for tissue visualization). (F-H) Target validation by [ 3 H]- 1 binding to whole cell homogenate from transfected HEK293T cells. (F) [ 3 H]- 1 saturation binding to CaMKIIα ( n = 5), shown is one representative curve; means ± SD). (G) CaMKIIα competition with GHB ( n = 3), 1 ( n = 5) and 2 ( n = 3), pooled data (means ± SEM). (H) Subtype selectivity of [ 3 H]- 1 for CaMKIIα cf. CaMKIIβ/γ/δ. Data are pooled ( n = 3) for each subtype and depicted as specific binding (% of total).

Article Snippet: 6x Hub human protein, WT hub or full-length human recombinant CaMKIIα (#02-109, Carna Biosciences) were immobilized on to a biosensor surfaces by amine coupling using a 20 mM NaAc pH 5 immobilization buffer.

Techniques: Labeling, Affinity Purification, Western Blot, Liquid Chromatography with Mass Spectroscopy, Concentration Assay, Autoradiography, Knock-Out, Staining, Binding Assay, Transfection

Journal: bioRxiv

Article Title: GHB confers neuroprotection by stabilizing the CaMKIIα hub domain

doi: 10.1101/2020.09.28.310474

Figure Lengend Snippet: (A) Schematic of a single CaMKIIα subunit comprised of a kinase domain (gray), regulatory segment (green), linker (yellow), and hub domain (lilac). 12-14 hub domains oligomerize into the holoenzyme, shown here in an activated form with kinase domains displaced from the hub. (B) Concentration-dependent binding of 2 to immobilized CaMKIIα 6x Hub measured by SPR (top), and Langmuir binding isotherm (bottom), representative data. (C) Ball and stick model of key binding residues (bold), nearby residues, and hydrogen bonds in green-dashed lines. (D) X-ray crystal structure of 2 bound to the CaMKIIα 6x (14-mer) Hub. (E) Close-up view of a single hub subunit showing the key molecular interactions, displacement (flip) of Trp403 with ligand bound highlighted. (F) Quenching of intrinsic fluorescence caused by Trp403 flip (6x Hub) with increasing concentrations of 2 ( n = 8), pooled data (means ± SEM). (G) Mutational analysis of key residues in the pocket using [ 3 H]- 1 equilibrium binding to CaMKIIα-HEK293T overexpressing cells. Compared to wildtype (WT), binding is completely obliterated in a construct lacking the hub as well as in R433Q, R453Q, R469Q, triple mutant R433/453/468/Q (RRR→QQQ) and H395A mutants ( n = 3).

Article Snippet: 6x Hub human protein, WT hub or full-length human recombinant CaMKIIα (#02-109, Carna Biosciences) were immobilized on to a biosensor surfaces by amine coupling using a 20 mM NaAc pH 5 immobilization buffer.

Techniques: Concentration Assay, Binding Assay, Fluorescence, Construct, Mutagenesis

Journal: bioRxiv

Article Title: GHB confers neuroprotection by stabilizing the CaMKIIα hub domain

doi: 10.1101/2020.09.28.310474

Figure Lengend Snippet: (A) Right-shifted thermal shift assay melting curves of CaMKIIα WT hub upon binding of GHB, 1 and 2 ( left ) and 1 concentration-dependence (right), representative data. (B) No effect of 1 on Ca 2+ -stimulated Thr286 phosphorylation. Shown is quantification of mean band intensities of Ca 2+ -stimulated pThr286 levels ( left ) normalized to total CaMKIIα expression of cultured cortical neurons (DIV 18-20) incubated with 50-100 μM Ca 2+ alone or together with 3 mM of 1 for 1 h and representative Western blots ( right ). GAPDH was used as loading control (C) Time-( left ) and concentration-dependent effects of 1 ( right ) on cell survival at 24 h in cultured cortical neurons (DIV 16-18) stimulated with 100-200/20 μM Glu/Gly for 1 h (tat- 4 as control). Cell death was normalized to maximum cell death as measured by LDH release. (One-way ANOVA, post-hoc Dunnett’s test). (D) Quantification of GluN2B-CaMKIIα co-localization in hippocampal neurons (DIV 14-19) exposed to Glu 400 μM for 2 min and immediately fixed ( left ) and representative immunostained images ( righ t). For B-D: Number in bar diagrams indicates number of experiments/individual cultures. Box plots (boxes, 25–75%; whiskers, minimum and maximum; lines, median). (One-way ANOVA, post-hoc Dunnett’s test).

Article Snippet: 6x Hub human protein, WT hub or full-length human recombinant CaMKIIα (#02-109, Carna Biosciences) were immobilized on to a biosensor surfaces by amine coupling using a 20 mM NaAc pH 5 immobilization buffer.

Techniques: Thermal Shift Assay, Binding Assay, Concentration Assay, Expressing, Cell Culture, Incubation, Western Blot

Journal: Molecular cancer therapeutics

Article Title: Structure-Based Screen Identification of a Mammalian Ste20-like Kinase 4 (MST4) Inhibitor with Therapeutic Potential for Pituitary Tumors

doi: 10.1158/1535-7163.MCT-15-0703

Figure Lengend Snippet: Predicted MST4 inhibitors as determined by computational modelling

Article Snippet: LANCE (Perkin Elmer) Europium TR-FRET kinase binding assays were performed in white 384-well plates (Perkin Elmer, OptiPlate #6007299) using recombinant MST4 kinase (Carna Biosciences #07-119), ULight PKC substrate (Perkin Elmer #TRF0108), ATP (Sigma Aldrich # {"type":"entrez-protein","attrs":{"text":"A26209","term_id":"93012","term_text":"pir||A26209"}} A26209 ) and LANCE Eu-anti-PKC (Ala25Ser) antibody (Perkin Elmer #TRF0207).

Techniques:

Journal: Molecular cancer therapeutics

Article Title: Structure-Based Screen Identification of a Mammalian Ste20-like Kinase 4 (MST4) Inhibitor with Therapeutic Potential for Pituitary Tumors

doi: 10.1158/1535-7163.MCT-15-0703

Figure Lengend Snippet: Hypoxia model confirms the effects of selected inhibitor candidates on cell survival under severe hypoxia. A, immunoblot analysis of MST4 protein levels in normal pituitary and pituitary adenomas [gonadotrope, prolactin (PRL), ACTH, and growth hormone]. GAPDH was used as a loading control. B, immunoblot analysis shows overexpression of MST4 in pcDNA3-MST4–stable transfectants. C, percentage of nonviable cells in vector control and MST4 cells in the presence of the mammalian target of rapamycin (mTOR) inhibitor PKI-587 (0, 0.1, 1, 10, 100, and 1,000 nmol/L). D, percentage of nonviable cells in vector control and MST4 cells in the presence of the Polo-like kinase 1 (PLK-1) inhibitor volasertib (0, 0.1, 1, 10, 100, and 1,000 nmol/L). E, percentage of nonviable cells in control and MST4 transfectants in the presence of hesperadin (0, 0.1, 1, 10, 100, and 1000 nmol/L). *, P < 0.01; #, P < 0.0001, MST4 transfectants compared with pcDNA3 vector control cells.

Article Snippet: LANCE (Perkin Elmer) Europium TR-FRET kinase binding assays were performed in white 384-well plates (Perkin Elmer, OptiPlate #6007299) using recombinant MST4 kinase (Carna Biosciences #07-119), ULight PKC substrate (Perkin Elmer #TRF0108), ATP (Sigma Aldrich # {"type":"entrez-protein","attrs":{"text":"A26209","term_id":"93012","term_text":"pir||A26209"}} A26209 ) and LANCE Eu-anti-PKC (Ala25Ser) antibody (Perkin Elmer #TRF0207).

Techniques: Western Blot, Over Expression, Plasmid Preparation

Journal: Molecular cancer therapeutics

Article Title: Structure-Based Screen Identification of a Mammalian Ste20-like Kinase 4 (MST4) Inhibitor with Therapeutic Potential for Pituitary Tumors

doi: 10.1158/1535-7163.MCT-15-0703

Figure Lengend Snippet: Hesperadin is identified as a potent inhibitor of MST4. A, the chemical structure of hesperadin. B, small molecule docking depicting the predicted interaction of hesperadin (cyan) with the ATP-binding domain of MST4. C, in vitro TR-FRET recombinant kinase assay demonstrates direct inhibition of the MST4 kinase by hesperadin at low nanomolar concentrations. PKI-587 and volasertib were also screened in the TR-FRET recombinant kinase assay and show no direct inhibition of MST4.

Article Snippet: LANCE (Perkin Elmer) Europium TR-FRET kinase binding assays were performed in white 384-well plates (Perkin Elmer, OptiPlate #6007299) using recombinant MST4 kinase (Carna Biosciences #07-119), ULight PKC substrate (Perkin Elmer #TRF0108), ATP (Sigma Aldrich # {"type":"entrez-protein","attrs":{"text":"A26209","term_id":"93012","term_text":"pir||A26209"}} A26209 ) and LANCE Eu-anti-PKC (Ala25Ser) antibody (Perkin Elmer #TRF0207).

Techniques: Binding Assay, In Vitro, Recombinant, Kinase Assay, Inhibition

Journal: Molecular cancer therapeutics

Article Title: Structure-Based Screen Identification of a Mammalian Ste20-like Kinase 4 (MST4) Inhibitor with Therapeutic Potential for Pituitary Tumors

doi: 10.1158/1535-7163.MCT-15-0703

Figure Lengend Snippet: Hesperadin blocks the effects of MST4 on cell survival under acute hypoxia (1% O2). A, representative immunocytochemistry of apoptotic cells as assessed by TUNEL in the presence or absence of hesperadin (40 nmol/L) under hypoxia for 17 hours. B, rates of apoptosis were expressed as a percentage of TUNEL-positive cells to total cells in the absence or presence of various concentrations of hesperadin (5, 10, 20, and 40 nmol/L). *, P = 0.01 (at 10 nmol/L); #, P = 0.04 (at 20 nmol/L); **, P < 0.001, MST4 transfectants compared with pcDNA3 control cells.

Article Snippet: LANCE (Perkin Elmer) Europium TR-FRET kinase binding assays were performed in white 384-well plates (Perkin Elmer, OptiPlate #6007299) using recombinant MST4 kinase (Carna Biosciences #07-119), ULight PKC substrate (Perkin Elmer #TRF0108), ATP (Sigma Aldrich # {"type":"entrez-protein","attrs":{"text":"A26209","term_id":"93012","term_text":"pir||A26209"}} A26209 ) and LANCE Eu-anti-PKC (Ala25Ser) antibody (Perkin Elmer #TRF0207).

Techniques: Immunocytochemistry, TUNEL Assay

Journal: Molecular cancer therapeutics

Article Title: Structure-Based Screen Identification of a Mammalian Ste20-like Kinase 4 (MST4) Inhibitor with Therapeutic Potential for Pituitary Tumors

doi: 10.1158/1535-7163.MCT-15-0703

Figure Lengend Snippet: Hesperadin blocks the effects of MST4 on proliferation and colony formation under chronic hypoxic stress (5% O2). A, representative immunocytochemistry of the rates of BrdUrd incorporation in pcDNA3 control and MST4 transfectants in the absence and presence of hesperadin (40 nmol/L) under chronic hypoxia (5% O2) for 7 days. B, hesperadin abolishes MST4 increased cell proliferation. In the absence or presence of various doses of hesperadin (0, 5, 10, 20, and 40 nmol/L), cell proliferation was measured by BrdUrd after exposure to chronic hypoxia (5% O2) for 7 days. C, photomicrograph of colony formation in vector and MST4 transfectants incubated with DMSO or hesperadin (20 nmol/L). D, hesperadin decreases the ability of MST4 to promote increased colony formation. Numbers of pcDNA3 and MST4 transfectant colonies were counted after exposure to chronic hypoxia (5%O2) for 7 days. *, P = 0.002; **, P < 0.001, MST4 transfectants compared with pcDNA3 vector cells; #, P < 0.01, MST4 transfectants with hesperadin treatment (20 nmol/L) compared with the cells treated with DMSO.

Article Snippet: LANCE (Perkin Elmer) Europium TR-FRET kinase binding assays were performed in white 384-well plates (Perkin Elmer, OptiPlate #6007299) using recombinant MST4 kinase (Carna Biosciences #07-119), ULight PKC substrate (Perkin Elmer #TRF0108), ATP (Sigma Aldrich # {"type":"entrez-protein","attrs":{"text":"A26209","term_id":"93012","term_text":"pir||A26209"}} A26209 ) and LANCE Eu-anti-PKC (Ala25Ser) antibody (Perkin Elmer #TRF0207).

Techniques: Immunocytochemistry, Plasmid Preparation, Incubation, Transfection

Journal: Molecular cancer therapeutics

Article Title: Structure-Based Screen Identification of a Mammalian Ste20-like Kinase 4 (MST4) Inhibitor with Therapeutic Potential for Pituitary Tumors

doi: 10.1158/1535-7163.MCT-15-0703

Figure Lengend Snippet: A, illustration of MST4 signaling pathways in response to hypoxic stress. B, hesperadin blocks MST4 downstream signaling effectors. Phosphorylation of AKT, p38 MAPK, and ERK were determined by immunoblot in the presence or absence of hesperadin (0, 20, and 40 nmol/L). C, MST4-induced HIF-1 activity is blocked by hesperadin. Control or MST4 cells were transfected with HRE-luciferase reporter constructs. After 24 hours of transfection, cells were subjected to normoxia or hypoxia (1% O2) for 17 hours. HRE-luciferase values were detected and normalized to Renilla control luciferase values. *, P < 0.001, MST4 transfectants compared with pcDNA3 vector cells; #, P = 0.002, MST4 transfectants with hesperadin treatment compared with DMSO-treated controls.

Article Snippet: LANCE (Perkin Elmer) Europium TR-FRET kinase binding assays were performed in white 384-well plates (Perkin Elmer, OptiPlate #6007299) using recombinant MST4 kinase (Carna Biosciences #07-119), ULight PKC substrate (Perkin Elmer #TRF0108), ATP (Sigma Aldrich # {"type":"entrez-protein","attrs":{"text":"A26209","term_id":"93012","term_text":"pir||A26209"}} A26209 ) and LANCE Eu-anti-PKC (Ala25Ser) antibody (Perkin Elmer #TRF0207).

Techniques: Western Blot, Activity Assay, Transfection, Luciferase, Construct, Plasmid Preparation