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BioAssay: AID 1997

qHTS for Inhibitors of CDC-like Kinase 4: Summary

Alternative splicing of pre-mRNAs plays a major role in the regulation of eukaryotic gene expression. Alternative splicing is now thought to be one of the primary reasons for the complexity of higher organisms leading to an average of three protein isoforms per gene. Aberrant splicing can lead to a number of diseases and is known to play a role in rare genetic diseases such as progeria syndrome. more ..
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 Tested Compounds
 Tested Compounds
All(29)
 
 
Probe(3)
 
 
Active(29)
 
 
 Tested Substances
 Tested Substances
All(29)
 
 
Probe(3)
 
 
Active(29)
 
 
AID: 1997
Data Source: NCGC (CLK905)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-10-20
Modify Date: 2011-09-07

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: Chemical Probe: 3    Active: 29
Related Experiments
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AIDNameTypeComment
1770qHTS Assay for Inhibitors of CDC-like Kinase 4 (Kinase-Glo Assay)Confirmatorydepositor-specified cross reference: Clk4 inhibition primary assay (Kinase-Glo)
1771qHTS Assay for Inhibitors of CDC-like Kinase 4 (ADP-Glo Assay)Confirmatorydepositor-specified cross reference: Clk4 inhibition primary assay (ADP-Glo)
1795qHTS Assay for Inhibitors of CDC-like Kinase 4 (ADP-FP Assay)Confirmatorydepositor-specified cross reference: Clk4 inhibition primary assay (ADP-FP)
1969Confirmaiton Assay for Inhibitors of CDC-like Kinase 4 (ADP-Glo Assay)Confirmatorydepositor-specified cross reference: Clk4 inhibition conformation assay (ADP-Glo)
1970Confirmation Assay for Inhibitors of CDC-like Kinase 4 (Kinase-Glo Assay)Confirmatorydepositor-specified cross reference: Clk4 inhibition conformation assay (ATP-Glo)
1983Confirmation Assay for Inhibitors of CDC-like Kinase 4 (ADP-FP Assay)Confirmatorydepositor-specified cross reference: Clk4 inhibition conformation assay (ADP-FP)
2705Assay for Inhibitors of Dual-Specificity Tyrosine-(Y)-Phosphorylation Regulated Kinase 1A (Kinase-Glo assay)Confirmatorydepositor-specified cross reference: Clk4 inhibition secondary assay: Dyrk1a Kinase-Glo assay
2710Kinase Inhibition Profile Study on Inhibitors of CDC-like Kinase 4Otherdepositor-specified cross reference: Clk4 inhibition secondary assay: Reaction Biology selectivity panel assay
493204Confirmation Assay for Inhibitors of CDC-like Kinase 4 (Kinase-Glo Assay): SAR round 2Confirmatorydepositor-specified cross reference
493206Assay for Inhibitors of Dual-Specificity Tyrosine-(Y)-Phosphorylation Regulated Kinase 1A (Kinase-Glo assay): round 2 SARConfirmatorydepositor-specified cross reference: Dyrk 1A Panel Assay
504421Kinase Inhibition Study on Inhibitors of CDC-like Kinase 4 (Reaction Biology data): SAR round 2Confirmatorydepositor-specified cross reference
504424Kinase Inhibition Study on Inhibitors of Dyrk1a (Reaction Biology data)Confirmatorydepositor-specified cross reference
504427Kinase Inhibition Study on Inhibitors of CDC-like Kinase 2 (Reaction Biology data)Confirmatorydepositor-specified cross reference
504428Kinase Inhibition Study on Inhibitors of CDC-like Kinase 3 (Reaction Biology data)Confirmatorydepositor-specified cross reference
504429Kinase Inhibition Study on Inhibitors of Dyrk1b (Reaction Biology data)Confirmatorydepositor-specified cross reference
504430Kinase Inhibition Study on Inhibitors of CDC-like Kinase 1 (Reaction Biology data)Confirmatorydepositor-specified cross reference
504534Secondary Assay for Inhibitors of Human Cdc-like kinase 4 (Clk4): HEK-293 Cell Viability AssayConfirmatorydepositor-specified cross reference
624034Reaction Biology data for inhibitors of CLK4: Probe SARConfirmatorydepositor-specified cross reference
624045Reaction Biology data for inhibitors of DYRK1A: Probe SARConfirmatorydepositor-specified cross reference
624046Reaction Biology data for inhibitors of DYRK1B: Probe SARConfirmatorydepositor-specified cross reference
624047Reaction Biology data for inhibitors of CLK1: Probe SARConfirmatorydepositor-specified cross reference
624048Reaction Biology data for inhibitors of CLK2: Probe SARConfirmatorydepositor-specified cross reference
624049Reaction Biology data for inhibitors of CLK3: Probe SARConfirmatorydepositor-specified cross reference
624093Confirmation Assay for Inhibitors of Dyrk1a (Kinase-Glo Assay): KU probeConfirmatorydepositor-specified cross reference
624094Confirmation Assay for Inhibitors of Clk4 (Kinase-Glo Assay): KU probeConfirmatorydepositor-specified cross reference
Description:
NIH Molecular Libraries Probe Production Centers Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLPCN Grant: 1R03MH084827-01
Assay Submitter (PI): Tom Misteli

Alternative splicing of pre-mRNAs plays a major role in the regulation of eukaryotic gene expression. Alternative splicing is now thought to be one of the primary reasons for the complexity of higher organisms leading to an average of three protein isoforms per gene. Aberrant splicing can lead to a number of diseases and is known to play a role in rare genetic diseases such as progeria syndrome. Hutchinson-Gilford Progeria Syndrome (HGPS) is a pediatric premature aging disease caused by a spontaneous mutation in the lamin A/C (LMNA) gene due to a single point mutation in the lamin A gene effecting 1 in 4 million people (1).
To identify both specific splicing modulators of the lamin A gene as well as general splicing modulators, a cell-based HTS assay was developed using a minigene reporter system (AIDs, 1459 & 1487). A quinazoline (CID: 16759567) showed weak activity as an inhibitor of aberrant splicing using the minigene reporter system (AID 1498). A preliminary kinase profile of this compound showed activity in splicing related kinases belonging to the CDC-like kinase (Clk) family particularly the Clk4. This AID summarizes the characterization of Clk4 inhibitors in three different assays and a protein kinase selectivity panel.
The quinazoline compound (CID_3232621, ML106) and the pyrimidine compound (CID_3234998, ML105) inhibit Clk4 with an IC50 = 0.06 uM. Inhibition was confirmed using purified Clk4 (Invitrogen, CA) employing three different assays: fluorescent polarization based detection of the ADP product (AID 1983), bioluminescent detection of ATP depletion (AID 1970), and bioluminescent detection of the ADP product (AID 1969; for further description of the bioluminescent assays see ref 1). Selectivity against other protein kinases was determined at Ambit Biosciences against 417 kinases. We also submitted TG003 to Ambit, which is the only other small molecule reported to inhibit Clk4 with a similar potency (2). TG003 was determined to have Kds of 19 nM, 95 nM, and 30 nM versus Clk1, Clk2, and Clk4, respectively. The Kd for TG003 versus Clk3 was 3 uM. It was also found that TG003 had activity versus CSNK1D (150 nM), CSNK1E (300 nM), Dyrk1A (12 nM), Dyrk1B (130 nM), PIM1 (130 nM), PIM3 (280 nM), and 170 Ysk4 (290 nM). The novel quinazoline CID 3232621 was found to have Kds of 37 nM, 50 nM and 27 nM versus Clk1, Clk4, and Dyrk1A, respectively. The only other locus of relevant activity (below 500 nM) was found for binding to the endothelial growth factor receptor (EGFR) (Kd = 230 nM; see ref 3).

A new probe compound (CID: 44968231, ML167) inhibits Clk4 with an IC50 = 0.14uM was identified later, it is selective against a panel of these related kinases: Clk1, 2 & 3, DYRK1/DYRK1A, and DYRK1B.
Protocol
Please refer to other AIDs (1459, 1487, 1498, 1770, 1771, 1795, 1969, 1970, 1983, 2705 and 2710) for detailed assay protocols.
Comment
This summary is written for the purposes of summarizing the activities of the chemical probe, identified in this project. MLSCN probes are given a score of 100. Less active molecules in the same chemical series as the probe molecules are given a score of 50.
Categorized Comment - additional comments and annotations
From MLP Probe Report:
Probe count: 4
MLP Probe ML# for probe 1: ML106
PubChem Substance ID (SID) for probe 1: 4237508
PubChem Compound ID (CID) for probe 1: 3232621
Probe type for probe 1: Modulator
IC50/EC50 (nM) for probe 1: 80
Target for probe 1: Clk4 (gi: 10190706)
Anti-target for probe 1: >400 kinases
Fold selectivity for probe 1: 0.014 selectivity score
NCBI Book chapter link for probe 1: http://www.ncbi.nlm.nih.gov/books/NBK47344/ (ID: 2358794)
Grant number for probe 1: MH084827-01
MLP Probe ML# for probe 2: ML167
PubChem Substance ID (SID) for probe 2: 90944997
PubChem Compound ID (CID) for probe 2: 44968231
Probe type for probe 2: Inhibitor
IC50/EC50 (nM) for probe 2: 136
Target for probe 2: Cdc2- like kinase 4 (Clk4) (gi: 10190706)
Disease relevance for probe 2: Gene splicing
Anti-target for probe 2: Clk1, Clk2, Clk3, Dyrk1A/1 B
Fold selectivity for probe 2: >10
NCBI Book chapter link for probe 2: http://www.ncbi.nlm.nih.gov/books/NBK56236/ (ID: 2509966)
Grant number for probe 2: MH084827-01
MLP Probe ML# for probe 3: ML315
PubChem Substance ID (SID) for probe 3: 124950388
PubChem Compound ID (CID) for probe 3: 46926514
Probe type for probe 3: Inhibitor
IC50/EC50 (nM) for probe 3: 68
Target for probe 3: Clk4 (gi: 10190706)
Disease relevance for probe 3: Down syndrome
Anti-target for probe 3: Clk1; Clk2; Clk3; Dyrk1A; Dyrk1B
Fold selectivity for probe 3: 1; 3.4; >147; 4.1; 17
Grant number for probe 3: MH084827
MLP Probe ML# for probe 4: ML105
PubChem Substance ID (SID) for probe 4: 4239891
PubChem Compound ID (CID) for probe 4: 3234998
Probe type for probe 4: Modulator
NCBI Book chapter link for probe 4: http://www.ncbi.nlm.nih.gov/books/NBK47344/ (ID: 2358794)
PubMed Publication ID (PMID) for probe 2: 19837585
PubMed Publication ID (PMID) for probe 3: 23642479
NCBI Book chapter title for probe 1: A high-throughput screen for pre-mRNA splicing modulators
NCBI Book chapter title for probe 2: An inhibitor of the Cdc2-like kinase 4 (Clk4)
NCBI Book chapter title for probe 4: A high-throughput screen for pre-mRNA splicing modulators
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1ML#Molecular library probe numberString
2PhenotypeIndicates type of activity observed based on the compound activity across various assays tested.String
3Potency ATP depletion_Clk4The concentration of sample yielding half-maximal inhibition in Kinase-glo assay in detection of ATP depletion.FloatμM
4Potency ADP formation_Clk4The concentration of sample yielding half-maximal inhibition in ADP-glo assay in detection of ADP product.FloatμM
5Potency ADP-FP_Clk4The concentration of sample yielding half-maximal inhibition in ADP-FP assay in fluorescent polarization based detection of the ADP product.FloatμM
6Potency ATP depletion_Dyrk1aThe concentration of sample yielding half-maximal inhibition for Dyrk1a in Kinase-glo assay in detection of ATP depletion.FloatμM
7Potency_RB_Clk4The concentration of sample yielding half-maximal inhibition for Clk4 in Reaction Biology panel assay.FloatμM
8Potency_RB_Clk1The concentration of sample yielding half-maximal inhibition for Clk1 in Reaction Biology panel assay.FloatμM
9Potency_RB_Clk2The concentration of sample yielding half-maximal inhibition for Clk2 in Reaction Biology panel assay.FloatμM
10Potency_RB_Clk3The concentration of sample yielding half-maximal inhibition for Clk3 in Reaction Biology panel assay.FloatμM
11Potency_RB_Dyrk1aThe concentration of sample yielding half-maximal inhibition for Dyrk1a in Reaction Biology panel assay.FloatμM
12Potency_RB_Dyrk1bThe concentration of sample yielding half-maximal inhibition for Dyrk1b in Reaction Biology panel assay.FloatμM
Additional Information
Grant Number: 1R03MH084827-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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