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.

Please refer to other AIDs (1459, 1487, 1498, 1770, 1771, 1795, 1969, 1970, 1983, 2705 and 2710) for detailed assay protocols.

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.

Grant Number: 1R03MH084827-01