Serine-arginine protein kinases (SPRKs) are a subfamily of serine-threonine kinases, regulating pre-mRNA splicing in response to extracellular stimuli by phosphorylating serine/arginine (SR)-rich splicing factors. The human genome encodes three SRPK genes, SRPK1, SRPK2 and SRPK3. While SRPK1 has been detected in many human tissues at varying expression levels, SRPK2 and SRPK3 exhibit a more tissue-specific expression. Most of SRPK1 is localized in the cytoplasm where it catalyses SR-domain phosphorylation of splicing-regulating proteins such as SRSFs to facilitate shuttling to the nucleus (Kataoka et al., 1999; Lai et al., 2001; Zhong et al., 2009). This process can be accelerated in response to extracellular stimuli (Nowak et al., 2010). Once in the nucleus, SRPK1 synergizes with other SR protein kinases, such as members of the CLK family of kinases, predominantly localized in the nucleus, to further phosphorylate SR proteins promoting spliceosome assembly (Aubol et al., 2016). During splicing, SR proteins are dephosphorylated by nuclear phosphatases. This highly coordinated process is crucial during development and it is often dysregulated in diseases. Alteration of SRPK expression has been found to induce a large number of aberrant alternative splicing events, leading to tumorigenesis (Corkery et al., 2015).
Merck KGaA in collaboration with the SGC has developed MSC2711186, a potent, cell active chemical probe for SRPK. MSC2711186 shows high in vitro as well as cellular potency for all three SRPK isoforms and does not show any activity towards the CLKs. MSC2711186 is accompanied by a negative control (MSC2705360), which is structurally closely related to the probe molecule.
Potency Against Target Family
MSC2711186 had an IC50 of 2.7 nM, 81 nM, and 0.59 nM to SRPK1, SRPK2 and SRPK3, respectively in the biochemical biochemical activity assay performed at Reaction Biology.
MSC2711186 exhibited a Kd of 145 nM on SRPK2 in ITC.
MSC2711186 was selective in an in vitro kinase panel from Reaction Biology at 1 µM against 395 Kinases, followed by cellular NanoBRET assays.
Based on the potency and the selectivity of the chemical probe and to minimize the risk of unspecific cytotoxicity, we recommend a concentration of no higher than 1 µM for cell-based assays. After 48 hours of 1 µM of compound exposure to human osteosarcoma cells (U2OS) and human embryoic kidney cells (HEK293T), there was no detectable effect on cell viability compared to 0.1 % DMSO. At 10 µM we discovered off-target effects including modulation of tubulin function. Therefore, usage at higher concentrations is not recommended.
MSC2711186 displayed an IC50 of 98 nM on SRPK1 and 40 nM on SRPK3 in intact cells and 44 nM on SRPK1, 149 nM on SRPK2 and 40nM on SRPK3 in lysed cells in NanoBRETTM assay.