MSC1186 A chemical probe for the SRPK family.

The chemical probe is available from Tocris and Sigma.

overview
Probe Negative control

 

MSC1186

 

MSC5360

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 MSC1186, a potent, cell active chemical probe for SRPK. MSC1186 shows high in vitro as well as cellular potency for all three SRPK isoforms and does not show any activity towards the CLKs. MSC1186 is accompanied by a negative control (MSC5360), which is structurally closely related to the probe molecule.

properties
Probe Negative control

 

MSC1186

 

MSC5360

Physical and chemical properties MSC1186

Molecular weight461.90
Molecular formulaC19H17ClFN7O2S
IUPAC nameN-(3-(((2-(5-chloro-4-fluoro-1H-benzo[d]imidazol-2-yl)pyrimidin-4-yl)amino)methyl)pyridin-2-yl)-N-methylmethanesulfonamide
clogP3.4
tPSA125.1
No. of chiral centres0
No. of rotatable bonds7
No. of hydrogen bond acceptors8
No. of hydrogen bond donors2
Storager. t.
Dissolution<0.0010 mg/ml

 

SMILES: CN(C1=NC=CC=C1CNC1=NC(=NC=C1)C1=NC2=C(N1)C=CC(Cl)=C2F)S(C)(=O)=O

InChI: 1S/C19H17ClFN7O2S/c1-28(31(2,29)30)19-11(4-3-8-23-19)10-24-14-7-9-22-17(26-14)18-25-13-6-5-12(20)15(21)16(13)27-18/h3-9H,10H2,1-2H3,(H,25,27)(H,22,24,26)

InChIKey: WBFJDKLBAAHKIL-UHFFFAOYSA-N

Physical and chemical properties MSC5360

Molecular weight442,92
Molecular formulaC20H19ClN6O2S
IUPAC nameN-(3-(((2-(5-chloro-1H-indol-2-yl)pyrimidin-4-yl)amino)methyl)pyridin-2-yl)-N-methylmethanesulfonamide
clogP4.3
tPSA112.2
No. of chiral centres0
No. of rotatable bonds7
No. of hydrogen bond acceptors8
No. of hydrogen bond donors2
Storager. t.

SMILES: CN(S(C)(=O)=O)C1=NC=CC=C1CNC2=NC(C3=CC4=C(C=CC(Cl)=C4)N3)=NC=C2

InChI: InChI=1S/C20H19ClN6O2S/c1-27(30(2,28)29)20-13(4-3-8-23-20)12-24-18-7-9-22-19(26-18)17-11-14-10-15(21)5-6-16(14)25-17/h3-11,25H,12H2,1-2H3,(H,22,24,26)

InChIKey: InChIKey=MXAVRMOXZMBKGE-UHFFFAOYSA-N

selectivity profile

Kinome-wide selectivity profile of MSC1186 was determined at Reaction Biology at 1 µM and on- and off targets were evaluated with in cellulo IC50 with NanoBRET assay.

CLK1 IC50 [nM]

>10000

CLK2 IC50 [nM]

>10000

CLK3 IC50 [nM]

>10000

CLK4 IC50 [nM]

>10000

DYRK1A IC50 [nM]

>10000

DYRK1B IC50 [nM]

>10000

DYRK2 IC50 [nM]

>10000

DYRK3 IC50 [nM]

>10000

The negative control MSC5360 showed no activity in biochemical assays and no activity was detected by NanoBRET assays in intact and in lysed cells.

in vitro potency

Potency Against Target Family

Biochemical assay

MSC1186 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.

ITC

MSC1186 exhibited a Kd of 145 nM on SRPK2 in ITC.

Selectivity

MSC1186 was selective in an in vitro kinase panel from Reaction Biology at 1 µM against 395 Kinases, followed by cellular NanoBRET assays.

Dosage

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.

Cellular Activity

MSC1186 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.

cell based assay data

MSC1186 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.

references

MSC-1186, a Highly Selective Pan-SRPK Inhibitor Based on an Exceptionally Decorated Benzimidazole-Pyrimidine Core. M Schröder et al. J. Med. Chem. 2023. 837–854 https://doi.org/10.1021/acs.jmedchem.2c01705

Martin Schröder, Matthias Leiendecker, Ulrich Grädler, Juliane Braun, Andreas Blumet al. MSC-1186, a Highly Selective Pan-SRPK Inhibitor Based on an Exceptionally Decorated Benzimidazole-Pyrimidine Core. J. Med. Chem. 66, 837−854 (2023).

Kataoka N, Bachorik JL, Dreyfuss G. Transportin-SR, a nuclear import receptor for SR proteins. J Cell Biol. 145:1145–1152 (1999).

Lai MC, Lin RI, Tarn WY. Transportin-SR2 mediates nuclear import of phosphorylated SR proteins. Proc Natl Acad Sci U S A. 98:10154–10159 (2001).

Zhong XY, Ding JH, Adams JA, Ghosh G, Fu XD. Regulation of SR protein phosphorylation and alternative splicing by modulating kinetic interactions of SRPK1 with molecular chaperones. Genes Dev. 23:482–495 (2009).

Nowak DG, Amin EM, Rennel ES, Hoareau-Aveilla C, Gammons M, et al. Regulation of vascular endothelial growth factor (VEGF) splicing from pro-angiogenic to anti-angiogenic isoforms: a novel therapeutic strategy for angiogenesis. J Biol Chem. 285:5532–5540 (2010).

Aubol BE, Wu G, Keshwani MM, Movassat M, Fattet L, et al. Release of SR Proteins from CLK1 by SRPK1: A Symbiotic Kinase System for Phosphorylation Control of Pre-mRNA Splicing. Mol Cell. 63: 218–228 (2016).

Corkery DP, Holly AC, Lahsaee S, Dellaire G. Connecting the speckles: Splicing kinases and their role in tumorigenesis and treatment response. Nucleus . 6:279-88 (2015).

pk properties
co-crystal structures

Co-crystal structure of SRPK1 in complex with MSC1186 (PDB ID: 7PQS).

synthetic schemes
materials and methods

SGC-PIKFYVE-1 Chemical probe for PIKfyve

Click here to obtain this probe.

overview
Probe Negative control

 

SGC-PIKFYVE-1

 

SGC-PIKFYVE-1N

From a library of indolyl pyrimidinamines, we identified a highly potent and cell-active chemical probe (SGC-PIKFYVE-1) that inhibits phosphatidylinositol-3-phosphate 5-kinase (PIKfyve). Comprehensive evaluation of kinome-wide selectivity confirmed that this PIKfyve probe demonstrates excellent selectivity. A structurally similar indolyl pyrimidinamine (SGC-PIKFYVE-1N) was characterized as a negative control that does not inhibit PIKfyve and exhibits exceptional selectivity when profiled broadly. Our PIKfyve chemical probe disrupts multiple phases of the β-coronavirus lifecycle: viral replication and viral entry. Versus published PIKfyve inhibitors, our scaffold is a distinct chemotype that lacks the canonical morpholine hinge-binder of classical lipid kinase inhibitors and has non-overlapping kinase off-targets. Our chemical probe set can be used by the community to further characterize the role of PIKfyve in virology and explore its other roles as well.

Figure 1: Kinome tree with PIKfyve highlighted as a red circle. Illustration is reproduced courtesy of Eurofins DiscoverX (http://treespot.discoverx.com)

Biological activity summary:
• Enzymatic assay (SignalChem): PIKfyve IC50 =  6.9 nM
• Cellular data (NanoBRET): PIKfyve IC50 = 4.0 nM
• Only 8/403 kinases with PoC <10 when screened at 1 μM
 

properties
Probe

SGC-PIKFYVE-1

Physical and chemical properties for SGC-PIKFYVE-1 
Molecular weight 331.4230 
Molecular formula C20H21N5 
IUPAC name 11-(3-(dimethylamino)prop-1-yn-1-yl)-5,6,7,8-tetrahydropyrimido[4',5':3,4]cyclohepta[1,2-b]indol-2-amine 
MollogP 1.52 
PSA 66.01 
No. of chiral centers 
No. of rotatable bonds 
No. of hydrogen bond acceptors 
No. of hydrogen bond donors 
Storage Stable as a solid at room temperature. DMSO stock solutions (up to 10 mM) are stable at -20oC 
Dissolution Soluble in DMSO up to 10 mM 
Negative control

SGC-PIKFYVE-1N

 

Physical and chemical properties for SGC-PIKFYVE-1N 
Molecular weight 304.3970 
Molecular formula C19H20N4 
IUPAC name 12-cyclopropyl-6,7,8,9-tetrahydro-5H-pyrimido[4',5':3,4]cycloocta[1,2-b]indol-2-amine 
MollogP 2.47 
PSA 62.77 
No. of chiral centers 
No. of rotatable bonds 
No. of hydrogen bond acceptors 
No. of hydrogen bond donors 
Storage Stable as a solid at room temperature. DMSO stock solutions (up to 10 mM) are stable at -20oC 
Dissolution Soluble in DMSO up to 10 mM 

SMILES: 

SGC-PIKFYVE-1: CN(C)CC#CC1=CC2=C(C=C1)NC3=C2C4=C(CCC3)C=NC(N)=N4

SGC-PIKFYVE-1N: NC1=NC2=C(C=N1)CCCCC3=C2C4=C(N3)C=CC(C5CC5)=C4 

InChI: 

SGC-PIKFYVE-1: InChI=1S/C20H21N5/c1-25(2)10-4-5-13-8-9-16-15(11-13)18-17(23-16)7-3-6-14-12-22-20(21)24-19(14)18/h8-9,11-12,23H,3,6-7,10H2,1-2H3,(H2,21,22,24) 

SGC-PIKFYVE-1N: InChI=1S/C19H20N4/c20-19-21-10-13-3-1-2-4-16-17(18(13)23-19)14-9-12(11-5-6-11)7-8-15(14)22-16/h7-11,22H,1-6H2,(H2,20,21,23) 

InChIKey: 

SGC-PIKFYVE-1: DORZPJWJOBMQKC-UHFFFAOYSA-N 

SGC-PIKFYVE-1N: XIVFOAKTTGQHLJ-UHFFFAOYSA-N 

selectivity profile

SGC-PIKFYVE-1 was profiled in the KINOMEscan assay against 403 wild-type kinases at 1 μM. Only 8 kinases showed PoC <10 giving an S10(1 μM) = 0.02. When the PoC <35 fraction was examined, 20 kinases were included (S35(1 μM) = 0.05). Potential off-targets within the S35(1 μM) fraction as well as PIKfyve were tested using a NanoBRET target engagement assay and via biochemical enzymatic assays where an assay was available. Data corresponding with off-target kinase activity is shown in the table below.

Kinase DiscoverX PoC value Assay format IC50 or Kd value (nM) 
PIKfyve 0.1 Enzymatic and NB 6.9 and 4.0 
MYLK4 1.8 Enzymatic and NB 66 and 270 
MEK1 4.5 Enzymatic >10000 
RIPK5 5.2 Enzymatic >10000 
IRAK3 9.2 NT 
MEK2 9.6 Enzymatic >10000 
DYRK1A 9.9 Enzymatic 2040 
YSK4 9.9 Enzymatic 4020 
ULK3 11 Enzymatic >10000 
MEK4 12 Enzymatic >10000 
HASPIN 13 Enzymatic 1400 
STK16 14 Enzymatic 560 
CLK2 20 Enzymatic 290 
CDK7 20 Enzymatic >10000 
IRAK4 21 Enzymatic 4500 
AURKB 23 Enzymatic 1400 
DYRK1B 26 Enzymatic 380 
CLK1 30 Enzymatic 420 
RIOK2 32 NT 
CLK4 34 Enzymatic 440 
PIP4K2C 53 NB and binding >10000 and 1900 
MAP4K5 96 Enzymatic and NB 89 and >10000 

Figure 2: SGC-PIKFYVE-1 was profiled in the KINOMEscan assay against 403 wild-type kinases at 1 μM and off-target kinases inhibited PoC <35 were tested in an orthogonal assay. Rows colored green are PIKfyve and the two kinases with enzymatic IC50 values within 30-fold of the PIKfyve enzymatic IC50 value: MYLK4 and MAP4K5. 

SGC-PIKFYVE-1N was also tested in the DiscoverX panel and 1 kinase had a PoC <35 (S35(1 μM) = 0.002). The negative control was sent to SignalChem for testing in enzyme assay for PIKfyve. All results are in the table below. 

Kinase DiscoverX PoC value Assay format IC50 value (nM) 
MAST1 32 NT NT 
PIKfyve 88 Enzymatic and NB 715 and >10000 

Figure 3: SGC-PIKFYVE-1N was profiled in the KINOMEscan assay against 403 wild-type kinases at 1 μM and a follow-up PIKfyve enzymatic assay was done to confirm no activity. 

in vitro potency
cell based assay data

A NanoBRET assay was utilized to assess the binding affinity of SGC-PIKFYVE-1 to PIKfyve. The negative control shows no binding affinity for PIKfyve. 

Figure 4: SGC-PIKFYVE-1 and SGC-PIKFYVE-1N were profiled in the PIKfyve NanoBRET assay. 

references

Drewry, D. H.; Potjewyd, F. M.; Smith, J. L.; Dickmander, R. J.; Bayati, A.; Howell, S.; Taft-Benz, S.; Min, S. M.; Hossain, M. A.; Heise, M.; McPherson, P. S.; Moorman, N. J.; Axtman, A. D. Identification and utilization of a chemical probe to interrogate the roles of PIKfyve in the lifecycle of β-coronaviruses. J Med Chem 2022, ASAP; doi: 10.1021/acs.jmedchem.2c00697. 

Drewry, D. H.; Potjewyd, F. M.; Smith, J. L.; Dickmander, R. J.; Bayati, A.; Howell, S.; Taft-Benz, S. A.; Hossain, M. A.; Heise, M. T.; McPherson, P. S.; Moorman, N. J.; Axtman, A. D. Identification and utilization of a chemical probe to interrogate the roles of PIKfyve in the lifecycle of β-coronaviruses. ChemRxiv 2022. doi: 10.26434/chemrxiv-2022-bj274. 

pk properties
co-crystal structures
synthetic schemes
materials and methods

MRK-990 Dual activity chemical probe for PRMT9 and PRMT5

The probe and control may be requested here.

overview
 
Probe Negative control

 

MRK-990

 

MRK-990-NC

MSD in collaboration with the SGC has developed a dual activity chemical probe MRK-990 for PRMT9 and PRMT5. When used in parallel with selective chemical probes for PRMT5 (e.g., GSK591 and LLY-283), MRK-990 can be used to study the biological role of PRMT9.

MRK-990 inhibits PRMT9 with IC50 = 10 nM and PRMT5 with IC50 = 30 nM in a radioactivity based methyltranserase assay. In an in-cell western assay, MRK-990 inhibits the methylation of SAP145 (PRMT9) with IC50 = 145 nM, and dimethylarginine (PRMT5) with IC50 = 519 nM. MRK-990-NC is a closely related negative control with IC50 > 10 micromolar.

properties
selectivity profile
in vitro potency
cell based assay data
references
pk properties
co-crystal structures
synthetic schemes
materials and methods

MRK-952 Chemical probe for NUDT5

The probe may be requested here.

overview
Probe Negative control

 

MRK-952

 

MRK-952-NC

MSD in collaboration with the SGC has developed a chemical probe MRK-952 for NUDT5. This chemotype complements that of TH5427 as reported by Helleday et al [1]. 

MRK-952 inhibits NUDT5 with IC50 = 85 nM in an AMP-Glo assay with the substrate ADP-ribose. To study the cellular on-target engagement an energy transfer probe (NU074573a) was developed. In the nanoBRET assay MRK-952 inhibited NUDT5 with EC50 = 23.5 ± 4 nM. MRK-952-NC is a closely related negative control with IC50 = 10 micromolar.

properties
Probe Negative control

 

MRK-952

 

MRK-952-NC

selectivity profile
in vitro potency
cell based assay data
references

[1] https://www.nature.com/articles/s41467-017-01642-w#Fig1

pk properties
co-crystal structures
synthetic schemes
materials and methods

BAY-805 Chemical probe for USP21

This probe is available from Sigma and Tocris, and the control may be requested here.

overview
Probe Negative control

 

BAY-805

 

BAY-728

Bayer in collaboration with the SGC has developed a first-in-class chemical probe BAY-805 for USP21. BAY-805 inhibits USP21 with IC50 < 10 nM in HTRF and Ub-Rhodamine assays. BAY-728 is a closely related negative control with IC50 > 12 micromolar.

properties
Probe Negative control

 

BAY-805

 

BAY-728

BAY-805 is selective for USP21 in a Ub-Rhodamine assay.

selectivity profile
in vitro potency
cell based assay data

BAY-805 binds USP21 in a HiBiT based target engagement assay with an EC50 = 95 nM.

references

1. Discovery and Characterization of BAY-805, a Potent and Selective Inhibitor of Ubiquitin-Specific Protease USP21. Fabian Göricke, Victoria Vu, Leanna Smith, Ulrike Scheib, Raphael Böhm, Namik Akkilic, Gerd Wohlfahrt, Jörg Weiske, Ulf Bömer, Krzysztof Brzezinka, Niels Lindner, Philip Lienau, Stefan Gradl, Hartmut Beck, Peter J. Brown, Vijayaratnam Santhakumar, Masoud Vedadi, Dalia Barsyte-Lovejoy, Cheryl H. Arrowsmith, Norbert Schmees, and Kirstin Petersen. https://pubs.acs.org/doi/pdf/10.1021/acs.jmedchem.2c01933

pk properties
co-crystal structures
synthetic schemes
materials and methods

MU1742 Chemical probe for CK1δ and CK1ε protein kinases

The probe is available from Tocris and Sigma.

overview
Probe Negative control

 

MU1742

 

MU2027

Casein kinases 1 (CK1) belong to the family of serine/threonine kinases.1 There are six CK1 isoforms (CK1α, CK1γ1, CK1γ2, CK1γ3, CK1δ, CK1ε) and several splice variants in humans.1,2 All CK1 isoforms share highly conserved kinase domain, with the highest homology displayed by CK1δ and CK1ε.3 They are generally cofactor-independent, and depend on N-terminal acidic and/or phosphorylated amino acids for substrate recognition.4 The cellular activity of CK1 can be regulated by sub-cellular localization, post-translational modifications or interaction with other proteins. The major post-translational modifications comprise activating and inhibitory phosphorylation which can be performed site-selectively by other kinases or via autophosphorylation.1,3 For instance, inhibitory autophosphorylation at C-terminus has been described for all human CK1 isoforms.1,3 It was found that CK1δ forms a dimer, which could possibly have negative regulatory effect on the CK1δ kinase activity in vivo.3 Substrate recognition motifs for CK1 kinases are widely distributed in cellular proteins and more than 140 substrates for CK1 isoforms have been reported, indicating their pleiotropic character.3 CK1 regulates Wnt, Hh and Hippo pathways, which are important for growth, development and homeostasis.3,5,6 Thus, CK1s are involved in the regulation of various cellular states, processes and functions such as chromosome segregation, gene expression, cellular morphology, immune response and inflammation, membrane trafficking, cytokinesis, autophagy, cell stemness and differentiation, cell survival, proliferation and apoptosis.1–3,7 Although CK1 isoforms are similar in their structure and function (especially CK1δ/ε), they have also distinct and specific functions.

SGC has developed in collaboration with Prof. Kamil Paruch (Masaryk University, Brno, Czech Republic) and Prof. Vitezslav Bryja (Masaryk University, Brno, Czech Republic) quality chemical probe MU1742 for CK1δ and CK1ε protein kinases, including the corresponding negative control compound MU2027. The chemical probe MU1742 exhibits excellent kinome-wide selectivity, high potency against CK1δ/ε in vitro and in cellulo. Moreover, MU1742 has suitable PK profile which allows utilization in vivo.

properties
Probe Negative control

 

MU1742

 

MU2027

Physical and chemical properties MU1742  
Molecular weight 408.46 
Molecular formula C22H22F2N6 
IUPAC name 4-(1-((4-fluoro-1-methylpiperidin-4-yl)methyl)-4-(5-fluoropyridin-2-yl)-1H-imidazol-5-yl)-1H-pyrrolo[2,3-b]pyridine 
ClogP 2.39 
PSA 55.59 
No. of chiral centres 
No. of rotatable bonds 
No. of hydrogen bond acceptors 
No. of hydrogen bond donors 
Storage 

Stability not tested.  

Recommendation: 

Long term storage at -20 °C. 

Short term storage at room temperature. 

Dissolution 

It is possible to prepare at least 10 mM DMSO solution. 

For aqueous solutions (in vivo experiments) we recommend to formulate MU1742 as a dihydrochloride salt (.2HCl). 

SMILES: FC1=CN=C(C2=C(N(C=N2)CC3(CCN(CC3)C)F)C4=CC=NC5=C4C=CN5)C=C1

InChI: InChI=1S/C22H22F2N6/c1-29-10-6-22(24,7-11-29)13-30-14-28-19(18-3-2-15(23)12-27-18)20(30)16-4-8-25-21-17(16)5-9-26-21/h2-5,8-9,12,14H,6-7,10-11,13H2,1H3,(H,25,26)

InChIKey: SWOIFXHMBKFCRM-UHFFFAOYSA-N

 

Physical and chemical properties MU2027 
Molecular weight 418.518 
Molecular formula C24H27N6F 
IUPAC name 4-(4-(5-ethylpyridin-2-yl)-1-((4-fluoro-1-methylpiperidin-4-yl)methyl)-1H-imidazol-5-yl)-1H-pyrrolo[2,3-b]pyridine 
ClogP 3.05 
PSA 62.63 
No. of chiral centres 
No. of rotatable bonds 
No. of hydrogen bond acceptors 
No. of hydrogen bond donors 
Storage 

Stability not tested.  

ecommendation: 

 

ong term storage at -20 °C. 

 

hort term storage at room temperature. 

 

Dissolution It is possible to prepare at least 10 mM DMSO solution. 

SMILES: FC1(CN2C(C3=CC=NC4=C3C=CN4)=C(N=C2)C5=NC=C(C=C5)CC)CCN(CC1)C

InChI: InChI=1S/C24H27FN6/c1-3-17-4-5-20(28-14-17)21-22(18-6-10-26-23-19(18)7-11-27-23)31(16-29-21)15-24(25)8-12-30(2)13-9-24/h4-7,10-11,14,16H,3,8-9,12-13,15H2,1-2H3,(H,26,27)

InChIKey: SRSOVGVHVNSXTC-UHFFFAOYSA-N

selectivity profile

The selectivity profile of MU1742 was determined by a kinome-wide screening against 415 protein kinases at 1 µM concentration (Reaction biology). The result shows that only CK1 kinases were strongly inhibited and no off target were observed below the threshold of 40 % residual activity.

Top hits from kinome-wide profiling (415 kinases):

Kinome tree representation of kinome-wide profiling:

in vitro potency

The in vitro potency and selectivity of MU1742 (chemical probe) and MU2027 (negative control compound) was further assessed by a determination of IC50 values (at Reaction Biology, 10 mM ATP conc.) against CK1α, CK1α1L, CK1δ, CK1ε and p38 α which is a common off target of CK1 inhibitors.

Biochemical assay:

*Tested in Reaction Biology

Concentration of ATP: 10 µM

Enzyme conc. used in the assays: CK1α1: 2.5 nM, CK1δ: 15 nM, CK1ε: 45 nM, p38a: 20 nM

The cellular potency of MU1742 was subsequently tested using NanoBRET assay with intact cells (HEK 293), demonstrating in cellulo target engagement. Interestingly, the cellular potency towards CK1α1 was more than 2 orders lower in cellulo than in vitro.

Cellular target engagement assay:

cell based assay data

The cellular activity has been further demonstrated using additional orthogonal assays such as western blotting. The inhibition of CK1δ and CK1ε was monitored via their effect on autophosphorylation. Since CK1δ and CK1ε are functionally redundant, CK1δ- and CK1ε-knock out cell lines were generated and used to differentiate the inhibitory activity on individual isoforms. As a readout for CK1α inhibition, we monitored an effect on B-catenin stabilization, compound BTX-A51 was used as the positive control. MU1742 was tested together with structurally analogous CK1 inhibitors MU1250 and MU1500 which exhibit different isoform selectivity.

In addition, the in cellulo CK1δ/ε inhibition was evaluated via an effect on phosphorylation of DVL3 as the phosphorylation dependent mobility shift on the Western blot. The effect on modulation of Wnt signaling was also confirmed by TopFlash reporter system upon overexpression of DVL3 and CK1ε. Furthermore, the inhibition of Wnt signaling and chemotaxis of leukemic cells by MU1742 was documented using trans-well assay and CCL19 chemokine. The in vivo activity of MU1742 was demonstrated via effect on phosphorylation of DVL2 from lung tissue after per oral administration of MU1742 (100 mg/kg).

The crude cytotoxic effect was tested in JURKAT and HEK 293 cell lines over 24 hours using Alamar blue as an indicator.

references

1. Schittek, B. & Sinnberg, T. Biological functions of casein kinase 1 isoforms and putative roles in tumorigenesis. Mol Cancer 13, 231 (2014).

2. Qiao, Y. et al. Small molecule modulators targeting protein kinase CK1 and CK2. European Journal of Medicinal Chemistry 181, 111581 (2019).

3. Knippschild, U. et al. The CK1 Family: Contribution to Cellular Stress Response and Its Role in Carcinogenesis. Front. Oncol. 4, (2014).

4. Behrend, L. Interaction of casein kinase 1 delta (CK1d) with post-Golgi structures, microtubules and the spindle apparatus. European Journal of Cell Biology 79, 240–251 (2000).

5. Yang, K. et al. The evolving roles of canonical WNT signaling in stem cells and tumorigenesis: implications in targeted cancer therapies. Lab Invest 96, 116–136 (2016).

6. Jiang, J. CK1 in Developmental Signaling. in Current Topics in Developmental Biology vol. 123 303–329 (Elsevier, 2017).

7. Jiang, S., Zhang, M., Sun, J. & Yang, X. Casein kinase 1α: biological mechanisms and theranostic potential. Cell Commun Signal 16, 23 (2018).

pk properties

The in vivo pharmacokinetic (PK) profile of MU1742 has been evaluated in mice. After a PO administration of 20 mg/kg of MU1742 .2HCl (formulated as dihydrochloride salt), compound exhibits 57 % PO bioavailability and relatively stable plasma concentrations. Additional PK parameters are summarized in the table, indicating that MU1742 is a suitable chemical probe for in vivo experiments.

co-crystal structures

Several analogues, which are structurally similar to MU1742, have been successfully co-crystalized with CK1δ. All structurally related compounds share a very similar binding mode which is characterized by, for instance, the hinge interaction with the pyrrolopyridine moiety, the back pocket interaction with the (hetero)aryl moiety and the interaction with water molecule (in green). Based these observations we assume that MU1742 interacts analogously with CK1α/δ/ε. One example of the co-crystal structure with early lead-CK1δ co-crystal structure is located below (PDB: 7QRA).

synthetic schemes
materials and methods
03.08.2022

New federation aims to transform biology with protein tools spanning proteome by 2035

by: SGC

SGC CEO, Aled Edwards, and Cheryl Arrowsmith, Chief Scientist for the Structural Genomics Consortium (SGC) Toronto laboratories are featured in a piece by the Institute for Protein Innovation discussing Target 2035- a remarkable initiative connecting scientists globally in the quest to study every human protein by 2035.

05.07.2022

A conversation on using chemical probes to study protein function in cells and organisms

by: SGC

Cheryl Arrowsmith, Chief Scientist for the Structural Genomics Consortium (SGC) Toronto laboratories, and Paul Workman, Professor of Pharmacology and Therapeutics at the Institute of Cancer Research (ICR) in London, talked to Nature Communications about chemical probes, their respective paths to leadership positions in the field, the online resources available to those interested in the topic and the promise and value of open — collaborative — science.

16.05.2022

Understanding antitumor activity opens new possibilities for future breast cancer treatments

by: SGC

Toronto, May 16, 2022 – An article published today in the journal Nature Chemical Biology reveals a mechanism of antitumor activity for triple negative breast cancer, which is the most aggressive breast cancer subtype with less promising prognosis and with few effective therapies.

08.03.2022

Structural Genomics Consortium Welcomes New Members - Bristol Myers Squibb, Genentech and Janssen

by: SGC

Toronto, March  8, 2022 - The Structural Genomics Consortium (SGC) is pleased to announce three new members of the open science consortium: Bristol Myers Squibb, Genentech, a member of the Roche Group, and Janssen Pharmaceutica NV.