The University of Toronto's Acceleration Consortium has received a record-breaking $200 million Canada First Research Excellence Fund (CFREF) to advance the discovery of new materials and molecules. This award marks the largest single federal research grant to a university in Canadian history.
By Sofia Melliou
Photo credits: Helen Li
The Structural Genomics Consortium is turning 20 this year and what is a better way to celebrate two decades of open science and innovation than organizing a symposium to highlight the past successes, focus on the present progress and look forward to the future with more ideas towards a common goal; to uncover the dark proteome!
| Probe | Negative control | |
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|  |
LIJTF500025 |
| LIJTF500120 |
LIM kinases belong to the family of cytoplasmic tyrosine-like kinases with dual specificity (serine/threonine and tyrosine). However, known LIMK substrate are usually phosphorylated at serine and threonine residues LIM kinases comprises LIM kinase 1 (LIMK1) and LIM kinase 2 (LIMK2) which show 50% sequence identity in human. Both LIMK1 and LIMK2 present with a unique domain organization containing two N-terminal LIM domains, a PDZ domain, a proline/serine-rich domain and a C-terminal kinase domain [1].
Both proteins are expressed widely in embryonic and adult tissues, but show some cell-type specific expression. Accordingly, the two kinases have overlapping functions, but appear non-redundant. Knockout studies in mice show that LIMK1 is required for development of the central nervous system [2], whereas LIMK2 knockout impairs the activity of testicular germ cells [3].
LIMKs are effectors of cell morphology and motility and apoptosis by regulating the actin cytoskeleton. The LIMKs signal downstream from Rho GTPases and are activated by phosphorylation of the activation loop by upstream kinases, including Rho kinase (ROCK), PAK1/2/4 and MRCKα. The best characterized LIMK substrates are cofilin1 (non-muscle cofilin), cofilin2 (muscle cofilin) and destrin (actin depolymerizing factor, ADF). Phosphorylation of cofilin serine-3 inactivates the actin severing ability promoting F-actin polymerization, stress fibre formation and focal adhesion formation [4].
LIM kinases can shuttle between the cytoplasm and the nuclear compartment of a cell, a process tightly regulated by association with other partners such as p57kip2 and phosphorylation in the activation segment by PAK kinases [1]. Inhibition of LIMK hyper-stabilizes mitotic spindles inducing a G2/M cell cycle block suggesting an important role for these kinases in microtubule dynamics [5].
Increased phosphorylation of LIMK1 has been reported in neurons in areas affected with Alzheimer Disease [6]. LIM kinases play important roles in cancer metastasis like highly invasive prostate and breast cancer, which is reversed by gene silencing [7, 8]. LIMK1 overexpression is also found in malignant melanoma, as well as most tumour cell lines. Other applications for LIMK inhibitors are open-angle glaucoma [9]. In addition, LIMK1 interacts with the long isoform of the type II bonemorphogenetic protein (BMP) receptor contributing to the pathology of Fragile X syndrome, a common inherited form of intellectual disability [10].
Takeda in collaboration with the SGC has developed LIJTF500025, a potent and selective inhibitor for the LIMK1/2. LIJTF500025 binding to LIMK1/2 was confirmed by DSF and ITC. Its crystall structure revealed an allosteric binding mode (Type III) that explains he high selectivity within the humane kinome. The cellular activity was determined by NanoBRET and revealed EC50 values of 82 nM on LIMK1 and 52 nM on LIMK2 was just one additional off-target (RIPK1 EC50 = 6 nM). The chemical probe (LIJTF500025) is accompanied by a negative control (LIJTF500120) that is structurally closely related to the probe molecule.
LIJTF500025 had an KD Value of 37 nM on LIMK1 determined by ITC.
LIJTF500025 was selective in an in-house DSF kinase panel against 107 kinases. In addition, the selectivity was confirmed in the ScanMAX Kinase Panel from Eurofins (DiscoverY) against 468 kinases at a screening concentration of 1 µM.
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.
LIJTF500025 displayed an EC50 of 82 nM, 52 nM and 6.3 nM on LIMK1, LIMK2 and RIPK1 respectively in intact cells in the NanoBRET assay.
| Probe | Negative control | |
|
| |
LIJTF500025 |
| LIJTF500120 |
| Physical and chemical properties LIJTF500025 | |
| Molecular weight | 479.92 |
| Molecular formula | C24H22ClN5O4 |
| IUPAC name | (S)-2-benzyl-6-(8-chloro-5-methyl-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)-7-oxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine-3-carboxamide |
| clogP | 1.92 |
| tPSA | 110.8 |
| No. of chiral centres | 1 |
| No. of rotatable bonds | 4 |
| No. of hydrogen bond acceptors | 9 |
| No. of hydrogen bond donors | 2 |
| Storage | r. t. |
SMILES: CN1C([C@@H](N2CCC3=C(N(N=C3C2=O)CC4=CC=CC=C4)C(N)=O)COC5=CC(Cl)=CC=C51)=O
InChI:InChI=1S/C24H22ClN5O4/c1-28-17-8-7-15(25)11-19(17)34-13-18(23(28)32)29-10-9-16-20(24(29)33)27-30(21(16)22(26)31)12-14-5-3-2-4-6-14/h2-8,11,18H,9-10,12-13H2,1H3,(H2,26,31)/t18-/m0/s1
InChIKey: KIBQDIDFPAQGOU-SFHVURJKSA-N
| Physical and chemical properties LIJTF500120 | |
| Molecular weight | 445,48 |
| Molecular formula | C24H23N5O4 |
| IUPAC name | (S)-1-benzyl-6-(5-methyl-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)-7-oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide |
| clogP | 1.06 |
| tPSA | 108.5 |
| No. of chiral centres | 1 |
| No. of rotatable bonds | 4 |
| No. of hydrogen bond acceptors | 9 |
| No. of hydrogen bond donors | 2 |
| Storage | r. t. |
SMILES: CN1C2=CC=CC=C2OC[C@H](N3CCC4=C(N(CC5=CC=CC=C5)N=C4C(N)=O)C3=O)C1=O
InChI: InChI=1S/C24H23N5O4/c1-27-17-9-5-6-10-19(17)33-14-18(23(27)31)28-12-11-16-20(22(25)30)26-29(21(16)24(28)32)13-15-7-3-2-4-8-15/h2-10,18H,11-14H2,1H3,(H2,25,30)/t18-/m0/s1
InChIKey: NQMLLMFMFLFBAI-SFHVURJKSA-N
Kinome-wide selectivity profile of LIJTF500025 was determined in our in-house kinase DSF-panel comprising 107 kinases and at Eurofins (DiscoverY) at 1 µM comprising 368 kinases.
DSF-Panel against 107 kinases:
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Eurofins DiscoverY (468 kinases) @ 1µM:
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The negative control LIJTF500120 showed no stabilization against 107 kinases screened in our in-house DSF-Panel.
DSF-Panel against 107 kinases:
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LIJTF500025 displayed EC50 values of 82 nM on LIMK1; 52 nM on LIMK2; and 6.3 nM on RIPK1, determined by NanoBRETTM assay.
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The negative control compound LIJTF500120 displayed a EC50 value of > 50 µM on LIMK1/2 and 3.5 µM on RIPK1, determined by NanoBRETTM assay.
LIJTF500025 showed a dose dependent inhibition of the phosphorylation of cofilin in LN229 cells:
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| Probe | Negative control | |
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|  |
CS640 |
| CS640s |
CAMK1D (calcium/calmodulin-dependent protein kinase ID) is a member of the CAMK family of protein kinases. These enzymes play a crucial role in intracellular signaling pathways, and are activated by the binding of calcium ions and calmodulin. CAMK1D is activated has been shown to phosphorylate a variety of substrates, including other kinases and transcription factors.
CAMK1D is widely expressed, and recent studies have suggested that CAMK1D may play a role in the development of triple negative breast cancer is also involved in regulation of insulin release and glucose homeostasis.
Overall, CAMK1D is a multifunctional protein kinase that is involved in a wide range of cellular processes and pathways, making it a potential target for the development of therapeutic drugs for a variety of disease conditions.
| Probe | Negative control | |
|
| |
CS640 |
| CS640s |
Physical and chemical properties CS640 | |
| Molecular weight | 397.527 |
| Molecular formula | C21H31N7O |
| IUPAC name | (S)-2-(3-Aminopiperidin-1-yl)-4-((2,6-diisopropylpyridin-4-yl)amino)pyrimidine-5-carboxamide |
| logP | 2.4992 |
| PSA | 0.29147 |
| No. of chiral centres | 0 |
| No. of rotatable bonds | 6 |
| No. of hydrogen bond acceptors | 8 |
| No. of hydrogen bond donors | 3 |
| Storage | stable as solid and as DMSO solution. Can handle thaw and freeze cycle, but it is not recommended |
| Dissolution | soluble in DMSO in a concentration of 50 mM |
SMILES:
CC(C1=CC(NC2=NC(N3CCC[C@@H](C3)N)=NC=C2C(N)=O)=CC(C(C)C)=N1)C
InChI:
1/C21H31N7O/c1-12(2)17-8-15(9-18(26-17)13(3)4)25-20-16(19(23)29)10-24-21(27-20)28-7-5-6-14(22)11-28/h8-10,12-14H,5-7,11,22H2,1-4H3,(H2,23,29)(H,24,25,26,27)/t14-/m0/s1/f/h25H,23H2
InChIKey:
BWBUPDTUXQDHSX-AWEZNQCLSA-N
Physical and chemical properties CS640s | |
| Molecular weight | 411.554 |
| Molecular formula | C22H33N7O |
| IUPAC name | (S)-2-(3-Aminopiperidin-1-yl)-4-((2,6-diisopropylpyridin-4-yl)amino)-N-methylpyrimidine-5-carboxamide |
| logP | 2.8507 |
| PSA | 0.26643 |
| No. of chiral centres | 0 |
| No. of rotatable bonds | 6 |
| No. of hydrogen bond acceptors | 8 |
| No. of hydrogen bond donors | 3 |
| Storage | stable as solid and as DMSO solution. Can handle thaw and freeze cycle, but it is not recommended |
Selectivity profile of CS640 was determined via the Eurofins DiscoverX screen activity assay and followed up by a IC50 determination at Reaction Biology (RB) and an in house nanoBRET assay.
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| Kinase | Percent of control(%) | RB IC50 (nM) | NanoBRET IC50 (nM) |
| CAMK1D | 0.9 | 8 | 29 |
| CAMK1B | 0 | 3 | 8.2 |
| CAMK1A | 0 | 1 | 23 |
| CAMK1G | 0 | 1 | 55 |
| PIP5K1C | 0.95 | 11200 | - |
| RIPK4 | 4.5 | 5690 | - |
The negative control CS640 with its blocked hinge-binding amine showed no activity on a DSF assay for 100 kinases and low activity on target on NanoBRET assay.
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CS640 shows potent activity on CAMK1D IC50 of 8 nM (ADP-Glow assay) and 11 nM (pCAMK1D phosphorylation assay).
CS640 shows excellent selectivity in a Eurofins DiscoverX screen (468 Kinases). Only minor activity outside the CAMK1 subfamily found. Other possible off-targets from Discover X screen were not confirmed in nanoBRET or WesternBlot assays.
| Kinase | Percent of control(%) | RB IC50 (nM) | NanoBRET IC50 (nM) |
| CAMK1D | 0.9 | 8 | 29 |
| CAMK1B | 0 | 3 | 8.2 |
| CAMK1A | 0 | 1 | 23 |
| CAMK1G | 0 | 1 | 55 |
CS640 shows potent activity on CAMK1D IC50 of 8 nM (ADP-Glow assay) and 11 nM (pCAMK1D phosphorylation assay).
CS640 shows excellent selectivity in a Eurofins DiscoverX screen (468 Kinases). Only minor activity outside the CAMK1 subfamily found. Other possible off-targets from DX screen were not confirmed in orthogonal assay such as nanoBRET or Western Blot.
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The negative control CS640s was tested in an in house kinase DSF Panel, showing no Tm shift on any kinase.
To minimize the chance of off-target effects, we recommend a concentration of no higher than 1 µM for cell-based assays. Some toxicity was found at conc. of 10 µM at some cell lines. For in vivo studies the probe was tested up to 40 mg/kg in in Diet-Induced Obesity in vivo mouse models.
In NanoBRET assay using HEK293T cells CS640 binds all Kinase from the CAMK1 subfamily:
CAMK1A IC50 = 23 nM, CAMK1B IC50 = 8.2 nM, CAMK1D IC50 = 29 nM, CAMK1G IC50 = 55 nM
In the Cerep-Panlabs Saftey Screens, CS640 showed no critical off target effects
Discovery of Highly Selective Inhibitors of Calmodulin-Dependent Kinases That Restore Insulin Sensitivity in the Diet-Induced Obesity in Vivo Mouse Model
J. Med. Chem. 2020, 63, 13, 6784–6801 / https://doi.org/10.1021/acs.jmedchem.9b01803
In vivo pharmacokinetic profile of 19 in male CD-1 mice (25−40 g) and male Crl:CD Sprague−Dawley rats (250−400 g) dosed in 10% DMSO/90% hydroxypropyl-β-cyclodextrin (20% w/v), n = 3 per group. Free cell IC50 calculated by dividing measured cellular IC50 by free fraction in mouse plasma.
Binding mode of CS640 in complex with CAMK1D (PDB 6T28). The inhibitor binds to the ATP pocket as a Typ1 inhibitor.
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| Probe | Negative control | |
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|  |
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.
| Probe | Negative control | |
|
| |
MSC1186 |
| MSC5360 |
Physical and chemical properties MSC1186 | |
| Molecular weight | 461.90 |
| Molecular formula | C19H17ClFN7O2S |
| IUPAC name | N-(3-(((2-(5-chloro-4-fluoro-1H-benzo[d]imidazol-2-yl)pyrimidin-4-yl)amino)methyl)pyridin-2-yl)-N-methylmethanesulfonamide |
| clogP | 3.4 |
| tPSA | 125.1 |
| No. of chiral centres | 0 |
| No. of rotatable bonds | 7 |
| No. of hydrogen bond acceptors | 8 |
| No. of hydrogen bond donors | 2 |
| Storage | r. 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 weight | 442,92 |
| Molecular formula | C20H19ClN6O2S |
| IUPAC name | N-(3-(((2-(5-chloro-1H-indol-2-yl)pyrimidin-4-yl)amino)methyl)pyridin-2-yl)-N-methylmethanesulfonamide |
| clogP | 4.3 |
| tPSA | 112.2 |
| No. of chiral centres | 0 |
| No. of rotatable bonds | 7 |
| No. of hydrogen bond acceptors | 8 |
| No. of hydrogen bond donors | 2 |
| Storage | r. 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
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.
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The negative control MSC5360 showed no activity in biochemical assays and no activity was detected by NanoBRET assays in intact and in lysed cells.
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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.
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.
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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).
Co-crystal structure of SRPK1 in complex with MSC1186 (PDB ID: 7PQS).
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Click here to obtain this probe.
| Probe | Negative control | |
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|  |
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.
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| 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
| 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 | 0 |
| No. of rotatable bonds | 1 |
| No. of hydrogen bond acceptors | 3 |
| No. of hydrogen bond donors | 2 |
| 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 |
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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 | 0 |
| No. of rotatable bonds | 1 |
| No. of hydrogen bond acceptors | 2 |
| No. of hydrogen bond donors | 2 |
| 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
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.
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| 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.
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| 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.
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.
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.
The probe and control may be requested here.
| 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.
The probe may be requested here.
| 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.
| Probe | Negative control | |
|
| |
MRK-952 |
| MRK-952-NC |
[1] https://www.nature.com/articles/s41467-017-01642-w#Fig1
| 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.
| Probe | Negative control | |
|
| |
BAY-805 |
| BAY-728 |
BAY-805 is selective for USP21 in a Ub-Rhodamine assay.
BAY-805 binds USP21 in a HiBiT based target engagement assay with an EC50 = 95 nM.
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
| 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.
| 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 | 0 |
| No. of rotatable bonds | 4 |
| No. of hydrogen bond acceptors | 6 |
| No. of hydrogen bond donors | 1 |
| 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 | 0 |
| No. of rotatable bonds | 5 |
| No. of hydrogen bond acceptors | 6 |
| No. of hydrogen bond donors | 1 |
| 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
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:
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:
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.
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).
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.
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).
