Toronto, February 22, 2022 – The roadmap for CACHE, an open science benchmarking initiative to enable the development of computational methods for hit-finding, has been published in Nature Reviews Chemistry.
The probe is available at Sigma and Cayman Chemical.
The negative control ist available at Sigma.
| Probe | Negative control | |
 |
|  |
M4K2234 |
| M4K2234NC |
ALK1/2 are receptor serine/threonine protein kinases belonging to a group of tyrosine kinase-like kinases (TLK) and it are encoded by ACVRL1 and ACVR1 genes. It is composed of extracellular domain, transmembrane domain, glycine-serine rich (GS) domain and kinase domain.1,2 ALK1/2, as well as others related kinases ALK1-7, are so-called transforming growth factor β (TGF-β) type I receptors. ALK1-7 kinases form hetero tetrameric complexes with TGF-β type II receptors that are stabilized by corresponding ligands from TGF-β superfamily. Upon formation of hetero-tetrameric complex and binding of ligand, constitutively active TGF-β type II receptors phosphorylate TGF-β type I receptors (ALK1-7) on several Ser/Thr residues of GS domain which leads to stabilization of kinase domain in the active state.2,3 ALKs mediate SMAD independent as well as SMAD depended signaling pathways. ALK2 has emerged in the literature as a promising therapeutic target for treatment of fibrodysplasia ossificans progressiva (FOP), diffuse intrinsic pontine glioma (DIPG)1,4 and, more recently, also multiple sclerosis (MS).5,6
SGC has developed in collaboration with M4K Pharma, OICR and the Oxford University the chemical probe M4K2234 for ALK1 and ALK2 protein kinases, including the corresponding negative control compound M4K2234NC. Probe compound M4K2234 exhibits potent inhibitory activity against ALK1/2 in vitro as well as in cellulo. M4K2234 has a favorable in vivo ADMET profile, therefore, is promising chemical probe also for in vivo applications.
| Probe | Negative control | |
|
| |
M4K2234 |
| M4K2234NC |
| Physical and chemical properties M4K2234 | |
| Molecular weight | 462.57 |
| Molecular formula | C27H31FN4O2 |
| IUPAC name | 2-fluoro-4-(5-(4-(4-isopropylpiperazin-1-yl)phenyl)-4-methylpyridin-3-yl)-6-methoxybenzamide |
| ClogP | 3.9831 |
| PSA | 71.69 |
| No. of chiral centres | 0 |
| No. of rotatable bonds | 6 |
| No. of hydrogen bond acceptors | 6 |
| No. of hydrogen bond donors | 1 |
| Storage | Stability not tested. Recommendation: -20 °C for long term storage. |
| Dissolution | 10 mM DMSO solution is possible |
SMILES: O=C(N)C1=C(OC)C=C(C2=C(C)C(C3=CC=C(N4CCN(C(C)C)CC4)C=C3)=CN=C2)C=C1F
InChI: InChI=1S/C27H31FN4O2/c1-17(2)31-9-11-32(12-10-31)21-7-5-19(6-8-21)22-15-30-16-23(18(22)3)20-13-24(28)26(27(29)33)25(14-20)34-4/h5-8,13-17H,9-12H2,1-4H3,(H2,29,33)
InChIKey: RIWTUJFFSTVYPW-UHFFFAOYSA-N
| Physical and chemical properties M4K2234NC | |
| Molecular weight | 516.659 |
| Molecular formula | C31H37FN4O2 |
| IUPAC name | (2-fluoro-4-(5-(4-(4-isopropylpiperazin-1-yl)phenyl)-4-methylpyridin-3-yl)-6-methoxyphenyl)(pyrrolidin-1-yl)methanone |
| ClogP | 5.3921 |
| PSA | 48.91 |
| No. of chiral centres | 0 |
| No. of rotatable bonds | 6 |
| No. of hydrogen bond acceptors | 6 |
| No. of hydrogen bond donors | 0 |
| Storage | Stability not tested. Recommendation: -20 °C for long term storage. |
| Dissolution | 10 mM DMSO solution is possible |
SMILES: O=C(N1CCCC1)C2=C(OC)C=C(C3=C(C)C(C4=CC=C(N5CCN(C(C)C)CC5)C=C4)=CN=C3)C=C2F
InChI: InChI=1S/C31H37FN4O2/c1-21(2)34-13-15-35(16-14-34)25-9-7-23(8-10-25)26-19-33-20-27(22(26)3)24-17-28(32)30(29(18-24)38-4)31(37)36-11-5-6-12-36/h7-10,17-21H,5-6,11-16H2,1-4H3
InChIKey: WFJOMSKCDAOJBT-UHFFFAOYSA-N
Selectivity profile of M4K2234 was determined by kinome-wide screening against 375 protein kinases at 1 µM concentration (Reaction Biology). M4K2234 inhibits only ALK1/2 and one additional off target (TNIK), when threshold of 25 % residual enzyme activity is applied.
  |
In vitro selectivity was subsequently refined by determination of IC50 values (at Reaction Biology, at 10 mM ATP conc.) against ALK1-6 and the most significant off target (TNIK) from the kinome-wide screening.
| M4K2234 | ||
| Kinase | IC50 (nM) | Selectivity |
| ALK1/ACVRL1 | 7 | 0.5 |
| ALK2/ACVR1 | 14 | 1.00 |
| ALK3/BMPR1A | 168 | 12 |
| ALK4/ACVR1B | 1660 | 119 |
| ALK5/TGFBR1 | 1950 | 375 |
| ALK6/BMPR1B | 88 | 6.3 |
| TNIK | 41 | 2.9 |
The high potency of M4K2234 towards ALK2 has also been demonstrated in cell based target engagement assays with an IC50 of 83nM for ALK1 and 13nM for ALK2. Importantly, M4K2234 exhibited only very weak potency against ALK4/5.
Cellular activity has been demonstrated on modulation of SMAD phosphorylation by Western blotting. M4K2234 affects phosphorylation of SMAD1/5/8 that corresponds to BMP branch of signalling which is mediated, besides others, also by ALK1/2 kinases. On the other hand, M4K2234 has only a very weak effect on SMAD2/3 phosphorylation that corresponds to TGF beta branch of signalling which is mediated mostly via ALK4/5/7. This observation is consistent with NanoBRET cellular target engagement assay.
Cytotoxicity was evaluated after 24 hour incubation with U2OS cell line and using alamar blue as staning agent. Cytotoxic effect have been observed above 50 µM concentration. Considering the cellular activity towards the main targets (ALK1/2), we recomend to use the probe at 1 µM concentration as the maximum concentration in cellular assays.
M4K2234 exhibits comparable potency towards mutant variants as well as towards wild type ALK2 kinase.
Potency against mutant variants is further documented in grow inhibition assays with DIPG patient-derived cell lines containing ALK2 mutation.
1. Cao, H. et al. Differential kinase activity of ACVR1 G328V and R206H mutations with implications to possible TβRI cross-talk in diffuse intrinsic pontine glioma. Sci Rep 10, 6140 (2020).
2. Schmierer, B. & Hill, C. S. TGFβ–SMAD signal transduction: molecular specificity and functional flexibility. Nat Rev Mol Cell Biol 8, 970–982 (2007).
3. Chaikuad, A. et al. Structure of the Bone Morphogenetic Protein Receptor ALK2 and Implications for Fibrodysplasia Ossificans Progressiva. Journal of Biological Chemistry 287, 36990–36998 (2012).
4. Sekimata, K., Sato, T. & Sakai, N. ALK2: A Therapeutic Target for Fibrodysplasia Ossificans Progressiva and Diffuse Intrinsic Pontine Glioma. Chem. Pharm. Bull. 68, 194–200 (2020).
5. Eixarch, H., Calvo-Barreiro, L., Montalban, X. & Espejo, C. Bone morphogenetic proteins in multiple sclerosis: Role in neuroinflammation. Brain, Behavior, and Immunity 68, 1–10 (2018).
6. Sotiropoulos, M. G. & Chitnis, T. Opposing and potentially antagonistic effects of BMP and TGF-β in multiple sclerosis: The “Yin and Yang” of neuro-immune Signaling. Journal of Neuroimmunology 347, 577358 (2020).
In vivo pharmacokinetic (PK) profile of M4K2234 has been evaluated in mice. After PO administration of 10 mg/kg, compound exhibits PK parameters that are summarized in the table, indicating that M4K2234 is a suitable chemical probe for in vivo application.
Of note, M4K2234 has much lower brain penetrance than the chemical probe MU1700 (mice, PO, 100 mg/kg).
| M4K2234 |
In Vitro ADMET |
|
MLM (% @ 1h) | 87 |
HLM (% @ 1h) | 93 |
Caco-2 AB (x10-6 cm/s) | 6 |
Caco-2 BA/AB | 2.65 |
CYP inhibition (5 isoforms), IC50 | > 10 µM |
hERG inhibition, IC50 | >30 µM |
In Vivo ADME/PK (mice) |
|
Dose | 10 mg/kg |
Bioavailability | 71 % |
Cl (ml/min/kg) | 31 (PO) |
T1/2 | 1.3 h |
Cmax (PO) | 3000 nM |
Vss (L/kg) | 3 |
Cbrain/Cplasma (4h, 100 mg/kg) | 0.13 |
A close analogue of M4K2234 has been successfully co-crystalized with ALK2 (PDB:6T6D).
 |
| PDB: 6T6D (analogue of M4K2149) |
| Probe | Negative control | |
 |
|  |
MU1700 |
| MU1700NC |
ALK1/2 are receptor serine/threonine protein kinases belonging to a group of tyrosine kinase-like kinases (TLK) and it are encoded by ACVRL1 and ACVR1 genes. It is composed of extracellular domain, transmembrane domain, glycine-serine rich (GS) domain and kinase domain.1,2 ALK1/2, as well as others related kinases ALK1-7, are so-called transforming growth factor β (TGF-β) type I receptors. ALK1-7 kinases form hetero tetrameric complexes with TGF-β type II receptors that are stabilized by corresponding ligands from TGF-β superfamily. Upon formation of hetero-tetrameric complex and binding of ligand, constitutively active TGF-β type II receptors phosphorylate TGF-β type I receptors (ALK1-7) on several Ser/Thr residues of GS domain which leads to stabilization of kinase domain in the active state.2,3 ALKs mediate SMAD independent as well as SMAD depended signaling pathways. ALK2 has emerged in the literature as a promising therapeutic target for treatment of fibrodysplasia ossificans progressiva (FOP), diffuse intrinsic pontine glioma (DIPG)1,4 and, more recently, also multiple sclerosis (MS).5,6
SGC has developed in collaboration with Prof. Kamil Paruch (Masaryk University, Brno, Czech Republic) quality chemical probe MU1700 for ALK1 and ALK2 protein kinases, including the corresponding negative control compound MU1700NC. Probe compound MU1700 exhibits potent inhibitory activity against ALK1/2 in vitro and in cellulo. MU1700 has favorable in vivo profile and exceptionally high brain penetrance. Therefore, MU1700 is promising chemical probe also for in vivo applications.
| Probe | Negative control | |
|
| |
MU1700 |
| MU1700NC |
Formulation: It is recommended to use MU1700 and MU1700NC in a salt form (e.g. .2HCl) since free bases have limited solubility.
Physical and chemical properties MU1700 (free base) | |
| Molecular weight | 406.49 |
| Molecular formula | C26H22N4O |
| IUPAC name | 6-(4-(piperazin-1-yl)phenyl)-3-(quinolin-4-yl)furo[3,2-b]pyridine |
| ClogP | 4.39 |
| PSA | 54.19 |
| No. of chiral centres | 0 |
| No. of rotatable bonds | 3 |
| No. of hydrogen bond acceptors | 5 |
| No. of hydrogen bond donors | 1 |
| Storage | Stability not tested. Recommendation: -20 °C for long term storage. |
| Dissolution | Free base: ca. 1 mM DMSO solution possible Salt form (.2HCl): 10 mM DMSO solution possible |
SMILES: C12=C(N=CC=C2C3=COC4=C3N=CC(C5=CC=C(N6CCNCC6)C=C5)=C4)C=CC=C1
InChI: InChI=1S/C26H22N4O/c1-2-4-24-22(3-1)21(9-10-28-24)23-17-31-25-15-19(16-29-26(23)25)18-5-7-20(8-6-18)30-13-11-27-12-14-30/h1-10,15-17,27H,11-14H2
InChIKey: FFLJVBPCONQSQW-UHFFFAOYSA-N
Physical and chemical properties MU1700NC (free base) | |
| Molecular weight | 406.49 |
| Molecular formula | C26H22N4O |
| IUPAC name | 6-(4-(piperazin-1-yl)phenyl)-3-(quinolin-8-yl)furo[3,2-b]pyridine |
| ClogP | 4.39 |
| PSA | 54.19 |
| No. of chiral centres | 0 |
| No. of rotatable bonds | 3 |
| No. of hydrogen bond acceptors | 5 |
| No. of hydrogen bond donors | 1 |
| Storage | Stability not tested. Recommendation: -20 °C for long term storage. |
| Dissolution | Free base: ca. 1 mM DMSO solution possible Salt form (.2HCl): 10 mM DMSO solution possible |
SMILES: C12=C(C=CC=C2C3=COC4=C3N=CC(C5=CC=C(N6CCNCC6)C=C5)=C4)C=CC=N1
InChI: InChI=1S/C26H22N4O/c1-3-19-4-2-10-28-25(19)22(5-1)23-17-31-24-15-20(16-29-26(23)24)18-6-8-21(9-7-18)30-13-11-27-12-14-30/h1-10,15-17,27H,11-14H2
InChIKey: KNGDSLDIOAICSE-UHFFFAOYSA-N
Selectivity profile of MU1700 was determined by kinome-wide screening against 369 protein kinases at 1 µM concentration (Reaction biology). MU1700 inhibits only ALK1/2/6 and no additional off targets when threshold of 25 % residual enzyme activity is applied.
 |
In vitro selectivity was subsequently confirmed by determination of IC50 values (at Reaction Biology, 10 mM ATP conc.) against ALK1-6 and the most significant off targets from kinome-wide screening.
| Biochemical assay | ||
| MU1700 | |
Kinase | IC50 (nM) | Selectivity (fold) |
ALK1/ACVRL1 | 13 | 2.22 |
ALK2/ACVR1 | 6 | 1.00 |
ALK3/BMPR1A | 425 | 72.08 |
ALK4/ACVR1B | inactive | - |
ALK5/TGFBR1 | inactive | - |
ALK6/BMPR1B | 41 | 6.88 |
DDR1 | 501 | 85.03 |
FLT3 | 751 | 127.33 |
KHS/MAP4K5 | 539 | 91.33 |
The potency in biochemical assays translates well into cell based assays for both probe and negative control compounds as it was demonstrated in NanoBRET target engagement assay with intact cells. From NanoBRET IC50 values we can see that MU1700 has sufficient permeability through cell membrane and inhibit ALK1/2 with high potency in cellulo. Regarding ALK2 inhibition, the cellular potency of MU1700 is comparable to current state-of-the-art ALK2 inhibitor LDN-193189, but their selectivity profile within ALK1-7 subfamily (as well as kinome-wide selectivity) is significantly improved. Importantly, MU1700 is inert towards ALK4/5 also in cellulo. For comparison, M4K2234 and M4K2234NC is also displayed in the table.
| Cellular target engagement assay for ALK1-6 |
 |
Cellular activity has been demonstrated on modulation of SMAD phosphorylation using wertern blotting. MU1700 affects phosphorylation of SMAD1/5/8 that corresponds to BMP branch of signalling wchich is mediated also by ALK1/2 kinases. On the other hand we see only very weak effect on SMAD2/3 phosphorylation that corresponds to TGF beta branch of signalling which is mediated via ALK4/5/7. This observation is consistent with NanoBRET cellular target engagement assay.
Cytotoxicity was evaluated by 24 hour incubation with U2OS cell line and using alamar blue as staning agent. Cytotoxic effect starts to be significant above 2.5 µM concentration. Considering the cellular activity towards main targets (ALK1/2) and general cytotoxic effect, we dont recomend to go higher than 1 µM for cellular assays.
1. Cao, H. et al. Differential kinase activity of ACVR1 G328V and R206H mutations with implications to possible TβRI cross-talk in diffuse intrinsic pontine glioma. Sci Rep 10, 6140 (2020).
2. Schmierer, B. & Hill, C. S. TGFβ–SMAD signal transduction: molecular specificity and functional flexibility. Nat Rev Mol Cell Biol 8, 970–982 (2007).
3. Chaikuad, A. et al. Structure of the Bone Morphogenetic Protein Receptor ALK2 and Implications for Fibrodysplasia Ossificans Progressiva. Journal of Biological Chemistry 287, 36990–36998 (2012).
4. Sekimata, K., Sato, T. & Sakai, N. ALK2: A Therapeutic Target for Fibrodysplasia Ossificans Progressiva and Diffuse Intrinsic Pontine Glioma. Chem. Pharm. Bull. 68, 194–200 (2020).
5. Eixarch, H., Calvo-Barreiro, L., Montalban, X. & Espejo, C. Bone morphogenetic proteins in multiple sclerosis: Role in neuroinflammation. Brain, Behavior, and Immunity 68, 1–10 (2018).
6. Sotiropoulos, M. G. & Chitnis, T. Opposing and potentially antagonistic effects of BMP and TGF-β in multiple sclerosis: The “Yin and Yang” of neuro-immune Signaling. Journal of Neuroimmunology 347, 577358 (2020).
In vivo pharmacokinetic (PK) profile of MU1700 has been evaluated in mice. After PO administration of 20 mg/kg, compound exhibits PK parameters that are summarized in the table, indicating that MU1700 is suitable chemical probe for in vivo disease models. Of note, MU1700 has remarkably high brain penetrance (mice, PO, 100 mg/kg).
Dose | 20 mg/kg |
Bioavailability | 79 % |
Cl (ml/min/kg) | 30 (IV) |
T1/2 | 2.5 h |
Cmax (PO) | 3697 nM |
Cbrain/Cplasma (4h, 100 mg/kg) | 25 |
| Probe | Negative control | |
 |
|  |
SGC-UBD253 |
| SGC-UBD253N |
SGC in collaboration with Dr Mark Lautens’ at the University of Toronto’s Department of Chemistry has developed the first chemical probe SGC-UBD253 for the ubiquitin binding domain (UBD) of HDAC6. SGC-UBD253 binds potently to HDAC6 UBD with KD = 84 nM (SPR) and inhibits the HDAC6-ISG15 interaction with EC50 = 1.9 micromolar (nanoBRET). SGC-UBD253N is a closely related negative control with KD = 32 micromolar (SPR).
Using sequence alignment of Zf-UBD pocket, 10 UBD-containing proteins in addition to HDAC6 were purified and tested for direct binding by SPR.
Three biophysical methods clearly show SGC-UBD253 potently (≤100 nM) binds HDAC6-UBD.
Using a nanoBRET assay, the chemical probe SGC-UBD253 considerably decreases the interaction between full-length HDAC6 with ISG15 relative to SGC-UBD253N.
Toronto, November 16, 2021 – The Structural Genomics Consortium (SGC) is pleased to announce its inaugural initiative in reproductive biology, funded by the Bill & Melinda Gates Foundation. This initiative will be the first in SGC’s new open science Women’s and Children’s Health Program, focusing on the advancement of the field of drug discovery in reproductive biology and disease, child development, and childhood diseases.
11 November, 2021, Toronto, Canada - Cyclica, the partner of choice for data-driven drug discovery, and the Structural Genomics Consortium (SGC), a global public-private partnership dedicated to open science, have collaborated on a project in support of Target 2035, an initiative to discover probe molecules in support of developing medicines for all.
New look for Target 2035
SGC’s Target 2035 initiative just launched a new website with a fresh look and feel. Users will be able to access information about the project, upcoming webinars, and general updates. Check the Target 2035 Twitter and Linked In accounts for the latest news.
What is Target 2035
| Probe | Negative control | |
 |
|  |
CK156 |
| CKJB71 |
DAP Kinase-Related Apoptosis-Inducing Protein Kinase 1 (DRAK1) is part of the DAPK (death-associated protein kinases) family, which comprises of five members (DAPK1–3 and DRAK2). Both, DRAK´s (DRAK1 & 2) belong to the so-called dark kinome and their cellular functions are largely unknown [1–3]. However, recent findings indicate that DRAK1 might play a role in different cancers such as glioblastoma multiforme (GBM) [4], head and neck squamous cell carcinoma (HNSCC) [5] or in testicular cancer [6]. In addition, DRAK1 has been identified to be a direct target gene of p53, and vice versa DRAK1 might regulate the transcriptional activity of p53 [6, 7]. More recently, Park et al demonstrated that DRAK1 might also act as a negative regulator of TRAF6, which is a central player in inflammatory signalling pathway in cervical cancer [8]. However, the precise role of DRAK1 in different cancers remains elusive and chemical tools are urgently needed to determine the role of DRAK1 in these diseases.
SGC has developed CK156, a potent and selective DRAK1 inhibitor with an IC50 of 49 nM for DRAK1 determined by a radiometric assay and a KD of 21 nM determined by ITC. Cellular activity was examined by NanoBRET and CK156 revealed an IC50 of 181 nM on DRAK1. The chemical probe (CK156) is accompanied by a negative control (CKJB71), which is structurally closely related to the probe molecule.
Potency Against Target Family
| Kinase | 33PanQinase IC50 (nM) |
| DRAK1 | 49 |
ITC: KD = 21 nM
Selectivity
CK156 has been shown to be selective in an in vitro kinase panel from DiscoverX (scanMAX®) against 468 Kinases followed by cellular NanoBRET assays.
Dosage
To minimize the chance of any unspecific cytotoxicity, we recommend a concentration of no higher than 5 µM for cell-based assays.
Cellular Activity
CK156 displayed an IC50 of 181 nM in NanoBRETTM assay.
| Probe | Negative control | |
|
| |
CK156 |
| CKJB71 |
Physical and chemical properties CK156 | |
| Molecular weight | 395.46
|
| Molecular formula | C21H25N5O3 |
| IUPAC name | N-tert-butyl-7,10-dioxa-13,17,18,21-tetraazatetracyclo[12.5.2.1²,⁶.0¹⁷,²⁰]docosa-1(20),2,4,6(22),14(21),15,18-heptaene-5-carboxamide |
| logP | 1.89 |
| PSA | 89.78 |
| No. of chiral centres | 0 |
| No. of rotatable bonds | 2 |
| No. of hydrogen bond acceptors | 8 |
| No. of hydrogen bond donors | 2 |
| Storage | stable as solid in the dark at -20°C. NB making aliquots rather than freeze-thawing is recommended |
| Dissolution | soluble in DMSO in a concentration of 10 mM |
SMILES: O=C(C(C=C1)=C(OCCOCCNC2=N3)C=C1C4=C3N(C=C2)N=C4)NC(C)(C)C
InChI: InChI=1S/C21H25N5O3/c1-21(2,3)25-20(27)15-5-4-14-12-17(15)29-11-10-28-9-7-22-18-6-8-26-19(24-18)16(14)13-23-26/h4-6,8,12-13H,7,9-11H2,1-3H3,(H,22,24)(H,25,27)
InChIKey: YCFFONJEHNSECY-UHFFFAOYSA-N
Physical and chemical properties CKJB71 | |
| Molecular weight | 422.49 |
| Molecular formula | C22H26N6O3 |
| IUPAC name | 5-(4-methylpiperazine-1-carbonyl)-7,10-dioxa-13,17,18,21-tetraazatetracyclo[12.5.2.1²,⁶.0¹⁷,²⁰]docosa-1(20),2,4,6(22),14(21),15,18-heptaene |
| logP | 1.00 |
| PSA | 84.23 |
| No. of chiral centres | 0 |
| No. of rotatable bonds | 1 |
| No. of hydrogen bond acceptors | 9 |
| No. of hydrogen bond donors | 1 |
| Storage | stable as solid in the dark at -20°C. NB making aliquots rather than freeze-thawing is recommended |
| Dissolution | soluble in DMSO in a concentration of 10 mM |
SMILES: O=C(C1=CC=C2C=C1OCCOCCNC3=NC4=C2C=NN4C=C3)N5CCN(CC5)C
InChI: InChI=1S/C22H26N6O3/c1-26-7-9-27(10-8-26)22(29)17-3-2-16-14-19(17)31-13-12-30-11-5-23-20-4-6-28-21(25-20)18(16)15-24-28/h2-4,6,14-15H,5,7-13H2,1H3,(H,23,25)
InChIKey:
DDXOMWWURGFLKK-UHFFFAOYSA-N
Selectivity profile of CK156 was determined with the scanMAX® assay from DiscoverX at 1000 and 100 nM and on- and off targets were evaluated with in vitro IC50 with the 33PanQinase activity assay from ProQinase and in cellulo IC50 with NanoBRET assay.
Kinase | Percent of control(%) @ 1000 nM | 33PanQinase IC50 (nM) | NanoBRET IC50 (nM) |
| DRAK1 | 0.3 | 49 | 181 |
| CSNK2A2 | 1.5 | 380 | 39000 |
| CSNK2A1 | 3.9 | 950 | 34000 |
| GAK | 23 | n.d. | 24000 |
| BIKE | 25 | n.d. | 8000 |
| JAK3(JH1domain-catalytic) | 28 | n.d. | n.d |
| RIOK3 | 28 | n.d. | n.d. |
| BUB1 | 30 | n.d. | n.d. |
| ERK8 | 33 | n.d. | n.d |
| DRAK2 | 39 | n.d. | 8000 |
The negative control CKJB71 showed no activity in a DSF assay for 90 kinases and no on-target activity determined by NanoBRET in intact cells and in lysed cells.
CK156 displayed no general cytotoxicity in three different cell lines (HEK293T, U2OS and MRC9-Fibroblasts) at 1µM and only slight toxicity at 10 µM. The cells were analysed after 18 and 36h.
[1] Berginski ME, Moret N, Liu C, Goldfarb D, Sorger PK, G. S. The Dark Kinase Knowledgebase: An Online Compendium of Knowledge and Experimental Results of Understudied Kinases. Nucleic Acids Res. 2020. https://doi.org/10.1093/nar/gkaa853.
[2] Farag, A. K.; Roh, E. J. Death-Associated Protein Kinase (DAPK) Family Modulators: Current and Future Therapeutic Outcomes. Medicinal Research Reviews. John Wiley and Sons Inc. January 1, 2019, pp 349–385. https://doi.org/10.1002/med.21518.
[3] Bialik, S.; Kimchi, A. The Death-Associated Protein Kinases: Structure, Function, and Beyond. Annual Review of Biochemistry. Annu Rev Biochem 2006, pp 189–210. https://doi.org/10.1146/annurev.biochem.75.103004.142615.
[4] Mao, P.; Hever-Jardine, M. P.; Rahme, G. J.; Yang, E.; Tam, J.; Kodali, A.; Biswal, B.; Fadul, C. E.; Gaur, A.; Israel, M. A.; Spinella, M. J. Serine/Threonine Kinase 17A Is a Novel Candidate for Therapeutic Targeting in Glioblastoma. PLoS One 2013, 8 (11). https://doi.org/10.1371/journal.pone.0081803.
[5] Park, Y.; Kim, W.; Lee, J. M.; Park, J.; Cho, J. K.; Pang, K.; Lee, J.; Kim, D.; Park, S. W.; Yang, K. M.; Kim, S. J. Cytoplasmic DRAK1 Overexpressed in Head and Neck Cancers Inhibits TGF-Β1 Tumor Suppressor Activity by Binding to Smad3 to Interrupt Its Complex Formation with Smad4. Oncogene 2015, 34 (39), 5037–5045. https://doi.org/10.1038/onc.2014.423.
[6] Mao, P.; Hever, M. P.; Niemaszyk, L. M.; Haghkerdar, J. M.; Yanco, E. G.; Desai, D.; Beyrouthy, M. J.; Kerley-Hamilton, J. S.; Freemantle, S. J.; Spinella, M. J. Serine/Threonine Kinase 17A Is a Novel P53 Target Gene and Modulator of Cisplatin Toxicity and Reactive Oxygen Species in Testicular Cancer Cells. J. Biol. Chem. 2011, 286 (22), 19381–19391. https://doi.org/10.1074/jbc.M111.218040.
[7] Oue, Y.; Murakami, S.; Isshiki, K.; Tsuji, A.; Yuasa, K. Intracellular Localization and Binding Partners of Death Associated Protein Kinase-Related Apoptosis-Inducing Protein Kinase 1. Biochem. Biophys. Res. Commun. 2018, 496 (4), 1222–1228. https://doi.org/10.1016/j.bbrc.2018.01.175.
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The probe PFI-7 (hydrochloride) is available at Sigma and Tocris.
The inactive control PFI-7N (hydrochloride) is available at Sigma.
| Probe | Negative control | |
 |
|  |
PFI-7 |
| PFI-7N |
Pfizer in collaboration with the SGC have developed PFI-7, a potent, cell active chemical probe for the E3 ligase GID4. PFI-7 binds potently to GID4 with KD = 0.08 μM (SPR) and displaces the known degron1 peptide in a NanoBRETTM assay with EC50 = 0.6 μM. PFI-7N is a closely related negative control with KD = 5 μM (SPR). A co-crystal structure has been deposited.
We have further developed a handle PFI-E3H1 and a PEGylated analogue to show that the handle tolerates a substitution. These findings offers opportunities to synthesize proximity-inducing or degrader modalities2.
 |  |
 |   |
[PFI-7] (μM) | [PFI-7] (μM) |
A NanoBRET assay was used to show target engagement in cells.
The interaction was between NanoLuc® tagged degrons and full-length GID4.
 |
Main features
| Probe | Negative control | |
 |
|  |
MRIA9 |
| MR7 |
Salt-inducible kinases (SIK1-3) are members of the AMP-activated protein kinase (AMPK) family which is part of the calcium/calmodulin-dependent kinase (CaMK) group. These serine/threonine kinases act as regulators of energy homeostasis and metabolic stress. The SIK family member SIK2, for example, is activated in cells recovering from starvation, leading to phosphorylation and hence activation of the transcription factor cAMP response element-binding protein (CREB1) [1-3]. In addition to the key function of SIK in regulating metabolism, imbalance of SIK has been observed in the context of several diseases, especially in cancer, with both tumor promoting and tumor suppressive roles being reported [4]. SIK2 is often deleted in breast cancer, and downregulation of SIK1 has been linked to a tumor suppressor role but also the development of metastasis by promoting p53-dependent anoikis [5]. We have recently shown that in biopsies from patients with gastric cancer, there were increased levels of SIK2 mRNA and protein in advanced stages of the tumor compared to lower grade tumors, independent of its metastatic stage [6]. Overall, these data highlight the complex roles of SIK family proteins in different types of cancer and they may emerge as important therapeutic targets. To clarify the multifaceted roles of these kinases in disease and normal physiology, chemical tools targeting SIK are urgently needed.
SGC has developed MRIA9, a potent and selective pan SIK inhibitor with a IC50 determined by a radiometric assay of 55, 48 and 22 nM for SIK1, SIK2 and SIK3 respectively and IC50 of 516, 180 and 127 nM on NanoBRET™ assay. [7] MRIA9 has been developed based on PAK1 inhibitor G-5555 published by Genentech. Group I PAK (PAK1, PAK2 and PAK3) remains off targets with respectively in vitro IC50s of 580, 41 and 140 nM. However, due to lack of accessibility of a NanoBRET™ assay for Group I PAKs, MRIA9 is considered a SIK and Group I PAK probe. In addition, the chemical probe (MRIA9) is accompanied by a negative control (MR7), which is structurally similar to the probe molecule.
Potency Against Target Family
| Kinase | 33PanQinase IC50 (nM) |
| SIK1 | 55 |
| SIK2 | 48 |
| SIK3 | 22 |
MRIA9 has been shown to be selective in an in vitro kinase panel from Reaction Biology followed by cellular NanoBRET assays. The selectivity outside target family revealed Group I PAKs as closest off-target.
To minimize the chance of off-target effects, we recommend a concentration of no higher than 10 µM for cell-based assays.
In NanoBRET assay using HEK923T cells MRIA9 shows an IC50 of 516, 180 and 127 nM for SIK1, SIK2 and SIK3 respectively.
| Probe | Negative control | |
|
| |
MRIA9 |
| MR7 |
| Physical and chemical properties MRIA9 | |
| Molecular weight | 496.93 |
| Molecular formula | C24H22ClFN6O3 |
| IUPAC name | 8-(((2r,5r)-5-amino-1,3-dioxan-2-yl)methyl)-6-(2-chloro-4-(3-fluoropyridin-2-yl)phenyl)-2-(methylamino)pyrido[2,3-d]pyrimidin-7(8H)-one |
| logP | 2.16 |
| PSA | 113.9 |
| No. of chiral centre | 0 |
| No. of rotatable bonds | 7 |
| No. of hydrogen bond acceptors | 7 |
| No. of hydrogen bond donors | 2 |
| Storage | stable as solid in the dark at -20°C. NB making aliquots rather than freeze-thawing is recommended |
| Dissolution | soluble in DMSO in a concentration of 50 mM |
SMILES:
CNC1=NC=C2C(N(C(C(C3=CC=C(C=C3Cl)C4=NC=CC=C4F)=C2)=O)C[C@H]5OC[C@@H](CO5)N)=N1
InChI: InChI=1S/C24H22ClFN6O3/c1-28-24-30-9-14-7-17(16-5-4-13(8-18(16)25)21-19(26)3-2-6-29-21)23(33)32(22(14)31-24)10-20-34-11-15(27)12-35-20/h2-9,15,20H,10-12,27H2,1H3,(H,28,30,31)/t15-,20-
InChIKey:
QKNBRNSGPNCARD-SGNKCFNYSA-N
| Physical and chemical properties MR7 | |
| Molecular weight | 510.95 |
| Molecular formula | C25H24ClFN6O3 |
| IUPAC name | 8-(((2r,5r)-5-amino-1,3-dioxan-2-yl)methyl)-6-(2-chloro-4-(3-fluoropyridin-2-yl)phenyl)-2-(dimethylamino)pyrido[2,3-d]pyrimidin-7(8H)-one |
| logP | 2.95 |
| PSA | 105.11 |
| No. of chiral centre | 0 |
| No. of rotatable bonds | 8 |
| No. of hydrogen bond acceptors | 8 |
| No. of hydrogen bond donors | 1 |
| Storage | stable as solid in the dark at -20°C. NB making aliquots rather than freeze-thawing is recommended |
| Dissolution | soluble in DMSO in a concentration of 50 mM |
SMILES:
CN(C1=NC=C2C(N(C(C(C3=CC=C(C=C3Cl)C4=NC=CC=C4F)=C2)=O)C[C@H]5OC[C@@H](CO5)N)=N1)C
InChI: InChI=1S/C25H24ClFN6O3/c1-32(2)25-30-10-15-8-18(17-6-5-14(9-19(17)26)22-20(27)4-3-7-29-22)24(34)33(23(15)31-25)11-21-35-12-16(28)13-36-21/h3-10,16,21H,11-13,28H2,1-2H3/t16-,21-
InChIKey:
RRFFBKGCCWPMLK-OQIWPSSASA-N
Selectivity profile of MRIA9 was determined with the 33PanQinase activity assay from Reaction Biology at 1uM and off targets were confirmed with in vitro IC50 with the same assay and in cellulo IC50 with NanoBRET™ assay. MRIA9 is a pan SIK and group I PAK inhibitor.
| Kinase | Percent of control(%) | 33 PanQinase IC 50 (nM) | NanoBRET IC 50 (nM) |
| SIK2 | 1 | 48 | 180 |
| SIK3 | 2 | 22 | 127 |
| SIK1 | 4 | 55 | 516 |
| KHS1 | 8 | 210 | 13000 |
| PAK3 | 9 | 140 | n.d |
| PAK2 | 10 | 41 | n.d |
| NLK | 13 | 250 | 3100 |
| PKN3 | 35 | 1400 | 6700 |
| PAK1 | 36 | 580 | n.d |
| MAP2K4 | 37 | 830 | n.d |
| TIE2 | 39 | 3100 | 6000 |
| MST4 | 45 | 1600 | 34000 |
| MELK | 48 | 2200 | n.d |
The negative control MR7 with its blocked hinge-binding amine showed no activity on a DSF assay for 100 kinases and low activity on target on NanoBRET assay.
 |
MRIA9 modulated endogenous substrates linked to SIK activity. In the ovarian cancer cell SKOV-3 in which the PI3K/AKT/MTOR pathway was activated by rapamycine, MRIA9 abrogated the phosphorylation of AKT in a dose-dependent manner. In addition, SIK2 auto-phosphorylation activity was completely inhibited whereas the negative control did not influence SIK2 activity.
| SKOV-3 cell line |
 |
MRIA9 replicated a known phenotype of SIK inhibition, displacing the centrosome from the nucleous in ovarian cancer cell line SKOV-3, similar to the phenotype seen when silence RNA is used to knock down SIK2.
| SKOV-3 cell line |
 |
In the NCI-60 screen, which is a human tumor cell line screen, MRIA9 showed only modest cell toxicity or growth inhibition. It was tested in a single high dose of 10 µM in the full NCI-60 panel. https://dtp.cancer.gov/discovery_development/nci-60/methodology.htm
(1) Sun, Z.; Jiang, Q.; Li, J.; Guo, J. The potent roles of salt-inducible kinases (SIKs) in metabolic homeostasis and tumorigenesis. Signal transduction and targeted therapy 2020, 5 (1), 150.
(2) Conkright, M. D.; Canettieri, G.; Screaton, R.; Guzman, E.; Miraglia, L.; Hogenesch, J. B.; Montminy, M. TORCs: transducers of regulated CREB activity. Molecular cell 2003, 12 (2), 413–423.
(3) Katoh, Y.; Takemori, H.; Lin, X.-Z.; Tamura, M.; Muraoka, M.; Satoh, T.; Tsuchiya, Y.; Min, L.; Doi, J.; Miyauchi, A.; Witters, L. A.; Nakamura, H.; Okamoto, M. Silencing the constitutive active transcription factor CREB by the LKB1-SIK signaling cascade. The FEBS journal 2006, 273 (12), 2730–2748.
(4) Chen, F.; Chen, L.; Qin, Q.; Sun, X. Salt-inducible kinase 2: an oncogenic signal transmitter and potential target for cancer therapy. Frontiers in oncology 2019, 9, 18.
(5) Cheng, H.; Liu, P.; Wang, Z. C.; Zou, L.; Santiago, S.; Garbitt, V.; Gjoerup, O. V.; Iglehart, J. D.; Miron, A.; Richardson, A. L.; Hahn, W. C.; Zhao, J. J. SIK1 couples LKB1 to p53-dependent anoikis and suppresses metastasis. Science signaling 2009, 2 (80), ra35.
(6) Montenegro, R. C.; Howarth, A.; Ceroni, A.; Fedele, V.; Farran, B.; Mesquita, F. P.; Frejno, M.; Berger, B.-T.; Heinzlmeir, S.; Sailem, H. Z.; Tesch, R.; Ebner, D.; Knapp, S.; Burbano, R.; Kuster, B.; Müller, S. Identification of molecular targets for the targeted treatment of gastric cancer using dasatinib. Oncotarget 2020, 11 (5), 535–549.
(7) Tesch, R.; Rak, M.; Raab, M.; Berger, L. M.; Kronenberger, T.; Joerger, A. C.; Berger, B.-T.; Abdi, I.; Hanke, T.; Poso, A.; Strebhardt, K.; Sanhaji, M.; Knapp, S. Structure-Based Design of Selective Salt-Inducible Kinase Inhibitors. Journal of Medicinal Chemistry 2021, 64 (12), 8142-8160.
Binding mode of MRIA9 in complex with the crystallographic surrogate model MST3 (PDB 7B31). The inhibitor binds to the ATP pocket and different stability on the P-loop region of MST3 and SIK2 studied by molecular dynamics, explain the selectivity of MRIA9 towards SIK2. [7]
