| 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).
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.
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.
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.
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.
Toronto, February 25, 2022 - The Structural Genomics Consortium (SGC) is excited to welcome the Montreal Neurological Institute-Hospital (The Neuro) at McGill University and Open Chemistry Networks (OCN) based at University College London (UCL) as its latest academic partners.
SGC is pleased to have The Neuro and UCL officially contribute to our academic network, adopting SGC’s “patent-free” open science model for their SGC-associated teams.
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 |
