The probe is available from Sigma.
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.
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: C1(C=CC=C2)=C2N=CC=C1C3=COC4=C3N=CC(C5=CC=C(C=C5)N6CCNCC6)=C4
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: C1(N=CC=C2)=C2C=CC=C1C3=COC4=C3N=CC(C5=CC=C(C=C5)N6CCNCC6)=C4
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.
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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 |
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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 |
