05.07.2022

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

by: SGC

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

16.05.2022

Understanding antitumor activity opens new possibilities for future breast cancer treatments

by: SGC

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

08.03.2022

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

by: SGC

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

25.02.2022

Structural Genomics Consortium announces two new academic partners, the Montreal Neurological Institute-Hospital and University College London

by: SGC

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.

22.02.2022

CACHE Research Paper in Nature Reviews Chemistry Outlines Project’s Method for Generating Better Computational Methods for Molecule Hit-Finding

by: SGC

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.

M4K2234 Chemical probe for ALK1 and ALK2 protein kinases

The probe is available at Sigma and Cayman Chemical.

The negative control ist available at Sigma.

  • overview
  • properties
  • in vitro potency
  • cell based assay data
  • references
  • pk properties
  • co-crystal structures
overview
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.

properties
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
in vitro potency

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 
cell based assay data

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.

references

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).

pk properties

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

co-crystal structures

A close analogue of M4K2234 has been successfully co-crystalized with ALK2 (PDB:6T6D).

PDB: 6T6D (analogue of M4K2149)
synthetic schemes
materials and methods

MU1700 Chemical probe for ALK1 and ALK2 protein kinases

The probe is available from Sigma and Tocris (as HCl salts).

  • overview
  • properties
  • selectivity profile
  • cell based assay data
  • references
  • pk properties
overview
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. 

properties
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 weight406.49
Molecular formulaC26H22N4O
IUPAC name6-(4-(piperazin-1-yl)phenyl)-3-(quinolin-4-yl)furo[3,2-b]pyridine
ClogP4.39
PSA54.19
No. of chiral centres0
No. of rotatable bonds3
No. of hydrogen bond acceptors5
No. of hydrogen bond donors1
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 weight406.49
Molecular formulaC26H22N4O
IUPAC name6-(4-(piperazin-1-yl)phenyl)-3-(quinolin-8-yl)furo[3,2-b]pyridine
ClogP4.39
PSA54.19
No. of chiral centres0
No. of rotatable bonds3
No. of hydrogen bond acceptors5
No. of hydrogen bond donors1
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

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
in vitro potency
cell based assay data

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.

references

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).

pk properties

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

co-crystal structures
synthetic schemes
materials and methods

SGC-UBD253 Chemical probe for the ubiquitin binding domain of HDAC6.

The probe and control are now available at Millipore-Sigma.

  • overview
  • selectivity profile
  • in vitro potency
  • cell based assay data
  • references
overview
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).

properties
selectivity profile

Using sequence alignment of Zf-UBD pocket, 10 UBD-containing proteins in addition to HDAC6 were purified and tested for direct binding by SPR.

in vitro potency

Three biophysical methods clearly show SGC-UBD253 potently (≤100 nM) binds HDAC6-UBD.

cell based assay data

Using a nanoBRET assay, the chemical probe SGC-UBD253 considerably decreases the interaction between full-length HDAC6 with ISG15 relative to SGC-UBD253N.

references
  1. Ferreira de Freitas R, Harding RJ, Franzoni I, Ravichandran M, Mann MK, Ouyang H, Lautens M, Santhakumar V, Arrowsmith CH, Schapira M. Identification and Structure-Activity Relationship of HDAC6 Zinc-Finger Ubiquitin Binding Domain Inhibitors. J Med Chem. 2018 May 24;61(10):4517-4527. doi: 10.1021/acs.jmedchem.8b00258. PMID: 29741882
  2. Harding RJ, Ferreira de Freitas R, Collins P, Franzoni I, Ravichandran M, Ouyang H, Juarez-Ornelas KA, Lautens M, Schapira M, von Delft F, Santhakumar V, Arrowsmith CH. Small Molecule Antagonists of the Interaction between the Histone Deacetylase 6 Zinc-Finger Domain and Ubiquitin. J Med Chem. 2017 Nov 9;60(21):9090-9096. doi: 10.1021/acs.jmedchem.7b00933. PMID: 29019676
pk properties
co-crystal structures
synthetic schemes
materials and methods
16.11.2021

Structural Genomics Consortium Receives Grant for Women’s Health Discovery

by: SGC

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.

11.11.2021

Cyclica and Structural Genomics Consortium co-crystallize DCAF1

by: SGC

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.

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