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.

07.10.2021

New look for Target 2035

by: SGC

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

CK156 Chemical probe for DRAK1 (DAPK family) which is also a member of the dark kinome.

The probe CK156 is available at Sigma.

The negative control CKJB71 is available at Sigma.

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

Kinase33PanQinase IC50 (nM)
DRAK149

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.

properties
Probe Negative control

 

CK156

 

CKJB71

Physical and chemical properties CK156

Molecular weight

395.46

 

Molecular formulaC21H25N5O3
IUPAC nameN-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
logP1.89
PSA89.78
No. of chiral centres0
No. of rotatable bonds2
No. of hydrogen bond acceptors8
No. of hydrogen bond donors2
Storagestable as solid in the dark at -20°C. NB making aliquots rather than freeze-thawing is recommended
Dissolutionsoluble 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 weight422.49
Molecular formulaC22H26N6O3
IUPAC name5-(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
logP1.00
PSA84.23
No. of chiral centres0
No. of rotatable bonds1
No. of hydrogen bond acceptors9
No. of hydrogen bond donors1
Storagestable as solid in the dark at -20°C. NB making aliquots rather than freeze-thawing is recommended
Dissolutionsoluble 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

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

CK156

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.

in vitro potency
cell based assay data

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.

references

[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.

[8] Park Y, Pang K, Park J, Hong E, Lee J, Ooshima A, Kim HS, Cho JH, Han Y, Lee C, Song YS, Park KS, Yang KM, Kim SJ. Destablilization of TRAF6 by DRAK1 Suppresses Tumor Growth and Metastasis in Cervical Cancer Cells. Cancer Res. 2020 Jun 15;80(12):2537-2549. doi: 10.1158/0008-5472.CAN-19-3428.

pk properties
co-crystal structures
synthetic schemes
materials and methods

PFI-7 Chemical probe for GID4, substrate-recognition subunit of the CTLH E3 ubiquitin-protein ligase complex

The probe PFI-7 (hydrochloride) is available at Sigma and Tocris.

The inactive control PFI-7N (hydrochloride) is available at Sigma.

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

properties
selectivity profile
in vitro potency

[PFI-7] (μM)

[PFI-7] (μM)

cell based assay data

A NanoBRET assay was used to show target engagement in cells. 

The interaction was between NanoLuc® tagged degrons and full-length GID4.

references
  1. Cheng Dong, Heng Zhang, Li Li, Wolfram Tempel, Peter Loppnau & Jinrong Min. Molecular basis of GID4-mediated recognition of degrons for the Pro/N-end rule pathway. Nature Chemical Biology 14, 466-473 (2018).​
  2. Aleša Bricelj, Christian Steinebach, Robert Kuchta, Michael Gütschow, and Izidor Sosič. E3 Ligase Ligands in Successful PROTACs: An Overview of Syntheses and Linker Attachment Points, https://doi.org/10.3389/fchem.2021.707317 ; Milka Kostic. Targeted Protein Degradation and Proximity-Based Pharmacology, https://doi.org/10.5281/zenodo.5534371 .
  3. Dominic D.G. Owens et al., A chemical probe to modulate human GID4 Pro/N-degron interactions. https://pubmed.ncbi.nlm.nih.gov/38773330/
  4. Aliakbar K Yazdi et al., Chemical tools for the Gid4 subunit of the human E3 ligase C-terminal to LisH (CTLH) degradation complex. https://pubmed.ncbi.nlm.nih.gov/38516600/
pk properties
co-crystal structures

Main features

  • PFI-7 bound to GID4 substrate-binding pocket
  • Structure overview
  • Overlap with substrate peptide
synthetic schemes
materials and methods

MRIA9 A pan chemical probe for SIK1/2/3.

The probe MRIA9 (Trifluoroacetate) is available from Sigma.

Click here to obtain the control.

  • overview
  • properties
  • selectivity profile
  • cell based assay data
  • references
  • co-crystal structures
overview
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

Kinase33PanQinase IC50 (nM)
SIK155
SIK248
SIK322

Selectivity

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.

Dosage

To minimize the chance of off-target effects, we recommend a concentration of no higher than 10 µM for cell-based assays.

Cellular Activity

In NanoBRET assay using HEK923T cells MRIA9 shows an IC50 of 516, 180 and 127 nM for SIK1, SIK2 and SIK3 respectively.

properties
Probe Negative control

 

MRIA9

 

MR7

Physical and chemical properties MRIA9
Molecular weight496.93
Molecular formulaC24H22ClFN6O3
IUPAC name8-(((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
logP2.16
PSA113.9
No. of chiral centre0
No. of rotatable bonds7
No. of hydrogen bond acceptors7
No. of hydrogen bond donors2
Storagestable as solid in the dark at -20°C. NB making aliquots rather than freeze-thawing is recommended
Dissolutionsoluble 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 weight510.95
Molecular formulaC25H24ClFN6O3
IUPAC name8-(((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
logP2.95
PSA105.11
No. of chiral centre0
No. of rotatable bonds8
No. of hydrogen bond acceptors8
No. of hydrogen bond donors1
Storagestable as solid in the dark at -20°C. NB making aliquots rather than freeze-thawing is recommended
Dissolutionsoluble 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

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.

KinasePercent of control(%)33 PanQinase IC 50 (nM)NanoBRET IC 50 (nM)
SIK2148180
SIK3222127
SIK1455516
KHS1821013000
PAK39140n.d
PAK21041n.d
NLK132503100
PKN33514006700
PAK136580n.d
MAP2K437830n.d
TIE23931006000
MST445160034000
MELK482200n.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.

in vitro potency
cell based assay data

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

references

(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.

pk properties
co-crystal structures

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]

synthetic schemes
materials and methods

Homer

The probe is available from Sigma and Cayman Chemicals.

The control may be requested here.

  • overview
  • properties
  • selectivity profile
  • cell based assay data
  • references
  • co-crystal structures
overview
Probe Negative control Negative control

 

 

Homer

 

nc_WDR5

 

nc_VHL

The Histone Lysine Methyltransferase (HMT) complex MLL1 is a crucial epigenetic writer to control DNA accessibility and promote gene transcription [1,2]. The complex incorporates WDR5 (WD40-repeat containing protein 5) that acts as scaffolding component and enhances HMT activity of MLL1. [2] The propeller-shaped WDR5 protein contains two binding sites that interact not only with oncogenic drivers like MLL1, but also with various other proteins like the oncoprotein MYC. [3]

Here, we present a new chemical degrader probe for the WDR5 protein, Homer, that was initially designed by the OICR and the SGC Toronto [4, 5] and then further advanced into a PROTAC (Proteolysis targeting chimera) chemical probe by the SGC Frankfurt [6]. This scaffold represents the first chemical degrader probe for WDR5.

Homer binds potently to WDR5 with a K D (ITC) of 18 nM. It is selective as shown in quantitative proteomic studies. This compound is non-toxic as demonstrated by the AlarmarBlue assay in U2OS cells. Homer shows remarkably cellular activity in MV4-11 cells by inducing WDR5 degradation in a catalytic manner a DC 50 of 53 nM. The DC max of Homer is 58% and observed at cellular concentrations of 1 µM.

In addition, two negative control compounds, nc_WDR5 and nc_VHL, are provided, which showed no degradational activity.

Summary of Potency and efficacy of Homer

WDR5Homer KD
DSF13.2 K 
ITC18 nM 
NanoBRET13.6 µM 
HiBit

DCmax: 58% at 1 µM 

DC50: 53 nM 

quantitative Proteomics (-log10p > 3, log2FC < -0.5) 

Selectivity 

Selectivity of the first WDR5 probe OICR-9429 was shown previously [5, 6]. Selectivity for the Homer probe was confirmed by quantitative proteomics.

Dosage 

For optimal degradation and to overcome the Hook effect, we recommend that a concentration of 1 µM should be used in cell-based assays. 

Cellular Activity 

Homer induces depletion of endogenous WDR5 in various cancer cell lines by inducing protein ubiquitylation and degradation. 

properties
Probe Negative control Negative control

 

 

Homer

 

nc_WDR5

 

nc_VHL

Homer
Physical and Chemical Properties
Molecular weight1012.16
Molecular formulaC52H60F3N9O7S
IUPAC nameN-(4'-((5-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-5-oxopentyl)carbamoyl)-4-(4-methylpiperazin-1-yl)-[1,1'-biphenyl]-3-yl)-6-oxo-4-(trifluoromethyl)-1,6-dihydropyridine-3-carboxamide
clogPo/w (SwissADME)5.31
TPSA (SwissADME)237.41 Å2
No. of chiral centres3
No. of rotatable bonds23
No. of hydrogen bond acceptors12
No. of hydrogen bond donors6
Storagestable as powder at -20°C. NB making aliquots rather than freeze-thawing is recommended
Dissolutionsoluble in DMSO at 50 mM

 

nc_VHL
Physical and Chemical Properties
Molecular weight1012.16
Molecular formulaC52H60F3N9O7S
IUPAC nameN-(4'-((5-(((S)-1-((2S,4S)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-5-oxopentyl)carbamoyl)-4-(4-methylpiperazin-1-yl)-[1,1'-biphenyl]-3-yl)-6-oxo-4-(trifluoromethyl)-1,6-dihydropyridine-3-carboxamide 
clogPo/w (SwissADME)5.25
TPSA (SwissADME)237.41 Å2
No. of chiral centres3
No. of rotatable bonds23
No. of hydrogen bond acceptors12
No. of hydrogen bond donors6
Storagestable as powder at -20°C. NB making aliquots rather than freeze-thawing is recommended
Dissolutionsoluble in DMSO at 50 mM
nc_WDR5 
Physical and Chemical Properties
Molecular weight999.12
Molecular formulaC51H57F3N8O9S
IUPAC nameN-(4'-((5-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-5-oxopentyl)carbamoyl)-4-morpholino-[1,1'-biphenyl]-3-yl)-6-oxo-4-(trifluoromethyl)-1,6-dihydropyridine-3-carboxamide 
clogPo/w (SwissADME)5.41
TPSA (SwissADME)243.40 Å2
No. of chiral centres3
No. of rotatable bonds23
No. of hydrogen bond acceptors12
No. of hydrogen bond donors6
Storagestable as powder at -20°C. NB making aliquots rather than freeze-thawing is recommended
Dissolutionsoluble in DMSO at 50 mM

SMILES: 

Homer: CC1=C(SC=N1)C2=CC=C(C=C2)CNC([C@@H]3C[C@H](CN3C([C@@H](NC(CCCCNC(C4=CC=C(C=C4)C5=CC=C(C(NC(C6=CNC(C=C6C(F)(F)F)=O)=O)=C5)N7CCN(CC7)C)=O)=O)C(C)(C)C)=O)O)=O

nc_VHL: CC1=C(C2=CC=C(CNC([C@@H]3C[C@H](O)CN3C([C@H](C(C)(C)C)NC(CCCCNC(C4=CC=C(C5=CC=C(N6CCN(C)CC6)C(NC(C7=CNC(C=C7C(F)(F)F)=O)=O)=C5)C=C4)=O)=O)=O)=O)C=C2)SC=N1 

nc_WDR5: CC1=C(C2=CC=C(CNC([C@@H]3C[C@@H](O)CN3C([C@H](C(C)(C)C)NC(CCCCNC(C4=CC=C(C5=CC=C(N6CCOCC6)C(NC(C7=CNC(C=C7C(F)(F)F)=O)=O)=C5)C=C4)=O)=O)=O)=O)C=C2)SC=N1 

InChI: 

Homer: InChI=1S/C52H60F3N9O7S/c1-31-45(72-30-59-31)34-11-9-32(10-12-34)27-58-49(70)42-25-37(65)29-64(42)50(71)46(51(2,3)4)61-43(66)8-6-7-19-56-47(68)35-15-13-33(14-16-35)36-17-18-41(63-22-20-62(5)21-23-63)40(24-36)60-48(69)38-28-57-44(67)26-39(38)52(53,54)55/h9-18,24,26,28,30,37,42,46,65H,6-8,19-23,25,27,29H2,1-5H3,(H,56,68)(H,57,67)(H,58,70)(H,60,69)(H,61,66)/t37-,42+,46-/m1/s1 

nc_VHL: InChI=1S/C52H60F3N9O7S/c1-31-45(72-30-59-31)34-11-9-32(10-12-34)27-58-49(70)42-25-37(65)29-64(42)50(71)46(51(2,3)4)61-43(66)8-6-7-19-56-47(68)35-15-13-33(14-16-35)36-17-18-41(63-22-20-62(5)21-23-63)40(24-36)60-48(69)38-28-57-44(67)26-39(38)52(53,54)55/h9-18,24,26,28,30,37,42,46,65H,6-8,19-23,25,27,29H2,1-5H3,(H,56,68)(H,57,67)(H,58,70)(H,60,69)(H,61,66)/t37-,42-,46+/m0/s1 

nc_WDR5: InChI=1S/C51H57F3N8O8S/c1-30-44(71-29-58-30)33-10-8-31(9-11-33)26-57-48(68)41-24-36(63)28-62(41)49(69)45(50(2,3)4)60-42(64)7-5-6-18-55-46(66)34-14-12-32(13-15-34)35-16-17-40(61-19-21-70-22-20-61)39(23-35)59-47(67)37-27-56-43(65)25-38(37)51(52,53)54/h8-17,23,25,27,29,36,41,45,63H,5-7,18-22,24,26,28H2,1-4H3,(H,55,66)(H,56,65)(H,57,68)(H,59,67)(H,60,64)/t36-,41+,45-/m1/s1 

InChIKey: 

Homer: OFNZESNEBSQKSE-BQGOKDIQSA-N 

nc_VHL: OFNZESNEBSQKSE-CCVFZADKSA-N 

nc_WDR5: BFXBMIZHEIFXOC-LTJJNQLXSA-N 

selectivity profile
Quantitative Proteomics revealed that only WDR5 is significally and substantially depleted by Homer treatment. 
Homer binds potently to WDR5 as determined by ITC. 
in vitro potency
cell based assay data

Homer is cell permeable and degrades WDR5 in MV4-11 cells. The control compounds nc_WDR5 and nc_VHL are not active. 

NanoBRET data of Homer in HEX293 cells show Homer is cell permeable. 

(up) HiBiT assay curves from Homer (black) and both negative controls (red) as well as from parent WDR5 compound (blue) show that Homer degrades WDR5 in MV4-11 cells after 24 h. The most effective degradation (58% WDR5 depletion) is observed at 1 µM. (down) WesternBlots validate HiBiT data and show degradation of WDR5 by Homer treatment in MV4-11 cells after 24 h. 

Homer decreases WDR5 protein stability as shown in the Cycloheximide chase assay. 

Rescue experiments show that WDR5 depletion requires binding of Homer. WDR5 levels can be restored by excess of WDR5 ligand. 

qPCR shows that Homer does not affect WDR5 transcription.

references

[1] Wu, Shu, “MLL1/WDR5 complex in leukemogenesis and epigenetic regulation”, Chin. J. Cancer 2011, 30(4), 240-246.
[2] Jiang, “The complex activities of the SET1/ MLL complex core subunits in development and disease“, BBA – Gene Regulatory Mechanisms 2020, 1863, 194560.
[3] Thomas et al., “Interaction with WDR5 promotes target gene recognition and tumorigenesis by MYC”, Mol. Cell 2015, 58, 440-452.
[4] Grebien et al., “Pharmacological targeting of the Wdr5-MLL interaction in C/EBPa N-terminal leukemia”, Nat Chem Biol. 2015, 11, 571.
[5] Getlik et al., “Structure-Based Optimization of a Small Molecule Antagonist of the Interaction Between WD Repeat-Containing Protein 5 (WDR5) and Mixed-Lineage Leukemia 1 (MLL1)”, J Med Chem. 2016, 59, 2478.
[6] Dölle, Adhikari et al., “Design, Synthesis, and Evaluation of WD-Repeat-Containing Protein 5 (WDR5) Degraders”, J Med Chem. 2021, May 13. doi: 10.1021/acs.jmedchem.1c00146. Epub ahead of print. PMID: 33980013.
[7] A.C. Wallace, R.A. Laskowski, J.M. Thornton, “LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions”, Protein Eng. 8, 1995, 127-134.

pk properties
co-crystal structures

PDB ID 7Q2J

Left panel: Quaternary complex of WDR5:VHL:ElonginB:ElonginC:Homer bound to chemical probe PROTAC Homer in surface representation.

Right panel: Surface slice with focus on the binding pocket of WDR5 and VHL. Homer is shown in stick representation

Observed electron density of Homer countered at 1 sigma.

Left panel: Contact surface area between WDR5 (wheat) and VHL (green) in surface representation

Right panel: Close up on the detailed interaction between WDR5 and VHL in cartoon/stick representation. The orientation is the same as in the left Panel.

WDR5 (D)       
VHL (C)

Detailed interaction between Homer and VHL:WDR5. The figure was created with LIGPLOT [7]

synthetic schemes
materials and methods
12.02.2021

Promising new approach to stop growth of brain cancer cells

by: SGC

Inhibiting a key enzyme that controls a large network of proteins important in cell division and growth paves the way for a new class of drugs that could stop glioblastoma, a deadly brain cancer, from growing.

Researchers at Princess Margaret Cancer Centre, the Hospital for Sick Children (SickKids) and University of Toronto, showed that chemically inhibiting the enzyme PRMT5 can suppress the growth of glioblastoma cells.

SGC-SMARCA-BRDVIII A Chemical Probe for SMARCA2/4 and PB1(5)

This probe is available from Cayman, Sigma, and Tocris.

The control is available from Sigma.

  • overview
  • properties
  • selectivity profile
  • cell based assay data
  • references
  • co-crystal structures
overview
Probe Negative control

 

SGC-SMARCA-BRDVIII

 

SGC-BRDVIII-NC

The SWI/SNF (switch/sucrose non-fermenting) chromatin remodelling complexes, BAF (BRG1/BRM-associated factor) and PBAF (polybromo-associated BAF), are crucial epigenetic regulators to control DNA accessibility, and thus largely contribute to cell proliferation and differentiation mechanisms [1]. The complexes incorporate either the related subunit SMARCA2 or SMARCA4 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 2/4), which both consist of a mutually exclusive catalytic ATPase domain and a bromodomain that reads acetylated histones on the chromatin. A unique feature of PBAF is the subunit PBRM1 (polybromo-1) containing six tandem-acting bromodomains. SWI/SNF complexes are known to be strong tumor suppressors and their dysfunctions trigger substantial oncogenic programs or deregulate cell lineage differentiation mechanisms [1].

Here, we present a new chemical probe for the SMARCA2/4 and PB1(5) bromodomains of BAF/PBAF, SGC-SMARCA-BRDVIII, that was initially designed by Genentech/Constellation [2] and then further advanced into the chemical probe platform by the SGC Frankfurt [3]. This scaffold represents the second chemical probe for the SMARCA2/4 and PB1(5) bromodomains that is based on a different chemotype than our first generation bromodomain inhibitor PFI-3 [4,5]. Moreover, this inhibitor has also recently been used to develop the PROTAC (proteolysis targeting chimera) ACBI-1 [6].

SGC-SMARCA-BRDVIII binds potently to the SMARCA2/4 and PB1(5) bromodomains with a KD(ITC) of 35, 36 and 13 nM. It is selective within the other bromodomain families and shows no off-targets activity on 85 protein kinases screened using temperature shift binding assays. This compound is non-toxic as demonstrated by the NCI-60 human tumor cell lines screen, but it shows remarkably cellular activity in an adipogenesis cell differentiation assay with an EC50 of < 1.0 µM.

Of special note is that SGC-SMARCA-BRDVIII outperformed the chemical probe PFI-3 in that particular assay, and therefore, we advise to use SGC-SMARCA-BRDVIII and PFI-3 to confirm the results, when elucidating the biological role of the SWI/SNF bromodomains.

In addition, a negative control compound, SGC-BRDVIII-NC, is provided, which showed no cellular activity. If interested, a chemogenomic tool compound, SGC-pan-BRDVIII, that additionally hits the PB1(2,3) members with a KD(ITC) of 200-400 nM can be inquired [3].

Potency Against Target Family

Bromodomain SGC-SMARCA-BRDVIII KD (nM)
SMARCA235
SMARCA436
PB1(5)13
PB1(2)3655
PB1(3)1963

Table 1: Screening SGC-SMARCA-BRDVIII against selected targets.

Selectivity 
Selectivity of the SGC-SMARCA-BRDVIII probe within the bromodomain families was confirmed by an in-house thermal shift panel containing 25 bromodomains. No activity was also observed on 85 protein kinases screened in an in-house DSF panel.

Selectivity Dosage
To minimize the chance of off-target effects, we recommend that a concentration of no higher than 10 µM should be used in cell-based assays.

Cellular Activity
The formation from 3T3-L1 mouse fibroblasts into adipocytes was impaired with an EC50 below 1.0 µM upon treatment with SGC-SMARCA-BRDVIII.

properties
Probe Negative control

 

SGC-SMARCA-BRDVIII

 

SGC-BRDVIII-NC

 

 

 

 

 

SGC-SMARCA-BRDVIII
Physical and chemical properties
Molecular weight380.45 g/mol
Molecular formulaC19H25N5O3
IUPAC nametert-butyl 4-[3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl]piperazine-1-carboxylate
logP1.82
TPSA104.8
No. of chiral centres0
No. of rotatable bonds5
No. of hydrogen bond acceptors5
No. of hydrogen bond donors2
StorageStable as powder at -20°C. NB making aliquots rather than freeze-thawing is recommended
DissolutionDMSO (up to 50 mM)
SGC-BRDVIII-NC
Physical and chemical properties
Molecular weight385.21 g/mol
Molecular formulaC20H27N5O3
IUPAC nametert-butyl-4-[3-amino-6-(2-methoxyphenyl)yridazine-4-yl]piperazine-1-carboxylate
clogP2.02
TPSA93.8
No. of chiral centres0
No. of rotatable bonds6
No. of hydrogen bond acceptors5
No. of hydrogen bond donors1
StorageStable as powder at -20°C. NB making aliquots rather than freeze-thawing is recommended
DissolutionDMSO (up to 50 mM)

SMILES:
SGC-SMARCA-BRDVIII: OC1=CC=CC=C1C2=CC(N3CCN(CC3)C(OC(C)(C)C)=O)=C(N=N2)N
SGC-BRDVIII-NC: NC(N=NC(C1=CC=CC=C1OC)=C2)=C2N3CCN(CC3)C(OC(C)(C)C)=O

InChI:
SGC-SMARCA-BRDVIII: InChI=1S/C19H25N5O3/c1-19(2,3)27-18(26)24-10-8-23(9-11-24)15-12-14(21-22-17(15)20)13-6-4-5-7-16(13)25/h4-7,12,25H,8-11H2,1-3H3,(H2,20,22)
SGC-BRDVIII-NC:InChI=1S/C20H27N5O3/c1-20(2,3)28-19(26)25-11-9-24(10-12-25)16-13-15(22-23-18(16)21)14-7-5-6-8-17(14)27-4/h5-8,13H,9-12H2,1-4H3,(H2,21,23)

InChIKey:
SGC-SMARCA-BRDVIII: AQTNUGRRZDRZIA-UHFFFAOYSA-N
SGC-BRDVIII-NC: YOBVMDSDYHOOLN-UHFFFAOYSA-N
 

selectivity profile

In-house DSF panel revealed no off-targets for SGC-SMARCA-BRDVIII outside the BRD subfamily VIII. The negative control compound, SGC-BRDVIII-NC is completely inactive on all tested targets.

Bromodomains 

SGC-SMARCA-BRDVIII

(ΔTm°C)a,b

TAF1L(1)-0.2± 0.1ATAD20.4± 0.1
TAF1L(2)-0.1± 0.1EP3000.0± 0.1
BRD2(1)0.0± 0.2BRD4(1)0.7± 0.1
PB1(1)0.9± 0.1BRD3(2)-0.1± 0.1
PB1(2)2.5± 0.1BRPF1A0.2± 0.1
PB1(3)2.7± 0.2BRD10.2± 0.1
PB1(4)0.7± 0.1TRIM33B0.1± 0.1
PB111.3± 0.3SP110A-0.1± 0.1
PB10.3± 0.1PCAF0.0± 0.1
SMARCA27.7± 0.2WDR9-0.4± 0.1
SMARCA47.4± 0.1BRDT(1_2)0.1± 0.1
CREBBP-0.1± 0.1  

Table 1: Screening SGC-SMARCA-BRDVIII against a panel of bromodomains. 

Figure 1: SGC-SMARCA-BRDVIII (22) binds potently to SMARCA2/4 and PB1(5) as determined by ITC and shows weak affinity for the highly conserved homologues PB1(2) and PB1(3).

in vitro potency
cell based assay data

SGC-SMARCA-BRDVIII (22) is cell active and it impairs the formation of adipocytes from 3T3-L1 fibroblasts by reducing the expression levels of adipocyte-related genes. The control compound SGC-BRDVIII-NC (35) is not active.

Figure 1: Cell based assay data for SGC-SMARCA-BRDVIII

SGC-SMARCA-BRDVIII is non-toxic and does not influence cell growth at a dose of 10 µM as indicated in the NCI-60 human tumor cell lines screen, and can therefore ideally used in cell differentiation assays.

references
  1. Kadoch, C., Crabtree, GR. Mammalian SWI/SNF chromatin remodeling complexes and cancer: Mechanistic insights gained from human genomics. Sci. Adv., 2015, 1, 5, e1500447.
  2. Albrecht, BK., Cote, A., Crawford, TD., Duplessis, M., Good, AC., LeBlanc, Y., Magnuson, SR., Nasveschuk, CG., Romero, AF., Tang, Y., Taylor, AM. Therapeutic pyridazine compounds and uses thereof. US Patent, WO 2016/138114 A1.
  3. Wanior, M., Preuss, F., Ni, X., Krämer, A., Mathea, S., Göbel, T., Heidenreich, D., Simonyi, S., Kahnt, AS., Joerger, AC., Knapp, S. Pan-SMARCA/PB1 bromodomain inhibitors and their role in regulating adipogenesis. J. Med. Chem., 2020, 63, 23, 14680–14699.
  4. Gerstenberger, B.S., Trzupek, JD., Tallant, C., Fedorov, O., Filippakopoulos, P., Brennan, PE., Fedele, V., Martin, S., Picaud, S., Rogers, C., Parikh, M., Taylor, A., Samas, B., O'Mahony, A., Berg, E., Pallares, G., Torrey, AD., Treiber, DK., Samardjiev, IJ., Nasipak, BT., Padilla-Benavides, T., Wu, Q., Imbalzano, AN., Nickerson, JA., Bunnage, ME., Müller, S., Knapp, S., Owen, DR. Identification of a chemical probe for family VIII bromodomains through optimization of a fragment hit. J. Med. Chem. 2016, 59, 4800–4811.
  5. Fedorov, O., Castex, J., Tallant, C., Owen, DR., Martin, S., Aldeghi, M., Monteiro, O., Filippakopoulos, P., Picaud, S., Trzupek, JD., Gerstenberger, BS., Bountra, C., Willmann, D., Wells, C., Philpott, M., Rogers, C., Biggin, PC., Brennan, PE., Bunnage, ME., Schüle, R., Günther, T., Knapp, S., Müller, S. Selective targeting of the BRG/PB1 bromodomains impairs embryonic and trophoblast stem cell maintenance. Sci. Adv. 2015, 1, 10, e1500723.
  6. Farnaby, W., Koegl, M., Roy, MJ., Whitworth, C., Diers, E., Trainor, N., Zollman, D., Steurer, S., Karolyi-Oezguer, J., Riedmueller, C., Gmaschitz, T., Wachter, J., Dank, C., Galant, M., Sharps, B., Rumpel, K., Traxler, E., Gerstberger, T., Schnitzer, R., Petermann, O., Greb, P., Weinstabl, H., Bader, G., Zoephel, A., Weiss-Puxbaum, A., Ehrenhöfer-Wölfer, K., Wöhrle, S., Boehmelt, G., Rinnenthal, J., Arnhof, H., Wiechens, N., Wu, MY., Owen-Hughes, T., Ettmayer, P., Pearson, M., McConnell, DB., Ciulli, A. BAF complex vulnerabilities in cancer demonstrated via structure-based PROTAC design. Nat. Chem. Biol. 2019, 15, 672–680.
pk properties
co-crystal structures

Binding mode of SGC-SMARCA-BRDVIII in complex with PB1(5)-BRD (PDB-ID 6ZS4). The inhibitor binds to the highly conserved Asn739 and Tyr696. The methylation of the hydroxy group in SGC-BRDVIII-NC blocks its binding to the bromodomain K(ac) binding pocket.

synthetic schemes
materials and methods
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