11 November, 2021, Toronto, Canada - Cyclica, the partner of choice for data-driven drug discovery, and the Structural Genomics Consortium (SGC), a global public-private partnership dedicated to open science, have collaborated on a project in support of Target 2035, an initiative to discover probe molecules in support of developing medicines for all.
New look for Target 2035
SGC’s Target 2035 initiative just launched a new website with a fresh look and feel. Users will be able to access information about the project, upcoming webinars, and general updates. Check the Target 2035 Twitter and Linked In accounts for the latest news.
What is Target 2035
| Probe | Negative control | |
 |
|  |
CK156 |
| CKJB71 |
DAP Kinase-Related Apoptosis-Inducing Protein Kinase 1 (DRAK1) is part of the DAPK (death-associated protein kinases) family, which comprises of five members (DAPK1–3 and DRAK2). Both, DRAK´s (DRAK1 & 2) belong to the so-called dark kinome and their cellular functions are largely unknown [1–3]. However, recent findings indicate that DRAK1 might play a role in different cancers such as glioblastoma multiforme (GBM) [4], head and neck squamous cell carcinoma (HNSCC) [5] or in testicular cancer [6]. In addition, DRAK1 has been identified to be a direct target gene of p53, and vice versa DRAK1 might regulate the transcriptional activity of p53 [6, 7]. More recently, Park et al demonstrated that DRAK1 might also act as a negative regulator of TRAF6, which is a central player in inflammatory signalling pathway in cervical cancer [8]. However, the precise role of DRAK1 in different cancers remains elusive and chemical tools are urgently needed to determine the role of DRAK1 in these diseases.
SGC has developed CK156, a potent and selective DRAK1 inhibitor with an IC50 of 49 nM for DRAK1 determined by a radiometric assay and a KD of 21 nM determined by ITC. Cellular activity was examined by NanoBRET and CK156 revealed an IC50 of 181 nM on DRAK1. The chemical probe (CK156) is accompanied by a negative control (CKJB71), which is structurally closely related to the probe molecule.
Potency Against Target Family
| Kinase | 33PanQinase IC50 (nM) |
| DRAK1 | 49 |
ITC: KD = 21 nM
Selectivity
CK156 has been shown to be selective in an in vitro kinase panel from DiscoverX (scanMAX®) against 468 Kinases followed by cellular NanoBRET assays.
Dosage
To minimize the chance of any unspecific cytotoxicity, we recommend a concentration of no higher than 5 µM for cell-based assays.
Cellular Activity
CK156 displayed an IC50 of 181 nM in NanoBRETTM assay.
| Probe | Negative control | |
|
| |
CK156 |
| CKJB71 |
Physical and chemical properties CK156 | |
| Molecular weight | 395.46
|
| Molecular formula | C21H25N5O3 |
| IUPAC name | N-tert-butyl-7,10-dioxa-13,17,18,21-tetraazatetracyclo[12.5.2.1²,⁶.0¹⁷,²⁰]docosa-1(20),2,4,6(22),14(21),15,18-heptaene-5-carboxamide |
| logP | 1.89 |
| PSA | 89.78 |
| No. of chiral centres | 0 |
| No. of rotatable bonds | 2 |
| No. of hydrogen bond acceptors | 8 |
| No. of hydrogen bond donors | 2 |
| Storage | stable as solid in the dark at -20°C. NB making aliquots rather than freeze-thawing is recommended |
| Dissolution | soluble in DMSO in a concentration of 10 mM |
SMILES: O=C(C(C=C1)=C(OCCOCCNC2=N3)C=C1C4=C3N(C=C2)N=C4)NC(C)(C)C
InChI: InChI=1S/C21H25N5O3/c1-21(2,3)25-20(27)15-5-4-14-12-17(15)29-11-10-28-9-7-22-18-6-8-26-19(24-18)16(14)13-23-26/h4-6,8,12-13H,7,9-11H2,1-3H3,(H,22,24)(H,25,27)
InChIKey: YCFFONJEHNSECY-UHFFFAOYSA-N
Physical and chemical properties CKJB71 | |
| Molecular weight | 422.49 |
| Molecular formula | C22H26N6O3 |
| IUPAC name | 5-(4-methylpiperazine-1-carbonyl)-7,10-dioxa-13,17,18,21-tetraazatetracyclo[12.5.2.1²,⁶.0¹⁷,²⁰]docosa-1(20),2,4,6(22),14(21),15,18-heptaene |
| logP | 1.00 |
| PSA | 84.23 |
| No. of chiral centres | 0 |
| No. of rotatable bonds | 1 |
| No. of hydrogen bond acceptors | 9 |
| No. of hydrogen bond donors | 1 |
| Storage | stable as solid in the dark at -20°C. NB making aliquots rather than freeze-thawing is recommended |
| Dissolution | soluble in DMSO in a concentration of 10 mM |
SMILES: O=C(C1=CC=C2C=C1OCCOCCNC3=NC4=C2C=NN4C=C3)N5CCN(CC5)C
InChI: InChI=1S/C22H26N6O3/c1-26-7-9-27(10-8-26)22(29)17-3-2-16-14-19(17)31-13-12-30-11-5-23-20-4-6-28-21(25-20)18(16)15-24-28/h2-4,6,14-15H,5,7-13H2,1H3,(H,23,25)
InChIKey:
DDXOMWWURGFLKK-UHFFFAOYSA-N
Selectivity profile of CK156 was determined with the scanMAX® assay from DiscoverX at 1000 and 100 nM and on- and off targets were evaluated with in vitro IC50 with the 33PanQinase activity assay from ProQinase and in cellulo IC50 with NanoBRET assay.
Kinase | Percent of control(%) @ 1000 nM | 33PanQinase IC50 (nM) | NanoBRET IC50 (nM) |
| DRAK1 | 0.3 | 49 | 181 |
| CSNK2A2 | 1.5 | 380 | 39000 |
| CSNK2A1 | 3.9 | 950 | 34000 |
| GAK | 23 | n.d. | 24000 |
| BIKE | 25 | n.d. | 8000 |
| JAK3(JH1domain-catalytic) | 28 | n.d. | n.d |
| RIOK3 | 28 | n.d. | n.d. |
| BUB1 | 30 | n.d. | n.d. |
| ERK8 | 33 | n.d. | n.d |
| DRAK2 | 39 | n.d. | 8000 |
The negative control CKJB71 showed no activity in a DSF assay for 90 kinases and no on-target activity determined by NanoBRET in intact cells and in lysed cells.
CK156 displayed no general cytotoxicity in three different cell lines (HEK293T, U2OS and MRC9-Fibroblasts) at 1µM and only slight toxicity at 10 µM. The cells were analysed after 18 and 36h.
[1] Berginski ME, Moret N, Liu C, Goldfarb D, Sorger PK, G. S. The Dark Kinase Knowledgebase: An Online Compendium of Knowledge and Experimental Results of Understudied Kinases. Nucleic Acids Res. 2020. https://doi.org/10.1093/nar/gkaa853.
[2] Farag, A. K.; Roh, E. J. Death-Associated Protein Kinase (DAPK) Family Modulators: Current and Future Therapeutic Outcomes. Medicinal Research Reviews. John Wiley and Sons Inc. January 1, 2019, pp 349–385. https://doi.org/10.1002/med.21518.
[3] Bialik, S.; Kimchi, A. The Death-Associated Protein Kinases: Structure, Function, and Beyond. Annual Review of Biochemistry. Annu Rev Biochem 2006, pp 189–210. https://doi.org/10.1146/annurev.biochem.75.103004.142615.
[4] Mao, P.; Hever-Jardine, M. P.; Rahme, G. J.; Yang, E.; Tam, J.; Kodali, A.; Biswal, B.; Fadul, C. E.; Gaur, A.; Israel, M. A.; Spinella, M. J. Serine/Threonine Kinase 17A Is a Novel Candidate for Therapeutic Targeting in Glioblastoma. PLoS One 2013, 8 (11). https://doi.org/10.1371/journal.pone.0081803.
[5] Park, Y.; Kim, W.; Lee, J. M.; Park, J.; Cho, J. K.; Pang, K.; Lee, J.; Kim, D.; Park, S. W.; Yang, K. M.; Kim, S. J. Cytoplasmic DRAK1 Overexpressed in Head and Neck Cancers Inhibits TGF-Β1 Tumor Suppressor Activity by Binding to Smad3 to Interrupt Its Complex Formation with Smad4. Oncogene 2015, 34 (39), 5037–5045. https://doi.org/10.1038/onc.2014.423.
[6] Mao, P.; Hever, M. P.; Niemaszyk, L. M.; Haghkerdar, J. M.; Yanco, E. G.; Desai, D.; Beyrouthy, M. J.; Kerley-Hamilton, J. S.; Freemantle, S. J.; Spinella, M. J. Serine/Threonine Kinase 17A Is a Novel P53 Target Gene and Modulator of Cisplatin Toxicity and Reactive Oxygen Species in Testicular Cancer Cells. J. Biol. Chem. 2011, 286 (22), 19381–19391. https://doi.org/10.1074/jbc.M111.218040.
[7] Oue, Y.; Murakami, S.; Isshiki, K.; Tsuji, A.; Yuasa, K. Intracellular Localization and Binding Partners of Death Associated Protein Kinase-Related Apoptosis-Inducing Protein Kinase 1. Biochem. Biophys. Res. Commun. 2018, 496 (4), 1222–1228. https://doi.org/10.1016/j.bbrc.2018.01.175.
[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.
The probe PFI-7 (hydrochloride) is available at Sigma and Tocris.
The inactive control PFI-7N (hydrochloride) is available at Sigma.
| Probe | Negative control | |
 |
|  |
PFI-7 |
| PFI-7N |
Pfizer in collaboration with the SGC have developed PFI-7, a potent, cell active chemical probe for the E3 ligase GID4. PFI-7 binds potently to GID4 with KD = 0.08 μM (SPR) and displaces the known degron1 peptide in a NanoBRETTM assay with EC50 = 0.6 μM. PFI-7N is a closely related negative control with KD = 5 μM (SPR). A co-crystal structure has been deposited.
We have further developed a handle PFI-E3H1 and a PEGylated analogue to show that the handle tolerates a substitution. These findings offers opportunities to synthesize proximity-inducing or degrader modalities2.
 |  |
 |   |
[PFI-7] (μM) | [PFI-7] (μM) |
A NanoBRET assay was used to show target engagement in cells.
The interaction was between NanoLuc® tagged degrons and full-length GID4.
 |
Main features
| Probe | Negative control | |
 |
|  |
MRIA9 |
| MR7 |
Salt-inducible kinases (SIK1-3) are members of the AMP-activated protein kinase (AMPK) family which is part of the calcium/calmodulin-dependent kinase (CaMK) group. These serine/threonine kinases act as regulators of energy homeostasis and metabolic stress. The SIK family member SIK2, for example, is activated in cells recovering from starvation, leading to phosphorylation and hence activation of the transcription factor cAMP response element-binding protein (CREB1) [1-3]. In addition to the key function of SIK in regulating metabolism, imbalance of SIK has been observed in the context of several diseases, especially in cancer, with both tumor promoting and tumor suppressive roles being reported [4]. SIK2 is often deleted in breast cancer, and downregulation of SIK1 has been linked to a tumor suppressor role but also the development of metastasis by promoting p53-dependent anoikis [5]. We have recently shown that in biopsies from patients with gastric cancer, there were increased levels of SIK2 mRNA and protein in advanced stages of the tumor compared to lower grade tumors, independent of its metastatic stage [6]. Overall, these data highlight the complex roles of SIK family proteins in different types of cancer and they may emerge as important therapeutic targets. To clarify the multifaceted roles of these kinases in disease and normal physiology, chemical tools targeting SIK are urgently needed.
SGC has developed MRIA9, a potent and selective pan SIK inhibitor with a IC50 determined by a radiometric assay of 55, 48 and 22 nM for SIK1, SIK2 and SIK3 respectively and IC50 of 516, 180 and 127 nM on NanoBRET™ assay. [7] MRIA9 has been developed based on PAK1 inhibitor G-5555 published by Genentech. Group I PAK (PAK1, PAK2 and PAK3) remains off targets with respectively in vitro IC50s of 580, 41 and 140 nM. However, due to lack of accessibility of a NanoBRET™ assay for Group I PAKs, MRIA9 is considered a SIK and Group I PAK probe. In addition, the chemical probe (MRIA9) is accompanied by a negative control (MR7), which is structurally similar to the probe molecule.
Potency Against Target Family
| Kinase | 33PanQinase IC50 (nM) |
| SIK1 | 55 |
| SIK2 | 48 |
| SIK3 | 22 |
MRIA9 has been shown to be selective in an in vitro kinase panel from Reaction Biology followed by cellular NanoBRET assays. The selectivity outside target family revealed Group I PAKs as closest off-target.
To minimize the chance of off-target effects, we recommend a concentration of no higher than 10 µM for cell-based assays.
In NanoBRET assay using HEK923T cells MRIA9 shows an IC50 of 516, 180 and 127 nM for SIK1, SIK2 and SIK3 respectively.
| Probe | Negative control | |
|
| |
MRIA9 |
| MR7 |
| Physical and chemical properties MRIA9 | |
| Molecular weight | 496.93 |
| Molecular formula | C24H22ClFN6O3 |
| IUPAC name | 8-(((2r,5r)-5-amino-1,3-dioxan-2-yl)methyl)-6-(2-chloro-4-(3-fluoropyridin-2-yl)phenyl)-2-(methylamino)pyrido[2,3-d]pyrimidin-7(8H)-one |
| logP | 2.16 |
| PSA | 113.9 |
| No. of chiral centre | 0 |
| No. of rotatable bonds | 7 |
| No. of hydrogen bond acceptors | 7 |
| No. of hydrogen bond donors | 2 |
| Storage | stable as solid in the dark at -20°C. NB making aliquots rather than freeze-thawing is recommended |
| Dissolution | soluble in DMSO in a concentration of 50 mM |
SMILES:
CNC1=NC=C2C(N(C(C(C3=CC=C(C=C3Cl)C4=NC=CC=C4F)=C2)=O)C[C@H]5OC[C@@H](CO5)N)=N1
InChI: InChI=1S/C24H22ClFN6O3/c1-28-24-30-9-14-7-17(16-5-4-13(8-18(16)25)21-19(26)3-2-6-29-21)23(33)32(22(14)31-24)10-20-34-11-15(27)12-35-20/h2-9,15,20H,10-12,27H2,1H3,(H,28,30,31)/t15-,20-
InChIKey:
QKNBRNSGPNCARD-SGNKCFNYSA-N
| Physical and chemical properties MR7 | |
| Molecular weight | 510.95 |
| Molecular formula | C25H24ClFN6O3 |
| IUPAC name | 8-(((2r,5r)-5-amino-1,3-dioxan-2-yl)methyl)-6-(2-chloro-4-(3-fluoropyridin-2-yl)phenyl)-2-(dimethylamino)pyrido[2,3-d]pyrimidin-7(8H)-one |
| logP | 2.95 |
| PSA | 105.11 |
| No. of chiral centre | 0 |
| No. of rotatable bonds | 8 |
| No. of hydrogen bond acceptors | 8 |
| No. of hydrogen bond donors | 1 |
| Storage | stable as solid in the dark at -20°C. NB making aliquots rather than freeze-thawing is recommended |
| Dissolution | soluble in DMSO in a concentration of 50 mM |
SMILES:
CN(C1=NC=C2C(N(C(C(C3=CC=C(C=C3Cl)C4=NC=CC=C4F)=C2)=O)C[C@H]5OC[C@@H](CO5)N)=N1)C
InChI: InChI=1S/C25H24ClFN6O3/c1-32(2)25-30-10-15-8-18(17-6-5-14(9-19(17)26)22-20(27)4-3-7-29-22)24(34)33(23(15)31-25)11-21-35-12-16(28)13-36-21/h3-10,16,21H,11-13,28H2,1-2H3/t16-,21-
InChIKey:
RRFFBKGCCWPMLK-OQIWPSSASA-N
Selectivity profile of MRIA9 was determined with the 33PanQinase activity assay from Reaction Biology at 1uM and off targets were confirmed with in vitro IC50 with the same assay and in cellulo IC50 with NanoBRET™ assay. MRIA9 is a pan SIK and group I PAK inhibitor.
| Kinase | Percent of control(%) | 33 PanQinase IC 50 (nM) | NanoBRET IC 50 (nM) |
| SIK2 | 1 | 48 | 180 |
| SIK3 | 2 | 22 | 127 |
| SIK1 | 4 | 55 | 516 |
| KHS1 | 8 | 210 | 13000 |
| PAK3 | 9 | 140 | n.d |
| PAK2 | 10 | 41 | n.d |
| NLK | 13 | 250 | 3100 |
| PKN3 | 35 | 1400 | 6700 |
| PAK1 | 36 | 580 | n.d |
| MAP2K4 | 37 | 830 | n.d |
| TIE2 | 39 | 3100 | 6000 |
| MST4 | 45 | 1600 | 34000 |
| MELK | 48 | 2200 | n.d |
The negative control MR7 with its blocked hinge-binding amine showed no activity on a DSF assay for 100 kinases and low activity on target on NanoBRET assay.
 |
MRIA9 modulated endogenous substrates linked to SIK activity. In the ovarian cancer cell SKOV-3 in which the PI3K/AKT/MTOR pathway was activated by rapamycine, MRIA9 abrogated the phosphorylation of AKT in a dose-dependent manner. In addition, SIK2 auto-phosphorylation activity was completely inhibited whereas the negative control did not influence SIK2 activity.
| SKOV-3 cell line |
 |
MRIA9 replicated a known phenotype of SIK inhibition, displacing the centrosome from the nucleous in ovarian cancer cell line SKOV-3, similar to the phenotype seen when silence RNA is used to knock down SIK2.
| SKOV-3 cell line |
 |
In the NCI-60 screen, which is a human tumor cell line screen, MRIA9 showed only modest cell toxicity or growth inhibition. It was tested in a single high dose of 10 µM in the full NCI-60 panel. https://dtp.cancer.gov/discovery_development/nci-60/methodology.htm
(1) Sun, Z.; Jiang, Q.; Li, J.; Guo, J. The potent roles of salt-inducible kinases (SIKs) in metabolic homeostasis and tumorigenesis. Signal transduction and targeted therapy 2020, 5 (1), 150.
(2) Conkright, M. D.; Canettieri, G.; Screaton, R.; Guzman, E.; Miraglia, L.; Hogenesch, J. B.; Montminy, M. TORCs: transducers of regulated CREB activity. Molecular cell 2003, 12 (2), 413–423.
(3) Katoh, Y.; Takemori, H.; Lin, X.-Z.; Tamura, M.; Muraoka, M.; Satoh, T.; Tsuchiya, Y.; Min, L.; Doi, J.; Miyauchi, A.; Witters, L. A.; Nakamura, H.; Okamoto, M. Silencing the constitutive active transcription factor CREB by the LKB1-SIK signaling cascade. The FEBS journal 2006, 273 (12), 2730–2748.
(4) Chen, F.; Chen, L.; Qin, Q.; Sun, X. Salt-inducible kinase 2: an oncogenic signal transmitter and potential target for cancer therapy. Frontiers in oncology 2019, 9, 18.
(5) Cheng, H.; Liu, P.; Wang, Z. C.; Zou, L.; Santiago, S.; Garbitt, V.; Gjoerup, O. V.; Iglehart, J. D.; Miron, A.; Richardson, A. L.; Hahn, W. C.; Zhao, J. J. SIK1 couples LKB1 to p53-dependent anoikis and suppresses metastasis. Science signaling 2009, 2 (80), ra35.
(6) Montenegro, R. C.; Howarth, A.; Ceroni, A.; Fedele, V.; Farran, B.; Mesquita, F. P.; Frejno, M.; Berger, B.-T.; Heinzlmeir, S.; Sailem, H. Z.; Tesch, R.; Ebner, D.; Knapp, S.; Burbano, R.; Kuster, B.; Müller, S. Identification of molecular targets for the targeted treatment of gastric cancer using dasatinib. Oncotarget 2020, 11 (5), 535–549.
(7) Tesch, R.; Rak, M.; Raab, M.; Berger, L. M.; Kronenberger, T.; Joerger, A. C.; Berger, B.-T.; Abdi, I.; Hanke, T.; Poso, A.; Strebhardt, K.; Sanhaji, M.; Knapp, S. Structure-Based Design of Selective Salt-Inducible Kinase Inhibitors. Journal of Medicinal Chemistry 2021, 64 (12), 8142-8160.
Binding mode of MRIA9 in complex with the crystallographic surrogate model MST3 (PDB 7B31). The inhibitor binds to the ATP pocket and different stability on the P-loop region of MST3 and SIK2 studied by molecular dynamics, explain the selectivity of MRIA9 towards SIK2. [7]
The probe is available from Sigma and Cayman Chemicals.
The control may be requested here.
| 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
| WDR5 | Homer KD |
| DSF | 13.2 K |
| ITC | 18 nM |
| NanoBRET | 13.6 µM |
| HiBit | DCmax: 58% at 1 µM DC50: 53 nM |
| quantitative Proteomics | (-log10p > 3, log2FC < -0.5) |
Selectivity of the first WDR5 probe OICR-9429 was shown previously [5, 6]. Selectivity for the Homer probe was confirmed by quantitative proteomics.
For optimal degradation and to overcome the Hook effect, we recommend that a concentration of 1 µM should be used in cell-based assays.
Homer induces depletion of endogenous WDR5 in various cancer cell lines by inducing protein ubiquitylation and degradation.
| Probe | Negative control | Negative control | ||
|
| |
| |
Homer |
| nc_WDR5 |
| nc_VHL |
| Homer | |
| Physical and Chemical Properties | |
| Molecular weight | 1012.16 |
| Molecular formula | C52H60F3N9O7S |
| IUPAC name | N-(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 centres | 3 |
| No. of rotatable bonds | 23 |
| No. of hydrogen bond acceptors | 12 |
| No. of hydrogen bond donors | 6 |
| Storage | stable as powder at -20°C. NB making aliquots rather than freeze-thawing is recommended |
| Dissolution | soluble in DMSO at 50 mM |
| nc_VHL | |
| Physical and Chemical Properties | |
| Molecular weight | 1012.16 |
| Molecular formula | C52H60F3N9O7S |
| IUPAC name | N-(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 centres | 3 |
| No. of rotatable bonds | 23 |
| No. of hydrogen bond acceptors | 12 |
| No. of hydrogen bond donors | 6 |
| Storage | stable as powder at -20°C. NB making aliquots rather than freeze-thawing is recommended |
| Dissolution | soluble in DMSO at 50 mM |
| nc_WDR5 | |
| Physical and Chemical Properties | |
| Molecular weight | 999.12 |
| Molecular formula | C51H57F3N8O9S |
| IUPAC name | N-(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 centres | 3 |
| No. of rotatable bonds | 23 |
| No. of hydrogen bond acceptors | 12 |
| No. of hydrogen bond donors | 6 |
| Storage | stable as powder at -20°C. NB making aliquots rather than freeze-thawing is recommended |
| Dissolution | soluble in DMSO at 50 mM |
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
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
Homer: OFNZESNEBSQKSE-BQGOKDIQSA-N
nc_VHL: OFNZESNEBSQKSE-CCVFZADKSA-N
nc_WDR5: BFXBMIZHEIFXOC-LTJJNQLXSA-N
 |
| Quantitative Proteomics revealed that only WDR5 is significally and substantially depleted by Homer treatment. |
 |
| Homer binds potently to WDR5 as determined by ITC. |
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.
[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.
 |  |
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]
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.
| 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) |
| SMARCA2 | 35 |
| SMARCA4 | 36 |
| 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.
| Probe | Negative control | |
|
| |
SGC-SMARCA-BRDVIII |
| SGC-BRDVIII-NC |
| SGC-SMARCA-BRDVIII | |
| Physical and chemical properties | |
| Molecular weight | 380.45 g/mol |
| Molecular formula | C19H25N5O3 |
| IUPAC name | tert-butyl 4-[3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl]piperazine-1-carboxylate |
| logP | 1.82 |
| TPSA | 104.8 |
| No. of chiral centres | 0 |
| No. of rotatable bonds | 5 |
| No. of hydrogen bond acceptors | 5 |
| No. of hydrogen bond donors | 2 |
| Storage | Stable as powder at -20°C. NB making aliquots rather than freeze-thawing is recommended |
| Dissolution | DMSO (up to 50 mM) |
| SGC-BRDVIII-NC | |
| Physical and chemical properties | |
| Molecular weight | 385.21 g/mol |
| Molecular formula | C20H27N5O3 |
| IUPAC name | tert-butyl-4-[3-amino-6-(2-methoxyphenyl)yridazine-4-yl]piperazine-1-carboxylate |
| clogP | 2.02 |
| TPSA | 93.8 |
| No. of chiral centres | 0 |
| No. of rotatable bonds | 6 |
| No. of hydrogen bond acceptors | 5 |
| No. of hydrogen bond donors | 1 |
| Storage | Stable as powder at -20°C. NB making aliquots rather than freeze-thawing is recommended |
| Dissolution | DMSO (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
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± 0.1 | ATAD2 | 0.4± 0.1 |
| TAF1L(2) | -0.1± 0.1 | EP300 | 0.0± 0.1 |
| BRD2(1) | 0.0± 0.2 | BRD4(1) | 0.7± 0.1 |
| PB1(1) | 0.9± 0.1 | BRD3(2) | -0.1± 0.1 |
| PB1(2) | 2.5± 0.1 | BRPF1A | 0.2± 0.1 |
| PB1(3) | 2.7± 0.2 | BRD1 | 0.2± 0.1 |
| PB1(4) | 0.7± 0.1 | TRIM33B | 0.1± 0.1 |
| PB1 | 11.3± 0.3 | SP110A | -0.1± 0.1 |
| PB1 | 0.3± 0.1 | PCAF | 0.0± 0.1 |
| SMARCA2 | 7.7± 0.2 | WDR9 | -0.4± 0.1 |
| SMARCA4 | 7.4± 0.1 | BRDT(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).
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.
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.
On September 8th, 2020, the Royal Society of Canada (RSC) elected Dr. Cheryl Arrowsmith, SGC-Toronto's Chief Scientist, as an RSC Fellow, the highest honour an individual can achieve in the Arts, Social Sciences and Sciences.
| Probe | Negative control | |
 |
|  |
NR162 |
| NR187 |
CASK (Ca2+/calmodulin-dependent Ser/Thr kinase) is a multidomain scaffolding protein, containing several protein-protein interaction domains. From the N-to the C-terminus CASK consists of a CAMK-like domain, two L27 domains, a PDZ domain, a SH3 domain and a guanylate kinase domain1. CASK is a member of the MAGUK (membrane-associated guanylate kinase) family that functions as a Mg2+-independent neurexin kinase with implicated roles in neuronal synapses and trafficking2. Furthermore, CASK has been categorized as a pseudokinase as it lacks the typical DFG motif and shows a GFG-motif instead. Pseudokinases carry a mutation at least in one of three highly conserved motifs within the catalytic kinase domain (VAIK-, DGF and Y/HRD motif) resulting in inactivation of catalytic activity. All in all 48 kinases of the kinome are pseudokinases3. However, CASK has been shown to maintain some catalytic activity in the absence of metal ions. High expression levels and mutations in CASK have been linked to diverse diseases, including colorectal cancer4, Parkinson's disease5 and X-linked mental retardation6, making CASK a potential drug target.
Phylogenetic kinase tree, CASK highlighted with red circle. Illustration is reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com).
SGC has developed NR162, a potent and selective CASK inhibitor with a Kd of 22 nM in ITC and IC50 of 80 nM in NanoBRET assay. NR162 has been developed based on a series of TYRO3 inhibitors first published by Pfizer7. TYRO3 is a weak off target, but NR162 is 47-fold selective against TYRO3 (IC50 of 3.8 µM (NanoBRET). In addition, the chemical probe (NR162) is accompanied by a negative control (NR187), which is structurally similar to the probe molecule.
Potency Against Target Family
NR162 is a chemical probe for CASK with a Kd of 22 nM in ITC and an IC50 of 80 nM in NanoBRET assay. The closest off-target within the target family is ERBB3 with an IC50 of 18.2 µM (227 fold selective).
| Kinase | NR162 (µM) |
| CASK | 0.08 |
| ERBB3 | 18.2 |
Selectivity
NR162 has been shown to be selective in an in vitro kinase panel followed by cellular NanoBRET assays. Selectivity has been also confirmed by a DiscoverX KINOMEScan at 1000 nM. Off-targets were assessed as false positive in a follow-up NanoBRET. The selectivity outside target family revealed TYRO3 with an IC50 of 3.8 µM (47-fold) 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 NR162 shows an IC50 of 80 nM against on target.
| NR162 |
|
Click here to download the SDF file. |
| NR162 | |
| Physical and chemical properties | |
| Molecular weight | 608.07 |
| Molecular formula | C24H30Br2N6O3 |
| IUPAC name | 4-(cyclopentylamino)-2-((2,5-dibromo-4-methylbenzyl)amino)-N-(3-(2-oxooxazolidin-3-yl)propyl)pyrimidine-5-carboxamide |
| logP | 4.2 |
| TPSA | 107.42 |
| No. of chiral centres | 0 |
| No. of rotatable bonds | 10 |
| No. of hydrogen bond acceptors | 6 |
| No. of hydrogen bond donors | 3 |
| Storage | stable as solid in the dark at -20°C. NB making aliquots rather than freeze-thawing is recommended |
| Dissolution | soluble in DMSO |
| NR187 |
|
Click here to download the SDF file. |
| NR187 | |
| Physical and chemical properties | |
| Molecular weight | 499.06 |
| Molecular formula | C26H35ClN6O2 |
| IUPAC name | 2-((4-chlorobenzyl)(ethyl)amino)-4-(cyclopentylamino)-N-(3-(2-oxopyrrolidin-1-yl)propyl)pyrimidine-5-carboxamide |
| clogP | 3.53 |
| TPSA | 89.4 |
| No. of chiral centres | 0 |
| No. of rotatable bonds | 11 |
| No. of hydrogen bond acceptors | 6 |
| No. of hydrogen bond donors | 2 |
| Storage | stable as solid in the dark at -20°C. NB making aliquots rather than freeze-thawing is recommended |
| Dissolution | soluble in DMSO |
SMILES:
NR162: O=C(C1=CN=C(N=C1NC2CCCC2)NCC3=CC(Br)=C(C=C3Br)C)NCCCN4CCOC4=O
NR187: O=C(C1=CN=C(N=C1NC2CCCC2)N(CC3=CC=C(C=C3)Cl)CC)NCCCN4CCCC4=O
InChI:
NR162: InChI=1S/C24H30Br2N6O3/c1-15-11-20(26)16(12-19(15)25)13-28-23-29-14-18(21(31-23)30-17-5-2-3-6-17)22(33)27-7-4-8-32-9-10-35-24(32)34/h11-12,14,17H,2-10,13H2,1H3,(H,27,33)(H2,28,29,30,31)
NR187: InChI=1S/C26H35ClN6O2/c1-2-32(18-19-10-12-20(27)13-11-19)26-29-17-22(24(31-26)30-21-7-3-4-8-21)25(35)28-14-6-16-33-15-5-9-23(33)34/h10-13,17,21H,2-9,14-16,18H2,1H3,(H,28,35)(H,29,30,31)
InChIKey:
NR162: BPLPIBNWDLPUKP-UHFFFAOYSA-N
NR187: AYRUNBYNKCLGFI-UHFFFAOYSA-N
A DiscoverX KINOMEScan of NR162 at 1000 nM revealed very few off-targets.
| Target | NR162 (% Control) |
| CASK | 0 |
| AURKC | 0 |
| ABL1 | 0 |
| RPSKA6 | 15 |
| PIKFYVE | 30 |
| ERBB3 | 35 |
| RPS6KA5 | 35 |
| LRRK2 | 41 |
| STK38L | 42 |
Hits from the KINOMEScan were assessed in a cellular NanoBRET assay in HEK293T cells and IC50 of 18.2 µM for ERBB3 (227 fold selective) was determined. All other off-targets were not confirmed in the follow-up NanoBRET assay.
The negative control NR187 with its blocked hinge-binding amine showed no activity in NanoBRET and KINOMEScan was also clean.
In NanoBRET assay perfomed in HEK923T cells NR162 displayed the following IC50 value against CASK 80 nM and 3.8 µM against TYRO3.
In the NCI-60 screen, which is a human tumor cell line screen, NR162 showed no significant cell toxicity as well as 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
Crystallography revealed that due to the missing aspartate in CASK the inhibitor shows an improved selectivity pattern and is no longer active on MERTK, AXL and ABL1 and affinity is reduced to TYRO3 (off-targets of the lead structure PFE-PKIS12).
Figure 1: Overview of NR162 in complex with CASK (right) and details of the interaction of NR162 with the CASK hinge region (left).
Figure 2: Co-crystal structure of NR26_162 in complex with CASK (GFG-pocket) and alignment with MERTK (DFG-pocket).
DSF assay
Recombinant proteins were assessed as described in (Fedorov O, Niesen FH, Knapp S. Kinase inhibitor selectivity profiling using differential scanning fluorimetry. Methods Mol Biol. 2012;795:109-18.)
ITC
The ITC measurement was performed on a NanoITC (TA Instruments) at 25°C in buffer containing 30 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM TCEP and 5% Glycerol. CASK at 141 µM was injected into the cell, containing compounds at 2-6 µM. The integrated heat of titration was calculated and fitted to a single, independent binding model using the software provided by the manufacture. The thermodynamic parameters (ΔH and TΔS), equilibrium association and dissociation constants (Ka and KD), and stoichiometry (n) were calculated.
NanoBRET (Promega)
N-terminal NanoLuc and C-terminal NanoLuc/ kinase fusions, encoded in pFC32K expression vectors (Promega), were used. For cellular BRET target engagement experiments, HEK-293T were transfected with NLuc/target fusion constructs using FuGENE HD (Promega) according to the manufacturer’s protocol. Briefly, Nluc/target fusion constructs were diluted into Transfection Carrier DNA (Promega) at a mass ratio of 1:10 (mass/mass), diluted with OptiMEM media to a twentieth part of the volume of the HEK cells. FuGENE HD was added at a ratio of 1:3 (μg DNA: μL FuGENE HD). One part (vol) of FuGENE HD complexes was combined with 20 parts (vol) of HEK-293 cells suspended at a density of 2 x 105 cells/ml and afterwards the mixture was incubated in a humidified, 37°C/5% CO2 incubator for 24 h.
After this, the cells were washed and resuspendend in OptiMEM medium. For Target engagement assays 4 x 103 cells/well were plated out in a 384-well plate (Greiner). For all experiments the recommended energy transfer probes (Promega) were used if possible, at a final concentration of the Kd of the tracer on the target. In some cases, higher tracer concentrations were used for better signal-to-noise ratios. Compounds and the energy transfer probe were added to the cells and incubated for 2h in humidified, 37°C/5% CO2 incubator. The chemical inhibitors were prepared as concentrated stock solutions in DMSO (Sigma-Aldrich) and diluted with OptiMEM for this experiment. Straight before the measurement NanoGlo Substrate and Extracellular NanoLuc Inhibitor (Promega) were mixed carefully with the supernatant.
Luminescence was measured on a BMP PheraStar with 450 nm (donor) and 600 nm filters (acceptor) using 0.5 s integration time. Milli-BRET units (mBU) are calculated by multiplying the raw BRET values by 1000. Tracer and DMSO controls were used to calculate a normalized signal.
mBU=1000*I[600 nm]/I[450 nm]
Inhibitory constants were calculated by using the sigmoidal dose-response (four parameters) equation in GraphPad Prism.
Y=Bottom+((Top-Bottom) )/(1+〖10〗^(X-LogIC50*HillSlope) )