A DiscoverX KINOMEScan of NR162 at 1000 nM revealed very few off-targets.

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

1.  Y.-P. Hsueh, Annals of neurology 2009, 66, 438.
2. K. Mukherjee, M. Sharma, H. Urlaub, G.P. Bourenkov, R. Jahn, T.C. Südhof, M.C. Wahl, Cell 2008, 133, 328.
3. J.Bourdeau, D. Miranda-Saavedra, G.J. Barton, D.R. Alessi, Trends in cell biology 2006, 16, 443.
4. J.-L. Wei, Z.-X. Fu, M. Fang, Q.-Y. Zhou, Q.-N. Zhao, J.-B. Guo, W.-D. Lu, H. Wang, Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 2014, 35, 9185.
5. L. Fallon, F. Moreau, B. G. Croft, N. Labib, W.-J. Gu, E. A. Fon, The Journal of biological chemistry 2002, 277, 486.
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7. N.A. Powell, J.K.Hoffman, F.L.Ciske, M.D.Kaufman, et al., Highly selective 2,4-diaminopyrimidine-5-carboxamide inhibitors of Sky kinase. Bioorganic & Medicinal Chemistry Letters. 2013 https://doi.org/10.1016/j.bmcl.2012.12.013

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revealed that due to the missing aspartate in CASK the inhibitor shows an improved selectivity pattern and is no longer active on MERTK, AXL and ABL1 and affinity is reduced to TYRO3 (off-targets of the lead structure PFE-PKIS12).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) ) 

SGC-CK2-1 was profiled in the KINOMEscan assay against 403 wild-type kinases at 1 μM. Only 11 kinases showed PoC <35 giving an S(35) at 1 μM = 0.027. Potential off-targets were tested in the nanoBRET target engagement assay (DYRK2) and via Eurofins radiometric and LANCE kinase assays. Data corresponding with off-target kinase activity is shown in the table below.

A NanoBRET assay was utilized to assess the binding affinity of SGC-CK2-1 to CK2α and CK2α'. The negative control shows no binding affinity for CK2α'.

Figure 1: SGC-CK2-1 and SGC-CK2-1N were profiled in the CK2 NanoBRET assays.

1. Wells, C., Drewry, D. H., Pickett, J. E. & Axtman, A. D. SGC-CK2-1: the first selective chemical probe for the pleiotropic kinase CK2. ChemRxiv, 10.26434/chemrxiv.12296180.v12296181 (2020).
2. Rabalski, A. J., Gyenis, L. & Litchfield, D. W. Molecular Pathways: Emergence of Protein Kinase CK2 (CSNK2) as a Potential Target to Inhibit Survival and DNA Damage Response and Repair Pathways in Cancer Cells. Clinical Cancer Research 22, 2840-2847, doi:10.1158/1078-0432.Ccr-15-1314 (2016).
3. Meggio, F. & Pinna, L. A. One-thousand-and-one substrates of protein kinase CK2? FASEB journal : official publication of the Federation of American Societies for Experimental Biology 17, 349-368, doi:10.1096/fj.02-0473rev (2003).
4. Nuñez de Villavicencio-Diaz, T., Rabalski, A. J. & Litchfield, D. W. Protein Kinase CK2: Intricate Relationships within Regulatory Cellular Networks. Pharmaceuticals 10, 27 DOI: 10.3390/ph10010027 (2017).
5. Ahmed, K., Gerber, D. A. & Cochet, C. Joining the cell survival squad: an emerging role for protein kinase CK2. Trends in Cell Biology 12, 226-230, doi:10.1016/S0962-8924(02)02279-1 (2002).

Related Links: https://www.tocris.com/products/sgc-ck2-1_7450#product-literature

PFI-6 shows potent activity on MLLT1/3 (Table 1). PFI-6 is universally inactive in the SGC Bromodomain panel, and showed no activity in an Invitrogen panel of 40 kinases (screening conducted at 10µM). Additionally, PFI-6 showed no activity in a panel of 25 PDEs, ion channels and GPCRs >50µM. The negative control, PFI-6N, was not reactive against MLLT1 >30µM, MLLT3 >30µM, YEATS2 >30µM and YEATS4 >30µM.

In the NanoBRET cellular target engagement assay, PFI-6 displayed potent inhibition, with an average IC50 value of 0.76 μM (±0.1) (Figure 1). In comparison, PFI-6N had no inhibitory properties (up to 30 μM) (Figure 1). Further in cell validation using a Fluorescence Recovery After Photobleaching (FRAP) assay was used to confirm target inhibition by PFI-6  (Figure 2) . 

The figure below shows PFI-6 is soaked into the YEATS domain. PDB codes will be added when they become available. 

The AlphaScreen assays were employed to screen NVS-MLLT-1 and NVS-MLLT-C against a panel of bromodomains. NVS-MLLT-1 shows potent activity on MLLT1/3 whilst being selective for YEATS2/4. NVS-MLLT-1 shows selectivity across all bromodomains (CERC2 IC50 > 40 μM). NVS-MLLT-1 shows no activity on kinases tested, NVS-MLLT-1 has some activity on ACES and E3 binding. NVS-MLLT-C shows no activity in a panel of kinases, and no activties on other targets tested. 

General Selectivity- NVS-MLLT-1

NVS-MLLT-1 shows no activity on kinases tested. NVS-MLLT-1 has some activity on ACES and H3 binding.

General Selectivity- NVS-MLLT-C

NVS-MLLT-C shows no activity on kinases tested. NVS-MLLT-C shows no activities on other targets tested

Cellular target Engagement

In the NanoBRET cellular target engagement assay, NVS-MLLT-1 displayed potent inhibition, with an average IC50 value of 0.5 μM (±0.12) (Figure 1). In comparison, NVS-MLLT-C had no inhibitory properties (up to 30 μM) (Figure 1). Further in cell validation using a Fluorescence Recovery After Photobleaching (FRAP) assay was used to confirm target inhibition by NVS-MLLT-1  (Figure 2) . 

Figure 1: A NanoBRET assay was used in HEK293 cells to determine target engagement in cells. All probes were tested for 24h in the presence of 2.5 μM SAHA (24h). Graph represents Mean±SEM, n=3 independent replicates, with n=4 technical replicates.  mBU: BRET units

Figure 2: Further confirmation of MLLT1 target engagement in cells.  Fluorescence Recovery After Photobleaching analysis shows NVS-MLLT-1 decreases the half-life recovery time of cells. n=3 independent experiments, Mean±SEM, fold change from SAHA treatment alone. One way ANOVA, Dunnett's multiple comparisons.

Materials and Methods

NanoLuciferase Bioluminescent Resonance Energy Transfer (NanoBRET) Assay 
Cellular activity against MLLT3 was assessed using a NanoBRET assay. HEK293 cell (8 x 10⁵) were plated a 6-well plate, after 6h cells were co-transfected with C-terminal HaloTag-Histone 3.3 (NM_002107) and an N-terminal NanoLuciferase fusion of MLLT3 (original MLLT3 WT sequences from Promega HaloTag® human ORF in pFN21A or MLLT3 MUT - Y78A Tyrosine is changed to an Alanine) at a 1:10 (NanoLuc® to HaloTag®) ratio respectively with FuGENE HD transfection reagent. Sixteen hours post-transfection, cells were collected, washed with PBS, and exchanged into media containing phenol red-free DMEM and 4% FBS in the absence (control sample) or the presence (experimental sample) of 100 nM NanoBRET 618 fluorescent ligand (Promega). Cells were then re-plated in a 96-well assay white plate (Corning Costar #3917) at 2x10⁴ cells per well. Compounds were then added directly to the cells (in the presence of SAHA 2.5 µM) at final concentrations 0-30 μM or an equivalent amount of DMSO as a vehicle control, and the plates were incubated for 24 h at 37°C in the presence of 5% CO2. NanoBRET Nano-Glo substrate (Promega) was added to both control and experimental samples at a final concentration of 10 µM. Readings were performed within 10 minutes using a ClarioSTAR (BMG Labtech) equipped with 460 nm and 610 nm filters. A corrected BRET ratio was calculated and is defined as the ratio of the emission at 610 nm/460 nm for experimental samples minus the emission at 610 nm/460 nm for control samples (without NanoBRET fluorescent ligand). BRET ratios are expressed as milliBRET units (mBU), where 1 mBU corresponds to the corrected BRET ratio multiplied by 1000.

Peptide Displacement Assay
Peptide displacement assays were set up with biotinylated peptides (chosen based on ChIPseq data from the literature and purchased from LifeTein) and 6His tagged protein (Table 2).  For detection, two orthogonal technologies were used, i. AlphaScreen® technology from Perkin Elmer and ii. HTRF from Cisbio.  Compounds were dispensed in duplicate at single concentration (100 µM) for the initial screen and as 11-point dose response curves starting from 200 µM for IC₅₀ value determination.

Table 2: Peptides used for the peptide displacement assays

To determine optimal assay conditions for each protein prep, proteins and peptides were titrated against each other in a 16 by 16 matrix in 1:1 dilutions, starting from 3.2 µM. For the final ratio of protein and peptide to use in the assay, the point representing the EC₉₀ in the two-dimensional titration was chosen.  Typically, final assay concentrations for protein and peptide fell between 25 and 200 nM.  For AlphaScreen®, AlphaScreen Histidine (Nickel Chelate) Detection Kit donor and acceptor beads were used at a 1:2500 dilution from purchased stock; for HTRF, SA-XL665 and anti-6His antibody were used at 1:2000 and 1:10000 dilution from purchased stock, respectively.  Assays were performed on 384 well ProxiPlates (Perkin Elmer) at a final volume of 20 µl and plates were read using a Pherastar FSX plate reader (BMG Labtech).

Isothermal Titration Calorimetry
Isothermal titration calorimetry (ITC) was carried out using a TA NanoITC (standard volume) instrument.  Protein was prepared by dialysis (overnight at 4°C) against a ~1000 times excess of buffer (20 mM Tris at pH 7.5, 500 mM NaCl, 5% (v/v) glycerol, 2 mM DTT) using SnakeSkin® Dialysis Tubing with a 7 kDa MWCO and then concentrated to 300 µM.  The experiment was carried out at 20°C in reverse mode with the compound in the cell at 50 µM and the protein in the syringe at 300 µM due to the solubility of the compound with the first injection at 4 µl and the following 30 at 8 µl. Data was analysed using the NanoAnalyze software package by TA Instruments.

Differential Scanning Fluorimetry
Differential scanning fluorimetry (DSF) to determine the effect of compounds on the thermal stability of proteins (DT_m) was carried out on 384 well PCR plates using a LightCycler 480 (Roche).  Protein at 10 µM was buffered in 10 mM HEPES at pH 7.5 and 500 mM NaCl.  The experiment was carried out from 25 to 95°C with three acquisitions per degree.  Compounds were added at 50 µM final concentration and DMSO reference and no-addition controls were also collected.

References

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4. Yokoyama, A., et al., A higher-order complex containing AF4 and ENL family proteins with P-TEFb facilitates oncogenic and physiologic MLL-dependent transcription. Cancer Cell, 2010. 17(2): p. 198-212.DOI: 10.1016/j.ccr.2009.12.040.https://www.ncbi.nlm.nih.gov/pubmed/20153263

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6. Monroe, S.C., et al., MLL-AF9 and MLL-ENL alter the dynamic association of transcriptional regulators with genes critical for leukemia. Exp Hematol, 2011. 39(1): p. 77-86 e1-5.DOI: 10.1016/j.exphem.2010.09.003.https://www.ncbi.nlm.nih.gov/pubmed/20854876

7. Erb, M.A., et al., Transcription control by the ENL YEATS domain in acute leukaemia. Nature, 2017. 543(7644): p. 270-274.DOI: 10.1038/nature21688.https://www.ncbi.nlm.nih.gov/pubmed/28241139

8. Wan, L., et al., ENL links histone acetylation to oncogenic gene expression in acute myeloid leukaemia. Nature, 2017. 543(7644): p. 265-269.DOI: 10.1038/nature21687.https://www.ncbi.nlm.nih.gov/pubmed/28241141

9. Christott, T. et al., Discovery of a selective inhibitor for the YEATS domains of ENL/AF9. SLAS Discov, 2019. 24(2):133-141. DOI: 10.1177/2472555218809904. https://www.ncbi.nlm.nih.gov/pubmed/30359161

10. Chaikuad, A., S. Knapp, and F. von Delft, Defined PEG smears as an alternative approach to enhance the search for crystallization conditions and crystal-quality improvement in reduced screens. Acta Crystallogr D Biol Crystallogr, 2015. 71(Pt 8): p. 1627-39.DOI: 10.1107/S1399004715007968.https://www.ncbi.nlm.nih.gov/pubmed/26249344

11. Moustakim, M., et al., Discovery of an MLLT1/3 YEATS Domain Chemical Probe. Angew Chem Int Ed Engl, 2018. 57(50): p. 16302-16307.DOI: 10.1002/anie.201810617.https://www.ncbi.nlm.nih.gov/pubmed/30288907

12. Heidenreich, D., et al., Structure-Based Approach toward Identification of Inhibitory Fragments for Eleven-Nineteen-Leukemia Protein (ENL). J Med Chem, 2018. 61(23): p. 10929-10934.DOI: 10.1021/acs.jmedchem.8b01457.https://www.ncbi.nlm.nih.gov/pubmed/30407816

In vitro Metabolic stability of NVS-MLLT-1

To establish the in cell half life of NVS-MLLT-1, metabolic stability studies were carried out exposing nominated compounds to samples of primary human hepatocytes. Results from ADME and phys chem experiments are summarised below. 

The figures below shows MLLT1 co-crystal strutcure at 1.7 Å. NVS-MLLT-1 is soaked into the YEATS domain. PDB codes will be added when they become available. 

SGC-CLK-1 was profiled in the KINOMEscan assay against of 403 wild-type kinases at 1 µM. Only 6 kinases showed PoC < 35 giving an S(35) at 1 µM =0.02. Potential off-targets were tested in the nanoBRET target engagement assay where possible and the data are shown in the table.

A Nanobret assay was utilised to assess the binding affinity of SGC-CLK-1 to CLK1, CLK2 and CLK4 (Figure 1). The negative control shows no binding affinity. 

1. Araki, S., et al., Inhibitors of CLK protein kinases suppress cell growth and induce apoptosis by modulating pre-mRNA splicing. PLoS One, 2015. 10(1): p. e0116929.DOI: 10.1371/journal.pone.0116929. https://www.ncbi.nlm.nih.gov/pubmed/25581376

2. Bullock, A.N., et al., Kinase domain insertions define distinct roles of CLK kinases in SR protein phosphorylation. Structure, 2009. 17(3): p. 352-62.DOI: 10.1016/j.str.2008.12.023. https://www.ncbi.nlm.nih.gov/pubmed/19278650

3. Corkery, D.P., et al., Connecting the speckles: Splicing kinases and their role in tumorigenesis and treatment response. Nucleus, 2015. 6(4): p. 279-88.DOI: 10.1080/19491034.2015.1062194. https://www.ncbi.nlm.nih.gov/pubmed/26098145

4. Funnell, T., et al., CLK-dependent exon recognition and conjoined gene formation revealed with a novel small molecule inhibitor. Nat Commun, 2017. 8(1): p. 7.DOI: 10.1038/s41467-016-0008-7. https://www.ncbi.nlm.nih.gov/pubmed/28232751

5. Iwai, K., et al., Anti-tumor efficacy of a novel CLK inhibitor via targeting RNA splicing and MYC-dependent vulnerability. EMBO Mol Med, 2018. 10(6).DOI: 10.15252/emmm.201708289. https://www.ncbi.nlm.nih.gov/pubmed/29769258

6. jain, P., et al., Human CDC2-like kinase 1 (CLK1): a novel target for Alzheimer's disease. Curr Drug Targets, 2014. 15(5): p. 539-50.DOI: 10.2174/1389450115666140226112321. https://www.ncbi.nlm.nih.gov/pubmed/24568585

7.  Koedoot, E., et al., Splicing regulatory factors in breast cancer hallmarks and disease progression. Oncotarget, 2019. 10(57): p. 6021-6037.DOI: 10.18632/oncotarget.27215. https://www.ncbi.nlm.nih.gov/pubmed/31666932

8.  Sako, Y., et al., Development of an orally available inhibitor of CLK1 for skipping a mutated dystrophin exon in Duchenne muscular dystrophy. Sci Rep, 2017. 7: p. 46126.DOI: 10.1038/srep46126. https://www.ncbi.nlm.nih.gov/pubmed/28555643

The KINOMEscan of SGC-STK17B-1 at 1mM showed few off-targets, Figure 1. With cutoff 10% control (90% inhibition), MET, NEK6, PIM2, WEE1, and CAMKK2 are shown in red circles in the TREEspot. This screening was followed by K_d determination and NanoBRET assays for several potential off-targets (Table 1).

Figure 1:  Kinome selectivity and TREEspot analysis of 403 wild-type kinases for SGC-STK17B-1.

Results in Table 1 confirmed the high selectivity of SGC-STK17B-1 towards STK17B/DRAK2.  Non-off targets were found for this chemical probe at concentrations shown.

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2. Lan, Y., et al., STK17B promotes carcinogenesis and metastasis via AKT/GSK-3beta/Snail signaling in hepatocellular carcinoma. Cell Death Dis, 2018. 9(2): p. 236.DOI: 10.1038/s41419-018-0262-1. https://www.ncbi.nlm.nih.gov/pubmed/29445189
3. McGargill, M.A., et al., Drak2 regulates the survival of activated T cells and is required for organ-specific autoimmune disease. J Immunol, 2008. 181(11): p. 7593-605.DOI: 10.4049/jimmunol.181.11.7593. https://www.ncbi.nlm.nih.gov/pubmed/19017948
4. Ni, Y., et al., Identification and SAR of a new series of thieno[3,2-d]pyrimidines as Tpl2 kinase inhibitors. Bioorg Med Chem Lett, 2011. 21(19): p. 5952-6.DOI: 10.1016/j.bmcl.2011.07.069. https://www.ncbi.nlm.nih.gov/pubmed/21862328
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6. Picado et al. A Chemical Probe for Dark Kinase STK17B Derives Its Potency and High Selectivity through a Unique P-Loop Conformation. J Med Chem 2020. 63 (23): p14626. https://doi.org/10.1021/acs.jmedchem.0c01174. https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01174.

The co-crystal structure shown an unusual flip of R41 (P-loop) forming salt bridge network with inhibitor COOH group, R41 and E117 in one of the two experimentally observed conformations is a likely mechanism of selectivity. PDB depositions for the chemical probe are 6Y6F and 6Y6H (links will be updated as soon as they become available). 

Important features:

• Type-I binding mode
• Back pocket interaction of COOH with lysine (K62)
• Additional ethylene diol fills back pocket

Acknowledgments.  Co-crystal structure was obtained by Apirat Chaikuad and Stefan Knapp at SGC-Frankfurt.