Probe		Negative control
PFI-5		PFI-5N

• SMILES:
• PFI-5: [H][C@@]1(N2CCC(CN3C=CC(C(N4CC(C4)(N5C=NC6=C5C=C(C)N=C6N)C)=O)=C3)CC2)CCCCC=C1
• PFI-5N: [H][C@]1(N2CCC(CN3C=CC(C(N4CC(C4)(N5C=NC6=C5C=C(C)N=C6N)C)=O)=C3C)CC2)CCCCC=C1
• InChI:
• PFI-5: InChI=1S/C29H39N7O.C2H4O2/c1-21-15-25-26(27(30)32-21)31-20-36(25)29(2)18-35(19-29)28(37)23-11-12-33(17-23)16-22-9-13-34(14-10-22)24-7-5-3-4-6-8-24;1-2(3)4/h5,7,11-12,15,17,20,22,24H,3-4,6,8-10,13-14,16,18-19H2,1-2H3,(H2,30,32);1H3,(H,3,4)/t24-;/m0./s1
• PFI-5N: InChI=1S/C30H41N7O.C2H4O2/c1-21-16-26-27(28(31)33-21)32-20-37(26)30(3)18-36(19-30)29(38)25-12-15-35(22(25)2)17-23-10-13-34(14-11-23)24-8-6-4-5-7-9-24;1-2(3)4/h6,8,12,15-16,20,23-24H,4-5,7,9-11,13-14,17-19H2,1-3H3,(H2,31,33);1H3,(H,3,4)/t24-;/m1./s1
• InChIKey:
• PFI-5: AVKAVKZKOFSSMY-DEOSSOPVSA-N
• PFI-5N: RSNWUCXHNLZHPY-XMMPIXPASA-N

Main features

• SMYD2 structure with peptide substrate and SAM
• Structures of BAY-598 and AZ-505 showing these bind in the substrate pocket
• PFI-5 occupies both the SAM and peptide pockets
• Overall view of PFI-5 binding to SMYD2
• Key interactions of PFI-5 with SMYD2

Selectivity screening of MU1210 was determined against 210 kinases in an activity-based assay (Invitrogen) at 1 µM (Figure 1). Top hits of the screen are indicated in Table 1.

Figure 1:  Visual representation of the top hits from MU1210 screened against a panel of 210 kinases.

Table 1: Potency Against Target Family. Please note DYRKs are not in this panel.

MU1210 is selective in a panel of 210 kinases in an activity-based assay (Invitrogen) at 1 µM. The closest off-target in the activity based screen HIPK2 was not inhibited in the cellular NanoBRET assays at 10 µM (Table 2).

Table 2: Confirmation of hit CLK2 in the cellular NanoBRET assay.

Hits from the kinase panel were assessed in a NanoBRET assay. MU1210 was much less active against HIPK2 and GSK3A/B in cells than in vitro (Figure 2). Kd values of Staurosporine are shown for comparison (Table 3) (6, 7).

Figure 2: In vitro DSF assay of MU1210 and Staurosporine on potential off-targets.

Table 3: In vitro DSF assay of MU1210 and Staurosporine on potential off-targets.

Materials and Methods

In vitro phophorylation assays
In vitro phosphorylation assays are described in (5).

NanoBRET assay
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.

Inhibitory constants were calculated by using the sigmoidal dose-response (four parameters) equation in GraphPad Prism.

For a better comparison in between assays of different kinases Ki values were also calculated using the Cheng-Prusoff equation with the corresponding tracer Kds and the used tracer concentrations.

DSF assay
Purified, recombinant proteins were measured as described in (8).

In the NanoBRET assay MU1210 the following IC₅₀ values were observed:

Figure 1: IC₅₀ values from screening MU1210 and the negative control MU140 in the NanoBRET assay. 

The K_i values as determined by NanoBRET assays in HEK293T cells were:

Table 1: Ki vales from screening MU1210 and the negative control MU140 in the NanoBRET assay.

After treatment of HeLa cells with MU1210 (VN339) for 3 hours the phosphorylation state of SR proteins was altered (Figure 2). MU1210 inhibited the phosphorylation of SRSF proteins in a dose-dependend manner, while the negative control MU140 had no effect on SRSF-phosphorylation.

Figure 2: Western blot analysis of phosphorylation state of SR proteins following treatment with MU1210 and its negative control MU140. 

Treatment with MU1210 at 10 µM affected the alternative splicing of Mdm4 in MCF7 cells leading to an accumulation of the shorter Mdm4 form (Mdm4-S) (Figure 3). No changes were observed compared to the DMSO control, as well as the negative control MU140.

Figure 3: PCR showing alternative splicing following treatment with MU1210 (10 µM).

In vitro toxicity was assessed using a MTT assay. MU1210 was not toxic in cells at >1 µM after 24 hours. The negative control MU140 showed no significant toxicity after 24 hours, up to 35 µM (5). MU1210 toxicity was further assessed following 72 hours of treatment (Table 2).  

Table 2: Cellular toxicity in selected cell lines. Cells were treated with MU1210 for 72 hours. Cellular toxicity was assessed using a MTT assay.

MU1210 induced severe impairment of cell proliferation at >1µM over a longer time period (Figure 4). The high initial concentration of 5 µM in MEF cells caused precipitations of the probe in the wells due to the limited solubility of the compound.

Figure 4: Cell proliferation following treatment with MU1210 in selected cell lines. 

Materials and Methods

Western blot analysis
For the dose dependence change of phosphor-SRSF proteins in Hela cells, 200,000 cells in 3 ml DMEM containing 10% FBS and Penicillin/Streptomycin were seeded in 6 well plates for 24h. The in DMSO diluted compounds were added and incubated for the stated time. Afterwards the cells were lysed mixed with SDS loading buffer, briefly heated and loaded onto a 12% SDS gel. After running the gel and blotting onto a nitrocellulose membrane, anti-phospho-SR antibody (Merck-Millipore, MABE50) was used to analyse the level of phosphorylated SRSF proteins. After washing steps, the membrane was incubated with anti-mouse antibodies coupled with horseradish peroxidase and analysed. Tubulin acted as a loading control.

Splicing analysis
The Mdm4 splicing in MCF7 cells is described in (5).

Cellular proliferation
For the proliferation assay cells were seeded between 100-200 cells/well in a 384 clear bottom plate (Nunc) in DMEM containing 10 % FBS and Penicillin/Streptomycin. After 24h compounds were added by an acoustic dispenser (ECHO) directly in each well. Proliferation was measured by determining the confluency over time in an Incucyte S3 automated microscope.

Work on this probe has been published in Furo[3,2-b]pyridine: A Privileged Scaffold for Highly Selective Kinase Inhibitors and Effective Modulators of the Hedgehog Pathway

1. Aubol, B.E., et al., Release of SR Proteins from CLK1 by SRPK1: A Symbiotic Kinase System for Phosphorylation Control of Pre-mRNA Splicing. Mol Cell, 2016. 63(2): p. 218-228.DOI: 10.1016/j.molcel.2016.05.034. https://www.ncbi.nlm.nih.gov/pubmed/27397683
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. Davis, M.I., et al., Comprehensive analysis of kinase inhibitor selectivity. Nat Biotechnol, 2011. 29(11): p. 1046-51.DOI: 10.1038/nbt.1990. https://www.ncbi.nlm.nih.gov/pubmed/22037378
4.  Fedorov, O., F.H. Niesen, and S. Knapp, Kinase inhibitor selectivity profiling using differential scanning fluorimetry. Methods Mol Biol, 2012. 795: p. 109-18.DOI: 10.1007/978-1-61779-337-0_7. https://www.ncbi.nlm.nih.gov/pubmed/21960218
5. Manning, G., et al., The protein kinase complement of the human genome. Science, 2002. 298(5600): p. 1912-34.DOI: 10.1126/science.1075762. https://www.ncbi.nlm.nih.gov/pubmed/12471243
6. Nayler, O., S. Stamm, and A. Ullrich, Characterization and comparison of four serine- and arginine-rich (SR) protein kinases. Biochem J, 1997. 326 ( Pt 3): p. 693-700.DOI: 10.1042/bj3260693. https://www.ncbi.nlm.nih.gov/pubmed/9307018
7. Nemec, V., et al., Furo[3,2-b]pyridine: A Privileged Scaffold for Highly Selective Kinase Inhibitors and Effective Modulators of the Hedgehog Pathway. Angew Chem Int Ed Engl, 2019. 58(4): p. 1062-1066.DOI: 10.1002/anie.201810312. https://www.ncbi.nlm.nih.gov/pubmed/3056960
8. Wodicka, L.M., et al., Activation state-dependent binding of small molecule kinase inhibitors: structural insights from biochemistry. Chem Biol, 2010. 17(11): p. 1241-9.DOI: 10.1016/j.chembiol.2010.09.010. https://www.ncbi.nlm.nih.gov/pubmed/21095574

Co-structures of CLK1 with closely related compounds (PDB IDs: 6I5I, 6I5L, 6I5K, 6I58), are published in (5).

A DSF screen against Human bromodomains reveals no significant off-targets (Table 1). A BROMOscan with NVS-BPTF-1 similarly showed good selectivity. BPTF exhibited a K_D of 3nM, BRPF 37nM, CECR2 66nM, GCN5L2 62nM and PCAF 74nM in BROMOscan (Table 2)

Table 1: DSF screen Table 2: BromoScan 

NVS-BPTF-1 was also tested against the NIBR principal panel which showed no binding to 12 GPCRs, 3 nuclear receptors, 3 transporters and 7 other enzymes with an IC₅₀<10µM.

The control compound, NVS-BPTF-C showed no binding to 14 GPCRs, 3 nuclear receptors, 3 transporters and 5 enzymes with an IC₅₀<10µM in the NIBR principal panel.

Against the NIBR kinase panel, NVS-BPTF-1 showed no binding against 48 kinases with an IC₅₀<30µM.

Against the NIBR kinase panel, NVS-BPTF-C showed no binding against 59 kinases with an IC₅₀<30µM.

Materials and Methods

Thermal Stability Assay

Thermal melting experiments were carried out using an Mx3005p Real Time PCR machine (Agilent). Proteins were prepared in 10 mM HEPES pH 7.5, 500 mM NaCl and assayed in a 96-well plate at a final concentration of 2 μM in 20 μL volume. Compounds final concentration is 10 μM.

AlphaScreen

All bromodomain proteins were prepared according to the published procedures (Filippakopoulos at al, 2012). Assay was performed as described previously (Philpott et al, 2011). All reagents were pre-diluted in 25 mM HEPES, 100 mM NaCl, 0.1 % BSA, pH 7.4 and 0.05 % CHAPS and allowed to equilibrate to room temperature prior to addition to plates.  Plates filled with 5 uL of the assay buffer followed by 7 µL of biotinylated peptide [H-YSGRGKacGGKacGLGKacGGAKacRHRK(Biotin)-OH and His-tagged protein to achieve final assay concentrations of 25 nM. Plates were sealed and incubated for a further 60 minutes, before the addition of 8 μl of the mixture of streptavidin-coated donor beads (12.5 μg/ml) and nickel chelate acceptor beads (12.5 μg/ml) under low light conditions. Plates were foil-sealed to protect from light, incubated at room temperature for 60 minutes and read on a PHERAstar FS plate reader (BMG Labtech, Germany) using an AlphaScreen 680 excitation/570 emission filter set.

BioLayer Interferometry

BioLayer Interferometry (BLI) experiments were performed on a 16-channel ForteBio Octet RED384 instrument at 25 °C in 25mM HEPES, pH 7.5, and 100mM NaCl buffer. Proteins were biotinylated in vivo using the  construct with C-terminal AviTag. Proteins were immobilized on SSA sensors. Measurements were performed using 240 sec association step followed by 240 sec dissociation step on black tilted low volume 384-well plate (ForteBio). Baseline was stabilized for 120 sec prior to association step. Signal from the reference sensors was subtracted prior to Kd calculations using Analysis software (ForteBio).

To determine cellular target engagement, a NanoBRET assay based on a bespoke tracer (5961) was developed to measure the binding of the chemical probe and negative control to the NanoLuc-tagged BPTF bromodomain.

An EC₅₀ of 16 nM was observed in this assay for NVS-BPTF-1 whereas the negative control NVS-BPTF-C was significantly less potent.

The putative cellular off-targets CECR2, PCAF and GCN5L2 were similarly tested in the NanoBRET assay with no activity detected.

Materials and Methods

NanoBRET

Dose-response experiments were conducted in 384 well format using HEK293T cells expressing NanoLuc fused to the N-terminus of the BPTF bromodomain, using the 5961 tracer at a final concentration of 1 µM.

1. Wysocka, J., Swigut, T., Xiao, H., Milne, T. A., Kwon, S. Y., Landry, J., Kauer, M., Tackett, A. J., Chait, B. T., Badenhorst, P., Wu, C., and Allis, C. D. (2006) A PHD finger of NURF couples histone H3 lysine 4 trimethylation with chromatin remodelling. Nature 442, 86-90
2. Goller, T., Vauti, F., Ramasamy, S., and Arnold, H. H. (2008) Transcriptional regulator BPTF/FAC1 is essential for trophoblast differentiation during early mouse development. Molecular and cellular biology 28, 6819-6827
3. Landry, J., Sharov, A. A., Piao, Y., Sharova, L. V., Xiao, H., Southon, E., Matta, J., Tessarollo, L., Zhang, Y. E., Ko, M. S., Kuehn, M. R., Yamaguchi, T. P., and Wu, C. (2008) Essential role of chromatin remodeling protein Bptf in early mouse embryos and embryonic stem cells. PLoS genetics 4, e1000241
4. Ngeow, K. C., Friedrichsen, H. J., Li, L., Zeng, Z., Andrews, S., Volpon, L., Brunsdon, H., Berridge, G., Picaud, S., Fischer, R., Lisle, R., Knapp, S., Filippakopoulos, P., Knowles, H., Steingrimsson, E., Borden, K. L. B., Patton, E. E., and Goding, C. R. (2018) BRAF/MAPK and GSK3 signaling converges to control MITF nuclear export. Proceedings of the National Academy of Sciences of the United States of America 115, E8668-e8677
5. Dar, A. A.; Majid, S.; Bezrookove, V.; Phan, B.; Ursu, S.; Nosrati, M.; De Semir, D.; Sagebiel, R. W.; Miller, J. R.; Debs, R.; Cleaver, J. E.; Kashani-Sabet, M., BPTF transduces MITF-driven prosurvival signals in melanoma cells. Proc. Natl. Acad. Sci. U. S. A., 2016, 113 (22), 6254–6258.

Novartis have solved the structure of NVS-BPTF-1 in complex with BPTF at a resolution of 1.76Å. This structure is not currently deposited in the PDB.

Black dotted-line: H-Bond

Orange dotted-line: pi-pi interaction

Red sphere: water molecule

A DSF screen against Human bromodomains reveals only one significant off-target; BRD9:

ITC showed good potency and sufficient selectivity against BRD9 to meet SGC probe criteria:

Materials and Methods

Thermal stability assay

Thermal melting experiments were carried out using an Mx3005p Real Time PCR machine (Stratagene). Proteins were buffered in 10 mM HEPES pH 7.5, 500 mM NaCl and assayed in a 96-well plate at a final concentration of 2 μM in 20 μL volume. Compounds were added at a final concentration of 10 μM. SYPRO Orange (Molecular Probes) was added as a fluorescence probe at a dilution of 1:1000. Excitation and emission filters for the SYPRO-Orange dye were set to 465 nm and 590 nm, respectively. The temperature was raised with a step of 3 °C per minute from 25 °C to 96 °C and fluorescence readings were taken at each interval.

AlphaScreen

All bromodomain proteins were prepared according to the published procedures (Filippakopoulos at al, 2012). Assay was performed as described previously (Philpott et al, 2011). All reagents were pre-diluted in 25 mM HEPES, 100 mM NaCl, 0.1 % BSA, pH 7.4 and 0.05 % CHAPS and allowed to equilibrate to room temperature prior to addition to plates.  Plates filled with 5 uL of the assay buffer followed by 7 uL of biotinylated peptide [H-YSGRGKacGGKacGLGKacGGAKacRHRK(Biotin)-OH and His-tagged protein to achieve final assay concentrations of 25 nM. Plates were sealed and incubated for a further 60 minutes, before the addition of 8 μl of the mixture of streptavidin-coated donor beads (12.5 μg/ml) and nickel chelate acceptor beads (12.5 μg/ml) under low light conditions. Plates were foil-sealed to protect from light, incubated at room temperature for 60 minutes and read on a PHERAstar FS plate reader (BMG Labtech, Germany) using an AlphaScreen 680 excitation/570 emission filter set.

Isothermal Titration Calorimetry (ITC)

Experiments were carried out on a VP-ITC microcalorimeter (MicroCal™). All experiments were performed at 15 °C in 25 mM HEPES pH 7.4, 150 mM NaCl, 500 μM TCEP. 50 mM stocks of compound was thawed and diluted in 2 mL of buffer to a final concentration of 10 µM in the ITC cell. The protein titrations were conducted using an initial injection of 2 µl followed by 30 identical injections of 6 µl. The dilution heats were measured on separate experiments and were subtracted from the titration data. Thermodynamic parameters were calculated using ∆G = ∆H - T∆S = -RTlnKB, where ∆G, ∆H and ∆S are the changes in free energy, enthalpy and entropy of binding respectively. In all cases a single binding site model was employed.

TP-238 and its negative control were tested for target engagement in HEK cells using NanoBRET^TM. Significant inhibition by TP-238 against BPTF (n = 3, EC₅₀ = 228 nM) and CECR2 (n = 3, EC₅₀ = 289 nM) whereas the negative control TP-422 exhibited no significant activity at the tested concentrations:

Further, no significant inhibition against BRD9 was found in a NanoBRET^TM assay in HEK293 cells:

FRAP assays were also performed which showed significant inhibition by TP-238 against BPTF and CECR2:

FL-BPTF FRAP:

FL-CECR2 FRAP:

Materials and Methods

HEK cells were reverse transfected with NL-FL CECR2 or NL-BPTF BD. Cells were treated with a concentration range of TP-238, TP-422 (negative control), with BD070434a tracer (1 µM) for BPTF or BRD-02 tracer (0.5 µM) for CECR2 for 3 hrs before BRET measurements were taken.

For BRD9, the full length protein was used. HEK2983 cells were transfected with NL-BRD9 and treated with tracer (2 µM) and the respective compounds, followed by addition of Nano-Glo substrate and extracellular inhibitor. BRET was determined at 450 and 610nM.

FRAP assays were performed using U2OS cells reverse transfected with GFP-FL FALZ WT or #PHE or GFP-FL CECR2 WT or #ALA for 6-8 hrs. Transfection reagents were replaced with media or 2.5 µM SAHA + incubated O/N. Cells were treated with 1 µM test cpds for 1 hr before imaging. 6 repeat assays were performed for each protein.

1. Footz, TK., Brinkman-Mills, P et al.  Analysis of the cat eye syndrome critical region in humans and the region of conserved synteny in mice: a search for candidate genes at or near the human chromosome 22 pericentromere. Genome Res, 2001,11: 1053–1070. 
2. Lee SK, Park EJ et al. Genome-wide screen of human bromodomain-containing proteins identifies Cecr2 as a novel DNA damage response protein. Mol Cells, 2012, 34(1):85-91
3. Wu B, Wang Y, Wang C, Wang GG, Wu J, Wan YY. BPTF Is Essential for T Cell Homeostasis and Function. J Immunol. 2016, 197(11):4325-4333.
4. Richart L, Real FX, Sanchez-Arevalo Lobo VJ., c-MYC partners with BPTF in human cancer. Mol Cell Oncol. 2016, 3(3)

A close homologue to TP-248 was co-crystallised to investigate the possible binding mode of the probe: