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

The SYPRO Orange thermal shift assay was employed to screen VinSpinIn and VinSpinIC against a panel of methyl binding domains (MBDs), which included four of the five Spin family members (Table 1). VinSpinIn induced a large shift in thermal stability of Spin1, Spin2B, Spin3, Spin4. No other significant shift in thermal stability was observed for VinSpinIn or the inactive VinSpinIC.

Table 1: Screening on a panel of MBDs.^a protein = 0.05 to 0.2 mg/ml, compound = 200 µM; ^b protein = 2 µM, compound = 20 µM; ^c Intrinsic tryptophan fluorescence used

A fluorescence polarization displacement assays was employed to screen the compounds against L3MBTL1 and L3MBTL3 (MBT methyl lysine readers), which gave respective K_disp.s of 27 and 8 µM for VinSpinIn, and 28 and 14 µM for the inactive VinSpinIC. Therefore, VinSpinIn is approximately 267 times less potent towards L3MBTL1 than Spin1.

Selectivity screening was also performed against a panel of methyl transferases domains (MTDs) including protein, DNA and RNA methyltransferases using a Scintillation Proximity Assay (SPA) (Figure 1).

Figure 1: (Top) Methyltransferase activity in presence of VinSpinIn at 10 & 50 µM
(Bottom) Methyltransferase activity in presence of VinSpinIC at 10 and 50 µM.

For selected targets IC₅₀s were determined (Table 2). The lowest IC₅₀ of VinSpinIn (PRMT4) was approximately 300 times greater than the AlphaScreen IC₅₀ on Spin1.

Table 2: IC₅₀s of selected methyltransferases.^a Not determined

Materials and Methods

Effects of VinSpinIn  and VinSpinIC on methyltransferase activity of G9a, GLP, SUV39H1, SUV39H2, SETDB1, SETD8, SUV420H1, SUV420H2, SETD7, MLL1 trimeric complex, MLL3 pentameric complex, EZH2 trimeric complex, PRMT1, PRMT3, PRMT4, PRMT5-MEP50 complex, PRMT6, PRMT7, PRMT8, PRMT9, PRDM9, SETD2, SMYD2, SMYD3, and DNMT1 was assessed by monitoring the incorporation of tritium-labeled methyl group to lysine or arginine residues of peptide substrates using Scintillation Proximity Assay (SPA) as previously described¹⁴.  Assays were performed in a 10 µl reaction mixture containing ³H-SAM (Cat.# NET155V250UC; Perkin Elmer; www.perkinelmer.com) at substrate concentrations close to K_m values for each enzyme.  Two concentrations (10µM and 50 µM) of VinSpinIn or  VinSpinIC  were used in all selectivity assays.  To stop the enzymatic reactions, 10 µl of 7.5 M guanidine hydrochloride was added, followed by 180 µl of buffer (20 mM Tris, pH 8.0), mixed and then transferred to a FlashPlate (Cat.# SMP103; Perkin Elmer; www.perkinelmer.com). After mixing, the reaction mixtures in Flash plates were incubated for 2 hours and the CPM were measured using Topcount plate reader (Perkin Elmer, www.perkinelmer.com). The CPM counts in the absence of compound for each data set were defined as 100% activity. In the absence of the enzyme, the CPM counts in each data set were defined as background (0%).

For DOT1L, NSD1, NSD2, NSD3, ASH1L, DNMT3A/3L, and DNMT3B/3L, a filter-based assay was used. In this assay, 10 µl of reaction mixtures were incubated at 23 ^oC for 1 hour, 50 µl of 10% trichloroacetic acid (TCA) was added, mixed and transferred to filter-plates (Millipore; cat.# MSFBN6B10; www.millipore.com). Plates were centrifuged at 2000 rpm (Allegra X-15R - Beckman Coulter, Inc.) for 2 min followed by 2 additional 10% TCA wash and one ethanol wash followed by centrifugation. Plates were dried and 30 µl MicroO (MicroScint-O; Cat.# 6013611, Perkin Elmer; www.perkinelmer.com) was added to each well, centrifuged and removed. 50 µl of MicroO was added again and CPM was measured using Topcount plate reader.

IC₅₀ Determinations:
IC₅₀ values were determined for inhibition of methyltransferase activity of the following enzymes:

• PRMT4 (20 nM PRMT4, 1 µM of biotinlated-H3 1-25, 2 µM of ³H-SAM)
• SETD2 (150 nM SETD2, 0.5 µM of biotinylated-H3 21-44, 5 µM of ³H-SAM)
• PRMT7 (25 nM PRMT7, 0.3 µM of biotinylated-H2B(23-37), 1 µM of ³H-SAM)
• SUV39H1 (20 nM SUV30H1, 0.2 µM of biotinlated-H3 1-25, 5 µM of ³H-SAM)
• PRMT6 (20 nM PRMT6, 1 µM of biotinlated-H4 1-24, 2 µM of ³H-SAM)
• PRC2 (20 nM PRC2, 1 µM of biotinlated-H3 21-44, 2 µM of ³H-SAM)
• SMYD2 (30 nM SMYD2, 3 µM of biotinlated-P⁵³peptide, 0.3 µM of ³H-SAM)
• PRDM9 (5 nM PRDM9, 4 µM of biotinlated-H3 1-25, 71 µM of SAM)
• PRMT1 (15 nM PRMT1, 0.13 µM of biotinlated-H4 1-24, 5 µM of ³H-SAM)
• PRMT8 (20 nM PRMT8, 1 µM of biotinlated-H4 1-24, 2 µM of ³H-SAM)
• DNMT3A/3L (20 nM DNMT3A/3L, 0.5 µM of poly DI-DC, 1 µM of ³H-SAM)
• DNMT3B/3L (50 nM DNMT3B/3L, 0.5 µM of poly DI-DC, 1 µM of ³H-SAM)
• G9a (5 nM G9A, 0.8 µM of biotinlated-H3 1-25, 10 µM of SAM)
• GLP (5 nM Glp, 0.8 µM of biotinlated-H3 1-25, 10 µM of SAM).

To stop the enzymatic reactions, 7.5 M Guanidine hydrochloride was added, followed by 180 µL of buffer (20 mM Tris, pH 8.0), mixed and then transferred to a FlashPlate (Cat.# SMP103; Perkin Elmer; www.perkinelmer.com). After mixing, the reaction mixtures in Flash plate were incubated for 2 hour and the CPM counts were measured using Topcount plate reader (Perkin Elmer, www.perkinelmer.com). The CPM counts in the absence of compound for each data set were defined as 100% activity. In the absence of the enzyme, the CPM counts in each data set were defined as background (0%). The IC₅₀ values were calculated using GraphPad Prism 7 software.

In a NanoBRET cellular target engagement assay VinSpinIn displayed dose dependant inhibition of the Spin1-H3 interaction, with an IC₅₀ of 270 nM. The inactive VinSpinIC showed no inhibition (Figure 1).

Figure 1: NanoBRET cellular target engagement assay performed in U2OS cells. Full length SPIN1 with N-terminal NanoLuc; Histone 3.3 with C-terminal Halotag; 24h incubation with VinSpinIn and VinSpinIC

Materials and Methods

U20S cell (2.8 x 10⁵) were plated in each well of 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 full length SPIN1 at a 1:500 (NanoLuc® to HaloTag®) ratio respectively with FuGENE HD transfection regent¹⁵.

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 384-well assay white plate (Greiner #3570) at 2.7x10³ cells per well.  VinSpinIn and VinSpinIC were then added directly to media at final concentrations 0-30μM or an equivalent amount of DMSO as a vehicle control, and the plates were incubated for 24 h at 37^oC 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.

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The co-crystal structure of Spin1 with VinSpinIn confirms that VinSpinIn binds to both domain 1 and 2 of Spin1, and therefore is referred to as a bidentate inhibitor (Figure 1).

Figure 1: Co-crystal structure of Spin1 (domain 1 = purple; domain 2 = green; domain 3 = yellow) with VinSpinIn (Cyan).

A KINOMEscan of DDR-TRK-1 at 1μM revealed very few off-targets:

An activity assay at RBC (10μM ATP) revealed the following within the DDR/TRK family:

Dose-response curves for the probe (and where collected, the negative control) are shown below:

In a NanoBRET^TM cellular target engagement assay DDR1-TRK-1 displayed dose dependant inhibition as follows:

NanoBRET assay
Dose-response experiments were conducted in 96 well format using HEK293T cells expressing NanoLuc fused to the C-terminus of full-length protein kinases for DDR2, TRKA and TRKB or isoform 2 for DDR1, using Promega tracer  as indicated below.

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Structures of DDR1 complexes. A. Overall view of DDR1. Structural features are labelled for clarity. N-lobe is coloured orange,  A loop is marked in yellow and C-lobe is coloured wheat. B. D2099 compound electron density (PDB 5FDP). C. D2164 compound electron density (PDB 5FDX). D. Molecular docking of DDR-TRK-1 compound.

Inhibition of DDR1, DDR2, TRKA, TRKB and TRKC kinase activity was measured using radiometric assay (Reaction Biology Corporation). Compounds were tested in 10-dose IC50 singlicate mode with a 3-fold serial dilution starting at 1 or 10 μM at [ATP] = 10 µM.

Kinome-wide profiling was performed at DiscoverX using the KINOMEscan assay.

Probe		Negative control
SKI-73		SKI-73N

• SMILES:
• SKI-73: CC1=C(C(C(C(C)(CC(N[C@H](C(NCCC2=CC=C(OC)C=C2)=O)CC[C@H](CNCC3=CC=CC=C3)C[C@@H]4[C@@H](O)[C@@H](O)[C@@H](O4)N5C=NC6=C5N=CN=C6N)=O)C)=C(C1=O)C)=O)C
• SKI-73N: CC1=C(C(C(C(C)(CC(N[C@H](C(NCCC2=CC=C(OC)C=C2)=O)CC[C@H](NCC3=CC=CC=C3)C[C@@H]4[C@@H](O)[C@@H](O)[C@@H](O4)N5C=NC6=C5N=CN=C6N)=O)C)=C(C1=O)C)=O)C
• InChI:
• SKI-73: InChI=1S/C46H58N8O8/c1-26-27(2)39(57)36(28(3)38(26)56)46(4,5)21-35(55)53-33(44(60)49-19-18-29-12-15-32(61-6)16-13-29)17-14-31(23-48-22-30-10-8-7-9-11-30)20-34-40(58)41(59)45(62-34)54-25-52-37-42(47)50-24-51-43(37)54/h7-13,15-16,24-25,31,33-34,40-41,45,48,58-59H,14,17-23H2,1-6H3,(H,49,60)(H,53,55)(H2,47,50,51)/t31-,33-,34+,40+,41+,45+/m0/s1
• SKI-73N: InChI=1S/C45H56N8O8/c1-25-26(2)38(56)35(27(3)37(25)55)45(4,5)21-34(54)52-32(43(59)47-19-18-28-12-15-31(60-6)16-13-28)17-14-30(48-22-29-10-8-7-9-11-29)20-33-39(57)40(58)44(61-33)53-24-51-36-41(46)49-23-50-42(36)53/h7-13,15-16,23-24,30,32-33,39-40,44,48,57-58H,14,17-22H2,1-6H3,(H,47,59)(H,52,54)(H2,46,49,50)/t30-,32-,33+,39+,40+,44+/m0/s1
• InChIKey:
• SKI-73: HZHUKQLWLNDVDZ-IBDVCRJISA-N
• SKI-73N: NEMZJATUXVLJBZ-AEPDLXTASA-N

Main features

• Overall structure of (S)-SKI-72 with PRMT4 showing dimeric crystal structure
• PRMT4 with sinefungin and H3R17me
• (S)-SKI-72 occupies both the SAM and peptide pockets
• (S)-SKI-72 binding interactions

• Böttcher, J., Dilworth, D., Reiser, U., Neumüller, R., Schleicher, M., & Petronczki, M. et al. (2019). Fragment-based discovery of a chemical probe for the PWWP1 domain of NSD3. Nature Chemical Biology. doi: 10.1038/s41589-019-0310-x

Main features

• Predicted binding mode with H3K36me3
• Overlay with BI-9321 structure
• Zoomed BI-9321 structure showing hydrogen bonds

Main features

• Active form of mPRDM9 showing H3 peptide stabilized by C-terminus of SET domain (orange).
• Overlay with inactive form of hPRDM9 which does not contain SAM or H3 peptide. Note the displacement of the helix (blue) which is stabilized in the active form (orange).
• Binding of MRK-740 destabilizes the helix in similar manner to that of the apo-form (blue).
• Overview of MRK-740 bound to mPRDM9 with SAM.
• Detailed view of MRK-740 binding.