lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): N#CCN1CN([C@H]2CC[C@H](CNCC(F)F)CC2)c2c(cnc3[nH]ccc23)C1=O
InChIKey: OWZUPJBYJRUXLY-HDJSIYSDSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Assay: The inhibitory activity of the compounds of the present invention against JAK was measured.The respective enzymes (JAK1, JAK2, JAK3 and Tyk2) were purchased from Carna Biosciences, Inc.As a substrate of the enzymes (hereinafter referred to as substrate), LANCE Ultra ULight-JAK-1 (Tyr1023) Peptide (manufactured by PerkinElmer Inc.) was used.As an antibody for detecting phosphorylation of the substrate, LANCE Ultra Europium-anti-phospho tyrosine antibody (PT66) (manufactured by PerkinElmer Inc.) was used.The other reagents were purchased from the following.Adenosine triphosphate (ATP): Sigma-Aldrich4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES): DOJINDO LABORATORIESGlycol ether diamine tetraacetic acid (EGTA): DOJINDO LABORATORIES Magnesium chloride (MgCl2): Wako Pure Chemical Industries, Ltd.Dithiothreitol (DTT): Wako Pure Chemical Industries, Ltd.Tween 20: Sigma-AldrichEthylenediaminetetraacetic acid (EDTA): DOJINDO LABORATORIESThe compounds of the present invention, the enzymes (JAK1, JAK2, JAK3 and Tyk2), the substrate and ATP were used for assay as diluted with assay buffer.Â  As the assay buffer, one having the following composition was used.Â  HEPES (pH7.5): 50 mM EGTA: 1 mM MgCl2: 10 mM DTT: 2 mM Tween 20: 0.01% (wt/wt). The dilute concentration and the addition amount on a well plate as described hereinafter were adjusted so that the following final concentrations were achieved on the well plate.
Measurement: IC50 = 41 nM (pChEMBL 7.389999866485596)
Descriptors: MW 402.45, ALogP 2.72, HBA 5, HBD 2, PSA 88.05, QED 0.73
Compound (SMILES): CC(C(=O)Nc1ccc2nc[nH]c2c1)n1ccc(-c2cc(Cl)ccc2C#N)cc1=O
InChIKey: FYWRBGOWASHFQJ-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Measurement of FXIa Inhibition: To determine the factor XIa inhibition of the substances according to the invention, a biochemical test system is used which utilizes the reaction of a peptidic factor XIa substrate to determine the enzymatic activity of human factor XIa. Here, factor XIa cleaves from the peptic factor XIa substrate the C-terminal aminomethylcoumarin (AMC), the fluorescence of which is measured. The determinations are carried out in microtitre plates.Test substances are dissolved in dimethyl sulphoxide and serially diluted in dimethyl sulphoxide (3000 μM to 0.0078 μM; resulting final concentrations in the test: 50 μM to 0.00013 μM). In each case 1 μl of the diluted substance solutions are placed into the wells of white microtitre plates from Greiner (384 wells). 20 μl of assay buffer (50 mM of Tris/HCl pH 7.4; 100 mM of sodium chloride solution; 5 mM of calcium chloride solution; 0.1% of bovine serum albumin) and 20 μl of factor XIa from Kordia (0.45 nM in assay buffer) are then added successively. After 15 min of incubation, the enzyme reaction is started by addition of 20 μl of the factor XIa substrate Boc-Glu(OBzl)-Ala-Arg-AMC dissolved in assay buffer (10 μM in assay buffer) from Bachem, the mixture is incubated at room temperature (22° C.) for 30 min and fluorescence is then measured (excitation: 360 nm, emission: 460 nm). The measured emissions of the test batches with test substance are compared to those of control batches without test substance (only dimethyl sulphoxide instead of test substance in dimethyl sulphoxide), and the IC50 values are calculated from the concentration/activity relationships.
Measurement: IC50 = 490 nM (pChEMBL 6.309999942779541)
Descriptors: MW 417.86, ALogP 4.12, HBA 5, HBD 2, PSA 103.57, QED 0.52
Compound (SMILES): CNC(C)C(=O)Nc1ccc(-c2c(-c3ccncc3)nc3cc(C)ccn23)c(C#Cc2ccc3c(ccc(=O)n3C)c2)n1
InChIKey: ADGXZHKQDHRJQW-UHFFFAOYSA-N
Target: Cytochrome P450 2C9 (SINGLE PROTEIN), Homo sapiens
UniProt: P11712
Assay: Inhibition Assays: The final incubation volume contains TRIS buffer (0.1 M), MgCl2 (5 mM), a certain concentration of human liver microsomes dependent on the P450 isoenzyme measured (P450 2C9, P450 3A4: 0.1 mg/ml; P450 2D6: 0.2 mg/ml; P450 2C19: 0.5 mg/ml; P450 2C8: 0.05 mg/ml) and a certain concentration of the individual substrate for each isoenzyme (P450 2C9: Diclofenac 10 μM; P450 3A4: Midazolam 5 μM; P450 2D6: Dextromethorphan 5 μM; P450 2C19: S-Mephenytoin 70 μM; P450 2C8: Amodiaquine 1 μM).
Measurement: IC50 = 900 nM (pChEMBL 6.050000190734863)
Descriptors: MW 567.65, ALogP 4.56, HBA 8, HBD 2, PSA 106.21, QED 0.3
Compound (SMILES): COc1ccc(CNC(=O)N2[C@H](C)CN(Cc3ccccc3)c3ccccc3[C@@H]2C)cc1
InChIKey: ZHYCMILLXCWHNK-RTWAWAEBSA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1I (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P0X4
Assay: Channel blocking activity at recombinant human CaV 3.3 channel expressed in HEK293 cells assessed as inhibition of Ca2+ flux preincubated for 3 mins followed by CaCl2 addition by fluo-4 dye-based FLIPR assay
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 429.56, ALogP 5.38, HBA 3, HBD 1, PSA 44.81, QED 0.59
Compound (SMILES): O=C(O)c1ccc(-c2ccc(OCCCCO)c(-c3ccc(N4CCCC4)c(C(F)(F)F)c3)c2)cc1
InChIKey: FCKTUZDSGYGGDB-UHFFFAOYSA-N
Target: Retinoic acid receptor gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P13631
Assay: Activation of RARgamma (unknown origin) expressed in human HeLa cells co-expressing ERE-beta-Glob-Luc-SV-Neo incubated for 18 hrs by luciferase reporter gene assay
Measurement: Kd = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 499.53, ALogP 6.49, HBA 4, HBD 2, PSA 70, QED 0.33
Compound (SMILES): COc1cc(N2CCC(N3CCC(C(F)(F)F)CC3)CC2)ccc1Nc1nccc(-c2c(-c3cccc(C(=O)Nc4c(F)cccc4F)c3)nc3ccccn23)n1
InChIKey: KJPGZMLTGYOXBT-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human full length EGFR expressed in baculovirus expression system assessed as decrease in transfer of gamma-phosphate from ATP onto tyrosine residue of biotinylated peptide substrate preincubated for 1 hr followed by substrate addition measured after 90 mins by HTRF method
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 782.82, ALogP 8.98, HBA 9, HBD 2, PSA 99.92, QED 0.14
Compound (SMILES): CCN(CC)CCCC[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](Cc1ccccc1)NC(=O)c1ccc(C(C)C)cc1)C(=O)N[C@@H](CO)C(=O)OC
InChIKey: ROYGCJFTRGCLIU-JCXBGQGISA-N
Target: Chromobox protein homolog 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HC52
Assay: Antagonist activity at recombinant human N-terminal His-tagged CBX8 chromodomain (8 to 61 residues) expressed in Escherichia coli Rosetta BL21(DE3)pLysS using biotin-labeled H3K9me3 measured after 30 mins by AlphaScreen assay
Measurement: IC50 = 1900 nM (pChEMBL 5.71999979019165)
Descriptors: MW 781.01, ALogP 2.83, HBA 9, HBD 6, PSA 195.27, QED 0.07
Compound (SMILES): Cc1cc2ccc1[C@@H](C)COC(=O)Nc1ccc(C3CC3)c(c1)CN(C)C(=O)[C@@H]2Nc1ccc2c(N)ncc(F)c2c1
InChIKey: YBLXUAHRYXEFCL-WWOZWPLTSA-N
Target: Prothrombin (SINGLE PROTEIN), Homo sapiens
UniProt: P00734
Assay: Inhibition of thrombin (unknown origin) at 25 degC by chromogenic substrate assay
Measurement: Ki = 18000 nM (pChEMBL 4.75)
Descriptors: MW 567.67, ALogP 6.62, HBA 6, HBD 3, PSA 109.58, QED 0.26
Compound (SMILES): CCN(CC)c1nc2ccc(C(O)(c3ccc(C(F)(F)F)nc3)c3cncn3C)cc2c(Cl)c1Oc1ccc(F)cc1
InChIKey: AXVBVFNCJWQPMU-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor® Assay: ThermoFluor® experiments were carried out using instruments owned by Janssen Research and Discovery, L.L.C. through its acquisition of 3-Dimensional Pharmaceuticals, Inc. 1,8-ANS (Invitrogen) was used as a fluorescent dye. Protein and compound solutions are dispensed into black 384-well polypropylene PCR microplates (Abgene) and overlayed with silicone oil (1 μL, Fluka, type DC 200) to prevent evaporation. Bar-coded assay plates are robotically loaded onto a thermostatically controlled PCR-type thermal block and then heated at a typical ramp-rate of 1 °C./min for all experiments. Fluorescence was measured by continuous illumination with UV light (Hamamatsu LC6) supplied via fiber optic and filtered through a band-pass filter (380-400 nm; >60D cutoff). Fluorescence emission of the entire 384-well plate was detected by measuring light intensity using a CCD camera (Sensys, Roper Scientific) filtered to detect 500±25 nm, resulting in simultaneous and independent readings of all 384 wells. Images were collected at each temperature, and the sum of the pixel intensity in a given area of the assay plate was recorded versus temperature. Reference wells contained RORγt without compounds, and the assay conditions were as follows: 0.065 mg/mL RORγt, 60 μM 1,8-ANS, 100 mM Hepes, pH 7.0, 10 mM NaCl, 2.5 mM GSH, 0.002% Tween-20. Project compounds were arranged in a pre-dosed mother plate (Greiner Bio-one) wherein compounds are serially diluted in 100% DMSO by 1:2 from a high concentration of 10 mM over 12 columns within a series (column 12 is a reference well containing DMSO, no compound). The compounds were robotically dispensed directly into assay plates (1x=46 mL) using a Hummingbird capillary liquid handling instrument (Digilab). Following compound dispense, protein and dye in buffer was added to achieve the final assay volume of 3 μL, followed by 1 μL of silicone oil.
Measurement: Kd = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 600.02, ALogP 7.1, HBA 7, HBD 1, PSA 76.3, QED 0.19
Compound (SMILES): O=C[C@H]1CC[C@@H](c2nnn3cnc4[nH]ccc4c23)CC1
InChIKey: TWVLDWHLQMIHJW-AOOOYVTPSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 130 nM (pChEMBL 6.889999866485596)
Descriptors: MW 269.31, ALogP 2.08, HBA 5, HBD 1, PSA 75.94, QED 0.72
Compound (SMILES): FC(F)Oc1ccc(CN2CCC(c3nnn4cnc5[nH]ccc5c34)CC2)cc1
InChIKey: RJMHXFHAHXHNIV-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 620 nM (pChEMBL 6.210000038146973)
Descriptors: MW 398.42, ALogP 3.59, HBA 6, HBD 1, PSA 71.34, QED 0.56
Compound (SMILES): CC(C)(O)C(=O)N(CCN1[C@@H]2CC[C@H]1C[C@@H](c1cccc(C(N)=O)c1)C2)Cc1c(F)cccc1F.O=C(O)C(F)(F)F
InChIKey: MPTYGOKGRHKOTN-RMRMZDFSSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Displacement of [3H]-diprenorphine from human mu opioid receptor expressed in CHOK1 cell membranes
Measurement: Ki = 10 nM (pChEMBL 8)
Descriptors: MW 485.58, ALogP 3.57, HBA 4, HBD 2, PSA 86.87, QED 0.6
Compound (SMILES): COc1ccc(Cc2c(O[C@@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@H]3O)n[nH]c2C)cc1
InChIKey: ZXZRLHXGESMVDH-QFXBJFAPSA-N
Target: Sodium/glucose cotransporter 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P13866
Assay: Inhibition of human SGLT1 expressed in COS7 cells assessed as reduction in [14C]-AMG uptake
Measurement: IC50 = 626 nM (pChEMBL 6.199999809265137)
Descriptors: MW 380.4, ALogP -0.5, HBA 8, HBD 5, PSA 137.29, QED 0.45
Compound (SMILES): CC(C)C[C@@H](/C=C/[N+](=O)[O-])NC(=O)[C@H](Cc1ccccc1)NC(=O)OCc1ccccc1
InChIKey: KRBOYQNUSRICHT-WQHODERWSA-N
Target: Cathepsin B (SINGLE PROTEIN), Homo sapiens
UniProt: P07858
Assay: Inhibition of human cathepsin B
Measurement: Ki = 68 nM (pChEMBL 7.170000076293945)
Descriptors: MW 439.51, ALogP 3.85, HBA 5, HBD 2, PSA 110.57, QED 0.41
Compound (SMILES): COCCOCCOCC#Cc1cccc2nc(Cn3nc(-c4cc(O)cc(F)c4)c4c(N)ncnc43)n(Cc3ccccc3C(F)(F)F)c(=O)c12
InChIKey: JTACVHJPXOKMTI-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Enzyme Inhibition Assay: The PI3K enzymes catalyse the phosphorylation of phosphatidylinositol 4,5-biphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3) in the presence of ATP and Mg2+ ions. The PIP3 product can be detected by displacement of biotin-PIP3 from energy transfer complexes consisting of europium labelled anti-GST monoclonal antibody, a GST-tagged Pleckstrin homology (PH) domain, biotinylated PIP3 and streptavidin-allophycocyanin (APC) by the time-resolved fluorescence resonance energy transfer (TR-FRET) (HTRF PI3K enzyme assay, Millipore). Excitation, at 330 nm, of europium in the complex results in an energy transfer to the APC and a fluorescent emission at 665 nm although europium itself emits at its characteristic wavelength of 620 nm. The PIP3 product formed by PI3K activity displaces biotin-PIP3 from the complex and results in a loss of energy transfer (decreasing signal).The compound to be tested was added, at the desired final concentrations, to a mixture of PIP2 substrate.
Measurement: IC50 = 2.5 nM (pChEMBL 8.600000381469727)
Descriptors: MW 717.68, ALogP 4.78, HBA 12, HBD 2, PSA 152.43, QED 0.1
Compound (SMILES): CC(Oc1cncc(-c2c[nH]c3ccc(-c4nnc(N)s4)cc23)n1)C(F)(F)F
InChIKey: DGBIUYHHQKNLEE-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Enzyme Assay: The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction.Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 uM. A reference compound was included on each assay plate in order to validate that plate; on
Measurement: IC50 = 13.22 nM (pChEMBL 7.880000114440918)
Descriptors: MW 406.39, ALogP 4.06, HBA 7, HBD 2, PSA 102.6, QED 0.53
Compound (SMILES): CCCSc1nnc2c(n1)OC(C(=O)c1ccc(OC)cc1)Nc1ccccc1-2
InChIKey: DHNRRYABRJSZFA-UHFFFAOYSA-N
Target: Aldehyde dehydrogenase, dimeric NADP-preferring (SINGLE PROTEIN), Homo sapiens
UniProt: P30838
Assay: Inhibiton Assay: IC50 values were determined for CB29 and its analogs using propionaldehyde as the substrate for ALDH1A1 and ALDH2 or benzaldehyde as the substrate for ALDH3A1. The assays were performed on a Beckman DU-640 spectrophotometer at various concentrations of inhibitors ranging from 50 nM to 250 μM following a 1 minute pre-incubation. There was no pre-incubation time-dependence to the inhibition. All reactions were initiated by the addition of the aldehyde substrate. The inhibition curves were fit to the Logistic four parameter IC50 equation using the SigmaPlot (v11, StatSys). We characterized the mode of inhibition using steady-state kinetics through co-variation of inhibitor and substrate concentrations. The steady state kinetic measurements were performed in 100 mM Na2HPO4 buffer, pH 7.5. The reaction mixture contained 10 nM ALDH3A1, varied benzaldehyde (50 μM-800 μM; fixed NADP+, 1.5 mM) and varied inhibitor concentrations. In all cases−including the control reactions lacking inhibitors, the final reaction mixture contained 2% (v/v) DMSO. The reaction was initiated by addition of substrate and the initial rate of product formation was determined on a Beckman DU-640.
Measurement: IC50 = 2500 nM (pChEMBL 5.599999904632568)
Descriptors: MW 408.48, ALogP 4.06, HBA 8, HBD 1, PSA 86.23, QED 0.48
Compound (SMILES): CC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@H](C(=O)N[C@@H](CCC(=O)O)C(=O)NCC(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(N)=O)C(C)C)C(C)C
InChIKey: YRXYMTGSRURMBH-FCUNHWGASA-N
Target: Tissue factor pathway inhibitor (SINGLE PROTEIN), Homo sapiens
UniProt: P10646
Assay: Binding affinity to TFPI (unknown origin) incubated for 1.5 hrs using tracer peptide JBT0131 by screening ELISA
Measurement: IC50 = 39 nM (pChEMBL 7.409999847412109)
Descriptors: MW 2500.94
Compound (SMILES): Cc1cncc(-c2cc3c(cn2)cnn3-c2cnc(C)c(N3CCC[C@H](N)C3)n2)n1
InChIKey: PUJNJBCBFYLKKR-INIZCTEOSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 0.047 nM (pChEMBL 10.329999923706055)
Descriptors: MW 401.48, ALogP 2.21, HBA 9, HBD 1, PSA 111.53, QED 0.56
Compound (SMILES): COc1ccc(-n2nc(C(C)(C)C)cc2NC(=O)Nc2ccc(Oc3ccnc(Nc4ccc5c(cnn5C)c4)n3)c3ccccc23)cc1
InChIKey: HWLWBINIXVPIHY-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: c-Src and Syk Enzyme Inhibition Assay: The inhibitory activities of compounds of the invention against c-Src and Syk enzymes (Invitrogen), are evaluated in a similar fashion to that described hereinabove. The relevant enzyme (3000 ng/mL or 2000 ng/mL respectively, 2.5 uL) is incubated with the test compound (either 4 ug/mL, 0.4 ug/mL, 0.04 ug/mL, or 0.004 ug/mL, 2.5 uL each) for 2 hr at RT. The FRET peptides (8 uM, 2.5 uL), and appropriate ATP solutions (2.5 uL, 800 uM for c-Src, and 60 uM ATP for Syk) are then added to the enzymes/compound mixtures and incubated for 1 hr. Development reagent (protease, 5 uL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan Flash, ThermoFisher Scientific).
Measurement: IC50 = 81 nM (pChEMBL 7.090000152587891)
Descriptors: MW 653.75, ALogP 8.19, HBA 10, HBD 3, PSA 133.04, QED 0.15
Compound (SMILES): CN(C)CCCOc1ccc(-c2ccc3ncc4c(c3c2)n([C@H]2CCCOC2)c(=O)n4C)c(F)n1
InChIKey: SBWOIJRPFUUCGS-SFHVURJKSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Cellular Assay: Assay c) BT474 cells (human breast ductal carcinoma, ATCC HTB-20) were seeded into black 384 well plates (Costar, #3712) at a density of 5600 cells/well in DMEM containing 10% FBS and 1% glutamine and allowed to adhere overnight. The following morning compounds in 100% DMSO were added to assay plates by acoustic dispensing. After a 2 h incubation at 37° C. and 5% CO2, the medium was aspirated and the cells were lysed with a buffer containing 25 mM Tris, 3 mM EDTA, 3 mM EGTA, 50 mM sodium fluoride, 2 mM Sodium orthovanadate, 0.27 M sucrose, 10 mM β-glycerophosphate, 5 mM sodium pyrophosphate, 0.5% Triton X-100 and complete protease inhibitor cocktail tablets (Roche #04 693 116 001, used 1 tab per 50 ml lysis buffer). After 20 minutes, the cell lysates were transferred into ELISA plates (Greiner #781077) which had been pre-coated with an anti total-AKT antibody in PBS buffer and non-specific binding was blocked with 1% BSA in PBS containing 0.05% Tween 20. Plates were incubated over night at 4° C. The next day the plates were washed with PBS buffer containing 0.05% Tween 20 and further incubated with a mouse monoclonal anti-phospho AKT T308 for 2 h. Plates were washed again as above before addition of a horse anti-mouse-HRP conjugated secondary antibody. Following a 2 h incubation at r.t., plates were washed and QuantaBlu substrate working solution (Thermo Scientific #15169, prepared according to provider's instructions) was added to each well. The developed fluorescent product was stopped after 60 minutes by addition of Stop solution to the wells. Plates were read using a Tecan Satire plate reader using 325 nm excitation and 420 nm emission wavelengths respectively. Except where specified, reagents contained in the Path Scan Phospho AKT (Thr308) sandwich ELISA kit from Cell Signalling (#7144) were used in this ELISA assay.
Measurement: IC50 = 1030 nM (pChEMBL 5.989999771118164)
Descriptors: MW 479.56, ALogP 3.77, HBA 8, HBD 0, PSA 74.41, QED 0.3
Compound (SMILES): C=CC(=O)N1CCC(C2CCNc3c(C(N)=O)c(-c4ccc(Oc5ccc(F)cc5F)cc4)nn32)CC1
InChIKey: DFUIAUMNSYRTOL-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Time-Resolved Fluorescence Resonance Energy Transfer Assay: Compounds disclosed herein were tested for inhibition of Btk kinase activity in an assay based on time-resolved fluorescence resonance energy transfer methodology. Recombinant Btk was pre-incubated with the compounds disclosed herein at room temperature for 1 hour in an assay buffer containing 50 mM Tris pH7.4, 10 mM MgCl2, 2 mM MnCl2, 0.1 mM EDTA, 1 mM DT, 20 nM SEB, 0.1% BSA, 0.005% tween-20. The reactions were initiated by the addition of ATP (at the concentration of ATP Km) and peptide substrate (Biotin-AVLESEEELYSSARQ-NH2). After incubating at room temperature for 1 h, an equal volume of stop solution containing 50 mM HEPES pH7.0, 800 mM KF, 20 mM EDTA, 0.1% BSA, Eu cryptate-conjugated p-Tyr66 antibody and streptavidin-labeled XL665 was added to stop the reaction. Plates were further incubated at room temperature for 1 hour, and then the TR-FRET signals (ex337 nm, em 620 nm/665 nm) were read on BMG PHERAstar FS instrument. The residual enzyme activity in presence of increasing concentrations of compounds was calculated based on the ratio of fluorescence at 615 nm to that at 665 nm. The IC50 for each compound was derived from fitting the data to the four-parameter logistic equation by Graphpad Prism software.
Measurement: IC50 = 1.2 nM (pChEMBL 8.920000076293945)
Descriptors: MW 507.54, ALogP 4.5, HBA 6, HBD 2, PSA 102.48, QED 0.48
Compound (SMILES): CCCN(Cc1ccc(C(=O)O)cc1)C(=O)c1ncc(Oc2ccccc2OC)cn1
InChIKey: WBAUSFZQQFMBCS-UHFFFAOYSA-N
Target: Lysophosphatidic acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92633
Assay: In Vitro LPA1 Receptor Assay: Engagement of the LPA1 receptor by its ligand, oleoyl-L-α-lysophosphatidic acid (LPA), leads to the release of intracellular stores of calcium into the cytoplasm mediated through a Gq-driven increase in inositol triphosphate (IP3) levels. Gq is a heterotrimeric G protein subunit that activates phospholipase C.The ability of test compounds to prevent LPA-driven intracellular release of stored calcium from RH7777 cells expressing human LPA1 receptors was assayed by stimulating the cells with LPA in the presence of various test compounds to measure the test compounds' antagonism, i.e. activity in vitro. Approximately 10,000 cells in normal culture medium (Dulbecco's Modified Eagle Media, 10% Foetal Calf Serum) were dispensed per assay well in a black clear bottom collagen coated 384 well plate (Beckton Dickinson). Cells were allowed to settle for thirty minutes at room temperature before being incubated overnight at 37° C., 5% CO2. Sixteen to twenty hours after dispensing, the cells were loaded with a calcium-sensitive fluorescent dye by replacing the culture medium with assay buffer (1× Hanks buffered saline, 25 mM HEPES, 0.1% w/v fatty acid free BSA (bovine serum albumin), pH7.4) containing 1.25 mM probenecid and 1× Calcium 5 Reagent (Molecular Devices). Cells were incubated at 37° C. for one hour to allow for dye uptake. To test for antagonist activity, test compounds at a final concentration range between 0.32 nM-10 μM (diluted in assay buffer) were added to the assay wells and allowed to incubate for twenty five minutes. After incubation with test compounds the assay plate was placed in a FLIPR Tetra system (Molecular Devices) and LPA (diluted in assay buffer) was added to give a final concentration equivalent to its determined EC80 against LPA1 receptor. Ligand-dependent changes in intracellular calcium levels were determined by measuring changes in fluorescence of the dye over 515-575 nM following excitation over 470-495 nM.
Measurement: IC50 = 120 nM (pChEMBL 6.920000076293945)
Descriptors: MW 421.45, ALogP 4.03, HBA 6, HBD 1, PSA 101.85, QED 0.56
Compound (SMILES): Cc1cnn(C)c1-c1cc(C(=O)N[C@H](CN)Cc2cccc(F)c2)sc1Cl
InChIKey: ATGGXNWSNRBMNN-AWEZNQCLSA-N
Target: RAC-gamma serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y243
Assay: Inhibition of full length human AKT3 expressed in Sf9 cells assessed as reduction in substrate phosphorylation using biotin-ahx-ARKRERAYSFGHHA-amide substrate and [gamma-33P]ATP incubated for 40 mins by top count microplate scintillation counting method
Measurement: IC50 <= 25.12 nM (pChEMBL 7.599999904632568)
Descriptors: MW 406.91, ALogP 3.55, HBA 5, HBD 2, PSA 72.94, QED 0.66
Compound (SMILES): Cc1cccc(-c2c(-c3cccc(OC(F)(F)F)c3)cc3n2[C@@H](CCN)CNC3=O)c1
InChIKey: QRVJKXOAMVIRHV-KRWDZBQOSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Trans-Phosphorylation Assay: The inhibitory activity of putative kinase inhibitors and the potency of selected compounds were determined using a trans-phosphorylation assay. Specific peptide or protein substrates were trans-phosphorylated by their specific ser-thr or tyr kinase in the presence of ATP traced with 33P-gamma-ATP, and in the presence of their own optimal buffer and cofactors.At the end of the phosphorylation reaction, more than 98% unlabeled ATP and radioactive ATP were captured by an excess of the ion exchange dowex resin; the resin then settled down to the bottom of the reaction plate by gravity. Supernatant was subsequently withdrawn and transferred into a counting plate, then evaluated by beta-counting. Kinase Buffer (KB):The buffer for PIM1 assay was composed of HEPES 50 mM, at pH 7.5, with 10 mM MgCl2, 1 mM DTT, 3 uM NaVO3, and 0.2 mg/mL BSA.Â  Full-length human PIM1 was expressed and purified as described in Bullock A N, et al., J. Biol. Chem. 2005, 280, 41675-82.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 429.44, ALogP 4.66, HBA 4, HBD 2, PSA 69.28, QED 0.62
Compound (SMILES): O=C(C1CCCCC1)N(CCN1[C@@H]2CC[C@H]1C[C@@H](c1cccc(O)c1)C2)CC1CCCCC1
InChIKey: DKYJPWSZAUPZNF-LGKQTMLJSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Cavia porcellus
UniProt: P41144
Assay: Displacement of [3H]-diprenorphine from guinea pig kappa opioid receptor expressed in CHOK1 cell membranes
Measurement: Ki = 100 nM (pChEMBL 7)
Descriptors: MW 452.68, ALogP 6.09, HBA 3, HBD 1, PSA 43.78, QED 0.54
Compound (SMILES): Cc1cncc(-c2cc3c(cn2)cnn3-c2cccc(N3CCC[C@H](N)C3)n2)n1
InChIKey: OTCBQVWPAUVEEA-INIZCTEOSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 0.038 nM (pChEMBL 10.420000076293945)
Descriptors: MW 386.46, ALogP 2.51, HBA 8, HBD 1, PSA 98.64, QED 0.58
Compound (SMILES): CCCS(=O)(=O)Nc1ccc(F)c(C(=O)c2c[nH]c3ncc(-c4ccc(S(=O)(=O)N(C)C)cc4)cc23)c1F
InChIKey: GCKBWWMVGNBVHY-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Inhibition of C-Raf1 (unknown origin) assessed as decrease in biotin-tagged MEK1 phosphorylation after 45 mins
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 562.62, ALogP 4.14, HBA 6, HBD 2, PSA 129.3, QED 0.3
Compound (SMILES): CN1CCN(Cc2ccc(-c3cn4c5nc(ncc35)NCCCCCCNC(=O)[C@@H](N)CCC4)cc2)CC1
InChIKey: VNERMGVCIRNEMM-SANMLTNESA-N
Target: Tyrosine-protein kinase receptor TYRO3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06418
Assay: Inhibition of Tyro3 (unknown origin) by microfluidic capillary electrophoresis assay
Measurement: IC50 = 110 nM (pChEMBL 6.960000038146973)
Descriptors: MW 518.71, ALogP 3.06, HBA 8, HBD 3, PSA 104.34, QED 0.49
Compound (SMILES): CC(=O)N(CCCN1[C@@H]2CC[C@H]1C[C@@H](c1cccc(C(N)=O)c1)C2)CC1CCCCC1
InChIKey: UIEJLBLSVRXOJC-QEIWAGNOSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Displacement of [3H]-diprenorphine from human mu opioid receptor expressed in CHOK1 cell membranes
Measurement: Ki = 10 nM (pChEMBL 8)
Descriptors: MW 425.62, ALogP 4.31, HBA 3, HBD 1, PSA 66.64, QED 0.67
Compound (SMILES): COc1ccc(C(=O)C2CCN(CC(=O)N(Cc3nc4c(c(=O)[nH]3)COCC4)CC(F)F)CC2)cc1
InChIKey: WDZBHMSPANOWRC-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: Biochemical Assay: The autoparsylation activity of the TNKS 1/2 or PARP1/2 enzymes was measured by the liquid chromatography-mass spectrometry (LC/MS) detection of nicotinamide as readout. Compound activity in inhibiting the TNKS and PARP autoparsylation was evaluated by IC50 measurements. In the compound screening assays, the reaction is composed of 5 μL of compound in 8-point serial dilutions with concentrations ranging from 0.0086 to 18.75 μM, 20 nM of purified enzyme, and 250 μM of β-NAD+ in the 1× Assay Buffer. After 60 min incubation at room temperature, the reactions were quenched by the addition of 10 μL of 5× quenching solution (20% formic acid and 500 nM [d]-nicotinamide in water). For the background control wells, 10 μL of the 5× quenching solution per well was added prior to the addition of β-NAD+.
Measurement: IC50 > 19000 nM (pChEMBL 4.7210001945495605)
Descriptors: MW 504.53, ALogP 2.04, HBA 7, HBD 1, PSA 104.83, QED 0.52
Compound (SMILES): Cl.O=C(CC(c1ccccc1)c1ccccc1)N1CCN(C(c2ccccc2)c2c(Cl)cccc2Cl)CC1
InChIKey: PACAERJLZUDWEA-UHFFFAOYSA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1G (SINGLE PROTEIN), Homo sapiens
UniProt: O43497
Assay: Inhibition of human alpha1G T-type calcium channel expressed in HEK293 cells assessed as current dependent channel blocking at -110 mV holding potential with pre-pulse at -100 mV for 1 sec prior to test pulse at -40 mV for 50 ms by whole cell patch clamp assay
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 529.51, ALogP 7.45, HBA 2, HBD 0, PSA 23.55, QED 0.25
Compound (SMILES): Cc1ccc(F)c2nc([C@H](C)Nc3ncnc(N)c3C#Cc3ccc(F)cn3)n(-c3ccccc3)c(=O)c12
InChIKey: PDYXAKCUXSFVDT-KRWDZBQOSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2xKm ATP (100-300 uM), 10 uM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 1% (v/v) DMSO at the following concentrations for each isoform: PI3K alpha, beta, delta, and gamma at 50 picomolar (pM) and PI3Kgamma at 2 nanomolar (nM). After an assay reaction time of 30 minutes at 25° C., reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 us delay and 500 us read window).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 509.52, ALogP 4.31, HBA 8, HBD 2, PSA 111.61, QED 0.35
Compound (SMILES): COc1cc(OCCO)cc(N(CC2CC2)c2ccc3ncc(-c4cnn(CCS(C)(=O)=O)c4)nc3c2)c1
InChIKey: BAZVFUXBEXJILA-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 537.64, ALogP 3.47, HBA 10, HBD 1, PSA 119.67, QED 0.29
Compound (SMILES): CC(C)c1cc(NC(=O)Nc2ccc(Oc3cc(Nc4ccc5[nH]ncc5c4)ncn3)c3ccccc23)n(-c2ccc(=O)n(CCO)c2)n1
InChIKey: YBFJEMNJNCMXOO-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: c-Src and Syk Enzyme Inhibition Assay: The inhibitory activities of compounds of the invention against c-Src and Syk enzymes (Invitrogen), are evaluated in a similar fashion to that described hereinabove. The relevant enzyme (3000 ng/mL or 2000 ng/mL respectively, 2.5 uL) is incubated with the test compound (either 4 ug/mL, 0.4 ug/mL, 0.04 ug/mL, or 0.004 ug/mL, 2.5 uL each) for 2 hr at RT. The FRET peptides (8 uM, 2.5 uL), and appropriate ATP solutions (2.5 uL, 800 uM for c-Src, and 60 uM ATP for Syk) are then added to the enzymes/compound mixtures and incubated for 1 hr. Development reagent (protease, 5 uL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan Flash, ThermoFisher Scientific).
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 656.71, ALogP 6.15, HBA 11, HBD 5, PSA 176.9, QED 0.12
Compound (SMILES): Cc1nc2cc(-c3c(C)noc3C)cc(-c3c(C)n[nH]c3C)c2[nH]1
InChIKey: VOTGXBKUYUWTOH-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Amplified Luminescent Proximity Homogeneous Assay (ALPHA): Binding of the bromodomain BRD4-I to an acetylated histone H4 peptide was measured using a bead-based Amplified Luminescent Proximity Homogeneous Assay (ALPHA). The synthetic peptide containing amino acids 1-18 of histone H4 was acetylated at lysine 5, 8, 12, 16 and conjugated to biotin (SGRGACKGGACKGLGACKGGAACKRH-GSGSK-biotin[SEQ ID NO:1]) was purchased from Millipore. BRD4-I was expressed and purified from Escherichia coli as an N-terminal His6-tagged protein. Nickel-Chelate ALPHA acceptor beads (Perkin Elmer) were used to specifically bind BRD4-1 and ALPHA streptavidin donor beads (Perkin Elmer) were used because they specifically recognized the biotinylated H4 peptide. Binding of BRD4-1 to the peptide resulted in proximity of the donor and acceptor beads which leads to an increase in ALPHA signal whereas disruption of this protein-peptide interaction with a small molecule inhibitor resulted in a decrease in ALPHA signal. Assays were performed in 50 mM Hepes (pH 7.5), 150 mM NaCl, 0.1 mg/ml BSA, 0.01% (v/v) Brij, 0.5% (v/v) DMSO, 200 nM H4 peptide and 15 nM of BRD4-1 protein. After an assay reaction time of 60 minutes at 25° C., binding was measured with 20 ug/ml streptavidin donor beads and 20 ug/ml nickle-chelate acceptor beads. ALPHA signal was detected on an Envision plate reader (Ex: 320 nm; Em: 570 nm; Ex time: 180 ms). Data were normalized based on a positive (2 uM I-BET) and negative (DMSO) controls and IC50 values were calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations.
Measurement: IC50 = 42.7 nM (pChEMBL 7.369999885559082)
Descriptors: MW 321.38, ALogP 4.15, HBA 4, HBD 2, PSA 83.39, QED 0.58
Compound (SMILES): O=C(NC[C@@H](C(=O)NO)N1CCCCC1)c1ccc(OCc2ccncc2)cc1
InChIKey: ZTDRQXAWZXLROA-IBGZPJMESA-N
Target: Stromelysin-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P08254
Assay: Inhibition Assay: The molecules are tested in dose-response studies on the following enzymes MMP1, MMP3, MMP9, ADAM9 and ADAM10 according to the same protocol as that described for the TACE enzyme in example 28 but with different substrates (MMP R&D system reference: P126-990 and ADAM R&D system reference: ES003).
Measurement: IC50 = 6125 nM (pChEMBL 5.210000038146973)
Descriptors: MW 398.46, ALogP 1.75, HBA 6, HBD 3, PSA 103.79, QED 0.46
Compound (SMILES): Cn1c(=O)c2c(CC(=O)Nc3nc(-c4ccc(C(F)(F)F)c(F)c4)cs3)cccc2n(C)c1=O
InChIKey: FMIIIXHTXHNZIP-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NET8
Assay: Radiometric Calcium Assay: The inhibition of TRPA1 receptor activation was measured as inhibition of allylisothiocyanate (AITC) induced cellular uptake of radioactive calcium. Test compounds were dissolved in DMSO to prepare 10 mM stock solution and then diluted using plain medium with 0.1% BSA and 1.8 mM CaCl2 to get desired concentration. Final concentration of DMSO in the reaction was 0.5% (v/v). Human TRPA1 expressing CHO cells were grown in F-12 DMEM medium with 10% FBS, 1% penicillin-streptomycin solution, and 400 ug/ml of G-418. Cells were seeded 24 h prior to the assay in 96 well plates so as to get 50,000 cells per well on the day of experiment. Cells were treated with test compounds for 10 min followed by addition of AITC at a final concentration of 30 micromolar and 5 uCi/ml 45Ca+2 for 3 min at 25° C. Cells were washed and lysed using buffer containing 1% Triton X-100, 0.1% deoxycholate and 0.1% SDS. Radioactivity in the lysate was measured in Packard Top count.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 492.45, ALogP 3.7, HBA 7, HBD 1, PSA 85.99, QED 0.44
Compound (SMILES): CCCc1nn(C)c(C)c1NS(=O)(=O)c1ccc(CCCC2CCN(C)CC2)cc1
InChIKey: XJHUAHFZFJZMEE-UHFFFAOYSA-N
Target: Glycylpeptide N-tetradecanoyltransferase (SINGLE PROTEIN), Leishmania major
UniProt: Q4Q5S8
Assay: Enzyme Inhibition Assay: Measurements were performed using a modification of the scintillation proximity assay platform described previously by Georgopapadakou, N. H. et al. (22nd International Congress on Chemotherapy, 2001, Abstract P16.001). Compounds were solubilised in DMSO at a top concentration of 10 mM and serially diluted in half log steps to achieve a range of final assay concentrations of 100 uM to 1 nM. Compound at each concentration (100-fold final) was added to white 384 well plates in a volume of 0.5 ml. Human, A. fumigatus, T. brucei or L. major N-myristoyl transferase enzyme, dissolved to a working concentration of 10 nM in assay buffer (30 mM Tris/HCl pH 7.4, 0.5 mM EGTA, 0.5 mM EDTA, 1.25 mM DTT, 0.1% Triton X-100), was then added to columns 1 to 11 and 13 to 23 of the plates in a volume of 20 ml. To columns 12 and 24, 20 ml assay buffer was added to provide a no enzyme control. Following a 5 minute incubation at room temperature the substrates (GCGGSKVKPQPPQAK(Biotin)-Amide and myristoyl coenzyme A), dissolved in assay buffer, were added to all wells in a volume of 20 ml to start the reaction. The final concentrations of peptide and 3H-myristoyl coenzyme A were 0.5 mM and 125 nM respectively and the specific activity of the radiolabel was 8 Ci/mmol. Plates were then incubated at room temperature for up to 50 minutes (dependant upon the period of linearity for the different enzyme species) before SPA beads, suspended to 1 mg/ml in a stop solution (200 mM Phosphoric Acid/NaOH pH 4, 750 mM MgCl2), were added in a volume of 40 ml.
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 432.63, ALogP 4.15, HBA 5, HBD 1, PSA 67.23, QED 0.65
Compound (SMILES): CC1(C)CNc2ncc(-c3c[nH]c4ccc(-c5nnc(N)s5)cc34)nc21
InChIKey: MABMEAYNQCJUKG-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Enzyme Assay: The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction.Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 uM. A reference compound was included on each assay plate in order to validate that plate; on
Measurement: IC50 = 4.928 nM (pChEMBL 8.3100004196167)
Descriptors: MW 363.45, ALogP 3.43, HBA 7, HBD 3, PSA 105.4, QED 0.5
Compound (SMILES): Clc1ccc(NC2CCC(c3nnn4cnc5[nH]ccc5c34)CC2)cc1
InChIKey: VTTAHOGLSIALQG-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 366.86, ALogP 4.4, HBA 5, HBD 2, PSA 70.9, QED 0.56
Compound (SMILES): COc1cc(O)cc(N(CCCNCC(F)(F)F)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: UCUKGOVQHPVFAN-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 5370.32 nM (pChEMBL 5.269999980926514)
Descriptors: MW 486.5, ALogP 4.42, HBA 8, HBD 2, PSA 88.33, QED 0.34
Compound (SMILES): CC(C)(O)c1cn(-c2ccc(-c3cc(F)c(CO)c(S(C)(=O)=O)c3)cc2)c(C(C)(C)c2c(Cl)cccc2Cl)n1
InChIKey: LODWYHGIAQYCAL-UHFFFAOYSA-N
Target: Oxysterols receptor LXR-beta (SINGLE PROTEIN), Homo sapiens
UniProt: P55055
Assay: Agonist activity at human LXR-beta expressed in African green monkey CV1 cells measured after 18 to 20 hrs by luciferase reporter gene assay
Measurement: EC50 = 42 nM (pChEMBL 7.380000114440918)
Descriptors: MW 591.53, ALogP 6.43, HBA 6, HBD 2, PSA 92.42, QED 0.26