lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COC(=O)CNC(=O)c1ccc(Nc2ncnc3cc(OCCN4CCCCC4)c(OC)cc23)cc1
InChIKey: ISUVLTISVOPDLS-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of N-terminal GST-tagged human VEGFR-2 cytoplasmic domain (790 to 1356 residues) expressed in baculovirus expression system preincubated for 10 mins followed by FAM-labelled peptide substrate addition by caliper mobility shift assay
Measurement: IC50 = 966 nM (pChEMBL 6.010000228881836)
Descriptors: MW 493.56, ALogP 3.15, HBA 9, HBD 2, PSA 114.91, QED 0.41
Compound (SMILES): CC1Cc2c(nnn2-c2cc[nH]n2)CN1C(=O)c1cccc(C(F)(F)F)c1F
InChIKey: JWJNXWJNUVFQCA-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: FLIPR Assay: 1321 N1 cells expressing the recombinant human, rat or mouse P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 ul volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250Ã¿ the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 uL of the compound into 300 uL of assay buffer. A further 3x dilution occurred when transferring 50 uL/well of the compound plate to 100 uL/well in the cell plate. Cells were incubateÂ  with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 uL/well of BzATP (final concentration is 250 uM BzATP (human and rat) or 600 (mouse)). The fluorescence change was measured 180 seconds after adding the agonist.
Measurement: IC50 = 3.9 nM (pChEMBL 8.40999984741211)
Descriptors: MW 394.33, ALogP 2.74, HBA 5, HBD 1, PSA 79.7, QED 0.68
Compound (SMILES): Cn1cc(-c2ccc3nc(NC(=O)C4CCCCCC4)cn3n2)c(-c2ccc(F)cc2)n1
InChIKey: JWEPBVPDMDUEMS-UHFFFAOYSA-N
Target: Casein kinase I isoform delta (SINGLE PROTEIN), Homo sapiens
UniProt: P48730
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 ul prepared from 15 ul additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours and terminated by adding 45 ul of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIPÂ®3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the unphosphorylated substrate and phosphorylated product Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 uM ATP, 1.5 uM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 4.2 nM (pChEMBL 8.380000114440918)
Descriptors: MW 432.5, ALogP 4.84, HBA 6, HBD 1, PSA 77.11, QED 0.47
Compound (SMILES): C=CC(=O)Nc1ccccc1C1CCNc2c(C(N)=O)c(-c3ccc(Oc4ccccc4)cc3)nn21
InChIKey: TZAAHFAAEVJUJO-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Inhibition of BTK in human Ramos cells assessed as reduction in BTK phosphorylation at Tyr223 residue preincubated for 1 hr followed by pervanadate or Na3VO4 stimulation for 20 mins by HTRF assay
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 479.54, ALogP 4.97, HBA 6, HBD 3, PSA 111.27, QED 0.32
Compound (SMILES): COc1cn(C(CC2COCCO2)C(=O)Nc2ccc(C(=O)O)cc2)c(=O)cc1-c1cc(Cl)ccc1C#N
InChIKey: BHLQGSGEFNRQIY-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Inhibition Assay: 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 Tris/HCl pH 7.4; 100 mM sodium chloride solution; 5 mM of calcium chloride solution; 0.1% of bovine serum albumin) and 20 μl of plasma kallikrein from Kordia (0.6 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 substrate H-Pro-Phe-Arg-AMC dissolved in assay buffer (10 μl 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).
Measurement: IC50 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 537.96, ALogP 3.73, HBA 8, HBD 2, PSA 139.88, QED 0.44
Compound (SMILES): Cc1cc(-c2cccc(F)c2)cc(CNc2c(C)ccc(OCC(=O)O)c2C)c1C
InChIKey: JDRDZBXKNDKRIF-UHFFFAOYSA-N
Target: Prostaglandin E2 receptor EP4 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P35408
Assay: Receptor Binding Assay: The competition reaction was initiated by incubation of membrane protein homogenate (20 μg protein) for 120 min at 22° C. with 0.5 nM [3H]PGE2 ligand in the absence or presence of the test compound in a buffer containing 10 mM MES/KOH (pH 6.0), 10 mM MgCl2 and 1 mM EDTA.Non-specific binding was determined in the presence of 10 μM PGE2 (the corresponding non-radioactive prostanoid). Affinity of the compound binding to hEP4 receptor was measured by displacement of the radiolabeled ligand in the presence of varying doses of tested compound.Incubations were terminated by rapid filtration under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and washed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard).
Measurement: IC50 > 300 nM (pChEMBL 6.5229997634887695)
Descriptors: MW 407.49, ALogP 5.8, HBA 3, HBD 2, PSA 58.56, QED 0.52
Compound (SMILES): COc1ccc(C[C@H]2C(=O)C[C@@H]3CCN(CCC(C)C)[C@@H]4CCC[C@H]2[C@@H]34)cc1
InChIKey: NOXURLMMPFLQQR-ANYFMGCHSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: Displacement of [3H]U69593 from KOR (unknown origin) expressed in HEKT cells measured after 90 mins by microbeta scintillation counting method
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 383.58, ALogP 4.98, HBA 3, HBD 0, PSA 29.54, QED 0.69
Compound (SMILES): CCOc1cc2c(N)nccc2cc1C[C@H](C(=O)O)N1CC[C@H](NS(=O)(=O)c2cnc(N3CCCC3)s2)C1=O
InChIKey: DEPXLHRQEGGSSJ-RBUKOAKNSA-N
Target: Coagulation factor X (SINGLE PROTEIN), Homo sapiens
UniProt: P00742
Assay: Inhibition Assay: The compounds according to the invention are tested in a range of concentrations (10pM to 10 µM final) in assay buffer (50 mM TRIS, 100 mM NaCl, 0.1 % BSA, pH 7,5) with 0.1% maximal final DMSO concentration and deposited in a proportion of 25 µl per well, on 25 µL enzyme (human coagulation factor Xa: Enzyme Research Laboratories HFXa, final concentration 0.003Ul/ml). The reactive were mixed, centrifuged and incubated 10 minutes at 37°C in a 96 well microtiter plate. The enzyme reaction was started with 50 µL substrate (S-2765, Biogenic ref 821413 in a final concentration of 62.5µM final). The time course of the reaction was monitored at 405 nm in a microtiter plate reader (Tecan M200) for 20 minutes at 37°C. The percentage of inhibition of enzymatic activity (expressed as maximum rate of cleavage of the substrate) is calculated with respect to the enzymatic activity in the absence of inhibitor.
Measurement: IC50 = 8.4 nM (pChEMBL 8.079999923706055)
Descriptors: MW 574.69, ALogP 1.85, HBA 10, HBD 3, PSA 168.05, QED 0.32
Compound (SMILES): COc1ccc(OC)c2c1C[C@H]1c3cc(O)c(O)cc3CCN1C2
InChIKey: QNEFEUWSOPMZJT-HNNXBMFYSA-N
Target: D(1A) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P21728
Assay: Reception Binding Assay: Different concentrations (10^−5 M-10^−11 M) of the compound of the invention and corresponding isotope receptor ligand as well as receptor protein were loaded into the reaction tube and incubated in 30° C. water bath for 60 minutes. The reaction was terminated in a refrigerator. The reaction mixture was put in a Millipore filter (millipore) cell sample collector, filtered through suction filtration using GF/C glass fiber filter paper, and dried. The resulting sample was placed into 0.5 mL tube. 500 μL liquid scintillation fluid was added and intensity of radioactivity was determined by counting. For dopamine D1 receptor: isotope receptor ligands [3H] SCH23390 (85.0 Ci/mmol) (D1-selective, purchased from Amersham Corporation), D1 receptor protein expressed in HEK-293 cells; For D2 dopamine receptor: isotope receptor ligands [3H] Spiperone (77.0 Ci/mmol) (D2-selective, purchased from Amersham Corporation); D2 receptor protein expressed in HEK-293 cells; For 5-HT1A receptor: isotope receptor ligands [3H] 8-OH-DPAT; 5-HT1A receptor protein expressed in HEK-293 cells; For 5-HT2A receptor: isotope receptor ligands [3H]-Ketanserin; 5-HT2A receptor protein expressed in HEK-293 cells.
Measurement: IC50 = 13.27 nM (pChEMBL 7.880000114440918)
Descriptors: MW 327.38, ALogP 2.77, HBA 5, HBD 2, PSA 62.16, QED 0.83
Compound (SMILES): O=c1n(Cc2cncc(-c3ccccc3Cl)c2)nc(-c2ccc(Cl)cc2)n1CC(O)C(F)(F)F
InChIKey: DFUJBPWYCJPYLU-UHFFFAOYSA-N
Target: Vasopressin V2 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P30518
Assay: Cellular In Vitro Assay: On the day before the assay, the cells are plated out in culture medium (DMEM, 10% FCS, 2 mM glutamine, 10 mM HEPES) in 384-well microtiter plates and kept in a cell incubator (96% humidity, 5% v/v CO2, 37° C.). On the day of the assay, the culture medium is replaced by a Tyrode solution (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 20 mM glucose, 20 mM HEPES), which additionally contains the cofactor coelenterazine (50 μM), and the microtiter plate is then incubated for a further 3-4 hours. The test substances in various concentrations are placed for 10 to 20 minutes in the wells of the microtiter plate before the agonist [Arg8]-vasopressin is added, and the resulting light signal is measured immediately in the luminometer.
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 509.32, ALogP 5.05, HBA 6, HBD 1, PSA 72.94, QED 0.4
Compound (SMILES): NC(=O)CCCCc1cccc(Nc2nccc(Nc3cccc(C(F)(F)F)c3)n2)c1
InChIKey: NPTRODVWHGMFRC-UHFFFAOYSA-N
Target: Aurora kinase A (SINGLE PROTEIN), Homo sapiens
UniProt: O14965
Assay: Enzyme Inhibition Assay: The phosphorylation of L-Hse was monitored by coupling the formation of ADP with pyruvate kinase and lactate dehydrogenase (PK/LDH). The resulting oxidation of NADH was monitored at 340 nm by using a SpectraMax plate reader in a 96-well format.
Measurement: IC50 = 7460 nM (pChEMBL 5.130000114440918)
Descriptors: MW 429.45, ALogP 5.18, HBA 5, HBD 3, PSA 92.93, QED 0.41
Compound (SMILES): c1cc2c(ncn3nnc(C4CCCCCC4)c23)[nH]1
InChIKey: QCVMWZJIMQPVCO-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
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 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 255.32, ALogP 3.04, HBA 4, HBD 1, PSA 58.87, QED 0.68
Compound (SMILES): COC(c1ccccc1C)C1CNC1
InChIKey: MHSDMSZHDYBCRG-UHFFFAOYSA-N
Target: Sodium-dependent noradrenaline transporter (SINGLE PROTEIN), Homo sapiens
UniProt: P23975
Assay: Binding Assay: NE transporter binding assay for evaluating binding of the compound to the norepinephrine transporter was carried out using human recombinant norepinephrine transporter membrane (PerkinElmer Life and Analytical Sciences, USA) expressed in MDCK cells and radioisotope [3H]Nisoxetine (PerkinElmer).That is, the test drug, 6 nM [3H]Nisoxetine, norepinephrine transporter membrane (11 ug/well), 120 mM NaCl and 50 mM Tris-HCl buffer (pH 7.4) containing 5 mM KCl were added to obtain a reaction mixture with a final volume of 0.25 ml. After incubation for 60 minutes at 4° C., the mixture was quickly passed through a Filtermat A glass fiber filter pre-soaked with 0.5% (w/v) PEI (polyethyleneimine) using Inotech Harvester (Inotech) to terminate the reaction. After washing with cold washing buffer (50 mM Tris-HCl, pH 7.4, 0.9% NaCl) solution, the filter was covered with MeltiLex and sealed in a sample bag. After drying in an oven, radioactivity was counted using MicroBeta Plus (Wallac).
Measurement: IC50 = 1660 nM (pChEMBL 5.78000020980835)
Descriptors: MW 191.27, ALogP 1.9, HBA 2, HBD 1, PSA 21.26, QED 0.79
Compound (SMILES): CN[C@@H](C)C(=O)Nc1ccc2c(n1)CN(C1CCC(C)(C)CC1)C(=O)N2c1cccc(Cl)c1
InChIKey: HTFGTGMFHNJNHU-INIZCTEOSA-N
Target: Baculoviral IAP repeat-containing protein 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13490
Assay: In Vitro Competitive Displacement Binding Assay: Modified SMAC peptides and compounds were tested for their ability to displace the fluorescent tracer from either XIAP, cIAP-1 or cIAP-2. BIR3 domains of cIAP-1, cIAP-2 and XIAP were incubated with test compounds or SMAC based peptides and their respective peptide probes (Peptide Protein Research) in assay buffer (50 mM Hepes pH 7.5, 0.025% Tween-20, 0.01% BSA, and 1 mM DTT). Positive controls consisted of BIR3 proteins and tracer (no inhibition) and negative controls contained tracer only (100% inhibition). The samples were incubated at room temperature for 1 hr (XIAP and cIAP-2) or 3 hrs (cIAP-1) prior to being read in the BMG Pherastar in Fluorescence Polarization mode (FP 485 nm, 520 nm, 520 nm).
Measurement: IC50 = 505 nM (pChEMBL 6.300000190734863)
Descriptors: MW 470.02, ALogP 5.32, HBA 4, HBD 2, PSA 77.57, QED 0.62
Compound (SMILES): c1cc(-c2ncon2)cc(-c2c[nH]c3ncnc(N4CCOCC4)c23)c1
InChIKey: MMCZXHZCSBHBOJ-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Lantha Screen Technology Assay: LRRK2 kinase activity was measured using Lantha Screen technology from Invitrogen. GST-tagged truncated LRRK2 from Invitrogen (Cat # PV4874) was incubated with a fluorescein-labeled peptide substrate based upon ezrin/radixin/moesin (ERM), also known as LRRKtide (Invitrogen cat #PR8976A), in the presence of a dose response of compound. Upon completion, the assay was stopped and detected with a terbium labeled anti-phospho-ERM antibody (Invitrogen, cat #PR8975A). The assay was carried out under the following protocol: 3 uL of a working solution of substrate (233 nM LRRKtide, 117 uM ATP) prepared in assay buffer (50 mM HEEPES, pH 7.5, 3 mM MgCl2, with 2 mM DTT and 0.01% Brij35 added fresh) was added to a low volume Greiner 384-well plate. The compound dose response was prepared by diluting compound to a top concentration of 3.16 mM in 100% DMSO and serial diluted by half-log in DMSO 11 times. Aliquots (3.5 uL) of the 100% DMSO dose response were mixed with 46.5 uL water.
Measurement: IC50 = 475 nM (pChEMBL 6.320000171661377)
Descriptors: MW 348.37, ALogP 2.51, HBA 7, HBD 1, PSA 92.96, QED 0.61
Compound (SMILES): N#C/C(=C\N1CCC(c2nnn3cnc4[nH]ccc4c23)CC1)c1ccc(Cl)cc1
InChIKey: OIZPIQJMCWMTLP-FOWTUZBSSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
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 = 370 nM (pChEMBL 6.429999828338623)
Descriptors: MW 403.88, ALogP 4, HBA 6, HBD 1, PSA 85.9, QED 0.52
Compound (SMILES): Cc1n[nH]c(C)c1-c1cc(-c2c(C)noc2C)cc2nc(N)[nH]c12
InChIKey: PUWUOAONKKOSFC-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Homogeneous Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Binding of the two tandem bromodomains, BRD4-1 and BRD4-2, to an acetylated histone H4 peptide was measured using a homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay. 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. BRD4-1 and BRD4-2 were expressed and purified from Escherichia coli as N-terminal His6-tagged proteins. An XL665 labeled anti-His antibody (Cisbio) was used to specifically bind BRD4 and a cryptate labeled streptavidin protein was used because it specifically recognized the biotinylated H4 peptide. Binding of BRD4 to the peptide resulted in an increase in FRET signal whereas disruption of this protein-peptide interaction with a small molecule inhibitor resulted in a decrease in FRET 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 and 200 nM H4 peptide at the following concentrations for each BRD4 isoform: 60 nM BRD4-1 and 120 nM BRD4-2. After an assay reaction time of 60 minutes at 25° C., binding was measured with 2 nM crytptate labeled streptavidin and 10 nM anti-His-XL665 antibody. TR-FRET signal was detected on an Envision plate reader (Ex: 320 nm; Em: 615/665 nm; 100 As delay and 200 Î¼s read window). Data were normalized based on a positive (2 Î¼M I-BET) and negative (DMSO) controls and IC50values 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 = 96.9 nM (pChEMBL 7.010000228881836)
Descriptors: MW 322.37, ALogP 3.42, HBA 5, HBD 3, PSA 109.41, QED 0.52
Compound (SMILES): CN[C@@H](C)C(=O)N[C@@H]1C(=O)N(Cc2c(C3CC3)cnc3ccccc23)c2ccccc2N(C(C)=O)[C@H]1C
InChIKey: YNDJSCYVSRFGDS-VFUKXHBFSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay: The peptide AVPIAQKSEK-(ε-biotin)-OH 1:2 TFA ("Peptide A") was identified as a substrate for the TR-FRET assay by screening the 6× Histidine-tagged BIR2 domain and BIR3 domain of XIAP against a set of 29 peptides synthesized based on sequences reported by Sweeny et al. (Biochemistry, 2006, 45, 14740 14748). The peptides were labeled with the fluorescent tags FITC or TAMRA and Kd values were determined by fluorescence polarization assay. The sequence AVPIAQKSEK was identified as optimal for using in an assay. The peptide sequence was derivatized with biotin to provide AVPIAQKSEK-(ε-biotin)-OH 1:2 TFA as the substrate for the TR-FRET assay. Ten nanomolar of 6× Histidine-tagged BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(ε-biotin)-OH 1:2 TFA, in the presence of 50 mM Tris-C1, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/mL bovine serum albumin (BSA). Following a 45 mM incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations.
Measurement: IC50 = 18090 nM (pChEMBL 4.739999771118164)
Descriptors: MW 499.62, ALogP 3.49, HBA 5, HBD 2, PSA 94.64, QED 0.54
Compound (SMILES): COc1ccc(C(=O)Nc2ccc(F)c([C@]3(C)CS(=O)(=O)C4(CN(c5ccc([N+](=O)[O-])cc5)C4)C(=N)N3)c2)nc1
InChIKey: RMHMIIGNGBLSOY-VWLOTQADSA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: Time-Resolved Endpoint Proteolysis Assay: Inhibitor IC50s at purified human autoBACE-2 are determined in a time-resolved endpoint proteolysis assay that measures hydrolysis of the QSY7-EISEVNLDAEFC-Eu-amide FRET peptide substrate (BACE-HTRF assay). BACE-mediated hydrolysis of this peptide results in an increase in relative fluorescence (RFU) at 620 nm after excitation with 320 nm light. Inhibitor compounds, prepared at 3x the desired final concentration in 1Ã¿BACE assay buffer (20 mM sodium acetate pH 5.0, 10% glycerol, 0.1% Brij-35) supplemented with 7.5% DMSO are pre-incubated with an equal volume of autoBACE-2 enzyme diluted in 1xBACE assay buffer (final enzyme concentration 1 nM) in black 384-well NUNC plates for 30 minutes at 30° C. The assay is initiated by addition of an equal volume of the QSY7-EISEVNLDAEFC-Eu-amide substrate (200 nM final concentration, Km=8 uM for 4 uM for autoBACE-2) prepared in 1xBACE assay buffer supplemented with 7.5% DMSO and incubated for 90 minutes at 30° C. DMSO is present at 5% final concentration in the assay. Following laser excitation of sample wells at 320 nm, the fluorescence signal at 620 nm is collected for 400 ms following a 50 us delay on a RUBYstar HTRF plate reader (BMG Labtechnologies). Raw RFU data is normalized to maximum (1.0 nM BACE/DMSO) and minimum (no enzyme/DMSO) RFU values. IC50s are determined by nonlinear regression analysis (sigmoidal dose response, variable slope) of percent inhibition data with minimum and maximum values set to 0 and 100 percent respectively. Similar IC50s are obtained when using raw RFU data. The Ki values are calculated from the IC50 using the Cheng-Prusoff equation.
Measurement: Ki = 0.32 nM (pChEMBL 9.489999771118164)
Descriptors: MW 568.59, ALogP 2.86, HBA 9, HBD 3, PSA 167.62, QED 0.3
Compound (SMILES): COc1ccc2[nH]cc(C(=O)OCC34CCN(CC3)CC4)c2c1Cl
InChIKey: JGMCXSXPMCYLRN-UHFFFAOYSA-N
Target: Neuronal acetylcholine receptor subunit alpha-7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q05941
Assay: Radioligand Binding Assay: Protocol 1: The assay was conducted following the literature reference Meyer E. M., et al., Analysis of 3-(4-Hydroxy, 2-Methoxybenzylidene) Anabaseine Selectivity and Activity at Human and Rat Alpha-7 Nicotinic Receptors, J. Pharmacol. Exp. Ther., 287: 918-925, 1998. The assay was conducted using the following materials: receptor source: rat brain, radioligand: [125I] α-Bungarotoxin (2200 Ci/mmol) with a final concentration of 1 nM, non-specific determinant: Methyllycaconitine (MLA) [1 μM], reference compound: Methyllycaconitine (MLA), and positive control: Methyllycaconitine (MLA). Incubation Conditions: Reactions were carried out in 20 mM HEPES (pH 7.4) containing 120 mM NaCl, 5 mM KCl, 2.5 mM CaCl2 and 1.2 mM MgSO4 at 37° C. for 90 minutes. The reaction was terminated by rapid vacuum filtration onto glass fiber filters. Radioactivity trapped onto the filters was determined by liquid scintillation counting and compared to control values in order to ascertain any interactions of test compound with the nicotinic, ganglionic binding site.
Measurement: IC50 = 23.9 nM (pChEMBL 7.619999885559082)
Descriptors: MW 348.83, ALogP 3.47, HBA 4, HBD 1, PSA 54.56, QED 0.86
Compound (SMILES): O=C(O)c1ccc2cccc(N3CCN(CCc4ccc(OCCCN5CCCCCC5)cc4)CC3)c2n1
InChIKey: VGLVSBTUAYHUEF-UHFFFAOYSA-N
Target: Histamine H3 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5N1
Assay: Antagonist activity at human Histamine H3 receptor expressed in CHO cell membranes assessed as inhibition of histamine-induced [35S]GTPgammaS binding after 2 to 6 hrs by scintillation proximity assay
Measurement: Ki = 0.5012 nM (pChEMBL 9.300000190734863)
Descriptors: MW 516.69, ALogP 4.94, HBA 6, HBD 1, PSA 69.14, QED 0.38
Compound (SMILES): CC1Cc2c(F)cccc2N1C(=O)Cc1nc(N2CCOCC2)c(F)c(=O)[nH]1
InChIKey: NZYUXPSXEQAJKB-UHFFFAOYSA-N
Target: RAC-alpha serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P31749
Assay: Sandwich Immunoassay: This test is based on measurement of the expression of AKT protein phosphorylated on serine 473 (P-AKT-S473) in the PC3 human prostate carcinoma line, by the technique based on a sandwich immunoassay using the Kit MSD Multi-spot Biomarker Detection of Meso Scale Discovery: phospho-Akt kits (Ser473) whole cell lysate (# K151 CAD) or phospho-Akt (Ser473) / Akt whole cell lysate Total (# K15100D). The primary antibody specific for P-AKT-S473 (Kit # K151 CAD) is coated onto an electrode in each well of 96-well plates of the MSD kit: after adding a protein lysate in each well, the revelation of the signal done by the addition of a secondary antibody labeled with an electrochemiluminescent detection compound. The procedure is described in the kit.On day 1, the PC3 cells are inoculated in 96-well plates (TPP # 92096) at a concentration of 35000 cells / well in 200 uL DMEM medium (DMEM Gibco # 1 1960-044) containing 10% fetal calf serum (FBS Gibco, # 10500-056) and 1% glutamine.
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 390.39, ALogP 1.4, HBA 5, HBD 1, PSA 78.53, QED 0.86
Compound (SMILES): O=C(O)C1C2CN(C(=O)c3ccc(N4CC(F)(F)C4)c(OCC4CC4)n3)CC21
InChIKey: LOISQIXNOQGNEZ-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1×HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37 °C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30 °C. for 30 min. Compounds were added to a final assay volume of 100 μl and incubated for 30 min at 30 °C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 μl detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 393.39, ALogP 1.73, HBA 5, HBD 1, PSA 82.97, QED 0.79
Compound (SMILES): COCCNC(=O)c1cccc(Nc2nn(CC3CC3)c3c2CN(C(C)=O)CC3)c1
InChIKey: IMKSEBVXLVCTHC-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: TR-FRET Binding Assay: His/Flag epitope tagged CBP was cloned, expressed, and purified to homogeneity. CBP binding and inhibition was assessed by monitoring the engagement of a biotinylated small molecule compound with the target using the TR-FRET assay technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate CBP (4 nM final) was combined with biotin-ligand (60 nM final) in 50 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.2% DMSO) or compound dilution series in DMSO. After 10 minutes incubation at room temperature, a mixture Eu-W1024 Anti-6×His antibody (6×His disclosed as SEO ID NO: 3) (Perkin Elmer AD0110) and SureLight Allophycocyanin-Streptavidin (APC-SA, Perkin Elmer CR130-100) were added to a final concentrations of 0.2 nMolar antibody and 50 nMolar APC-SA, respectively. After twenty minutes of equilibration, the plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 3280 nM (pChEMBL 5.480000019073486)
Descriptors: MW 411.51, ALogP 2.32, HBA 6, HBD 2, PSA 88.49, QED 0.65
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)c3cc(CN4CC[C@H](F)C4)nc(C(F)(F)F)n3)CC2)C1.O=C(O)C(F)(F)F.O=C(O)C(F)(F)F.O=C(O)C(F)(F)F.O=C(O)C(F)(F)F
InChIKey: UUSPOTZYBATORE-YAVGSXOASA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Inhibition of human N-terminal His-tagged JAK2 catalytic domain (828 to 1132 residues) expressed in baculovirus infected insect cells assessed as decrease in phosphorylation of biotinylated peptide substrate after 1 hr in presence of 1 mM ATP by HTRF method
Measurement: IC50 > 10 nM (pChEMBL 8)
Descriptors: MW 637.65, ALogP 3.4, HBA 10, HBD 1, PSA 135.75, QED 0.3
Compound (SMILES): N#Cc1nc(-c2ccnc(Nc3ccc(OCCN4CC[C@H](F)C4)nc3)n2)ccc1N1CC[C@H](O)C1
InChIKey: HFHNIKCXAOFZEN-HKUYNNGSSA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: Time-Resolved Fluorescence-based Lanthascreen Assay: Kinase inhibition assays (10 μL) were performed at 20° C. in 384-well plate format. Compound IC50 values were determined at the apparent Km for ATP (20 μM) based on a radiometric assay (Invitrogen) using 8 or 10 point curves in duplicate. The final reaction conditions contained 400 nM fluorescein-IkBα substrate (DRHDSGLDSMKDE), 20 μM ATP, 2 nM or 8 nM IKKε or TBK1 kinase respectively, and 3% DMSO in kinase assay buffer consisting of 50 mM HEPES (pH 7.5), 10 mM MgCl, 1 mM EGTA, 0.01% Brij-35.Compound dilutions were prepared from 10 mM DMSO stocks by dilution into DMSO. Compound dilution series were further diluted in kinase assay buffer to give a 12% DMSO stock, the final concentration in the assay being 3% DMSO.The kinase phosphorylation assay was initiated by the addition of the kinase and the reaction was allowed to proceed for 1 h or 2.5 h for IKKε and TBK-1 kinase respectively.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 490.54, ALogP 2.54, HBA 10, HBD 2, PSA 123.32, QED 0.49
Compound (SMILES): CC(C)(C)OC(=O)c1cc(OC2CCC(NC(=O)Nc3ccc(Cl)c(C(F)(F)F)c3)CC2)ccn1
InChIKey: GFTAEBJAMHCGNW-UHFFFAOYSA-N
Target: Bifunctional epoxide hydrolase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34913
Assay: sEH Assay: See reference (Jones, P. D.; Wolf, N. M.; Morisseau, C.; Whetstone, P.; Hock, B.; Hammock, B. D. Anal. Biochem. 343:66-75; 2005) for sEH assay.
Measurement: IC50 = 0.9 nM (pChEMBL 9.050000190734863)
Descriptors: MW 513.94, ALogP 6.22, HBA 5, HBD 2, PSA 89.55, QED 0.46
Compound (SMILES): CCOc1ccc(C)nc1C(=O)N[C@H]1CCC[C@@H]1Nc1cnc(C(F)(F)F)cn1
InChIKey: QBYFBRDXXSWPGL-STQMWFEESA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Fluorescent Imaging Plate Reader (FLIPR) Assay: Orexin antagonist activity was determined by measuring changes in intracellular calcium levels using a Ca2+ sensitive fluorescent dye. The changes in fluorescent signal were monitored by Fluorescent Imaging Plate Reader (FLIPR®) technology available from Molecular Devices, LLC, U.S.A. Orexin mediated increases in intracellular Ca2+ concentration were readily detected upon activation with orexin-A. Twenty-four hours prior to the assay, RBL-2H3 cells stably expressing either human orexin receptor 1 or human orexin receptor 2 were seeded in cell culture medium in black, clear-bottom 384-well plates (commercially available from Corning Inc., U.S.A.) and grown overnight at 37 °C., 5% CO2. On the day of the assay, cell culture media was removed and cells were loaded with Calcium 5 Dye (commercially sold by Molecular Devices, LLC, U.S.A.) for 1 hour at 37 °C., 5% CO2.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 409.41, ALogP 3.36, HBA 6, HBD 2, PSA 89.03, QED 0.76
Compound (SMILES): O=C(Nc1ccc(CCC2CCCCN2)cc1)c1ccc(C(F)(F)F)cc1
InChIKey: WWSJDMMBOIQNOG-UHFFFAOYSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q923Y9
Assay: Radioligand Binding Assay: HEK-293 cells stably expressing rat TAAR1 were maintained at 37° C. and 5% CO2 in DMEM high glucose medium, containing fetal calf serum (10%, heat inactivated for 30 min at 56° C.), penicillin/streptomycin (1%), and 375 ug/ml geneticin (Gibco). Cells were released from culture flasks using trypsin/EDTA, harvested, washed twice with ice-cold PBS (without Ca2+ and Mg2+), pelleted at 1,000 rpm for 5 min at 4° C., frozen and stored at -80° C. Frozen pellets were suspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 10 mM EDTA and homogenized with a Polytron (PT 6000, Kinematica) at 14,000 rpm for 20 s. The homogenate was centrifuged at 48,000Ã¿g for 30 min at 4° C. Subsequently, the supernatant was removed and discarded, and the pellet resuspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 0.1 mM EDTA using the Polytron (20 s at 14,000 rpm). This procedure was repeated and the final pellet resuspended in HEPES-NaOH containing 0.1 mM EDTA and homogenized using the Polytron. Typically, aliquots of 2 ml membrane portions were stored at -80° C. With each new membrane batch the dissociation constant (Kd) was determined via a saturation curve. The TAAR1 radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine (described in WO 2008/098857) was used at a concentration equal to the calculated Kd value, that was usually around 2.3 nM, resulting in the binding of approximately 0.2% of the radioligand and a specific binding representing approximately 85% of the total binding. Nonspecific binding was defined as the amount of 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine bound in the presence of 10 uM unlabeled ligand. All compounds were tested at a broad range of concentrations (10 pM to 10 uM) in duplicates. The test compounds (20 ul/well) were transferred into a 96 deep well plate (TreffLab), and 180 ul of HEPES-NaOH (20 mM, pH 7.4) containing MgCl2 (10 mM) and CaCl2 (2 mM) (binding buffer), 300 ul of the radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine at a concentration of 3.3xKd in nM and 500 ul of the membranes (resuspended at 50 ug protein per ml) added. The 96 deep well plates were incubated for 1 hr at 4° C. Incubations were terminated by rapid filtration through Unifilter-96 plates (Packard Instrument Company) and glass filters GF/C (Perkin Elmer) presoaked for 1 hr in polyethylenimine (0.3%) and washed 3 times with 1 ml of cold binding buffer. After addition of 45 ul of Microscint 40 (PerkinElmer) the Unifilter-96 plate was sealed and after 1 hr the ratioactivity counted using a TopCount Microplate Scintillation Counter (Packard Instrument Company).
Measurement: Ki = 2.7 nM (pChEMBL 8.569999694824219)
Descriptors: MW 376.42, ALogP 5.03, HBA 2, HBD 2, PSA 41.13, QED 0.77
Compound (SMILES): COc1cccc2c1[C@H](C)N(C(=O)Nc1cccnc1)[C@H](C)CN2Cc1ccccc1
InChIKey: AIGFEVGWAQPOEK-MOPGFXCFSA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1H (SINGLE PROTEIN), Homo sapiens
UniProt: O95180
Assay: Channel blocking activity at recombinant human CaV 3.2 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 = 305 nM (pChEMBL 6.519999980926514)
Descriptors: MW 416.53, ALogP 5.09, HBA 4, HBD 1, PSA 57.7, QED 0.64
Compound (SMILES): C=CC(=O)Nc1ccccc1C1CCNc2c(C(N)=O)c(-c3ccc(C)c(OC)c3)nn21
InChIKey: PBPKJBZMRXWDIR-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase 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 DTT, 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.
Measurement: IC50 = 0.23 nM (pChEMBL 9.640000343322754)
Descriptors: MW 431.5, ALogP 3.5, HBA 6, HBD 3, PSA 111.27, QED 0.52
Compound (SMILES): CC(C)c1cc(CNc2nc(Nc3cc(C(C)C)[nH]n3)cc(OCCN(C)C)n2)on1
InChIKey: OJZDPBNYFYWVOT-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human IGF1R kinase domain (M954 to C1367 residues) tri-phosphorylated enzyme form expressed in baculovirus based expression system using poly-EY peptide incubated for 2 hrs by luciferase-luciferin coupled chemiluminescent assay
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 428.54, ALogP 3.73, HBA 9, HBD 3, PSA 117.02, QED 0.42
Compound (SMILES): c1ccc2c(N3CCC4(CCCO4)CC3)cncc2c1
InChIKey: WXLPPFKUYINVII-UHFFFAOYSA-N
Target: Cytochrome P450 11B1, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P15538
Assay: Inhibition of human CYP11B1-8C7 expressed in chinese hamster V79 cells using 11-deoxycortisol as substrate preincubated for 1 hr followed by substrate addition measured for 3 hrs by HTRF assay
Measurement: IC50 = 787 nM (pChEMBL 6.099999904632568)
Descriptors: MW 268.36, ALogP 3.38, HBA 3, HBD 0, PSA 25.36, QED 0.79
Compound (SMILES): Nc1nn2cccnc2c1C(=O)Nc1cn(C2CCCNCC2)nc1-c1cc(Cl)ccc1OC(F)F
InChIKey: KTYHBLBCZOBNJZ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 0.17 nM (pChEMBL 9.770000457763672)
Descriptors: MW 516.94, ALogP 4, HBA 9, HBD 3, PSA 124.39, QED 0.35
Compound (SMILES): OC(CNC[C@H]1CC[C@H](c2nnn3cnc4[nH]ccc4c23)CC1)(c1ccccc1)C(F)(F)F
InChIKey: PPWMFRBJDTVYQO-STFKQGPRSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
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 = 93 nM (pChEMBL 7.03000020980835)
Descriptors: MW 458.49, ALogP 3.92, HBA 6, HBD 3, PSA 91.13, QED 0.41
Compound (SMILES): Nc1ccccc1NC(=O)CCCCNC(=O)C1=NN=C(c2ccccc2)C1
InChIKey: WFOGZMRGGQSKIS-UHFFFAOYSA-N
Target: Histone deacetylase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O15379
Assay: HDAC enzyme inhibitionAssay: HDAC enzyme inhibition assays were performed using purified HDACs 1-10 essentially as described in Beckers et al., 2007, Int. J. Cancer., 121:1138-48 and Perez-Balado et al., 2007, J. Med. Chem., 50:2497-2505. Inhibition assays using nuclear extract were performed essentially as described in Herman et al., 2006, Nat. Chem. Biol., 2:551-558. Briefly, the purified HDACs or nuclear extract were incubated with an acetylated substrate in the absence of the compound to be assayed and with increasing concentrations of the compound. The rate of substrate deacetylation was measured under each condition, and half-maximal inhibitory concentration with regard to each HDAC was determined by standard means.
Measurement: IC50 = 1303 nM (pChEMBL 5.880000114440918)
Descriptors: MW 377.45, ALogP 2.74, HBA 5, HBD 3, PSA 108.94, QED 0.49
Compound (SMILES): CC(C)(C#N)COC(=O)N1CC[C@@H](C#N)[C@H](n2cc(C(N)=O)c(Nc3ccc(F)cc3)n2)C1
InChIKey: OITVIBANKHICLY-KBXCAEBGSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biochemical HTRF Assay: The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at ambient temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at ambient temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at ambient temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading.
Measurement: IC50 = 41 nM (pChEMBL 7.389999866485596)
Descriptors: MW 453.48, ALogP 2.94, HBA 8, HBD 2, PSA 150.06, QED 0.68
Compound (SMILES): COC[C@@H]1CCCN1c1nc2c(c(Nc3ccc(C(F)(F)F)cc3)n1)CCN(c1ncccc1C(F)(F)F)CC2
InChIKey: GNMAZHJFXALBHQ-IBGZPJMESA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: O35433
Assay: Functional Assay: The assay was performed as described below, using HEK293 cells transfected with rat TRPV1 (rTRPV1/HEK293). These cells had a geneticin selection marker and were grown in Dulbecco's Modified Eagle Medium (DMEM, Gibco BRL) supplemented with 10% fetal bovine serum, penicillin/streptomycin (50 units/mL), and 500 μg/mL geneticin in 5% CO2 at 37° C. Cells were passaged frequently, every 3-5 days, to avoid overgrowth, depletion of essential medium components, or acidic medium exposure. Cells were passaged using a brief wash in 0.05% trypsin with 1 mM EDTA, followed by dissociation in divalent-free phosphate-buffered saline (Hyclone #SH30028.02). Dissociated cells were seeded onto poly-D-lysine coated black-walled 96-well plates (Biocoat; Becton Dickinson #354640) at about 40,000 cells per well and grown for approximately 1 day in culture medium to near confluency. The assay buffer was composed of 130 mM NaCl, 2 mM KCl, 2 mM MgCl2, 10 mM HEPES, 5 mM glucose, and either 2 mM or 20 μM CaCl2. On the day of the experiment, the culture medium was replaced with 2 mM calcium assay buffer using an automated plate washer (ELx405; Biotek, VT). The cells were incubated in 100 μL/well Fluo-3/AM (2 μM; TEFLabs #0116) with Pluronic F127 (100 μg/mL; Sigma #P2443) for 1 h at rt in the dark. After loading the cells, the dye solution was replaced with 50 μL/well of 20 μM calcium assay buffer using the ELx405 plate washer. Test compounds (50 μL/well) were added to the plate and incubated for 30 min. Intracellular Ca2+ levels were subsequently assayed using a Fluorometric Imaging Plate Reader (FLIPR™ instrument, Molecular Devices, CA) to simultaneously monitor Fluo-3 fluorescence in all wells (λexcitation=488 nm, λemission=540 nm) during challenge with agonist (capsaicin). The IC50 values were determined. Cells were challenged with 150 nM capsaicin and the fluorescence counts were captured following agonist addition at a sampling rate of 0.33 Hz. The contents of the wells were mixed 3 times (40 μL mix volume) immediately after the additions were made. Concentration dependence of block was determined by exposing each well of cells in duplicate rows of a 96 well plate to a serial dilution of test compound. The concentration series usually started at 10 Î¼M with a three-fold serial decrement in concentration. The magnitude of the capsaicin response was determined by measuring the change in fluo3 fluorescence before and 100 seconds after the addition of the agonist.
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 566.55, ALogP 5.87, HBA 7, HBD 1, PSA 66.41, QED 0.37