lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCCCN(CCCC)C(=O)c1cc(C)n(-c2ccc(N[S+]([O-])c3c(-c4ccccc4)noc3C)cc2C(=O)N2Cc3ccccc3C[C@H]2CO)n1
InChIKey: CUKVCPXQZDATEX-UPHSDLEZSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Surface plasmon resonance (SPR): Methods for Determining IC50s.The present method includes utility of a Surface plasmon resonance (SPR)- based biosensor (Biacore GE Healthcare, Uppsala, Sweden) to characterize BCL-2 inhibitors.Biacore utilizes the phenomenon of surface plasmon resonance (SPR) to detect and measure binding interactions. In a typical Biacore experiment, one of the interacting molecules (ligand) is immobilized on a flexible dextran matrix while the interacting partner (analyte) is allowed to flow across that surface. A binding interaction results in an increase in mass on the sensor surface and a corresponding direct change in the refractive index of the medium in the vicinity of the sensor surface. Changes in refractive index or signal are recorded in resonance units (R.U.) Signal changes due to association and dissociation of complexes are monitored in a non-invasive manner, continuously and in real-time, the results of which are reported in the form of a sensorgram.
Measurement: IC50 = 664.26 nM (pChEMBL 6.179999828338623)
Descriptors: MW 722.91, ALogP 6.88, HBA 9, HBD 2, PSA 139.79, QED 0.12
Compound (SMILES): COC(=O)Nc1nc2cc(C3(O)c4ccccc4C(=O)N3c3cccc(C(C)C)c3)ccc2[nH]1
InChIKey: KWVFTCMYWXMQHR-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Inhibition of c-Raf1 (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 456.5, ALogP 4.72, HBA 5, HBD 3, PSA 107.55, QED 0.41
Compound (SMILES): CC[C@H](C)[C@]1(C)NCCOc2ccccc2CCCNC(=O)[C@@H](CC(C)C)NC(=O)[C@@H](C)N(C)C1=O
InChIKey: DQRYTWNZMVCUJH-VUZRLSQNSA-N
Target: Growth hormone secretagogue receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92847
Assay: Agonist activity at human GSHR1a transfected in CHOK1 cells co-expressing Galpha16 assessed as increase in Ca2+ release by aequorin assay
Measurement: EC50 = 1 nM (pChEMBL 9)
Descriptors: MW 502.7, ALogP 2.9, HBA 5, HBD 3, PSA 99.77, QED 0.59
Compound (SMILES): Nc1nnc(-c2ccc3[nH]cc(-c4ccnc(N5CCOCC5)n4)c3c2)s1
InChIKey: SNHNWCQKOINDPL-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
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 = 19.41 nM (pChEMBL 7.710000038146973)
Descriptors: MW 379.45, ALogP 2.56, HBA 8, HBD 2, PSA 105.84, QED 0.56
Compound (SMILES): O=S(=O)(NCc1ccccc1)c1ccc(Nc2ccc(Cl)cc2Cl)cc1C(F)(F)F
InChIKey: VATRHEAELYURGF-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: Binding Assay: The binding assays were performed by testing the ability of the new compounds to displace the radiolabeled synthetic non-selective cannabinoid agonist [3H]CP55940 (168 Ci/mmol; PerkinElmer) from the human CB1 (hCB1) or human CB2 (hCB2) receptors on membranes derived from stably transfected HEK-293 cells (PerkinElmer). Membranes were diluted in assay buffer (50 mM Tris-HCl, 1 mM EDTA, 1 mM MgCl2, 1 mg/ml BSA, pH=7.4). The amount of membrane was determined for each batch of membranes according to protein binding assay. The minimum amount of membrane that gave 50% specific binding was used for the binding assay. In most assays, binding was tested using 8 μg and 4 μg protein of hCB1 and hCB2 membranes, respectively. The tested compounds were dissolved in DMSO and diluted in assay buffer to a final concentration of 0.1% solvent. Total binding of [3H]CP55940 was evaluated with 1.5 nM to hCB1 and with 0.5 nM to hCB2, according to Kd affinity of [3H]CP55940 for the respective membranes. The ability of the tested compounds to displace [3H]CP55940 was evaluated first at a single concentration point of 100 nM for binding toward hCB2 or hCB1. In certain cases, the displacement was tested at compound concentrations ranging from 0.03 nM to 10 μM. Non-specific binding was measured by the addition of 1 μM of unlabelled CP55940 to the tubes. Binding assays were performed in triplicate in a total volume of 500 μl for 60 minutes at 30° C., in a shaking bath. Free and bound radioligands were separated by rapid filtration through GF/C filter plates (PerkinElmer) that had been presoaked with 0.1% Polyethylenimine (Sigma). Filters were shaken for 1 hour in 7 ml scintillation fluid (PerkinElmer) and radioactivity was determined by liquid scintillation counter (Wallac; PerkinElmer).
Measurement: IC50 = 6000 nM (pChEMBL 5.21999979019165)
Descriptors: MW 475.32, ALogP 6.23, HBA 3, HBD 2, PSA 58.2, QED 0.44
Compound (SMILES): O=C(NC1(C(F)(F)F)CC1)c1ccc(N2CC(F)(F)C2)c(OCC2CC2)n1
InChIKey: IPAVPWDHCDYXJQ-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.times.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.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul 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 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM 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.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 391.34, ALogP 3.15, HBA 4, HBD 1, PSA 54.46, QED 0.76
Compound (SMILES): CN(Cc1cccc(-c2ccncc2)c1)C(=O)OC(C(F)(F)F)C(F)(F)F
InChIKey: RBZKWMMBPOHSDF-UHFFFAOYSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: Inhibition of recombinant human full length MAGL expressed in HEK293 cells preincubated for 30 mins followed by FP-Rh addition after 30 mins by gel-based ABPP assay
Measurement: IC50 = 6.9 nM (pChEMBL 8.15999984741211)
Descriptors: MW 392.3, ALogP 4.81, HBA 3, HBD 0, PSA 42.43, QED 0.7
Compound (SMILES): CC[C@H](C)[C@H](NC(=O)[C@H](CC1CCCCC1)NC(=O)c1ccno1)C(=O)N1CCC2(CCc3ccccc3O2)CC1
InChIKey: SORNFWZMJMAMOD-ULNCZQIZSA-N
Target: Proteinase-activated receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P55085
Assay: Antagonist activity at PAR2 in human HT-29 cells assessed as inhibition of 2f-LIGRLO-NH2-stimulated intracellular calcium release preincubated for 30 mins followed by 2f-LIGRLO-NH2 stimulation measured for 60 sec by Fluo-3 AM dye based FLIPR assay
Measurement: IC50 = 1995.26 nM (pChEMBL 5.699999809265137)
Descriptors: MW 564.73, ALogP 4.66, HBA 6, HBD 2, PSA 113.77, QED 0.46
Compound (SMILES): CCOc1ncccc1-c1nnc([C@@H]2CCN2C(=O)c2cc(C)ccc2-n2nccn2)[nH]1
InChIKey: PKOKFVHTQBGJKS-SFHVURJKSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Intracellular Calcium Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 μg/mL G418, 100 U/mL penicillin, 100 μg/mL streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2. Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH:water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES for use in the assay at a final concentration of 3 nM. Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES. On the day of the assay, 50 μL of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/L, 5 mM probenecid (Sigma) and 3 μM of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at rt for 30 min before measurement. Within the Fluorescent Imaging Plate Reader (FLIPR Tetra, Molecular Devices), antagonists are added to the plate in a volume of 10 μL/well, incubated for 120 min and finally 10 μL/well of agonist is added. Fluorescence is measured for each well at 1 second intervals, and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 3 nM orexin-A with vehicle in place of antagonist.
Measurement: IC50 = 92 nM (pChEMBL 7.039999961853027)
Descriptors: MW 430.47, ALogP 2.74, HBA 8, HBD 1, PSA 114.71, QED 0.5
Compound (SMILES): Cc1c(N(C)S(=O)(=O)c2ccc(-c3ccc4c(c3)CNCC4)cc2)c(CC(C)C)nn1C
InChIKey: RSFRJVKGQDTUAK-UHFFFAOYSA-N
Target: Glycylpeptide N-tetradecanoyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30419
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 = 37 nM (pChEMBL 7.429999828338623)
Descriptors: MW 452.62, ALogP 4.06, HBA 5, HBD 1, PSA 67.23, QED 0.61
Compound (SMILES): CC(O)C(=O)N(CCN1[C@@H]2CC[C@H]1C[C@@H](c1cccc(C(N)=O)c1)C2)Cc1ccccc1.O=C(O)C(F)(F)F
InChIKey: MMRXOEQFTJHHBF-QTIXCXGXSA-N
Target: Delta-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41143
Assay: Displacement of [3H]-diprenorphine from human delta opioid receptor expressed in cell membranes
Measurement: Ki = 100 nM (pChEMBL 7)
Descriptors: MW 435.57, ALogP 2.91, HBA 4, HBD 2, PSA 86.87, QED 0.67
Compound (SMILES): O=C(NC1CC(F)(F)CC1Nc1ccc(C(F)(F)F)cn1)c1ccccc1-n1nccn1
InChIKey: KDDGILRGTBBNDE-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
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 = 633 nM (pChEMBL 6.199999809265137)
Descriptors: MW 452.39, ALogP 3.69, HBA 6, HBD 2, PSA 84.73, QED 0.58
Compound (SMILES): O=C(O)CC[C@H]1CCc2cc(-c3cc(OC(F)(F)F)cc(Cl)c3F)ccc2O1
InChIKey: RANJMKMDNWDGRM-GFCCVEGCSA-N
Target: Free fatty acid receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5NUL3
Assay: Agonist activity at human GPR120 short isoform expressed in CHOK1 cells assessed as increase in IP1 accumulation after 60 mins by HTRF assay
Measurement: EC50 = 72 nM (pChEMBL 7.139999866485596)
Descriptors: MW 418.77, ALogP 5.6, HBA 3, HBD 1, PSA 55.76, QED 0.64
Compound (SMILES): CCOc1cc(N2CCN(CCS(C)(=O)=O)CC2)c(C)cc1Nc1nccc(-c2c(-c3ccc(OC)c(C(=O)Nc4c(F)cccc4F)c3)nc3ccccn23)n1
InChIKey: PGQXZUDLYVXHJH-UHFFFAOYSA-N
Target: Insulin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P06213
Assay: Inhibition of human GST-rTEV-tagged insulin receptor (979 to 1382 residues) expressed in baculovirus infected insect Sf9 cells assessed as decrease in biotin-aminohexyl-AEEEEYMMMMAKKKK-NH2 phosphorylation after 1 hr by fluorometer
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 796.9, ALogP 6.61, HBA 12, HBD 2, PSA 143.29, QED 0.13
Compound (SMILES): O=C(O)c1c[nH]c2cc(F)c(-c3ccc(C4(O)CCC4)cc3)c(F)c12
InChIKey: VPUNMIRZXILGLE-UHFFFAOYSA-N
Target: AMP-activated protein kinase (AMPK) alpha-1/beta-1/gamma-1 (PROTEIN COMPLEX), Homo sapiens
UniProt: Q13131
Assay: Binding affinity to human BAP-tagged AMPK alpha1/beta1/gamma1 by SPR assay
Measurement: Kd = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 343.33, ALogP 4.18, HBA 2, HBD 3, PSA 73.32, QED 0.67
Compound (SMILES): Cn1ccc2cc(N(C(=O)c3cc(-c4cc5c(cc4C(=O)N4Cc6ccccc6C[C@H]4CN4CCOCC4)OCO5)n4c3CCCC4)c3ccc(O)cc3)ccc21
InChIKey: ZQDDAXHWZXNRGJ-DHUJRADRSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Mus musculus
UniProt: P10417
Assay: Fluorescence Polarisation Assay: The fluorescence polarisation tests were carried out on microplates (384 wells). The Bcl-2 protein, at a final concentration of 2.50×10−8 M, is mixed with a fluorescent peptide (Fluorescein-REIGAQLRRMADDLNAQY), at a final concentration of 1.00×10−8 M in a buffer solution (Hepes 10 mM, NaCl 150 mM, Tween20 0.05%, pH 7.4), in the presence or in the absence of increasing concentrations of test compounds. After incubation for 2 hours, the fluorescence polarisation is measured.
Measurement: IC50 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 763.89, ALogP 7.3, HBA 9, HBD 1, PSA 101.64, QED 0.18
Compound (SMILES): COC(=O)c1ccnc(N2CCC(OC(=O)N3CCN(C(C)C)CC3)CC2)c1
InChIKey: DGEOGEMCIBNLJF-UHFFFAOYSA-N
Target: Histamine H3 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5N1
Assay: Radioligand Binding Assay: The affinity of compounds of the invention to the H3 receptor can be assessed by measuring displacement of binding of the radioligand [3H]-N-Î±-Methylhistamine (PerkinElmer, #NET1027250UC) to membranes containing human H3 receptors (PerkinElmer, #ES-392-M400UA; GenBank: NM-007232.2; Hill S J et al, International Union of Pharmacology XIII. Classification of histamine receptors, Pharmacol Rev, 49(3), 253-278, 1997).Protocol: 1. Preparation of binding assay buffer (500 ml): 25 ml 1 M Tris-HCl pH 7.5 (Invitrogen, #15567-027), 2.5 ml 1 M MgCl2 (Sigma, #M1028-100ML), 472.5 ml ddH2O. 2. Compound serial dilution: Dilution was performed by BioTek Precision on compound dilution plate. Compound concentrations start at 5 or 10 uM, 10 point dose titrations with 3- or 5-fold serial dilutions. 3. Preparation of 2x membrane solution (25 ml): 1.25 ml human Histamine H3 receptor stock, 23.75 ml assay buffer. 4. Preparation of 2x solution of [3H]-N-Î±-methylhistamine (25 ml).
Measurement: Ki = 68 nM (pChEMBL 7.170000076293945)
Descriptors: MW 390.48, ALogP 2, HBA 7, HBD 0, PSA 75.21, QED 0.73
Compound (SMILES): Cc1c(-c2c(F)cc(C(N)=O)c3[nH]c4cc(CCO)ccc4c23)cccc1-n1c(=O)cc2c(F)cccn2c1=O
InChIKey: RLAPDPXPNISUNN-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Jak2 Tyrosine Kinase Assay: The assays were performed in V-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (100 mM HEPES pH 7.4, 10 mM MgCl2, 25 mM beta-glycerolphosphate, 0.015% Brij 35 surfactant and 4 mM DTT). The reaction was initiated by the combination of Jak2 tyrosine kinase with substrates and test compounds. The reaction mixture was incubated at room temperature for 60 minutes and terminated by adding 45 μL of 35 mM EDTA to each sample.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 540.53, ALogP 3.97, HBA 6, HBD 3, PSA 122.59, QED 0.31
Compound (SMILES): COc1ccc(-c2cccc(OCC(O)CN3CCc4ccccc4C3)c2)cc1Cl
InChIKey: RDAZZHZFRAMBDO-UHFFFAOYSA-N
Target: Protein arginine N-methyltransferase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: O14744
Assay: Inhibition of PRMT5 in human Z138 cells assessed as decrease in di-methyl arginine levels after 96 hrs by Western blot method
Measurement: EC50 = 10001 nM (pChEMBL 5)
Descriptors: MW 423.94, ALogP 4.81, HBA 4, HBD 1, PSA 41.93, QED 0.59
Compound (SMILES): Cn1cc(-c2ccc3nc(NC(=O)c4cncc(O)c4)cn3n2)c(-c2ccc(F)cc2)n1
InChIKey: SQQJDSGSPIIHFX-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 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 429.42, ALogP 3.29, HBA 8, HBD 2, PSA 110.23, QED 0.45
Compound (SMILES): COc1cc2c(Nc3cccc(Cl)c3C)ncnc2cc1O[C@H]1CO[C@H]2[C@@H]1OC[C@@H]2OC
InChIKey: ZTASYFQODPYQIW-FNAHDJPLSA-N
Target: Ephrin type-A receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29317
Assay: Inhibition of recombinant human N-terminal His6-tagged EphA2 (N598 to R890 residues) expressed in baculovirus using poly-EY as substrate after 180 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 457.91, ALogP 3.9, HBA 8, HBD 1, PSA 83.96, QED 0.6
Compound (SMILES): COc1cc(N2CCN(C(C)=O)CC2)ccc1Nc1ncc(Cl)c(Nc2ccc(N3CCN(C(C)=O)CC3)cc2F)n1
InChIKey: XARGOQFTRRRXGT-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Homogeneous Time-resolved fluorescence (HTRF) Assay: In order to measure the inhibitory activity against ALK, a Grainer 96-well round type bottom plate was added with the compounds (2 μl) prepared in Examples 1 to 89, and mixed with ALK enzyme (1 ul) and a peptide substrate (2 μl) with biotin attached thereto for 15 minutes and cultured thereafter. Here, ATP solution (5 μl) was added thereto and kinase reaction was performed at room temperature for 30 minutes. XL 665 (5 μl), to which Streptavidin dissolved in ethylenediaminetetraacetic acid solution was attached, anti-phosphotyrosine antibody (5 μl), to which europium (Eu3+) was attached, were added to a reaction solution to stop the reaction, cultured for 1 hour, and analyzed using Homogeneous Time-resolved fluorescence (HTRF, Cisbio). The result was read by a Wallac Envision 2103 device at the wavelength range of 615/665 nm.
Measurement: IC50 = 280 nM (pChEMBL 6.550000190734863)
Descriptors: MW 597.1, ALogP 4.1, HBA 9, HBD 2, PSA 106.17, QED 0.42
Compound (SMILES): CN[C@@H](C)C(=O)N[C@@H]1C(=O)N(Cc2c(OC)ccc3cc(Br)ccc23)c2ccccc2N(C(=O)c2ccc(C#N)cc2)[C@H]1C
InChIKey: IHQDYZPHPDSPOE-IHPVIADISA-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 = 14710 nM (pChEMBL 4.829999923706055)
Descriptors: MW 654.57, ALogP 5.16, HBA 6, HBD 2, PSA 114.77, QED 0.28
Compound (SMILES): O=C1NC(=S)S/C1=C/c1ccc(OCc2ccc(F)cc2)c(Cl)c1
InChIKey: ZVERSDWSMSUDNZ-OVCLIPMQSA-N
Target: CDGSH iron-sulfur domain-containing protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZ45
Assay: Displacement of [3H]rosiglitazone from recombinant human C-terminal His-tagged MitoNEET cytosolic domain (32 to 108 residues) expressed in Escherichia coli BL21 by Cheng-Prusoff analysis
Measurement: Ki = 40 nM (pChEMBL 7.400000095367432)
Descriptors: MW 379.87, ALogP 4.55, HBA 4, HBD 1, PSA 38.33, QED 0.62
Compound (SMILES): CN1C(=O)S/C(=C\c2cnn3c(NC4CC4)cc(Nc4cccc(Cl)c4)nc23)C1=O
InChIKey: KLCOGJOUUQJTDZ-CHHVJCJISA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Inhibition Assay: 30 uM ATP: In a final reaction volume of 50 ul, recombinant PIM-1 (1 ng) was incubated with 12 mM MOPS pH 7.0, 0.4 mM EDTA, glycerol 1%, brij 35 0.002%, 2-mercaptoethanol 0.02%, BSA 0.2 mg/ml, 100 uM KKRNRTLTK (SEQ ID NO.: 5), 10 mM MgAcetate, 30 uM ATP, [γ-33P-ATP] (specific activity approx. 500 cpm/pmol), DMSO 4% and test inhibitor compound at the required concentration. The reaction was initiated by the addition of the Magnesium ATP mixture. After 40 min incubation at 23° C., the reactions were quenched by the addition of 100 ul 0.75% Phosphoric acid, and the labeled peptide collected by filtration through a phosphocellulose filter plate. The plate was washed 4 times with 0.075% phosphoric acid (100 ul per well) and then, after the addition of scintillation fluid (20 ul per well), the counts were measured by a scintillation counter.
Measurement: IC50 > 2500 nM (pChEMBL 5.6020002365112305)
Descriptors: MW 440.92, ALogP 4.37, HBA 8, HBD 2, PSA 91.63, QED 0.57
Compound (SMILES): COc1cc(N2CCN(CCS(C)(=O)=O)CC2)ccc1Nc1nccc(-c2c(-c3ccc(OC)c(C(=O)Nc4c(F)cccc4F)c3)nc3ccccn23)n1
InChIKey: RYENXMLACXHUQY-UHFFFAOYSA-N
Target: Insulin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P06213
Assay: Inhibition of human GST-rTEV-tagged insulin receptor (979 to 1382 residues) expressed in baculovirus infected insect Sf9 cells assessed as decrease in biotin-aminohexyl-AEEEEYMMMMAKKKK-NH2 phosphorylation after 1 hr by fluorometer
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 768.85, ALogP 5.92, HBA 12, HBD 2, PSA 143.29, QED 0.15
Compound (SMILES): CNC(=O)c1ccccc1Nc1nc(Nc2ccc(N3CCOCC3)c3c2OCCO3)nc2c1CCN2
InChIKey: GMWQBALBFOYABZ-UHFFFAOYSA-N
Target: Protein-tyrosine kinase 2-beta (SINGLE PROTEIN), Homo sapiens
UniProt: Q14289
Assay: Biochemical Assay: To measure inhibitory activity of FAK/PYK2 inhibitors, compounds were first prepare as a 1 mM stock in 100% DMSO and 3-fold serial dilution was performed in a 96-well plate (Corning, 3897) to generate 12 different concentration of 100× stock. A 5 μl of 100× stock of each concentration was added to wells containing 95 μl of 1× reaction buffer (40 mM Tris, pH7.5, mM MgCl2, 1 mM DTT and 1 mM CHAPS) to generate 5× stock. Then 2 μl of 5× stock of each concentration was added to a 384 wells-OptiPlate (PerkinElmer, 6007299). For FAK, 4 μl of 2.5 nM FAK, 1 μl of 8 μg/μl BSA and 1.5 μl of 666 nM ULight-poly Glu, Ala, Tyr (1:1:1) peptide substrate, prepared in above reaction buffer, were added to each well. The reaction was initiated by adding 1.5 μl of 33.3 μM ATP. The reaction was allowed to proceed for 120 min before being quenched with 5 μl of 40 mM EDTA stop buffer prepared in 1×LANCE detection buffer (PerkinElmer, CR97-100). For PYK2, 5 μl of 2 nM PYK2 and 1.5 μl of 666 nM ULight-poly Glu, Ala, Tyr (1:1:1) peptide substrate, prepared in above reaction buffer, were added to each well. The reaction was initiated by adding 1.5 μl of 46.6 μM ATP. The reaction was allowed to proceed for 40 min before being quenched with 5 μl of 40 mM EDTA stop buffer prepared in 1×LANCE detection buffer (PerkinElmer, CR97-100). Upon quenching of the reaction, 5 μl of 8 nM anti-phosphotyrosine antibody was added to each well and incubated for 60 minutes.
Measurement: IC50 < 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 503.56, ALogP 2.9, HBA 10, HBD 4, PSA 121.9, QED 0.4
Compound (SMILES): CC(C)C[C@H](NC(=O)c1cnc(C2CC2)c(OCC2CC2)n1)C(=O)N(C)C
InChIKey: DZRUYZCURLYRHS-HNNXBMFYSA-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 10s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 374.49, ALogP 2.38, HBA 5, HBD 1, PSA 84.42, QED 0.72
Compound (SMILES): COc1cc(OCCF)cc(N(CCO)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: POHLKOAYIRHFFP-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in presence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 437.48, ALogP 3.52, HBA 8, HBD 1, PSA 85.53, QED 0.43
Compound (SMILES): CCN1CCC2N=C(COc3cc4ncnc(Nc5ccc(Cl)c(Cl)c5)c4cc3OC)SC2C1
InChIKey: JJHKGTMOROVPCK-UHFFFAOYSA-N
Target: Ephrin type-A receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29317
Assay: Inhibition of recombinant human N-terminal His6-tagged EphA2 (N598 to R890 residues) expressed in baculovirus using poly-EY as substrate after 180 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 518.47, ALogP 5.68, HBA 8, HBD 1, PSA 71.87, QED 0.43
Compound (SMILES): COc1cc(OC)cc(N(CCN2CCC[C@H]2C(=O)O)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1.Cl
InChIKey: MPNAFJKVNZCMBJ-SNYZSRNZSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in presence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 502.58, ALogP 3.73, HBA 9, HBD 1, PSA 105.84, QED 0.37
Compound (SMILES): Cl.Cl.c1ccc2c(c1)CCN(CCCc1nc3ccccc3[nH]1)C2
InChIKey: VKHTZIMQLQAZHP-UHFFFAOYSA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Displacement of [3H]-N-methylspiperone from human dopamine D3 receptor by liquid scintillation counting method
Measurement: Ki = 2176 nM (pChEMBL 5.659999847412109)
Descriptors: MW 291.4, ALogP 3.55, HBA 2, HBD 1, PSA 31.92, QED 0.8
Compound (SMILES): CN(C)c1nc(Cl)nc2c1ncn2[C@H]1[C@H](O)[C@H](O)[C@]2(CO)C[C@H]12
InChIKey: BSBLYPMZOLQTAO-RUVJYUCVSA-N
Target: 5-hydroxytryptamine receptor 2B (SINGLE PROTEIN), Homo sapiens
UniProt: P41595
Assay: Displacement of [3H]lysergic from human 5-HT2BR expressed in HEK cell membranes incubated in dark for 90 mins by microbeta scintillation counting method
Measurement: Ki = 4800 nM (pChEMBL 5.320000171661377)
Descriptors: MW 339.78, ALogP -0.18, HBA 8, HBD 3, PSA 107.53, QED 0.67
Compound (SMILES): O=C(Nc1ccc(C2CCNC2)cc1)c1cnc(Cl)cn1
InChIKey: KBECWDSFYCOIGS-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 = 12.1 nM (pChEMBL 7.920000076293945)
Descriptors: MW 302.77, ALogP 2.46, HBA 4, HBD 2, PSA 66.91, QED 0.91
Compound (SMILES): Cc1nc(C(C)(C)NC(=O)c2ccc(N3CC(=O)C3)c(OCC3CC3)n2)no1
InChIKey: GLMWYKNAHRMHIF-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.times.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.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul 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 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM 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.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 385.42, ALogP 1.62, HBA 8, HBD 1, PSA 110.45, QED 0.77
Compound (SMILES): COc1cc(NC(=N)NC(=N)N2CCN(C(=N)NC(=N)Nc3cc(OC)c(OC)c(OC)c3)CC2)cc(OC)c1OC
InChIKey: HJXLFJWQLBRGLE-UHFFFAOYSA-N
Target: Acetylcholinesterase (SINGLE PROTEIN), Electrophorus electricus
UniProt: O42275
Assay: Non-competitive inhibition of Electrophorus electricus acetylcholinesterase in presence of varying acetylthiocholine iodide substrate level preincubated for 20 mins followed by substrate addition measured every 1 min for 30 mins
Measurement: Ki = 41210 nM (pChEMBL 4.380000114440918)
Descriptors: MW 586.65, ALogP 1.8, HBA 10, HBD 8, PSA 205.38, QED 0.16
Compound (SMILES): CCN(CC)CCNc1cc(Nc2cc(C3CC3)[nH]n2)nc(NCc2cc(C(C)C)no2)n1
InChIKey: HYLOXIBTLUPZHP-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 453.6, ALogP 4.3, HBA 9, HBD 4, PSA 119.82, QED 0.3
Compound (SMILES): CS(=O)(=O)Nc1ccc2c3c(ccc2c1)O[C@H](c1cc(F)c(F)cc1F)[C@@H](N)C3
InChIKey: WVIFQSHXWACPEI-AZUAARDMSA-N
Target: Dipeptidyl peptidase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P27487
Assay: Inhibition of DPP4 (unknown origin) expressed in Sf9 cells using Gly-Pro-AMC substrate
Measurement: IC50 = 8.91 nM (pChEMBL 8.050000190734863)
Descriptors: MW 422.43, ALogP 3.63, HBA 4, HBD 2, PSA 81.42, QED 0.63
Compound (SMILES): Cc1c(C(=O)Nc2cccc(C(F)(F)F)c2)cc(C(C)(C)C)n1CC(F)(F)CN1CCOCC1
InChIKey: ZIHIAFOGWPAXRL-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBA0
Assay: Inhibition Assay: Test Example 1: (1) The TRPV4 inhibitory activity was measured using CHO-K1 cells stably expressing human TRPV4 (hTRPV4/CHO cells). (2) In the day before the experimental day, frozen cell were thawed and washed with the culture medium (MEM-α, 10% FBS, 2 mmol/L GlutaMax, 50 unit Penicillin, 50 μg/mL Streptomycin). Then cells were suspended in the culture medium. (3) The 384-well plates which hTRPV4/CHO cells were seeded at densities of 4000 cells/well in the culture medium and cultured in a CO2 incubator in the presence of 5% CO2 at 37° C. for overnight was used as assay plates. (4) The assay plate was washed with assay buffer (Hanks, 20 mmol/L HEPES, 2.5 mmol/L probenecid, pH7.4), and the buffer were remained at 20 μL/well. (5) 10 μl of dye loading buffer (9 μmol/L Fluo 4-AM, 0.09% Pluronic F-127/assay buffer) was added to each well, then the assay plate was incubated in a CO2 incubator in the presence of 5% CO2 at 37° C. for one hour. (final 3 μmol/L Fluo4-AM). (6) The assay plate was washed with the assay buffer, and the buffers were remained at 20 μL/well. Then the assay plate was incubated for 10 min at room temperature. (7) Diluted compound solution was dispensed to each well in the compound plate, and mixed with built-in Pipette and Mixer of the Fluorescence analysis system FLIPR TETRA (Molecular devices). (8) After incubation for 5 min, 20 μL of 4α-PDD solution were applied to each well of assay plate and mixed with the FLIPR TETRA. (final 1 Î¼mol/L 4α-PDD). (9) The fluorescent intensity was measured with FLIPR TETRA system for 10 min from the point of time addition the compound solution, at Ex 470-495 nm, Em 515-575 nm wavelength.
Measurement: IC50 = 780 nM (pChEMBL 6.110000133514404)
Descriptors: MW 487.51, ALogP 5.33, HBA 4, HBD 1, PSA 46.5, QED 0.56
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccc(S(=O)(=O)C(F)F)cc3)n2)CCC(N2CCC(O)CC2)CC1
InChIKey: SEPBKVAHZCODHG-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: HTRF 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 uL of 1.11x enzyme and 1.11x substrate in 1x 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 room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x 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.
Measurement: IC50 = 37 nM (pChEMBL 7.429999828338623)
Descriptors: MW 536.61, ALogP 2.73, HBA 9, HBD 3, PSA 154.34, QED 0.47
Compound (SMILES): Cn1c(SCCCN2CC[C@]3(C[C@@H]3c3ccc(C(F)(F)F)cc3)C2)nnc1-c1ccc(S(N)(=O)=O)cc1
InChIKey: HZISGJVOPZSKRZ-QPPBQGQZSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Displacement of [3H]-spiperone from human dopamine D2 receptor expressed in CHO-K1 cell membranes coexpressing Galpha16 after 120 mins by liquid scintillation counting
Measurement: Ki = 218.78 nM (pChEMBL 6.659999847412109)
Descriptors: MW 551.66, ALogP 4.51, HBA 7, HBD 1, PSA 94.11, QED 0.33
Compound (SMILES): COCCOc1cc2ncnc(Nc3ccc4c(cnn4Cc4ccccc4)c3)c2cc1NC(=O)/C=C/CN(C)C
InChIKey: CNRMVFRKCDVXKW-JXMROGBWSA-N
Target: Receptor tyrosine-protein kinase erbB-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P04626
Assay: Irreversible inhibition of GST-tagged ERBB2 (unknown origin) (Ile-675 to Val-1256 residues) expressed in baculovirus infected Sf9 insect cells assessed as reduction in Glu/Tyr copolymer phosphorylation after 6 mins by ELISA
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 551.65, ALogP 4.85, HBA 9, HBD 2, PSA 106.43, QED 0.17
Compound (SMILES): CCN1CCC(c2nc(COc3cc4ncnc(Nc5ccc(Cl)c(Cl)c5)c4cc3OC)no2)CC1
InChIKey: QWTRRGLWVSSIML-UHFFFAOYSA-N
Target: Ephrin type-A receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29317
Assay: Inhibition of recombinant human N-terminal His6-tagged EphA2 (N598 to R890 residues) expressed in baculovirus using poly-EY as substrate after 180 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 529.43, ALogP 5.85, HBA 9, HBD 1, PSA 98.43, QED 0.3
Compound (SMILES): COc1cc(OC)cc(N(CC(C)NCC(F)(F)F)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: IQQVSAGBFNJDMB-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in presence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 500.53, ALogP 4.73, HBA 8, HBD 1, PSA 77.33, QED 0.36
Compound (SMILES): CN(c1ccc(S(=O)(=O)N2CCCC2)cn1)[C@@H]1CCN(c2ncnc3[nH]ccc23)C1
InChIKey: WULROJCEUFDOMA-OAHLLOKOSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzymatic Assay: JAK1/2/3 kinase assay are performed in vitro using Kit-Tyr 6 Peptide (Invitrogen, Cat. No. PV4122). TYK2 kinase assay are performed in vitro using Z′-LYTE Kinase Assay Kit-Tyr 3 Peptide (Invitrogen, Cat. No. PV3192). Recombinant human JAK1/2/3 or TYK2 catalytic domains are from Invitrogen (Cat No. PV4774/PV4210/PV3855/PV4790); All reactions (20 μL) are started by adding 2.5 μL of the testing compound in 4% DMSO solution, 5 μL of Kinase/Peptide substrate Mixture (3.2, 0.04, 0.2 or 8 μg/mL for Recombinant human JAK1/2/3 catalytic domains, 4 μM for Z-LYTE Tyr 6 peptide or Z-LYTE Tyr 3 peptide) or Phospho-Peptide solution (Invitrogen, Cat. No. PV3192, diluted with 1.33× Kinase Buffer), 2.5 μL ATP Solution (300/100/40/100 μM, JAK1/JAK2/JAK3/TYK2) or 1.33× Kinase Buffer (Invitrogen, Cat. No. PV3189, 5× diluted with distilled water). The 384-well assay plate (Corning, Cat. No. 3575) is mixed and incubated at room temperature for 1 hour. 5 μL of the Development Solution (Dilute Development Reagent A (Cat. No. PV3297) is diluted to 1/64 with Development Buffer (Cat. No. PV3127) for JAK1, JAK2 and JAK3 assay; Development Reagent A (Cat. No. PV3297) is diluted to 1/2048 with Development Buffer (Cat. No. PV3127) for TYK2 assay. The diluted Development Solution is then added to each well, mixed and incubated at room temperature for another 1 hour. The kinase reaction is then stopped by adding 5 μL of the Stop Reagent (Invitrogen, Cat. No. PV3094), and the plate is read with Wallac 1420 VICTOR3 Multilabel Counter (PerkinElmer) at 445 nm and 520 nm fluorescence. All compounds are initially tested at 8 different concentrations (1 μM down to 0.0003 μM) using a 1:3 serial dilution scheme.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 427.53, ALogP 1.85, HBA 7, HBD 1, PSA 98.32, QED 0.66
Compound (SMILES): Cc1c(-c2ccc(F)cc2)nnc(N2CCN(c3cnc(C(C)(C)O)cn3)[C@H](C)C2)c1C
InChIKey: LOCCROWNKPBTNX-OAHLLOKOSA-N
Target: Protein smoothened (SINGLE PROTEIN), Mus musculus
UniProt: P56726
Assay: Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 436.54, ALogP 3.63, HBA 7, HBD 1, PSA 78.27, QED 0.67
Compound (SMILES): O=C(Nc1ccc(-c2n[nH]c(=O)c3cccc(F)c23)cc1)N1Cc2ccccc2C1
InChIKey: QJQSUURRBCTTST-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: ROCK2 Kinase Activity Assays: HTRF assay: This assay used the CisBio HTRF KinEASE kit (kit 62ST2PEZ) and the kinase reaction containing 0.2 uM biotinylated substrate peptide (S2, CisBio), 5 uM, 100 uM or 1 mM of ATP, inhibitors (0.1-10,000 nM in 2% DMSO) and enzymes as in the 33P-ATP assay below. [33P]-ATP assay: ROCK2 activity was initially determined using a radioactive FlashPlate-based assay. In a 96-well format, biotinylated peptide substrates (2 mM final; S6-short for ROCK2), γ-[33P]-ATP (5 uM, 2 mCi/mmol), compounds (0.1-10000 nM in 2% DMSO), and ROCK2 (0.2 nM; Upstate) catalytic domains.
Measurement: Ki = 2.37 nM (pChEMBL 8.619999885559082)
Descriptors: MW 400.41, ALogP 4.28, HBA 3, HBD 2, PSA 78.09, QED 0.53
Compound (SMILES): N#C[C@@H]1CCN(C[C@H]2CC[C@H](c3nnn4cnc5[nH]ccc5c34)CC2)C1
InChIKey: NXMXNRRGEASZTB-KKUMJFAQSA-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 = 960 nM (pChEMBL 6.019999980926514)
Descriptors: MW 349.44, ALogP 2.72, HBA 6, HBD 1, PSA 85.9, QED 0.79