lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): C[C@@H](Cc1cccc(N)c1)NC[C@@H](O)c1cc(O)cc(O)c1
InChIKey: YSDUJIGQJOLYND-APPDUMDISA-N
Target: Beta-2 adrenergic receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P07550
Assay: Beta1-AR Binding Assay: Beta1-AR binding was done on rat cortical membrane following a previously described procedure (Beer et al., Biochem. Pharmacol. 37: 1145-1151, 1988). In brief, male Sprague-Dawley rats weighing 250-350 g were decapitated and their brains quickly removed. The cerebral cortices were dissected on ice, weighed and promptly transferred to a 50 ml test tube containing approximately 30 ml of 50 mM Tris-HCl, pH 7.8 (at room temperature). The tissues were homogenized with a polytron and centrifuged at 20,000xg for 12 min at 4° C. The pellet was washed again in the same manner and resuspended at a concentration of 20 mg (original wet wt) per 1 ml in the assay buffer (20 mM Tris-HCl, 10 mM MgCl2, 1 mM EDTA, 0.1 mM ascorbic acid at pH 7.8). To block the beta2 sites present in the cortical membrane preparation, 30 nM ICI 118-551 was also added to the assay buffer. To wells containing 100 ul of the test drug and 100 ul of [3H]CGP-12177 (1.4 nM final concentration), 0.8 ml of tissue homogenate was added. After 2 hours at 25° C., the incubation was terminated by rapid filtration. Nonspecific binding was determined by 10 uM propranolol.
Measurement: Ki = 28749 nM (pChEMBL 4.539999961853027)
Descriptors: MW 302.37, ALogP 1.93, HBA 5, HBD 5, PSA 98.74, QED 0.53
Compound (SMILES): Cc1cc(Nc2cc(C3CC3)[nH]n2)nc(NCc2cc(C(C)C)no2)n1
InChIKey: PXDGNXYOKGCMEM-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 353.43, ALogP 3.85, HBA 7, HBD 3, PSA 104.55, QED 0.59
Compound (SMILES): CC1CN(C(=O)c2cc(Cc3n[nH]c(=O)c4ccccc34)ccc2F)Cc2nnc(C(F)F)n21
InChIKey: KLEMFAOWDQXMRO-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: Cellular Assay: Based on the preliminary PARP1 inhibition evaluation of compounds at molecular level by ELISA, compounds were further evaluated for their cellular inhibition against PARP1 using a proliferation inhibition model.
Measurement: IC50 = 24.77 nM (pChEMBL 7.610000133514404)
Descriptors: MW 468.44, ALogP 3.4, HBA 6, HBD 1, PSA 96.77, QED 0.5
Compound (SMILES): Cc1cc(Nc2cc(C)c(C#N)c(NCCOc3ccccc3F)n2)n[nH]1
InChIKey: AWLBOIUGNGQJSU-UHFFFAOYSA-N
Target: Cytochrome P450 1A2 (SINGLE PROTEIN), Homo sapiens
UniProt: P05177
Assay: Inhibition of CYP1A2 (unknown origin)
Measurement: IC50 < 400 nM (pChEMBL 6.3979997634887695)
Descriptors: MW 366.4, ALogP 3.67, HBA 6, HBD 3, PSA 98.65, QED 0.55
Compound (SMILES): Cc1ncc(C)n2nc(CCc3ccc4ccc(F)cc4n3)nc12
InChIKey: VOASYVQFJORMOW-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Inhibition Assay: A PDE10A assay may for example, be performed as follows: The assay is performed in 60 uL samples containing a fixed amount of the relevant PDE enzyme (sufficient to convert 20-25% of the cyclic nucleotide substrate), a buffer (50 mM HEPES7.6; 10 mM MgCl2; 0.02% Tween20), 0.1 mg/ml BSA, 225 pCi of 3H-labelled cyclic nucleotide substrate, tritium labeled cAMP to a final concentration of 5 nM and varying amounts of inhibitors. Reactions are initiated by addition of the cyclic nucleotide substrate, and reactions are allowed to proceed for one hr at room temperature before being terminated through mixing with 15 uL 8 mg/mL yttrium silicate SPA beads (Amersham). The beads are allowed to settle for one hr in the dark before the plates are counted in a Wallac 1450 Microbeta counter. The measured signal can be converted to activity relative to an uninhibited control (100%) and IC50 values can be calculated using the Xlfit extension to EXCEL.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 321.36, ALogP 3.21, HBA 5, HBD 0, PSA 55.97, QED 0.58
Compound (SMILES): C[C@@H](CC(=O)O)NC(=O)c1nc(C#N)c2cc(Oc3ccccc3)ccc2c1O
InChIKey: CTADYWAQKGRMMW-LBPRGKRZSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZT9
Assay: HIF-PH Assay: Ketoglutaric acid α-[1-14C]-sodium salt, alpha-ketoglutaric acid sodium salt, and HPLC purified peptide may be obtained from commercial sources, e.g., Perkin-Elmer (Wellesley Mass.), Sigma-Aldrich, and SynPep Corp. (Dublin Calif.), respectively. Peptides for use in the assay may be fragments of HIFα as described above or as disclosed in International Publication WO 2005/118836, incorporated by reference herein. HIF-pH, e.g., PHD1 (EGLN2) or PHD2 (EGLN1), can be expressed in, e.g., insect Hi5 cells, and partially purified, e.g., through a SP ion exchange chromatography column. Enzyme activity is determined by capturing 14CO2 using an assay described by Kivirikko and Myllyla (1982, Methods Enzymol. 82:245-304). Assay reactions contain 50 mM HEPES (pH 7.4), 100 μM α-ketoglutaric acid sodium salt, 0.30 mCi/mL ketoglutaric acid α-[1-14C]-sodium salt, 40 μM FeSO4, 1 mM ascorbate, 1541.8 units/mL Catalase, with or without 50 μM peptide substrate and various concentrations of compound of the invention. Reactions are initiated by addition of HIF-PH enzyme.
Measurement: IC50 = 96750 nM (pChEMBL 4.010000228881836)
Descriptors: MW 391.38, ALogP 3.2, HBA 6, HBD 3, PSA 132.54, QED 0.59
Compound (SMILES): O=C(Nc1ccncc1)N1CCN(Cc2ccc3c(c2)OC(F)(F)O3)CC1
InChIKey: NOLNVTCPLFOTPX-UHFFFAOYSA-N
Target: Fatty-acid amide hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O00519
Assay: FAAH Assay: T84 frozen cell pellets or transfected SK-N-MC cells (contents of 1x15 cm culture dishes) were homogenized in 50 mL of FAAH assay buffer (125 mM Tris, 1 mM EDTA, 0.2% Glycerol, 0.02% Triton X-100, 0.4 mM Hepes, pH 9). The assay mixture consisted of 50 uL of the cell homogenate, 10 uL of the test compound, and 40 uL of anandamide [1-3H-ethanolamine] (3H-AEA, Perkin-Elmer, 10.3 Ci/mmol), which was added last, for a final tracer concentration of 80 nM. The reaction mixture was incubated at rt for 1 h. During the incubation, 96-well Multiscreen filter plates (catalog number MAFCNOB50; Millipore, Bedford, Mass., USA) were loaded with 25 uL of activated charcoal (Multiscreen column loader, catalog number MACL09625, Millipore) and washed once with 100 uL of MeOH. Also during the incubation, 96-well DYNEX MicroLite plates (catalog number NL510410) were loaded with 100 uL of MicroScint40 (catalog number 6013641, Packard Bioscience, Meriden, Conn., USA).
Measurement: IC50 = 2000 nM (pChEMBL 5.699999809265137)
Descriptors: MW 376.36, ALogP 2.75, HBA 5, HBD 1, PSA 66.93, QED 0.89
Compound (SMILES): N#Cc1cc(-c2ccnc(Nc3ccc(CN4CCOCC4)nc3)n2)cnc1N1CCC(OC(F)F)C1
InChIKey: ZSJKDURWVGXFJO-UHFFFAOYSA-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 508.53, ALogP 3.2, HBA 10, HBD 1, PSA 112.32, QED 0.49
Compound (SMILES): COc1cc2ncccc2cc1NC(=O)c1csc2cnc(NC3CC3)nc12
InChIKey: JFKRMJRQGVELFG-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P51617
Assay: In vitro IRAK-1 and IRAK-4 Assay: Purified recombinant IRAK-4 protein was incubated with 250 uM synthetic peptide(KKARFSRFAGSSPSQSSMVAR) in 30 ul of kinase buffer including (20 mM MOPS pH7.2, 25 mM beta glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM DTT, 50 uM ATP, 20 mM MgCl2, 10 uCi gamma-33P, 0.1% BSA) for the indicated time. For purified recombinant IRAK-1 protein kinase assay, 50 uM ATP was used. A 25 ul aliquot of the reaction mixture was transferred on to p81 phosphocellulose squares (Upstate Biotechnology, Lake Placid, N.Y.). The assay squares were washed three times with 0.75% phosphoric acid and once with acetone. Enzyme activity was measured by determining the bound radioactivity by liquid scintillation counting.
Measurement: IC50 = 575 nM (pChEMBL 6.239999771118164)
Descriptors: MW 391.46, ALogP 4.07, HBA 7, HBD 2, PSA 89.03, QED 0.53
Compound (SMILES): c1ccc(-c2cc(CNc3nccc(Nc4cc(C5CCCC5)n[nH]4)n3)on2)cc1
InChIKey: CMAQGLFHNAGBSI-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 7.300000190734863)
Descriptors: MW 401.47, ALogP 4.87, HBA 7, HBD 3, PSA 104.55, QED 0.41
Compound (SMILES): CC(=O)N[C@H]1CCN(C[C@H]2CC[C@H](c3nnn4cnc5[nH]ccc5c34)CC2)C1
InChIKey: VAMYQOUPUFFEEZ-JYJNAYRXSA-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 = 230 nM (pChEMBL 6.639999866485596)
Descriptors: MW 381.48, ALogP 2.09, HBA 6, HBD 2, PSA 91.21, QED 0.72
Compound (SMILES): Cc1ccc2c(c1)N(Cc1ccccc1)C[C@@H](C)N(C(=O)Nc1cccnc1)[C@H]2C
InChIKey: SHBCBFDPLIFTBC-UXHICEINSA-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 = 231 nM (pChEMBL 6.639999866485596)
Descriptors: MW 400.53, ALogP 5.39, HBA 3, HBD 1, PSA 48.47, QED 0.64
Compound (SMILES): CCc1cc(CNc2nc(Nc3cc(C4CC4)n[nH]3)cc(OCCN(C)C)n2)on1
InChIKey: JAMDHEBMJGXSJP-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 412.5, ALogP 2.92, HBA 9, HBD 3, PSA 117.02, QED 0.44
Compound (SMILES): Cc1ncc(C(CNC(=O)c2cccc(F)c2Cl)N2CCCC(F)(F)C2)cn1
InChIKey: FVXYBJRQYPAMFI-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Fluorescent Imaging Plate Reader (FLIPR) Assay: Briefly, 293-human or mouse P2X7 stable cells were incubated in sucrose buffer, pH 7.4 [KCl (5 mM), NaH2PO42H2O (9.6 mM), HEPES (25 mM), sucrose (280 mM), glucose (5 mM), CaCl2 (0.5 mM), and probenecid (0.1425 g in 3 mL 1N NaOH was added for 500 mL solution)] in 384-well plates. 93-rat P2X7 stable cells were incubated in HHPB (pH 7.4) [consisting of Hank's BSS (1X); HEPES (pH 7.4) (20 mM) (Sigma); probenecid (0.710 g/5 mL 1N NaOH) (Sigma); and BSA (0.05%) (Roche) which was added after the pH had been adjusted] in 384-well plates. Fluo-4 NW dye mix (Molecular Probes, Inc., Eugene, Oreg., USA) was prepared in buffer (see manufacturer's instructions). Cell plates were removed from the 37° C. incubator, the media discarded and then 30 μL of dye was added to each well. Plates were placed in the 37° C., non-CO2 incubator for 30 minutes and then room temperature for 30 minutes. Two sets of drug plates were prepared: A) Mixtures of compound plus agonist were prepared as follows, in order to determine close response: BzATP: 11 point ½ log, diluted in buffer, starting from 1 mM. Testing compounds: 11 point ½ log, diluted in 2% DMSO buffer starting from 10 μM. B) Agonist only mixture was prepared with BzATP at a single concentration in buffer (concentration determined by dose response). Compound mixtures (A) were added to assay plates containing cells and placed at room temperature for 30 minutes, then BzATP (B) was added. Fluorescence was read using the Tetra FLIPR® (Molecular Devices, Inc., Sunnyvale, Calif., USA) and IC50 values were calculated by standard methods to determine antagonist activity.
Measurement: IC50 = 41 nM (pChEMBL 7.389999866485596)
Descriptors: MW 412.84, ALogP 3.78, HBA 4, HBD 1, PSA 58.12, QED 0.81
Compound (SMILES): Cc1cnc(Nc2ccc(N3CCOCC3)cc2)nc1-c1cc2cc(COCC(F)(F)F)ccc2o1
InChIKey: CTCWNBMNJSPICY-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Inhibition of GST-tagged recombinant human cytoplasmic JAK3 expressed in baculovirus using TK-substrate-biotin as substrate preincubated for 5 mins followed by substrate addition after 30 mins by HTRF assay
Measurement: IC50 = 836.5 nM (pChEMBL 6.079999923706055)
Descriptors: MW 498.51, ALogP 5.86, HBA 7, HBD 1, PSA 72.65, QED 0.34
Compound (SMILES): N#Cc1cnc2c(Nc3ccc(F)c([C@]4(CF)C[C@@H](C(F)(F)F)OC(N)=N4)c3)ncc(F)c2c1
InChIKey: LWXHQLQQXIEDPY-OXJNMPFZSA-N
Target: Cathepsin D (SINGLE PROTEIN), Homo sapiens
UniProt: P07339
Assay: In Vitro Enzymatic Cathepsin D FRET Assay: Recombinant Cat D was expressed in CHO cells. The assay buffer for CathepsinD is 0.05 M citrate pH 3.5, 10% DMSO final, 5 mM CHAPS. The Cat D enzyme (9 nM) is pre-incubated for one hour with inhibitors, typically in about 1 uL of DMSO according to a serial dilution, is added thereto. The assays are effectively started by the addition of different FRET substrates (20 nM for Cat D) and the combination is incubated for one hour. The FRET assay is terminated with by addition of Tris buffer, which raises the pH to neutrality, and the fluorescence is determined. The FRET substrate is a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. The Cat D substrate peptide sequence is based on sequence #1 of Table 1 from Gulnik et al. FEBS Letters v413 p 379-384 1997. Proteolytic cleavage of the FRET substrate releases quenching of fluorescence (Cat D excitation 500 nm and emission 580 nm).
Measurement: IC50 > 44000 nM (pChEMBL 4.35699987411499)
Descriptors: MW 480.37, ALogP 4.35, HBA 7, HBD 2, PSA 109.21, QED 0.54
Compound (SMILES): Br.CCOc1cc2c(c(F)c1OCC)C(=N)N(CC(=O)c1cc(N3CCC(O)CC3)c(OC)c(C(C)(C)C)c1)C2
InChIKey: FXZOBSNOHJFGBM-UHFFFAOYSA-N
Target: Proteinase-activated receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P25116
Assay: Displacement of [3H] Ala-(4fluoro)Phe-Arg-(cy- clohexyl)Ala-(homo)Arg-Tyr-NH2 from thrombin receptor in human platelet membranes after 1 hr by liquid scintillation counting method
Measurement: IC50 = 29 nM (pChEMBL 7.539999961853027)
Descriptors: MW 541.66, ALogP 4.91, HBA 7, HBD 2, PSA 95.32, QED 0.44
Compound (SMILES): Cc1ccccc1-n1c(-c2cc(C(=O)N(C)Cc3ccccc3)c(O)cc2O)n[nH]c1=O
InChIKey: XOWIZECDQINJIB-UHFFFAOYSA-N
Target: Heat shock protein HSP 90-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P07900
Assay: Protein was prepared by dialysis for 24 hours against 1 liter of 30 mM Hepes pH 7.5, 150mM NaCl and 0.5mM TCEP. The final protein concentration was determined by Bradford Assay. The final protein concentration in the sample cell was 50 uM. Ligand stock solutions of 10 mM in DMSO were diluted to 5uM concentrations with ITC buffer and adjusted to 2% (v/v) DMSO. Both the titrate and titrant solutions were degassed prior to loading the calorimeter cell and injection syringe.The integrated heat data were fit with a one-site binding model using the Origin-7 software provided with the MicroCal VP-ITC. Protein concentration was corrected by titration of a reference compound and normalized for the concentration of each ligand. All ligands bound to N-HSP90 have a stoichiometry of approximately unity.
Measurement: Kd = 12.14 nM (pChEMBL 7.920000076293945)
Descriptors: MW 430.46, ALogP 3.22, HBA 6, HBD 3, PSA 111.45, QED 0.45
Compound (SMILES): CCOc1ccc(C(=O)C2CCN(CC(=O)NCc3nc4c(c(=O)[nH]3)COCC4)CC2)cc1
InChIKey: DBNHRWOXIIAGGS-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase tankyrase-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H2K2
Assay: Biochemical Assay: The autoparsylation activity of the TNKS 1/2 or PARP1/2 enzymes was measured by the liquid chromatography-mass spectrometry (LC/MS) detection of nicotinamide as readout. Compound activity in inhibiting the TNKS and PARP autoparsylation was evaluated by IC50 measurements. In the compound screening assays, the reaction is composed of 5 μL of compound in 8-point serial dilutions with concentrations ranging from 0.0086 to 18.75 μM, 20 nM of purified enzyme, and 250 μM of β-NAD+ in the 1× Assay Buffer. After 60 min incubation at room temperature, the reactions were quenched by the addition of 10 μL of 5× quenching solution (20% formic acid and 500 nM [d]-nicotinamide in water). For the background control wells, 10 μL of the 5× quenching solution per well was added prior to the addition of β-NAD+.
Measurement: IC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 454.53, ALogP 1.45, HBA 7, HBD 2, PSA 113.62, QED 0.58
Compound (SMILES): O=C(CC1CCCC1)Nc1ccc(NC(=O)N2Cc3ccc(Br)cc3C2)cc1
InChIKey: RAQCFPXHODXSTQ-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 2:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1287128.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 3.61 nM (pChEMBL 8.4399995803833)
Descriptors: MW 442.36, ALogP 5.52, HBA 2, HBD 2, PSA 61.44, QED 0.66
Compound (SMILES): CN1CCN(c2c(-c3ccc4nc(NC(=O)c5ccnc(F)c5)cn4n3)c(-c3ccc(F)cc3)nn2C)CC1
InChIKey: GZKXLOLAQZXUSN-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 = 0.7 nM (pChEMBL 9.149999618530273)
Descriptors: MW 529.56, ALogP 3.47, HBA 9, HBD 1, PSA 96.48, QED 0.35
Compound (SMILES): O=C1COc2ccc(C3(O)c4ccccc4C(=O)N3c3ccccc3Cl)cc2N1
InChIKey: RUKPVCRQZBPQLI-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition of B-Raf (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 406.83, ALogP 3.52, HBA 4, HBD 2, PSA 78.87, QED 0.68
Compound (SMILES): Cc1c(-c2c(Cl)cc(C(N)=O)c3[nH]c4cc(C(C)(C)O)ccc4c23)cccc1-n1c(=O)c2cccc(C)c2n(C)c1=O
InChIKey: SMULHNDEQVVOCA-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 = 350 nM (pChEMBL 6.460000038146973)
Descriptors: MW 581.07, ALogP 5.59, HBA 6, HBD 3, PSA 123.11, QED 0.25
Compound (SMILES): COc1cc(OC)c(F)c(N(CCO)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1F
InChIKey: JCQODDIJVHORMY-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 441.44, ALogP 3.46, HBA 8, HBD 1, PSA 85.53, QED 0.47
Compound (SMILES): CN(C)CCCNc1ccc(S(=O)(=O)NC(=O)c2ccc(N3CCN(Cc4ccccc4-c4ccc(Cl)cc4)CC3)cc2Oc2ccccc2)cc1C#N
InChIKey: HTYMZBPCAZNWAL-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q64373
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The inhibition constant (Ki) is the dissociation constant of an enzyme-inhibitor complex or a protein/small molecule complex, wherein the small molecule is inhibiting binding of one protein to another protein or peptide. Where the Ki for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-XL) is greater than the limits of detection of the assay used. Where the binding selectivity ratio for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the selectivity of a particular compound for Bcl-2 over Bcl-XL is at least as great as the number indicated. Where the Ki for a compound is represented as < (less than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-2) is lower than the limit of detection of the assay used. Inhibition constants were determined using Wang's equation (Wang Z-X).
Measurement: Ki = 283.84 nM (pChEMBL 6.550000190734863)
Descriptors: MW 763.36, ALogP 7.48, HBA 9, HBD 2, PSA 118.01, QED 0.11
Compound (SMILES): Cc1ccn2c(C(=O)Nc3cc(-c4noc(C5CC(F)(F)C5)n4)ccc3C)cnc2c1
InChIKey: PZQWOZMFFXZLHP-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Cell Proliferation Assay: The compounds was tested for inhibition of SCF dependent proliferation using human Mo7e cells which endogenously express c-kit in a 384 well format. Three-fold serially diluted test compounds (Cmax=10 mM) were evaluated for their antiproliferative activity of Mo7e cells stimulated with human recombinant SCF. After 48 hours of incubation at 37° C., cell viability was measured by adding 25 uL of CellTiter Glo (Promega) to the cells and the luminescence was measured by a CLIPR CCD camera (Molecular Devices).
Measurement: IC50 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 423.42, ALogP 4.77, HBA 6, HBD 1, PSA 85.32, QED 0.51
Compound (SMILES): O=C(Cn1c2c(c(-c3cccc(Cl)c3)cc1=O)C(=O)NC2)Nc1ccc(-c2nnn[nH]2)cc1
InChIKey: BUVUMAPNDQFBAF-UHFFFAOYSA-N
Target: Prothrombin (SINGLE PROTEIN), Homo sapiens
UniProt: P00734
Assay: Inhibition of human plasma thrombin using Boc-Asp(OBzl)-Pro-Arg-AMC as substrate measured for 30 mins by fluorometric assay
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 461.87, ALogP 2.23, HBA 7, HBD 3, PSA 134.66, QED 0.42
Compound (SMILES): CC1Cc2c(-c3ccnn3C)nn(C)c2CN1C(=O)c1cccc(C(F)(F)F)c1Cl
InChIKey: WNKFRMPTORTTKZ-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 = 12.7 nM (pChEMBL 7.900000095367432)
Descriptors: MW 437.85, ALogP 4.08, HBA 5, HBD 0, PSA 55.95, QED 0.61
Compound (SMILES): CC1(C)CCC(CN2CCN(c3ccc(C(=O)NS(=O)(=O)c4cccc(C(=O)NCC5CCOCC5)c4)c(Oc4ccc5[nH]ccc5c4)c3)CC2)=C(c2ccc(Cl)cc2)C1
InChIKey: HFDOAPBMMXPZDJ-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q64373
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The inhibition constant (Ki) is the dissociation constant of an enzyme-inhibitor complex or a protein/small molecule complex, wherein the small molecule is inhibiting binding of one protein to another protein or peptide. Where the Ki for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-XL) is greater than the limits of detection of the assay used. Where the binding selectivity ratio for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the selectivity of a particular compound for Bcl-2 over Bcl-XL is at least as great as the number indicated. Where the Ki for a compound is represented as < (less than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-2) is lower than the limit of detection of the assay used. Inhibition constants were determined using Wang's equation (Wang Z-X).
Measurement: Ki > 660 nM (pChEMBL 6.179999828338623)
Descriptors: MW 850.48, ALogP 8.67, HBA 8, HBD 3, PSA 133.07, QED 0.11
Compound (SMILES): COc1cccc(OC)c1C1CCC(=O)N1Cc1cccc(-n2cccn2)c1
InChIKey: ZJDAEGIIHPTLQD-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: In Vitro Assay: Intracellular Calcium Measurements : In Vitro Assay: Intracellular Calcium Measurements: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 g/ml G418, 100 U/ml penicillin, 100 g/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 M 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.
Measurement: IC50 = 839 nM (pChEMBL 6.079999923706055)
Descriptors: MW 377.44, ALogP 3.75, HBA 5, HBD 0, PSA 56.59, QED 0.66
Compound (SMILES): O=C(O)c1ccc(CN(CCC2CC2)C(=O)c2ccc(Oc3c(F)cccc3Cl)cc2)cc1
InChIKey: FPIPBUXXFQRFKI-UHFFFAOYSA-N
Target: Lysophosphatidic acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92633
Assay: In Vitro LPA1 Receptor Assay: Engagement of the LPA1 receptor by its ligand, oleoyl-L-α-lysophosphatidic acid (LPA), leads to the release of intracellular stores of calcium into the cytoplasm mediated through a Gq-driven increase in inositol triphosphate (IP3) levels. Gq is a heterotrimeric G protein subunit that activates phospholipase C.The ability of test compounds to prevent LPA-driven intracellular release of stored calcium from RH7777 cells expressing human LPA1 receptors was assayed by stimulating the cells with LPA in the presence of various test compounds to measure the test compounds' antagonism, i.e. activity in vitro. Approximately 10,000 cells in normal culture medium (Dulbecco's Modified Eagle Media, 10% Foetal Calf Serum) were dispensed per assay well in a black clear bottom collagen coated 384 well plate (Beckton Dickinson). Cells were allowed to settle for thirty minutes at room temperature before being incubated overnight at 37° C., 5% CO2. Sixteen to twenty hours after dispensing, the cells were loaded with a calcium-sensitive fluorescent dye by replacing the culture medium with assay buffer (1× Hanks buffered saline, 25 mM HEPES, 0.1% w/v fatty acid free BSA (bovine serum albumin), pH7.4) containing 1.25 mM probenecid and 1× Calcium 5 Reagent (Molecular Devices). Cells were incubated at 37° C. for one hour to allow for dye uptake. To test for antagonist activity, test compounds at a final concentration range between 0.32 nM-10 μM (diluted in assay buffer) were added to the assay wells and allowed to incubate for twenty five minutes. After incubation with test compounds the assay plate was placed in a FLIPR Tetra system (Molecular Devices) and LPA (diluted in assay buffer) was added to give a final concentration equivalent to its determined EC80 against LPA1 receptor. Ligand-dependent changes in intracellular calcium levels were determined by measuring changes in fluorescence of the dye over 515-575 nM following excitation over 470-495 nM.
Measurement: IC50 = 58 nM (pChEMBL 7.239999771118164)
Descriptors: MW 467.92, ALogP 6.41, HBA 3, HBD 1, PSA 66.84, QED 0.39
Compound (SMILES): CC1(C)CCC(CN2CCN(c3ccc(C(=O)NS(=O)(=O)c4ccc(NCC5CCOCC5)c(S(=O)(=O)C(F)(F)F)c4)c(Oc4ccc5[nH]ccc5c4)c3)CC2)=C(c2ccc(Cl)cc2)C1
InChIKey: QCQAZPIRDQPYEX-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q64373
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The inhibition constant (Ki) is the dissociation constant of an enzyme-inhibitor complex or a protein/small molecule complex, wherein the small molecule is inhibiting binding of one protein to another protein or peptide. Where the Ki for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-XL) is greater than the limits of detection of the assay used. Where the binding selectivity ratio for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the selectivity of a particular compound for Bcl-2 over Bcl-XL is at least as great as the number indicated. Where the Ki for a compound is represented as < (less than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-2) is lower than the limit of detection of the assay used. Inhibition constants were determined using Wang's equation (Wang Z-X).
Measurement: Ki > 660 nM (pChEMBL 6.179999828338623)
Descriptors: MW 954.53, ALogP 9.65, HBA 10, HBD 3, PSA 150.14, QED 0.1
Compound (SMILES): CCOc1ccc(F)c(F)c1[C@@H]1C[C@H](F)C(=O)N1Cc1ccc(OC(F)(F)F)cc1
InChIKey: HFVRPEZRBSJJJI-GJZGRUSLSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: In Vitro Assay: Intracellular Calcium Measurements : In Vitro Assay: Intracellular Calcium Measurements: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 g/ml G418, 100 U/ml penicillin, 100 g/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 M 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.
Measurement: IC50 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 433.35, ALogP 5.07, HBA 3, HBD 0, PSA 38.77, QED 0.6
Compound (SMILES): CC1C(=O)Nc2ccccc2N1C(=O)c1cccc(-c2ccccc2)c1
InChIKey: KFKFNYAICGALSE-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Inhibition of human BRD4 BD2 (342 to 460 residues) at 30 uM preincubated for 30 mins followed by substrate addition measured after 30 mins by TR-FRET assay
Measurement: IC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 342.4, ALogP 4.34, HBA 2, HBD 1, PSA 49.41, QED 0.75
Compound (SMILES): CC(C(=O)Nc1ccc(C2CCNC2)cc1)c1cccc(OC(F)(F)F)c1
InChIKey: WVSFHQCMHLKFOQ-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 = 4.6 nM (pChEMBL 8.34000015258789)
Descriptors: MW 378.39, ALogP 4.4, HBA 3, HBD 2, PSA 50.36, QED 0.81
Compound (SMILES): CSCC[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc1cnc[nH]1)NC(=O)CNC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@@H](N)Cc1c[nH]c2ccccc12)C(C)C)C(N)=O
InChIKey: FDJOMAIZVIIHGU-XLJSZMODSA-N
Target: Neuromedin-B receptor (SINGLE PROTEIN), Rattus norvegicus
UniProt: P24053
Assay: Displacement of [125I]-NMB from NMB receptor in rat C6 cells
Measurement: IC50 > 800 nM (pChEMBL 6.0970001220703125)
Descriptors: MW 812.01, ALogP -0.5, HBA 10, HBD 10, PSA 288.18, QED 0.06
Compound (SMILES): CN1CCN(c2ccc(-c3ccc4[nH]c5nccc(/C=C/c6cccc(F)c6)c5c4c3)cc2)CC1
InChIKey: OYUSBNCAAKVRRW-AATRIKPKSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: ELISA-Based In Vitro Kinase Assay: GST-tagged recombinant wild-type or L1196M mutated ALK kinase (rALK) was expressed in Sf9 insect cells using the pBacPAK baculovirus vector system (Clontech) and purified using Glutathione Sepharose 4B affinity beads (GE Healthcare). Recombinant 3C protease was used to remove the GST tag. Purified ALK was used to screen inhibitors in the ELISA-based kinase assay, as follows: Nunc-Immuno 96-well plates were incubated overnight at 30° C. with coating solution containing 2 μg of a specific ALK peptide substrate (ARDIYRASFFRKGGCAMLPVK) in PBS. Wells were then washed with 200 μL of wash buffer (PBS-Tween 0.05%) and incubated with 4% BSA in PBS for at least 2 h at 30° C. The kinase reaction was performed in the presence of 50 mM Tris pH 7.5, 5 mM MnCl2, 5 mM MgCl2, 0.3 mM ATP and purified rALK in a total volume of 100 μL/well at 30° C. for 15 min. For inhibitor testing the reaction mix was preincubated with inhibitor or vehicle for 10 min at room temperature before transferring to the ELISA plate. After the reaction, the wells were washed 5 times with 200 μL of wash buffer. Phosphorylated peptide was detected using 100 μL/well of a mouse monoclonal anti-phosphotyrosine antibody (clone 4G10 UpstateBiotech Ltd) diluted 1:2000 in PBS+4% BSA. After 30 min incubation at room temperature the antibody was removed and wells were washed as described above. 100 μL of a secondary antibody (anti-mouse IgG, Horseradish Peroxidase linked whole antibody, Amersham Pharmacia Biotech) diluted 1:1000 in PBS+4% BSA was added to each well and the plate was incubated again for 30 min at room temperature before washing as above. The plate was developed using 100 μL/well TMB Substrate Solution (Pierce) and the reaction was stopped by adding an equal volume of 1M H2SO4. Finally, the absorbance was read at 450 nm using an ELISA plate reader (Bio-Rad).
Measurement: IC50 = 320 nM (pChEMBL 6.5)
Descriptors: MW 462.57, ALogP 6.44, HBA 3, HBD 1, PSA 35.16, QED 0.33
Compound (SMILES): CC1(C)CCC(CN2CCN(c3ccc(C(=O)NS(=O)(=O)c4ccc(NCC5CCOCC5)c(C#N)c4)c(Oc4cccc5[nH]ccc45)c3)CC2)=C(c2ccc(Cl)cc2)C1
InChIKey: QNRMTVLRUDENQS-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q64373
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The inhibition constant (Ki) is the dissociation constant of an enzyme-inhibitor complex or a protein/small molecule complex, wherein the small molecule is inhibiting binding of one protein to another protein or peptide. Where the Ki for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-XL) is greater than the limits of detection of the assay used. Where the binding selectivity ratio for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the selectivity of a particular compound for Bcl-2 over Bcl-XL is at least as great as the number indicated. Where the Ki for a compound is represented as < (less than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-2) is lower than the limit of detection of the assay used. Inhibition constants were determined using Wang's equation (Wang Z-X).
Measurement: Ki > 660 nM (pChEMBL 6.179999828338623)
Descriptors: MW 847.48, ALogP 9.23, HBA 9, HBD 3, PSA 139.79, QED 0.11
Compound (SMILES): Cn1cc(-c2ccc3nc(NC(=O)c4ccccc4)c(C#N)n3n2)c(-c2ccc(F)cc2)n1
InChIKey: KTBHATQLILJZGE-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 437.44, ALogP 4.06, HBA 7, HBD 1, PSA 100.9, QED 0.46
Compound (SMILES): CN(C)C(=O)c1ccc(Nc2nn(C3(CC#N)CCN(C(=O)OC(C)(C)C)CC3)cc2C(N)=O)cc1
InChIKey: BWPZULCTUUHPIG-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
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 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 495.58, ALogP 3.07, HBA 8, HBD 2, PSA 146.58, QED 0.63
Compound (SMILES): O=C1NCCc2[nH]c(-c3cccc4ccc(OC5CCNCC5)nc34)cc21
InChIKey: UOOCRVJWAMBPLA-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Homogeneous Time Resolved Fluorescence Assay (HTRF): 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 μM. A reference compound was included on each assay plate in order to validate that plate; on one plate of every assay run, two additional reference compounds were included. The final buffer conditions were as follows: 60 mM Hepes, pH 7.0, 0.05% BSA, 2 mM DTT. Incubations were carried out at RT (22° C.) for 2 h for Pim-1, 1 hour and 30 min for Pim-3, and 45 min for Pim-2. The reaction was stopped by the addition of 3 mM EDTA, and fluorescence was measured by an HTRF® Rubystar microplate reader.
Measurement: IC50 = 95.6 nM (pChEMBL 7.019999980926514)
Descriptors: MW 362.43, ALogP 2.65, HBA 4, HBD 3, PSA 79.04, QED 0.67