lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1ncc(NC(=O)c2cccc(C(F)(F)F)c2)cc1-c1cc(N2CCOCC2=O)nc(N2CCOCC2)n1
InChIKey: PTVOALIUHBHIKV-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: In Vitro Enzyme Assay: Final assay conditions were 4 pM cRaf, 3 uM ATP, 10 nM biotin tagged MEK1 kinase dead protein substrate. Reactions were performed in Greiner384 well plates, catalog #784075. Reaction buffer was 50 mM Tris, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 0.01% Tween-20, 0.05% BSA and 1 mM DTT. 5 ul of 2×cRaf plus MEK1 were dispensed into plates containing 0.25 ul of compound in 100% DMSO and incubated at room temperature for 30 minutes. Reactions were started by addition of 5 ul of 2×ATP. Reactions were run for two hours and terminated by addition of 10 ul of 2× antibody and Alphascreen bead mixture in Stop Buffer. Stop Buffer was 50 mM Tris, pH 7.5, 0.005% Tween-20, 25 mM EDTA.
Measurement: IC50 = 2.996 nM (pChEMBL 8.520000457763672)
Descriptors: MW 542.52, ALogP 3.32, HBA 8, HBD 1, PSA 109.78, QED 0.52
Compound (SMILES): CCCC(=O)N(Cc1ccccc1)C(C(=O)NC1CCCCC1)c1c(C(=O)OCC)n(C(=O)OC(C)(C)C)c2cc(Cl)ccc12
InChIKey: WAAULTIISNKQIU-UHFFFAOYSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: Fluorescence Polarization (FP) Assay: Fluorescent polarization experiments were performed as follows. Briefly, the fluorescence polarization experiments were read on an Ultra Evolution 384-well plate reader (Tecan) with the 485 nm excitation and 535 nm emission filters. The fluorescence intensities parallel (Intparallel) and perpendicular (Intperpedicular) to the plane of excitation were measured in parallel perpendicular black 384-well NBS assay plates (Corning) at room temperature (20° C). The background fluorescence intensities of blank samples containing the references buffer were subtracted and steady-state fluorescence polarization was calculated using the equation: P=(Intparallel Gintperpendicular)/(Intparallel+Gintperpendicular), and the correction factor G (G=0.998 determined empirically) was introduced to eliminate differences in the transmission of vertically and horizontally polarized light. All fluorescence polarization values were expressed in millipolarization units (mP).
Measurement: Ki = 2.5 nM (pChEMBL 8.600000381469727)
Descriptors: MW 638.21, ALogP 7.57, HBA 7, HBD 1, PSA 106.94, QED 0.23
Compound (SMILES): O=C(Nc1cccc(Cl)c1)N1CCN(Cc2ccc(Br)c(Br)c2)CC1
InChIKey: QTKUCHXTYSOIAW-UHFFFAOYSA-N
Target: Fatty-acid amide hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O00519
Assay: Biological Assay: Assay Method 1: T84 frozen pellets (contents of 1-4x15 cm culture dishes) were homogenized in 300 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 was prepared from 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 200 nM. The reaction mixture was incubated at rt for 1 hour (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 = 240 nM (pChEMBL 6.619999885559082)
Descriptors: MW 487.62, ALogP 5.21, HBA 2, HBD 1, PSA 35.58, QED 0.64
Compound (SMILES): O=C1CN(C(=O)CNc2ccc(S(=O)(=O)NC(=O)NC3CCCCC3)cc2)c2ccccc2N1
InChIKey: NPSGOHATJRNPHR-UHFFFAOYSA-N
Target: Peroxisome proliferator-activated receptor gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P37231
Assay: Displacement of fluormone-PPARgamma Green from recombinant human N-terminal GST-tagged PPARgamma-LBD by fluorescence polarization assay
Measurement: IC50 = 1141 nM (pChEMBL 5.940000057220459)
Descriptors: MW 485.57, ALogP 2.4, HBA 6, HBD 4, PSA 136.71, QED 0.5
Compound (SMILES): Nc1cnccc1CNC(=O)c1cc(NC(=O)c2cc(-c3ccccn3)c(F)cc2Cl)n(-c2ccccc2)n1
InChIKey: FDXXSKLTTLBVQS-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Cell Based Assay: TRKA co-expressed with p75: The assays are based upon DiscoveRx's proprietary Enzyme Fragment Complementation (EFC) technology. In the case of the TRK cell lines, the enzyme acceptor (EA) protein is fused to a SH2 protein and the TRK receptor of interest has been tagged with a Prolink tag. Cryopreserved PathHunter cells are used from either in-house produced batches or bulk batches bought directly from DiscoveRx. Cryopreserved cells are resuscitated, spun 1000 rpm for 4 min to remove freezing media, and resuspended in MEM+0.5% horse serum (both Invitrogen) to 5e^5 cells/ml. The cells are then plated using a Multidrop into Greiner white tissue culture treated plates at 20 μl/well and incubated for 24 h at 37 °C., 5% CO2, high humidity. On the day of the assay, the cell plates are allowed to cool to room temperature for 30 min prior to the assay. 4 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using the compound of Example 135, WO2005/116035 at a top concentration of 150 μM is also prepared on each test plate. High percentage effect (HPE) is defined by 150 μM of the compound of Example 135, WO2005/116035 and 0% effect (ZPE) is defined by 100% DMSO. Plates containing 1 μl of serially diluted compound, standard and HPE/ZPE are diluted 1/66 in assay buffer (PBS minus Ca2+, minus Mg2+ with 0.05% pluronic F127) using a Wellmate. Using a Platemate Plus, 5 μl of 1/66 diluted test compounds is then transferred to the cell plate and allowed to reach equilibrium by incubating for 30 min at room temperature before addition of agonist stimulus: 10 μl/well of 2 nM (0.571 nM FAC) of the cognate neurotrophin (Peprotech) diluted in agonist buffer (HBSS with 0.25% BSA). Final assay concentration of the test compounds is 8.66 μM, (the compound of Example 135, WO2005/116035 FAC is 0.325 μM). The plates are left at room temperature for a further 2 hours before addition of 10 μl of the DiscoveRx PathHunter detection reagent (made up by adding 1 part Galacton Star, 5 parts Emerald II and 19 parts Cell Assay Buffer as per the manufacturer's instructions). After reagent addition, plates are covered and incubated at room temperature for 60 minutes. Luminescence signal is read using an Envision.
Measurement: IC50 = 11.8 nM (pChEMBL 7.929999828338623)
Descriptors: MW 541.97, ALogP 4.89, HBA 7, HBD 3, PSA 127.82, QED 0.27
Compound (SMILES): Cc1cc(NC(=O)Nc2cc(OC(C)C)cc(C(F)(F)F)c2)cnc1Oc1ccnc(NC(=O)C2CC2)c1
InChIKey: WHCYNEVUNVSFSN-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition Assay: [Experimental method]: Use Lance@Ultra Ulight™-TK assay kit from PerKinElmer to evaluate the inhibitory activity of compounds at KDR kinase. [Instrument]: PerKinElmer's ENVISION plate reader. [Materials]: Optiplate-384 well plate (PerKinElmer), kinase buffer (50 mM Hepes pH7.5, 0.25 mM EGTA, 2 mM DTT, 0.01% Tween 20, 10 mM Mg2+, 0.5 mM Mn2+), KDR kinase (790-1356 AA, Crown Bioscience), KDR kinase substrate (PerKinElmer catalogue # TRF0127-M), Lance@Eu-W1024-anti-phosphotyrosine (PT66) (PerKinElmer, catalogue #AD0068), ATP (Invitrogen), DMSO (Sigma, catalogue #34869), purified water (Millipore, type: Milli-Q). [Study conditions]: Mix KDR (final concentration: 20 nM) and compound (final DMSO: 0.5%), pre-incubate for 20 min at 30° C.; then add ATP (final concentration: 90 μM) and the substrate (final concentration 50 μM). React for 2 hrs at 30° C. After the reaction, add antibody and reaction for 60 min at 30° C. Read the plate (615 nm, 665 nm), calculate the ratio of the value at 665 nm vs 615 nm and analyze the data.
Measurement: IC50 = 30.2 nM (pChEMBL 7.519999980926514)
Descriptors: MW 529.52, ALogP 6.38, HBA 6, HBD 3, PSA 114.47, QED 0.31
Compound (SMILES): COC(=O)NCCCc1cc([C@H](C)N(C(=O)[C@H]2CNCCO2)C2CC2)cnc1OC
InChIKey: IZAPORQANWABNM-KBXCAEBGSA-N
Target: Renin (SINGLE PROTEIN), Homo sapiens
UniProt: P00797
Assay: Inhibitory Assay: A substrate of synthetic peptide (Nma-KHPFHLVIHK(Dnp)-NH2) and test compound were mixed, and fluorescence intensity was assayed using a fluorophotometer before staring an enzymatic reaction (exciting wavelength: 340 nm, measuring wavelength: 460 nm). Recombinant human renin was added and the mixture was incubated at 37° C. for 1 hour, and the fluorescence intensity was measured after the reaction using a fluorophotometer (exciting wavelength: 340 nm, measuring wavelength: 460 nm). Renin activity was evaluated on the ground of fluorescence intensity which was obtained by deduction of the intensity before the reaction from the intensity after the reaction, and 50% inhibitory concentration (IC50) was calculated from renin activities under the existence of various concentration of the test compound.
Measurement: IC50 = 6.7 nM (pChEMBL 8.170000076293945)
Descriptors: MW 420.51, ALogP 1.42, HBA 7, HBD 2, PSA 102.02, QED 0.58
Compound (SMILES): COC(=O)Cc1nnc(N[C@@H](Cc2ccc(NS(=O)(=O)O)cc2)c2csc(-c3cccs3)n2)s1
InChIKey: FKTZFMQOSAFOBX-AWEZNQCLSA-N
Target: Receptor-type tyrosine-protein phosphatase beta (SINGLE PROTEIN), Homo sapiens
UniProt: P23467
Assay: Inhibition of recombinant HPTPbeta (unknown origin) preincubated for 10 mins followed by substrate addition measured after 15 mins by DiFMUP probe-based fluorescence assay
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 537.67, ALogP 4.05, HBA 11, HBD 3, PSA 143.4, QED 0.2
Compound (SMILES): O=C(Nc1ccc(C(F)(F)F)cc1)N1CCC(F)(c2ncc([C@@H](O)CO)cc2F)CC1
InChIKey: PTRZACAJEQTXEI-INIZCTEOSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: Capsaicin-Based Assay: Two days prior to performing this assay, cells are seeded in poly-D-lysine-coated 96-well clear-bottom black plates (50,000 cells/well) in growth media containing 5 μM PonA (commercially available from Invitrogen) to induce expression of TRPV1. On the day of the assay, the plates are washed with 0.2 mL 1× Hank's Balanced Salt Solution (commercially available from Life Technologies) containing 1 mM CaCl2 and 20 mM HEPES, pH 7.4, and cells are loaded using 0.1 mL of wash buffer containing Fluo-4 (3 μM final). After one hour, the cells are washed twice with 0.2 mL of wash buffer and resuspended in 0.1 mL of wash buffer. The plates are transferred to a FLIPR for assay. 50 μL of test compound diluted with assay buffer (1× Hank's Balanced Salt Solution containing 1 mM CaCl2 and 20 mM HEPES, pH 7.4) are added to the cell plates and incubated for 2 min. The final concentration of the compound is adjusted to range from about 50 picoM to about 3 μM. Human TRPV1 is activated by the addition of 50 μL of capsaicin (400 nM), and the plates are incubated for an additional 3 min.
Measurement: IC50 = 38.4 nM (pChEMBL 7.420000076293945)
Descriptors: MW 445.39, ALogP 3.76, HBA 4, HBD 3, PSA 85.69, QED 0.63
Compound (SMILES): COc1cc2c(Nc3cc(Cl)c(Cl)cc3F)ncnc2cc1OC[C@H]1CN2CCOC[C@H]2CO1
InChIKey: KHSJGHHCLKKKKU-UONOGXRCSA-N
Target: Ephrin type-B receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P54760
Assay: Inhibition of recombinant human N-terminal His6-tagged EphB4 kinase domain (E605 to E890 residues) expressed in baculovirus using poly-AEKY as substrate after 150 mins in presence of gamma33P-ATP by liquid scintillation spectrometry
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 509.37, ALogP 4.31, HBA 8, HBD 1, PSA 77.97, QED 0.49
Compound (SMILES): CCNc1nc(-n2cc(-c3ccncn3)cn2)nc(NCc2nc3ccccc3[nH]2)c1Br
InChIKey: IGUPBCSERVLCJN-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: [3H] cAMP SPA Assay: The analysis of the activity of the enzyme Phosphodiesterase [3H] cAMP SPA (Perkin Elmer #7090 TRKQ) kit was used for the study of Phosphodiesterase 10. The kit contains:Yttrium scintillation proximity assay (SPA) in beads: yttrium silicate in microspheres resuspended pellet in MiliQ water containing 18 mM zinc sulfate.    10×PDE assay buffer: 500 mM Tris/HCl; 83 mM MgCl2; 17 mM EGTA; pH 7.5.    Tracer [3H] cAMP: substrate for enzymatic reaction.The test is based on the preferential binding of linear nucleotides to the SPA beads in the presence of zinc sulfate, with respect to the binding of cyclic nucleotides, which is almost imperceptible. Therefore, in optimum conditions, the product of the enzymatic reaction binds directly to SPA, and the substrate of the enzyme does not.
Measurement: IC50 = 29.3 nM (pChEMBL 7.53000020980835)
Descriptors: MW 491.36, ALogP 3.8, HBA 9, HBD 3, PSA 122.12, QED 0.31
Compound (SMILES): CN(c1ccc(C(=O)N[C@@H](CO)c2ccccc2)cc1F)[C@@H]1CCN(c2ncnc3[nH]ccc23)C1
InChIKey: ATKQNWIMTWIHAX-KNQAVFIVSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
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 (300/100/40/100M, 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 = 0.055 nM (pChEMBL 10.260000228881836)
Descriptors: MW 474.54, ALogP 3.28, HBA 6, HBD 3, PSA 97.38, QED 0.38
Compound (SMILES): COc1cc(N2CCN(C(C)=O)CC2)ccc1Nc1ncc(F)c(Nc2ccc(N3CCN(C)CC3)cc2OC)n1
InChIKey: NXBJPBANKLPBQD-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 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 564.67, ALogP 3.54, HBA 10, HBD 2, PSA 98.33, QED 0.42
Compound (SMILES): N#Cc1ccc(-c2cccc(Nc3nccc(C(F)(F)F)n3)c2)cc1
InChIKey: BLHOSORLVFUFCN-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Homogeneous Time-Resolved Fluorescence (HTRF) Assay: A recombinant GST-hSYK fusion protein was used to measure potency of compounds to inhibit human Syk activity. The recombinant human GST-SYK (Carna Biosciences #08-176) (5 μM final concentration) was incubated with various concentrations of the inhibitor diluted in DMSO (0.1% final concentration) for 10 minutes at room temperature in 15 mM Tris-HCl (pH 7.5), 0.01% tween 20, 2 mM DTT in 384 well plate format. To initiate the reaction the biotinylated substrate peptide (250 nM final concentration) that contains the phosphorylation site for Syk was added with magnesium (5 mM final concentration) and ATP (25 μM final concentration). Final volume of the reaction was 10 μL. Phosphorylation of the peptide was allowed to proceed for 45' at room temperature. To quench the reaction and detect the phosphorylated product, 2 nM of a Europium-anti-phosphotyrosine antibody (Perkin Elmer #AD0161) and 70 nM SA-APC (Perkin-Elmer #CR130-100) were added together in 15 mM Tris pH 7.5, 40 mM EDTA, 0.01% tween 20. Final volume of the quenching solution was 10 μL. The resulting HTRF signal was measured after 30 minutes on an EnVision (Perkin-Elmer) reader using a time-resolved fluorescence protocol.
Measurement: IC50 = 681 nM (pChEMBL 6.170000076293945)
Descriptors: MW 340.31, ALogP 4.78, HBA 4, HBD 1, PSA 61.6, QED 0.75
Compound (SMILES): COC(=O)N[C@H](C(=O)N1CCCC1C1=Nc2cc(-c3ccc(-c4ccc(-c5cnc([C@@H]6CCCN6C(=O)[C@@H](NC(=O)OC)C(C)C)[nH]5)cc4)c4c3CC3(CCCC3)C4)ccc2S(=O)(=O)N1)C(C)C
InChIKey: JNDLFUFJVVHFDM-FCFMZPAXSA-N
Target: Nonstructural protein 5A (SINGLE PROTEIN), Hepatitis C virus
UniProt: Q5L478
Assay: Luciferase Assay: The Huh-7 cells transfected with HCV replicons system were seeded into 96-well plates (8,000 cells in 125 μL/well) respectively; each test compound was diluted to desired concentration using 5-fold serial dilutions protocol, 10 doses in duplicate, and added to wells with POD™ 810 Plate Assembler. The plates were incubated in a CO2 incubator for 72 hours; after that, 40 μL of Luciferase assay substrate (Promega Bright-Glo) was added to each well, and detected by a chemiluminescence detection system (Topcount Microplate Scintillation and Luminescence Counter) 5 minutes later; the EC50 (half-maximal effective concentration, concentration for 50% of maximal effect) values of test compounds were analyzed by GraphPad Prism software, respectively. In this paper, experiments were repeated twice and set the holes without compounds as negative control.
Measurement: EC50 = 0.078 nM (pChEMBL 10.109999656677246)
Descriptors: MW 947.17, ALogP 7.81, HBA 10, HBD 4, PSA 204.49, QED 0.12
Compound (SMILES): COc1cccc(CNC(=O)c2nc(N)nc3c(OC)cccc23)n1
InChIKey: FFQXVWCDSUWPDY-UHFFFAOYSA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: Displacement of [3H]5-amino-7-[2- phenethyl]-2-(furan-2-yl)-7H-pyrazolo[4,3-e][l,2,4]triazolo[l,5-c]pyrimidine from human adenosine A2A receptor expressed in HEK293 cell membranes measured after 1 hr by TopCount scintillation counting method
Measurement: Ki = 0.8 nM (pChEMBL 9.100000381469727)
Descriptors: MW 339.36, ALogP 1.55, HBA 7, HBD 2, PSA 112.25, QED 0.72
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)c3ccc(-c4ccc(F)c(F)c4)c(F)c3)CC2)C1
InChIKey: NZFKMGISVBMCOH-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: In Vitro JAK Kinase Assay: Compounds herein were tested for inhibitory activity of JAK targets according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 596.62, ALogP 5.14, HBA 7, HBD 1, PSA 106.73, QED 0.29
Compound (SMILES): COC(=O)c1cnc(N2CCN(c3nnc(-c4ccc(F)c(C#N)c4)c(C)c3C)C[C@H]2C)cn1
InChIKey: MKMMPXPZIKKINZ-CQSZACIVSA-N
Target: Protein smoothened (SINGLE PROTEIN), Mus musculus
UniProt: P56726
Assay: Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 461.5, ALogP 3.06, HBA 9, HBD 0, PSA 108.13, QED 0.54
Compound (SMILES): CC(C)[C@@H](NC(=O)c1cc(=O)[nH]c(N(C)C)n1)c1ccc(SC(F)(F)F)cc1
InChIKey: RTAPSDDIHRJROQ-OAHLLOKOSA-N
Target: GMP-stimulated 3',5'-cyclic nucleotide phosphodiesterase (SINGLE PROTEIN), Macaca mulatta
UniProt: B3VTJ2
Assay: Inhibition of rhesus monkey PDE2A3 expressed in AD293 cells using FAM-labeled cAMP as substrate after 60 mins in presence of cGMP by florescence polarization assay
Measurement: Ki = 0.19 nM (pChEMBL 9.720000267028809)
Descriptors: MW 414.45, ALogP 3.57, HBA 5, HBD 2, PSA 78.09, QED 0.71
Compound (SMILES): CNC(=O)c1c(-c2ccc(F)cc2)oc2cc(N(C)S(C)(=O)=O)c(-c3ccc4c(n3)-c3cc5c(F)cccc5n3CCC4)cc12
InChIKey: AQTBQCHUFYSOQT-UHFFFAOYSA-N
Target: RNA-directed RNA polymerase (SINGLE PROTEIN), Hepacivirus hominis
UniProt: O39930
Assay: Inhibition of NS5B polymerase in HCV genotype 1a infected in HuH7 cells after 40 hrs by Taqman probe based RT-PCR analysis
Measurement: EC50 = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 626.69, ALogP 6.76, HBA 6, HBD 1, PSA 97.44, QED 0.23
Compound (SMILES): CCOCCN(C)Cc1c(-c2ccc(NC(=O)NOC)cc2)sc2c1c(=O)n(-c1ccccn1)c(=O)n2Cc1c(F)cccc1F
InChIKey: MNXSZYSLNRUFIA-UHFFFAOYSA-N
Target: Gonadotropin-releasing hormone receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P30968
Assay: 125I-Leuprorelin Binding Assay: The monkey and human membrane fractions prepared were diluted with the assay buffer to yield a 200 g/ml dilution, each of which was then dispensed at 188 μl per tube. To a tube containing the cell membrane fraction of the CHO with monkey GnRH receptors expressed were added 2 μl of a solution of 20 mM compound in 60% DMSO and 10 μl of 38 nM 125I-leuprorelin. To a tube containing the cell membrane fraction of the CHO with human GnRH receptors expressed were added 2 μl of a solution of 2 mM compound in 60% DMSO and 10 μl of 38 nM 125I-leuprorelin. To determine maximum binding quantity, a reaction mixture containing 2 μl of 60% DMSO and 10 μl of 38 nM 125I-leuprorelin was prepared. To determine non-specific binding amount, a reaction mixture containing 2 μl of a solution of 100 μM leuprorelin in 60% DMSO and 10 μl of 38 nM 125I-leuprorelin was prepared.
Measurement: IC50 = 0.1 nM (pChEMBL 10)
Descriptors: MW 650.71, ALogP 4.75, HBA 10, HBD 2, PSA 119.72, QED 0.15
Compound (SMILES): CC(c1ccc(F)cc1)c1cc(O)c(=O)[nH]n1
InChIKey: OUIUZIBDTPYHBB-UHFFFAOYSA-N
Target: D-amino-acid oxidase (SINGLE PROTEIN), Homo sapiens
UniProt: P14920
Assay: In Vitro DAAO Enzyme Assay: Human DAAO enzyme was supplied by the Takeda Pharmaceutical Company (Osaka) and each batch was tested and used at concentrations giving comparable levels of activity. The Km of D-Serine was measured for each enzyme batch to maintain consistency; this Km was used in subsequent assays.On the day of the assay compounds were serially diluted in DMSO before being diluted 1:20 with assay buffer (20 mM Tris ph 7.4). A 5 ul portion of assay buffer was added to the wells of a 384 clear base black-walled plate (Corning), 5 ul of diluted compound was then added via automated plate to plate transfer using the Bravo liquid handler (Agilent technologies) followed by 5 ul of human DAAO enzyme and then 5 ul D-Serine 50 mM was added to all but the negative control wells (final concentration of 10 mM). Finally 5 ul Amplex red reagent (Invitrogen) was added to all wells as per manufacturer's protocol. The plate was incubated for 60 minutes in the dark at 25° C., and the fluorescence in each well was measured in the Envision plate reader.
Measurement: IC50 = 26 nM (pChEMBL 7.579999923706055)
Descriptors: MW 234.23, ALogP 1.77, HBA 3, HBD 2, PSA 65.98, QED 0.83
Compound (SMILES): CC(C)(O)CN1C[C@H](c2cccc(F)c2F)CC[C@@H](NC(=O)c2ccc3c(c2)CC2(C3)C(=O)Nc3ncccc32)C1=O
InChIKey: WAZDMGGJAWEMTD-ZKUVRKPUSA-N
Target: Calcitonin-gene-related peptide receptor, CALCRL/RAMP1 (PROTEIN COMPLEX), Homo sapiens
UniProt: Q16602
Assay: Displacement of [125I]hCGRP from human recombinant CGRP receptor expressed in HEK293 cell membranes incubated for 3 hrs by scintillation counting method
Measurement: Ki = 1.7 nM (pChEMBL 8.770000457763672)
Descriptors: MW 574.63, ALogP 3.62, HBA 5, HBD 3, PSA 111.63, QED 0.43
Compound (SMILES): COc1cc(OC)cc(C(=O)N2CCN(c3ccc(NC(=O)N4Cc5ccccc5C4)cc3)CC2)c1
InChIKey: ZBJLXYVRAUPLHB-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 1: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-1251667.0. 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 = 187 nM (pChEMBL 6.730000019073486)
Descriptors: MW 486.57, ALogP 4.21, HBA 5, HBD 1, PSA 74.35, QED 0.58
Compound (SMILES): CN1C(=O)C2(N=C1N)c1cc(CCC3CC3)ccc1C[C@]21CC[C@@H](OC(F)F)CC1
InChIKey: ICEWIUVLYPPZQQ-DRXONWLQSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE Enzyme Assay (Ex.435): For each compound being tested, the BACE activity was monitored in a fluorescence quenching assay (FRET) using the ectodomain of BACE (aa 1-454) fused to a myc-his tag and secreted from HEK293/BACEect. cells into OptiMEM (Invitrogen) as enzyme source and a substrate peptide derived from the APP-Swedish mutation which possesses a Cy3-fluorophore at the N-terminus and a Cy5Q-quencher at the C-terminus (Cy3-SEVNLDAEFK-Cy5Q-NH2; Amersham). The substrate was dissolved at 1 mg/mL in DMSO.The assay was performed in the presence of 5 ul OptiMEM (supernatant collected over 24 hours and cleared from cellular debris by centrifugation) containing the ectodomain of BACE, 25 ul water containing the desired concentration of test compound and 1% DMSO, substrate peptide, 20 mM NaOAc, pH 4.4 and 0.04% Triton-X100 in a total assay volume of 50 ul in a 384 well plate. In general, 25 ul of compound dilution were given to the plate followed by the addition of 10 ul of BACE.
Measurement: IC50 = 13.5 nM (pChEMBL 7.869999885559082)
Descriptors: MW 417.5, ALogP 3.74, HBA 4, HBD 1, PSA 67.92, QED 0.79
Compound (SMILES): COc1ccc2c(c1)[C@@H](Nc1cc(O[C@@H]3C[C@@H](COS(N)(=O)=O)[C@@H](O)C3)ncn1)[C@@H](OC)CC2
InChIKey: MCALKOUCNUORLI-BVFBRMCBSA-N
Target: NEDD8 activating enzyme (PROTEIN COMPLEX), Homo sapiens
UniProt: Q13564
Assay: Inhibition of recombinant human NAEalpha/N-terminal His-tagged NAEbeta expressed in baculovirus infected insect Sf9 cells using N-terminal FLAG tagged Nedd8 as substrate incubated for 105 mins in presence of N-terminal GST-tagged Ubc12 by FRET assay
Measurement: IC50 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 494.57, ALogP 1.34, HBA 10, HBD 3, PSA 155.12, QED 0.46
Compound (SMILES): O=c1[nH]nc2c3c(c(Cl)ccc13)NC1=C2CCCCC1
InChIKey: WYAMVMZUUAYLTF-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: HT F Homogeneous PARP Inhibition Assay: PARP1 enzymatic assay was conducted using a method modified from HT F Homogeneous PARP Inhibition Assay Kit (Trevigen). 8.8 nM PARP1 was pre-incubated with different concentrations of compounds in a buffer containing 100 mM Tris-HCl pH 8.0, 100 mM NaCl, 20 mM MgCl2, and 1% DMSO for 30 min at RT. The auto-PARsylation reaction was initiated by addition of 500 nM NAD and 20 ng/ul activated DNA (Sigma) and incubated at RT for 40 min. The remaining NAD was detected by incubation with cycling assay solution containing 1% ethanol, 0.30 U/ml alcohol dehydrogenase, 25 uM resazurin, and 0.25 U/ml diaphorase for 50 min at RT. The concentration of NAD is proportional to the fluorescence signal at Ex540 nm/Em 590 nm.
Measurement: IC50 = 52.5 nM (pChEMBL 7.28000020980835)
Descriptors: MW 287.75, ALogP 3.68, HBA 3, HBD 2, PSA 57.78, QED 0.78
Compound (SMILES): O=C(NCCCN1CCCC1=O)c1ccc(NC(=O)N2Cc3ccc(F)cc3C2)cc1
InChIKey: BCABICAUTOKZAU-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 1: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-1251667.0. 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 = 12.3 nM (pChEMBL 7.909999847412109)
Descriptors: MW 424.48, ALogP 3.12, HBA 3, HBD 2, PSA 81.75, QED 0.7
Compound (SMILES): Cc1ccc2c(NCc3ccc(NC(=O)Nc4ccc(F)cc4)cc3)nc(N(C)C)nc2c1
InChIKey: OYUPNTBJUKVCPL-UHFFFAOYSA-N
Target: TCF4/beta-catenin (PROTEIN COMPLEX), Homo sapiens
UniProt: P35222
Assay: Inhibition of TCF-4/beta catenin in human Tcf33.13 cells expressing Tcf-4/SV40 binding sites driving firefly luciferase reporter gene after 24 hrs by luminometer
Measurement: IC50 = 2025 nM (pChEMBL 5.690000057220459)
Descriptors: MW 444.51, ALogP 5.4, HBA 5, HBD 3, PSA 82.18, QED 0.37
Compound (SMILES): O=C(N[C@H]1CCC[C@@H]1Nc1ncc(C(F)(F)F)cc1Cl)c1ncccc1-n1nccn1
InChIKey: ROTVISRNQQAPMT-KBPBESRZSA-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 = 32 nM (pChEMBL 7.5)
Descriptors: MW 451.84, ALogP 3.49, HBA 7, HBD 2, PSA 97.62, QED 0.62
Compound (SMILES): COc1cc(C)c(CNC(=O)c2cc(-c3cncc(C)c3)nc3onc(-c4cccs4)c23)nc1OC
InChIKey: GDSCVDPVLSCSCV-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: FLIPR Ca2+ Flux Assay: In a typical experiment the OX1 and OX2 receptor antagonistic activity of the compounds of the present invention was determined in accordance with the following experimental method. For intracellular calcium measurements, Chinese hamster ovary (CHO) cells expressing the rat orexin-1 receptor or the human orexin-2 receptor, are grown in Iscove's modified DMEM containing 2 mM L-glutamine, 0.5 g/ml G418, 1% hypoxanthine-thymidine supplement, 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 Becton-Dickinson black 384-well clear bottom sterile plates coated with poly-D-lysine. All reagents were from GIBCO-Invitrogen Corp. The seeded plates are incubated overnight at 37° C. and 5% CO2. Ala-6,12 human orexin-A as the agonist is prepared as a 1 mM stock solution in 1% bovine serum albumin (BSA) and diluted in assay buffer (HBSS containing 20 mM HEPES, 0.1% BSA and 2.5 mM probenecid, pH7.4) for diluted in 384-well plates, first in DMSO, then assay buffer.
Measurement: Ki = 0.7 nM (pChEMBL 9.149999618530273)
Descriptors: MW 501.57, ALogP 4.97, HBA 9, HBD 1, PSA 112.26, QED 0.33
Compound (SMILES): Cn1cc(C(=O)Nc2cn3nc(-c4cn(C)nc4-c4ccc(F)cc4)ccc3n2)cn1
InChIKey: RJXLNSVQUXQPFQ-UHFFFAOYSA-N
Target: Casein kinase I isoform epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: P49674
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 = 6.1 nM (pChEMBL 8.210000038146973)
Descriptors: MW 416.42, ALogP 2.92, HBA 8, HBD 1, PSA 94.93, QED 0.49
Compound (SMILES): CC(C)Oc1ccc2[nH]nc(-c3cc(N4CCOC5(CC5)C4)ncn3)c2c1
InChIKey: FCIAFJJSYNLEDK-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LanthaScreen™ Assay (5 mM ATP): a) 400 nl of a 1:2.15 serial dilution of test compound(98 μM top assay concentration) is spotted via Labcyte Echo to certain wells in a 384 well black, untreated plate. Control wells contain 400 nl of either DMSO or 400 nl of a known inhibitor in DMSO. b) 10 μl of a 2.5 nM LRRK2(G2019S mutation, GST-LRRK2(amino acids 970-2527)) enzyme solution in 1× assay buffer(50 mM Tris pH 8.5, 10 mM MgCl2, 0.01% Brij-35, 1.0 mM EGTA, 2 mM DTT, 0.05 mM NaVO4) is added to all wells. c) A 30 minute room temperature incubation is followed by addition of 10 μl of 800 nM fluorescein labeled LRRKtide peptide substrate and 10 mM ATP solution in 1× assay buffer to all wells. d) After a 35 minute room temperature incubation, 20 μl of TR-FRET Dilution Buffer(Invitrogen PV3756B) containing 4 nM Tb-labeled anti-phospho LRRKtide antibody and 20 mM EDTA is added to all wells. e) Plates are incubated at room temperature for 1 hour and read on an Envision™ multi-mode plate reader with LanthaScreen™ settings. Results are analysed using Assay Data Analyzer.
Measurement: IC50 = 4.04 nM (pChEMBL 8.390000343322754)
Descriptors: MW 365.44, ALogP 3.18, HBA 6, HBD 1, PSA 76.16, QED 0.77
Compound (SMILES): CNC(=O)[C@H](CC(C)(C)C)NC(=O)c1ccc(C2CC2)c(OCC(F)(F)C(F)(F)F)n1
InChIKey: MNVJXLACEBDMRB-AWEZNQCLSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
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 = 27.6 nM (pChEMBL 7.559999942779541)
Descriptors: MW 451.44, ALogP 3.82, HBA 4, HBD 2, PSA 80.32, QED 0.59
Compound (SMILES): COC(=O)N[C@H](C(=O)NCc1ccc(C(CO)N(CCC(C)C)S(=O)(=O)c2ccc(CO)cc2)s1)C(c1ccccc1)c1ccccc1
InChIKey: KXLOOBSPABGQIE-AEHIRBQFSA-N
Target: Gag-Pol polyprotein (SINGLE PROTEIN), Human immunodeficiency virus type 1 group M subtype B (isolate HXB2)(HIV-1)
UniProt: P04585
Assay: Inhibition Assay: The inhibition of WT HIV-1 protease was studied using the reaction of the protease (expressed in Eschericia coli) with a peptide substrate [Val-Ser-Gln-Asn-(betanaphthyl)Ala-Pro-Ile-Val (SEQ ID NO:1)]. Compounds were tested using Procedure A. In Procedure A, the test compound was first preincubated with the enzyme in assay buffer (50 mM sodium acetate, pH 5.5, 100 mM NaCl, and 0.1% BSA) for 30 minutes at room temperature. Substrate was then added to 400 micromolar in a total volume of 80 microliters containing 10 picomolar HIV-1 protease and the reaction was incubated for 1 hour at 30° C. The reaction was quenched with the addition of 120 microliters of 10% phosphoric acid. The product formation was determined after separation of product and substrate on a Zorbax Eclipse XDB-C18 column (Agilent Technologies, Santa Clara, Calif.) connected to an Agilent 1100 high performance liquid chromatography system with fluorescence detection (excitation 270 nanometer and emission 330 nanometer).
Measurement: IC50 = 0.54 nM (pChEMBL 9.270000457763672)
Descriptors: MW 693.89, ALogP 5.18, HBA 8, HBD 4, PSA 145.27, QED 0.13
Compound (SMILES): Cc1cccc2c1NC(=O)[C@@H](NC(=O)[C@H](CCC(F)(F)F)[C@H](CCCC(F)(F)F)C(N)=O)N=C2c1cccc(Cl)c1
InChIKey: HVLALDWMJAWZQF-YZZKKUAISA-N
Target: Neurogenic locus notch homolog protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P46531
Assay: Biological Assay: The pharmacological properties of the compounds of this invention may be confirmed by a number of biological assays.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 604.98, ALogP 5.67, HBA 4, HBD 3, PSA 113.65, QED 0.3
Compound (SMILES): CC(=O)O[C@H]1CC[C@@]2(C)C(=CC[C@H]3C4=CC=C(n5ccnc5)[C@@]4(C)CC[C@@H]32)C1
InChIKey: CSILUOLVYYWEFH-VATUJKLESA-N
Target: Steroid 17-alpha-hydroxylase/17,20 lyase (SINGLE PROTEIN), Homo sapiens
UniProt: P05093
Assay: Inhibition of CYP17 in human testicular microsomes using 17alpha-hydroxypregnenolone as substrate assessed as decrease in DHEA levels after 90 mins by ELISA
Measurement: IC50 <= 11 nM (pChEMBL 7.959000110626221)
Descriptors: MW 378.52, ALogP 5.15, HBA 4, HBD 0, PSA 44.12, QED 0.53
Compound (SMILES): CN(C)C(=O)/C(C#N)=C/c1ccc(Nc2cnc3[nH]cc(C(=O)C(C)(C)C)c3n2)cn1
InChIKey: OGMLTZUTACBZHC-MDWZMJQESA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Phosphorylation Assay: Phosphorylation of the appropriate peptide substrate (fluorescently labeled Srctide) by recombinant JAK3 (catalytic domain, aa 781-1124) was measured in the presence and absence of different concentrations of test compounds. Test compound, enzyme, fluorescently labeled substrate, and cofactors (ATP and Mg2+) were combined in a well of a microtiter plate and incubated for 2 hours at 25° C. The composition of the reaction buffer was 100 mM HEPES pH 7.5, 5 mM MgCl2, 0.01% Triton-X 100, 0.1% Bovine Serum Albumin, and 1% DMSO. Enzyme concentration in the final reaction mixture was 0.5 nM while peptide substrate was at 1 uM. ATP was used at 1xKm concentration corresponding to 2 uM. Serial dilutions (3-fold steps) of compounds were prepared in DMSO. At the end of the incubation, the reaction was stopped by the addition of 20 mM EDTA. Substrate and product (i.e. phosphorylated substrate) were separated electrophoretically and both were quantified by fluorescence intensity.
Measurement: IC50 = 0.9 nM (pChEMBL 9.050000190734863)
Descriptors: MW 417.47, ALogP 3.32, HBA 7, HBD 2, PSA 127.66, QED 0.37
Compound (SMILES): CC(=C(c1ccc(O)cc1)c1ccc(O)cc1)c1ccc(N)cc1
InChIKey: WIAPZXQYCVTONE-UHFFFAOYSA-N
Target: Estrogen receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P03372
Assay: Displacement of fluorescent ES2 from recombinant human ERalpha after 2 hrs in absence of light by fluorometric analysis
Measurement: EC50 = 11036 nM (pChEMBL 4.960000038146973)
Descriptors: MW 317.39, ALogP 4.66, HBA 3, HBD 3, PSA 66.48, QED 0.49
Compound (SMILES): CC(C)Nc1cc(-n2ccc3cc(C#N)cnc32)ncc1C(=O)NCCC1(O)CCCC1
InChIKey: RQEQIZKLRBHQCU-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 L prepared from 15 L additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij 35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction mixture was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 432.53, ALogP 3.54, HBA 7, HBD 3, PSA 115.86, QED 0.53
Compound (SMILES): O=C(O)c1ccc(-c2cnc(N3CCOCC3)c3nc(C#Cc4ccccn4)cn23)cc1
InChIKey: CZVDHEDVFMJBMC-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q9QYJ6
Assay: In Vitro Assay rPDE10: Rat recombinant PDE10a (rPDE10a) was expressed in Sf9 cells using a recombinant rPDE10a baculovirus construct. Cells were harvested after 48 h of infection and the rPDE10a protein was purified by metal chelate chromatography on Ni-sepharose 6FF. Tested compounds were dissolved and diluted in 100% DMSO to a concentration 100 fold of the final concentration in the assay. Compound dilutions (0.4 uL) were added in 384 well plates to 20 uL of incubation buffer (50 mM Tris pH 7.8, 8.3 mM MgCl2, 1.7 mM EGTA). 10 uL of rPDE10a enzyme in incubation buffer was added and the reaction was started by addition of 10 uL substrate to a final concentration of 60 nM cAMP and 0.008 uCi 3H-cAMP. The reaction was incubated for 60 min at rt. After incubation, the reaction was stopped with 20 uL of 17.8 mg/mL PDE SPA beads. After sedimentation of the beads during 30 min the radioactivity was measured in a Perkin Elmer Topcount scintillation counter and results were expressed as cpm. For blank values the enz
Measurement: IC50 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 425.45, ALogP 2.73, HBA 7, HBD 1, PSA 92.85, QED 0.5
Compound (SMILES): c1cc2c(ncn3nnc(C4CCCNC4)c23)[nH]1
InChIKey: SKZWRLQORDYXQT-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
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 = 290 nM (pChEMBL 6.539999961853027)
Descriptors: MW 242.29, ALogP 1.07, HBA 5, HBD 2, PSA 70.9, QED 0.67
Compound (SMILES): COC(=O)Nc1ccc(S(=O)(=O)C(C)C)c([C@H]2CCCN2C(=O)[C@H](Nc2ccc3c(F)c[nH]c(=O)c3c2)c2ccc(OC)c(OC)c2)c1
InChIKey: OWZJHFYKSGTIJZ-DLFZDVPBSA-N
Target: Coagulation factor VII/tissue factor (PROTEIN COMPLEX), Homo sapiens
UniProt: P13726
Assay: Inhibition of recombinant human factor-7a/TF using S2288 as substrate measured after 60 mins
Measurement: Ki = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 680.75, ALogP 5.56, HBA 9, HBD 3, PSA 156.13, QED 0.2
Compound (SMILES): Cc1nc(C)c(-c2csc(Nc3ccccc3)n2)s1
InChIKey: NWRDDAWJDQVGFS-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBA0
Assay: Antagonist activity at human recombinant TRPV4 expressed in CHO-K1 cells assessed as blockade of 1000 nM 4alphaPDD-induced calcium mobilization by FLIPR assay
Measurement: IC50 = 31 nM (pChEMBL 7.510000228881836)
Descriptors: MW 287.41, ALogP 4.63, HBA 5, HBD 1, PSA 37.81, QED 0.76
Compound (SMILES): COCCOCC(=O)N1CC=C(c2ccc(NC(=O)N3Cc4ccccc4C3)cc2)CC1
InChIKey: ZGYLWBLLDXRZCT-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 1: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-1251667.0. 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 = 21.5 nM (pChEMBL 7.670000076293945)
Descriptors: MW 435.52, ALogP 3.51, HBA 4, HBD 1, PSA 71.11, QED 0.68