lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCC(CC)n1cc(-c2cnc(N)c(-c3ccc(=O)n(-c4cc(OC)cc(OC)c4)n3)n2)cn1
InChIKey: SWJBPGCYSKADTC-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Enzyme Assay: FGFR1, 2 and 3 kinase activity was measured by the Invitrogen LanthaScreen Assay technology which directly measures the amount of substrate phosphorylation by TR-FRET using a fluorescein-labeled peptide and Europium-labeled antibody.To measure FGFR1 kinase activity, 200 pM His-tagged recombinant human FGFR1 catalytic domain (amino acids 308-731) (Life Technologies Cat. No. PR4660A) was incubated with 100 nM Alexa Fluor® 647-Poly-GT Peptide Substrate (Life Technologies Cat. No. PV5836) and ATP in the presence of Mg++, along with test compound in a buffer consisting of 250 mM HEPES, 25 mM MgCl2, 0.05% TritonX-100, pH 7.5, and 2% DMSO. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After 20 minutes incubation at 22° C., an equal volume of 2 nM LanthaScreen® Eu-PY20 Antibody (Life Technologies Cat. No. PV5691) and EDTA were added to quench the kinase reaction and start the detection reaction. After an additional 60 minute incubation at 22° C., the reaction was measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) calculated using a ratiometric emission factor. 100 POC is determined using no test compound and 0 POC is determined using no enzyme. The POC values were fit to a 4 parameter logistic curve and the IC50 value is point where the curve crosses 50 POC.To measure FGFR2 kinase activity: 200 pM His-tagged recombinant human FGFR2 cytoplasmic domain (amino acids 403-822), (Life Technologies Cat. No. PR5332A); 20 minutes incubation at 22° C., 60 minute detection incubation at 22° C.To measure FGFR3 kinase activity: 750 pM N-terminal GST-HIS6 fusion protein with a 3C cleavage site recombinant human FGFR3 (amino acids R397-T806) (ProQinase Cat. No. 1068-0000-1); 10 minutes incubation at 22° C., 60 minute detection incubation at 22° C.
Measurement: IC50 = 10.6 nM (pChEMBL 7.96999979019165)
Descriptors: MW 461.53, ALogP 3.51, HBA 10, HBD 1, PSA 122.97, QED 0.42
Compound (SMILES): CCC(CC)n1cc(-c2nc(-c3cnn([C@@H]4COC[C@H]4O)c3)cn3nccc23)cn1
InChIKey: QUUKMKOJWSAHRW-WOJBJXKFSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK2 Inhibition Assay: Compounds of Formula I were screened for their ability to inhibit JAK2 using the general enzyme inhibition assay method, in which the assay mixture contained 1 mM ATP, 10 μM Omnia® Y7 peptide (Catalog # IVGN KNZ3071C, Invitrogen Corporation, Carlsbad, Calif.) and 4 nM JAK2 in a total volume of 25 μL. JAK2 was purchased from Invitrogen Corporation, Carlsbad, Calif. (catalog # IVGN PV4288).
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 407.48, ALogP 2.75, HBA 9, HBD 1, PSA 95.29, QED 0.53
Compound (SMILES): C[C@H]1CN(c2ccncc2NC(=O)c2ccc3ccc(N4CCN(C)CC4)cc3n2)C[C@@H](N)[C@@H]1O
InChIKey: UECVFMBXOWMGOD-WVFSIPRWSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Enzyme Assay: Pim-1 and Pim-3 kinase assays-20 μL reactions were run in white 384 well polystyrene plates dotted with 0.8 μL compound/DMSO in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM mgCl2, 0.01% BSA, 5 mM DTT), containing 0.05 μM Biotin-labeled BAD peptide substrate (AnaSpec 62269), 1 mM ATP, and 2.5 pM (Pim-1, Invitrogen PV3503) or 1.25 pM (Pim-3, Millipore 14-738) enzyme for 1 h at 25° C. Reactions were stopped by addition of 10 μL STOP Buffer (150 mM Tris, pH=7.5, 150 mM NaCl, 75 mM EDTA, 0.01% Tween-20, 0.3% BSA,) supplemented with Phospho-Bad (Ser112) Antibody (Cell Signaling 9291) diluted 666-fold, and Streptavidin donor beads (PerkinElmer 6760002) along with Protein-A acceptor beads (PerkinElmer 6760137) at 15 μg/mL each. Supplementation of the STOP buffer with beads and stopping the reactions were done under reduced light. Prior to the stopping reactions STOP buffer with beads was preincubated for 1 h in the dark at room temperature. After stopping the reactions, plates were incubated for 1 h in the dark at room temperature before reading on a PHERAstar FS plate reader (BMG Labtech) under reduced light.Pim-2 kinase assay-20 μL reactions were run in white 384 well polystyrene plates dotted with 0.8 μL compound/DMSO in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM mgCl2, 0.01% BSA, 5 mM DTT), containing 0.05 μM Fluorescein-labeled CREBtide peptide substrate (Invitrogen PV3508), 1 mM ATP, and 1 nM enzyme (Invitrogen PV3649) for 2 h at 25° C. Reactions were stopped by addition of 10 μL TR-FRET Dilution Buffer (Invitrogen PV3574) with 30 mM EDTA and 1.5 nM LanthaScreen Tb-CREB pSer133 antibody (Invitrogen PV3566). After 30 min. incubation at room temperature, plates were read on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 475.6, ALogP 1.78, HBA 8, HBD 3, PSA 110.85, QED 0.52
Compound (SMILES): C=CC(=O)N1CCC(Oc2cc3c(Nc4cccc(-c5cnco5)c4)ncnc3cc2OC)CC1
InChIKey: AURZPIANIDXZEM-UHFFFAOYSA-N
Target: Receptor tyrosine-protein kinase erbB-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P04626
Assay: Cell Viability Assay and IC50 Estimation: Cell viability was determined using the Cell Titer Glo assay (Promega) as previously described. Briefly, 2000-3000 cells per well were plated in 384-well plates (Greiner Bio-One) in technical triplicate. Cells were treated with seven different concentrations of tyrosine kinase inhibitors or vehicle alone at a final volume of 40 μL per well. After 3 days hours, 11 μL of Cell Titer Glo was added to each well. Plates were shaken for 15 minutes, and bioluminescence was determined using a FLUOstar OPTIMA multi-mode micro-plate reader (BMG LABTECH). Bioluminescence values were normalized to DMSO treated cells, and normalized values were plotted in GraphPad Prism using non-linear regression fit to normalized data with a variable slope. IC50 values were calculated by GraphPad Prism at 50% inhibition.
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 471.52, ALogP 4.59, HBA 8, HBD 1, PSA 102.61, QED 0.39
Compound (SMILES): CNC1CCC(c2ccc3[nH]c(-c4cn(CC#N)c5ncccc45)c(C(C)C)c3n2)CC1
InChIKey: AVFZBJVZXBCCQT-UHFFFAOYSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
Assay: TLR7/8/9 Inhibition Reporter Assays: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 7.6 nM (pChEMBL 8.119999885559082)
Descriptors: MW 426.57, ALogP 5.47, HBA 5, HBD 2, PSA 82.32, QED 0.44
Compound (SMILES): Cc1cccc(-c2nc(-c3ccc(C(=O)O)cc3)[nH]c2-c2ccncc2)c1
InChIKey: KSIHALPJIJZKBN-UHFFFAOYSA-N
Target: Casein kinase I isoform delta (SINGLE PROTEIN), Homo sapiens
UniProt: P48730
Assay: LanthaScreen Eu Kinase Binding Assay: Table 3: Small molecules were tested for their inhibitory activities towards casein kinase 1 delta (CK1δ) and 1 epsilon (CK1ϵ). The in vitro LanthaScreen Eu kinase binding assay was used to determine the IC50 values for 15 of the synthesized compounds and 5 commercial inhibitors including SB203580 (Table 3). Tested compounds displayed a wide range of affinity between 6.8 nM (high affinity=strong inhibition) to values over 1000 nM (low affinity=very weak inhibition).
Measurement: IC50 = 4713 nM (pChEMBL 5.329999923706055)
Descriptors: MW 355.4, ALogP 4.81, HBA 3, HBD 2, PSA 78.87, QED 0.55
Compound (SMILES): FC(F)(F)c1ccc(C(F)(F)CN2CCC(Nc3ncnc4[nH]ncc34)CC2)cc1
InChIKey: GXJBVCKDEHELEL-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: CYP P450 Enzyme Inhibition: Inhibitory activities of test compounds on 5 major isoforms of CYP P450 were evaluated by using pooled human liver microsome (HLM, purchased from BD Gentest) and selective substrates for those isoforms. Those CYP isoforms and their corresponding probe substrates are as follows: CYP1A2 (phenacetin, 30 μM), CYP2C9 (tolutamide, 100 μM), CYP2C19 (S-mephenytoin, 40 μM), CYP2D6 (dextromethorphan, 5 μM) and CYP3A4 (midazolam, 1 μM). All probe substrates were used at concentrations near or below their Kms. For experiment, a reaction mixture of test compound at 10 uM or in serial dilution, CYP probe substrate described above and 0.2 mg/mL pooled HLM in phosphate buffer, pH 7.4 in a final volume of 200 μL was pre-incubated at 37° C. for 10 minutes in triplicate. The reaction was initiated by addition of NADPH at final concentration of 1 mM. The reaction was terminated after 10 minutes (CYP1A2, CYP2D6 and CYP3A4) or 30 minutes (CYP2C9 and CYP2C19) by addition of 100 μL ice-cold acetonitrile with internal standard (IS). The samples were then centrifuged at 13,000 rpm and the supernatants were injected to LC-MS/MS (Agilent Technologies) to quantify the concentration of the specific metabolites of the probe substrates formed by individual CYP450 isoforms.
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 426.39, ALogP 4.04, HBA 5, HBD 2, PSA 69.73, QED 0.6
Compound (SMILES): COc1ccc(C(=O)N[C@@H]2CCCN(c3cnc(C(N)=O)c(Nc4ccc(C5CCN(C=O)CC5)cc4)n3)C2)cc1
InChIKey: CWUHDORBMBHXJQ-XMMPIXPASA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: HotSpot kinase Assay: The degree of inhibition of a panel of kinases is determined using the in vitro HotSpot kinase assay (purified enzymes, 33P-ATP, an appropriate substrate and 1 uM ATP).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 557.66, ALogP 3.06, HBA 8, HBD 3, PSA 142.78, QED 0.34
Compound (SMILES): CC(C)c1ccc(-c2nc3ncccn3c2CN2C3CCC2CN(C(=O)c2ccccc2F)CC3)cc1
InChIKey: RYFIOJQQPUATAL-UHFFFAOYSA-N
Target: Potassium channel subfamily K member 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NPC2
Assay: Inhibition of Recombinant TASK-1 and TASK-3 In Vitro: The investigations on the inhibition of the recombinant TASK-1 and TASK-3 channels were conducted using stably transfected CHO cells. The compounds of the invention were tested here with administration of 40 mM of potassium chloride in the presence of a voltage-sensitive dye using the method described in detail in the following references [Whiteaker et al., Validation of FLIPR membrane potential dye for high-throughput screening of potassium channel modulators, J. Biomol. Screen. 6 (5), 305-312 (2001); Molecular Devices FLIPR Application Note: Measuring membrane potential using the FLIPR® membrane potential assay kit on Fluorometric Imaging Plate Reader (FLIPR®) systems, http://www.moleculardevices.com/reagents-supplies/assay-kits/ion-channels/flipr-membrane-potential-assay-kits]. The activity of the test substances was determined as their ability to inhibit a depolarization induced in the recombinant cells by 40 mM potassium chloride. The concentration which can block half of this depolarization is referred to as IC50.
Measurement: IC50 = 1800 nM (pChEMBL 5.75)
Descriptors: MW 497.62, ALogP 5.54, HBA 5, HBD 0, PSA 53.74, QED 0.36
Compound (SMILES): CC(=O)N1CCC(c2ccc(Nc3nc(N4CCC[C@@H](NC(=O)N5CCCCC5)C4)nnc3C(N)=O)cc2)CC1
InChIKey: PTGDURRRTYAKSP-HSZRJFAPSA-N
Target: Tyrosine-protein kinase Lck (SINGLE PROTEIN), Homo sapiens
UniProt: P06239
Assay: HotSpot kinase Assay: The degree of inhibition of a panel of kinases is determined using the in vitro HotSpot kinase assay (purified enzymes, 33P-ATP, an appropriate substrate and 1 uM ATP).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 549.68, ALogP 2.6, HBA 8, HBD 3, PSA 149.68, QED 0.5
Compound (SMILES): COc1ncccc1-c1nc2c(n1C(C)C)[C@@H](c1ccc(Cl)cc1)N(c1cc(C)c3nnn(C)c3n1)C2=O
InChIKey: VQVSQBFKHGXUFK-JOCHJYFZSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: AlphaScreen In-Vitro Binding Assay : In order to assess bromodomain selectivity, we set up a binding assay using the bromodomain encoded by the CREBBP gene. Compounds were tested in the CREBBP assay with a similar protocol, however using AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay, Perkin Elmer) as detection readout instead of TR-FRET. The assay was started by stepwise addition of 4.5μl per well of bromo domain protein (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.02% BSA, 150 mM NaCl, 324 nM His-CREBBP(1081-1197) (custom production at Viva Biotech Ltd.)) and 4.5μl per well of peptide solution (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.02% BSA, 150 mM NaCl, 120 nM acetyl-histone H4 (AcK 5, 8, 12) (Biosyntan GmbH)). Reactions were incubated at 30° C. for 35 minutes. Subsequently 4.5 μl per well detection mix (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.02% BSA, 150 mM NaCl, 45 μg/ml Ni-chelate acceptor beads, 45 μg/mIstreptavidin-donor beads) (Perkin Elmer)) were added. After 60 minutes incubation at room temperature, plates were measured in a Perkin Elmer EnVision multilabel reader. IC50 values were determined from percent inhibition values at different compound concentrations by non-linear regression analysis.
Measurement: IC50 > 37000 nM (pChEMBL 4.432000160217285)
Descriptors: MW 529, ALogP 4.92, HBA 9, HBD 0, PSA 103.85, QED 0.32
Compound (SMILES): CC(C)[C@H](C)n1cc(-c2nc(-c3cnn(C[C@@H](O)CO)c3)cn3nccc23)cn1
InChIKey: SSCCJQJLIGWQAF-WMLDXEAASA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK3 Inhibition Assay: Compounds of Formula I were screened for their ability to inhibit JAK3 using the general enzyme inhibition assay method, in which the assay mixture contained 1 mM ATP, 10 μM Omnia® Y7 peptide (Catalog # IVGN KNZ3071C, Invitrogen Corporation, Carlsbad, Calif.) and 2 nM JAK3 in a total volume of 25 μL. JAK3 was purchased from Invitrogen Corporation, Carlsbad, Calif. (catalog # IVGN PV4080).
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 395.47, ALogP 2.03, HBA 9, HBD 2, PSA 106.29, QED 0.5
Compound (SMILES): CNC(=O)c1ccc(N2CCN(Cc3ccc4nc(CC(F)(F)F)c(=O)[nH]c4c3)CC2)cn1
InChIKey: GWWKGCCMZJXPIM-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase tankyrase-1 (SINGLE PROTEIN), Homo sapiens
UniProt: O95271
Assay: Fluorescence Anisotropy Binding Assay: PARP5a: Recombinant PARP5a binding domain was diluted to 160 nM with 50 mM Tris pH 8, 0.001% Triton X100, 10 mM MgCl2, 150 mM NaCl and incubated for four hours with an equivalent volume of 6 nM fluorescent probe diluted with 50 mM Tris pH 8, 0.001% Triton X100, 10 mM MgCl2, 150 mM NaCl. The final DMSO concentration of the probe was kept below 1% (v/v).
Measurement: IC50 = 71000 nM (pChEMBL 4.150000095367432)
Descriptors: MW 460.46, ALogP 2.1, HBA 6, HBD 2, PSA 94.22, QED 0.61
Compound (SMILES): CNC[C@H](c1ccc(Cl)c(Cl)c1)[C@H](O)c1cccc(N(C)C)c1
InChIKey: XKVFRZWDKQJABG-CRAIPNDOSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: Inhibition Assay: Cytochrome P450 3A4 and 2D6:Recombinant enzymes, 3A4 and 2D6, generated using the ABL yeast expression system were used. For CYP3A4, the enzyme amount was 2 pmol per well, and the substrate (7-benzyloxy-4-(trifluoromethyl) coumarin; BFC) was used at 15 μM. The assay mixture also included K2HPO4 (pH 7.4; conc. 0.1 M) and NADPH (conc. 1 mM). The incubation time was 30 minutes, and the reference inhibitor was A-naphthoflavone. For CYP2D6, the enzyme amount was 2 pmol per well, and the substrate (7-methoxy-4-(aminomethyl)-coumarin; MAMC) was used at 20 μM. The assay mixture also included K2HPO4 (pH 7.4; conc. 0.1 M) and NADPH (conc. 0.06 mM). The incubation time was 35 minutes, and the reference inhibitor was quinidine.Briefly, 0.6 μL of serially diluted compound was added to 75 μL water. 10 μL of diluted compound in water was then added to each assay plate. 20 μL of a K2HPO4, enzyme, and substrate mixture was added. After 10 minutes of room temperature pre-incubation, 10 μL of NADPH was added, and the plates were placed at 37° C. for 30 or 35 minutes depending on the enzyme being tested. After the incubation was complete, 20 μL of 0.1% Tris/ACN was added to the plate. The plate was read on a FluorStar fluorescence plate reader.
Measurement: IC50 = 4600 nM (pChEMBL 5.340000152587891)
Descriptors: MW 353.29, ALogP 4.1, HBA 3, HBD 2, PSA 35.5, QED 0.82
Compound (SMILES): CCc1cc2c(C)c(-c3nc(C)no3)c(N3CCC(NC4CCOCC4)CC3)nc2c(F)c1F
InChIKey: REIUVHSKIZRZGF-UHFFFAOYSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: OPR Mu Antagonist Assay: The purpose of this assay is to confirm the potency and selectivity of compounds synthesized to be OPRK1 Antagonists. This assay monitors the OPRMu1 activation, in membrane recruitment of β-arrestin. The assay monitors GPCR-β-arrestin proximity using low affinity fragment complementation of beta-galactosidase (beta-gal). It employs U20S cells which express OPRMu1 fused to the complementary beta-gal fragment (enzyme acceptor). As designed, compounds that act as antagonists will prevent receptor activation resulting in reduce well luminescence. Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 10 micromolar.The Discover X OPRMu1-U20S cell line was routinely cultured in T175 flasks at 37° C., 5% CO2 and 95% relative humidity (RH). The growth media consisted of DMEM/F12 1:1 Media supplemented with 10% v/v heat inactivated fetal bovine serum, 25 mM HEPES, Non-essential amino acids, 1 mM Sodium Pyruvate, 1× antibiotic mix (penicillin streptomycin) plus 500 ug/mL Geneticin and 300 ug/mL Hygromycin (selection antibiotics).On Day 1 of the assay, 5000 cells in 20 ul of assay buffer (Discover X's Cell Plating Reagent 5) were seeded into each well of a 384 Corning 3570 standard white plate, and incubated 16-24 hours at 37° C., 5% CO2 and 95% (RH). On Day 2, 100 nl of test compound in DMSO was added to the appropriate wells, 100 nl of DMSO added to control wells and plates were incubated for 30 min at 37° C., 5% CO2 and 95% (RH). Next 100 nl of DAMGO OPRMu1 or DMSO in assay media (EC80 Challenge consists of 100 nl of 50 uM DAMGO, final assay concentration=250 nM, 100% Response wells receive 100 nl 200 uM DAMGO). After incubation for 3 hours at 37° C., 5% CO2 and 95% (RH), 10 ul of Path Hunter Detection Mix is added to each well, plate placed on a plate rotator/mixer for 10 minutes and then incubated at room temperature, in the dark for 1 hour. Well luminescence was measured on Perkin Elmer's Envision.The Percent Inhibition was calculated from the median ratio as follows:% ⁢ ⁢ Inhibition = 100 - ( 1 ⁢ 0 ⁢ 0 ⁢ ( Compound ⁢ ⁢ Well - 0 ⁢ % ⁢ ⁢ Response ⁢ ⁢ Well EC ⁢ ⁢ 80 ⁢ ⁢ Control ⁢ ⁢ Well - 0 ⁢ % ⁢ ⁢ Response ⁢ ⁢ Well ) )where:    COMPOUND WELL is defined as the well containing test compound;    EC80 CONTROL WELL is defined as wells containing DAMGO challenge (250 nM final)=0% inhibition; and    0% RESPONSE WELL is defined as wells containing DMSO=100% inhibition.IC50 is defined as the concentration of compound required to achieve 50% inhibition.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 471.55, ALogP 4.48, HBA 7, HBD 1, PSA 76.31, QED 0.59
Compound (SMILES): Nc1nonc1C(=O)N1CCC2CCC1CN2Cc1c(-c2ccc(Cl)cc2)nc2ccccn12
InChIKey: WTKLDUIAXFXSFO-UHFFFAOYSA-N
Target: Potassium channel subfamily K member 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NPC2
Assay: Inhibition of Recombinant TASK-1 and TASK-3 In Vitro: The investigations on the inhibition of the recombinant TASK-1 and TASK-3 channels were conducted using stably transfected CHO cells. The compounds of the invention were tested here with administration of 40 mM of potassium chloride in the presence of a voltage-sensitive dye using the method described in detail in the following references [Whiteaker et al., Validation of FLIPR membrane potential dye for high-throughput screening of potassium channel modulators, J. Biomol. Screen. 6 (5), 305-312 (2001); Molecular Devices FLIPR Application Note: Measuring membrane potential using the FLIPR® membrane potential assay kit on Fluorometric Imaging Plate Reader (FLIPR®) systems, http://www.moleculardevices.com/reagents-supplies/assay-kits/ion-channels/flipr-membrane-potential-assay-kits]. The activity of the test substances was determined as their ability to inhibit a depolarization induced in the recombinant cells by 40 mM potassium chloride. The concentration which can block half of this depolarization is referred to as IC50.
Measurement: IC50 = 29 nM (pChEMBL 7.539999961853027)
Descriptors: MW 477.96, ALogP 3.5, HBA 8, HBD 1, PSA 105.79, QED 0.48
Compound (SMILES): CC(C)C1CCN(C(=O)N[C@@H]2CCCN(c3nnc(C(N)=O)c(Nc4ccc(C(=O)N5CCOCC5)cc4)n3)C2)CC1
InChIKey: GNUGYSKRAVVTRC-HSZRJFAPSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: HotSpot kinase Assay: The degree of inhibition of a panel of kinases is determined using the in vitro HotSpot kinase assay (purified enzymes, 33P-ATP, an appropriate substrate and 1 uM ATP).
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 579.71, ALogP 2.23, HBA 9, HBD 3, PSA 158.91, QED 0.45
Compound (SMILES): CCc1cc(O)c(F)cc1-c1cc2[nH]nc(-c3ncc[nH]3)c2c(NCc2ccccc2N(C)S(C)(=O)=O)n1
InChIKey: BEEJICRDGFYLFA-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: JAK Caliper Enzyme Assay at 1 mM ATP: Test article was solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. An 11-point half log dilution series was created in DMSO with a top concentration of 600 μM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 resulting in a top final assay compound concentration of 10 μM and a 2% DMSO concentration.Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK3 or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 11.7 nM (pChEMBL 7.929999828338623)
Descriptors: MW 535.61, ALogP 4.43, HBA 7, HBD 4, PSA 139.89, QED 0.23
Compound (SMILES): CNC(=O)c1n[nH]c2nc(-c3cc(F)c(O)cc3CC(F)(F)F)nc(NCc3ccccc3N(C)S(N)(=O)=O)c12
InChIKey: BRQCTFOAORTYKM-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: JAK Caliper Enzyme Assay at 1 mM ATP: Test article was solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. An 11-point half log dilution series was created in DMSO with a top concentration of 600 μM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 resulting in a top final assay compound concentration of 10 μM and a 2% DMSO concentration.Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK3 or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 674 nM (pChEMBL 6.170000076293945)
Descriptors: MW 582.54, ALogP 2.58, HBA 8, HBD 5, PSA 179.22, QED 0.2
Compound (SMILES): C=C(C)c1cnc(-c2cc(Cl)ccc2F)cc1Nc1ccncc1C(=O)N[C@@H](CC)CO
InChIKey: CKLULXBYHFYPHR-INIZCTEOSA-N
Target: Bone morphogenetic protein receptor type-1A (SINGLE PROTEIN), Homo sapiens
UniProt: P36894
Assay: In Vitro Kinase Assay Inhibition of ALK1/2/3/4/5/6 Kinases: Compound of the invention were screened in an in vitro kinase assay against several members of the TGFβ family of Ser/Thr kinases. The kinases tested were ALK1 (ACVRL1), ALK2 (ACVR1), ALK3 (BMPR1A), ALK4 (ACVR1B), ALK5 (TGFBR1), and ALK6 (BMPR1B). Standard kinase testing conditions and techniques were employed. For each case, specific kinase/substrate pairs along with required cofactors were prepared in reaction buffer. Compound of the invention were delivered into the reaction, followed 15-20 min later by addition of a mixture of ATP and 33P ATP to a final concentration of 10 μM. Reactions were carried out at RT for 120 min, followed by spotting of the reactions onto P81 ion exchange filter paper. Unbound phosphate was removed by extensive washing of filters in 0.75% phosphoric acid. Kinase activity data was expressed as the percent of remaining kinase activity in test samples compared to vehicle. IC50 values were generated from activity values performed at multiple concentrations.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 454.93, ALogP 5.21, HBA 5, HBD 3, PSA 87.14, QED 0.43
Compound (SMILES): O=C(NCc1cccnc1)C1CCN(c2ncnc3[nH]ccc23)CC12CC2
InChIKey: RVRZIGYIYUQOHO-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: JAK Kinase Assays: Human baculovirus-expressed JAK1, 2, 3 and TYK2 were purchased from Carna Biosciences, Inc. All four purified enzymes contain only the catalytic domain. JAK1 (aa 850-1154) and TYK2 (aa 871-1187) are expressed with an N-terminally fused GST-tag, and JAK2 and JAK3 with an N-terminally fused His-tag.Inhibition of phosphorylation of a synthetic peptide was measured in an HTRF-based assay using the TK substrate-Biotin from the Cisbio HTRFKinEASE TK kit. First, 2 μl of TK solution (TK substrate-biotin in kinase buffer [1× enzymatic buffer from HTRFKinEASE TK kit, 1 mM DTT]) is added to a plate containing 1 μl prediluted compound (final assay concentration DMSO: 0.75%). Then, 5 μl kinase-ATP mix (prepared in kinase buffer) is added to the wells and the plates are incubated at RT for 20-30 min. For all four kinases a concentration of ATP that corresponded to the Km for ATP was used. The final concentrations of buffers, substrate, kinase and ATP were: JAK1: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 10 mM MgCl2, 1 mM DTT, 7 μM ATP, 50 nM SEB, 1 μM TK Substrate-Biotin and 5 ng JAK1; JAK2: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 4 μM ATP, 1 μM TK Substrate-Biotin and 0.1 ng JAK2; JAK3: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 2 μM ATP, 1 μM TK Substrate-Biotin and 0.3 ng JAK3; TYK2: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 13 μM ATP, 50 nM SEB, 1 μM TK Substrate-Biotin and 0.8 ng TYK2. Thereafter, the kinase reaction is stopped by adding 4 μl detection mix (final concentrations: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 0.8 M KF, 20 mM EDTA, 42 nM Streptavidin-XL665 and 1:400 STK Ab Cryptate) and the plates are incubated overnight in the dark. The HTRF signal is read using an Envision plate reader.
Measurement: EC50 = 1410 nM (pChEMBL 5.849999904632568)
Descriptors: MW 362.44, ALogP 2.28, HBA 5, HBD 2, PSA 86.8, QED 0.74
Compound (SMILES): CN1Cc2cc(Cl)ccc2-n2c(nnc2C2CC3(C2)CN(c2ncccc2F)C3)C1
InChIKey: IJLMXOYQKXELBB-UHFFFAOYSA-N
Target: Oxytocin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P30559
Assay: Oxytocin Receptor Antagonist Assay: This assay was performed in CHEM-1 cells expressing the human Oxytocin Receptor (hOTR) to determine the inhibitory effect of the compounds of the invention on the human Oxytocin receptor. Oxytocin evokes an increase in intracellular calcium in CHEM-1-hOTR cells which is measured in a fluorescence assay on the FLIPRTETRA using calcium sensitive dyes. Test compounds were assessed for their ability to affect the magnitude of the response to oxytocin, with antagonists showing a concentration-dependent reduction in the oxytocin-mediated fluorescence. Compounds displaying potency at the vasopressin V1a receptor of <100 nM were progressed to selectivity testing against hOTR and were tested in triplicate in a 10-point, 1:3 dilution series starting at a nominal concentration of 3 μM in the assay.CHEM-1-hOTR ready was used to assay frozen cells (Eurofins #HTS090RTA) which are supplied with a proprietary Media Component.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 424.91, ALogP 3.78, HBA 6, HBD 0, PSA 50.08, QED 0.63
Compound (SMILES): COc1c(F)cccc1COC[C@@H]1CCc2cc([C@H]3CC[C@](N)(COP(=O)(O)O)C3)ccc2C1
InChIKey: UYOYUPNHHAKWJZ-FTGWLVSGSA-N
Target: Sphingosine 1-phosphate receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99500
Assay: Receptor [35S] GTPgammaS Binding Assays: (S1P1 GTPgammaS/S1P3 GTPgammaS): Compounds were loaded in a 384 Falcon v-bottom plate (0.5 l/well in a 11 point, 3-fold dilution). Membranes prepared from S1P1/CHO cells or EDG3-Gal5-bla HEK293T cells (EDG3 equivalent S1P3) were added to the compound plate (40 l/well, final protein 3 μg/well) with MULTIDROP®. [35S]GTP (1250 Ci/mmol, Perkin Elmer) was diluted in assay buffer: 20 mM HEPES, pH7.5, 10 mM MgCl2, 150 mM NaCl, 1 mM EGTA (ethylene glycol tetraacetic acid), 1 mM DTT (Dithiothreitol), 10 μM GDP, 0.1% fatty acid free BSA, and 10 μg/ml Saponin to 0.4 nM. 40 μl of the [35S] GTP solution was added to the compound plate with a final concentration of 0.2 nM. The reaction was kept at room temperature for 45 min. At the end of incubation, all the mixtures in the compound plate were transferred to Millipore 384-well FB filter plates via the VELOCITY11® Vprep liquid handler. The filter plate was washed with water 4 times by using the manifold Embla plate washer and dried at 60° C. for 45 min. MicroScint 20 scintillation fluid (30 μl) was added to each well for counting on the Packard TOPCOUNT®. EC50 is defined as the agonist concentration that corresponds to 50% of the Ymax (maximal response) obtained for each individual compound tested. The EC50 for Example 689 was determined to be 5.7 nM in the assay utilizing membranes prepared from S1P1/CHO cells. The EC50 for Example 689 was determined to be >2000 nM in the assay utilizing membranes prepared from EDG3-Gal5-bla HEK293T cells.A smaller value for GTPγS S1P1 EC50 value indicated greater activity for the compound in the GTPγS S1P1 binding assay. A larger value for the GTPγS S1P3 EC50 value indicated less activity in the GTPγS S1P3 binding assay. Example 689, which is the phosphate ester of Example 672, possessed activity as an agonist of S1P1 and is selective over S1P3. Example 697, which is the phosphate ester of Example 681, possessed activity as an agonist of S1P1 and is selective over S1P3. Thus the compounds of the present invention may be used in treating, preventing, or curing various S1P1 receptor-related conditions while reducing or minimizing the side effects due to S1P3 activity. The selectivity of the compounds of the present invention indicate their potential use in treating, preventing, or curing autoimmune and inflammatory diseases such as multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, lupus, psoriasis, or vascular diseases, while reducing or minimizing possible side effects due to S1P3 activity. Other potential uses of the compounds of the present invention include minimizing or reducing rejection of transplanted organs, while reducing or minimizing side effects due to S1P3 activity.
Measurement: EC50 > 625 nM (pChEMBL 6.203999996185303)
Descriptors: MW 493.51, ALogP 4.23, HBA 5, HBD 3, PSA 111.24, QED 0.45
Compound (SMILES): FC(F)(F)c1cccc(CSc2nnc(Cc3c[nH]c4ccccc34)n2CCCc2c[nH]cn2)c1
InChIKey: DHMMKEWJQVUGEM-UHFFFAOYSA-N
Target: Somatostatin receptor type 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P31391
Assay: Agonist activity at human SST4 expressed in CHO-K1 cells assessed as inhibition of forskolin-stimulated cAMP production measured at 2.5 to 20 hrs by TR-FRET LANCE assay
Measurement: EC50 = 38.5 nM (pChEMBL 7.420000076293945)
Descriptors: MW 496.56, ALogP 6.02, HBA 5, HBD 2, PSA 75.18, QED 0.24
Compound (SMILES): CN(c1ncnc2[nH]ccc12)[C@H]1C[C@@H](CS(=O)(=O)N2CCOCC2)C1
InChIKey: GODNEHJKBMGCML-BETUJISGSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Caliper Enzyme Assay: Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK3 or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 1586 nM (pChEMBL 5.800000190734863)
Descriptors: MW 365.46, ALogP 0.83, HBA 6, HBD 1, PSA 91.42, QED 0.84
Compound (SMILES): CCCCCOC1CCc2cc([C@H]3CC[C@@](N)(COP(=O)(O)O)C3)ccc2C1
InChIKey: DLLAKINNLSLSAC-YSTUSHMSSA-N
Target: Sphingosine 1-phosphate receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99500
Assay: Receptor [35S] GTPgammaS Binding Assays: (S1P1 GTPgammaS/S1P3 GTPgammaS): Compounds were loaded in a 384 Falcon v-bottom plate (0.5 l/well in a 11 point, 3-fold dilution). Membranes prepared from S1P1/CHO cells or EDG3-Gal5-bla HEK293T cells (EDG3 equivalent S1P3) were added to the compound plate (40 l/well, final protein 3 μg/well) with MULTIDROP®. [35S]GTP (1250 Ci/mmol, Perkin Elmer) was diluted in assay buffer: 20 mM HEPES, pH7.5, 10 mM MgCl2, 150 mM NaCl, 1 mM EGTA (ethylene glycol tetraacetic acid), 1 mM DTT (Dithiothreitol), 10 μM GDP, 0.1% fatty acid free BSA, and 10 μg/ml Saponin to 0.4 nM. 40 μl of the [35S] GTP solution was added to the compound plate with a final concentration of 0.2 nM. The reaction was kept at room temperature for 45 min. At the end of incubation, all the mixtures in the compound plate were transferred to Millipore 384-well FB filter plates via the VELOCITY11® Vprep liquid handler. The filter plate was washed with water 4 times by using the manifold Embla plate washer and dried at 60° C. for 45 min. MicroScint 20 scintillation fluid (30 μl) was added to each well for counting on the Packard TOPCOUNT®. EC50 is defined as the agonist concentration that corresponds to 50% of the Ymax (maximal response) obtained for each individual compound tested. The EC50 for Example 689 was determined to be 5.7 nM in the assay utilizing membranes prepared from S1P1/CHO cells. The EC50 for Example 689 was determined to be >2000 nM in the assay utilizing membranes prepared from EDG3-Gal5-bla HEK293T cells.A smaller value for GTPγS S1P1 EC50 value indicated greater activity for the compound in the GTPγS S1P1 binding assay. A larger value for the GTPγS S1P3 EC50 value indicated less activity in the GTPγS S1P3 binding assay. Example 689, which is the phosphate ester of Example 672, possessed activity as an agonist of S1P1 and is selective over S1P3. Example 697, which is the phosphate ester of Example 681, possessed activity as an agonist of S1P1 and is selective over S1P3. Thus the compounds of the present invention may be used in treating, preventing, or curing various S1P1 receptor-related conditions while reducing or minimizing the side effects due to S1P3 activity. The selectivity of the compounds of the present invention indicate their potential use in treating, preventing, or curing autoimmune and inflammatory diseases such as multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, lupus, psoriasis, or vascular diseases, while reducing or minimizing possible side effects due to S1P3 activity. Other potential uses of the compounds of the present invention include minimizing or reducing rejection of transplanted organs, while reducing or minimizing side effects due to S1P3 activity.
Measurement: EC50 > 625 nM (pChEMBL 6.203999996185303)
Descriptors: MW 411.48, ALogP 3.83, HBA 4, HBD 3, PSA 102.01, QED 0.42
Compound (SMILES): Cc1cc(-c2[nH]c3ccc(C4CCC(NC5COC5)CC4)nc3c2C(C)C)cn2ncnc12
InChIKey: CJZJVAINTSUGMQ-UHFFFAOYSA-N
Target: Toll-like receptor 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR96
Assay: TLR7/8/9 Inhibition Reporter Assays: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 4038 nM (pChEMBL 5.389999866485596)
Descriptors: MW 444.58, ALogP 4.72, HBA 6, HBD 2, PSA 80.13, QED 0.47
Compound (SMILES): N#CCC1(n2ccc(-c3nc(-c4ccncc4F)cc4ncccc34)n2)CN(CC(F)(F)F)C1
InChIKey: WZXKOUZFRBIINU-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK Caliper Enzyme Assay at 1mM ATP: Compounds were added to a 384-well plate. Reaction mixtures contained 10 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% BSA, 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20% to 30% phosphorylation. The assays were stopped with 15 μL of 180 mM HEPES, pH 7.4, 20 mM EDTA, and 0.2% Coating Reagent 3. The assay plates were placed on a Caliper Life Science LC3000 instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 467.43, ALogP 4.18, HBA 7, HBD 0, PSA 83.52, QED 0.41
Compound (SMILES): CCOc1cc(-c2ccc(N3CCC(C)(NC(=O)C4(N(C)C)CCC4)CC3)nc2)c2c(C#N)cnn2c1
InChIKey: FWZXZSNQQKXLJH-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of Formula I were screened for their ability to inhibit wildtype and V804M mutant RET kinase using CisBio's HTRF KinEASE-TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Catalog No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Catalog No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 44.6 nM (pChEMBL 7.349999904632568)
Descriptors: MW 501.64, ALogP 3.63, HBA 8, HBD 1, PSA 98.79, QED 0.53
Compound (SMILES): Nc1nc(-c2ccc(F)cc2)c(-c2ccc(=O)n(C3CS(=O)(=O)C3)c2)c2nc(Cc3c(F)cccc3F)nn12
InChIKey: SALOGHGNVNNDIX-UHFFFAOYSA-N
Target: Adenosine receptor A1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30542
Assay: FLIPR and cAMP Inhibition Assay: hADORA1/CHO (hA1 expressing) cells (Genscript) were plated at 1×104 cells/well into 384-well polystyrene plates one day before starting the experiment. On the day of experiment, the supernatant was discard and replaced with 40 L of dye (FLIPR calcium 5 Assay Kit) per well and the plates were incubated for 60 mins at 37° C. plus 5% CO2. Then testing compounds were added at different concentrations for FLIPR inhibition assay. After a 400 s incubation with compound, 10 M adenosine was added into the cells, and the signal was captured by FLIPR.hA2a/CHO, hA2b/CHO and mA2a/CHO (Genscript) were plated at 5×103 cells/well into 384-well polystyrene plates at the day of experiment. Compounds were pre-incubated with cells for 30 min at 37° C., 5% CO2. Then 10 M adenosine was added to the cells and incubated for 30 min at 37° C., 5% CO2. Detection reagent (CISBIO) was added and the plates were incubated for 60 min at room temperature. The signal was captured by Envision.
Measurement: IC50 = 40 nM (pChEMBL 7.400000095367432)
Descriptors: MW 552.54, ALogP 3.18, HBA 9, HBD 1, PSA 125.24, QED 0.36
Compound (SMILES): Cc1ccc(-n2nccn2)c(C(=O)N2C3CC(C3)[C@@H](C)C2CNc2nc3cccnc3s2)n1
InChIKey: SFHUAJBMKRKKSN-XYLATYTLSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Scintillation Proximity Assay (SPA) Binding Assay: CHO cells stably transfected with human Orexin type 1 receptors (CHO-hOX1) or HEK-293 cells transiently transfected with human Orexin type 2 receptors (HEK-hOX2) were collected after 16 h induction with 5 mM sodium butyrate. The cell pellets were re-suspended, homogenized in 15 mM Tris/HCl pH=7.5, 1 mM EGTA, 0.3 mM EDTA, 2 mM MgCl2, protease inhibitors and centrifuged at 40,000 g (20 min, 4° C.). After re-suspension, homogenization and centrifugation as above, the final pellets were re-suspended in 75 mM Tris/HCl pH=7.5, 1 mM EGTA, 0.3 mM EDTA, 12.5 mM MgCl2, 250 mM Sucrose, protease inhibitors, divided into aliquots and frozen down at −80° C.Compounds of invention were serially diluted in neat DMSO at 100-fold concentrations (1% DMSO final in the assay) and 2 μl/well were plated into 96-well Isoplates (Perkin Elmer).
Measurement: Ki = 4365 nM (pChEMBL 5.360000133514404)
Descriptors: MW 460.57, ALogP 3.33, HBA 9, HBD 1, PSA 101.72, QED 0.49
Compound (SMILES): Cn1cc(-c2c(-c3ncco3)nc(N)n3nc(Cc4ncccc4F)nc23)ccc1=O
InChIKey: ANVNFQJDCKBOSL-UHFFFAOYSA-N
Target: Adenosine receptor A2b (SINGLE PROTEIN), Homo sapiens
UniProt: P29275
Assay: FLIPR and cAMP Inhibition Assay: hADORA1/CHO (hA1 expressing) cells (Genscript) were plated at 1×104 cells/well into 384-well polystyrene plates one day before starting the experiment. On the day of experiment, the supernatant was discard and replaced with 40 L of dye (FLIPR calcium 5 Assay Kit) per well and the plates were incubated for 60 mins at 37° C. plus 5% CO2. Then testing compounds were added at different concentrations for FLIPR inhibition assay. After a 400 s incubation with compound, 10 M adenosine was added into the cells, and the signal was captured by FLIPR.hA2a/CHO, hA2b/CHO and mA2a/CHO (Genscript) were plated at 5×103 cells/well into 384-well polystyrene plates at the day of experiment. Compounds were pre-incubated with cells for 30 min at 37° C., 5% CO2. Then 10 M adenosine was added to the cells and incubated for 30 min at 37° C., 5% CO2. Detection reagent (CISBIO) was added and the plates were incubated for 60 min at room temperature. The signal was captured by Envision.
Measurement: IC50 = 270 nM (pChEMBL 6.570000171661377)
Descriptors: MW 418.39, ALogP 1.85, HBA 10, HBD 1, PSA 130.02, QED 0.47