lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CNC(=O)CN[C@@H]1CCc2c(-c3noc(-c4ccc(OC(C)C)c(C#N)c4)n3)cccc21
InChIKey: FNPKADWHWBGWMW-HXUWFJFHSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: Agonist activity at human S1P1 expressed in CRE-bla CHOK1 cells assessed as inhibition of forskolin-induced cAMP formation preincubated for 4 hrs followed by FRET-based beta-lactamase fluorescent substrate addition measured after 2 hrs
Measurement: EC50 = 0.05 nM (pChEMBL 10.300000190734863)
Descriptors: MW 431.5, ALogP 3.39, HBA 7, HBD 2, PSA 113.07, QED 0.59
Compound (SMILES): C/C=C\c1ccc2c(NCc3ccc(NC(=O)C4CCN(Cc5ccccc5)CC4)cc3)nc(N(C)C)nc2c1
InChIKey: CSQCUQGTDYPDOZ-YWEYNIOJSA-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 = 305 nM (pChEMBL 6.519999980926514)
Descriptors: MW 534.71, ALogP 6.19, HBA 6, HBD 2, PSA 73.39, QED 0.27
Compound (SMILES): C[C@@H]1CCC[C@H](C)N1CCNC(=O)c1ccc(F)c(NC(=O)c2cnc3cc(-c4ccnn4C)ccn23)c1
InChIKey: ZSKVYLYUXZNFEG-KDURUIRLSA-N
Target: Platelet-derived growth factor receptor beta (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q05030
Assay: Inhibition of PDGFR-beta driven proliferation of rat A10 cells after 68 hrs in presence of rat recombinant PDGF-BB by cell titer-glo luminescence assay
Measurement: IC50 = 8.7 nM (pChEMBL 8.0600004196167)
Descriptors: MW 517.61, ALogP 4.12, HBA 7, HBD 2, PSA 96.56, QED 0.38
Compound (SMILES): COc1ncc(C(C)C)cc1-c1cnc(N2CCC[C@@H]2C)nc1CN1C(=O)O[C@H](c2cc(C(F)(F)F)cc(C(F)(F)F)c2)[C@@H]1C
InChIKey: JSWPBEFVLQPJBX-XWXLMPLOSA-N
Target: Cholesteryl ester transfer protein (SINGLE PROTEIN), Homo sapiens
UniProt: P11597
Assay: In Vitro Radioactive Assays of CETP-Catalyzed CE and TG Transfer (RTA Assay): Radiolabeled donor particles are generated by first combining 100 μl of 200 μM butylated hydroxyl toluene in CHCl3, 216 μL of 21.57 mM DOPC in EtOH, and either 500 μCi [3H]-triolein (Perkin Elmer #NET-431) or 500 μCi [3H]-cholesteryl oleate (GE #TRK886) in a glass tube. Reagents are mixed, dried under nitrogen, and then resuspended in 2 mL of 50 mM Tris, 27 μM EDTA at pH 7.4. After a brief vortex, the solution is sonicated until clear and mixed with 20 mL of fresh human serum. The mixture is incubated overnight at 37° C. The [3H] labeled LDL substrate is separated at 1.063 g/ml density by sequential ultracentrifugal flotation in NaBr according to the method by (Havel, Eder et al. 1955; Chapman, Goldstein et al. 1981). Once isolated the particles are dialyzed 3× in CETP buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA). Human HDL is purchased from Intracel and used as the acceptor particles. Transfer assays are performed in a 96 or 384-well v-bottom polypropylene plate. For the RTA using recombinant CETP (2% RTA), an assay cocktail is prepared with the final concentrations 128 μg/mL HDL, 20 nM rCETP, 2% human serum, and 1×CETP buffer. 1 μL of each test compound diluted in DMSO is added to 47 μL of assay cocktail per well and incubated at 37° C. for 1 hour. To initiate the transfer reaction, 2 μL radiolabeled LDL is added. After an additional 60 min of incubation at 37° C., the transfer action is terminated by precipitation of LDL with an equal volume of 20% W/V PEG 8000. The plates are centrifuged at 2000 rpm for 30 minutes at 4° C. A 40 μL aliquot of the HDL-containing supernatant is transferred to a Packard Optiplate™ with 200 μL of MicroScint™ 20. After mixing, plates are counted by liquid scintillation.
Measurement: IC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 637.62, ALogP 7.78, HBA 7, HBD 0, PSA 80.68, QED 0.24
Compound (SMILES): COc1cc(C2CCOCC2)c(F)cc1-n1c(=O)ccc2cc(S(=O)(=O)Nc3ccon3)ccc21
InChIKey: QWVZWBBJFGPQPP-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Nav1.7 In Vitro PX Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to -10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes.
Measurement: IC50 = 3070 nM (pChEMBL 5.510000228881836)
Descriptors: MW 499.52, ALogP 3.82, HBA 8, HBD 1, PSA 112.66, QED 0.43
Compound (SMILES): COCCO[C@H]1C=CC2C3Cc4ccc(OC)c5c4[C@@]2(CCN3C)C1O5
InChIKey: CCFZKAPEUYIPRY-CSHLKKEHSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Scintillation Proximity Assay (SPA): Briefly, serial dilutions of the test compounds were placed in a 96-well plate to which were added SPA beads, membrane and radioligand. The assay conditions for each opioid receptor subtype are described in Table 10 below. The plates were incubated for 8 hours-overnight at room temperature, spun at 1000 rpm to pellet the SPA beads, and radioactivity was measured using the TopCount microplate Scintillation counter. Specific binding at each concentration of test compound was calculated by subtracting the non-specific binding measured in the presence of excess cold ligand. IC50 values were obtained by non-linear regression of specific binding versus concentration curves and Ki values were calculated using Kd values that were experimentally pre-determined for each lot of membrane preparations.
Measurement: Ki = 1821.51 nM (pChEMBL 5.739999771118164)
Descriptors: MW 357.45, ALogP 2.17, HBA 5, HBD 0, PSA 40.16, QED 0.6
Compound (SMILES): CS(=O)(=O)c1ccc(-n2nc(C(N)=O)c3c2-c2cc(NC(=O)c4cccc([N+](=O)[O-])c4Cl)ccc2CC3)cc1
InChIKey: KIMFJVAZTQMQKK-UHFFFAOYSA-N
Target: Inhibitor of nuclear factor kappa-B kinase subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O14920
Assay: Inhibition of human recombinant C-terminal FLAG-epitope-tagged IKK2 expressed in baculovirus using biotinylated IkappaBalpha peptide as substrate assessed as decrease in [gamma-33P]-ATP incorporation in substrate after 30 mins by scintillation counting method
Measurement: IC50 <= 1000 nM (pChEMBL 6)
Descriptors: MW 566, ALogP 3.95, HBA 8, HBD 2, PSA 167.29, QED 0.26
Compound (SMILES): CN[C@@H](C)C(=O)N[C@H](C(=O)N1Cc2ccccc2C1C(=O)NCc1ccccc1)C(C)C
InChIKey: DWYXOSQSORZHMJ-MNDAUZPPSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay: The peptide AVPIAQKSEK-(ε-biotin)-OH 1:2 TFA ("Peptide A") was identified as a substrate for the TR-FRET assay by screening the 6× Histidine-tagged BIR2 domain and BIR3 domain of XIAP against a set of 29 peptides synthesized based on sequences reported by Sweeny et al. (Biochemistry, 2006, 45, 14740 14748). The peptides were labeled with the fluorescent tags FITC or TAMRA and Kd values were determined by fluorescence polarization assay. The sequence AVPIAQKSEK was identified as optimal for using in an assay. The peptide sequence was derivatized with biotin to provide AVPIAQKSEK-(Îµ-biotin)-OH 1:2 TFA as the substrate for the TR-FRET assay. Ten nanomolar of 6× Histidine-tagged BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(ε-biotin)-OH 1:2 TFA, in the presence of 50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/mL bovine serum albumin (BSA). Following a 45 min. incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations.
Measurement: IC50 = 210 nM (pChEMBL 6.679999828338623)
Descriptors: MW 436.56, ALogP 2.14, HBA 4, HBD 3, PSA 90.54, QED 0.59
Compound (SMILES): COC1CN(C(=O)c2ccc(-c3cc4c(-c5cccc(N6CCOc7cc(C8CC8)ccc7C6=O)c5C)ncnc4[nH]3)cc2)C1
InChIKey: WZPBWQPDZTYPQA-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Recombinant Human BTK Inhibition Assay: The inhibition assays were performed using the generic tyrosine kinase assay kit from C is Bio (HTRF KinEASE-TK #62TK0PEB). The assay is based on the principle of fluorescence resonance energy transfer (FRET) between the Europium Cryptate labeled anti-phosphotyrosine antibody and streptavidin labeled fluorophore, XL-665. In an uninhibited kinase reaction the biotinylated peptide substrate (Tk-Biotin) gets phosphorylated at an intermediate tyrosine site which is captured by anti-phosphotyrosine antibody which eventually acts as a FRET donor. The streptavidin labeled XL-665 binds strongly to the Tk-Biotin substrate and act as a FRET acceptor. The resultant FRET signal is measured at 665 nm. A ratio of this specific fluorescence (665 nm) to the background fluorescence (620 nm), conferred by the free antibody, is directly proportional to the kinase activity. Inhibition of the kinase activity by a pharmacological intervention such as a Btk inhibitor of the present disclosure will result into decreased FRET signal.
Measurement: IC50 = 162 nM (pChEMBL 6.789999961853027)
Descriptors: MW 599.69, ALogP 5.99, HBA 6, HBD 1, PSA 100.65, QED 0.26
Compound (SMILES): CCCN(Cc1ccc(OCCN2C(=O)CCC2=O)c(OC)c1)C(CC(=O)O)c1cc(C)sc1C
InChIKey: OJFWWDLILJKVHT-UHFFFAOYSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: Radioligand Binding Assay: The composition of the binding assay buffer is determined in a course of detailed optimization procedure. This resulted in a binding assay buffer constituted by the following components: 25 mM Hepes (pH=7.4), 5 mM MgCl2, 1 mM CaCl2, 100 mM NaCl, supplemented with 0.1% of protease free BSA (as a final concentration). Competition binding assay is performed using 125I-CXCL10 (PerkinElmer, NEX348, specific activity 2200 Ci/mmol) radioligand in a final concentration of 50-70 pM. The nonspecific binding is defined by 150 pM of hr-CXCL10 (R&D Systems, Cat No 266-IP). The total assay volume is equal to 150 ul and contained 1% of DMSO (final concentration). Binding reaction is initiated by adding of membranes (10-20 ug proteins, approximately 5x105 cell equivalents) to the reaction mixture. After 60 minutes of incubation at 25° C. the reaction is terminated by rapid filtration over GF/B glass fibre filters that are pre-soaked with 0.5% polyethyleneimine (Fluka Analytical, P3143) for 1 hour, using a Skatron cell harvester device. Filters then are washed with 8 ml of ice-cold wash buffer (modified binding buffer in which BSA is omitted and the concentration of NaCl is adjusted to 500 mM concentration). The radioactivity retained on the filters is measured by a Wizard 1470 Automatic Gamma counter.
Measurement: IC50 = 197 nM (pChEMBL 6.710000038146973)
Descriptors: MW 502.63, ALogP 4.33, HBA 7, HBD 1, PSA 96.38, QED 0.41
Compound (SMILES): Cn1cnc(S(=O)(=O)NCc2cccc(C(Cc3ccccc3)C(=O)N3CCOCC3)c2)c1
InChIKey: RGZQWLFZGIRJMF-UHFFFAOYSA-N
Target: Sodium- and chloride-dependent glycine transporter 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P48067
Assay: Radioligand Binding Assay: Radioligand binding to human GlyT1c transporter-expressing membranes was determined as described in Mezler et al., Molecular Pharmacology 74:1705-1715, 2008.
Measurement: Ki < 10 nM (pChEMBL 8)
Descriptors: MW 468.58, ALogP 2.08, HBA 6, HBD 1, PSA 93.53, QED 0.55
Compound (SMILES): CCOc1ccc(CC2CCN(c3ncc(C(=O)NO)cn3)CC2)cc1-c1nc2c(CC)nn(C)c2c(=O)[nH]1
InChIKey: VLMYLGPYYNFPET-UHFFFAOYSA-N
Target: cGMP-specific 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O76074
Assay: Inhibition of full length recombinant human N-terminal GST-tagged PDE5A1 expressed in baculovirus infected Sf9 cells using cGMP as substrate after 30 mins by HTRF assay
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 532.61, ALogP 2.65, HBA 10, HBD 3, PSA 151.15, QED 0.23
Compound (SMILES): CNS(=O)(=O)c1ccc(N(C)C)c(Nc2ncnc3[nH]cc(-c4ccco4)c23)c1
InChIKey: POFMJJCZZYDTJE-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase TNNI3K (SINGLE PROTEIN), Homo sapiens
UniProt: Q59H18
Assay: Displacement of 5-({[2-({[3-({4-[(5-hydroxy-2-methylphenyl)amino]-2-pyrimidinyl}amino)phenyl]carbonyl}amino)-ethyl]amino}carbonyl)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid from His6-MBP-TEV-full length human TNNI3K expressed in Baculovirus expression system after 60 mins by fluorescence polarization assay
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 412.48, ALogP 2.94, HBA 7, HBD 3, PSA 116.15, QED 0.45
Compound (SMILES): C=CCN1CCC(c2ccc(C(OCc3c(F)cccc3F)(C(F)(F)F)C(F)(F)F)cc2)(S(=O)(=O)c2ccc(F)cc2)C1
InChIKey: YEGUUVCZFBVYDH-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Scintillation Proximity Assay (SPA): The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 ug/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 uM to 0.28 nM for 10 min at room temperature in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% Glycerol (Sigma Cat#G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat #RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce [3H] 25-hydroxycholesterol specific binding by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 51 nM (pChEMBL 7.289999961853027)
Descriptors: MW 637.56, ALogP 7.2, HBA 4, HBD 0, PSA 46.61, QED 0.14
Compound (SMILES): CC(C)(C)CN1CCC2(CC1)CN(c1ccccc1NC(=O)Nc1csc(C(F)(F)F)n1)c1c(O)ccc(-c3nc4ccc(F)cc4s3)c12
InChIKey: LXHOMCBPMYHVFB-UHFFFAOYSA-N
Target: P2Y purinoceptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P47900
Assay: Scintillation Proximity Assay (SPA): A SPA membrane binding assay was used to identify inhibitors of [33P] 2MeS-ADP binding to cloned human P2Y1 receptors (The P2Y1 receptor membranes were provided by Biology and the cloning of the receptor and P2Y1 receptor membrane preparation is same as described by Biology). Binding reactions were performed in 384-well OptiPlates (PerkinElmer Life Sciences, Cat #6007299) in a volume of 50 uL containing 15 fmol of P2Y1 receptor (1.7 ug of total protein), 0.3 nM [33P] 2MeS-ADP (PerkinElmer; 2,000 Ci/mmol), various concentrations of the test compound (usually between 10 uM and 160 pM) in Buffer B containing 1% DMSO in assay buffer (15 mM, HEPES, 145 mM potassium chloride, 5 mM sodium Chloride, 5 mM EDTA, 0.1 mM MgCl2, pH 7.4) and 100 ug of SPA bead (WGA polystyrene Image beads, #RPNQ 0260V, Amersham). Reactions were allowed to proceed to completion at room temperature for 1 hour followed by centrifugation of the plate for 5 min. About 40 uL of the aqueous soluttion was aspirated. Plates were sealed and the [33P] 2MeS-ADP bound to the P2Y1 receptor membranes that were bound to the SPA bead were determined in a Gen 4 LEADSEEKERSM (Amersham) Image Reader. Dose-response curves (IC50) were fit by non-linear regression (Toolset an in house data processing program) and binding constants (Ki) calculated using the Cheng-Prusoff relationship (Ki=IC50/(1+L/Kd) in which a Kd for 2MeS-ADP to the P2Y1 receptor was determined to be 14 nM.
Measurement: Ki = 12.4 nM (pChEMBL 7.909999847412109)
Descriptors: MW 710.82, ALogP 9.46, HBA 8, HBD 3, PSA 93.62, QED 0.16
Compound (SMILES): Cc1nonc1CNc1nc(NC2CC2)n2ncc(/C=C3\NC(=O)NC3=O)c2n1
InChIKey: QEYGPZQLNLVJCT-WMZJFQQLSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: 5 uM ATP: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 5 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and
Measurement: IC50 > 2500 nM (pChEMBL 5.6020002365112305)
Descriptors: MW 396.37, ALogP 0.18, HBA 11, HBD 4, PSA 164.26, QED 0.33
Compound (SMILES): COc1cc(F)cc(N2CCC(CC#N)(n3cc(C(N)=O)c(Nc4ccc(F)cc4)n3)CC2)c1
InChIKey: PEKVQDACCJXPJP-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 466.49, ALogP 3.92, HBA 7, HBD 2, PSA 109.2, QED 0.55
Compound (SMILES): CC#CCOc1cnc(C(=O)Nc2csc([C@]34CO[C@@H](C)C[C@H]3CSC(N)=N4)n2)cn1
InChIKey: VBTNLZCJDCSFOZ-QAJFTPDKSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE1 Cell-Free Assay: The substrate is Biotin-GLTNIKTEEISEISY^EVEFR-C[Oregon Green]KK-OH. The BACE1 enzyme is affinity purified material from conditioned media of CHO-K1 cells that have been transfected with a soluble BACE construct (BACE1deltaTM96His). Compounds are incubated in a ½ log dose response curve from a top concentration of 100 μM with BACE1 enzyme and the biotinylated fluorescent peptide in 384-well black plates (Thermo Scientific #4318). BACE1 is at a final concentration of 0.1 nM with a final concentration of peptide substrate of 150 nM in a reaction volume of 30 μL assay buffer [100 mM sodium acetate, pH 4.5 (brought to pH with acetic acid), and 0.001% Tween-20]. Plates are covered and incubated for 3 hours at 37 °C. The reaction is stopped with the addition of 30 μL of 1.5 μM Streptavidin (Pierce, #21125). After a 10 minute incubation at room temperature, plates are read on a PerkinElmer EnVision for fluorescence polarization (Ex485 nm/Em530 nm).
Measurement: IC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 458.57, ALogP 2.27, HBA 10, HBD 2, PSA 124.61, QED 0.65
Compound (SMILES): CN1CCC(Cc2ccccc2)(c2cccc(OCCN(C)S(=O)(=O)c3cn(C)cn3)c2)C1
InChIKey: PKUNQFTZTCBZGM-UHFFFAOYSA-N
Target: Sodium- and chloride-dependent glycine transporter 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P48067
Assay: Radioligand Binding Assay: Radioligand binding to human GlyT1c transporter-expressing membranes was determined as described in Mezler et al., Molecular Pharmacology 74:1705-1715, 2008.
Measurement: Ki < 1 nM (pChEMBL 9)
Descriptors: MW 468.62, ALogP 2.94, HBA 6, HBD 0, PSA 67.67, QED 0.48
Compound (SMILES): COc1ncc(-c2ccc3nccc(N4CCOCC4)c3c2)cc1NS(=O)(=O)c1ccc(F)cc1F
InChIKey: AYHRETHQQQYRCJ-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Inhibition of PI3Kalpha (unknown origin) using PIP2 as substrate after 1 hr in presence of ATP by kinase-glo luminescence assay
Measurement: IC50 = 1.4 nM (pChEMBL 8.850000381469727)
Descriptors: MW 512.54, ALogP 4.22, HBA 7, HBD 1, PSA 93.65, QED 0.41
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3)ncnc2cc1OCc1nccn1Cc1ccccc1
InChIKey: PVTTWYOJLADNCZ-UHFFFAOYSA-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 = 1000 nM (pChEMBL 6)
Descriptors: MW 506.39, ALogP 6.51, HBA 7, HBD 1, PSA 74.09, QED 0.26
Compound (SMILES): Cc1c(-c2cccc(C(F)(F)F)c2)c(=O)c(C(=O)NCc2ccc(S(C)(=O)=NC#N)cc2)cn1C1CCOC1
InChIKey: AOIFTRNKICFEAI-UHFFFAOYSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Human Neutrophil Elastase Assay: The following buffers were used: Compound buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5; Assay buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5, containing 0.01% BSA.Assay conditions: Test compounds were prediluted in DMSO and subsequently in compound buffer (5% DMSO final). 5 μL of these compound dilutions were mixed with 10 μl Neutrophil elastase (9 ng/ml in assay buffer) in a black 384 well OptiPlate (Perkin Elmer, Cat No.: 6007270) and incubated for 15 min at room temperature. Subsequently 10 μL substrate solution in assay buffer were added (250 μM final concentration) and the plates were incubated for 60 min at room temperature. After inactivation of the enzyme, fluorescence intensities were measured at 380 nm excitation and 460 nm emission wavelengths.
Measurement: IC50 = 2.3 nM (pChEMBL 8.640000343322754)
Descriptors: MW 558.58, ALogP 4.67, HBA 7, HBD 1, PSA 113.55, QED 0.44
Compound (SMILES): CN(C)CCNC(=O)Cn1ccc(Nc2ncc3c(n2)-c2c(nn(C)c2Cc2cccc(-c4cnn(C)c4)c2)CC3)n1
InChIKey: RVIMGPNKJMIZJW-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: DELFIA Assay: The kinase activity is measured by DELFIA assay (dissociation-enhanced lanthanide fluorescence immunoassay, Perkin Elmer). The cytoplasmic kinase domain of human IGF-1R (amino acids 964-1370) is expressed as a fusion protein with a glutathione-S-transferase tag (IGF-1R-GST) in High Five.TM. Cells (Invitrogen). Enzyme activity is measured in the presence of substances and a control substance. Poly-glutamate-tyrosine peptide (pEY, Sigma Aldrich) and biotinylated pEY (bio-pEY) are used as reaction substrates.10 uL of substance in 25% DMSO are mixed with 30 uL of IGF-1R-GST solution (67 mM HEPES pH 7.4, 15 ug/mL pEY, 1.7 ug/mL bio-pEY, 13.3 mM MgCl2, 3.3 mM dithiothreitol, 0.0033% Brij 35, 2 ng IGF-1R-GST) in 96-well plates. The reactions are started with 10 uL of a 750 uM ATP solution. After 40 min at RT the reactions are stopped with 50 .mu.L of stop solution (250 mM EDTA, 20 mM HEPES pH 7.4). 90 uL from each reaction are transferred.
Measurement: IC50 = 0.57 nM (pChEMBL 9.239999771118164)
Descriptors: MW 565.69, ALogP 2.58, HBA 11, HBD 2, PSA 123.61, QED 0.26
Compound (SMILES): CN(C)C(=O)Oc1cccc(/C=C/C(=O)c2cccc(O)c2)c1
InChIKey: DMIPCQXUJFTZCN-MDZDMXLPSA-N
Target: Cholinesterase (SINGLE PROTEIN), Homo sapiens
UniProt: P06276
Assay: Inhibition of human serum BChE using S-Butyrylthiocholine iodide as substrate preincubated for 15 mins followed by substrate addition by Ellman's method
Measurement: IC50 = 1910 nM (pChEMBL 5.71999979019165)
Descriptors: MW 311.34, ALogP 3.35, HBA 4, HBD 1, PSA 66.84, QED 0.69
Compound (SMILES): Cc1nccn1-c1ccc(-c2cn(CC(=O)N(C)c3cccc(Cl)c3Cl)nn2)cc1
InChIKey: RBSGOJXGJTVMRX-UHFFFAOYSA-N
Target: Probable G-protein coupled receptor 142 (SINGLE PROTEIN), Mus musculus
UniProt: Q7TQN9
Assay: Agonist activity at mouse GPR142 expressed in CHO cells measured for 3 to 5 mins by Fluo-4AM dye-based FLIPR assay
Measurement: EC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 441.32, ALogP 4.41, HBA 6, HBD 0, PSA 68.84, QED 0.46
Compound (SMILES): Cc1ncc(-c2cc(C(N)=O)nc3cc(CN4CCOC(C(F)(F)F)C4)ccc23)o1
InChIKey: QWYGBQLZCIUSCA-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: Biological Assay: The utility of the compounds in accordance with the present invention as antagonists of metabotropic glutamate receptor activity, in particular mGluR2 activity, may be demonstrated by methodology known in the art. Antagonist constants are determined as follows. The compounds of the present invention were tested in a fluorescence laser imaging plate reader based assay. This assay is a common functional assay to monitor Ca2+ mobilization in whole cells expressing recombinant receptor coupled with a promiscuous G-protein. CHO dhfr- cells stably expressing recombinant human mGluR2 and Gα16 loaded with Fluo-4 AM (Invitrogen, Carlsbad Calif.) are treated with various concentrations of antagonists of compounds and the Ca2+ response is monitored on a FLIPR384 (Molecular Devices, Sunnydale Calif.) for agonist activity.
Measurement: IC50 = 1629 nM (pChEMBL 5.789999961853027)
Descriptors: MW 420.39, ALogP 3.06, HBA 6, HBD 1, PSA 94.48, QED 0.7
Compound (SMILES): Cc1cc(C)c(Oc2nc(NC3CCN(Cc4cccc(F)c4)CC3)nc3ccsc23)c(C)c1
InChIKey: IWJKUKULINTISM-UHFFFAOYSA-N
Target: Human immunodeficiency virus type 1 reverse transcriptase (SINGLE PROTEIN), Human immunodeficiency virus 1
UniProt: Q72547
Assay: Inhibition of recombinant HIV1 reverse transcriptase K103N/Y181C double mutant using DIG-dUTP/biotin-dUTP/dTTP assessed as suppression of biotin-dUTP incorporation after 1 hr by ELISA
Measurement: IC50 = 40400 nM (pChEMBL 4.389999866485596)
Descriptors: MW 476.62, ALogP 6.62, HBA 6, HBD 1, PSA 50.28, QED 0.34
Compound (SMILES): NC(=O)C12CC3CC(C1)C(NC(=O)c1cccc(NS(=O)(=O)c4ccccc4C(F)(F)F)c1)C(C3)C2
InChIKey: QEUFHIFPOKFCOD-UHFFFAOYSA-N
Target: 11-beta-hydroxysteroid dehydrogenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28845
Assay: HTRF Assay : The inhibiting activity of 11β-HSD1 derived from microsomal fractions was measured using the HTRF assay (62CO2PEB, Cisbio). Different concentrations of compounds were added to 96-well plates, followed by the addition of TE buffer (20 mM Tris buffer and 5 mM EDTA, pH 6.0) containing 200 μM NADPH (N1630, Sigma) and 160 nM cortisone (C2755, Sigma), The reactions were initiated by the addition of human microsomal fractions (M0317, Sigma), and were allowed to incubate for 2 h at 37° C. Europium (Eu3+) cryptate and XL665-conjugated cortisol were then added to each well and incubated for an additional 2 h at room temperature.
Measurement: IC50 = 19.1 nM (pChEMBL 7.71999979019165)
Descriptors: MW 521.56, ALogP 3.92, HBA 4, HBD 3, PSA 118.36, QED 0.53
Compound (SMILES): CC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@H](C(=O)N[C@@H](Cc1cnc[nH]1)C(=O)NCC(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(N)=O)C(C)C)C(C)C
InChIKey: MRYHHQIDXHBLJV-PLCDDLMESA-N
Target: Tissue factor pathway inhibitor (SINGLE PROTEIN), Homo sapiens
UniProt: P10646
Assay: Binding affinity to TFPI (unknown origin) incubated for 1.5 hrs using tracer peptide JBT0131 by screening ELISA
Measurement: IC50 = 101 nM (pChEMBL 7)
Descriptors: MW 2423.82
Compound (SMILES): Cc1c(N(CCCCC(F)(F)F)S(=O)(=O)c2ccc(C(=O)O)cc2F)sc2ccccc12
InChIKey: XYLNIVZECDDWBK-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Functional Assay: The functional activity of compounds of the formula (I) was determined by measuring changes in intracellular calcium concentration using a Ca2+-sensitive fluorescent dye. The changes in fluorescent signal were monitored by a fluorescence plate reader, either a FLIPR (Molecular Devices) or FDSS (Hamamatsu). Increases in intracellular Ca2+ concentration were readily detected upon activation with icilin.At 24 hrs prior to assay, HEK293 cells stably expressing canine TRPM8 were seeded in culture medium in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences, NJ, USA) and grown overnight in 5% CO2 at 37° C. On assay day, growth media was removed and cells were loaded with Calcium 3 Dye (Molecular Devices) for 35 min at 37° C., under 5% CO2 and then for 25 min at room temperature and atmosphere. Subsequently, cells were tested for agonist-induced increases in intracellular Ca2+ levels using FLIPR or FDSS. Cells were challenged with a compound of the Formula (I) (at varying concentrations) and intracellular Ca2+ was measured for 5 min prior to the addition of icilin to all wells to achieve a final concentration that produces approximately an 80% maximal response. EC50 or IC50 values for compounds of the present invention were determined from eight-point dose-response studies.
Measurement: IC50 = 4.2 nM (pChEMBL 8.380000114440918)
Descriptors: MW 489.51, ALogP 5.97, HBA 4, HBD 1, PSA 74.68, QED 0.31
Compound (SMILES): CCOc1ccc(-c2ccc(C(=O)NO)o2)cc1-c1nc2c(CC)nn(C)c2c(=O)[nH]1
InChIKey: PLRVHXKDQUSQCB-UHFFFAOYSA-N
Target: cGMP-specific 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O76074
Assay: Inhibition of full length recombinant human N-terminal GST-tagged PDE5A1 expressed in baculovirus infected Sf9 cells using cGMP as substrate after 30 mins by HTRF assay
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 423.43, ALogP 2.66, HBA 8, HBD 3, PSA 135.27, QED 0.32
Compound (SMILES): COC[C@@H]1CN(C(=O)c2ccc3cc(Oc4ccc(C(F)(F)F)cn4)ccc3n2)CCN1
InChIKey: BCOHYAVYZWPAHD-INIZCTEOSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FRET-Based Membrane Potential Assay: Two days prior to the experiment, frozen HEK293 cells stably expressing recombinant human Nav1.7 were quickly thawed and plated at 25,000 cells/well in growth medium [DMEM (Invitrogen #11965) with 10% HI FBS (Invitrogen #10082), 2 mM glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin (PSG, Sigma #G1146), and 500 ug/mL Geneticin (Invitrogen #10131)] in black-walled, clear-bottom 384-well poly-D-lysine-coated assay plates (Greiner Bio-One, Frickenhausen, Germany) and incubated in a humidified 5% CO2 incubator at 37° C. On the day of the assay, medium was removed by aspiration, and cells were washed with assay buffer [HBSS (Invitrogen, Carlsbad, Calif.) containing 20 mM HEPES (Invitrogen, Carlsbad, Calif.)]. After washing, 30 uL assay buffer containing the fluorescent voltage-sensor probe CC2-DMPE (Invitrogen, Carlsbad, Calif.) at 20 uM and 0.01% pluronic F-127 (Invitrogen, Carlsbad, Calif.) was added to the cells. Cells were incubated for 40 minutes at room temperature in the dark. Following the incubation, the cells were washed and 30 uL assay buffer containing 2.5 uM DiSBAC2(3) substrate (Invitrogen, Carlsbad, Calif.) and 0.5 mM VABSC-1 (Invitrogen, Carlsbad, Calif.) was added to the cells. The cells were incubated for 90 minutes at room temperature in the dark. Fluorescence readings were made using a FLIPR TETRA (Molecular Devices, Sunnyvale Calif.) equipped with voltage-sensor probe optics. At the start of each experiment the optimal (EC80) concentration of depolarizing agent (veratridine) was determined by testing a dilution curve of assay buffer containing veratridine (Sigma-Aldrich, St. Louis, Mo.) and 1 mg/mL scorpion venom (SVqq, from Leiurus quinquestriatus; Sigma-Aldrich, St. Louis, Mo.). Compounds were dissolved in dimethyl sulfoxide, and 8-point, 1:3 dilution concentration-response curves were prepared in duplicate in dimethyl sulfoxide, followed by preparation of 0.8 uL/well daughter plates of the dilutions. Test compounds in the daughter plate were diluted to (Ë¿3x) solutions in assay buffer immediately before assaying. Using the FLIPR TETRA, 20 uL of the (3x) compound solutions were first added to the cells, then 20 uL of depolarizing solution (3xEC80 veratridine+SVqq) were added 3 minutes later to activate the channel. Changes in fluorescence were measured at wavelengths of 440-480 nm and 565-625 nm over the course of the experimental run. Membrane depolarization was expressed as a ratio of the maximum F440-480 nm/F565-625 nm reading above average baseline F440-480 nm/F565-625 nm reading. IC50 values were calculated from curve fits of the ratio data using a four-parameter logistic Hill equation (Accelrys Assay Explorer 3.3 Client, Accelrys, San Diego, Calif.) with percent inhibition plotted against compound concentration.
Measurement: IC50 = 238 nM (pChEMBL 6.619999885559082)
Descriptors: MW 446.43, ALogP 3.5, HBA 6, HBD 1, PSA 76.58, QED 0.65
Compound (SMILES): C#CCO[C@@H]1CC[C@H](Cc2ccccc2)N(C(=O)n2ncc(C(O)(c3ccc(F)cc3)c3ccc(F)cc3)n2)C1
InChIKey: GVLOFRPVRYAFKI-VSGBNLITSA-N
Target: Diacylglycerol lipase-alpha (SINGLE PROTEIN), Mus musculus
UniProt: Q6WQJ1
Assay: Inhibition of mouse DAGL-alpha expressed in HEK293T cell membranes pre-incubated for 20 mins before PNP-butyrate substrate addition and measured after 30 mins by surrogate substrate assay
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 542.59, ALogP 4.53, HBA 6, HBD 1, PSA 80.48, QED 0.35
Compound (SMILES): Cc1oncc1S(=O)(=O)NCCN1CC2CN(CCCOc3ccc(C#N)cc3)CC(C1)O2
InChIKey: DIJXFMJAYGGVBL-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Inhibition of human ERG expressed in CHO cells incubated for 10 mins after 3 hrs incubation with Rb+-load buffer by atomic absorption spectrometry based Rb+ efflux assay
Measurement: IC50 < 31622.78 nM (pChEMBL 4.5)
Descriptors: MW 475.57, ALogP 0.99, HBA 9, HBD 1, PSA 120.93, QED 0.5
Compound (SMILES): COc1ccc(S(=O)(=O)N(Cc2ccc(F)c(C(F)(F)F)c2)c2sc3ccccc3c2Br)cc1
InChIKey: NKNBLYXGWNDOHG-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Functional Assay: The functional activity of compounds of the formula (I) was determined by measuring changes in intracellular calcium concentration using a Ca2+-sensitive fluorescent dye. The changes in fluorescent signal were monitored by a fluorescence plate reader, either a FLIPR (Molecular Devices) or FDSS (Hamamatsu). Increases in intracellular Ca2+ concentration were readily detected upon activation with icilin.At 24 hrs prior to assay, HEK293 cells stably expressing canine TRPM8 were seeded in culture medium in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences, NJ, USA) and grown overnight in 5% CO2 at 37° C. On assay day, growth media was removed and cells were loaded with Calcium 3 Dye (Molecular Devices) for 35 min at 37° C., under 5% CO2 and then for 25 min at room temperature and atmosphere. Subsequently, cells were tested for agonist-induced increases in intracellular Ca2+ levels using FLIPR or FDSS. Cells were challenged with a compound of the Formula (I) (at varying concentrations) and intracellular Ca2+ was measured for 5 min prior to the addition of icilin to all wells to achieve a final concentration that produces approximately an 80% maximal response. EC50 or IC50 values for compounds of the present invention were determined from eight-point dose-response studies.
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 574.42, ALogP 7.23, HBA 4, HBD 0, PSA 46.61, QED 0.23
Compound (SMILES): O=C(Nc1ccc(OC2CCN(C(=O)c3ccc[nH]3)CC2)cc1)N1Cc2ccncc2C1
InChIKey: VZDZSLMHVUAMOZ-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 2:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1287128.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 431.5, ALogP 3.64, HBA 4, HBD 2, PSA 90.56, QED 0.66
Compound (SMILES): CN1CCN(CCCOC(=O)Nc2nc3ccc(C4(O)c5ccccc5C(=O)N4c4ccc(F)cc4)cc3[nH]2)CC1
InChIKey: ODFQIFPIQVUBCG-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition of B-Raf (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 558.61, ALogP 3.74, HBA 7, HBD 3, PSA 114.03, QED 0.3
Compound (SMILES): N#CCC1(n2cc(-c3ccnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)Nc3ccc(C#N)cc3C(F)(F)F)CC2)C1
InChIKey: ASWDIUCIMSRFHX-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Inhibition of human N-terminal His-tagged JAK1 catalytic domain (837 to 1142 residues) expressed in baculovirus infected insect cells assessed as decrease in phosphorylation of biotinylated peptide substrate after 1 hr in presence of 1 mM ATP by HTRF method
Measurement: IC50 > 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 573.58, ALogP 4.94, HBA 7, HBD 2, PSA 129.66, QED 0.35
Compound (SMILES): Nc1cn2nc(C#Cc3ccc(NC(=O)c4ccccc4)cc3)ccc2n1
InChIKey: KUSRQJDRZJPANQ-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: PDGFRbeta Kinase Assay: Biochemical KDR kinase assays were performed in 96 well microtiter plates that were coated overnight with 75 ug/well of poly-Glu-Tyr (4:1) in 10 mM Phosphate Buffered Saline (PBS), pH 7.4. The coated plates were washed with 2 mls per well PBS+0.05% Tween-20 (PBS-T), blocked by incubation with PBS containing 1% BSA, then washed with 2 mls per well PBS-T prior to starting the reaction. Reactions were carried out in 100 uL reaction volumes containing 2.7 uM ATP in kinase buffer (50 mM Hepes buffer pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2 and 0.2 mM Na3VO4). Test compounds were reconstituted in 100% DMSO and added to the reaction to give a final DMSO concentration of 5%. Reactions were initiated by the addition 20 ul per well of kinase buffer containing 200-300 ng purified cytoplasmic domain KDR protein (BPS Bioscience, San Diego, Calif.). Following a 15 minute incubation at 30° C., the reactions were washed 2 mls per well PBS-T.
Measurement: IC50 = 1248 nM (pChEMBL 5.900000095367432)
Descriptors: MW 353.39, ALogP 2.96, HBA 5, HBD 2, PSA 85.31, QED 0.54
Compound (SMILES): Cc1c(N(CCCF)S(=O)(=O)c2ccc(C(=O)O)cc2)sc2ccccc12
InChIKey: BHLJSLHGTBKRGE-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Functional Assay: The functional activity of compounds of the formula (I) was determined by measuring changes in intracellular calcium concentration using a Ca2+-sensitive fluorescent dye. The changes in fluorescent signal were monitored by a fluorescence plate reader, either a FLIPR (Molecular Devices) or FDSS (Hamamatsu). Increases in intracellular Ca2+ concentration were readily detected upon activation with icilin.At 24 hrs prior to assay, HEK293 cells stably expressing canine TRPM8 were seeded in culture medium in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences, NJ, USA) and grown overnight in 5% CO2 at 37° C. On assay day, growth media was removed and cells were loaded with Calcium 3 Dye (Molecular Devices) for 35 min at 37° C., under 5% CO2 and then for 25 min at room temperature and atmosphere. Subsequently, cells were tested for agonist-induced increases in intracellular Ca2+ levels using FLIPR or FDSS. Cells were challenged with a compound of the Formula (I) (at varying concentrations) and intracellular Ca2+ was measured for 5 min prior to the addition of icilin to all wells to achieve a final concentration that produces approximately an 80% maximal response. EC50 or IC50 values for compounds of the present invention were determined from eight-point dose-response studies.
Measurement: IC50 = 38 nM (pChEMBL 7.420000076293945)
Descriptors: MW 407.49, ALogP 4.46, HBA 4, HBD 1, PSA 74.68, QED 0.63
Compound (SMILES): C[C@H]1CCCN1C(=O)c1cc(C(=O)NCc2ccc(F)cc2Cl)c2n1CCOC2
InChIKey: VIDZHURHIIVMSA-ZDUSSCGKSA-N
Target: Potassium channel subfamily K member 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O14649
Assay: TASK-1 Inhibiition Assay: Human TASK-1 channels were expressed in Xenopus oocytes. For this purpose, oocytes were isolated from Xenopus laevis and defoliculated. Subsequently, TASK-1-encoding RNA synthesized in vitro was injected into the oocytes. After two days of TASK-1 protein expression, TASK-1 currents were measured by two-microelectrode voltage clamp. Data were acquired and analyzed using a TEC-10cx amplifier (NPI Electronic, Tamm, Germany) connected to an ITC-16 interface (Instrutech Corp., Long Island, USA) and Pulse software (HEKA Elektronik, Lambrecht, Germany). Oocytes were clamped to -90 mV and TASK-1 mediated currents were measured during 500 ms voltage pulses to 40 mV. Oocytes were continuously superfused with ND96 buffer containing 96 mM sodium chloride, 2 mM potassium chloride, 1.8 mM calcium chloride, 1 mM magnesium chloride, 5 mM 4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid (HEPES; pH adjusted to 7.4 with sodium hydroxide). All experiments were performed at room temperature.
Measurement: IC50 = 190 nM (pChEMBL 6.71999979019165)
Descriptors: MW 419.88, ALogP 3.37, HBA 4, HBD 1, PSA 63.57, QED 0.83
Compound (SMILES): O=C1NC(=O)/C(=C/c2cnn3c(NC4CC4)nc(NCc4ccc(F)cc4F)nc23)N1
InChIKey: OAIZOZZKJIRTFP-RZNTYIFUSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 75 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and the resid
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 426.39, ALogP 1.77, HBA 8, HBD 4, PSA 125.34, QED 0.35
Compound (SMILES): O=C(NCc1ccc(F)cc1)c1ccc(NC(=O)N2Cc3ccccc3C2)cc1
InChIKey: NJUXLFQNMVNUBR-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 = 14.1 nM (pChEMBL 7.849999904632568)
Descriptors: MW 389.43, ALogP 4.3, HBA 2, HBD 2, PSA 61.44, QED 0.7
Compound (SMILES): CN1C2CCC1CC(NC(=O)c1cn(S(C)(=O)=O)c3ccc(F)cc13)C2
InChIKey: CGQOHZLNGFYUML-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 3A (SINGLE PROTEIN), Homo sapiens
UniProt: P46098
Assay: In Vitro Inhibition of Ca Flux Assay: The 5HT3 antagonist activity of the compounds of the invention was determined by measuring the ability of the compounds to inhibit the calcium flux activity of 3HT3a receptor expressed in HEK-293T cells. HEK-293T cells were transfected with the 5HT3a expression construct using Xtreme Gene 9 (Roche) in 150 mm tissue culture treated plates and incubated for 24 hours at 37 °C. Cells were then split and plated at a density of 60K cells/well in poly-lysine coated, black 96-well plates with clear bottoms (BD BioSciences) and incubated overnight at 37 °C. Growth media was removed and cells loaded with 200 uL calcium indicator dye in HBSS containing 20 mM HEPES (Calcium 5 Assay kit, Molecular Devices) and incubated at 37 °C. for 1 hour. While cells were incubating, the 10× antagonist and agonist/antagonist addition plates were made. For 10× antagonist plate: half log serial dilutions (final concentrations range from 10−7 through 10−10 with the bottom well a negative, no ligand control) were made from test compounds in DMSO at a 1000× concentration and then diluted to 10× in HBSS/20 mM HEPES. For addition plate: 5HT was diluted to 100× in HBSS/20 mM HEPES (final concentration in the assay−216 nM) and 15 uL was added to each well of the addition plate, 15 uL of 10× compound was also added to the addition plate, and finally 120 uL of HBSS/20 mM HEPES (for a total of 150 uL). Cells were then removed from the incubator and equilibrated to room temperature for 10 minutes, then 22.5 uL of 10× test compounds were added in triplicate to the plates and incubated at room temperature for 10 minutes (Tropisetron was used as a positive control in every assay). Test plate and addition plate were loaded into the FlexStation III (Molecular Devices), and using the fluidics, 22.5 uL compound additions were made (at t=~17 seconds), and fluorescence was measured for 90 seconds, reading every 2.2 seconds.
Measurement: IC50 = 4.06 nM (pChEMBL 8.390000343322754)
Descriptors: MW 379.46, ALogP 1.94, HBA 5, HBD 1, PSA 71.41, QED 0.88