lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(OC(=O)N1CC[C@H](n2cc(C(N)=O)c(Nc3ccc(F)cc3)n2)[C@H](C#N)C1)C(F)(F)F
InChIKey: KWGWKRFTQMDLEZ-KMQIDDJNSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biochemical HTRF Assay: The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format 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 μL of 1.11× enzyme and 1.11× substrate in 1× 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 ambient temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at ambient temperature for 120 minutes. At the end of the incubation, 20 μL of 2× 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. Plates were shaken and centrifuged and then incubated 60 minutes at ambient temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading.
Measurement: IC50 > 1496 nM (pChEMBL 5.824999809265137)
Descriptors: MW 468.41, ALogP 3.34, HBA 7, HBD 2, PSA 126.27, QED 0.65
Compound (SMILES): CCOCc1c(-c2cc3cc(C)cc(OC)c3s2)c2c(N)ncnn2c1C(=O)N[C@@H]1CCNC1=O
InChIKey: DDNVTIXZSMOZTO-OAHLLOKOSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Kinase Assay: For the assay, 50 nl of each stock solution of the test compound in DMSO was pipetted into a black, low-volume 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany) 2 ul of a solution of the above FGFR-3 fusion protein in aqueous assay buffer [8 mM MOPS pH 7.0, 10 mM magnesium acetate, 1.0 mM dithiothreitol, 0.05% (w/v) bovine serum albumin (BSA), 0.07% (v/v) Tween-20, 0.2 mM EDTA] was added, and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compound to the enzyme. Then, the kinase reaction was started by the addition of 3 ul of a solution of adenosine triphosphate (ATP, 16.7 uM; final concentration in the 5 ul assay volume=10 uM) and substrate (0.8 uM; final concentration in the 5 ul assay volume=0.5 uM) in assay buffer, and the resulting mixture was incubated for a reaction time of 60 min at 22° C. The concentration of FGFR-3 fusion protein was adjusted depending on the activity of the enzyme lot.
Measurement: IC50 = 7.1 nM (pChEMBL 8.149999618530273)
Descriptors: MW 494.58, ALogP 2.67, HBA 9, HBD 3, PSA 132.87, QED 0.36
Compound (SMILES): CC[C@@H]1c2nnc(-c3nc(C)ns3)n2CCN1C(=O)c1ccc(F)cc1
InChIKey: OZKSTTVUQRWBIO-CYBMUJFWSA-N
Target: Neuromedin-K receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P29371
Assay: Competitive Binding Assay: The affinity of compounds of the invention for the human NK-3 receptor was determined by measuring the ability of compounds of the invention to competitively and reversibly displace a well-characterized NK-3 radioligand in a concentration-dependent manner.3H-SB222200 Binding Competition Assay with Human NK-3 ReceptorThe ability of compounds of the invention to inhibit the binding of the NK-3 receptor selective antagonist 3H-SB222200 was assessed by an in vitro radioligand binding assay. Membranes were prepared from Chinese hamster ovary recombinant cells stably expressing the human NK-3 receptor. The membranes were incubated with 5 nM 3H-SB222200 (ARC) in a HEPES 25 mM/NaCl 0.1M/CaCl2 1 mM/MgCl2 5 mM/BSA 0.5%/Saponin 10 μg/ml buffer at pH 7.4 and various concentrations of compounds of the invention. The amount of 3H-SB222200 bound to the receptor was determined after filtration by the quantification of membrane associated radioactivity using the TopCount-NXT reader (Packard).
Measurement: Ki = 69 nM (pChEMBL 7.159999847412109)
Descriptors: MW 372.43, ALogP 2.85, HBA 7, HBD 0, PSA 76.8, QED 0.71
Compound (SMILES): Cc1nc(-c2nnc3n2CCN(C(=O)c2ccc(-c4cccs4)cc2)[C@@H]3C)sc1C
InChIKey: KSIGHAFTVLYICN-CQSZACIVSA-N
Target: Neuromedin-K receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P29371
Assay: Binding Competition Assay: The ability of compounds of the invention to inhibit the binding of the NK-3 receptor selective antagonist 3H-SB222200 was assessed by an in vitro radioligand binding assay. Membranes were prepared from Chinese hamster ovary recombinant cells stably expressing the human NK3 receptor. The membranes were incubated with 5 nM 3H-SB222200(ARC) in a HEPES 25 mM/NaCl 0.1M/CaCl2 1 mM/MgCl2 5 Mm/BSA 0.5%/Saponin 10 μg/ml buffer at pH 7.4 and various concentrations of compounds of the invention. The amount of 3H-SB222200 bound to the receptor was determined after filtration by the quantification of membrane associated radioactivity using the TopCount-NXT reader (Packard).
Measurement: Ki = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 435.58, ALogP 4.96, HBA 7, HBD 0, PSA 63.91, QED 0.46
Compound (SMILES): COC[C@@H](NC(=O)c1ccc(-c2cn[nH]c2)c(OC)c1)c1ccccc1
InChIKey: SDWJCHAEHSUYNZ-GOSISDBHSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity.
Measurement: IC50 = 3.94 nM (pChEMBL 8.399999618530273)
Descriptors: MW 351.41, ALogP 3.2, HBA 4, HBD 2, PSA 76.24, QED 0.69
Compound (SMILES): CC12C[C@H]3C[C@@H](N4[C@@H]5CCC[C@H]4C[C@@H](n4c(=O)c(N6CCC[C@H]6C(=O)O)nc6ccccc64)C5)C[C@@H]1C[C@@H]2C3
InChIKey: TWOHYSGFUMRTOK-DJXZHOHSSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: In Vitro Mu-Opioid Receptor Binding Assay: Radioligand binding assays were conducted using freshly thawed membranes expressing human μ-receptors (Perkin Elmer, Shelton, Conn.). Radioligand dose-displacement binding assays for human μ-opioid receptors used 0.2 nM [3H]-diprenorphine (NEN, Boston, Mass.), with 5-20 mg membrane protein/well in a final volume of 500 μL binding buffer (10 mM MgCl2, 1 mM EDTA, 5% DMSO, 50 mM HEPES, pH 7.4). Reactions were carried out in the absence or presence of increasing concentrations of unlabeled naloxone. All reactions were conducted in 96-deep well polypropylene plates for 1-2 hr at about 25 °C. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Packard, Meriden, Conn.) presoaked in 0.5% polyethylenimine using a 96-well tissue harvester (Brandel, Gaithersburg, Md.) followed by performing three filtration washes with 500 μL of ice-cold binding buffer. Filter plates were subsequently dried at 50 °C. for 2-3 hours. BetaScint scintillation cocktail (Wallac, Turku, Finland) was added (50 μL/well), and plates were counted using a Packard Top-Count for 1 min/well.
Measurement: Ki = 86 nM (pChEMBL 7.070000171661377)
Descriptors: MW 530.71, ALogP 5.22, HBA 6, HBD 1, PSA 78.67, QED 0.58
Compound (SMILES): CC[C@@H](NC(=O)c1cc(C(=O)N[C@H](C)c2ccc(F)cc2)c2n1CCOC2C)c1ccccc1
InChIKey: HTYVUSVTXVUCTQ-JIUDAOPXSA-N
Target: Potassium channel subfamily K member 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O14649
Assay: Activity 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 98 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. Test compounds were consecutively added to the bath solution at rising concentrations.
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 463.55, ALogP 5.09, HBA 4, HBD 2, PSA 72.36, QED 0.51
Compound (SMILES): CN1CC[C@@](O)(C#Cc2ccc3c(c2)-c2nc(C(N)=O)sc2[C@]2(CO3)CC2(F)F)C1=O
InChIKey: KAEYMCZSUSJSJR-UXHICEINSA-N
Target: Mitogen-activated protein kinase kinase kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99558
Assay: Inhibition Assay: The ability of the nuclear factor-kappa B (NF-kB)-inducing kinase (NIK) to catalyze the hydrolysis of adenosine-5'-triphosphate (ATP) was monitored using the Transcreener ADP (adenosine-5'-diphosphate) assay (BellBrook Labs). Purified NIK (0.2-1 nM) derived from a baculovirus-infected insect cell expression system was incubated with test compounds for 1-3.5 hours in 50 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffer (pH 7.2) containing 10 mM MgCl.sub.2, 2 mM dithiothreitol, 10 uM ATP, 0.01% Triton X-100, 0.1% gamma-globulins from bovine blood, 1% dimethylsulfoxide (DMSO), 12 ug/mL ADP antibody and 4 nM ADP-AlexaFluor.RTM. 633 tracer. Reactions were quenched by the addition of 20 mM 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetic acid and 0.01% Brij 35. The tracer bound to the antibody was displaced by the ADP generated during the NIK reaction, which causes a decrease in fluorescence polarization that was measured by laser excitation.
Measurement: Ki = 1.3 nM (pChEMBL 8.890000343322754)
Descriptors: MW 445.45, ALogP 1.52, HBA 6, HBD 2, PSA 105.75, QED 0.65
Compound (SMILES): Cc1c(NS(=O)(=O)c2ccc(-c3cccc(CNCCc4ccccn4)c3)cc2)c(CC(C)C)nn1C
InChIKey: BAFTUTHSLRPADF-UHFFFAOYSA-N
Target: Glycylpeptide N-tetradecanoyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30419
Assay: Enzyme Inhibition Assay: Measurements were performed using a modification of the scintillation proximity assay platform described previously by Georgopapadakou, N. H. et al. (22nd International Congress on Chemotherapy, 2001, Abstract P16.001). Compounds were solubilised in DMSO at a top concentration of 10 mM and serially diluted in half log steps to achieve a range of final assay concentrations of 100 uM to 1 nM. Compound at each concentration (100-fold final) was added to white 384 well plates in a volume of 0.5 ml. Human, A. fumigatus, T. brucei or L. major N-myristoyl transferase enzyme, dissolved to a working concentration of 10 nM in assay buffer (30 mM Tris/HCl pH 7.4, 0.5 mM EGTA, 0.5 mM EDTA, 1.25 mM DTT, 0.1% Triton X-100), was then added to columns 1 to 11 and 13 to 23 of the plates in a volume of 20 ml. To columns 12 and 24, 20 ml assay buffer was added to provide a no enzyme control. Following a 5 minute incubation at room temperature the substrates (GCGGSKVKPQPPQAK(Biotin)-Amide and myristoyl coenzyme A), dissolved in assay buffer, were added to all wells in a volume of 20 ml to start the reaction. The final concentrations of peptide and 3H-myristoyl coenzyme A were 0.5 mM and 125 nM respectively and the specific activity of the radiolabel was 8 Ci/mmol. Plates were then incubated at room temperature for up to 50 minutes (dependant upon the period of linearity for the different enzyme species) before SPA beads, suspended to 1 mg/ml in a stop solution (200 mM Phosphoric Acid/NaOH pH 4, 750 mM MgCl2), were added in a volume of 40 ml.
Measurement: IC50 = 769 nM (pChEMBL 6.110000133514404)
Descriptors: MW 517.7, ALogP 5.12, HBA 6, HBD 2, PSA 88.91, QED 0.27
Compound (SMILES): NC1=N[C@]2(COCC1)c1cc(-c3cccnc3F)ccc1Oc1cnc(-c3ccnc(F)c3)cc12
InChIKey: ZASOWXQELAKJDI-AREMUKBSSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: Fluorescence Resonance Energy Transfer (FRET) Assay: The assay buffer used in this screen is 0.05 M acetate, pH 4.2, 10% DMSO final, 100 uM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The Beta Secretase enzyme (0.2 nM) is pre-incubated for one hour with inhibitors, typically in about 1 uL of DMSO according to a serial dilution, are added thereto. This assay is effectively started by the addition of FRET substrate (50 nM) and the combination is incubated for one hour. The FRET assay is terminated with by addition of Tris buffer, which raises the pH to neutrality, and the fluorescence is determined. The FRET substrate is a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. Proteolytic cleavage of the FRET substrate releases quenching of fluorescence (excitation 488 nm and emission 425 nm).
Measurement: IC50 = 139 nM (pChEMBL 6.860000133514404)
Descriptors: MW 471.47, ALogP 4.61, HBA 7, HBD 1, PSA 95.51, QED 0.43
Compound (SMILES): COc1ccc([C@H]2C[C@@H]2COc2nc(C)ncc2-c2cc(=O)n(C)cc2[S+](C)[O-])nc1
InChIKey: YQIQSDYCNDTPNS-YZNAOKNISA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: PDE10 Fluorescence Polarization (FP) Assay: The fluorescence polarization assay for cyclic nucleotide phosphodiesterases was performed using an IMAP FP kit supplied by Molecular Devices, Sunnyvale, Calif. (product # R8139). IMAPU technology has been applied previously to phosphodiesterase assays (Huang, W., et al., J. Biomol Screen, 2002, 7: 215). Assays were performed at room temperature in 384-well microtiter plates with an incubation volume of 20.2 uL. Solutions of test compounds were prepared in DMSO and serially diluted with DMSO to yield 8 uL of each of 10 solutions differing by 3-fold in concentration, at 32 serial dilutions per plate.
Measurement: Ki = 49.05 nM (pChEMBL 7.309999942779541)
Descriptors: MW 440.53, ALogP 2.47, HBA 8, HBD 0, PSA 102.19, QED 0.52
Compound (SMILES): CCOc1cc(=O)n(C)cc1-c1cc(NC(=O)CC2C[C@@H]3CC[C@H]2C3)ccc1Oc1ccc(F)cc1F
InChIKey: UMYWJFRKYLKZNL-YTYFACEESA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Assay method B: Compound dilution series were prepared in DMSO via an approximately 3-fold serial dilution from one of the following: 0.47 mM to 7.8 nM. Compound dilutions were added directly into white, low-volume assay plates (Perkin Elmer Proxiplate 384 Plus# 6008280) using a Labcyte Echo in conjunction with Labcyte Access and Thermo Multidrop CombinL robotics. Compounds were then suspended in eight microliters (μL) of assay buffer (20 mM Sodium Phosphate, pH 6.0, 50 mM NaCl, 1 mM Ethylenediaminetetraacetic acid disodium salt dihydrate, 0.01% Triton X-100, 1 mM DL-Dithiothreitol) containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and Alexa-647-conjugated probe. The final concentration of 1X assay mixture for assay methods A, B and C contains 2% DMSO, 8 nM His-tagged bromodomain, 1 nM Europium-conjugated anti-His-tag antibody and 100 nM or 30 nM probe (for BDI or BDII, respectively) and compound concentration in the range of: 49.02 μM-15.63 nM for method A, 9.19 μM 150 pM for method B, and 0.92 μM 15 pM for method C. After a one-hour equilibration at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 75.4 nM (pChEMBL 7.119999885559082)
Descriptors: MW 508.57, ALogP 6.29, HBA 5, HBD 1, PSA 69.56, QED 0.39
Compound (SMILES): CC(Nc1nccc(N2C(=O)OCC2[C@@H](C)O)n1)c1ncc(-c2cccc(C(F)(F)F)c2F)cn1
InChIKey: OMLKQWGXCCTPMV-BWBHSNQOSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: LC-MS Detection of 2-HG Assay: The biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Costar, Cat. No. 781201) using a final reaction volume of 30 μL and the following assay buffer conditions: 50 mM HEPES pH 7.4, 10 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.02% BSA, 5 uM NADPH and 100 uM alpha-KG. The final reaction mixture contained 3.3% DMSO and inhibitors with concentrations ranging 0.02-50 μM. The IDH1 enzyme was used at a final concentration of 0.25 nM. Following 45 minutes incubation, the reaction mixtures were quenched by the addition of 10 μL of 16% formic acid containing 800 nM of 5-carbon labeled 13C-2-HG). The protein was then precipitated by the addition of 2.5 volumes of acetonitrile followed by centrifugation (3000×g, minutes). The concentration of 2-HG in the resulting supernatants was measured by LC-MS (see below). LC-MS method. Reaction mixture supernatants were submitted to chromatographic separation on a BiobasicAX column (2.1 mm×20 mm, 5 μm particle, Thermo Scientific Inc.). The chromatographic mobile phases were A) 25 mM ammonium biocarbonate and B) acetonitrile (0.1% ammonium hydroxide). Nicotinamide was eluted at 1 ml/min using a 85-5% B gradient over 0.9 minutes (Agilent 1200SL LC system, Thermofisher LX-4 autosampler) and analyzed by multiple reaction monitoring (MRM) on a API4000 QTrap mass spectrometer (ABSciex, Framingham, Mass.) in the positive electrospray ionization (ESI+) mode. The mass transition for 2-HG and 13C-2-HG were 147→129 and 152→134, respectively.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 492.43, ALogP 3.97, HBA 8, HBD 2, PSA 113.36, QED 0.5
Compound (SMILES): C[C@@H]1CN(c2ccc(Nc3ncc4cc5n(c4n3)C3(CCCCC3)CNC5=O)nc2)CCN1C
InChIKey: TZQUSNSHSLUEPS-QGZVFWFLSA-N
Target: Cyclin-dependent kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P24941
Assay: Caliper Assay: Selected compounds disclosed herein were tested in CDK4/cyclinD1, CDK6/CycD3 CDK2/CycA and CDK2/cyclinE kinase assays by Nanosyn (Santa Clara, Calif.) to determine their inhibitory effect on these CDKs. The assays were performed using microfluidic kinase detection technology (Caliper Assay Platform). The compounds were tested in 12-point dose-response format in singlicate at Km for ATP. Phosphoacceptor substrate peptide concentration used was 1 μM for all assays and Staurosporine was used as the reference compound for all assays. Specifics of each assay are as described below:CDK2/CyclinA: Enzyme concentration: 0.2 nM; ATP concentration: 50 μM; Incubation time: 3 hr.CDK2/CyclinE: Enzyme concentration: 0.28 nM; ATP concentration: 100 μM; Incubation time: 1 hr.CDK4/CyclinD1: Enzyme concentration: 1 nM; ATP concentration: 200 μM; Incubation time: 10 hr.CDK6/CyclinD3: Enzyme concentration: 1 nM; ATP concentration: 300 μM; Incubation time: 3 hr.
Measurement: IC50 = 1560 nM (pChEMBL 5.809999942779541)
Descriptors: MW 460.59, ALogP 3.11, HBA 8, HBD 2, PSA 91.21, QED 0.62
Compound (SMILES): COC(=O)Nc1ccc2c(c1)NC(=O)[C@H](C)CCC[C@H](N1CCC(c3c(F)ccc(Cl)c3F)=CC1=O)c1nc-2c(C)[nH]1
InChIKey: UWCHLKPJXDNUJJ-CMJOXMDJSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Assay: Plasma kallikrein determinations were made in 0.1 M sodium phosphate buffer at a pH of 7.5 containing 0.1-0.2 M sodium chloride and 0.5% PEG 8000. Determinations were made using purified human plasma kallikrein (Enzyme Research Laboratories) at a final assay concentration of 200 pM and the synthetic substrate S-2302 (H-(D)-Pro-Phe-Arg-pNA; CHROMOGENIX®) at a concentration of 0.00008-0.0004 M.
Measurement: Ki = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 598.05, ALogP 6.61, HBA 5, HBD 3, PSA 116.42, QED 0.29
Compound (SMILES): C[C@@H](Oc1nc(NC2CC2)n2ncc(/C=C3\NC(=O)NC3=O)c2n1)c1ccccn1
InChIKey: TXZPHCDDLXLASA-DOQDJBIESA-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 = 49170 nM (pChEMBL 4.309999942779541)
Descriptors: MW 406.41, ALogP 1.41, HBA 9, HBD 3, PSA 135.43, QED 0.41
Compound (SMILES): CO[C@H]1CC[C@]2(CC1)Cc1ccc(C#CC3CCC3)cc1[C@]21N=C(C)C(N)=N1
InChIKey: HZHGVFUZMFVSNM-AGILITTLSA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Homo sapiens
UniProt: P05067
Assay: Release Assay: SH-SY5Y cells were cultured in DMEM/F-12 with Glutamax, 10% FCS and 1% non-essential amino acids and cryopreserved and stored at -140 C. at a concentration of 7.5-9.5x106 cells per vial. Thaw cells and seed at a conc. of around 10000 cells/well in DMEM/F-12 with Glutamax, 10% FCS and 1% non-essential amino acids to a 384-well tissue culture treated plate, 100 uL cell susp/well. The cell plates were then incubated for 7-24 h at 37 C., 5% CO2. The cell medium was removed, followed by addition of 30 uL compound diluted in DMEM/F-12 with Glutamax, 10% FCS, 1% non-essential amino acids and 1% PeSt to a final conc. of 1% DMSO. The compounds were incubated with the cells for 17 h (overnight) at 37 C., 5% CO2. Meso Scale Discovery (MSD) plates were used for the detection of sAPPbeta release. MSD sAPPbeta plates were blocked in 1% BSA in Tris wash buffer (40 uL/well) for 1 h on shake at r.t. and washed 1 time in Tris wash buffer (40 uL/well).
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 375.52, ALogP 3.95, HBA 4, HBD 1, PSA 59.97, QED 0.76
Compound (SMILES): N[C@H](CCP(=O)(O)C(O)c1ccc(OCC(=O)O)c(O)c1)C(=O)O
InChIKey: USSOEGIWLJVUKK-UOGPZTOASA-N
Target: Metabotropic glutamate receptor 8 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P70579
Assay: Pharmacological Assay: Metabotropic glutamate receptors were transiently transfected in HEK293 cells by electroporation as described elsewhere (Brabet I. et al., 1998) and plated in 96-well microplates. The high affinity glutamate transporter EAAC1 was co-transfected with the receptor in order to avoid any influence of glutamate released by the cells in the assay medium. In the experiments carried out by the inventors, Group-III mGluRs were co-transfected with a chimeric G-protein which couples the activation of the receptor to the phospholipase-C (PLC) pathway. Thus receptor activation induces production of inositol phosphate (IP) which in turn induces intracellular Ca2+ release. Receptor activity was then determined by measurement of the IP production or Ca release as already described (Goudet C. et al., PNAS 2004). For intracellular calcium measurements, cells expressing mGluRs were loaded with Ca2+-sensitive fluorescent dye Fluo-4 AM (Invitrogen, Cergy-Pontoise, France) dissolved in Hanks' balanced Salt Solution.
Measurement: EC50 = 34000 nM (pChEMBL 4.46999979019165)
Descriptors: MW 363.26, ALogP -0.08, HBA 7, HBD 6, PSA 187.61, QED 0.32
Compound (SMILES): COc1ccc2c3c1O[C@H]1[C@@]4(OC)CC[C@@]5(C[C@@H]4COCc4ccccc4)[C@@H](C2)NCC[C@]315
InChIKey: FDYCYWQDIVTVDR-RHRQCQTHSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Radioligand Dose-Displacement Binding Assay: Radioligand dose-displacement binding assays for t-opioid receptors used 0.3 nM [3H]-diprenorphine (Perkin Elmer, Shelton, Conn.), with 5 mg membrane protein/well in a final volume of 500 ul binding buffer (10 mM MgCl2, 1 mM EDTA, 5% DMSO, 50 mM HEPES, pH 7.4). Reactions were carried out in the absence or presence of increasing concentrations of unlabeled naloxone. All reactions were conducted in 96-deep well polypropylene plates for 2 hr at room temperature. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Perkin Elmer, Shelton, Conn.), presoaked in 0.5% polyethylenimine using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by performing three filtration washes with 500 ul of ice-cold binding buffer. Filter plates were subsequently dried at 50° C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 ul/well), and plates were counted using a Packard Top-Count.
Measurement: Ki = 20.7 nM (pChEMBL 7.679999828338623)
Descriptors: MW 447.58, ALogP 4.01, HBA 5, HBD 1, PSA 48.95, QED 0.73
Compound (SMILES): COc1ccc(C(=O)C2CCN([C@@H]3CCN(Cc4nc5ccccc(=O)c5[nH]4)C3=O)CC2)cc1C
InChIKey: DUCZLEBAUSAZOC-OAQYLSRUSA-N
Target: Poly [ADP-ribose] polymerase tankyrase-1 (SINGLE PROTEIN), Homo sapiens
UniProt: O95271
Assay: Biochemical Assay: The autoparsylation activity of the TNKS 1/2 or PARP1/2 enzymes was measured by the liquid chromatography-mass spectrometry (LC/MS) detection of nicotinamide as readout. Compound activity in inhibiting the TNKS and PARP autoparsylation was evaluated by IC50 measurements. In the compound screening assays, the reaction is composed of 5 uL of compound in 8-point serial dilutions with concentrations ranging from 0.0086 to 18.75 uM, 20 nM of purified enzyme, and 250 uM of p-NAD+ in the 1x Assay Buffer. After 60 min incubation at room temperature, the reactions were quenched by the addition of 10 uL of 5x quenching solution (20% formic acid and 500 nM [d]-nicotinamide in water). For the background control wells, 10 uL of the 5x quenching solution per well was added prior to the addition of p-NAD+. The % Inhibition was calculated as: (Control-Sample)/(Control-Background)*100. "Control" is the average value of 8 wells without compound; and "Background" is the average of 8 wells mixed with 5x quenching solution measured prior to initiation of the reaction.
Measurement: IC50 = 278 nM (pChEMBL 6.559999942779541)
Descriptors: MW 474.56, ALogP 2.94, HBA 6, HBD 1, PSA 95.6, QED 0.55
Compound (SMILES): C[C@@H](NC(=O)c1ccc(-c2cc(C3CCNC(=O)C3)cnc2N)cc1F)c1cccc(Cl)c1
InChIKey: WQUOXLFKKGSHNM-IURRXHLWSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: ERK2 (20 pM) Kinase Assay: Compound potency against activated ERK2 was determined using a kinase assay that measures ERK2-catalyzed phosphorylation of biotinylated ERKtide peptide substrate ([Biotin]-AHA-K-R-E-L-V-E-P-L-T-P-S-G-E-A-P-N-Q-A-L-L-R-[NH2], the peptide sequence derived from EGF receptor: SEQ ID NO:1). The assay was carried out in 20 mM HEPES [pH 7.5], 5 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 0.05% BSA using 0.02 nM ERK2, 400 nM ERKtide peptide and 35 uM ATP (all concentrations are final in the reaction) in a total volume of 10.25 uL. A 16-point, half-log dilution series of compounds at 41x final concentration was used for generating IC50 curves. Compound dilution series were prepared in 100% DMSO. ERK2 was preincubated with compounds for 30 minutes at ambient temperature. Reaction was initiated by addition of a substrate cocktail of ERKtide peptide and ATP and was allowed to proceed for 4 hours at ambient temperature. Reaction was terminated by addition of 10 uL of a 2x stop buffer consisting of 100 mM Tris-Cl [pH 7.5], 25 mM EDTA, 0.01% Tween 20, 20 ug/mL of AlphaScreen Protein A Acceptor Beads, 20 ug/mL of Streptavidin Donor Beads (PerkinElmer, Waltham, Mass.), and 1:1000 dilution phospho-EGF Receptor (Thr669) antibody (Cat #8808, Cell Signaling Technology, Danvers, Mass.). Terminated reactions were read, after overnight incubation in the dark, on an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, Mass.), with excitation and emission wavelengths set to 680 nm and 570 nm, respectively. IC50 values were determined using a four-parameter fit.
Measurement: IC50 = 0.536 nM (pChEMBL 9.270000457763672)
Descriptors: MW 466.94, ALogP 4.61, HBA 4, HBD 3, PSA 97.11, QED 0.51
Compound (SMILES): CO[C@@H]1CC[C@@H](CC(=O)N[C@H]2CC[C@H](CCN3CCC(c4noc5cc(F)c(F)cc45)CC3)CC2)C1
InChIKey: HJUAHZKICLIRDT-UGESXGAOSA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Radioligand Binding Assay: Aliquots of membrane preparations were thawed at RT, resuspended in assay buffer (D2, D3: 50 mM Tris-HCl, 120 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 5 mM KCl, 1.5 mM CaCl2, pH=7.4; 5-HT2A: 50 mM Tris-HCl, 10 mM MgCl2, 1 mM EGTA, pH=7.4), homogenized with a Polytron for 20-30 sec at 12,000 rpm and adjusted to a final concentration of approximately 7.5 ug protein/well (D2, D3) and 15 ug protein/well (5-HT2A), respectively.The binding affinity (Ki) of the compounds was determined using radioligand binding. Membranes were incubated in a total volume of 200 ul with a fixed concentration of radioligand (final concentration approximately 0.7 nM [3H]-spiperone for D2, 0.5 nM [3H]-spiperone for D3, and 1.1 nM [3H]-ketanserin for 5-HT2A) and ten concentrations of test compound in ranging between 10 uM-0.1 nM for 1 h at RT. At the end of the incubation, the reaction mixtures were filtered on to unifilter 96-well white microplates with bonded GF/C filters.
Measurement: Ki = 19.99 nM (pChEMBL 7.699999809265137)
Descriptors: MW 503.63, ALogP 5.56, HBA 5, HBD 1, PSA 67.6, QED 0.51
Compound (SMILES): COCCCOC(=O)N1CC[C@@H](n2cc(C(N)=O)c(Nc3ccc(F)cc3)n2)[C@H](C#N)C1
InChIKey: UHZKGARVKDTXJF-RDTXWAMCSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biochemical HTRF Assay: The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format 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 μL of 1.11× enzyme and 1.11× substrate in 1× 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 ambient temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at ambient temperature for 120 minutes. At the end of the incubation, 20 μL of 2× 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. Plates were shaken and centrifuged and then incubated 60 minutes at ambient temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading.
Measurement: IC50 = 275 nM (pChEMBL 6.559999942779541)
Descriptors: MW 444.47, ALogP 2.42, HBA 8, HBD 2, PSA 135.5, QED 0.6
Compound (SMILES): CNC(=O)[C@@H](NC(=O)c1cc(OCC(F)(F)F)c(C2CC2)cn1)C(C)(C)C
InChIKey: YWZGEGZSNRFLOJ-CQSZACIVSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1×HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min. Compounds were added to a final assay volume of 100 μl and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 μl detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.
Measurement: EC50 = 660.7 nM (pChEMBL 6.179999828338623)
Descriptors: MW 387.4, ALogP 2.79, HBA 4, HBD 2, PSA 80.32, QED 0.79
Compound (SMILES): CNC(=O)[C@@H](NC(=O)c1ccc(C2CC2)c(OCc2ccccn2)n1)C(C)(C)C
InChIKey: PTJUDNXMKSXQFH-GOSISDBHSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1.times.HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 396.49, ALogP 2.82, HBA 5, HBD 2, PSA 93.21, QED 0.75
Compound (SMILES): Nc1ncc(C2CCOCC2)nc1-c1ccc(C(=O)N[C@H](CO)c2cccc(F)c2)c(F)c1
InChIKey: LZLDDRMCZNBSBN-OAQYLSRUSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: ERK2 (20 pM) Kinase Assay: Compound potency against activated ERK2 was determined using a kinase assay that measures ERK2-catalyzed phosphorylation of biotinylated ERKtide peptide substrate ([Biotin]-AHA-K-R-E-L-V-E-P-L-T-P-S-G-E-A-P-N-Q-A-L-L-R-[NH2], the peptide sequence derived from EGF receptor: SEQ ID NO:1). The assay was carried out in 20 mM HEPES [pH 7.5], 5 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 0.05% BSA using 0.02 nM ERK2, 400 nM ERKtide peptide and 35 uM ATP (all concentrations are final in the reaction) in a total volume of 10.25 uL. A 16-point, half-log dilution series of compounds at 41x final concentration was used for generating IC50 curves. Compound dilution series were prepared in 100% DMSO. ERK2 was preincubated with compounds for 30 minutes at ambient temperature. Reaction was initiated by addition of a substrate cocktail of ERKtide peptide and ATP and was allowed to proceed for 4 hours at ambient temperature. Reaction was terminated by addition of 10 uL of a 2x stop buffer consisting of 100 mM Tris-Cl [pH 7.5], 25 mM EDTA, 0.01% Tween 20, 20 ug/mL of AlphaScreen Protein A Acceptor Beads, 20 ug/mL of Streptavidin Donor Beads (PerkinElmer, Waltham, Mass.), and 1:1000 dilution phospho-EGF Receptor (Thr669) antibody (Cat #8808, Cell Signaling Technology, Danvers, Mass.). Terminated reactions were read, after overnight incubation in the dark, on an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, Mass.), with excitation and emission wavelengths set to 680 nm and 570 nm, respectively. IC50 values were determined using a four-parameter fit.
Measurement: IC50 = 0.429 nM (pChEMBL 9.369999885559082)
Descriptors: MW 454.48, ALogP 3.36, HBA 6, HBD 3, PSA 110.36, QED 0.53
Compound (SMILES): Cc1cc([C@@H](C)Oc2cc(F)ccc2Nc2ncnc3cc(N=S(C)(C)=O)cc(C)c23)on1
InChIKey: GCMXEMBFFWKSQR-OAHLLOKOSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BUB5
Assay: In vitro Kinase Assay: The Z-LYTE biochemical assay employs a fluorescence-based, coupled-enzyme format and is based on the differential sensitivity of phosphorylated and non-phosphorylated peptides to proteolytic cleavage. The peptide substrate is labeled with two fluorophores one at each end that make up a FRET pair.In the primary reaction, the kinase transfers the gamma-phosphate of ATP to a single tyrosine, serine or threonine residue in a synthetic FRET-peptide. In the secondary reaction, a site-specific protease recognizes and cleaves non-phosphorylated FRET-peptides. Phosphorylation of FRET-peptides suppresses cleavage by the Development Reagent. Cleavage disrupts FRET between the donor (i.e., coumarin) and acceptor (i.e., fluorescein) fluorophores on the FRET-peptide, whereas uncleaved, phosphorylated FRET-peptides maintain FRET.
Measurement: IC50 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 469.54, ALogP 5.62, HBA 8, HBD 1, PSA 102.5, QED 0.39
Compound (SMILES): Cc1nn(C)c(C)c1NS(=O)(=O)c1ccc(CCCN2CCNCC2)cc1
InChIKey: JFVLKGVMXSZQKG-UHFFFAOYSA-N
Target: Glycylpeptide N-tetradecanoyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30419
Assay: Enzyme Inhibition Assay: Measurements were performed using a modification of the scintillation proximity assay platform described previously by Georgopapadakou, N. H. et al. (22nd International Congress on Chemotherapy, 2001, Abstract P16.001). Compounds were solubilised in DMSO at a top concentration of 10 mM and serially diluted in half log steps to achieve a range of final assay concentrations of 100 uM to 1 nM. Compound at each concentration (100-fold final) was added to white 384 well plates in a volume of 0.5 ml. Human, A. fumigatus, T. brucei or L. major N-myristoyl transferase enzyme, dissolved to a working concentration of 10 nM in assay buffer (30 mM Tris/HCl pH 7.4, 0.5 mM EGTA, 0.5 mM EDTA, 1.25 mM DTT, 0.1% Triton X-100), was then added to columns 1 to 11 and 13 to 23 of the plates in a volume of 20 ml. To columns 12 and 24, 20 ml assay buffer was added to provide a no enzyme control. Following a 5 minute incubation at room temperature the substrates (GCGGSKVKPQPPQAK(Biotin)-Amide and myristoyl coenzyme A), dissolved in assay buffer, were added to all wells in a volume of 20 ml to start the reaction. The final concentrations of peptide and 3H-myristoyl coenzyme A were 0.5 mM and 125 nM respectively and the specific activity of the radiolabel was 8 Ci/mmol. Plates were then incubated at room temperature for up to 50 minutes (dependant upon the period of linearity for the different enzyme species) before SPA beads, suspended to 1 mg/ml in a stop solution (200 mM Phosphoric Acid/NaOH pH 4, 750 mM MgCl2), were added in a volume of 40 ml.
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 391.54, ALogP 1.68, HBA 6, HBD 2, PSA 79.26, QED 0.75
Compound (SMILES): CN[C@H](C)C(=O)Nc1cc(-c2cccc3cnn(C)c23)c(OC)c(C#Cc2ccccc2)n1
InChIKey: OANSFXRLESWTDA-QGZVFWFLSA-N
Target: Baculoviral IAP repeat-containing protein 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13490
Assay: XIAP BIR3 & cIAP1 BIR3 Binding Assays (DELFIA): BIR3 domains (10 nM) were incubated with SMAC peptide (10 nM) in assay buffer (50 mM Tris, 120 mM NaCl, 0.1% BSA, 1 mM DTT, 0.05% TritonX100) for one hour at room temperature in the presence of inhibitory compounds. The assay mixture was transferred to a strepatvidin coated plate and incubated for one hour at room temperature to allow binding of the biotinylated peptide and associated BIR3 domains to the plate. After several washing steps Eu labeled anti-GST antibody (e.g. Perkin Elmer DELFIA Eu-N1-antiGST AD0250) was added to detect BIR3 domain-SMAC peptide interactions according to Perkin Elmer's instructions. Briefly, the antibody was added (dilution 1:5000 in Perkin Elmer DELFIA Assay Buffer 2013-01) and incubated for one hour. After 3 washing steps using Delfia Washing Buffer (Perkin Elmer DELFIA Wash 2013-05), Enhancement Solution (Perkin Elmer Enhancement Asolution 2013-02) was added and incubation continued for 10 minutes. Time resolved Europium fluorescence was measured in a Wallac Victor using Standard assay settings.
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 439.52, ALogP 3.59, HBA 6, HBD 2, PSA 81.07, QED 0.46
Compound (SMILES): CC(C)OC(=O)NC1CCN(c2ccc(-c3nc4[nH]c(O[C@@H]5COC6C5OC[C@H]6O)nc4cc3Cl)cc2)CC1
InChIKey: MKJXKJYNLHSTCB-WXYTXABISA-N
Target: 5'-AMP-activated protein kinase catalytic subunit alpha-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13131
Assay: AMPK Activation Assay: In the 384-well plates 1.25 ul of test compound in assay buffer (20 mM HEPES pH 7.0, 0.025% BSA, 15 mM MgCl2, 0.01% Brij) was mixed with 1.25 ul of AMPK and 1.25 ul of the peptide (final concentration of 1 uM) and 1.25 ul of ATP (final concentration of 30 uM), both dissolved in assay buffer. This step was followed by an incubation time of 60 min. Then 5 ul of ADP Glo Reagent was added. This was followed by 40 min of incubation. Then 10 ul of Kinase Detection Reagent was admixed. The plates were sealed and after an incubation period of 30 min, the luminescence signal was measured in an Envision reader. All incubation steps were accomplished at room temperature. Each assay microtiter plate contained wells with vehicle controls instead of compound (0.1% DMSO in water) as reference for the low signal (100% CTL, low signal), and wells with serial dilutions of AMP (final 30 uM) as reference for high signals. The luminescence signal generated was proportional to the ADP concentration produced and was correlated with AMPK activity.
Measurement: EC50 = 290 nM (pChEMBL 6.539999961853027)
Descriptors: MW 558.04, ALogP 3.29, HBA 9, HBD 3, PSA 131.06, QED 0.42
Compound (SMILES): Nc1ncc(-c2cc(C(=O)Nc3ccc(OC(F)(F)Cl)cc3)cnc2N2CC[C@@H](O)C2)cc1F
InChIKey: NSBCKEPOIMASLL-OAHLLOKOSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Caliper ABL : The assay plates were prepared by addition of 50 nL per well of compound solution in 90% DMSO. The kinase reactions were started by stepwise addition of 4.5 uL per well of peptide/ATP-solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 uM sodium orthovanadate, 20 mM MgCl2, 2 mM MnCl2, 4 uM ATP, 4 uM peptide (FITC-Ahx-EAIYAAPFAKKK-NH2)) and 4.5 uL per well of enzyme solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 uM sodium orthovanadate, 20 mM MgCl2, 2 mM MnCl2, 3.5 nM ABL (ABL(64-515), produced in-house from E. coli)). Kinase reactions were incubated at 30° C. for 60 minutes and subsequently terminated by addition of 16 uL per well of stop solution (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35). Plates with terminated kinase reactions were transferred to the Caliper LC3000 workstations for reading. Phosphorylated and unphosphorylated peptides were separated using the Caliper microfluidic mobility shift technology.
Measurement: IC50 = 0.7 nM (pChEMBL 9.149999618530273)
Descriptors: MW 493.87, ALogP 3.86, HBA 7, HBD 3, PSA 113.6, QED 0.45
Compound (SMILES): O=C(Nc1nc2ccc(Cl)cc2s1)c1ccc(-c2ncc(C[C@@H](O)CO)cc2F)cc1
InChIKey: AMUZKWZDRNKLNY-MRXNPFEDSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: pH Assay: Culture medium was removed using an 8-channel-pipette (Rainin, USA) from the 96-well plate and the wells were refilled with 100 μL of loading buffer (20 mM HEPES, 115 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 1.8 mM CaCl2, 13.8 mM D-glucose, 2.5 mM probenecid, pH 7.4) containing 5 Î¼M Fura-2 AM (Dojin, Japan). The 96-well plate was incubated at 37° C. for 45 min. The cells were washed twice with 150 μL of measuring buffer (mentioned in step 2.2.(3) of Protocol 2, no probenecid). The wells were subsequently refilled with 70 μL of measuring buffer. Either 10 μL of measuring buffer or 10 μL of 10× stock serial dilution of test compound (described in step 3.2. above) were applied to each well. Usually, only one test compound was tested per 96-well plate. The number of replicates per 96-well plate for a particular antagonist at a particular concentration was 2×7 since, as described for the "agonist plate," two different sulfuric acid concentrations were used per 96-well plate and seven lanes (A-C, E-H) per 96-well plate were used (N=2×7). After an incubation at 4° C. for 15 min, the 96-well plate was transferred to a model FDSS-3000 plate reader apparatus (Hamamatsu Photonics K.K., Japan). Fura-2 fluorescent intensity was monitored as described in step 2.2.(5) above. After 16 time points of baseline detection, 20 μL of agonist solution (measuring buffer titrated with H2SO4 to yield a pH in the range of from about 5.0 to about 5.1 when mixed 1:4 with the measuring buffer containing test compound) was added to each well (final volume 100 μL/well).
Measurement: IC50 = 18.8 nM (pChEMBL 7.730000019073486)
Descriptors: MW 457.91, ALogP 4.3, HBA 6, HBD 3, PSA 95.34, QED 0.4
Compound (SMILES): N[C@H](CCP(=O)(O)C(O)c1ccc(/C=C/C(=O)O)cc1)C(=O)O
InChIKey: QKKOTCXCHRJFNS-WQMQZYANSA-N
Target: Metabotropic glutamate receptor 8 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P70579
Assay: Pharmacological Assay: Metabotropic glutamate receptors were transiently transfected in HEK293 cells by electroporation as described elsewhere (Brabet I. et al., 1998) and plated in 96-well microplates. The high affinity glutamate transporter EAAC1 was co-transfected with the receptor in order to avoid any influence of glutamate released by the cells in the assay medium. In the experiments carried out by the inventors, Group-III mGluRs were co-transfected with a chimeric G-protein which couples the activation of the receptor to the phospholipase-C (PLC) pathway. Thus receptor activation induces production of inositol phosphate (IP) which in turn induces intracellular Ca2+ release. Receptor activity was then determined by measurement of the IP production or Ca release as already described (Goudet C. et al., PNAS 2004). For intracellular calcium measurements, cells expressing mGluRs were loaded with Ca2+-sensitive fluorescent dye Fluo-4 AM (Invitrogen, Cergy-Pontoise, France) dissolved in Hanks' balanced Salt Solution.
Measurement: EC50 = 49000 nM (pChEMBL 4.309999942779541)
Descriptors: MW 343.27, ALogP 0.85, HBA 5, HBD 5, PSA 158.15, QED 0.34