lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1cccc(OC)c1-n1c(NS(=O)(=O)[C@@H](C)[C@H](C)c2ncc(Cl)cn2)nnc1-c1cnn2c1CCC2
InChIKey: SKMSVZILJDBKDT-GJZGRUSLSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: [35S]GTPgammaS Binding: The human APJ receptor was cloned by polymerase chain reaction and the gene encoding the receptor was subcloned in pFLAG-CMV-3 expression vector (Sigma, Saint Louis, Mo. USA) in-house at Amgen. A GTPγS binding assay was performed on membranes prepared from CHO cells stably expressing human APJ receptor. The optimum experimental conditions for the concentrations of GDP, MgCl2, and NaCl in the assay buffer were initially determined. The assay was performed in 9 μL assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, 100 mM NaCl and 0.1% (w/v) BSA], 1 μL of diluted test compound (starting with 0.75 mM, 2-fold serial dilution with DMSO, total 22 points), 10 μL of 18 μM GDP (final concentration of 3 μM GDP), 20 μL of 0.25 μg/mL membrane protein expressing human APJ receptor captured with WGA PS beads (final concentration of 5 μg per well), and 20 μL of 0.3 nM [35S]GTPγS (final concentration is 0.1 nM [35S]GTPγS)(Perkin Elmer Life and Analytical Sciences, Waltham USA). One column of the plate was 1 μL of DMSO as background and another column of the plate was 1 μL of 180 μM Pyr-Apelin-13 which was used as control at a final concentration of 3 μM. Incubation was at RT for 90 min and the microplate was read using a ViewLux ultra HTS Microplate Imager (PerkinElmer, Inc.). All the results presented are means of several independent experiments and analyzed by non-linear regression methods using the commercially available program Prism (GraphPad, San Diego, Calif.) providing the EC50 values detailed in Table 40.
Measurement: EC50 = 0.089 nM (pChEMBL 10.050000190734863)
Descriptors: MW 559.05, ALogP 3.47, HBA 11, HBD 1, PSA 138.94, QED 0.33
Compound (SMILES): C/C=C/C[C@@H](C)[C@@H](O)[C@H]1C(=O)N[C@@H](CC)C(=O)N(C)[C@H](C)C(=O)N(C)[C@@H]([C@H](C)CCOC)C(=O)N[C@@H](C(C)C)C(=O)N(C)[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@H](C)C(=O)N(C)[C@@H](CC(C)C)C(=O)N(C)[C@@H](CC(C)C)C(=O)N(C)[C@@H](C(C)C)C(=O)N1C
InChIKey: IJENCMKWSSDLPI-ZKISSFAWSA-N
Target: Peptidyl-prolyl cis-trans isomerase A (SINGLE PROTEIN), Homo sapiens
UniProt: P62937
Assay: Surface Plasmon Resonance (SPR) Assay: Binding of inhibitors to expressed cyclophilins was determined using surface plasmon resonance (SPR) experiments. Briefly, avi-tagged cyclophilin proteins with mono-biotinylation were immobilized onto a Biotin CAPture chip (GE Healthcare, cat. #28920234). SPR experiments were carried out on an upgraded Biacore T200 system using a running buffer containing the inhibitor, 10 mM HEPES, pH 7.4, 150 mM NaCl, 0.05% P20, and 3% DMSO. Single-cycle kinetics measurements were used to study the interactions between cyclophilin inhibitors and different cyclophlins. Accordingly, compound at various concentrations was injected to the flow cells with a contact time of 1 min each at a flow rate of 50 μL/min. The final dissociation phase lasts for 10 min following the sample injection. Data was analyzed using Biacore T200 Evaluation Software Version 1.0 and a 1:1 binding model was applied to fit the data to obtain kon, koff, and KD.
Measurement: Kd = 1.7 nM (pChEMBL 8.770000457763672)
Descriptors: MW 1246.69
Compound (SMILES): Nc1nn2cc(F)cnc2c1C(=O)Nc1cncc(F)c1N1CCN(C(=O)CN2CCC2)CC1
InChIKey: DVXDCBWHYWOAPT-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Inhibition Assay: Compounds were screened for their ability to inhibit ATR kinase using a radioactive-phosphate incorporation assay. Assays were carried out in a mixture of 50 mM Tris/HCl (pH 7.5), 10 mM MgCl2 and 1 mM DTT. Final substrate concentrations were 10 μM [γ-33P]ATP (3mCi 33P ATP/mmol ATP, Perkin Elmer) and 800 μM target peptide (ASELPASQPQPFSAKKK) (SEQ ID NO:1).Assays were carried out at 25° C. in the presence of 5 nM full-length ATR. An assay stock buffer solution was prepared containing all of the reagents listed above, with the exception of ATP and the test compound of interest. 13.5 μL of the stock solution was placed in a 96 well plate followed by addition of 2 μL of DMSO stock containing serial dilutions of the test compound (typically starting from a final concentration of 15 μM with 3-fold serial dilutions) in duplicate (final DMSO concentration 7%). The plate was pre-incubated for 10 minutes at 25° C. and the reaction initiated by addition of 15 μL [γ-33P]ATP (final concentration 10 μM).The reaction was stopped after 24 hours by the addition of 30 μL 0.1M phosphoric acid containing 2 mM ATP. A multiscreen phosphocellulose filter 96-well plate (Millipore, Cat no. MAPHN0B50) was pretreated with 100 μL 0.2M phosphoric acid prior to the addition of 454 of the stopped assay mixture. The plate was washed with 5×200 μL 0.2M phosphoric acid. After drying, 100 μL Optiphase SuperMix liquid scintillation cocktail (Perkin Elmer) was added to the well prior to scintillation counting (1450 Microbeta Liquid Scintillation Counter, Wallac).
Measurement: Ki = 505 nM (pChEMBL 6.300000190734863)
Descriptors: MW 471.47, ALogP 0.59, HBA 9, HBD 2, PSA 124.99, QED 0.55
Compound (SMILES): Cc1nn(C)cc1Nc1nc(N)c2c(-c3ccc4occc4c3)nn(CC(F)(F)F)c2n1
InChIKey: QHMOKKDVPORQHE-UHFFFAOYSA-N
Target: Protein-tyrosine kinase 6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13882
Assay: Brk Inhibitory Activity Assay: Measurement of an inhibitory activity on Brk enzyme was performed by using LanthaScreen (registered trademark) system (Invitrogen) in accordance with the attached manual. Reagents used are described below.Reaction Buffer: A solution containing 50 mmol/L HEPES (pH 7.5), 0.01% Brij 35, 10 mmol/L MgCl2 and 1 mmol/L EGTA was prepared by using purified water.A solution of a test substance (the compound of the present invention): A solution of each concentration of a test compound in DMSO was diluted 20-fold with Reaction Buffer, and a solution containing a test compound at a concentration 5 times a final concentration was prepared.An enzyme solution: A solution containing 480 ng/mL of Brk enzyme was prepared by using Reaction Buffer.A substrate solution: A solution containing 57 μmol/L of ATP and 500 nmol/L of Fluorescein-Poly GT (Invitrogen) was prepared by using Reaction Buffer.A detection solution: A solution containing 20 mmol/L of EDTA and 4 nmol/L of PY20 (Invitrogen) was prepared by using Dilution B (Invitrogen).To a 96-well plate (Nunc), a solution of 10 mmol/L of a test compound in DMSO was dispensed, and further, a dilution series at a common ratio of three was prepared by using DMSO. To each of wells of the 96-well plate for the measurement, 5 μL of Reaction Buffer containing DMSO was added for a blank group and a vehicle group and 5 μL of a test substance solution was added for a test substance group. Next, 10 μL per well of Reaction Buffer was added for the blank group, and 10 μL per well of the enzyme solution was added for the vehicle group and the test compound group, and thereafter, the mixture was stirred at room temperature for 10 minutes. After completion of stirring, 10 μL of the substrate solution was added to each of wells, and the mixture was stirred at room temperature under a shading condition for 1 hour. After completion of the reaction, 25 μL of the detection solution was added to each well, and the mixture was left to stand at room temperature under a shading condition for 30 minutes. After being left standing, fluorescence intensities at 520 nm and 495 nm were measured by using Analyst GT (Molecular Devices, LLC) when being irradiated with an excitation light at 340 nm. The phosphorylation of the artificial substrate was quantified by Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET). With regard to each well, the TR-FRET ratio was calculated by divining the fluorescence signal at 520 nm by the fluorescence signal at 495 nm, and the inhibition rate (%) in the test compound group was calculated according to the following Numerical Formula 1.Inhibition rate (%)={1−(TR-FRET ratio of test compound group−A)/(B−A)}×100  [Numerical Formula 1]
Measurement: IC50 = 7.6 nM (pChEMBL 8.119999885559082)
Descriptors: MW 442.41, ALogP 4.17, HBA 9, HBD 2, PSA 112.61, QED 0.43
Compound (SMILES): Cc1c(C(N)=O)sc2ncn(Cc3nnn(C[C@H](O)c4cc5cc(F)ccc5o4)n3)c(=O)c12
InChIKey: CLCRTUFFZNMHLS-LBPRGKRZSA-N
Target: Transient receptor potential cation channel subfamily A member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75762
Assay: Filpr Assay: At the assay day cells were washed 3× with assay puffer, 20 μL buffer remained in the wells after washing. 10 μL Ca6 kit (Cat. R8191 Molecular Devices) loading buffer in HBSS/HEPES was added to the cells and the plates were incubated with lid for 120 minutes at 3775% CO2. 10 μL of compound or controls in HBSS/HEPES buffer/5% DMSO from the intermediate dilution plate were carefully added to the wells. Luminescence (indicating the calcium influx or release) was read on the FLIPR tetra device for 10 minutes to monitor the compound induced effects (e.g. agonism). Finally, 10 μL of the agonist AITC 50 μM dissolved in HBSS/HEPES buffer/0.05% DMSO (final concentration 10 μM) was added to the wells followed by an additional read on the FLIPR tetra device for 10 minutes. The area under the signal curve (AUC) after AITC addition was used for IC50/% inhibition calculations.
Measurement: IC50 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 469.46, ALogP 1.52, HBA 11, HBD 2, PSA 154.95, QED 0.38
Compound (SMILES): O=C(CNC(=O)C(c1nc2cc(F)c(-c3ccc(NC(=O)NCc4ccccc4)cc3)cc2s1)S(=O)(=O)CCC(F)(F)F)NC1CC1
InChIKey: FDRVTVUBEHSRKZ-UHFFFAOYSA-N
Target: Endothelial lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5X9
Assay: Biological Activity: Endothelial lipase (EL) and hepatic lipase (HL) activities were measured using a fluorescent substrate, A10070, (Invitrogen, CA) doped into an artificial vesicle containing DMPG (Avanti Polar Lipids) as the excipient. Vesicles were prepared by combining 571 μL of 29 mM DMPG in a 1:1 mixture of MeOH and CHCl3 with 2000 μL of 1 mM A10070 in a 1:1 mixture of MeOH and CHCl3. The mixture was dried under nitrogen in multiple vials then resuspended in 20 mL total volume of 50 mM HEPES pH 8.0 buffer containing 50 mM NaCl and 0.2 mM EDTA. The sample was allowed to sit at room temperature for 15 min and then was sonicated 3×4 mins on ice with a Branson Sonicator using duty cycle 1. This preparation provides vesicles with a mole fraction of 0.11 for the FRET substrate.The enzymatic assay was measured using 384-well white Optiplates. Each well contained 20 μL of assay buffer (50 mM HEPES pH 8.0, 50 mM NaCl and 1 mM CaCl2) and 0.25 μL of a DMSO solution containing a compound of interest. EL or HL (10 μL) was added and allowed to incubate with the compound for 30 min at 37° C. The source of EL was conditioned media obtained from HT-1080 cells that were transformed using RAGE technology (Athersys) to overexpress endogenous EL, and HL was partially purified from conditioned media obtained from COS cells overexpressing HL. The reaction was started by the addition of 10 μL of a 1:10 dilution of vesicles. The final total reaction volume was 20.25 μL. The reaction rates were measured on a Gemini plate reader with an excitation wavelength of 490 nm and an emission wavelength of 530 nm. Readings were taken over a period of 60 minutes, and the slope between 300 and 900 secs of the readout was used to calculate the rate of the reaction.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 691.73, ALogP 5.23, HBA 7, HBD 4, PSA 146.36, QED 0.15
Compound (SMILES): CCS(=O)(=O)Nc1ncc(Oc2ccc(F)cc2F)c(-c2cn(C)c(=O)c3c2CCCC3)n1
InChIKey: DXNWVHLNIBBJTH-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: In Vitro Enzyme Inhibition Assay: Determination of the IC50 for the heterocyclic derivative BRD4 inhibitors disclosed herein was performed as follows. His-tagged BRD4 was cloned, expressed and purified to homogeneity. Filipakopoulos et al., Nature 468:1067 (2010). BRD4 binding and inhibition was assessed by monitoring the interaction of biotinylated H4-tetraacetyl peptide (AnaSpec, H4K5/8/12/16(Ac), biotin-labeled) with the target using the AlphaScreen technology (Life Technologies). In a 384-well ProxiPlate BRD4(BD1) (2 nM final) was combined with peptide (15 nM final) in 50 mM HEPES (pH 7.3), 10 mM NaCl, 0.25 mM TCEP, 0.1% (w/v) BSA, and 0.005% (w/v) Brij-35 either in the presence of DMSO (final 0.4% DMSO) or compound dilution series in DMSO. After 20 min incubation at room temp, Alpha streptavidin donor beads and Nickel Chelate acceptor beads were added to a final concentration of 5 μg/mL. After 2 hr of equilibration, plates were read on an Envision instrument and the IC50 was calculated using a four parameter non-linear curve fit. Chemistry Example 1 (2-methyl-4-phenylisoquinolin-1-one) had an IC50 of 2.782 μM in this assay format.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 476.51, ALogP 3.55, HBA 7, HBD 1, PSA 103.18, QED 0.58
Compound (SMILES): Cn1ccc2c(-c3ncc(Nc4ccc(N5CCOCC5)cn4)c4c3CNC4=O)ccnc21
InChIKey: DTLNBTLCQQPKRD-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: Biochemical Enzyme Assay: HPK1 biochemical enzyme assay: HPK1 enzyme inhibition was measured using a microfluidic mobility shift assay. Reactions were performed in a 384-well plate, containing 1.5 nM HPK1 (Invitrogen), in assay buffer (Carna Biosciences; pH 7.4). Test compounds were titrated in ten point curves (top final assay concentration 3 μM), and preincubated with enzyme/substrate mix for 30 min prior to initiation of the reaction by addition of ATP (1 mM final concentration) and substrate (1 μM final concentration; Carna Biosciences) diluted in assay buffer supplemented by MgCl2 (final assay concentration of 5 mM). Following 60 min incubation at RT, the reaction was terminated by addition of 60 l/well termination buffer (Carna Biosciences) and signal determination using aCaliper EZ Reader (Perkin Elmer, UK).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 441.5, ALogP 2.85, HBA 8, HBD 2, PSA 97.2, QED 0.5
Compound (SMILES): CC(C(=O)N1CCOCC1)N1CCC(N[C@@H]2C[C@H]2c2ccccc2)CC1
InChIKey: ZEBFHUSPYBVICS-KHMGXFTDSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: LSD1 enzymatic activity: Briefly, compounds of the present invention were solubilized in DMSO and a series of 10, three-fold serial dilutions were made for each compound in 15% DMSO. The initial starting concentration for the serial dilutions of each compound was 10 μM. Control samples lacking compound, LSD1 enzyme or various reaction components also were prepared and processed in parallel with compound test samples.An aliquot of each serial dilution of test compound was added to a 96 well plate containing 50 nM purified N-truncated LSD1 enzyme (RBC Cat# PDM-11-350), 50 mM Tris-HCl, pH 7.5, 0.05% CHAPS and 1% DMSO in a 10 microliter reaction volume. The plate was pre-incubated at room temperature for 30 min to which 10 M of histone 3.3 peptide (aa 1-21) was added to initiate the enzymatic reaction. The reaction mixture was incubated at room temperature for one hour. After one hour, 10 μl of a detection mixture of horseradish peroxidase (Sigma Cat #P8375) and Amplex Red (InVitrogen A36006) was added and kinetic measurements were read at 5 minute intervals for a period of 30 minutes using an Envision Multiplate Reader (PerkinElmer; excitation at 535 nM and emmission read at 590 nM). The IC50 value for each compound was determined from each 10-point dose-response curve using GraphPad Prism 4 software with a sigmodial dose response.
Measurement: IC50 = 108 nM (pChEMBL 6.96999979019165)
Descriptors: MW 357.5, ALogP 1.84, HBA 4, HBD 1, PSA 44.81, QED 0.87
Compound (SMILES): COc1cc(-n2cnc(Nc3nc(N4CCC[C@H]4CO)nc4cc(N5CCNCC5)ccc34)c2)cc(OC)c1OC
InChIKey: CWTHFOSWHDJQNL-FQEVSTJZSA-N
Target: Activin receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q04771
Assay: Biochemical assay: Biochemical assays to measure the inhibitory effects of the compounds were performed by ThermoFisher Scientific (Life Technologies). ALK2 inhibition was tested using LanthaScreen Eu Kinase Binding Assay screening protocol.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 560.66, ALogP 2.96, HBA 12, HBD 3, PSA 122.06, QED 0.28
Compound (SMILES): CCn1c(-c2cccnc2[C@H](C)OC)c2c3cc(ccc31)-c1csc(n1)C[C@H](NC(=O)[C@@H](COC1CN(C(=O)C#CC(C)(C)N(C)C)C1)OC)C(=O)N1CCC[C@H](N1)C(=O)OCC(C)(C)C2
InChIKey: GNKMOCUQYMVVAM-NUMDHXJMSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Biochemical Assay: A variety of Ras proteins may be inhibited by compounds of the present invention (e.g., K-Ras, N-Ras, H-Ras, and mutants thereof at positions 12, 13 and 61, such as G12C, G12D, G12V, G12S, G13C, G13D, and Q61L, and others described herein). The purpose of this biochemical assay is to measure the ability of test compounds to covalently label nucleotide-loaded K-Ras isoforms. In assay buffer containing 12.5 mM HEPES pH 7.4, 75 mM NaCl, 1 mM MgCl2, 1 mM BME, 5 uM Cyclophilin A and 2 uM test compound, a 5 uM stock of GMP-PNP-loaded K-Ras (1-169) G12C is diluted 10-fold to yield a final concentration of 0.5 uM; with final sample volume being 100 uL. The sample is incubated at 25�� C. for a time period of up to 24 hours prior to quenching by the addition of 10 uL of 5% Formic Acid. Quenched samples are centrifuged at 15000 rpm for 15 minutes in a benchtop centrifuge before injecting a 10 uL aliquot onto a reverse phase C4 column and eluting into the mass spectrometer with an increasing acetonitrile gradient in the mobile phase. Analysis of raw data may be carried out using Waters MassLynx MS software, with % bound calculated from the deconvoluted protein peaks for labeled and unlabeled K-Ras.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 925.17, ALogP 4.79, HBA 14, HBD 2, PSA 169.69, QED 0.16
Compound (SMILES): Cc1sc(-c2cnn(C)c2C(=O)NC2CCn3cc(-c4cccc(F)c4)nc3C2)nc1CF
InChIKey: BDQNUILQZZKXTE-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Phosphodiersterase Evaluation Assay: IMAP TR-FRET Phosphodiersterase Evaluation Assay Kit (Molecular Device) was used for the measurement. Ten μL of a diluted test compound having each concentration and 5 μL of the enzyme PDE10 (BPA Bioscience) that had been diluted to 2 ng/mL with 1×IMAP Reaction Buffer (prepared with 5×included with the kit, 10 mM Tris-HCl, pH=7.2, 10 mM MgCl2, 0.05% NaN3, and 0.1% BSA) containing 0.1% BSA were added to a 384-well plate (Corning). The obtained mixture was then pre-incubated at room temperature for 5 minutes. Thereafter, 5 μL of cAMP substrate solution which was included with the kit and diluted to 400 nM with 1×IMAP Reaction Buffer containing 0.1% BSA, was added to the reaction solution, and the obtained mixture was reacted at room temperature for 60 minutes. Thereafter, 60 μL of an IMAP TR-FRET Binding solution included with the kit was further added to the reaction solution, and the obtained mixture was then left for 3 hours or more. Subsequently, using ARVO SX (PerkinElmer), the fluorescent intensity of Terbium (Emission=490 nm) and TR-FRET (Emission=520 nm) in the reaction solution were measured at an excitation wavelength of 340 nm, so as to calculate the amount of the generated 5′-AMP. The count of a well to which a solvent had been added instead of the test compound was set at 0%, and the count of a well to which the enzyme PDE10 had not been added was set at 100%. Thus, the inhibitory activity of each test compound was calculated.
Measurement: IC50 < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 468.53, ALogP 4.07, HBA 7, HBD 1, PSA 77.63, QED 0.48
Compound (SMILES): CCn1c(-c2cccnc2[C@H](C)OC)c2c3cc(ccc31)-c1cc(O)cc(c1)C[C@H](NC(=O)[C@H](C(C)C)N(C)C(=O)[C@H]1CCN(C(=O)/C=C/CN(C)C)C1)C(=O)N1CCC[C@H](N1)C(=O)OCC(C)(C)C2
InChIKey: XRIAUNACEBCYEO-AIQWZNECSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Biochemical Assay: A variety of Ras proteins may be inhibited by compounds of the present invention (e.g., K-Ras, N-Ras, H-Ras, and mutants thereof at positions 12, 13 and 61, such as G12C, G12D, G12V, G12S, G13C, G13D, and Q61L, and others described herein). The purpose of this biochemical assay is to measure the ability of test compounds to covalently label nucleotide-loaded K-Ras isoforms. In assay buffer containing 12.5 mM HEPES pH 7.4, 75 mM NaCl, 1 mM MgCl2, 1 mM BME, 5 uM Cyclophilin A and 2 uM test compound, a 5 uM stock of GMP-PNP-loaded K-Ras (1-169) G12C is diluted 10-fold to yield a final concentration of 0.5 uM; with final sample volume being 100 uL. The sample is incubated at 25�� C. for a time period of up to 24 hours prior to quenching by the addition of 10 uL of 5% Formic Acid. Quenched samples are centrifuged at 15000 rpm for 15 minutes in a benchtop centrifuge before injecting a 10 uL aliquot onto a reverse phase C4 column and eluting into the mass spectrometer with an increasing acetonitrile gradient in the mobile phase. Analysis of raw data may be carried out using Waters MassLynx MS software, with % bound calculated from the deconvoluted protein peaks for labeled and unlabeled K-Ras.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 961.22, ALogP 5.9, HBA 12, HBD 3, PSA 178.88, QED 0.11
Compound (SMILES): Cc1cc(NC(=O)NC2c3ccccc3C(C)(C)C[C@H]2O)c(-c2ccccc2)nc1-c1ccc(S(C)(=O)=O)nc1
InChIKey: BBMMNQCXEFUAFI-MIJJZIGMSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Evaluation of Binding Activity for Human TrkA Protein: Measurement was carried out using TrkA LanthaScreen (registered trademark) Eu Kinase Binding Assay (ThermoFisher SCIENTIFIC). To a 384 well plate (Corning), 2.5 μL of the test compound having various concentrations and diluted with Kinase buffer (ThermoFisher SCIENTIFIC) and 2.5 μL of 15 nM TrkA enzyme (ThermoFisher SCIENTIFIC) were added. Moreover, 5 μL of 3 nM Eu-anti-His Tag antibody (ThermoFisher SCIENTIFIC) and 5 μL of 30 nM Kinase (registered trademark) Tracer 236 (ThermoFisher SCIENTIFIC) were added, followed by reaction at room temperature for 60 minutes. After the reaction, the fluorescence intensity of Europium (Emission wavelength 615 nm) and TR-FRET (Emission wavelength 665 nm) with an excitation wavelength of 340 nm were measured with EnVision 2100 (PerkinElmer) to calculate the fluorescence ratio as the amount of the test compound and the TrkA enzyme bonded. The inhibitory activity (IC50 value) of each test compound was calculated with the fluorescence ratio of the well to which a solvent was added instead of the test compound being 0% and the fluorescence ratio of the well without addition of the TrkA protein being 100%.The TrkA inhibitory activity of the test compound can be evaluated by IC50 value, and Table 1 shows the compounds having an IC50 value of 50 nmol/L or less as A (activity is very high), the compounds having an IC50 value of more than 50 nmol/L and 1000 nmol/L or less as B (activity is high), and the compounds having an IC50 value of more than 1000 nmol/L as C (activity is low).
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 556.69, ALogP 5.43, HBA 6, HBD 3, PSA 121.28, QED 0.3
Compound (SMILES): O=C(NC1CCOCC1)N1CCC([C@@H](O)C[C@H]2c3ccccc3-c3cncn32)CC1
InChIKey: CRPBNHZIHKRNAA-UNMCSNQZSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: Enzymatic IDO Assay: The IC50 values for each compound were determined by testing the activity of IDO in a mixture containing 50 mM potassium phosphate buffer at pH 6.5; 70 nM purified human IDO protein, 200 μM L-tryptophan, 20 mM ascorbate, 20 μM methylene blue, 0.1% DMSO. The inhibitors were initially diluted in DMSO at 100 mM and were diluted in potassium phosphate 50 mM, added to the reaction mixture at final concentrations raging from 1 mM to 5 nM and preincubated with the enzyme for 5 min at 25° C. The reaction was started by addition of L-tryptophan to 200 μM and incubated 15 min at 37° C. The reaction was stopped by addition of 0.5 vol of 30% trichloroacetic acid and incubated 30 min at 60° C. to hydrolyze N-formylkynurenine to kynurenine. The reaction was centrifuged at 3400 g for 5 min to remove precipitated protein and the supernatant was reacted with 2% (w/v) of p-dimethylaminobenzaldehyde in acetic acid. The reaction was incubated 10 min at 25° C. and read at 480 nm in a spectrophotometer. Control samples with no IDO inhibitor, or with no IDO enzyme or with the reference inhibitors 1-methyl-tryptophan (200 μM) and menadione (1.2 μM) were used as controls to set the parameters for the non-linear regressions necessary for determination of the IC50 for each compound. Nonlinear regressions and determination of the IC50 values were performed using the GraphPad Prism 4 software. Compounds with an IC50 of less than 500 μM were considered as active inhibitors in this assay.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 410.52, ALogP 2.8, HBA 5, HBD 2, PSA 79.62, QED 0.81
Compound (SMILES): Cc1cc(N)nc(C)c1CNC(=O)c1ccc2c(c1)C1OC2c2ccc(-c3ccc(F)c(C(F)(F)F)c3)cc21
InChIKey: IPDRWCSRZZELEX-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Plasma Kallikrein Inhibition Assay: The chromogenic substrate D-Pro-Phe-Arg-pNa, 2HCl (BIOPHEN CS-31(02) from Hyphen BioMed, Neuville-Sur-oise, France) was dissolved in 5 mL deionized water and stored at 4° C. Concentration was determined in the spectrophotometer at 342 nm using an extinction coefficient of 8270. All other chemicals were of analytical grade.Human plasma kallikrein was purchased from Enzyme Research Labs (South Bend, Ind., USA, batch HPKa 2830). A stock solution of 7 μM in 50% glycerol was stored at −20° C.Enzyme reactions were conducted in assay buffer comprised of 20 mM HEPES at pH 7.4, 150 mM NaCl, 0.1% PEG-8000 and 0.01% Triton X-100.Both enzyme and substrate were diluted in assay buffer.The compound solutions as well as the enzyme and the substrate solutions were transferred to 96-well plates (Clear, UV-Star, Flat-bottom, Half-Area plates; cat. No. 675801 Greiner Bio-one, purchased from VWR International, Arlington Heights, Ill., USA) using a Rainin LTS 96-channel pipettor (Rainin, Columbus, Ohio, USA). Plate measurements were conducted using a SPECTROStar Nano reader (BMG Labtech, San Francisco, Calif., USA). The SPECTROStar Nano is a spectrophotometer and absorbance was measured at 405 nm. We used discrete wavelength, precise, kinetic reads of 15 cycles with a 60 sec cycle time.Determination of IC50 valuesFor the determination of IC50 values, the assays were performed at room temperature in 96-well plates with a total assay volume of 85 μL per well. The test compound was dissolved in 100% DMSO. The compounds were serially diluted in DMSO in a 7 point dose response. For the assays, 66.5 μL of protease solution (protease in assay buffer) was added per well followed by the addition of 8.5 μL of compound in 100% DMSO. The final assay concentration of the human plasma kallikrein was 250 μM. After 30 min. incubation at room temperature on an orbital shaker, the reactions were started by the addition of 10 μL substrate solution (in assay buffer, final assay concentration was 600 uM). After the addition of the substrate solution the final DMSO concentration was 10%. The plate was placed again on the shaker for 5 sec, spun at 2000 rpm for 5 sec and read on the spectrophotometer. The effect of the compound on the enzymatic activity was obtained from the linear part of the progress curves and determined after 15 minutes.
Measurement: IC50 = 437 nM (pChEMBL 6.360000133514404)
Descriptors: MW 533.53, ALogP 6.56, HBA 4, HBD 2, PSA 77.24, QED 0.29
Compound (SMILES): Cc1cc(-c2[nH]c3ccc(C4CCN(C(=O)CCN5CCOCC5)CC4)cc3c2C(C)C)cc(C)n1
InChIKey: NFAOXCQIPPYLJJ-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications.
Measurement: IC50 = 3.9 nM (pChEMBL 8.40999984741211)
Descriptors: MW 488.68, ALogP 5.4, HBA 4, HBD 1, PSA 61.46, QED 0.5
Compound (SMILES): CCCCc1nc(O)c(-c2nnc(CCc3nc(-c4ccccn4)no3)o2)c(O)c1-c1c(OC)cccc1OC
InChIKey: RJLSYFUCCNWJBU-UHFFFAOYSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: Intracellular cAMP Accumulation Assay: HEK293 cells stably expressing human APJ receptor were used to assess the activity of compounds. Cultured cells were detached and resuspended in the cAMP Homogeneous Time-Resolved Fluorescence (HTRF) assay buffer (Cisbio cat; #62AM4PEJ). The assay was performed in 384-well assay plates (Perkin-Elmer; cat #6008289) according to assay protocol provided by the manufacturer. Serial dilutions of a compound together with assay buffer containing 0.2 nM IBMX and 2 μM forskolin were added to each well containing 5,000 cells and incubated for 30 minutes at room temperature. Subsequently, cAMP D2 reagent was added in the lysis buffer followed by the EuK antibody (Cisbio; cat #62AM4PEJ) and incubated for 60 min. The fluorescence emission ratio was measured using fluorometer. The intracellular cAMP concentrations (compound-stimulated inhibition of forskolin-mediated cAMP production) were calculated by extrapolation from a standard curve using known cAMP concentrations. The EC50 values were obtained by fitting the data to a sigmoidal concentration-response curve with variable slope. The maximal achievable inhibition of forskolin-induced cAMP levels (Ymax) for each compound was expressed as relative percentage of inhibition attained using pyroglutamated apelin-13 ((Pyr1)apelin-13) peptide, which was set to 100%.The examples disclosed below were tested in the APJ in vitro assays described above and were found having human APJ cyclic AMP (hcAMP) activity. The EC50 value of each compound is presented at the end of the example description.
Measurement: EC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 544.57, ALogP 4.8, HBA 12, HBD 2, PSA 162.54, QED 0.23
Compound (SMILES): Cc1c(OCC2CCN(C)CC2)nn(-c2ccccc2)c1NC(=O)NC1c2ccccc2CC[C@H]1O
InChIKey: PJWZRKSGKLECQH-IKOFQBKESA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Kinase Binding Assay: TrkA binding activity was determined in a TrkA LanthaScreen Eu Kinase Binding Assay. 5 nM His-tagged recombinant human TrkA (6HIS tagged cytoplasmic domain from Invitrogen, Catalog No. PV3144) was incubated with 4 nM Alexa-Fluor® Tracer 236 (Invitrogen Cat. No. PV5592), 2 nM biotinylated anti-His (Invitrogen Cat. No. PV6090), and 2 nM europium-labeled Streptavidin (Invitrogen Cat. No. PV5899), in buffer (25 mM MOPS, pH 7.5, 5 mM MgCl2, 0.005% Triton X-100). Three fold serial dilutions of compounds of the invention in DMSO were added to a final percentage of 2% DMSO. After 60-minute incubation at 22° C., the reaction was measured using the EnVision mutlimode plate reader (PerkinElmer) via TR-FRET dual wavelength detection at 615 nM and 665 nM. The percent of control was calculated using a ratiometric emission factor.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 489.62, ALogP 4.07, HBA 6, HBD 3, PSA 91.65, QED 0.49
Compound (SMILES): CN(CC(C)(C)O)C(=O)C[C@@H]1CCc2cc(C(O)(C(F)(F)F)C(F)(F)F)ccc2N1S(=O)(=O)c1ccc(F)cc1
InChIKey: ITDBJRVRXPTXKJ-SFHVURJKSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Spa Binding Assay: The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol 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 chromotography. 50 nM RORγ (A262-S507) is incubated with test compound at varying concentrations for 15 min at room temperature in PBS buffer containing 0.5% fatty acid free BSA. 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 15 min. 4 mg/mL of Ysi Copper HIS-TAG SPA Beads (Perkin Elmer) are added, and the mixture is incubated for 30 min. The reaction is read on a MicroBeta Trilux scintillation plate reader (Perkin Elmer).
Measurement: IC50 = 29.3 nM (pChEMBL 7.53000020980835)
Descriptors: MW 600.55, ALogP 4.27, HBA 5, HBD 2, PSA 98.15, QED 0.46
Compound (SMILES): C=CC(=O)Nc1cccc(-c2cnc3[nH]cc(-c4ccnc(C)c4)c3c2)c1
InChIKey: SLPIDGQHMOOHRY-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ITK/TSK (SINGLE PROTEIN), Homo sapiens
UniProt: Q08881
Assay: ITK Inhibitor Binding Kinetics: This assay is based on the binding of a proprietary, Alexa Fluor 647-labeled, ATP-competitive kinase inhibitor (kinase tracer-236) to the ITK expression construct in the presence of a Europium-conjugated antibody, resulting in a FRET (fluorescence resonance energy transfer) signal. Displacement of the kinase tracer by compound results in a lower emission ratio upon excitation of the Europium chelate. Candidate compounds are designed as potential irreversible inhibitors of ITK, capable of ligating to an active site cysteine residue resulting in time dependent covalent binding. The time dependent nature of irreversible inhibition is investigated by performing the binding assay with and without a pre-incubation of compound and ITK.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 354.41, ALogP 4.72, HBA 3, HBD 2, PSA 70.67, QED 0.52
Compound (SMILES): CCS(=O)(=O)c1ccc(Cl)cc1Cn1cnc2c(Cl)c(CN3CC[C@@H](CNC)C3)c(C(F)(F)F)cc2c1=O
InChIKey: DLXBTGAHZYDKRB-HNNXBMFYSA-N
Target: Epithelial discoidin domain-containing receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q08345
Assay: LanthaScreen Assay: DDR1 binding activity was measured using the LanthaScreen (Registered trademark) Eu Kinase Binding Assay (manufactured by Life Technologies Corporation). The test compound and the Alexa Fluor® 647-labeled Kinase Tracer 178 (manufactured by Life Technologies Corporation) were added to a mixture of DDR1 and the LanthaScreen (Registered trademark) Eu-anti-GST antibody. After one-hour reaction at room temperature, the fluorescence resonance energy transfer was measured. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate relative to the test compound-free control.
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 591.48, ALogP 4.61, HBA 7, HBD 1, PSA 84.3, QED 0.41
Compound (SMILES): Cc1csc2c(-n3nc(Nc4ccc5c(c4)CCC(N(C)C4CC6CCC4C6)CC5)nc3N)nc(Cl)nc12
InChIKey: BNNQSSKJQPUHNC-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: PHOSPHO-AKT IN-CELL WESTERN ASSAY: The resulting chemiluminescence was read with a Luminomitor within 10 minutes to minimize changes in signal intensity. After reading the chemiluminescence, the cells were washed 1× with wash buffer and 1× with PBS by plate washer. The plate was tapped onto paper towels to remove excess liquid from wells and air-dried at ambient temperature for 5 minutes.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 549.15, ALogP 5.93, HBA 9, HBD 2, PSA 97.78, QED 0.24
Compound (SMILES): CCS(=O)(=O)NC(=O)c1ccc(COc2cc(F)cc(-c3ccc(OCC4CC4)nc3)c2)cc1
InChIKey: RTHGSPNVKIOGLY-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: EFS-FRET Assay: This screen is used to determine the effects of compounds on human Nav1.7 channels, utilizing electrical field stimulation (EFS) system in 96-well plate format on FDSS (Hamamatsu Photonics) platform. The changes of membrane potential are monitored with FRET dye pair, DiSBAC2(3) and PTS 18.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 484.55, ALogP 4.34, HBA 6, HBD 1, PSA 94.59, QED 0.46
Compound (SMILES): CCn1c([C@]2(C)CCCN2C(=O)c2cc(C)ccc2-n2nccn2)nc2cc(OC)c(OC)cc21
InChIKey: MCJXSALINKTKNY-SANMLTNESA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Inhibition Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 μg/ml G418, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2.Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 33 nM (pChEMBL 7.480000019073486)
Descriptors: MW 474.57, ALogP 4.11, HBA 8, HBD 0, PSA 87.3, QED 0.42
Compound (SMILES): CCS(=O)(=O)Nc1cc(-c2ccc3nc(-c4nc5ccccc5[nH]4)oc3c2)ccc1F
InChIKey: KOISVAZGRAWYOM-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Scintillation Proximity Assay (PI3K-γ): [γ-33P]ATP (10 mCi/mL) and Wheat Germ Agglutinin (WGA) YSi SPA Scintillation Beads was purchased from Perkin-Elmer (Waltham, Mass.). Lipid kinase substrate, D-myo-Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)D (+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho linked (PIP2), CAS 204858-53-7, was purchased from Echelon Biosciences (Salt Lake City, Utah). PI3Kγ (p110γ) Recombinant Human Protein was purchased from Life technology (Grand Island, N.Y.). ATP, MgCl2, DTT, EDTA, MOPS and CHAPS were purchased from Sigma-Aldrich (St. Louis, Mo.).The kinase reaction was conducted in polystyrene 384-well Greiner Bio-one white plate from Thermo Fisher Scientific in a final volume of 25 μL. Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 2%. The PI3Kγ assay was carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 μM PIP2, 2 μM ATP, 0.5 μCi [γ-33P] ATP, 13 nM PI3Kγ. Reactions were incubated for 120 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 163 mM potassium phosphate pH 7.8, 20% glycerol, 25 mM EDTA. The final concentration of SPA beads is 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1500 rpm for 10 min, the radioactivity of the product was determined by scintillation counting on Topcount (Perkin-Elmer).
Measurement: IC50 < 250 nM (pChEMBL 6.6020002365112305)
Descriptors: MW 436.47, ALogP 4.94, HBA 5, HBD 2, PSA 100.88, QED 0.41
Compound (SMILES): Cc1ccc(-c2cc(C(F)(F)F)c3c(N)ncnn23)cc1C(=O)Nc1cn(Cc2ccc(C(F)(F)F)cc2)cn1
InChIKey: DRSRAKSYPOKKPW-UHFFFAOYSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: RIPK1 HTRF Binding Assay: A solution was prepared containing 0.2 nM Anti GST-Tb (Cisbio, 61GSTTLB), 90.6 nM probe and 1 nM His-GST-TVMV-hRIPK1(1-324) in FRET Buffer (20 mM HEPES, 10 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 0.05 mg/mL BSA). Using Formulatrix Tempest, the detection antibody/enzyme/probe solution (2 mL) was dispensed into wells of a 1536 plate (Black Low Binding Polystyrene 1536 Plate (Corning, 3724)) containing 10 nL of compounds of interest at appropriate concentration in DMSO. The plate was incubated at rt for 1 h. FRET was measured using the EnVision plate reader (Excitation: 340 nM, Emission: 520 nM/495 nM). Total signal (0% inhibition) was calculated from wells containing 10 nL DMSO only. Blank signal (100% inhibition) calculated from wells containing 10 nL of 15 nM staurosporine and internal controls.
Measurement: IC50 = 27000 nM (pChEMBL 4.570000171661377)
Descriptors: MW 559.47, ALogP 5.82, HBA 7, HBD 2, PSA 103.13, QED 0.26
Compound (SMILES): CCn1nccc1-c1c(-c2cccc(C#N)c2)nc(N)c2nc(C(O)c3c(F)cccc3F)nn12
InChIKey: CFVWBRAURJJFJF-UHFFFAOYSA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: HTRF Assay: Assays were conducted in black low volume 384-well polystyrene plates (Greiner 784076-25) in a final volume of 10 μL. Test compounds were first serially diluted in DMSO and 100 nl added to the plate wells before the addition of other reaction components. The final concentration of DMSO was 1%. Tag-Lite® Adenosine A2A labeled cells (CisBio C1TT1A2A) were diluted 1:5 into Tag-lite buffer (CisBio LABMED) and spun 1200 g for 5 mins. The pellet was resuspended at a volume 10.4× the initial cell suspension volume in Tag-lite buffer, and Adenosine A2A Receptor Red antagonist fluorescent ligand (CisBio L0058RED) added at 12.5 nM final concentration. 10 ul of the cell and ligand mix was added to the assay wells and incubated at room temperature for 45 minutes before reading on a PHERAstar FS plate reader (BMG Labtech) with HTRF 337/620/665 optical module. Percent binding of the fluorescent ligand was calculated; where 100 nM of A2A antagonist control ZM 241385 (Tocris 1036) displaces the ligand 100% and 1% DMSO has 0% displacement. The % binding data versus the log of the inhibitor concentration was fitted to a one-site competitive binding model (GraphPad Prism version 7.02) where the ligand constant=12.5 nM and the ligand Kd=1.85 nM.
Measurement: Ki < 10 nM (pChEMBL 8)
Descriptors: MW 472.46, ALogP 3.49, HBA 9, HBD 2, PSA 130.94, QED 0.4
Compound (SMILES): CC(C)(O)c1cc2nccc(C(=O)O)c2[nH]1
InChIKey: CBVFSACWQSKTBS-UHFFFAOYSA-N
Target: Lysine-specific demethylase 5A (SINGLE PROTEIN), Homo sapiens
UniProt: P29375
Assay: Enzymatic Assay: The enzymatic assay of Jarid1A activity is based upon Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) detection. The ability of test compounds to inhibit the activity of Jarid1A was determined in 384-well plate format under the following reaction conditions: 1 nM Jarid1A, 300 nM H3K4me3-biotin labeled peptide (Anaspec cat #64357), 2 μM alpha-ketoglutaric acid in assay buffer of 50 mM HEPES, pH7.3, 0.005% Brij35, 0.5 mM TCEP, 0.2 mg/ml BSA, 50 μM sodium L-ascorbate, and 2 μM ammonium iron(II) sulfate. Reaction product was determined quantitatively by TR-FRET after the addition of detection reagent Phycolink Streptavidin-allophycocyanin (Prozyme) and Europium-anti-mono- or di-methylated histone H3 lysine 4 (H3K4me1-2) antibody (PerkinElmer) in the presence of 5 mM EDTA in LANCE detection buffer (PerkinElmer) at a final concentration of 25 nM and 1 nM, respectively.The assay reaction was initiated by the following: 2 μl of the mixture of 900 nM H3K4me3-biotin labeled peptide and 6 μM alpha-ketoglutaric acid with 2 μl of 11-point serial diluted inhibitor in 3% DMSO was added to each well of plate, followed by the addition of 2 μl of 3 nM Jarid1A to initiate the reaction.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 220.23, ALogP 1.49, HBA 3, HBD 3, PSA 86.21, QED 0.72
Compound (SMILES): O=C(O)c1nn[nH]c1Oc1ccc(C#Cc2cccnc2)cc1
InChIKey: BBQORQUXVPIBQY-UHFFFAOYSA-N
Target: 2-Hydroxyacid oxidase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UJM8
Assay: In-Vitro Human Glycolate Oxidase (hGOX) Assay: The in-vitro glycolate oxidase assay was performed using recombinant full-length human hydroxyacid oxidase 1 (HAO1), the equivalents of hGOX. The enzyme was obtained from AbCam (Catalog #ab113144) and was purified using conventional chromatography to >95% purity. Purified HAO1 was dissolved in assay buffer consisting of 10 mM NaCl, 110 mM KCl, 2 mM MgCl2, 50 mM HEPES (pH 7.4), and 0.01% Triton X-100. The assay used Corning 3575 384-well flat bottom, low flange, non-binding surface, black polystyrene plates.Test compounds in DMSO were preincubated at different concentrations with purified recombinant human GO (6 nM) for 10 min, followed by the addition of glycolate substrate (85 μM) to start the reaction. The plates were incubated for 10 min at room temperature, at which point Amplex red reagent (50 μM) was added.The fluorescence intensity signal was measured on a VariosKan LUX instrument using an excitation of 560 nm and an emission of 590 nm. The IC50 values were calculated using Graphpad Prism.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 306.28, ALogP 2.09, HBA 5, HBD 2, PSA 100.99, QED 0.72
Compound (SMILES): O=c1[nH]c2ncccc2c(=O)n1O
InChIKey: VRPVMTYMAHQMDP-UHFFFAOYSA-N
Target: Flap endonuclease 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P39748
Assay: Enzymatic Assay: Fluorogenic biochemical assays used recombinant full-length FEN-1 or catalytic domains of Exo1, XPG or GEN1 and 200 nM of a DNA substrate (formed by annealing three oligonucleotidesquencher (5′-CAC GTT GAC TAC CGC TCA ATC CTG ACG AAC ACA TC-BHQ2), flap (5′-TAMRA-GA TGT CAA GCA GTC CTA ACT TTG AGG CAG AGT CCG C) and template (5′-GC GGA CTC TGC CTC AAG ACG GTA GTC AAC GTG-3′) (PMID: 21062821)) in 50 mM Tris pH 8.0, 10 mM MgCl2, 1 mM DTT, and 0.01% Tween-20. Inhibitors arrayed in dose response were added from DMSO stocks (using a V&P 384-pintool head) to the enzyme solution in low volume, black polystyrene 384-well plates (Corning #3677) 15 minutes prior to the addition of the DNA substrate. Reactions were allowed to proceed for 2-4 hours at 25° C. or 37° C. Fluorescence data (Excitation: 530 nm/10 nm bandwidth; Emission: 590 nm/20 nm bandwith) were measured with an Infinite M1000 plate reader (Tecan).
Measurement: IC50 = 106 nM (pChEMBL 6.96999979019165)
Descriptors: MW 179.13, ALogP -0.68, HBA 5, HBD 2, PSA 87.98, QED 0.52