lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1cccc(OC)c1-n1c(NS(=O)(=O)CCN2C(=O)CCC[C@@H]2C)nnc1-c1ccco1
InChIKey: DUTMNZHRXNSITG-HNNXBMFYSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: GTPγS 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 assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, and 0.1% (w/v) BSA with 200 mM NaCl, 3 μM GDP] and membranes expressing human APJ receptor/well along with WGA PS beads. The reaction was initiated by addition of 0.2 nM [35S]GTPγS in the absence or presence of various ligands and incubated at RT for 90 min. Nonspecific binding was determined in the presence of 100 μM GTPγS and was always less than 0.2% of total binding.
Measurement: EC50 = 1 nM (pChEMBL 9)
Descriptors: MW 489.55, ALogP 2.69, HBA 9, HBD 1, PSA 128.79, QED 0.49
Compound (SMILES): COc1ccccc1CN1C[C@]2(CC[C@](c3ccccc3)(N(C)C)CC2)N(Cc2ccc(C#N)cc2)C1=O
InChIKey: OMKDSWPCDJXLHL-MEKGRNQZSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Receptor Binding Assay: Mu-Opioid Peptide (hMOP): The hMOP receptor binding assay was performed as homogeneous SPA-assay (scintillation proximity assay) using the assay buffer 50 mM TRIS-HCl (pH 7.4) supplemented with 0.052 mg/ml bovine serum albumin (Sigma-Aldrich Co. St. Louis. Mo.). The final assay volume (250 μl/well) included 1 nM of [N-allyl-2.3-3H]naloxone as ligand (PerkinElmer Life Sciences. Inc. Boston. Mass. USA). and either test compound in dilution series or 25 μM unlabelled naloxone for determination of unspecific binding. The test compound was diluted with 25% DMSO in H2O to yield a final 0.5% DMSO concentration. which also served as a respective vehicle control. The assay was started by adding wheat germ agglutinin coated SPA beads (GE Healthcare UK Ltd. Buckinghamshire. UK) which had been preloaded with hMOP receptor membranes (PerkinElmer Life Sciences. Inc. Boston. Mass. USA). After incubation for 90 minutes at RT and centrifugation for 20 minutes at 500 rpm the signal rate was measured by means of a 1450 Microbeta Trilux β-counter (PerkinElmer Life Sciences/Wallac. Turku. Finland). Half-maximal inhibitory concentration (IC50) values reflecting 50% displacement of [3H]naloxone-specific receptor binding were calculated by nonlinear regression analysis and Ki values were calculated by using the Cheng-Prusoff equation.
Measurement: Ki = 390 nM (pChEMBL 6.409999847412109)
Descriptors: MW 508.67, ALogP 5.77, HBA 4, HBD 0, PSA 59.81, QED 0.41
Compound (SMILES): CCS(=O)(=O)c1ccc(Cl)cc1Nn1cnc2cc(CN3CCCOCC3)c(Br)cc2c1=O
InChIKey: JGCPBKXGFDSOBA-UHFFFAOYSA-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 = 290 nM (pChEMBL 6.539999961853027)
Descriptors: MW 555.88, ALogP 3.7, HBA 8, HBD 1, PSA 93.53, QED 0.5
Compound (SMILES): CS(=O)(=O)Nc1cnn(C(=O)N2CCN(Cc3ccc(-c4ccccc4)cc3Cl)CC2)c1
InChIKey: VKBBTGTVJYGVQY-UHFFFAOYSA-N
Target: Fatty-acid amide hydrolase 1 (SINGLE PROTEIN), Mus musculus
UniProt: O08914
Assay: In Vitro Competitive Activity-Based Protein Profiling: Proteomes (mouse brain membrane fraction or cell lysates for mouse assays; human prefrontal cortex or cell membrane fractions for human assays) (50 μL, 1.0 mg/mL total protein concentration) were preincubated with varying concentrations of inhibitors at 37° C. After 30 min, FPRh or HT-01 (1.0 μL, 50 μM in DMSO) was added and the mixture was incubated for another 30 min at 37° C. Reactions were quenched with SDS loading buffer (15 μL-4×) and run on SDS-PAGE. Following gel imaging, serine hydrolase activity was determined by measuring fluorescent intensity of gel bands corresponding to MAGL and FAAH using ImageJ 1.43u software.Preparation of Mouse Brain Proteomes from Inhibitor Treated Mice.Inhibitors were administered to wild-type C57Bl/6J by oral gavage in a vehicle of polyethylene glycol. Each animal was sacrificed 4 h following administration and brain proteomes were prepared and analyzed according to previously established methods (See Niphakis, M. J., et al. (2011) ACS Chem. Neurosci. and Long, J. Z., et al. Nat. Chem. Biol. 5:37-44).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 473.99, ALogP 3.36, HBA 6, HBD 1, PSA 87.54, QED 0.61
Compound (SMILES): CCOC(=O)c1cccc(Sc2c(C)n(CC(=O)N(C)C)c3c(F)c(Cl)ccc23)c1
InChIKey: ZZABGPPTZWHNLK-UHFFFAOYSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Inhibition Assay: ATX activity is assayed in concentrated conditioned media from Hep3B human hepatocellular carcinoma cells by measuring the amount of choline released from the substrate, lysophosphatidylcholine (LPC) as it is cleaved to LPA. Conditioned media is collected from confluent Hep3B cells and concentrated 20-fold using Centriprep-30 filter devices (Millipore). To assay for autotaxin inhibition, 10-20 μL of the concentrated conditioned media is incubated with 2.5 μL of a test compound in DMSO and 72.5-82.5 μL lyso-PLD buffer (100 mM Tris pH 9, 500 mM NaCl, 5 mM MgCl2, 5 mM CaCl2, 0.05% Triton X-100 in the presence or absence of 0.2% fatty-acid-free human serum albumin) for 15 min at 37° C. After the 15 min incubation, 5 uL of 2 mM LPC (14:0; Avanti Polar Lipids Cat#855575C) diluted in lyso-PLD buffer is added for a final concentration of 100 uM and the incubation continues for 1.5-3 hours at 37° C. 100 μL of a color mix containing 4.5 mM 4-aminoantipyrine, 2.7 mM N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine, 21 units/ml horseradish peroxidase and 3 units/mL choline oxidase in 50 mM Tris, pH 8, 4.5 mM MgCl2 is added and the incubation continued for 15 minutes at room temperature before reading the absorbance at 555 nm.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 448.95, ALogP 5.16, HBA 5, HBD 0, PSA 51.54, QED 0.49
Compound (SMILES): NC1CCN(c2ccc(S(=O)(=O)N[C@@H]3CCNC3)c(S(N)(=O)=O)c2-c2nn[nH]n2)C(=O)C1
InChIKey: YFUQHFWRSXGVPJ-QVDQXJPCSA-N
Target: Metallo-beta-lactamase type 2 (SINGLE PROTEIN), Bacteria
UniProt: C7C422
Assay: Enzyme Activity: Determination of IC50 : The Class B enzyme activities were measured in the presence of the test inhibitor in a fluorescence assay against a commercially available substrate consisting of a cephalosporin core linking 7-hydroxycoumarin to fluorescein (CCF2-FA). The enzyme (NDM-1, IMP-1 or VIM-1) and the substrate were diluted in 100 mM KH2PO4 buffer (pH 7) containing 0.005% Tween-20 and 10 μM ZnSO4. In the assay, the final concentration of enzyme was 1 pM, 2 pM and 30 pM for NDM-1, IMP-1 and VIM-1, respectively, and the final concentration of CCF2-FA was 1.25 μM. The test inhibitor was dissolved in dimethylsulfoxide and diluted 1:50 in the assay, resulting in a final concentration range of 20 μM to 0.00063 μM. In a 384-well microplate, the test inhibitor was incubated with the metallo-β-lactamase enzyme and the substrate for 2 hours at 25° C. Fluorescence at 460 nm following excitation at 405 nm was measured. The IC50 value was determined from semi-logarithmic plots of enzyme inhibition versus inhibitor concentration, with a curve generated using a 4-parameter fit.
Measurement: IC50 = 7.19 nM (pChEMBL 8.140000343322754)
Descriptors: MW 485.55, ALogP -2.39, HBA 10, HBD 5, PSA 219.15, QED 0.29
Compound (SMILES): CC1=CC[C@@H]2[C@@H](C1)c1c(O)cc(C(C)(C)C(=O)OCCCN=[N+]=[N-])cc1OC2(C)C
InChIKey: PIFWOUKRNWDMOC-IAGOWNOFSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P20272
Assay: Binding Assay: All the cannabinoids discussed in this invention are tested for their ability to bind to CB1 and CB2 receptors using membranes from rat brain or HEK293 cells expressing either mCB2 or hCB2, respectively via competition-equilibrium binding using [3H]CP-55,940. Results are analyzed using nonlinear regression to determine ligand IC50 (Prism by GraphPad Software, Inc.).
Measurement: Ki = 1 nM (pChEMBL 9)
Descriptors: MW 413.52, ALogP 5.52, HBA 5, HBD 1, PSA 104.52, QED 0.17
Compound (SMILES): COc1cccc(OC)c1C(=O)N[C@@H]1CC(F)C[C@@H]1Nc1nc2ccc(Cl)cc2s1
InChIKey: AUWUTXUWNNCEMB-UCWKZMIHSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Fluorescent Imaging Plate Reader (FLIPR) Assay: Orexin antagonist activity was determined by measuring changes in intracellular calcium levels using a Ca2+ sensitive fluorescent dye. The changes in fluorescent signal were monitored by Fluorescent Imaging Plate Reader (FLIPR) technology available from Molecular Devices, LLC, U.S.A. Orexin mediated increases in intracellular Ca2+ concentration were readily detected upon activation with orexin-A. Twenty-four hours prior to the assay, RBL-2H3 cells stably expressing either human orexin receptor 1 or human orexin receptor 2 were seeded in cell culture medium in black, clear-bottom 384-well plates (commercially available from Corning Inc., U.S.A.) and grown overnight at 37° C., 5% CO2. On the day of the assay, cell culture media was removed and cells were loaded with Calcium 5 Dye (commercially sold by Molecular Devices, LLC, U.S.A.) for 1 hour at 37° C., 5% CO2. Test compounds (at 10 point half log concentration response curves from 10 uM) were added to cells for 15 minutes prior to the addition of orexin-A to all wells, to achieve a final concentration that produces approximately an 80% maximal response. The IC50 values were determined from ten point concentration response curves. Curves were generated using the average of two wells for each data point.
Measurement: IC50 = 64 nM (pChEMBL 7.190000057220459)
Descriptors: MW 449.94, ALogP 4.68, HBA 6, HBD 2, PSA 72.48, QED 0.57
Compound (SMILES): Cc1ccc(NC(=O)N2CC[C@@H](CC(F)(F)F)C2)cc1-c1cc(N[C@H](C)CO)nc(C2=CCOC2)c1
InChIKey: WFXADWBZLIDHEL-MSOLQXFVSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Enzymatic RAF1 Kinase: Small molecule inhibition of RAF1 kinases was measured using ADP-Glo assay. In the assay, ADP is converted to ATP in the presence of test kinase and substrate, resulting in luciferase reaction and luminescent readout with light generated proportional to the relative kinase activity. Compounds diluted in DMSO were used in 10-point, 3-fold dose curve for both assays. Final concentrations of 3 nM RAF1 (CarnaBio, Cat. 09-125) and 30 nM MEK1 substrate (Millipore, Cat. 14-420) were incubated with 3 μM ATP, 10 mM MgCl2, 0.003% Brij-35, 2 mM DTT, 0.05% BSA, 1 mM EGTA, and 50 mM HEPES for 90 minutes at room temp prior to addition of ADP-Glo reagent (Promega, Cat. V9102) for 40 minutes, and detection reagent for 45 minutes. Luminescence was read on an Envision plate reader (PerkinElmer) and percent remaining activity was used to calculate IC50 using a four-parameter fit model using Dotmatics Knowledge Solutions Studies curve fitting (Dotmatics, Bishops Stortford, UK, CM23).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 504.55, ALogP 5.07, HBA 5, HBD 3, PSA 86.72, QED 0.49
Compound (SMILES): CCCn1cc(-n2c(C#N)c(Sc3cccc(C(=O)O)c3)c3ccc(Cl)cc32)cn1
InChIKey: KOCAUNIBIIJPAY-UHFFFAOYSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Human Autotaxin Assay: ATX activity is assayed in concentrated conditioned media from Hep3B human hepatocellular carcinoma cells by measuring the amount of choline released from the substrate, lysophosphatidylcholine (LPC) as it is cleaved to LPA. Conditioned media is collected from confluent Hep3B cells and concentrated 20-fold using Centriprep-30 filter devices (Millipore). To assay for autotaxin inhibition, 10-20 μL of the concentrated conditioned media is incubated with 2.5 μL of a test compound in DMSO and 72.5-82.5 μL lyso-PLD buffer (100 mM Tris pH 9, 500 mM NaCl, 5 mM MgCl2, 5 mM CaCl2, 0.05% Triton X-100 in the presence or absence of 0.2% fatty-acid-free human serum albumin) for 15 min at 37° C. After the 15 min incubation, 5 ul of 2 mM LPC (14:0; Avanti Polar Lipids Cat #855575C) diluted in lyso-PLD buffer is added for a final concentration of 100 uM and the incubation continues for 1.5-3 hours at 37° C. 100 μl of a color mix containing 4.5 mM 4-aminoantipyrine, 2.7 mM N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine, 21 units/ml horseradish peroxidase and 3 units/ml choline oxidase in 50 mM Tris, pH 8, 4.5 mM MgCl2 is added and the incubation continued for 15 minutes at room temperature before reading the absorbance at 555 nm.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 436.92, ALogP 5.61, HBA 6, HBD 1, PSA 83.84, QED 0.42
Compound (SMILES): COc1cccc(OC)c1-n1c(NS(=O)(=O)[C@H](C)[C@H](O)c2ncc(C)cn2)nnc1-c1ccc(C)o1
InChIKey: SDZAJZRWNQFDLC-QRWLVFNGSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: GTPγS 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 assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, and 0.1% (w/v) BSA with 200 mM NaCl, 3 μM GDP] and membranes expressing human APJ receptor/well along with WGA PS beads. The reaction was initiated by addition of 0.2 nM [35S]GTPγS in the absence or presence of various ligands and incubated at RT for 90 min. Nonspecific binding was determined in the presence of 100 μM GTPγS and was always less than 0.2% of total binding.
Measurement: EC50 = 0.94 nM (pChEMBL 9.029999732971191)
Descriptors: MW 514.56, ALogP 2.82, HBA 11, HBD 2, PSA 154.49, QED 0.34
Compound (SMILES): COC(=O)NC[C@H]1CCN(c2nc3ccc(-c4c(C)noc4C)c4c3n2[C@@H](c2ccccn2)CO4)C1
InChIKey: NEMQZJKGNNTRIP-DYESRHJHSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: AlphaScreen Assay A2: BRD4-BD1 and BRD4-BD2 assays were conducted in white 384-well polystyrene plate in a final volume of 40 μL for BD1 and 60 μL for BD2. Inhibitors were first serially diluted in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 1.25% (BD1) and 0.83% (BD2). The assays were carried out at room temperature in the assay buffer (50 mM Tris-HCl, pH 7.5, 0.01% Tween-20, 0.01% BSA, 5 mM DTT), containing 50 nM Biotin-labeled tetra-acetylated histone H4 peptide (H4Ac4) and BRD4-BD1 or BRD4-BD2 protein at concentration less than 1 nM. The incubation for 75 min. was followed by the addition of 20 μL of assay buffer supplemented with Streptavidin donor beads (PerkinElmer 6760002) and GSH Acceptor beads (PerkinElmer-AL109C) at final concentration 2-4 μg/mL under reduced light. After plate sealing, the plate was incubated in the dark at room temperature for 75 min. before reading on a PHERAstar FS plate reader (BMG Labtech). IC50 determination was performed by fitting the curve of percent control activity versus the log of the inhibitor concentration using the GraphPad Prism 5.0 software.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 488.55, ALogP 3.87, HBA 9, HBD 1, PSA 107.54, QED 0.45
Compound (SMILES): CC(C)(C)CNc1c(C#N)cnc2c(Cl)cc(N[C@H](C3=CN(C4(C(F)(F)F)CC4)NN3)c3cccc4cnc(Cl)cc34)cc12
InChIKey: UQTVYKTVZLKKSU-NDEPHWFRSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: High Throughput Biochemical Assay: EGFR activity was measured using KinEASE (Cisbio), a time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay. In this assay, EGFR-catalyzes the phosphorylation of a universal Tyrosine kinase peptide substrate labeled with XL665. Europium conjugated phosphor-tyrosine specific antibody binds the resulting phosphorylated peptide. Formation of phosphorylated peptide is quantified by TR-FRET with Europium as the donor and XL665 the acceptor. The assay was performed in two main steps. The first step is the kinase reaction step and the second step is the detection step with TR-FRET reagents. In brief, test compounds 1:3 serially diluted in DMSO were delivered into Corning white, low volume, non-binding 384 well plates using the Echo 550 acoustic liquid dispenser (Labcyte®). EGFR enzyme (Human EGFR, cytoplasmic domain [669-1210] from Carna Biosciences Cat. No. 08-115) and substrates TK substrate-biotin (included in Cisbio HTRF KinEASE-TK kit Cat. No. 62TK0PEJ) were dispensed into assay plates using a Multi-Flo (Bio-Tek Instruments). The standard 10 μL reaction mixture contained 6 μM ATP (1×Km) or 12 μM ATP (2×Km), 1 μM biotinylated peptide, 0.3 nM EGFR (for 1×Km ATP) or 0.1 nM EGFR (for 2×Km ATP) in reaction buffer (10 mM MOPs, pH 7.0, 1.5% Glycerol, 0.5 mg/ml BSA, 10 mM Mg-Acetate, 1 mM DTT, 0.025% NP-40). After 60 min of incubation at room temperature, 10 μL of Stop and Detect Solution (1:400 Europium Cryptate labeled anti-phosphorylated peptide antibody solution and 125 nM strepavidin-XL665 Tracer in a 50 mM Hepes pH 7.0 detection buffer containing sufficient EDTA) was added. The plate was then further incubated for over 60 minutes at room temperature and read using an Envision 2103 Multilabeled reader (PerkinElmer) with excitation/emission/FRET emission at 340 nm/615 nm/665 nm, respectively.
Measurement: IC50 = 44 nM (pChEMBL 7.360000133514404)
Descriptors: MW 641.53, ALogP 7.83, HBA 8, HBD 4, PSA 100.93, QED 0.15
Compound (SMILES): CNC(=O)[C@@H]1CCCN1C1CC=C(c2cc3c(-c4cc(F)ccc4OC)ccnc3[nH]2)CC1
InChIKey: QYJGZZQYOHFFSZ-IMMUGOHXSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P50750
Assay: LANCE ULight TR-FRET Kinase Assay: CDK9 enzyme activities were measured using LANCE ULight TR-FRET kinase assay reagents (PerkinElmer, Waltham, Mass.). Compounds were directly added in 100% DMSO to white low volume assay plates (Perkin Elmer Proxiplate 6008289) using a Labcyte Echo acoustic dispenser. Assay reagents in serine/threonine kinase assay buffer containing 20 mM HEPES, 10 mM MgCl2, 100 mM Na3VO4, and 0.0075% Triton X-100. were added for final reaction mixture concentrations of 1000 μM ATP, 100 nM U-light MBP peptide (Perkin Elmer TRF0109M) and reaction initiated with 4 nM CDK9/Cyclin T1 (Carna Biosciences 04-110). The kinase reaction was carried out for 30 minutes before addition of stopping buffer to a final of 20 mM EDTA and 0.5 nM of LANCE Ultra Europium anti-phospho-MBP antibody (PerkinElmer TRF0201M) in LANCE detection buffer (PerkinElmer CR97-100). The reaction was equilibrated for 1 hour and the signal read in the Perkin Elmer Envision in TR-FRET mode (excitation at 320 nm and emission at 615/665 nm).
Measurement: IC50 = 200 nM (pChEMBL 6.699999809265137)
Descriptors: MW 448.54, ALogP 4.52, HBA 4, HBD 2, PSA 70.25, QED 0.6
Compound (SMILES): Cc1ccc(C(=O)NC2CC2)cc1-c1cnn(-c2cncc(NC(=O)C3CCC3)c2)c1
InChIKey: CQCOXOCESVFWLZ-UHFFFAOYSA-N
Target: Receptor-interacting serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43353
Assay: V9103X ADP-Glo Kinase Assay Custom: In a 384-well plate, test compound diluted in assay buffer (1% DMSO final) is mixed with 8His-RIPK2 FL enzyme (final concentration of 8 nM). After 15 minutes of pre-incubation at RT, ATP dissolved in assay buffer is added (final concentration 5 μM). The mixture is incubated for 60 minutes at 37° C. in a humidified incubator. Then, ADP Glo Reagent is added, followed by a 40 minute incubation at rt. Finally, Kinase Detection Reagent is added and the entire mixture is incubated for 40 min at RT. The luminescence signal is measured with an Envision reader to determine the amount of ADP produced. Assay buffer: 25 mM HEPES (4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid), 0.1% BSA (bovine serum albumin), 10 mM MgCl2, 5 mM MnCl2, 50 mM KCl, 0.01% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), 10 μM Na3VO4, 1 mM DTT (dithiothreitol), pH 7.5 All plates contain wells with vehicle controls instead of compound (1% DMSO) as reference for the high signal (100% CTL (100% of control), high signal), and wells without enzyme as reference for low signal (0% CTL, low signal). The luminescent signal generated is proportional to the ADP produced and is correlated with enzyme activity. The analysis of the data is performed by the calculation of the percentage of ADP production in the presence of the test compound and RIPK2 as compared to the ADP production in the presence of RIPK2 plus 50 μM Gefitinib.
Measurement: IC50 = 2.7 nM (pChEMBL 8.569999694824219)
Descriptors: MW 415.5, ALogP 3.87, HBA 5, HBD 2, PSA 88.91, QED 0.64
Compound (SMILES): O=C(NCc1cncc(C(F)(F)F)c1)c1cn(CCCCn2cc(C(=O)NCc3cc(C4CC4)ccn3)nn2)nn1
InChIKey: FHDIQXHCGKNWKK-UHFFFAOYSA-N
Target: Glutaminase kidney isoform, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: O94925
Assay: Enzymatic Activity Assay: The inhibition of purified recombinant human GAC by varying concentrations of inhibitors is assessed via a dual-coupled enzymatic assay. The glutamate produced by the glutaminase reaction is used by glutamate oxidase to produce α-ketoglutarate, ammonia, and hydrogen peroxide, with this hydrogen peroxide subsequently being used by horseradish peroxidase to produce resorufin in the presence of Amplex UltraRed. The assay buffer consisted of 50 mM Hepes (pH 7.4), 0.25 mM EDTA and 0.1 mM Triton X-100. GAC was incubated with potassium phosphate (10 minutes at room temperature) prior to incubation with inhibitor (10 minutes at room temperature). The final reaction conditions were as follows: 2 nM GAC, 50 mM potassium phosphate, 100 mU/mL glutamate oxidase (Sigma), 1 mM glutamine (Sigma), 100 mU/mL horseradish peroxidase (Sigma), 75 μM Amplex UltraRed (Life Technologies), and 1% (v/v) DMSO. The production of resorufin was monitored on a Perkin Elmer Envision plate reader (excitation 530 nm, emission 590 nm) either in a kinetics or endpoint mode (at 20 minutes). IC50 values were calculated using a four-parameter logistic curve fit.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 568.56, ALogP 2.9, HBA 10, HBD 2, PSA 145.4, QED 0.25
Compound (SMILES): CNc1ccc(Cc2ccc([C@H]3O[C@@H]4C[C@H]5[C@@H]6C[C@H](F)C7=CC(=O)C=C[C@]7(C)[C@@]6(F)[C@@H](O)C[C@]5(C)[C@]4(C(=O)CO)O3)cc2)cc1OC
InChIKey: MYTSRIFZTRZXOK-BXLNGJGJSA-N
Target: Glucocorticoid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04150
Assay: Glucocorticoid Receptor Binding Assay: Small molecules were tested for glucocorticoid receptor (GR) binding using the Polarscreen Glucocorticoid Receptor Assay Kit, Red (ThermoFisher A 15898) according to the manufacturer's protocol. Briefly, compounds were serially diluted in DMSO then transferred into assay kit buffer at a 1:10 dilution. Compounds were further diluted 1:5 in assay kit buffer, and 10 μl was transferred to a 384 well low volume black walled plate (Corning 4514). 5 μl of 4×Fluormone GS Red stock solution and 5 ul of 4×GR full length stock solution were added to each well containing test compound, and plates were incubated protected from light at room temperature for 4 hours. Fluorescence Polarization (mP) was measured for each plate using an EnVision Multilabel Plate Reader (Perkinelmer #2104-0010), and data were analyzed using a four parameter curve fit to generate EC50 values.
Measurement: IC50 = 16.9 nM (pChEMBL 7.769999980926514)
Descriptors: MW 649.73, ALogP 4.97, HBA 8, HBD 3, PSA 114.32, QED 0.39
Compound (SMILES): Cc1cccc(CC(C)C)c1-n1nc2c(c1-c1ccc(NC(N)=O)cc1)CN(C(=O)OC(C)(C)C)CC2
InChIKey: LOTFQDNOWJVVKB-UHFFFAOYSA-N
Target: C5a anaphylatoxin chemotactic receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21730
Assay: chemotaxis assay: Optionally, compounds may be further assayed for their ability to inhibit chemotaxis in cells. Chemotaxis assays are performed using 5 μm pore polycarbonate, polyvinylpyrrolidone-coated filters in 96-well chemotaxis chambers (Neuroprobe; Gaithersburg, Md.) using chemotaxis buffer (Hank's balanced salt solution (HBSS) and 1% FBS). C5aR ligands (i.e., C5a, R&D Systems; Minneapolis, Minn.) are use to evaluate compound mediated inhibition of C5aR mediated migration. Other chemokines (i.e., SDF-1α; R&D Systems; Minneapolis, Minn.) are used as specificity controls. The lower chamber is loaded with 29 μl of chemokine (i.e., 0.03 nM C5a) and varying amounts of compound; the top chamber contains 100,000 U937 or neutrophil cells in 20 μl. The chambers are incubated 1.5 hours at 37° C., and the number of cells in the lower chamber quantified either by direct cell counts in five high powered fields per well or by the CyQuant assay (Molecular Probes), a fluorescent dye method that measures nucleic acid content and microscopic observation.
Measurement: IC50 = 275 nM (pChEMBL 6.559999942779541)
Descriptors: MW 503.65, ALogP 5.83, HBA 5, HBD 2, PSA 102.48, QED 0.46
Compound (SMILES): Cc1cccc(OCC(C)C)c1-n1nc2c(c1-c1ccc(NC(N)=O)cc1)CN(Cc1c(F)cccc1F)CC2
InChIKey: TVNBHSJWAMIZLO-UHFFFAOYSA-N
Target: C5a anaphylatoxin chemotactic receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21730
Assay: chemotaxis assay: Optionally, compounds may be further assayed for their ability to inhibit chemotaxis in cells. Chemotaxis assays are performed using 5 μm pore polycarbonate, polyvinylpyrrolidone-coated filters in 96-well chemotaxis chambers (Neuroprobe; Gaithersburg, Md.) using chemotaxis buffer (Hank's balanced salt solution (HBSS) and 1% FBS). C5aR ligands (i.e., C5a, R&D Systems; Minneapolis, Minn.) are use to evaluate compound mediated inhibition of C5aR mediated migration. Other chemokines (i.e., SDF-1α; R&D Systems; Minneapolis, Minn.) are used as specificity controls. The lower chamber is loaded with 29 μl of chemokine (i.e., 0.03 nM C5a) and varying amounts of compound; the top chamber contains 100,000 U937 or neutrophil cells in 20 μl. The chambers are incubated 1.5 hours at 37° C., and the number of cells in the lower chamber quantified either by direct cell counts in five high powered fields per well or by the CyQuant assay (Molecular Probes), a fluorescent dye method that measures nucleic acid content and microscopic observation.
Measurement: IC50 = 27.5 nM (pChEMBL 7.559999942779541)
Descriptors: MW 545.63, ALogP 6.21, HBA 5, HBD 2, PSA 85.41, QED 0.28
Compound (SMILES): Cn1cncc1-c1ccc2c(-c3ccc(C(=O)O)cc3)nn(C(=O)c3c(Cl)cccc3C(F)(F)F)c2c1
InChIKey: WFIGKMLTRWQOHK-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Biological Assays: The compounds of the invention inhibit RORgammaT activity. Activation of RORgammaT activity can be measured using, e.g., biochemical TR-FRET assay. In such an assay, interaction of cofactor-derived peptides with human RORgammaT-Ligand Binding Domain (LBD) can be measured. The TR-FRET technique is a sensitive biochemical proximity assay that will give information concerning the interaction of a ligand with the LBD, in the presence of cofactor-derived peptides (Zhou et al., Methods 25:54-61, 2001).To identify novel antagonists of RORgammaT, an assay was developed which employs the interaction of RORgammaT with its co-activator peptide SRC1_2. This peptide mimics the recruitment of co-activators to RORgammaT through its interaction with the LXXLL (SEQ ID NO:1) (e.g., NR box) motifs (Xie et al., J. Immunol. 175: 3800-09, 2005; Kurebayashi et al., Biochem. Biophys. Res. Commun. 315: 919-27, 2004; Jin et al., Mol. Endocrinology 24:923-29, 2010). The RORγ-Ligand Binding Domain TR-FRET Assay was run according to the following protocol.HIS-tagged RORγ-LBD protein was expressed in SF9 cells using a baculovirus expression system. The RORγ-LBD protein was purified by glutathione sepharose chromatography. Separately, SF9 cells not expressing any recombinant protein were lysed and the lysate was added to the purified RORγ-LBD at 0.25 μl lysate (from 10,000 SF9 cells)/nM purified protein. The mixture was then diluted in assay buffer (50 mM Tris pH 7.0, 50 mM KCl, 1 mM EDTA, 0.1 mM DTT) to obtain RORγ-LBD final concentration of 3 nM in 384-well assay plate.Compounds to be tested were injected to the assay plate using Acoustic Droplet Ejection technology by Echo 550 liquid handler (Labcyte, Calif.).A stock of biotinylated-LXXLL peptide from coactivator SRC1 (Biotin-CPSSHSSLTERHKILHRLLQEGSPS) (SEQ ID NO:2) was prepared in assay buffer and added to each well (100 nM final concentration). A solution of Europium tagged anti-HIS antibody (1.25 nM final concentration) and APC conjugated streptavidin (8 nM final concentration) were also added to each well.The final assay mixture was incubated overnight at 4° C., and the fluorescence signal was measured on an Envision plate reader: (Excitation filter=340 nm; APC emission=665 nm; Europium emission=615 nm; dichroic mirror=D400/D630; delay time=100 μs, integration time=200 μs). IC50 values for test compounds were calculated from the quotient of the fluorescence signal at 665 nm divided by the fluorescence signal at 615 nm.
Measurement: IC50 = 5606 nM (pChEMBL 5.25)
Descriptors: MW 524.89, ALogP 6.16, HBA 6, HBD 1, PSA 90.01, QED 0.3
Compound (SMILES): CCOc1cc([C@@H](CS(C)(=O)=O)n2c(=O)n(CCOC)c3cc(Br)cnc32)ccc1OC
InChIKey: RAACNIKUVLHWLG-QGZVFWFLSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4B (SINGLE PROTEIN), Homo sapiens
UniProt: Q07343
Assay: Inhibitory Effect on the Enzyme In Vitro Assay: The enzyme activity was indicated by measuring the AMP/GMP expression and tracing AMP/GMP antibody binding based on fluorescence polarization in the biological assay.Reagents:Experimental Buffer Solution:10 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 0.01% Brij 35, 1 mM DTT and 1% DMSO.Enzyme:Recombinant human PDE4B (Gen accession number: NM_002600; amino acid 305 end) was expressed by baculovirus in Sf9 insect cells using an N-terminal GST tag. MW=78 kDa.Enzyme Substrate:1 μM cAMPDetection:Transcreener® AMP2/GMP2 antibody and AMP2/GMP2 AlexaFluor633 tracerOperation Procedures:i) Recombinant human PDE4B and the enzyme substrate (1 μM cAMP) were dissolved in the newly-prepared buffer solution, respectively.ii) The PDE4B buffer solution defined as above was transferred into the reaction wells.iii) The compound which was dissolved in 100% DMSO was added to the reaction wells of the PDE4B buffer solution by ultrasonic oscillator (echo 550; nanoliter range) and incubated at room temperature for 10 minutes.iv) The enzymatic buffer solution was then added to the reaction wells defined as above to initiate the reaction.v) The reaction was incubated at room temperature for 1 hour.vi) The reaction was terminated by adding detecting mixture (Transcreener AMP2/GMP2 antibody and AMP2/GMP2 AlexaFluor633 tracer) and incubated for 90 minutes with slowly mixing. The measurement range of fluorescence polarization is Ex/Em=620/688.
Measurement: IC50 = 9.1 nM (pChEMBL 8.039999961853027)
Descriptors: MW 528.43, ALogP 2.65, HBA 9, HBD 0, PSA 101.65, QED 0.4
Compound (SMILES): O=C(O)Cc1cccc(CCOc2ccc(-c3ccn[nH]3)nc2)c1
InChIKey: VZNZRXYIWNCYDR-UHFFFAOYSA-N
Target: Cytochrome P450 4F2 (SINGLE PROTEIN), Homo sapiens
UniProt: P78329
Assay: Inhibition Test for Each Compound of the Present Invention Against 20-HETE Producing Enzymes (CYP4F2 and CYP4A11): In the CYP4F2 inhibition test, the reaction solution containing each compound [final concentration of 50 mM, KPO4 (pH 7.4), 2.5 μM luciferine derivative, and 1 mM NADPH] was added to an Escherichia coli membrane fraction (100 μg/mL protein) in which human CYP4F2 had been expressed. In the CYP4A11 inhibition test, the reaction solution containing each compound [final concentration of 100 mM, Tris-HCl (pH 7.5), 60 μM luciferine derivative, 1.3 mM NADP+, 3.3 mM Glucose 6-Phosphate, 3.3 mM MgCl2, and 0.4 U/mL Glucose 6-Phosphate dehydrogenase] was added to an Escherichia coli membrane fraction (100 μg/mL protein) in which human CYP4A11 had been expressed. Following this, the membrane fraction was left to stand at room temperature for 60 minutes to perform an enzymatic reaction. After the reaction, a luciferine detection reagent was added, and the luminescence value was measured using a plate reader. By using that value, the percent inhibition of 20-HETE producing enzyme (%) was calculated according to the equation described below, and the 50% inhibitory concentration (IC50 value) for each compound was calculated.
Measurement: IC50 = 7800 nM (pChEMBL 5.110000133514404)
Descriptors: MW 323.35, ALogP 2.72, HBA 4, HBD 2, PSA 88.1, QED 0.7
Compound (SMILES): COc1cc(F)ccc1-c1cnc2[nH]c(=O)n(CC(=O)NC3CC3)c2c1
InChIKey: AMMAFLSOCDVHRK-UHFFFAOYSA-N
Target: Glutamate receptor ionotropic, NMDA 2B (SINGLE PROTEIN), Homo sapiens
UniProt: Q13224
Assay: HTS cell-based calcium flux assay: NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 137 mM NaCl, 4 mM KCl, 2 mM CaCl2, 0.5 mM MgCl2 (standard assay) or 1.5 mM MgCl2 (HTS assay), 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (137 mM NaCl (standard assay) or 150 mM (HTS assay), 4 mM KCl (standard assay) or 3 mM (HTS assay), 2 mM CaCl2, 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to EC40 (HTS assay) is used to maximize the assay's signal window and ability to detect NMDA receptor antagonists and negative allosteric modulators. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 71 nM (pChEMBL 7.150000095367432)
Descriptors: MW 356.36, ALogP 1.82, HBA 5, HBD 2, PSA 89.01, QED 0.73
Compound (SMILES): COc1ncc(-c2cc(C(F)(F)F)c3c(N)ncnn23)cc1C(=O)NCCC(O)(c1ccccc1)C(F)(F)F
InChIKey: BVVRPWNBVFVCMQ-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 = 24000 nM (pChEMBL 4.619999885559082)
Descriptors: MW 554.45, ALogP 3.97, HBA 8, HBD 3, PSA 127.66, QED 0.3
Compound (SMILES): N[C@H]1C[C@@H]1c1cnc(-c2cccc(O)c2)s1
InChIKey: JRNQFIYVQYTRHZ-UWVGGRQHSA-N
Target: Amine oxidase [flavin-containing] A (SINGLE PROTEIN), Homo sapiens
UniProt: P21397
Assay: Monoamine Oxidase Assays: Assays were conducted in 96-well black plates with clear bottom (Corning) in a final volume of 100 μL. The assay buffer was 100 mM HEPES, pH 7.5. Each experiment was performed in triplicate within the same experiment. Briefly, a fixed amount of MAO (0.25 μg for MAO-A and 0.5 μg for MA-B) was incubated on ice for 15 minutes in the reaction buffer, in the absence and/or in the presence of various concentrations of inhibitor (e.g., from 0 to 50 μM, depending on the inhibitor strength). Tranylcypromine (Biomol International) was used as a control for inhibition. After leaving the enzyme(s) interacting with the inhibitor, 60 to 90 μM of kynuramine was added to each reaction for MAO-B and MAO-A assay respectively, and the reaction was left for 1 hour at 37° C. in the dark. The oxidative deamination of the substrate was stopped by adding 50 μL (v/v) of NaOH 2N. The conversion of kynuramine to 4-hydroxyquinoline, was monitored by fluorescence (excitation at 320 nm, emission at 360 nm) using a microplate reader (Infinite 200, Tecan).
Measurement: Ki = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 232.31, ALogP 2.33, HBA 4, HBD 2, PSA 59.14, QED 0.84
Compound (SMILES): Cc1nc(C)c(Cn2c(=O)n(-c3cscn3)c3cc(S(=O)(=O)NC4(C)CC4)ccc32)s1
InChIKey: FXEWVHWLPURWNO-UHFFFAOYSA-N
Target: Poly(ADP-ribose) glycohydrolase (SINGLE PROTEIN), Homo sapiens
UniProt: Q86W56
Assay: PARG Assay: PARG In vitro assays were conducted in a total volume of 15 ul in a standard 384 well format. 5 ul of Human Full Length PARG (Produced internally by Astra Zeneca), used at a final reaction concentration of 80 μM, was added to 5 ul of Ribosylated PARP substrate (also produced internally by Astra Zeneca) at final reaction concentration of 4.5 nM in assay buffer (50 mM Tris pH7.4, 0.1 mg/ml BSA, 3 mM EDTA, 0.4 mM EGTA, 1 mM DTT, 0.01% Tween 20, 50 mM KCl). The reaction was incubated at room temperature for 10 minutes and then 5 ul detection reagent was added. Detection Reagent consists of 42 nM MAb Anti-6HIS XL665 (CisBio: 61 HISXLB) and 2.25 nM Streptavidin Europium Cryptate (CisBio: 610SAKLB), both at 3× working stock concentrations (final concentrations of 14 nM and 0.75 nM respectively), in a detection buffer of 50 mM Tris pH7.4, BSA at 0.1 mg/ml and KF at 100 mM. Following incubation at room temperature for 60 minutes in the dark, TR-FRET signal was measured at Ex 340 and Em 665 and Em 620. A ratio was calculated as Em665/EM620×104 for each well and used to calculate percent inhibition for test compounds.
Measurement: EC50 = 740 nM (pChEMBL 6.130000114440918)
Descriptors: MW 475.62, ALogP 3.2, HBA 9, HBD 1, PSA 98.88, QED 0.46
Compound (SMILES): CC[S+]([O-])c1nc2n(n1)[C@H](c1ccccc1)C[C@@H]2F
InChIKey: COQPEMYQZSJEBH-JDSCLEBSSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: Biochemical Assay: The ability of the receptor interacting protein kinase (RIPK1) to catalyze the hydrolysis of adenosine-5′-triphosphate (ATP) is monitored using the Transcreener ADP (adenosine-5′-diphosphate) assay (BellBrook Labs). Purified human RIP1 kinase domain (2-375) (50 nM) derived from a baculovirus-infected insect cell expression system is incubated with test compounds for 2 hours in 50 mM Hepes buffer (pH 7.5) containing 30 mM MgCl2, 1 mM dithiothreitol, 50 uM ATP, 0.002% Brij-35, and 0.5% dimethyl sulfoxide (DMSO). Reactions are quenched by the addition of 1× Bell Brooks Stop buffer B (20 mM Hepes (ph7.5), 40 mM ethylenediaminetetraacetic acid and 0.02% Brij-35) containing an additional 12 mM EDTA and 55 ug/mL ADP2 antibody and 4 nM ADP-AlexaFluor® 633 tracer. The tracer bound to the antibody is displaced by the ADP generated during the RIP1K reaction, which causes a decrease in fluorescence polarization that is measured by laser excitation at 633 nm with a FP microplate reader M1000. Fractional activity was plotted against test article concentration. Using Genedata Screener software (Genedata; Basel, Switzerland), the data were fit to the tight-binding apparent inhibition constant (Ki app) Morrison equation [Williams, J. W. and Morrison, J. F. (1979) The kinetics of reversible tight-binding inhibition. Methods Enzymol 63: 437-67]. The following equation was used to calculate fractional activity and Ki app:Fractional ⁢ ⁢ activity = V i V o = 1 - ( [ E ] T + [ I ] T + K i app ) - ( [ E ] T + [ I ] T + K i app ) 2 - 4 ⁡ [ E ] T ⁡ [ I ] T 2 ⁡ [ E ] Twhere [E]T and [I]T are the total concentrations of active enzyme and test article, respectively.
Measurement: Ki = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 279.34, ALogP 2.41, HBA 4, HBD 0, PSA 53.77, QED 0.81
Compound (SMILES): C[C@@H](NC(=O)c1cc(=O)[nH]c(-c2ncccn2)n1)c1ccc(OC(F)(F)F)cc1F
InChIKey: RLQWOAAIGQBXTG-SECBINFHSA-N
Target: GMP-stimulated 3',5'-cyclic nucleotide phosphodiesterase (SINGLE PROTEIN), Macaca mulatta
UniProt: B3VTJ2
Assay: Fluorescence Polarization (FP) Assay: In a typical experiment the PDE2 inhibitory activity of the compounds of the present invention was determined in accordance with the following experimental method. Rhesus PDE2A3 was amplified from rhesus macaque brain cDNA (Biochain Institute, Hayward, Calif.) using primers based on human PDE2A sequence (accession NM_002599.3) where the forward primer containing a Kozak consensus was 5′-gccaccatggggcaggcatgtggc-3′ and the reverse primer was 5′-tcactcagcatcaaggctgca-3′. Amplification with Easy-A High-Fidelity PCR cloning enzyme (Stratagene, La Jolla, Calif.) was 95° C. for 2 minutes followed by thirty three cycles of 95° C. for 40 seconds, 52° C. for 30 seconds, and 72° C. for 2 minutes 48 seconds. Final extension was 72° C. for 7 minutes. The PCR product was TA cloned into pcDNA3.3-TOPO (Invitrogen, Carlsbad, Calif.) according to standard protocol. A consensus sequence was developed from multiple clones and then deposited into GenBank (EU812167). AD293 cells (Stratagene, La Jolla, Calif.) with 70-80% confluency were transiently transfected with rhesus PDE2A3/pcDNA3.3-TOPO using Lipofectamine 2000 according to manufacturer specifications (Invitrogen, Carlsbad, Calif.).
Measurement: Ki = 28 nM (pChEMBL 7.550000190734863)
Descriptors: MW 423.33, ALogP 2.76, HBA 6, HBD 2, PSA 109.86, QED 0.61
Compound (SMILES): CCc1ccc(N(CC(C)C)S(=O)(=O)c2ccc3c(c2)NC(=O)CS3)cc1
InChIKey: LMTYXNXTGSHOAX-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: GAL4-RORgamma Transactivation: The RORγ transactivation model was developed from the line HG5LN, which is a HeLa line that stably expresses a luciferase reporter gene controlled by a pentamer of the GAL4 recognition domain of yeast and of a β-globin promoter. The HG5LN line was stably transfected by the DNA-binding domain (DBD) of GAL4 fused to the ROR gamma ligand-binding domain (LBD). Molecules that inhibit the ROR gamma constitutive activity reduce the luciferase expression, thus leading to a reduction in the emitted luminescence.The cells are seeded in 384-well plates (5000 cells in 45 μL/well of culture medium containing 10% fetal calf serum) and incubated for 4 hours at 37° C., 5% CO2. 5 μL of the test molecules (compounds described in the tables described above) are then added to each well and the plates are incubated for 18 hours at a temperature of 37° C. under 5% of CO2. 20 μL of luciferase substrate (Promega) are added to each well and the luminescence emitted is read by a microplate reader.The luminescence units (RLU) are normalized by positive controls (POS containing a saturated concentration of benzenesulfonamide, N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl]) and negative controls (NEG containing DMSO): % inhibition=((RLU−NEG)*100)/(POS−NEG). The IC50 values are calculated from a 4-parameter logistic model using the XLFit software (IDBS).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 404.56, ALogP 4.14, HBA 4, HBD 1, PSA 66.48, QED 0.79
Compound (SMILES): CC(C)N1CCc2cc(Nc3cc4cc(-c5cncc6c5CC[C@H]6O)c(F)c(N)c4cn3)nn2CC1=O
InChIKey: WLOFDVHJCSFNQF-JOCHJYFZSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: HPK1-FL HTRF Enzymatic Assay: To a 384 well Proxiplate with 80 nL compound or DMSO spotted on was added 4 μl/well kinase mix. The mixture was preincubated for 30 minutes and then 4 μl/well substrate mix was added. The solution was incubated for 60 min and then 4 μl/well detection mix was added. The solution was incubated for another 60 min. The plates were then loaded onto a Perkin Elmer Envision and the TR-FRET signal was measured at 615 and 665 nm. A ratio of 665/620 was used to calculate the % activity at each concentration of compound.
Measurement: Ki = 5.1 nM (pChEMBL 8.289999961853027)
Descriptors: MW 501.57, ALogP 3.73, HBA 8, HBD 3, PSA 122.19, QED 0.36
Compound (SMILES): N#CCNC(=O)C1CC=C(c2cc(NC(=O)C3CC3)nc3[nH]ccc23)CC1
InChIKey: HQQXPHUIMMZFQY-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: ADP-Glo Kinase Assay: A control material and a test material were prepared for each concentration, in such a way that they were diluted by means of DMSO. At the same time, ATP (250 uM) and JAK substrate (JAK1, IRS-itide 40 ng/mL) were prepared, in such a way that they were diluted by means of kinase buffer (40 mM Tris-HCl pH 7.5, 20 mM MgCl2, 0.5 mg/mL BSA, 50 uM DTT).A test drug for each concentration, substrate, ATP and JAK enzyme were mixed in an eppendorf tube, and reacted in a 30° C. incubator for 40 minutes.ADP-Glo reagent contained in ADP-Glo Kinase Enzyme System (Promega, USA, V9571) was added into each eppendorf tube, and reacted in a 30° C. incubator for 40 minutes. A kinase detection reagent contained in ADP-Go Kinase Enzyme System was inserted into the eppendorf tube, then an integration time was set to 1 second by using Wallac Victor 2, then luminescence was measured, and then JAKs phosphorylation inhibition ability of the test material was analyzed. The concentration of the compound, which causes 50% of the JAK enzyme activity inhibition in comparison With the control group, was determined as IC50 (nM) of an inhibitor, wherein the results thereof are shown in a following table 1.
Measurement: IC50 = 110 nM (pChEMBL 6.960000038146973)
Descriptors: MW 363.42, ALogP 2.73, HBA 4, HBD 3, PSA 110.67, QED 0.71
Compound (SMILES): CCc1cc([C@]23C[C@H]2CN(CC[C@H]2CC[C@H](NC(=O)Cc4cc(C)no4)CC2)C3)no1
InChIKey: LSQJPMUMQFFZON-LYXWCMDISA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Binding Inhibition for Dopamine D3 Receptor: Buffer solution: 50 mM Tris-HCl (35409-45, Nacalai Tesque) (pH 7.4) containing 120 mM NaCl (31320-05, Nacalai Tesque), 1 mM MgCl2.6H2O (20909-55, Nacalai Tesque), 5 mM KCl (28514-75, Nacalai Tesque) and 2 mM CaCl2 (067-31, NAKARAI CHEMICALS, LTD.)Radioligand: (final concentration) 2 nM [3H]-Methylspiperone ([3HN-methyl-]-Methylspiperone, NET-856, 83.8 Ci/mmol, PerkinElmer)Non-specific ligand: (final concentration) 10 μM Butaclamol [(+)-Butaclamol Hydrochloride, D033, Sigma]SPA beads solution: SPA beads [WGA PVT SPA Scintillation Beads, RPNQ0001 (500 mg), RPNQ0060 (2 g), PerkinElmer] (0.2 mg/well)Incubation time and temperature: 120 min at 25° C.
Measurement: Ki = 0.23 nM (pChEMBL 9.640000343322754)
Descriptors: MW 426.56, ALogP 3.41, HBA 6, HBD 1, PSA 84.4, QED 0.7