lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1cc(-c2cc(F)cnc2OC)ccc1-c1nccc2cc(S(=O)(=O)Nc3nncs3)ccc12
InChIKey: OEKKEIOEYGMNMD-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: In Vitro PX Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to â¿¿10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes.
Measurement: IC50 = 80.1 nM (pChEMBL 7.099999904632568)
Descriptors: MW 523.57, ALogP 4.77, HBA 9, HBD 1, PSA 116.19, QED 0.32
Compound (SMILES): CN(c1ncnc2[nH]ccc12)[C@H]1C[C@@H]2CN(C(=O)OC(C)(C)C)C[C@@H]2C1
InChIKey: NZHSLDWIEHMESB-WDNDVIMCSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK1 Kinase Assay: TBDIn vitro kinase assays described below can be used to determine the activity of a test compound for inhibiting the activity of JAK1 kinase. The test compounds were dissolved in dimethyl sulfoxide and diluted with water to a serial concentration gradient as required in the experiment. JAK1 substrates (Cell Signaling Technology, Catalog Number: 1305s) and ATP (2 mM) solution were diluted with water to obtain a final concentration of 20 μM ATP and 1.2 μM substrate solution. The appropriate amount of JAK1 kinase (Invitrogen, Catalog Number: pv4774) was mixed with 4× buffer (prepared by user, and comprising 50 mM HEPES, pH 7.3, 125 mM NaCl, 24 mM MgCl2, 1.25 mM DTT) to a final concentration of 8 ng/μL. To each well of a microplate [DELFIA® Streptavidin-coated clear plate (Perkin Elmer, Catalog Number: AAAND-0005)]17.5 μL of ATP/substrate mixture, 5 μL of an aqueous solution of a test compound (5 μL of pure water only were added to the control and blank), and 7.5 μL of the kinase solution prepared above (4× buffer only was added to the control) were added. Each well was mixed sufficiently, then incubated at room temperature (27° C.) for 50 minutes, washed with wash buffer, and dried three times, then HRP conjugated antibody was added [Phospho-Tyrosine Mouse mAb (P-Tyr-100) (HRP Conjugate, Cell Signaling Technology, Catalog Number: 5465)], and incubated for 1 hour. The microplate was washed with wash buffer and dried three times, and then TMB (Sigma, Catalog Number: T4444) was added, and incubated for 5 to 15 minutes to allow for color change. Stop solution (1 N sulfuric acid solution) was added to stop the reaction.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 357.46, ALogP 3.04, HBA 5, HBD 1, PSA 74.35, QED 0.89
Compound (SMILES): CC(C)n1cnc2cc(-c3ccc4c(c3)N([C@H]3C[C@@H](N5CCC(C)(C)C5)C3)C(=O)C43CCN(C(=O)[C@@H]4CCO4)CC3)nc(Nc3ccncc3F)c21
InChIKey: ZHJJCNIHQSFIEO-MZXMJASYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: Enzymatic Activity Assay: Recombinant HPK1 kinase domain produced via baculovirus infection of insect cells was obtained from Proteros (Proteros Biostructures #PR-0322) and was pre-activated in the presence of 2 mM ATP (Sigma-Aldrich, cat #GE27-2056-01) and 2 mM magnesium chloride for 16 hours at 4° C. The protein reaction mixture was then loaded to a desalting column (Thermo Fisher Scientific, Cat #89889) to remove excess ATP. HPK1 was eluted with buffer containing 20 mM Tris (2-Amino-2-(hydroxymethyl)propane-1,3-diol) pH 8.0, 150 mM NaCl, 2 mM dithiothreitol and 5% glycerol, and was frozen at −80° C. for later use. HPK1 dual phosphorylation was confirmed by mass spectrometry.Ten nanoliters of test compounds dissolved in DMSO at various concentrations were dispensed into a 384-well ProxiPlate (PerkinElmer #6008289). Five microliters of a solution of recombinant HPK1 diluted in HPK1 kinase assay buffer (50 mM BES [N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid], pH 7.0; 10 mM magnesium chloride; 0.01% Triton X-100; 1 mM dithiothreitol; 0.01% bovine serum albumin; 0.1 mM sodium orthovanadate) was added to the compound-containing plate and was incubated for 15 minutes at 25° C. Five microliters of a mixture of ATP (Sigma-Aldrich #A6559) and peptide substrate STK S1 (Cisbio #61ST1BLC) diluted in HPK1 kinase assay buffer was then added to start the reaction. Final concentrations were 0.15 nM for HPK1, 10 μM for ATP, and 1 μM for the STK S1 peptide substrate. The reaction mixture was incubated at 25° C. for 3 hours and was stopped with the addition of 10 μl of an EDTA (Ethylenediaminetetraacetic acid)-containing detection buffer (Cisbio #62SDBRDF) supplemented with Europium cryptate-labeled anti-phospho-serine/threonine antibodies (Cisbio #62ST1PEJ) and XL665-labeled streptavidin (Cisbio #610SAXLG). The mixture was incubated for 16 hours at room temperature and peptide phosphorylation was measured by time-resolved fluorescence energy transfer (665 nm/620 nm) on an Envision plate reader (PerkinElmer).
Measurement: IC50 = 0.3 nM (pChEMBL 9.520000457763672)
Descriptors: MW 706.87, ALogP 6.22, HBA 9, HBD 1, PSA 108.72, QED 0.24
Compound (SMILES): O=C([C@@H]1CCCO1)N1CC2CCC(C1)N2Cc1ccc2cc(Oc3ccc(-c4ccn[nH]4)cn3)ccc2n1
InChIKey: XXYLZARRABFAOF-GMKWRKAISA-N
Target: Leukotriene A-4 hydrolase (SINGLE PROTEIN), Homo sapiens
UniProt: P09960
Assay: Enzymatic Assay: The compounds of the invention are assessed for the ability to interact with human LTA4 hydrolase in an enzymatic assay that measures the ability of the enzyme to cleave the peptide bond of arginyl-aminomethylcoumarin (Arg-AMC). LTA4H Enzyme (1 nM final), Arg-AMC substrate (50 μM final), and compound are combined in a reaction buffer (50 mM Tris-HCl (pH 7.5), 100 mM KCl, 0.5% bovine serum albumin) at room temperature for 1 h. The formation of product is assessed by measuring the fluorescence of aminomethylcoumarin product (excitation wavelength 380 nm/emission wavelength 460 nm).
Measurement: IC50 = 1.7 nM (pChEMBL 8.770000457763672)
Descriptors: MW 510.6, ALogP 4.17, HBA 7, HBD 1, PSA 96.47, QED 0.42
Compound (SMILES): Cc1ncsc1C(CNC(=O)c1cc(-c2ncc(F)cn2)ccc1Cl)N1CCOC(c2cnn(C)c2)C1
InChIKey: XMWGPEZLJZCAKC-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Fluo-8 No Wash Calcium Assa: Extracellular binding of Bz-ATP to P2X7 receptor opens the channel and allows Ca2+ influx into the cells. This Ca2+ entry was measured in HEK-293 cells stably transfected with P2X7 receptor using Screen Quest Fluo-8 No Wash Calcium Assay Kit (AAt Bioquest, cat. 36316). Once inside the cell, the lipophilic blocking groups of Fluo-8 are cleaved by non-specific cell esterases, resulting in a negatively-charged fluorescent dye that stays inside cells. Its fluorescence increases upon binding to calcium. When HEK-293/P2X7 cells are stimulated with Bz-ATP, Ca2+ enters the cells and the fluorescence of Fluo-8 NW increases. The dye has an absorption spectrum compatible with excitation at 488 nm by argon laser sources and its emission wavelength is in the range of 515-575 nm.HEK-293 cells stably transfected with P2X7 receptor were seeded overnight in growth medium at 10,000 to 20,000 cells/well in 384-well plate. 24 hours later, the medium was removed and the cells were pre-loaded at RT for 1 hour with 20 μL/w of Fluo-8 NW. Then 10 μL/w of test compounds and reference antagonist A438079 at 3×-concentration were injected with the FLIPRTETRA and the kinetic response over a period of five minutes was monitored. A second injection of 15 μL/w of 3× reference activator (Bz-ATP at EGO was performed with the FLIPRTETRA and the signal of the emitted fluorescence was recorded for additional three minutes. All the experiment was carried out in a Low Divalent Cation Assay Buffer (0.3 mM Ca2+ and 0 mM Mg2+). The effect of the test compounds was measured as percent inhibition vs the reference antagonist and IC50 values were calculated accordingly.
Measurement: IC50 = 139 nM (pChEMBL 6.860000133514404)
Descriptors: MW 542.04, ALogP 3.98, HBA 9, HBD 1, PSA 98.06, QED 0.38
Compound (SMILES): CC(C)CC(=O)Nc1cc(-c2ccc(OC[C@@](C)(N)CC(C)C)c(C(F)(F)F)c2)ccn1
InChIKey: KNKAABYFUJVMAY-QHCPKHFHSA-N
Target: AP2-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q2M2I8
Assay: AAK1 Kinase Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme and substrates (fluoresceinated peptide (5-FAM)-Aha-KEEQSQITSQVTGQIGWR-NH2 and ATP) and test compounds in assay buffer (10 mM Tris-HCL pH 7.4, 10 mM MgCl2, 0.01% Tween-20 and 1.0 mM DTT). The reactions were initiated by the combination of bacterially expressed, GST-Xa-hAAK1 with substrates and test compounds. The reactions were incubated at room temperature for 3 hours and terminated by adding 60 μl of 35 mM EDTA buffer to each sample. The reactions were analyzed on the Caliper LabChip 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to EDTA quenched control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assays are ATP, 22 μM; (5-FAM)-Aha-KEEQSQITSQVTGQIGWR-NH2, 1.5 μM; GST-Xa-hAAK1, 3.5 nM; and DMSO, 1.6%. Dose response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 0.7 nM (pChEMBL 9.149999618530273)
Descriptors: MW 451.53, ALogP 5.89, HBA 4, HBD 2, PSA 77.24, QED 0.5
Compound (SMILES): Cc1nn(C)cc1-c1nc(N2CCOCC2)c2nc(C(=O)NCC3(CO)COC3)[nH]c2n1
InChIKey: GUONHZRPEGDHMP-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: In Vitro Enzyme PI3K Assay: The Kinase-Glo luminescent kinase assay kit (from Promega) was used to measure kinase activity. In this assay, amount of ATP remaining in solution following a kinase reaction is measured. The effect of compounds on PI3Kδ inhibition was carried out by adding 2.29 μg/ml of recombinant PI3K δ enzyme (Proteros, Germany) to reaction mixture containing assay buffer (50 mM HEPES, pH 7.4, 50 mM NaCl, 0.05% CHAPS) supplemented with 10 mM MgCl2, 5 mM DTT, 60 μM Phosphatidyl inositol bisphosphate (PIP2) and 10 μM ATP in the absence or presence of different concentrations of the compounds in a final volume of 15 μl/well, in a 384 well plate. The reaction mixture was incubated for 2 hours at room temperature. At the end of incubation period the equal volume of Kinase-Glo plus (Promega, V3772), was added per well and the luminescence was measured after incubating for 10 minutes at room temperature in dark. Results were calculated by measuring the luminescence units of test samples to blanks containing no enzyme.In certain embodiments, the compounds showed IC50 of less than 1000 nM, in another embodiment, the IC50 values range from about 100 nM to 500 nM, and in yet another embodiment, it is even less than 30 nM for PI3Kδ as shown in Table 3 and 4.The compounds of the present invention were tested for their selectivity for PI3Kδ over PI3Kα, PI3Kβ and PI3Kγ following the above assay using specific recombinant enzymes (Proteros, Germany) for each kinase. Assay condition of kinase assay (PI3Kα, PI3Kβ, PI3Kγ and PI3Kδ) was as follows. Enzyme: 2.29 μg/mL; ATP: 10 μM; PIP2 substrate: 60 μM and Reaction Time: 2 hours.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 442.48, ALogP -0.36, HBA 10, HBD 3, PSA 143.31, QED 0.47
Compound (SMILES): COc1cc(OCc2cccc3c2CCN3C(=O)c2ccccc2F)c2cc(-c3cn4nc(OC)sc4n3)oc2c1
InChIKey: ODUPCRAOQFHNMS-UHFFFAOYSA-N
Target: Proteinase-activated receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96RI0
Assay: FLIPR Assay in PAR4-Expressing HEK293 Cells: FLIPR-based calcium mobilization assay in HEK293 cells was used to measure PAR4 antagonism, agonism, and selectivity against PAR1. The activity of the PAR4 antagonists of the present invention were tested in PAR4 expressing cells by monitoring H-Ala-Phe(4-F)-Pro-Gly-Trp-Leu-Val-Lys-Asn-Gly-NH2-induced intracellular calcium mobilization. Counter screens for agonist activity and PAR1 antagonist activity were also performed. Briefly, PAR1/PAR4-expressing HEK293 cells were grown in DMEM (Life Technology, Grand Island, N.Y.) containing 10% heat-inactivated FBS, 1% Penicillin-Streptomycin, 10 μg/mL blasticidin, and 100 μg/mL Zeocin at 37° C. with 5% CO2. Cells were plated overnight prior to the experiment in a black 384-well Purecoat Amine clear bottom plate (Becton Dickinson Biosciences, San Jose, Calif.) at 10,000 cells/well in 30 μL growth medium and incubated in a humidified chamber at 37° C. with 5% CO2 overnight. Prior to compound addition, the cell medium was replaced with 40 L of 1× calcium and magnesium-containing Hank's Balanced Saline Solution (HBSS) (with 20 mM HEPES) and 1:1000 diluted fluorescent calcium indicator (Codex Biosolutions, Gaithersburg, Md.). After a 30 minute incubation period at 37° C. and a further 30 minute incubation and equilibration period at room temperature, 20 μL test compound (diluted in 1× HBSS buffer) was added at various concentrations at 0.17% dimethyl sulfoxide (DMSO) final concentration. Changes in fluorescence intensity were measured using a Functional Drug Screening System (FDSS, Hamamatsu, Japan) to determine agonist activities. The cells were then incubated for 30 minutes at room temperature followed by addition of 20 μL of agonist peptide for antagonist activity measurement. The PAR4 agonist peptide (H-Ala-Phe(4-F)-Pro-Gly-Trp-Leu-Val-Lys-Asn-Gly-NH2) and the PAR1 agonist peptide (SFFLRR) were routinely tested to ensure a proper response at the EC50 value in the assay (5 μM for PAR4 agonist peptide and 2 μM for PAR1 agonist peptide). Compound potency was derived from 11-point concentration-response curves.
Measurement: EC50 = 4.2 nM (pChEMBL 8.380000114440918)
Descriptors: MW 570.6, ALogP 6.14, HBA 9, HBD 0, PSA 91.33, QED 0.23
Compound (SMILES): Cn1cc(-c2cc(-c3ccc(N4CCN(C(=O)N5CCCC5)CC4)nc3)c3c(C#N)cnn3c2)cn1
InChIKey: JHBCHOZYURDLBN-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of General Formula I were screened for their ability to inhibit wild type and V804M mutant RET kinase using CisBio's HTRF® KinEASE -TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Cat. No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Cat. No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Cat. No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 5.6 nM (pChEMBL 8.25)
Descriptors: MW 481.56, ALogP 3.01, HBA 8, HBD 0, PSA 98.59, QED 0.45
Compound (SMILES): CC(C)Nc1cc(Nc2ccc3ncsc3c2)ncc1C(=O)NC[C@@H](F)C(O)C(C)C
InChIKey: WYECJZZJXJDWGL-UJONTBEJSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 μL prepared from 15 μL additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 445.56, ALogP 4.34, HBA 7, HBD 4, PSA 99.17, QED 0.39
Compound (SMILES): C[C@@H](NC(=O)Nc1cc2cnn(-c3ccc(F)cc3)c2c(CN(C)C)n1)c1ccccc1
InChIKey: WZYFUXFQVMROME-MRXNPFEDSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 μM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 μL) was dispensed, followed by the addition of 15 μL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 μL kinase buffer containing 2.45 μM ERK2 IMAP substrate peptides and 75 μM ATP. The final reaction in each well of 25 μL consists of 0.5 nM hERK2, 900 nM unlabeled peptide, 80 nM labeled-peptide, and 30 μM ATP. Phosphorylation reactions were allowed to proceed for 60 minutes and were immediately quenched by the addition of 60 μL IMAP detection beads (1:1000 dilutions) in IMAP binding buffer (Molecular Devices) with 24 mM NaCl. Plates were read on EnVision reader after 60 minutes binding equilibration using Fluorescence Polarization protocol (Perkin Elmer).
Measurement: IC50 = 1577 nM (pChEMBL 5.800000190734863)
Descriptors: MW 432.5, ALogP 4.5, HBA 5, HBD 2, PSA 75.08, QED 0.47
Compound (SMILES): CC1(C)c2cnc(Nc3ccccc3)nc2CN1C(=O)NC1CCN(Cc2ccccc2)C1
InChIKey: RAPBSGQZKKKEHS-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Activated ERK2 Kinase Assay: Compound potency against activated ERK2 was determined using a kinase assay that measures ERK2-catalyzed phosphorylation of biotinylated ERKtide peptide substrate ([Biotin]-AHA-K-R-E-L-V-E-P-L-T-P-S-G-E-A-P-N-Q-A-L-L-R- [NH2], the peptide sequence derived from EGF receptor: SEQ ID NO:1). The assay was carried out in 50 mM HEPES [pH 7.5], 5 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 0.05% BSA using 0.25 nM ERK2, 200 nM ERKtide peptide and 35 μM ATP (all concentrations are final in the reaction) in a total volume of 10.25 μL. A 16-point, half-log dilution series of compounds at 41× final concentration was used for generating IC50 curves. Compound dilution series were prepared in 100% DMSO. ERK2 was preincubated with compounds for 30 minutes at ambient temperature. Reaction was initiated by addition of a substrate cocktail of ERKtide peptide and ATP and was allowed to proceed for 2-3 hours at ambient temperature. Reaction was terminated by addition of 10 μL of a 2× stop buffer consisting of 100 mM Tris-Cl [pH 7.5], 25 mM EDTA, 0.01% Tween 20, 10 μg/mL of AlphaScreen Protein A Acceptor Beads, 10 μg/mL of Streptavidin Donor Beads (PerkinElmer, Waltham, Mass.), and 1.4 μg/mL phospho-EGF Receptor (Thr669) antibody (Cat #3056, Cell Signaling Technology, Danvers, Mass.). Terminated reactions were read, after overnight incubation in the dark, on an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, Mass.), with excitation and emission wavelengths set to 680 nm and 570 nm, respectively.
Measurement: IC50 = 969 nM (pChEMBL 6.010000228881836)
Descriptors: MW 442.57, ALogP 4.26, HBA 5, HBD 2, PSA 73.39, QED 0.62
Compound (SMILES): C[C@H](Nc1nc(C(=O)N2CCC(C(=O)OC(C)(C)C)C2)c2sccc2n1)c1ccccn1
InChIKey: DBCZDYLDGWOYRQ-MLCCFXAWSA-N
Target: Adenosine receptor A2b (SINGLE PROTEIN), Homo sapiens
UniProt: P29275
Assay: Activity Assay: Compounds (Adenosine Receptor modulators) and activity assay.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 453.57, ALogP 4.06, HBA 8, HBD 1, PSA 97.31, QED 0.58
Compound (SMILES): CC(=O)c1nn(CC(=O)N2C[C@H](F)C[C@H]2C(=O)Nc2cccc(Br)n2)c2ccc(-c3cnc(N)nc3)cc12
InChIKey: XSODZDKXBFADNJ-APWZRJJASA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Human Factor D Assay: Human Factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration was incubated with test compound at various concentrations for 5 minutes at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBzl and DTNB (Ellman's reagent) are added to final concentrations of 100 μM each. Absorbance at 405 nm (A405) was recorded at 30 second intervals for 30 minutes using a microplate spectrophotometer. IC50 values are calculated by nonlinear regression of complement Factor D reaction rates as a function of test compound concentration.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 581.41, ALogP 3.01, HBA 9, HBD 2, PSA 148.99, QED 0.26
Compound (SMILES): Cc1ccc(Nc2ccc3nc(N)nc(C)c3n2)cc1
InChIKey: JPCCTWNTNCHKHX-UHFFFAOYSA-N
Target: Dihydrofolate reductase (SINGLE PROTEIN), Homo sapiens
UniProt: P00374
Assay: Inhibitory Activity Assay: These assays were carried out at 37° C. under 18 μM dihydrofolic acid concentration.
Measurement: IC50 = 174 nM (pChEMBL 6.760000228881836)
Descriptors: MW 265.32, ALogP 2.97, HBA 5, HBD 2, PSA 76.72, QED 0.74
Compound (SMILES): Cc1cccc(C(=O)n2c(=O)n(C)c3ccc(S(=O)(=O)NC4(C)CC4)cc32)c1
InChIKey: PAXBMYPGUMFRHZ-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 = 9568 nM (pChEMBL 5.019999980926514)
Descriptors: MW 399.47, ALogP 2.17, HBA 6, HBD 1, PSA 90.17, QED 0.73
Compound (SMILES): CC(C)(C)NC(=O)N1C[C@@H](c2ccc(C(O)(C(F)(F)F)C(F)(F)F)cc2)[C@@](C)(c2ccc(F)cc2)C1
InChIKey: QKJNUHDUWYDGIL-SIKLNZKXSA-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 (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 μg/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 μM to 0.28 nM for 10 min at room temperature in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% Glycerol (Sigma Cat # G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat # RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined.
Measurement: IC50 = 201 nM (pChEMBL 6.699999809265137)
Descriptors: MW 520.49, ALogP 6, HBA 2, HBD 2, PSA 52.57, QED 0.49
Compound (SMILES): COc1ncc(-c2nc3c(n2C(C)C)C(c2ccc(Cl)cc2)N(c2cc(Cl)cc4[nH]ncc24)C3=O)c(OC)n1
InChIKey: PUZLQFUMPKIPOQ-UHFFFAOYSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: Enzyme Level Activity Assay: In the present disclosure, the enzyme level activity the compounds is detected by using the MDM2/p53 protein binding experiment and using a TR-FRET method. The steps were specifically as follows: an Echo pipette (Labcyte) was used to perform a 3.162-fold serial dilution on the test compounds, and each compound was diluted in 11 concentrations and 250 nL for each concentration was transferred to a 384-well plate, with two replicate wells set for each compound concentration. The well with positive compound (100% inhibition) added was set as a positive control, and the well with DMSO only as a negative control. The GST-MDM2 protein (R & D-E3-202-050) was diluted to 0.625 nM with a buffer (125 mM NaCl, 1 mM DTT, 0.01% Gelatin (animal gelatin), 0.1% Pluronic f-127 (polyether), 1 PBS), and 20 μL of the diluent was added to the 384-well plate. After centrifugation and shaking, the 384-well plate was placed in a 23° C. incubator and incubated for 20 min. The His-p53 protein (R & D-SP-450-020) was diluted to 12.5 nM with a buffer, and 20 μL of the diluent was added to the 384-well plate. After centrifugation and shaking, the 384-well plate was placed in a 23° C. incubator and incubated for 60 min. The Eu2+ anti-GST antibody (Cisbio-61GSTKLB) and XL665 anti-His antibody (Cisbio-61HISXLB) were diluted with a buffer, and the diluted mixture contains 0.3 nM Eu2+ anti-GST antibody and 9 nM XL665 anti-His antibody. 10 μL of the mixture of the two antibodies was added to the 384-well plate. After centrifugation and shaking, the 384-well plate was placed in a 23° C. incubator and incubated for 20 h. Reading was performed on an Envision multifunctional microplate reader (PerkinElmer) (excitation light at 340 nm, and emission lights at 665 nm and 615 nm). Ratio=signal intensity at 665 nm/signal intensity at 615 nm×10000 is used to calculate the inhibition ratio, and the formula is as follows: inhibition ratio=(Ratio of the well with compounds added−Ratio of the negative control)/(Ratio of the positive control−Ratio of the negative control)*100%, and the IC50.
Measurement: IC50 = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 564.43, ALogP 5.87, HBA 8, HBD 1, PSA 111.05, QED 0.27
Compound (SMILES): O=c1[nH]c2cc(NCc3cc4cc(Cl)ccc4[nH]c3=O)ccc2o1
InChIKey: IZMZMGUSJJVYHV-UHFFFAOYSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Enzymatic Assay: Assays were performed in a 384-well black plate. An aliquot of 250 nL of compound was incubated with 10 μL of 30 nM IDH1-R132H or 10 nM IDH1-R132C recombinant protein in assay buffer (50 mM Tris pH=7.5, 150 mM NaCl, 5 mM MgCl2, 0.1% (w/v) Bovine Serum Albumin, and 0.01% Triton X-100) in each well at 25° C. for 15 minutes. After the plate was centrifuged briefly, an aliquot of 10 μL of 2 mM α-ketoglutarate and 20 μM NADPH solution prepared in assay buffer was then added to each well and the reaction was maintained at 25° C. for 45 minutes. An aliquot of 10 μL of diaphorase solution (0.15 U/mL diaphorase and 30 μM Resazurin in assay buffer) was added to each well. The plate was maintained at 25° C. for 15 minutes and then read on a plate reader with excitation and emission wavelengths at 535 nm and 590 nm, respectively. The IC50 of a given compound was calculated by fitting the dose response curve of inhibition of NADPH consumption at a given concentration with the four parameter logistic equation.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 341.75, ALogP 3.23, HBA 4, HBD 3, PSA 90.89, QED 0.53
Compound (SMILES): O=C(O)[C@@H]1c2ccccc2C(=O)N(c2ccc(Cl)c(Cl)c2)[C@H]1c1ccc2c(c1)OCCO2
InChIKey: XWALVQJPVULWLJ-YADHBBJMSA-N
Target: Stimulator of interferon genes protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q86WV6
Assay: H-cGAMP Filtration Binding Assay (HAQ STING): The ability of compounds to bind STING is quantified by their ability to compete with tritiated cGAMP ligand for human STING receptor membrane using a radioactive filter-binding assay. The binding assay employs STING receptor obtained from Trichoplusia ni (T. ni; Expression Systems, cat #94-002F, Expression Systems, Davis, Calif., USA) cell membranes overexpressing full-length HAQ STING prepared in-house and tritiated cGAMP ligand also purified in-house.
Measurement: IC50 = 483 nM (pChEMBL 6.320000171661377)
Descriptors: MW 470.31, ALogP 5.33, HBA 4, HBD 1, PSA 76.07, QED 0.56
Compound (SMILES): Cn1c(CCC(=O)NCCN2CCOCC2)nc2ccc(O)c(C=O)c21
InChIKey: RFEBUBOLFPMYCX-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase/endoribonuclease IRE1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75460
Assay: IRE-1alpha Assay: A fusion protein comprising glutathione S transferase (GST) and human IRE-1α (GST-IRE-1α) obtained from a 500 ml baculovirus-infected insect cell culture can be used to measure IRE-1α activity in vitro. Five μl of a reaction mixture comprising 1× reaction buffer (5× reaction buffer is 100 mM Hepes pH 7.5, 250 mM KOAc, 2.5 mM MgCl2), 3 mM DTT, and 0.4% polyethylene glycol water is added to each well of 384 well plates. Twenty-five nanoliters of a 1 mM test compound solution are added to test wells. Three μl of a 128 ng/ml IRE-1α preparation are added to each test well and to positive control wells (final concentration 5.82 ng/well). Negative control wells contain only reaction mixture and test compound. After spinning the plates at 1200 rpm for 30 seconds, 3 μl of an IRE-1α human mini-XBP-1 mRNA stem-loop substrate 5'-CAGUCCGCAGCACUG-3' (SEQ ID NO:1), labeled with the fluorescent dye Cy5 at the 5' end and Black Hole Quencher 2 (BH2) at the 3' end, are added to each well of a control plate. The plates are again spun at 1200 rpm for 30 seconds. Final concentrations for the assay are: 63 nM IRE-1α substrate, 5.82 ng IRE-1α protein, and 2.5 μM test compound. The plates are covered with lids and incubated for one hour at 30° C. The plates are then transferred to an ACQUEST™ microplate reader. Data is analyzed using data analysis software, and the percent activity of IRE-1α is calculated.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 360.41, ALogP 0.47, HBA 7, HBD 2, PSA 96.69, QED 0.7
Compound (SMILES): Cc1cc2nc(-c3cccc(CN4CCN(C)CC4)c3)[nH]c2cc1Nc1nccc(-c2cccnc2)n1
InChIKey: DFFPGUXEUXPNMD-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: In Vitro TK Inhibition Assay: Colorimetric cell proliferation and viability assay (reagent CellTiter-Blue purchased from Promega cat N°G8081) was performed on BaF3 Kit WT cell line.A total of 2.104 cells/50 μl were seeded per well of a 96-wells plate. Treatment was initiated by addition of a 2× drug solution of ½ serial dilutions ranging from 0 to 10 μM. After incubating for 48 hours at 37° C., 10 μl of a ½ dilution of CellTiter-Blue reagent was added to each well and the plates were returned to the incubator for an additional 4 hours. The fluorescence intensity from the CellTiter-Blue reagent is proportional to the number of viable cells and data were recorded (544Ex/590Em) using a POLARstar OMEGA microplate reader (BMG LabteckSarl). A background control without cells was used as a blank. The positive control of the assay corresponds to the cell proliferation obtained in the absence of drug treatment (100% proliferation). Each sample was done in triplicate. The results were expressed as a percentage of the proliferation obtained in absence of treatment. All drugs were prepared as 20 mM stock solutions in DMSO and conserved at −80° C. Drug dilutions were made fresh in medium before each experiment. A DMSO control was included in each experiment.
Measurement: EC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 490.62, ALogP 4.88, HBA 7, HBD 2, PSA 85.86, QED 0.35
Compound (SMILES): O=C(Nc1cc(-c2cc(CN3CCOCC3)ccc2F)ccc1N1CCN(CC(F)(F)F)CC1)c1c[nH]c(=O)cc1C(F)(F)F
InChIKey: VUXNDTHYMVUCTO-UHFFFAOYSA-N
Target: WD repeat-containing protein 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P61964
Assay: Surface Plasmon Resonance (SPR) Assay: Exemplary compounds of the application were dissolved in 100% DMSO at 10 mM, assayed fresh, and then stored at −20° C. for repeat studies and other experiments. Full length WDR5 with an N-terminal His tag and C-terminal AviTag (Avidity Inc.) was expressed in E. coli with coexpression of BirA to biotin label the protein in vivo. Purification was via Ni-NTA. The purified WDR5 protein has a molecular weight of 41976 Da.SPR studies were performed using a Biacore T200 instrument (GE Health Sciences Inc.). Biotinylated WDR5 protein (approximately 3000RU) was stably captured to streptavidin coupled SA chips according to the manufacture's protocol (GE Health Sciences Inc.). The running buffer used was HBS-EP (20 mM Hepes pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P-20) plus 5% DMSO with a flow rate of 40p1/min. For SPR analysis, 5 different concentrations of each exemplary compound of the application were sprayed into 96 or 384 well plates using an HP D300 digital dispenser. The concentration ranged from about 195 nM to about 12 nM in a two-fold series. Concentration ranges were adjusted higher or lower for weaker or more potent compounds, respectively, when necessary. For the KD determinations, single cycle kinetic analysis was performed with an on time of 60 seconds, and an off time of 300 or 600 seconds. Curve fitting and KD calculations were performed with the Biacore T200 Evaluation software (GE Health Sciences Inc).
Measurement: Kd > 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 641.59, ALogP 4.97, HBA 6, HBD 2, PSA 80.91, QED 0.36
Compound (SMILES): Cc1cccc2c(=O)[nH]c(C(C)SC3CCOCC3)nc12
InChIKey: QVNLKCMBHHDOKD-UHFFFAOYSA-N
Target: Protein mono-ADP-ribosyltransferase PARP14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q460N5
Assay: Enzymatic Inhibition Assay: Enzymatic inhibition of PARP14 was measured using a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) monitoring the auto-modification of PARP14 by biotinylated nicotinamide adenine dinucleotide (biotin-NAD). 1 μL of a dose response curve of each test compound was spotted in 384-well nickel-coated white microplates (Thermo) using a Mosquito (TTP Labtech). Reactions were performed in a 50 μL volume by adding 40 μL of PARP14 in assay buffer (20 mM HEPES pH==8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002%0 Tween20), incubating with test compound at 25° C. for 30 min, then adding 10 μL of biotin-NAD (Biolog). The final concentrations of PARP14 and biotin-NAD are 50 nM and 3 μM, respectively. Reactions proceeded at 25° C. for 3 h, then were quenched with 5 μL of 10 mM unmodified nicotinamide adenine dinucleotide (Sigma-Aldrich). The quenched reactions were washed 3 times with 100 μL of TBST wash buffer (50 mM Tris-HCl, 150 mM NaCl and 0.1% Tween20). Next, to the washed and dried plate was added 25 μL of DELFIA Europium-N1 streptavidin (Perkin Elmer) diluted in DELFIA assay buffer (Perkin Elmer). After a 30 min incubation at 25° C., the plate was washed 5 times with TBST wash buffer. Finally, 25 μL of DELFIA enhancement solution was added. After a 5 min incubation the plate was read on an Envision platereader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 340 nm and emission of 615 nm to measure the amount of Europium present in each well, informing on the amount of biotin-NAD that was transferred in the automodification reaction.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 304.42, ALogP 3.2, HBA 4, HBD 1, PSA 54.98, QED 0.95
Compound (SMILES): CC(C)Nc1cc(-n2ncc3cc(C#N)cnc32)ncc1-c1cn(C)nn1
InChIKey: QDFAFMOTRUAWKR-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IRAK4 Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 μL prepared from 15 μL additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij 35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction mixture was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 359.4, ALogP 2.3, HBA 9, HBD 1, PSA 110.13, QED 0.6
Compound (SMILES): C=CC(=O)N[C@H]1CCC[C@H]1NC(=O)c1sc2nccc3c2c1NC(=O)N3c1cnnc(-c2ccccc2)c1
InChIKey: OMAHGKITIOBWKH-ZWKOTPCHSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Lanthascreen Binding Assay: A BTK kinase lanthascreen binding assay monitors compound binding to unphosphorylated-BTK kinase domain (UP-BTK), by competing with a fluorescent labeled tracer. UP-BTK, consisting of the kinase domain of non-phosphorylated BTK protein (389-659aa), was produced in a Baculovirus/insect cell expression system. Into a 384-well plate, 2 ng of GST-tagged human BTK (389-659aa) was incubated with compound, 50 nM of Tracer 236 and 2 nM anti-GST antibody for 60 minutes using an optimized Lanthascreen assay. After 60 minutes, plates were read at 340 nM and 615/665 nM in an Infinite F500 (Tecan).
Measurement: IC50 = 8.71 nM (pChEMBL 8.0600004196167)
Descriptors: MW 525.59, ALogP 4.39, HBA 7, HBD 3, PSA 129.21, QED 0.32
Compound (SMILES): NCCC(=O)N[C@H]1CC[C@H](NC(=O)c2c[nH]c3ccccc23)CC1
InChIKey: YYGVFRMLRINURH-JOCQHMNTSA-N
Target: Histone-lysine N-methyltransferase SMYD3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H7B4
Assay: SMYD3 Biochemical Assay: The assays were all performed in a buffer consisting of 25 mM Tris-Cl pH 8.0, 1 mM TCEP, 0.005% BSG, and 0.005% Tween 20, prepared on the day of use. Compounds in 100% DMSO (1 ul) were spotted into a 384-well white opaque OptiPlate using a Bravo automated liquid handling platform outfitted with a 384-channel head (Agilent Technologies). DMSO (1 ul) was added to Columns 11, 12, 23, 24, rows A-H for the maximum signal control and 1 ul of SAH, a known product and inhibitor of SMYD3, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control. A cocktail (40 ul) containing the SMYD3 enzyme was added by Multidrop Combi (Thermo-Fisher). The compounds were allowed to incubate with SMYD3 for 30 min at room temperature, then a cocktail (10 ul) containing SAM and MEKK2 was added to initiate the reaction (final volume=51 ul). The final concentrations of the components were as follows: SMYD3 was 0.4 nM, 3H-SAM was 8 nM, MEKK2 was 12 nM, SAH in the minimum signal control wells was 1 mM, and the DMSO concentration was 2%. The assays were stopped by the addition of non-radiolabeled SAM (10 ul) to a final concentration of 100 uM, which dilutes the 3H-SAM to a level where its incorporation into MEKK2 is no longer detectable. Radiolabeled MEKK2 was detected using a scintillation proximity assay (SPA). 10 uL of a 10 mg/mL solution of SPA beads in 0.5 M citric acid was added and the plates centrifuged at 600 rpm for 1 min to precipitate the radiolabeled MEKK2 onto the SPA beads. The plates were then read in a PerkinElmer TopCount plate reader to measure the quantity of 3H-labeled MEKK2 as disintegrations per minute (dpm) or alternatively, referred to as counts per minute (cpm).
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 328.42, ALogP 1.67, HBA 3, HBD 4, PSA 100.01, QED 0.67
Compound (SMILES): CS(=O)(=O)N1CCC(Nc2ncc(C#N)c(-c3cnn(-c4ccc(CN[C@@H]5CCCC[C@@H]5O)cc4Cl)c3)n2)CC1
InChIKey: GPNCPLBDPVGQLQ-NOZRDPDXSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24864
Assay: CDK2/Cyclin E1 HERE Enzyme Activity Assay: CDK2/Cyclin E1 enzyme activity assays utilize full-length human CDK2 co-expressed as N-terminal GST-tagged protein with FLAG-Cyclin E1 in a baculovirus expression system (Cama Product Number 04-165). Assays are conducted in white 384-well polystyrene plates in a final reaction volume of 8 μL. CDK2/Cyclin E1 (0.25 nM) is incubated with compounds (40 nL serially diluted in DMSO) in the presence of ATP (50 μM or 1 mM) and 50 nM ULight-labeled eIF4E-binding protein 1 (THR37/46) peptide (PerkinElmer) in assay buffer (containing 50 mM HEPES pH 7.5, 1 mM EGTA, 10 mM MgCl2, 2 mM DTT, 0.05 mg/ml BSA, and 0.01% Tween 20) for 60 minutes at room temperature. The reactions are stopped by the addition of EDTA and Europium-labeled anti-phospho-4E-BP1 antibody (PerkinElmer), for a final concentration of 15 mM and 1.5 nM, respectively. HTRF signals are read after 1 hour at room temperature on a PHERAstar FS plate reader (BMG Labtech). Data is analyzed with IDBS XLFit and GraphPad Prism 5.0 software using a three or four parameter dose response curve to determine IC50 for each compound.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 585.13, ALogP 3.08, HBA 10, HBD 3, PSA 149.06, QED 0.36
Compound (SMILES): C#COCCOCCOCCOc1ccc2c3c1[C@H]1CCC[C@H]1N3CCNC2
InChIKey: GWSZWGIABIWWDG-RBUKOAKNSA-N
Target: 5-hydroxytryptamine receptor 2C (SINGLE PROTEIN), Homo sapiens
UniProt: P28335
Assay: Intracellular Calcium Assay: Cell Lines and Cell Culture.The PathHunter® U2OS HTR2C β-Arrestin cell line (5-HT2cR-U2OS; DiscoveRx) stably express the nonedited human 5-HT2CR isoform (h5-HT2CR). The 5-HT2CR-U2OS cells were grown in Assay Complete U2OS Medium 31 (DiscoveRx) at 37° C., 5% CO2 and 85% relative humidity according to manufacturer's recommendations utilizing AssayComplete Cell Detachment Reagent (DiscoveRx). Cells were passaged at 70-80% confluence and all experiments were conducted using cells in log phase growth.Intracellular Calcium Assay.The ability of the molecules to act as agonists to induce 5-HT2CR-mediated intracellular calcium (Cai ++) release was conducted in an U2OS cell line stably expressing the human 5-HT2CR. For all molecules examined, the observed potency was shifted rightward relative to 5-HT or WAY163909 (Table 1).Intracellular calcium (Cai ++) release was monitored using the FLIPR Calcium 4 Assay Kit (Molecular Devices) according to previously published protocols with minor modifications. See, e.g., Shashack, M. J., et al., ACS Chem Neurosci. 2 (11), 640-644 (2011); Seitz, P. K.; Bremer, N. M., et al., BMC Neurosci. 13, 25 (2012).Cells were plated at 5,000-7,000 cells/well in Assay Complete Cell Plating Reagent 16 (DiscoveRx) in black-sided, clear bottomed 96-well tissue culture plates and allowed to adhere overnight. Medium was removed and replaced with 40 μl Hank's balanced salt solution without calcium, magnesium and phenol red (HBSS; Corning) plus 40 μl Calcium 4 dye solution in Buffer B supplemented with 2.5 mM probenecid (Sigma-Aldrich) to inhibit extracellular dye transport. Plates were incubated for 60 min at 37° C. followed by 30 min at room temperature in the dark.Fluorescence (λex=485 nm, λem=525 nm) was measured using a FlexStation3 (Molecular Devices). Baseline was established for 17 secs before addition of 20 μl vehicle (HBSS without calcium or magnesium) or 5× concentrated compound. Addition of 5-HT, WAY163909, or ligand occurred at the 17-sec time point and fluorescence was recorded every 1.7 sec for 120 sec. Maximum peak height was determined using FlexStation software (SoftMax Pro 5.4). After the final readings, cells were fixed in 2% paraformaldehyde overnight.Data AnalysisPeak responses from each well were normalized to total cell mass as determined with crystal violet staining. See e.g., Ding, C. et al., ACS Chem. Neurosci 3 (7), 538-545 (2012).
Measurement: EC50 = 575 nM (pChEMBL 6.239999771118164)
Descriptors: MW 386.49, ALogP 2.27, HBA 6, HBD 1, PSA 52.19, QED 0.49
Compound (SMILES): C=CC(=O)N(C)[C@H]1C[C@H](N(C)C(=O)N(C)[C@H](C(=O)N[C@H]2Cc3nc(cs3)-c3ccc4c(c3)c(c(-c3cccnc3[C@H](C)OC)n4CC)CC(C)(C)COC(=O)[C@@H]3CCCN(N3)C2=O)C(C)C)C1
InChIKey: PLJXDULWCCRBOI-IZOSVWSKSA-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 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 938.21, ALogP 6.39, HBA 12, HBD 2, PSA 171.54, QED 0.13