lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cn1c(=O)c2cnc3[nH]ccc3c2n(C2CCCCC2)c1=O
InChIKey: DUPIQMAGYFFIBF-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS. From the plot of logarithm of a compound concentration and inhibitory activity, the IC50 was calculated.
Measurement: IC50 = 40 nM (pChEMBL 7.400000095367432)
Descriptors: MW 298.35, ALogP 2.08, HBA 5, HBD 1, PSA 72.68, QED 0.75
Compound (SMILES): CC#CC(=O)N1C2CN[C@@H](C2)C1c1nc(-c2ccc(C(=O)Nc3cc(C(F)(F)F)ccn3)cc2)c2c(N)nccn12
InChIKey: IBLFVRAFTKADBF-KPRXVCGWSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: In Vitro Assay for the Inhibition of BTK Kinase Activity: 1: Test Principle:Mobility-Shift Assay, which is microfluidic chip technology, applies the basic concept of capillary electrophoresis to microfluidic environments. The substrate used for the experiment is a polypeptide with a fluorescent label. Under the action of enzyme in the reaction system, the substrate is transformed into a product, and the charge the substrate carries also changes accordingly. The use of the charge difference between the substrate and the product involved in Mobility-Shift Assay achieves the separating of the two, and they are tested respectively. The test results are expressed by conversion rates.2: Test Method:(1) preparation of samples to be tested: diluted with 100% DMSO to 50 times the final concentration of the reaction, i.e. 25 μmol/L;(2) dilution: 25 μmol/L is the initial concentration, then diluted with 4 times the concentration and diluted with 10 concentration gradients;(3) 100% DMSO was added to the positive control well and the negative control well, respectively;(4) the prepared compounds with 10 concentrations were diluted 10-fold with 1 xkinase buffer, respectively; wherein, the kinase buffer contained hydroxyethyl piperazine ethanesulfonic acid at a concentration of 50 mmol/L and a pH of 7.5, 0.01% dodecyl polyethylene glycol ether, 10 mmol/L magnesium chloride, 2 mmol/L dithiothreitol;(5) preparation of 2.5×enzyme solution: the kinase was added to 1×kinase buffer to form 2.5×enzyme solution;(6) preparation of 2.5×substrate solution: FAM-labeled polypeptide and ATP were added to the 1 xkinase buffer to form 2.5×substrate solution; (7) addition of the enzyme solution to the 384-well plate: 5 μl of 5×compound dissolved in 10% DMSO contained in the 384-well reaction plate, then 10 μl of 2.5×enzyme solution was added, the obtained system was incubated for 10 minutes at room temperature;(8) addition of the substrate solution to the 384-well plate: 10 μl of 2.5×substrate solution was added to the 384-well reaction plate;(9) kinase reaction and termination: after incubation at 28° C. for 1 hour, 25 μl stop solution was added to terminate the reaction; wherein, the stop solution contained hydroxyethyl piperazine ethanesulfonic acid at a concentration of 100 mmol/L and a pH of 7.5, 0.015% dodecyl polyethylene glycol ether, 0.2% surface reagent No. 3, 20 mmol/L ethylenediaminetetraacetic acid; (10) Caliper data reading: conversion rate data was read from Caliper.
Measurement: IC50 = 1.8 nM (pChEMBL 8.739999771118164)
Descriptors: MW 560.54, ALogP 3.28, HBA 8, HBD 3, PSA 130.54, QED 0.33
Compound (SMILES): CC(C)(C)OC(=O)N1CCC(c2ccc3nc(-c4ccc(OCc5ccccc5)cc4)[nH]c3n2)CC1
InChIKey: ZXAMENOMEYDZBH-UHFFFAOYSA-N
Target: Protein arginine N-methyltransferase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: O14744
Assay: Biochemical Assay : The 6 μL assay reactions are run in Greiner brand black 384-well low volume plates. All reactions contained assay buffer (phosphate buffered saline, 0.01% (v/v) Tween-20, 0.01% (w/v) albumin from chicken egg white, 1 mM dithiothreitol, 200 nM peptide substrate, 1 μM S-Adenosyl methionine, and 15 ng/reaction enzyme, with the enzyme being omitted from negative control reactions. Compounds were added in a volume of 100 nL from dilution series prepared in DMSO, positive and negative control reactions receiving the same volume DMSO without compound. The plates were sealed with adhesive seals and incubated for 4 hours at 37° C. Reaction progress was measured using the Transcreener EPIGEN methyltransferase assay (BellBrook Labs, Madison, Wis.) as recommended by the manufacturer. To each reaction 2 μL detection mix were added, containing coupling enzymes, fluorescence polarisation tracer, and AMP antibody. Plates were incubated for 90 minutes before being read on a PerkinElmer EnVision plate reader in fluorescence polarisation mode.
Measurement: IC50 = 726 nM (pChEMBL 6.139999866485596)
Descriptors: MW 484.6, ALogP 6.32, HBA 5, HBD 1, PSA 80.34, QED 0.36
Compound (SMILES): COC(=O)N1CC[C@H](NS(C)(=O)=O)[C@@H]1CO[C@@H]1CC[C@]2(c3ncccn3)C[C@@H]2C1
InChIKey: JOMHLIUPHAKPTB-SPCJURDBSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: IP-One assay kit: IP-One, Cisbio, cat #621PAPEC, is a commercially available assay.
Measurement: IC50 = 2033 nM (pChEMBL 5.690000057220459)
Descriptors: MW 424.52, ALogP 1.06, HBA 7, HBD 1, PSA 110.72, QED 0.73
Compound (SMILES): O=C(NCCOc1nc(N2CC3CCC(C2)N3)c2cnc(-c3cccc4cccc(Cl)c34)c(F)c2n1)c1ccc(S(=O)(=O)F)cc1
InChIKey: MSAGSCKEPUKTHU-UHFFFAOYSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: KRas G12D Binding Assay: Table 2: The ability of a compound to bind to KRAS G12D was measured using a TR-FRET displacement assay. Biotinylated GDP-loaded recombinant human KRAS G12D (corresponding to amino acids 1:169, produced at Array BioPharma) was incubated with a custom-made Cy5 labelled tracer, europium labelled streptavidin and compound (2% DMSO final) in buffer (50 mM HEPES [pH 7.5], 5mM MgCl2, 0.005% Tween-20 & 1 mM DTT). After a 60 minute incubation at 22° C., the reaction was measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) calculated using a ratiometric emission factor. 100 POC is determined using no test compound and 0 POC is determined using a concentration of control compound that completely inhibits binding of the tracer to KRAS. The POC values were fit to a 4-parameter logistic curve and the IC50 value was determined as the concentration where the curve crosses 50 POC.
Measurement: IC50 = 4046 nM (pChEMBL 5.389999866485596)
Descriptors: MW 665.12, ALogP 5.05, HBA 9, HBD 2, PSA 126.41, QED 0.17
Compound (SMILES): O=C(COC(=O)C(NC(=O)c1ccccc1)c1ccccc1)Nc1ccccc1
InChIKey: MOTTVQFDOUIUBU-UHFFFAOYSA-N
Target: Kallikrein-7 (SINGLE PROTEIN), Homo sapiens
UniProt: P49862
Assay: Inhibition Assay: Substrate:(MeO-Suc-Arg-Pro-Tyr-pNA), acetate salt, Peptide international, lot #906841, previously named S-2586Enzyme: Active human KLK7 (SCCE) 0.62 mg/ml in 0.3 M NaCl, 10 mM NaAc, pH 4Buffer: 10 mM NaPhospate, 0.5 M NaCl, pH 7.2150 μl of Substrate (2.5 mM) in Buffer was added to 96 wells plate (F8 Polysorp Unfra, Nunc cat. no. 469957). 10 μl of DMSO was added to the blank and control wells.10 μl of substance in DMSO was added to wells giving final concentrations of 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.3125 μM, and 0.15625 μM.40 μl of active KLK7 (SCCE) (diluted to 12.5 μg/ml in activity buffer) was added all wells except blank to which activity buffer was added.Immediately after addition of SCCE the plate was transferred to a Spectramax 250 Microplate Reader (Molecular Devices) and enzyme activity (V) was measured as release of pNa measuring absorbance at 405 nm in 37° C. for 15 min with reading every 30 s.The mean value V of each sample was calculated (n=2 for the substances and n=4 for the control). From the values of V the % of total activity was calculated as (Vinhibitor/Vcontrol (no inhibitor))×100.
Measurement: IC50 = 1560 nM (pChEMBL 5.809999942779541)
Descriptors: MW 388.42, ALogP 3.34, HBA 4, HBD 2, PSA 84.5, QED 0.61
Compound (SMILES): CCn1c(=O)c(-c2ccccc2)cc2cnc(Nc3ccc(N(C)C)cn3)cc21
InChIKey: CQADGYMRJAVKJA-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Assays: A concentration of a drug that reduces the observed activity of an enzyme by 50%. An IC50 is not a true affinity constant and the specific value determined can vary depending on the particular assay used. Nonetheless the rank order of IC50 for a set of compounds will typically be similar from one assay to another. (The true affinity constant for an inhibitor binding to an enzyme, Ki, can of course also be used for the same purposes as described herein for IC50). With respect to the kinases that are targeted by the compounds disclosed herein, kinase activity can be assayed, without limitation, as consumption of ATP or as incorporation of phosphate into a substrate. For example, the assay used in Example 76 assesses consumption of ATP based on luminescent detection of ADP. As an alternate example, the assay used in Example 78 assesses incorporation of phosphate based on the radiologic detection of 33P-labeled artificial substrates. Myriad other kinase assays are known in the art many of which are commercially available either as a kit or a service and one of skill in the art will recognize their particular usefulness for generating IC50 data.
Measurement: IC50 = 3000 nM (pChEMBL 5.519999980926514)
Descriptors: MW 385.47, ALogP 4.29, HBA 6, HBD 1, PSA 63.05, QED 0.56
Compound (SMILES): CC(O)C(NC(=O)CN1Cc2ccc(-c3nc(NC4CCOCC4)ncc3Cl)cc2C1=O)c1ccccc1
InChIKey: LJGQCVMRWMHTGN-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: ERK2 In Vitro Inhibition Assay: Activity of ERK2 enzyme (Life Technologies) was determined using a time-resolved fluorescence format measuring the phosphorylation of a truncated version of Activating transcription factor 2 labelled with green fluorescent protein (ATF2-GFP) (Life Technologies). Assay reactions containing 50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Triton X-100, 1 mM DTT, 2.5% DMSO, 0.4 mM ATF2-GFP, 20 μM ATP and 0.25 nM ERK2 were set up in the presence of compound and allowed to proceed for 30 min at room temperature. Reactions were then stopped using TR-FRET dilution buffer (Life Technologies), 25 mM EDTA and 2 nM Tb-Anti-pATF2 (Thr71) (Life Technologies). After a further incubation period of at least 30 minutes, fluorescence was read on a Pherastar reader (Lanthascreen optic module; excitation 340 nm, emission 520 nm (channel A), 495 nm (channel B)). The ratio between A and B counts was used to calculate signal. IC50 values were calculated using a sigmoidal dose response equation (Prism GraphPad software, La Jolla, Calif., USA).
Measurement: IC50 = 4.2 nM (pChEMBL 8.380000114440918)
Descriptors: MW 536.03, ALogP 3.58, HBA 7, HBD 3, PSA 116.68, QED 0.4
Compound (SMILES): Cn1cnc(COc2nc(N3CC4CCC(C3)N4)c3cnc(-c4cc(O)cc5ccccc45)c(F)c3n2)c1
InChIKey: ABXLIHXSISTEJY-UHFFFAOYSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: KRas G12D Surface Plasmon Resonance (SPR) Binding Assay: Table 1: Briefly, 1 L of 1.05× HBS-Mg buffer (262.5 mM Bioultra Hepes, pH 7.5, 157.5 mM NaCl, 105 mM MgCl2, 0.525 mM TCEP, 0.0305% Brij-35) was prepared and filter sterilized using a 0.22 μm bottle top filter. Approximately 50 mL of 1.05× HBS-Mg buffer was removed and saved for future dilutions. A 50 mL aliquot of DMSO (Sigma Aldrich DMSO Lot. #SHBK2079) was added and continued to stir for 10 minutes, creating the final 1.0× HBS-Mg buffer (250 mM Bioultra Hepes pH 7.5, 150 mM NaCl, 100 mM MgCl2, 0.5mM TCEP, 0.03% Brij-35).
Measurement: Kd = 259 nM (pChEMBL 6.590000152587891)
Descriptors: MW 511.56, ALogP 3.94, HBA 9, HBD 2, PSA 101.22, QED 0.37
Compound (SMILES): CCC(O)(CC)c1nc(C(C)(C)C)nc2c1nnn2Cc1ccccc1Cl
InChIKey: FDIHBRZXMGYSAY-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1×HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min. Compounds were added to a final assay volume of 100 μl and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 μl detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.EC50 values were determined using Activity Base analysis (ID Business Solution, Limited). The EC50 values for a wide range of cannabinoid agonists generated from this assay were in agreement with the values published in the scientific literature.The compounds of the invention are CB2 receptor agonists with EC50 below 1 μM and selectivity versus CB1 in the corresponding assay of at least 10 fold. Particular compound of the invention are CB2 receptor agonists with EC50 below 0.05 μM and selectivity versus CB1 in the corresponding assay of at least 500 fold.
Measurement: EC50 = 45.3 nM (pChEMBL 7.340000152587891)
Descriptors: MW 387.92, ALogP 4.23, HBA 6, HBD 1, PSA 76.72, QED 0.71
Compound (SMILES): Cn1ccc(CN2C(=O)c3c(Cl)ccc(NS(=O)(=O)c4ccc(C(C)(C)C)cc4)c3C2=O)n1
InChIKey: AVAAVCBMOXEYBW-UHFFFAOYSA-N
Target: C-C chemokine receptor type 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P51686
Assay: FLIPR Assay: A calcium flux assay was used to determine the ability of the compounds to interfere with the binding between CCR9 and its chemokine ligand (TECK) in Chem1-hCCR9 overexpressing cells. hCCR9 overexpressing cells were seeded (25,000 cells/well) into black Poly-D-Lysine coated clear bottom 96-well plates (BD Biosciences, Cat #356640) and incubated overnight at 37° C./5% CO2 in a humidified incubator. Media was aspirated and cells washed twice with 100 μL assay buffer (1×HBSS, 20 mM HEPES) containing 2.5 mM Probenecid. A 0.3× Fluo-4 NW calcium dye was prepared in assay buffer containing 5 mM Probenecid and stored in the dark. Each well was loaded with 100 μL of 0.3× Fluo-4 NW calcium dye and incubated at 37° C./5% CO2 for 60 minutes and then at room temperature for 30 minutes. A half-log serially diluted concentration response curve was prepared at a 3× final assay concentration for each compound (10 μM-0.1 nM final assay concentration) and 50 μL of the compound then transferred to the cells (150 μL final volume) for 60 minutes prior to stimulation (30 minutes at 37° C./5% CO2 and 30 minutes at room temperature). TECK was diluted to 4× its EC80 in assay buffer (containing 0.1% [w/v] bovine serum albumin [BSA]) and 50 μL dispensed through the fluorometric imaging plate reader (FLIPR) instrument to stimulate the cells (200 μL final volume). The increase in intracellular calcium levels was measured with the FLIPR instrument.
Measurement: Ki = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 486.98, ALogP 3.97, HBA 6, HBD 1, PSA 101.37, QED 0.55
Compound (SMILES): CC(=O)N[C@@H](C)CO[C@H]1C[C@H](COc2cc(C)c3nc(OC4CC4)sc3n2)C1
InChIKey: LQBIYALHINSPJE-NOLJZWGESA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Inhibitory Activity on Human ACC1 and the ACC2: Recombinant human ACC1 and recombinant human ACC2, which were prepared by the method mentioned above, were preincubated with assay buffer solution (50 mM HEPES-KOH (pH 7.4), 10 mM magnesium chloride, 6-10 mM potassium citrate, 4 mM reduced form of glutathione, 1.5 mg/ml bovine serum albumin) for one hour. Then, 0.2 μL of each this invention compound solution (in DMSO) were dispensed to 384-well microplate, 5 μL of the preincubated enzyme solution and 5 μL of substrate solution (50 mM HEPES-KOH (pH 7.4), 1 mM ATP, 0.8 mM acetyl CoA and 25-50 mM potassium bicarbonate) were added to microplate. After centrifugation and shaking, the reaction mixtures were incubated in a humidified box at room temperature for 1 to 3 hours. After the incubation, the enzyme reactions were stopped by the addition of EDTA. Then, after the samples were cocrystallized with CHCA (α-cyano-4-hydroxy cinnamic acid) matrices on MALDI target plate, by using the matrix assist laser deionization time-of-flight mass spectrometer (MALDI-TOF MS), samples were measured in reflector negative mode. Deprotonated ions of acetyl CoA (AcCoA) of substrate and malonyl CoA (MalCoA) of the reaction product were detected, then, the conversion rates of acetyl CoA to malonyl CoA was calculated by the intensity of [MalCoA-H]−/(Intensity of [MalCoA-H]−+Intensity of [AcCoA-H]−) using each signal strength. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate of the enzymatic reaction at each concentration of the compounds. In addition, potassium citrate concentrations in assay buffer solution, potassium hydrogen carbonate concentrations in substrate solution and incubation time were adjusted by each lot of enzyme.
Measurement: IC50 = 99 nM (pChEMBL 7)
Descriptors: MW 405.52, ALogP 3.24, HBA 7, HBD 1, PSA 82.57, QED 0.69
Compound (SMILES): Clc1cccc2c(-n3c(CC4CC4)nc4c(-c5ncco5)cc(N5CCOCC5)cc43)ccnc12
InChIKey: AQQHYKVMCYCIAA-UHFFFAOYSA-N
Target: Phosphatidylinositol 3-kinase regulatory subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O00459
Assay: TR-FRET PI3K Beta Assay: Class I PI3K isoforms were expressed and purified as heterodimeric recombinant proteins. All assay reagents and buffers for the TR-FRET assay were purchased from Millipore. PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2× Km ATP (75-500 μM), 2 μM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, and 1% (v/v) DMSO at the following concentrations for each isoform: PI3Kα, PI3Kβ, and PI3Kδ between 25 and 50 μM, and PI3Kγ at 2 nM. The compounds were added to the assay solution and incubated for 30 minutes at 25° C. The reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 500 μs read window).The results were normalized based on positive (1 μM wortmanin) and negative (DMSO) controls, and the IC50 values for PI3K α, β, δ, and γ were calculated from the fit of the dose-response curves to a four-parameter equation. These assays generally produced results within 3-fold of the reported mean. As shown in Table 1, the compounds provided herein are inhibitors of PI3Kβ.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 485.98, ALogP 5.67, HBA 7, HBD 0, PSA 69.21, QED 0.32
Compound (SMILES): O=S(=O)(c1cc(Br)cc(Cl)c1O)n1c(-c2cccnc2)cc2cc(Cl)ccc21
InChIKey: IBAMEXTXUINFQX-UHFFFAOYSA-N
Target: ATP-citrate synthase (SINGLE PROTEIN), Homo sapiens
UniProt: P53396
Assay: ADP-Glo Assay: Compounds of the present invention were evaluated in an ADP-GLO assay as follows:a) Dilute cpd 1:3 in succession in DMSO by hand for each cpds for 12 ptsb) Add 0.1 μL diluted cpd solution to assay plate, each dose with 2 replicatesc) Centrifuge 1000 RPM for 1 mind) Add 5 μL ACL working solution to 384-well assay plate, centrifuge 1000 RPM for 1 mine) Incubate at 25° C. for 15 minf) Add 5 μL substrate working solution to initiate reactiong) Final ACL reaction concentrations: 3 nM ACL, 15 μM ATP, 3 μM CoA, 300 μM Citrate, 0.01% Brij35, 4 mM DTT, 1% DMSO;h) Reference final conc: 30 uM starting Conc, 3× dilution, 11+0 points. Test cpd conc: 30/100 uM starting Conc, 3× dilution, 11+0 points.i) Incubate at 25° C. for 60 minj) Add 10 μL ADP Glo reagent, centrifuge 1000 RPM for 1 mink) Incubate at 25° C. for 40 minl) Add 20 μL kinase detection reagent, centrifuge 1000 RPM for 1 minm) Incubate at 25° C. for 40 minn) Read on Envision for US LUM as RLU.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 498.19, ALogP 5.72, HBA 5, HBD 1, PSA 72.19, QED 0.39
Compound (SMILES): O=C(c1cccc(F)c1-n1cccn1)N1CCC[C@H]1c1nc(-c2ccccc2OC(F)(F)F)no1
InChIKey: DCZJANTVKPBBCM-KRWDZBQOSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: In Vitro Assay: Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES. On the day of the assay, 50 uL of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/L, 5 mM probenecid (Sigma) and 3 uM of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at rt for 30 min before measurement.Within the Fluorescent Imaging Plate Reader (FLIPR Tetra, Molecular Devices), antagonists are added to the plate in a volume of 10 uL/well, incubated for 120 min and finally 10 uL/well of agonist is added. Fluorescence is measured for each well at 1 second intervals, and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 3 nM orexin-A with vehicle in place of antagonist. The IC50 value (the concentration of compound needed to inhibit 50% of the agonistic response) is determined and may be normalized using the obtained IC50 value of an on-plate reference compound. Optimized conditions were achieved by adjustment of pipetting speed and cell splitting regime. The calculated IC50 values may fluctuate depending on the daily cellular assay performance. Fluctuations of this kind are known to those skilled in the art. In the case where IC50 values have been determined several times for the same compound, the geometric mean has been given.
Measurement: IC50 = 333 nM (pChEMBL 6.480000019073486)
Descriptors: MW 487.41, ALogP 4.94, HBA 7, HBD 0, PSA 86.28, QED 0.37
Compound (SMILES): CC(=O)N1CCCC(c2ccc(CN3CCN(C[C@H](O)CN4CCc5ccccc5C4)C3=O)nc2)C1
InChIKey: CQENAKGLCAFPAE-ZCJYOONXSA-N
Target: Protein arginine N-methyltransferase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: O14744
Assay: TR-FRET Assay: Protein arginine methyltransferase 5 (PRMT5) is a type II arginine methyltransferase that catalyze mono- and symmetric demethylation on arginine residues of histone or non-histone proteins in presence of S-adenosylmethionine (AdoMet or SAM) a cofactor responsible for donating the methyl group. PRMT5 is reported to be overexpressed in several human cancers. To identify compounds that inhibit the PRMT5 and decrease its activity, a TR-FRET based assay has been established. Time-resolved fluorescence resonance energy transfer (TR-FRET) HTS assays are homogeneous proximity assays where the interaction of two dye-labeled binding partners is detected by the energy transfer between a donor and an acceptor dye, and the subsequent light emission by the acceptor dye. PRMT5 catalyzes Histone H4 peptide [1-16] which is biotin tagged to the Lysine amino acid at carboxyl end, in presence of S-adenosyl-1-methionine (SAM) to methylate the peptide. The antibody specific to mono methylated H4 peptide (H4R3) with Ig conjugate binds to the methylated peptide, indirectly binding to the Europium lanthanide. SureLight Allophycocyanin-Streptavidin binds to the biotin tag of the peptide, therefore accepting the energy transferred from the Europium lanthanide. This energy transfer between Europium to SureLight Allophycocyanin is a direct measure of the activity/inhibition of the PRMT5 enzyme.
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 491.64, ALogP 2.46, HBA 5, HBD 1, PSA 80.22, QED 0.64
Compound (SMILES): Fc1ccc2nc(-n3ccc4ccncc43)ncc2c1
InChIKey: BPTNCXCYJAIUSO-UHFFFAOYSA-N
Target: Microtubule-associated protein tau (SINGLE PROTEIN), Homo sapiens
UniProt: P10636
Assay: Tau binding assay: For displacement tau binding assay, unlabeled test compounds were dissolved in DMSO at 1 mM. Dilutions of tests compounds to various concentrations were made in 100% DMSO at 1000× final assay concentration and 0.225 μl aliquots were dispensed into assay plates. Brain homogenates were diluted to 0.5 mg/mL from original 30 mg/mL volume in Assay Buffer, and 200 μl were added to the assay plate for a final concentration of 100 μg wet weight/assay tube. [3H]-6 was prepared at 100× final concentration in Assay Buffer plus 20% DMSO and 25 μl was added to the assay plate for final assay concentration of 0.25 nM. The assay plate was incubated at room temperature (25° C.) for 90 minutes. Unbound and bound ligand were separated by filtration of bound onto GF/C filter plates (pre-treated for 30 min with 0.2% polyethyleninamine) using a Packard UniFilter Harvester and washing away unbound with 2.5 ml per well ice cold 5 mM Tris at pH 7.4. Filter plates were dried for 1 hour in a vacuum oven and 50 μl/well MicroScint-20 were added. Plates were counted 1 min per well by Packard Topcount. Total amount of radioligand used in the assay was determined by counting 25 μl of 10× ligand stock. Data was analyzed using Activity Base software to generate 4 p fit of dose responses; and, Ki values were calculated.
Measurement: Ki = 2.55 nM (pChEMBL 8.59000015258789)
Descriptors: MW 264.26, ALogP 3.11, HBA 4, HBD 0, PSA 43.6, QED 0.53
Compound (SMILES): NS(=O)(=O)c1ccc(S(=O)(=O)C2CCN(C(=O)c3cnc(OC4CCC4)c(C4CC4)c3)CC2)c(F)c1
InChIKey: OHJCFUHWDONOCM-UHFFFAOYSA-N
Target: Carbonic anhydrase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P00918
Assay: Human Carbonic Anhydrase-II Inhibition Assay: Human carbonic anhydrase II (hCA-II) inhibition was measured by an absorbance method using 4-nitrophenyl acetate (4-NPA) as its substrate. 4-NPA can be catalyzed by active hCA II via a zinc-hydroxide mechanism. The nitrophenolate in the products can be ionized to generate a bright yellow anion with high absorbance at 348 to 400 nm, as reported in the literature (Armstrong et al., J. Biol. Chem. 1966, 241, 5137-5149). OD340 nm was chosen for detecting hCA II substrate conversion.Assay working solutions were made as follows:Assay buffer: 50 mM MOPS, 33 mM Na2SO4, 1 mM EDTA, 0.5 mg/mL BSA, pH 7.5;Enzyme solution: hCA-II (human, full length) stock solution (1.0 mg/mL in 20 mM HEPES, 50 mM NaCl, pH 7.4), diluted to 2133× final concentration in assay buffer;4-NPA substrate solution: 4-NPA substrate stock solution (250 mM in DMSO, stored at −20° C.), diluted to 50× final concentration in deionized water.Test compounds (10 mM stock in DMSO, 100 μL) were obtained in 96-well sample plates (Corning Costar #3655) and diluted to 0.5 mM. Column-wise serial dilutions were made by transferring 20 μL compound solutions to the next column, from column 3 up to 22. After this, 1.2 μL were transferred to 384 well assay plates (Corning Costar #3701). Then 30 μL of 16 nM hCA II solution was added (8 nM final concentration), mixed five times. 30 μL of 4-NPA substrate solution was added (2.5 mM final concentration), mixed five times. Absorbance at 340 nm was then measured immediately as time zero. The assay plates were incubated at room temperature for 1 hour and then measured as time 1 hour (Perkin Elmer EnVision 2103; Filter: Photometric 340; Light intensity 60%; Number of flashes: 10). IC50 values and Ki values were calculated from these readouts.
Measurement: IC50 = 2.9 nM (pChEMBL 8.539999961853027)
Descriptors: MW 537.64, ALogP 2.76, HBA 7, HBD 1, PSA 136.73, QED 0.57
Compound (SMILES): CNC(=O)[C@@H](CCCCNC(=N)N)NC(=O)c1csc([C@H]2C[C@@H](N)CN2C(=O)c2cn3cc(Cl)ccc3n2)n1
InChIKey: LLPPLVKHKYCFMU-IIDMSEBBSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Inhibitory Activity Assay: Plasma Kallikrein: The example compounds were evaluated for inhibition of the human activated kallikrein enzyme in two formats of an assay employing a fluorogenic peptide substrate. In one assay format, the concentrations of reagents were as follows: 20 mM Tris pH 7.5, 1 mM EDTA, 150 mM sodium chloride, 0.1% PEG-400, 0.1% Triton X-100, 500 pM activated kallikrein enzyme, 300 uM Pro-Phe-Arg-7-amido-4-methylcoumarin substrate. Prior to reaction initiation with substrate, enzyme and inhibitors were preincubated for 30 min at room temperature. After initiation with substrate, reactions were incubated for 10 min at room temperature and fluorescence emission at 460 nm from 380 nm excitation measured with a microplate reader. In another assay format, the concentrations of reagents were as follows: 20 mM Tris pH 7.5, 1 mM EDTA, 150 mM sodium chloride, 0.1% PEG-400, 0.1% Triton X-100, 5 pM activated kallikrein enzyme, 300 uM Pro-Phe-Arg-7-amido-4-methylcoumarin substrate. Prior to reaction initiation with substrate, enzyme and inhibitors were preincubated for 30 min at room temperature. After initiation with substrate, reactions were incubated for 18 hr at room temperature and fluorescence emission at 460 nm from 380 nm excitation measured with a microplate reader.
Measurement: EC50 > 9900 nM (pChEMBL 5.004000186920166)
Descriptors: MW 575.1, ALogP 0.86, HBA 9, HBD 6, PSA 196.62, QED 0.12
Compound (SMILES): COc1ccc(CN2C[C@]3(CC[C@](c4ccccc4)(N(C)C)CC3)N(CC(=O)N3CCCC3)C2=O)cc1
InChIKey: LEXWMPRAZSIKMK-RNPORBBMSA-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 = 20.5 nM (pChEMBL 7.690000057220459)
Descriptors: MW 504.68, ALogP 4.33, HBA 4, HBD 0, PSA 56.33, QED 0.56
Compound (SMILES): N#Cc1ccc(N2CCN(C(=O)c3cccc(OCCNc4cn[nH]c(=O)c4C(F)(F)F)c3)CC2)nc1
InChIKey: BVDBMRMPEYAAPE-UHFFFAOYSA-N
Target: Protein mono-ADP-ribosyltransferase TIPARP (SINGLE PROTEIN), Homo sapiens
UniProt: Q7Z3E1
Assay: Enzymatic Assay: Displacement of Probe A, a biotinylated probe binding to the TIPARP active site, was measured using a time-resolved fluorescence energy transfer (TR-FRET) assay. 20 nL of a dose response curve of each test compound was spotted in black 384-well polystyrene proxiplates (Perkin Elmer) using a Mosquito (TTP Labtech). Reactions were performed in a 8 μL volume by adding 6 JAL of TIPARP and Probe A in assay buffer (20 mM HEPES pH=8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002% Tween20), incubating with test compound at 25° C. for 30 min, then adding 2 μL of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin (Perkin Elmer). The final concentrations of TIPARP and Probe A were 6 nM and 2 nM, respectively. The final concentration of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin were 4 nM and 0.25 nM, respectively. Reactions were incubated at 25° C. for an additional 30 min, then read on an Envision plate reader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 320 nm and emission of 615 nm and 665 nM with a 90 μs delay. The ratio of the 665/615 nm emission were calculated for each well to determine the amount of complex of TIPARP and Probe A in each well.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 513.48, ALogP 2.51, HBA 8, HBD 2, PSA 127.24, QED 0.46
Compound (SMILES): CNc1cc(Nc2cccn([C@@H]3CCC[C@H](C#N)C3)c2=O)nc2c(C(=O)N[C@@H]3CC[C@H]3OC)cnn12
InChIKey: UMIPVBFTHQXQOE-IXIUHKBRSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: TYK2 JH2 Domain Binding Assay: The binding reaction was assembled by combining 16 μl of DNA-tagged kinase extract, 3.8 μl liganded affinity beads, and 0.18 μl test compound (PBS/0.05% Tween 20/10 mM DTT/0.1% BSA/2 μg/ml sonicated salmon sperm DNA)]. Extracts were used directly in binding assays without any enzyme purification steps at a ≥10,000-fold overall stock dilution (final DNA-tagged enzyme concentration <0.1 nM). Extracts were loaded with DNA-tag and diluted into the binding reaction in a two step process. First extracts were diluted 1:100 in 1× binding buffer (PBS/0.05% Tween 20/10 mM DTT/0.1% BSA/2 μg/ml sonicated salmon sperm DNA) containing 10 nM DNA-tag. This dilution was allowed to equilibrate at room temperature for 15 minutes and then subsequently diluted 1:100 in 1× binding buffer. Test compounds were prepared as 111× stocks in 100% DMSO. Kds were determined using an 11-point 3-fold compound dilution series with three DMSO control points. All compounds for Kd measurements are distributed by acoustic transfer (non-contact dispensing) in 100% DMSO
Measurement: Kd < 0.2 nM (pChEMBL 9.699000358581543)
Descriptors: MW 490.57, ALogP 2.84, HBA 10, HBD 3, PSA 138.37, QED 0.46
Compound (SMILES): Fc1ccc(-n2cc(COc3nccc(-c4ccc[nH]4)n3)nn2)cc1OC(F)F
InChIKey: HJUJKOIKBNNFOF-UHFFFAOYSA-N
Target: Retinoic acid receptor RXR-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P19793
Assay: NR2B standard 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 149 mM NaCl, 4 mM KCl, 2 mM CaCl2, and 1.5 mM MgCl2, 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 (149 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 ¿EC80 (standard assay) or EC40 (HTS assay) is used to maximize the assay's signal window or the ability to detect NMDA receptor antagonists and negative allosteric modulators, respectively. 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 = 681 nM (pChEMBL 6.170000076293945)
Descriptors: MW 402.34, ALogP 3.37, HBA 7, HBD 1, PSA 90.74, QED 0.51
Compound (SMILES): C[C@H](NC(=O)c1cc(C#N)cnc1NCc1ccc(-c2cnc3[nH]nc(N)c3c2)cc1)c1ccc(F)cc1
InChIKey: CIOUUENQNOJMOC-INIZCTEOSA-N
Target: 3-phosphoinositide-dependent protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O15530
Assay: PDK1 kinase assay: PDK1 (amino acids 51-359) and AKT2 (amino acids 140-467 fused to PIFtide, amino acids EEQEMFRDFDYIADW) were expressed as N-terminally tagged GST fusion proteins in insect cells and purified to homogeneity. Enzyme activity was determined in a coupled PDK1/AKT/FAM-crosstide assay and phosphorylation of FAM-crosstide was determined by standard IMAP protocol (Molecular Devices). For inhibition studies, compounds were titrated 4-fold in DMSO and diluted 40-fold into assay buffer (10 mM Tris HCl pH7.2; 10 mM MgCl2; 0.01% Triton X-100; 1 mM DTT) containing PDK1, AKT2, and FAM-crosstide (final concentrations: 0.75 nM PDK1, 10 nM unphosphorylated AKT2, and 100 nM crosstide substrate). The kinase reaction was initiated by adding ATP to a final concentration of 40 μM of PDK1 and incubated at 25° C. for 30 min. To detect assay product, the kinase reaction was combined with Progressive Binding Solution (1:600 Progressive Binding Reagent, 50% Buffer A, 50% Buffer B, Molecular Devices) in a 1:3 ratio. The mixture was incubated for 2 hours at 25° C. and the plate was scanned on an Analyst AD with excitation at 485 nm and emission at 530 nm. The fluorescence polarization value P is defined by the equation below. The value mP is generated by multiplying the P value for each reaction well by a factor of 1000. The value ΔmP for each well is the mP value for that well minus the mP value for the average negative control. P=(Fpar−Fperp)/(Fpar+Fperp)  Eq.:Where par is fluorescence intensity parallel to the excitation plane; and perp is fluorescence intensity perpendicular to the excitation plane. ΔmP values were plotted as a function of compound concentration and the data were analyzed with a 4-parameter fit using GraphPad Prism software.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 506.55, ALogP 4.72, HBA 7, HBD 4, PSA 145.4, QED 0.25
Compound (SMILES): CCn1cc(Nc2ncnc(-c3ccc(O[C@H]4CCN(C(=O)CO)C[C@H]4F)c(C#N)c3)n2)c(C)n1
InChIKey: ORHHPZBLKIDTBX-XLIONFOSSA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: TBK1 and IKKE Biological Assays: Enzymatic activity of IKKε and TBK1 was measured using a homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay that monitors enzyme dependent phosphorylation of a biotinylated serine/threonine peptide substrate. An increase in the amount of phosphorylated peptide results in an increase in TR-FRET signal. TBK1 and IKKε were expressed and purified as full length recombinant proteins. Detection reagents for the assay were purchased from Cisbio. TBK1 and IKKε enzymes were assayed under initial rate conditions in the presence of 2×Km ATP (40-80 μM) and 1 μM peptide, hepes (pH 7), 0.1 mM orthovanadate, 0.02% NaN3, 0.01% BSA, 10 mM MgCl2, 0.01% (v/v) tritonX, 1 mM dithiothreitol, 0.5% (v/v) DMSO at the following concentrations for each enzyme: TBK1 at 2.5 nM and IKKε at 0.3 nM. After an assay reaction time of 240 minutes at 25° C., reactions were terminated with EDTA.Amount of phosphorylated peptide was determined by the addition of 125 nM streptavidin XL665 and europium cryptate labeled anti-phospho monoclonal antibody and the resulting TR-FRET signal was recorded on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 200 μs read window). Data was normalized based on a positive (1 μM Staurosporine) and negative (DMSO) controls and IC50 values calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations.
Measurement: IC50 = 4000 nM (pChEMBL 5.400000095367432)
Descriptors: MW 480.5, ALogP 1.99, HBA 10, HBD 2, PSA 142.08, QED 0.52
Compound (SMILES): COc1cc2c(Oc3ccc(NC(=O)Nc4cc(C)ccc4F)c(Cl)c3)ccnc2cc1OCCCN1CCC2(CC1)COC2
InChIKey: LPBMTIMIYKRLLN-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: VEGFR2 Binding Assay: A competition binding assay (DISCOVERX KINOMESCAN) was used to measure the ability of a compound to compete for binding of an immobilized adenosine triphosphosphate (ATP) site directed ligand using a DNA-tagged vascular endothelial growth receptor 2 (VEGFR2) as the target. The ability of the test compound to compete with the immobilized ligand was measured using quantitative polymerase chain reaction (qPCR) of the DNA tag (Fabian, M. A. et al., 23 Nature Biotechnology 329-336 (2005); Karaman, M. W. et al., 26 Nature Biotechnology 127-132 (2008)).A VEGFR2 tagged T7 phage strain was prepared in an Escherichia coli (E. coli) derived from the BL21 strain. The E. coli were grown to log-phase, infected with VEGFR2 tagged T7 phage and then incubated with shaking at 32° C. until lysis. The lysate containing the kinase was then centrifuged and filtered to remove cell debris. Affinity resin for the VEGFR2 assay was prepared by treating Streptavidin-coated magnetic beads with a biotinylated small molecule ligand for 30 minutes at room temperature. The beads were blocked with excess biotin and then washed with blocking buffer (SEABLOCK (PIERCE), 1% bovine serum albumin, 0.17% phosphate buffered saline, 0.05% TWEEN 20, 6 mM dithiothreitol). The binding reaction was initiated by combining in a well of a polystyrene 96-well plate, DNA tagged VEGFR2, liganded affinity beads and the serial diluted test compound in 1× binding buffer (20% SEABLOCK, 0.17× phosphate buffered saline, 0.05% TWEEN 20, 6 mM dithiothreitol) in a final volume of 0.135 ml. The assay plates were incubated at room temperature with shaking for 1 hour and then the beads were washed with wash buffer (1× phosphate buffered saline, 0.05% TWEEN 20). The beads were re-suspended in elution buffer (1× phosphate buffered saline, 0.05% TWEEN 20, 0.05 μM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The VEGFR2 concentration in the eluate was measured using qPCR.An 11-point dose response curve of 3-fold serial diluted test compound starting at 1 μM was used to determine the VEGFR2 binding constant (Kd). The compounds were prepared in 100% DMSO at 100× the final test concentration and the diluted to 1× in the assay for final DMSO concentration of 1%. Binding constants were calculated with standard dose-response curve using the Hill equation with Hill slope set to −1. Curves were fit using a non-linear least square fit with the Levenberg-Marquardt algorithm.
Measurement: Kd = 4800 nM (pChEMBL 5.320000171661377)
Descriptors: MW 635.14, ALogP 7.66, HBA 7, HBD 2, PSA 94.18, QED 0.17
Compound (SMILES): Cc1cc(OCCCc2c3n(c4c(-c5c(C)nc(CN(C)CCN(C)C)nc5C)c(Cl)ccc24)[C@H](C)CN(c2cn(C)c4ccc(C(=O)O)cc24)C3=O)cc(C)c1Cl
InChIKey: PIGNMPCWVIGDNH-HHHXNRCGSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07820
Assay: Fluorescence Polarization Anisotropy Competition Assay: Fluorescence Polarization Anisotropy Competition Assay. Anisotropy measurements were carried out in 384-well, black, flat-bottom plates (Greiner Bio-one, Monroe, N.C., USA). The assay was run using either a fluorescein isothiocyanate-labeled BH3 peptide derived from Bak (FITC-AHx-GQVGRQLAIIGDDINR-NH2) or a fluorescein isothiocyanate-labeled BH3 peptide derived from Bim (FITC-AHx-EARIAQELRRIGDEFNETYTR-NH2)that were purchased from GenScript (Piscataway, N.J.) at >95% purity and used without further purification. 10 nM FITC-Bak peptide and 15 nM recombinant Mcl-1 (residues 172-327) were added to assay buffer (3 mM dithiothreitol, 50 mM NaCl, 20 mM Tris, pH 7.5). The Bim based assay was run with 1 nM FITC-Bim peptide and 1.5 nM recombinant Mcl-1 (residues 172-327) added to assay buffer (20 mM TRIS pH 7.5, 50 mM NaCl, 3 mM DTT, 0.01% CHAPS). For selectivity assays, 40 nM Bcl-2 (residues 1-207 A96T,G110R, Δ35-91, replaced with Bcl-xL35-50) or 4 nM Bcl-xL (residues 1-209, loop 45-86 deleted) were incubated with 10 nM FITC-Bak in assay buffer. Compounds are diluted in DMSO in a 10-point, 3-fold serial dilution scheme. For the FITC-BAK assay 2.5 uL compound is added to 47.5 μL of assay buffer containing FITC-Bak and protein, for a final DMSO concentration of 5% and a top concentration of 20 μM. A FITC-Bak peptide alone (100% inhibition) and peptide plus protein (0% inhibition) control is included on each assay plate. For the FITC-Bim assay, compound is added to 40 uL of assay buffer containing protein, 15 minutes prior to addition of 10 μL of the FITC-Bim peptide, for a final DMSO concentration of 0.165% and a top concentration of 200 nM. A FITC-Bim peptide alone (100% inhibition) and peptide plus protein (0% inhibition) control is included on each assay plate. The plate was mixed and incubated for 90 minutes at room temperature.
Measurement: Ki = 2.2 nM (pChEMBL 8.65999984741211)
Descriptors: MW 824.85, ALogP 9.06, HBA 9, HBD 1, PSA 108.96, QED 0.12
Compound (SMILES): Cc1ccncc1C(=O)N1CC(Nc2cncc(N3C(=O)c4ccc(Cl)cc4C3(C)C)c2)C1
InChIKey: FNXBNMLWVDZGIA-UHFFFAOYSA-N
Target: Cytochrome P450 11B2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P19099
Assay: Cellular Enzyme Assay: G-402 cells expressing CYP11 constructs were established as described above and maintained in McCoy's 5a Medium Modified, ATCC Catalog No. 30-2007 containing 10% FCS and 400 ug/ml G418 (Geneticin) at 37° C. under an atmosphere of 5% CO2/95% air. Cellular enzyme assays were performed in DMEM/F12 medium containing 2. 5% charcoal treated FCS and appropriate concentration of substrate (0.3-10 uM 11-Deoxycorticosterone, 11-Deoxycortisol or Corticosterone). For assaying enzymatic activity, cells were plated onto 96 well plates and incubated for 16 h. An aliquot of the supernatant is then transferred and analyzed for the concentration of the expected product (Aldosterone for CYP11B2; Cortisol for CYP11B1). The concentrations of these steroids can be determined using HTRF assays from CisBio analyzing either Aldosterone or Cortisol.
Measurement: EC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 461.95, ALogP 4.27, HBA 5, HBD 1, PSA 78.43, QED 0.63