lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCn1cc(-c2cn3ncc(C(N)=O)c(N[C@H]4CCN(c5ccc(C#N)nn5)CC4(C)C)c3n2)cn1
InChIKey: MOJWAWUGEIWENF-IBGZPJMESA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Kinase Assay: The assay reactions 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 (100 mM HEPES pH 7.4, 10 mM MgCl2, 25 mM beta-glycerol phosphate 0.015% Brij35 and 4 mM DTT). The reaction was initiated by the combination of GST-JAK3 enzyme 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 to each sample. Each 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 with no enzyme control reactions for 100% inhibition, and vehicle-only treated reactions for 0% inhibition. The final concentration of reagents in the assay was: ATP, 8 μM; fluoresceinated peptide, 1.5 μM; GST-JAK3, 4.5 nM; and DMSO, 1.6%.
Measurement: IC50 = 0.6 nM (pChEMBL 9.220000267028809)
Descriptors: MW 485.56, ALogP 2.09, HBA 11, HBD 2, PSA 155.94, QED 0.42
Compound (SMILES): Nc1ncc(-c2nc(N3CCOCC3)nc3ccoc23)cn1
InChIKey: IELPEGIEYWZOIT-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Enzymatic Inhibitory Activity Assay:   1. Buffer preparation: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS.  2. Compound was formulated in 100% DMSO in a concentration gradient, deposited to a 384-well plate to make final DMSO concentration of 1%.  3. PI3Kα and PI3Kδ enzymes were diluted to be the optimum concentration with the following buffer: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS, 2 mM DTT. Transferred to a 384-well plate and incubated with the compound for a certain time.  4. Substrate was diluted to an optimum concentration with following buffer: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS, 2 mM DTT, 50 μM PIP2, Km ATP. The reaction was performed in a 384-well plate for 1 h at room temperature for PI3Kα and 2 hrs at room temperature for PI3Kδ.  5. Read the conversion rate using Caliper Reader, and calculate the inhibition rate as the average of two tests.
Measurement: IC50 = 26 nM (pChEMBL 7.579999923706055)
Descriptors: MW 298.31, ALogP 1.1, HBA 8, HBD 1, PSA 103.19, QED 0.75
Compound (SMILES): Cc1cccc(N2C(=O)c3ccccc3C2(O)c2ccc3c(c2)NC(=O)CO3)c1
InChIKey: DZCBRTBEXDTPDC-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Inhibition of c-Raf1 (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 386.41, ALogP 3.18, HBA 4, HBD 2, PSA 78.87, QED 0.71
Compound (SMILES): CN1Cc2c(Cl)cc(Cl)cc2[C@@H](c2ccc(S(=O)(=O)NCCOCCOCCOCCNC(=O)C(O)C(O)C(=O)NCCOCCOCCOCCNS(=O)(=O)c3ccc([C@@H]4CN(C)Cc5c(Cl)cc(Cl)cc54)cc3)cc2)C1
InChIKey: SNTWQTFUQNUEKF-BYZHBIFJSA-N
Target: Sodium/hydrogen exchanger 3 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P26433
Assay: Inhibition of rat NHE-3-mediated Na+ dependent H+ antiport expressed in opossum kidney cells in presence of NHE-1 antagonist EIPA by BCECF-AM dye based fluorescence assay
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 1207.09
Compound (SMILES): CN(C)C1(c2ccccc2)CCC(c2ccccc2)(N(C)C(=O)c2cccc(C(F)(F)F)c2)CC1
InChIKey: UVDKKVMGQDSNHM-UHFFFAOYSA-N
Target: Nociceptin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41146
Assay: Binding Assay: The compounds were examined with membranes of recombinant CHO-ORL 1 cells in a receptor binding assay with 3H-nociceptin/orphanin FQ. This test system was conducted in accordance with the method outlined by Ardati et al. (Mol. Pharmacol., 51, 1997, pp. 816-824). The concentration of 3H-nociceptin/orphanin FQ amounted to 0.5 nM in these tests. The binding assays were conducted in each case on 20 ug of membrane protein per 200ul of preparation in 50 mM of HEPES, pH 7.4, 10 nM of MgCl2 and 1 mM of EDTA. The binding to the ORL 1-receptor was determined using 1 mg of WGA-SPA beads (Amersham-Pharmacia, Freiburg) in each case by incubating the preparation for one hour at RT and then conducting measurements in the Trilux scintillation counter (Wallac, Finland).
Measurement: Ki = 0.54 nM (pChEMBL 9.270000457763672)
Descriptors: MW 480.57, ALogP 6.7, HBA 2, HBD 0, PSA 23.55, QED 0.41
Compound (SMILES): CCCn1c(=O)c2cn[nH]c2c2ccc(-c3ccncc3)cc21
InChIKey: BPLNZOQGPAGRTJ-UHFFFAOYSA-N
Target: Methionine aminopeptidase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P50579
Assay: Enzymatic Screening Assay: The enzymatic test is a test in two steps.In a first step, it consists in placing in contact the compound according to the invention, the dialysed MetAP2 protein and the substrate (Met-Pro-Arg-pNa peptide synthesized by Neosystem), the N-terminal methionine of which can be cleaved with MetAP2, and which bears at the C-terminal end a para-nitroaniline (pNa) chromophore, which can itself be released by another peptidase only when the N-terminal methionine has been cleaved beforehand.Consequently, the second step consists in reacting the peptides cleaved in the preceding step with a second peptidase in order to release the chromophore. The peptidase used in this second step is cathepsin, which comes from the TagZyme DAPase kit (Quiagen, 34366).The MetAP2 activity is proportional to the amount of para-nitroaniline released, which is measured by absorbance at 405 nm.
Measurement: IC50 = 158 nM (pChEMBL 6.800000190734863)
Descriptors: MW 304.35, ALogP 3.35, HBA 4, HBD 1, PSA 63.57, QED 0.63
Compound (SMILES): Cc1ccc(-c2c(N)ncnc2N2CCC(c3nc(-c4ccc(F)c(C(F)(F)F)c4)cn3C)CC2)cc1
InChIKey: HDVJHZYNOODBLA-UHFFFAOYSA-N
Target: Ribosomal protein S6 kinase beta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23443
Assay: P70S6K Enzyme Assay: P70S6K inhibitor compounds are diluted and plated in 96 well plates. A reaction mixture including the following components is then added to the compound plate to initiate the enzyme reaction; P70S6K (3 nM, T412E mutant, Millipore) is mixed with 24 uM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 uM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction is incubated for 90 min at 25.degree. C., before the addition of 10 mM EDTA to stop the reaction. The proportion of substrate and product (phosphorylated) peptide is analysed on a Caliper Life Sciences Lab Chip 3000, using a pressure of -1.4 psi, and upstream and downstream voltages of -3000 and -700 respectively. Product peaks are resolved before substrate peaks on the resulting chromatograms.
Measurement: IC50 = 7.3 nM (pChEMBL 8.140000343322754)
Descriptors: MW 510.54, ALogP 5.98, HBA 6, HBD 1, PSA 72.86, QED 0.34
Compound (SMILES): CCOP(=O)(OCC)c1ccc2cc1CN(C)C(=O)[C@H](Nc1ccc3c(N)nccc3c1)c1ccc(c(C)c1)[C@@H](C)COC(=O)N2
InChIKey: YIXUXJDCYJOAHT-WKRVVKTRSA-N
Target: Coagulation factor VII (SINGLE PROTEIN), Homo sapiens
UniProt: P08709
Assay: In Vitro Assay: FVIIa-Xase Ki (37° C.): S2765 (0.5 mM), PCPS (25 μM), calcium chloride (5 mM), full-length human TF (3 nM), human FVIIa (5 pM), and FVIIa inhibitor were incubated for 15 min at 37° C. Reactions were initiated by the addition of human FX (range of concentrations). Preliminary experiments revealed that the plasma purified FX contains a residual amount of human FVIIa which could not be removed by affinity chromatography. The residual FVIIa is sufficient, when combined with PCPS, calcium and TF to catalyze the conversion of FX to Xa, and was increased by the addition of 5 pM human FVIIa. FXa in turn hydrolyses S2765, which was monitored for 60 min at 405 nm.
Measurement: Ki = 0.62 nM (pChEMBL 9.210000038146973)
Descriptors: MW 645.7, ALogP 6.49, HBA 9, HBD 3, PSA 145.11, QED 0.19
Compound (SMILES): CC1c2nc(NCC(F)F)c(N3CCC(Oc4ccc(F)cc4F)CC3)nc2CCN1S(C)(=O)=O
InChIKey: WWPMMCYXCUAIQH-UHFFFAOYSA-N
Target: G-protein coupled receptor 6 (SINGLE PROTEIN), Homo sapiens
UniProt: P46095
Assay: GPR6 In Vitro Assay (A.1): This cell based assay measures the ability of compounds to inhibit the constitutive cAMP activity of GPR6 receptor expressed in CHO-K1 cells. CHO cells were stably expressed with GPR6 receptor, whose expression is controlled by a tetracycline inducable element. The cells were cultured in medium containing F12K, 10% FBS, 1% PennStrep, 200 ug/mL Hygromycin. GPR6 receptor expression was induced for 20 hrs with 1 ug/ml doxycycline (sigma D9891) in growth media. After addition of doxycycline cells were plated at a density of 250-500 cells per well in half-volume black clear bottom plates (Costar) and place in an incubator (37° C, 5% C(O)2) for 20 hours prior to cAMP assays.Culture media was removed from cells and they were washed with 50 uL of Ringer's Buffer (MgCl2 0.047 mg/mL, NaH2PO4 0.18 mg/mL, Na2HPO4 0.1 mg/mL, KCl 0.34 mg/mL, NaHC(O)3 1.26 mg/mL, D-glucose 1.8 mg/mL, NaCl 7 mg/mL; pH=7.4). Compounds suspended in DMSO were diluted in Ringer's Buffer.
Measurement: IC50 = 0.087 nM (pChEMBL 10.0600004196167)
Descriptors: MW 517.55, ALogP 3.36, HBA 7, HBD 1, PSA 87.66, QED 0.56
Compound (SMILES): CC(=O)OC(C)(C)C(=O)Nc1cnc(Nc2ccc(Oc3ccc(C)nc3)cc2)n(Cc2ccc(Cl)cc2)c1=O
InChIKey: NUIIRMLVEBUJKP-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Inhibition Assay: Stably expressing cell line (C6BU-1 cell transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 in M MgCl2, 5.0 mM CaCl2, 5.6 D-glucose, 2.5 mM probenecid, 1.0% BSA, and 0.08% Pluronic F-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), and each well was added with 40 μL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.). Measurement of fluorescence inte
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 562.03, ALogP 5.46, HBA 9, HBD 2, PSA 124.44, QED 0.26
Compound (SMILES): CCC1CC(=O)NN=C1c1ccc2nc(-c3ccc(/C=C/c4ccccc4)cc3)oc2c1
InChIKey: YTUHUYVCPVMBIL-CMDGGOBGSA-N
Target: Glucose-dependent insulinotropic receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TDV5
Assay: AlphaScreen cAMP Assay: MIN6 cells [Miyazaki J et al. Endocrinology. 1990 July; 127(1):126-32] were stably transfected with an expression vector for human GPR119 cDNA (Acc. No. NP-848566). Min-6/hGPR119 cells are cultured in DMEM, 10% FBS, 50 uM beta-mercaptoethanol, 0.3 mg/mL Geniticin, 2 mM GlutaMAX at 37 C. 5% CO2. For the assay, the cells are placed in Optiplates (white, 384-well, 160W-barcoded, TC, sterile with lid, Cat. No. #6007688 (Perkin Elmer); 10000 cells/well; 50 ul). The plates covered with lids are then incubated for 24 hours at 37 C./5% CO2.After the medium has been sucked out of the wells completely, 10 ul of the test compound are added, the compounds are diluted beforehand with stimulating buffer (140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 10 mM Hepes, 5 mM NaHCO3; pH 7.4. 0.5 mM IBMX and 0.1% BSA, the final DMSO concentration is 1%). After 45 minutes' incubation at RT, the cAMP concentrations are determined using the AlphaScreen cAMP Assay.
Measurement: EC50 = 384 nM (pChEMBL 6.420000076293945)
Descriptors: MW 421.5, ALogP 5.92, HBA 4, HBD 1, PSA 67.49, QED 0.41
Compound (SMILES): Cn1cc(-c2cc3c(-c4cccc(N5CCC(N)CC5)n4)n[nH]c3cn2)cn1
InChIKey: OZNVWRLWXYBLRC-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM Kinase Binding Activity Assay: PIM-1, PIM-2, and PIM-3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.
Measurement: Ki = 0.086 nM (pChEMBL 10.069999694824219)
Descriptors: MW 374.45, ALogP 2.35, HBA 7, HBD 2, PSA 101.54, QED 0.57
Compound (SMILES): C=CC(=O)N1CC[C@H](Oc2cc3c(Nc4ccc(F)c(Cl)c4F)ncnc3cc2OC)C[C@H]1C(=O)NCCOC
InChIKey: HUPLQXTZJKDURX-YWZLYKJASA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of N-terminal GST-tagged recombinant human EGFR T790M mutant expressed in baculovirus infected Sf21 cells using poly (Glu,Tyr)4:1 as substrate preincubated for 10 mins followed by substrate addition measured after 1 hr by fluorescence polarization assay
Measurement: IC50 = 3.8 nM (pChEMBL 8.420000076293945)
Descriptors: MW 576, ALogP 4, HBA 8, HBD 2, PSA 114.91, QED 0.21
Compound (SMILES): Cc1ccc(CN(C)C2CCN(C(=O)Cn3cc(NC(=O)c4cnn5cccnc45)c(-c4cc(Cl)ccc4OC(F)F)n3)CC2)c(C)c1
InChIKey: IOWHKOBSYRHYSB-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 1.14 nM (pChEMBL 8.9399995803833)
Descriptors: MW 677.16, ALogP 5.84, HBA 9, HBD 1, PSA 109.89, QED 0.2
Compound (SMILES): Cc1cn(-c2cc(NC(=O)c3ccc(C)c(NC(=O)c4cccc5c(N)nccc45)c3)cc(C(F)(F)F)c2)cn1
InChIKey: QOXOALUVNITUHC-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Inhibition Assay: The compounds prepared in Examples were tested for inhibitory activities against three subtypes of RAF, i.e. RAF1 Y340D Y341D, BRAF normal type and BRAF V600E, using Kinase Profiling Service (Invitrogen) according to the manufacturer's instructions. The levels of the inhibitory activities of the compounds against the enzymes were calculated as % inhibitory activities at various concentrations. Based on the % inhibitory activities, dose-response curves were plotted and IC50 values were calculated using GraphPad Prism software.
Measurement: IC50 = 94.8 nM (pChEMBL 7.019999980926514)
Descriptors: MW 544.54, ALogP 6.14, HBA 6, HBD 3, PSA 114.93, QED 0.25
Compound (SMILES): CCNc1nc(Nc2cc(C)c(S(C)(=O)=O)cc2OC)ncc1C(F)(F)F
InChIKey: QHUPHGLWVVOXED-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: In vitro LRRK2 Lanthascreen Binding Assay: This assay was used to determine a compound's potency in inhibiting activity of LRRK2 by determining, Kiapp, IC50, or percent inhibition values. In 384 well proxiplates F black, shallow well plates LRRK2, Eu-anti-GST-antibody, Alexa Fluor Kinase tracer 236 and test compound were incubated together.Binding of the Alexa Fluor "tracer" to a kinase was detected by addition of a Eu-labeled anti-GST antibody. Binding of the tracer and antibody to a kinase results in a high degree of FRET, whereas displacement of the tracer with a kinase inhibitor results in a loss of FRET.Assay conditions and materials used were as follows: Final Assay Conditions:GST-LRRK2 G2019S 10 nM; Eu-anti-GST-antibody 2 nMKinase tracer 236 8.5 nM; Kinase reaction time: 1 hour; Temperature: ambient; Total volume: 15 ul; DMSO: 1%; Materials: 384 well proxiplates F black shallow well Perkin Elmer cat#6008260Kinase: LRRK2 G2019S Invitrogen cat #PV4882(LOT 567054A). Eu-labeled anti-GST antibody Invitrogen cat #PV5594Alexa Fluor.
Measurement: Ki = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 404.41, ALogP 3.39, HBA 7, HBD 2, PSA 93.21, QED 0.76
Compound (SMILES): COc1ccc(CC(=O)O)cc1-c1ccc(F)c2c1CN(C(=O)CCc1ccccc1)CC2
InChIKey: FEJLIRCYWJSLGQ-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Radioligand Displacement Assay: Binding assay was performed in a final assay volume of 250 ul. First, 25 ul of test compound, previously diluted in Binding-Buffer (Binding-Buffer: 50 mM Tris-Base, 100 mM NaCl, 1 mM EDTA, 0.1% BSA (protease free), 0.01% NaN3, 10 mM MnCl2, pH 7.0) was placed into each well. After addition of 75 ul Binding-Buffer, 50 ul of the radioligand 3H-PGD2 (at 2.5 nM (220.000 dpm/well) from ANAWA ART0662) was added to each well. Binding assay was started by addition of 100 ul CRTH2 membrane fragments, reaching a final concentration of 20 ug/well. For non-specific binding, PGD2 was added to the reaction mixture to 10 mM final concentration. This assay mix was incubated for 90 minutes at room temperature and then filtered through a GF/C filter 96-well plate which was pre-soaked for 3 hours in 0.5% polyethyleneimine (PEI). The filter-wells were washed three times with ice cold Binding-Buffer. Then, 40 ul of Microscint-40 (Packard) was added to each well and the retained radioactivity quantified in a Topcount (Packard).
Measurement: IC50 = 5.83 nM (pChEMBL 8.229999542236328)
Descriptors: MW 447.51, ALogP 4.65, HBA 3, HBD 1, PSA 66.84, QED 0.57
Compound (SMILES): CNC(=O)c1cccc(Nc2ncnc(N3CCN(c4ccc(F)cc4)CC3)n2)c1
InChIKey: ICGQDCDSIPCJMF-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Cell-Based Assay: Compounds were tested on human Nav1.7 (hNav1.7) expressed stably in 293 cells, which have no endogenous sodium channels. Two forms of whole-cell patch-clamp assays were used. In both, peak current through Nav1.7 was evoked by a depolarizing voltage pulse, and currents were monitored as a function of time in response to exposure to several different concentrations of drug. Automated electrophysiology uses the PatchXpress 7000A system. Up to sixteen cells at a time were voltage-clamped in parallel, and the steady-state voltage dependence of inactivation was determined experimentally for each cell. For each cell, fractional inactivation was set at 20%, and peak current through Nav1.7 as a function of time was measured in response to increasing concentrations of test compound. Manual electrophysiology refers to conventional whole-cell patch-clamping (Hamill et al., 1981). After formation of the whole-cell clamp, cells were lifted off the bottom of the dish with the patch pipette and positioned directly in front of an array of glass microperfusion tubes each with internal diameter about 250 microns, with individual compound concentrations flowing from each tube. Current as a function of time in response to increasing drug concentrations was measured with Nav1.7 fully in the resting state (holding voltage set to −140 mV) and on the same cells with the holding voltage set to produce about 20% inactivation. Compounds were not applied for a prespecified time, but for as long as required to achieve a stable level of block.
Measurement: IC50 = 6830 nM (pChEMBL 5.170000076293945)
Descriptors: MW 407.45, ALogP 2.44, HBA 7, HBD 2, PSA 86.28, QED 0.67
Compound (SMILES): O=C(Nc1cn(CC(=O)N2C[C@H]3CN(CCCN4C(=O)Cc5ccccc54)C[C@H]3C2)nc1-c1cc(Cl)ccc1OC(F)F)c1cnn2cccnc12
InChIKey: LBLCQZGEHUNKCK-PSWAGMNNSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 0.76 nM (pChEMBL 9.119999885559082)
Descriptors: MW 730.18, ALogP 4.47, HBA 10, HBD 1, PSA 130.2, QED 0.21
Compound (SMILES): CCCCN(Cc1ccc(OCCN2C(=O)CCC2=O)c(CC)c1)C(CC(=O)O)c1ccc(Cl)c(Cl)c1
InChIKey: NPRWPPHHOHQWNK-UHFFFAOYSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: Radioligand Binding Assay: The composition of the binding assay buffer is determined in a course of detailed optimization procedure. This resulted in a binding assay buffer constituted by the following components: 25 mM Hepes (pH=7.4), 5 mM MgCl2, 1 mM CaCl2, 100 mM NaCl, supplemented with 0.1% of protease free BSA (as a final concentration). Competition binding assay is performed using 125I-CXCL10 (PerkinElmer, NEX348, specific activity 2200 Ci/mmol) radioligand in a final concentration of 50-70 pM. The nonspecific binding is defined by 150 pM of hr-CXCL10 (R&D Systems, Cat No 266-IP). The total assay volume is equal to 150 ul and contained 1% of DMSO (final concentration). Binding reaction is initiated by adding of membranes (10-20 ug proteins, approximately 5x105 cell equivalents) to the reaction mixture. After 60 minutes of incubation at 25° C. the reaction is terminated by rapid filtration over GF/B glass fibre filters that are pre-soaked with 0.5% polyethyleneimine (Fluka Analytical, P3143) for 1 hour, using a Skatron cell harvester device. Filters then are washed with 8 ml of ice-cold wash buffer (modified binding buffer in which BSA is omitted and the concentration of NaCl is adjusted to 500 mM concentration). The radioactivity retained on the filters is measured by a Wizard 1470 Automatic Gamma counter.
Measurement: IC50 = 120 nM (pChEMBL 6.920000076293945)
Descriptors: MW 549.5, ALogP 5.9, HBA 5, HBD 1, PSA 87.15, QED 0.3
Compound (SMILES): CC1(C(=O)N[C@H]2CC[C@H](Nc3cc(NC4CC4)n4ncc(/C=C5\NC(=O)NC5=O)c4n3)CC2)CC1
InChIKey: RWPZDIBQLDPCCB-HKBWDPEPSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 75 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and the resid
Measurement: IC50 > 2500 nM (pChEMBL 5.6020002365112305)
Descriptors: MW 478.56, ALogP 2.12, HBA 8, HBD 5, PSA 141.55, QED 0.3
Compound (SMILES): Cc1nsc(-c2nnc3n2CCN(C(=O)c2ccc(F)c(F)c2)C3C)n1
InChIKey: VRVGLEWHAKZMDU-UHFFFAOYSA-N
Target: Neuromedin-K receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P29371
Assay: Functional Assay: Chinese Hamster Ovary recombinant cells expressing the human NK-3 receptor and a construct that encodes the photoprotein apoaequorin were used for this assay. The antagonist activity of compounds of the invention is measured following pre-incubation (3 minutes) of the compound (at various concentrations) with the cells, followed by addition of the reference agonist (NKA) at a final concentration equivalent to the EC80 (3 nM) and recording of emitted light (FDSS 6000 Hamamatsu) over the subsequent 90-second period. The intensity of the emitted light is integrated using the reader software. Compound antagonist activity is measured based on the concentration-dependent inhibition of the luminescence response to the addition of Neurokinin A.
Measurement: IC50 = 44 nM (pChEMBL 7.360000133514404)
Descriptors: MW 376.39, ALogP 2.6, HBA 7, HBD 0, PSA 76.8, QED 0.69
Compound (SMILES): COc1cccc2c(C(=O)NCCCCCC(=O)Nc3ccccc3N)c[nH]c12
InChIKey: SXQSGBIZTDJPQF-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: HDAC enzyme inhibitionAssay: HDAC enzyme inhibition assays were performed using purified HDACs 1-10 essentially as described in Beckers et al., 2007, Int. J. Cancer., 121:1138-48 and Perez-Balado et al., 2007, J. Med. Chem., 50:2497-2505. Inhibition assays using nuclear extract were performed essentially as described in Herman et al., 2006, Nat. Chem. Biol., 2:551-558. Briefly, the purified HDACs or nuclear extract were incubated with an acetylated substrate in the absence of the compound to be assayed and with increasing concentrations of the compound. The rate of substrate deacetylation was measured under each condition, and half-maximal inhibitory concentration with regard to each HDAC was determined by standard means.
Measurement: IC50 = 196 nM (pChEMBL 6.710000038146973)
Descriptors: MW 394.48, ALogP 3.69, HBA 4, HBD 4, PSA 109.24, QED 0.33
Compound (SMILES): COC1CCC2(CC1)Cc1ccc(-c3cc(F)cc(Cl)c3)cc1C21C=C(N)N(C)C1=O
InChIKey: OCHBPHCOGAPRRE-UHFFFAOYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE Enzyme Assay (Ex.434): Inhibitory activity of compounds was assessed by a fluorescence quench assay of BACE activity using commercially available substrate HiLyte Fluor 488-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys-(QXLT 520)-OH (AnaSpec, San Jose, Calif.) and truncated human beta-secretase (residues 1-458, His6-tagged at the C-terminus) expressed in insect cells D. melanogaster S2 using a baculovirus expression system (Mallender et al., Characterization of recombinant, soluble beta-secretase from an insect cell expression system., Mol Pharmacol 59:619-26, 2001). The assay was performed at room temperature in 96-well white opaque Optiplates aque Optiplates (PerkinElmer, Waltham, Mass.) in a total volume of 200 ul of the incubation mixture containing 50 mM sodium acetate buffer, pH 4.5, 0.4 uM FRET substrate, 2.4 nM enzyme, 5% DMSO, and 0.05% Brij-35. The tested compounds were serially diluted in DMSO and pre-incubated with the substrate. The reaction was started by addition of enzyme.
Measurement: IC50 = 11.1 nM (pChEMBL 7.960000038146973)
Descriptors: MW 440.95, ALogP 4.79, HBA 3, HBD 1, PSA 55.56, QED 0.73
Compound (SMILES): CC(=O)C1=C(C)N(c2cccc(C(F)(F)F)c2)C(=O)N(C)C1c1ccc(C#N)cc1-c1nnc(C)o1
InChIKey: QUCHLKNWWAXJJS-UHFFFAOYSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Human Neutrophil Elastase Assay: Materials: Human neutrophil elastase was purchased from Calbiochem (Cat. No.: 324681) and the elastase substrate MeOSuc-Ala-Ala-Pro-Val-AMC from Bachem (Cat. No.: I-1270). All other materials were of the highest grade commercially available.The following buffers were used: Compound buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5; Assay buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5, containing 0.01% BSA. Assay conditions: Test compounds were prediluted in DMSO and subsequently in compound buffer (5% DMSO final). 5 uL, of these compound dilutions were mixed with 10 uL Neutrophil elastase (9 ng/ml in assay buffer) in a black 384 well OptiPlate (Perkin Elmer, Cat No.: 6007270) and incubated for 15 min at room temperature. Subsequently 10 uL, substrate solution in assay buffer were added (250 uM final concentration) and the plates were incubated for 60 min at room temperature. After inactivation of the enzyme, fluorescence intensities were measured at 380 nm excitation and 460 nm emission wavelengths. Each plate contains wells with a high value control (DMSO+enzyme+substrate) and wells with a low value control (DMSO+inactivated enzyme+substrate). IC50 values were estimated using a sigmoidal concentration response curve with variable slope.
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 495.46, ALogP 5.41, HBA 6, HBD 0, PSA 103.33, QED 0.48
Compound (SMILES): CC(=O)NCc1ccc(-c2cccc(CNC(=O)C3=C(O)C(=O)N(C)C3)c2)cc1
InChIKey: XQNOQQSTRWQFMS-UHFFFAOYSA-N
Target: Endothelial lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5X9
Assay: Enzymatic Assay: Endothelial lipase activity was measured using a fluorescent substrate, A10070, (Invitrogen, CA) doped into an artificial vesicle containing DMPG (Avanti Polar Lipids) as the excipient. Vesicles were prepared by combining 285 uL of 1 mMDMPG in a 1:1 mixture of MeOH and CHCl3 with 15 uL of 1 mM A10070 in a 1:1 mixture of MeOH and CHCl3. The mixture was dried under nitrogen and resuspended in 150 uL of 50 mM HEPES pH 8.0 buffer containing 100 mM NaCl and 0.2 mM EDTA. The sample was allowed to sit at rt for 15 min and then was sonicated 3x4 mins on ice with a Branson Sonicator using duty cycle 1. This preparation provides vesicles with a mole fraction of 0.05 for the FRET substrate.The enzymatic assay was measured using white, opaque 96-well half area plates. Each well contained 60 uL of assay buffer (50 mM HEPES pH 8.0, 50 mM NaCl and 1 mM CaCl2) and 2 ul of a DMSO solution containing compound of interest. Conditioned media obtained from HT-1080 cells, which were transformed by RAGE technology (Athersys) to overexpress endogenous EL, was added and the reaction was allowed to incubate for 20 min at 37deg; C. with gentle agitation. The reaction was started by the addition of 20 uL of a 1:4 dilution of vesicles. The final total reaction volume was 100 uL. The reaction rates were measured on a Gemini plate reader with an excitation wavelength of 488 nm and a emission of 530 nm. Readings were taken every 20 seconds for 10 min with agitation between each reading. The slope of the linear portion of the readout was used to calculate the rate of the reaction.
Measurement: IC50 = 241.3 nM (pChEMBL 6.619999885559082)
Descriptors: MW 393.44, ALogP 1.89, HBA 4, HBD 3, PSA 98.74, QED 0.7
Compound (SMILES): C[C@H]1OC(=O)C2CC(F)(F)[C@@H](C)[C@H](/C=C/c3ccc(-c4ccccc4Cl)cn3)C21
InChIKey: WGXLZMPOJPJRDZ-WLUPFFAXSA-N
Target: Proteinase-activated receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P25116
Assay: FLIPR Assay: HEK 293 Cells were grown in media containing DMEM, 10% FBS pen/strep/L-Glutamine and non-essential amino acids. The cells were plated in 384-well PDL coated plates at 12000 cells/well and incubated overnight at 37° C./5% CO2. Media was then removed from the cells, which were then incubated with buffer (Hank's containing HEPES and Chaps) containing FLIPR calcium-5 dye, made with buffer containing probenecid, for 60 minutes at 37° C. Varying concentrations of compound in a final concentration of 5% DMSO were then added to the cells and incubated at 25° C. for 30 minutes. The plates were then added to the FLIPR Tetra and the device added a concentration of a PAR1 selective receptor-activating peptide with the sequence Ala-parafluoroPhe-Arg-Cha-Cit-Try-Nh2 (prepared in water) at a concentration equal to the effective concentration that achieves 80% activation of signaling on the day of the experiment.
Measurement: IC50 = 2.7 nM (pChEMBL 8.569999694824219)
Descriptors: MW 417.88, ALogP 5.88, HBA 3, HBD 0, PSA 39.19, QED 0.58
Compound (SMILES): Cc1ccn2nc([C@H](C)Nc3ncnc4[nH]cc(-c5cc(NS(C)(=O)=O)cc(C(=O)N(C)C)c5)c34)n(-c3ccccc3)c(=O)c12
InChIKey: UGSKCSOMFIYKJY-IBGZPJMESA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: HTRF Assay : PI-3 Kinase HTRF kit (ref. #33-037) and the different PI3K recombinant isoforms (ref. #14-602, ref. #14-603, ref. #14-604, ref. #15-558 for Alpha, Beta, Delta and Gamma respectively) were purchased at Millipore (expressed in insect cells). ATP was purchased at Sigma Aldrich (ref. #A7699).The compounds were pre-incubated with the enzyme for 30 min before starting of the catalytic reaction. [PIP2] was used at its Km. [ATP] was used at 15 μM for all isoforms for technical reasons (Km values varied between 10 and 20 μM depending on the isoform). Time of assay and [Enzyme] were optimized to work in the linear range. Stop and Detection mixtures were used as specified in the Millipore PI-3 Kinase kit.
Measurement: IC50 = 0.3 nM (pChEMBL 9.520000457763672)
Descriptors: MW 625.72, ALogP 3.98, HBA 10, HBD 3, PSA 159.38, QED 0.23
Compound (SMILES): [C-]#[N+]c1cccc(-c2ccc3c(c2)C2(CCSC(N)=N2)C2(CCc4ccccc4CC2)C3)c1
InChIKey: OBUDUTOJOHTGQA-UHFFFAOYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE Enzyme Assay (Ex.435): For each compound being tested, the BACE activity was monitored in a fluorescence quenching assay (FRET) using the ectodomain of BACE (aa 1-454) fused to a myc-his tag and secreted from HEK293/BACEect. cells into OptiMEM (Invitrogen) as enzyme source and a substrate peptide derived from the APP-Swedish mutation which possesses a Cy3-fluorophore at the N-terminus and a Cy5Q-quencher at the C-terminus (Cy3-SEVNLDAEFK-Cy5Q-NH2; Amersham). The substrate was dissolved at 1 mg/mL in DMSO.The assay was performed in the presence of 5 ul OptiMEM (supernatant collected over 24 hours and cleared from cellular debris by centrifugation) containing the ectodomain of BACE, 25 ul water containing the desired concentration of test compound and 1% DMSO, substrate peptide, 20 mM NaOAc, pH 4.4 and 0.04% Triton-X100 in a total assay volume of 50 ul in a 384 well plate. In general, 25 ul of compound dilution were given to the plate followed by the addition of 10 ul of BACE.
Measurement: IC50 = 1166.5 nM (pChEMBL 5.929999828338623)
Descriptors: MW 449.62, ALogP 6.67, HBA 3, HBD 1, PSA 42.74, QED 0.42
Compound (SMILES): O=S1(=O)Nc2c(cc(Cl)c3cccnc23)-c2cc(N3CCOCC3)ccc21
InChIKey: ARYICEGDNNUTHB-UHFFFAOYSA-N
Target: Ferroportin (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NP59
Assay: Antagonist activity at C-terminal halo-tag fused human ferroportin expressed in MDCK cells assessed as inhibition of hepcidin-induced ferroportin internalization after overnight incubation by Draq5 staining-based fluorescence assay
Measurement: IC50 = 1620 nM (pChEMBL 5.789999961853027)
Descriptors: MW 401.88, ALogP 3.51, HBA 5, HBD 1, PSA 71.53, QED 0.68
Compound (SMILES): CC1=C(C(=O)Nc2ccc(Cl)cc2)C(c2ccccc2Br)N=C(Nc2nc3ccccc3o2)N1
InChIKey: HEJCBLGVJCIGGQ-UHFFFAOYSA-N
Target: Galactokinase (SINGLE PROTEIN), Homo sapiens
UniProt: P51570
Assay: qHTS Assay: Assay details and protocol: The primary assay monitored ATP depletion using Promega's KinaseGlo technology, where ATP levels are measured through luminescence generated from firefly luciferase, a bioluminescent ATP-dependent enzymes. ATP was held at 35 μM, near its reported KM value, and the KM for galactose was determined under the 1536-well assay conditions to be 50-100M (FIG. 1A). As well, the IC50 for a commercially available CD45 inhibitor (N-(9,10-dioxo-9,10-dihydrophenanthren-2-yl)pivalamide) was confirmed (previously found to inhibit GALK (FIG. 1B)). This was used as the positive control for the assay. The assay used 5 nM GALK and a 1 hr incubation time, which gave sufficient signal:background and stability for the HTS.
Measurement: IC50 = 5168.2 nM (pChEMBL 5.289999961853027)
Descriptors: MW 536.82, ALogP 6.27, HBA 6, HBD 3, PSA 91.55, QED 0.29
Compound (SMILES): Brc1cccc(Oc2ccc3c(/C=C/c4ccccc4)n[nH]c3c2)c1
InChIKey: JULWISUJCDSSAO-FMIVXFBMSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of unphosphorylated human 50 residues truncated cytoplasmic domain of VEGFR2 E990V mutant expressed in baculovirus/insect expression system assessed as reduction in NADH oxidation using poly(E4Y1) as substrate by pyruvate kinase/lactate dehydrogenase coupled assay
Measurement: Ki = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 391.27, ALogP 6.29, HBA 2, HBD 1, PSA 37.91, QED 0.44
Compound (SMILES): CCN1CCC(CCOc2ccc(-c3nc(C#N)nc4c3ccn4C)cc2C(F)(F)F)C1
InChIKey: JEYNWVWDGPTOHT-UHFFFAOYSA-N
Target: Cathepsin S (SINGLE PROTEIN), Homo sapiens
UniProt: P25774
Assay: Inhibition Assay: 5 μL of a human cathepsin S enzyme (R&D1183-CY-010) was added to a 96-well plate so as to be 20 ng/well. Next, 10-fold dilution of the test compound (a 10 mM DMSO solution) was serially performed with a buffer solution for assay (a solution of 50 mM sodium methoxide, 250 mM sodium chloride and 5 mM dithiothreitol (DTT), adjusted to pH=4.5 with 1 N hydrochloric acid) six times such that the final concentration becomes 0.1 nM to 10 μM then, 20 μL of each of the solutions was added to the wells (the final DMSO concentration was 0.1%), and 25 μL of synthetic substrate VVR-AMC (3211-V manufactured by PEPTIDE INSTITUTE, INC.) was added thereto such that the final concentration becomes 40 μM, thereby starting a enzyme reaction. A fluorescence intensity (an excitation wavelength: 380 nm and a fluorescent wavelength: 460 nm) was measured at 37° C. for 10 minutes immediately after the start of the reaction using a fluorescence spectrophotometer (SPECTRA MAX GEMINI, manu
Measurement: IC50 = 2.1 nM (pChEMBL 8.680000305175781)
Descriptors: MW 443.47, ALogP 4.64, HBA 6, HBD 0, PSA 66.97, QED 0.56
Compound (SMILES): CCOc1cccc(F)c1C1CC(C)C(=O)N1Cc1cnc2ccccc2c1
InChIKey: VPRUQLQARPYIHN-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: In Vitro Assay: Intracellular Calcium Measurements : In Vitro Assay: Intracellular Calcium Measurements:Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 g/ml G418, 100 U/ml penicillin, 100 g/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2. Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH:water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 1640 nM (pChEMBL 5.789999961853027)
Descriptors: MW 378.45, ALogP 4.88, HBA 3, HBD 0, PSA 42.43, QED 0.64
Compound (SMILES): c1cnc2c(c1)CCC[C@@H]2N(CCCN1CCNCC1)Cc1nccc2c3ccccc3n(CCN3CCOCC3)c12
InChIKey: GXPPYEGXOSWLLT-YTTGMZPUSA-N
Target: C-X-C chemokine receptor type 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P61073
Assay: Calcium Mobilization Assay: Functional modulation of CXCR4 was determined by calcium mobilization assay using leukemic lymphoid CEM cells, which naturally express high levels of CXCR4. Generally, the assay is carried out as follows: Cells are grown in vitro to confluency. On the day of the assay, cells are removed from the incubator, culture medium removed and replaced with a calcium sensitive dye (Calcium3 assay kit; Molecular Devices, Sunnyvale, Calif.). The cells are allowed to dye load for 45-60 min at 37 °C. in the cell culture incubator and then allowed to equilibrate to room temperature for no more than 15 min before assay. Compound plates were generated containing up to 3% dimethyl sulfoxide in media kept at room temperature. Test compounds were added to the cells at a 1:3 dilution, and calcium mobilization was measured using a Flex Station fluorescence imager (Molecular Devices). This first read was used to determine direct agonist activation of the CXCR4 receptor by the test compounds.
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 567.78, ALogP 4.09, HBA 8, HBD 1, PSA 61.69, QED 0.31
Compound (SMILES): CN(C)C(=O)Oc1cccc(/C=C/C(=O)c2ccc(O)cc2)c1
InChIKey: CDYZIDMUJMIFTH-IZZDOVSWSA-N
Target: Cholinesterase (SINGLE PROTEIN), Homo sapiens
UniProt: P06276
Assay: Inhibition of human serum BChE using S-Butyrylthiocholine iodide as substrate preincubated for 15 mins followed by substrate addition by Ellman's method
Measurement: IC50 = 2190 nM (pChEMBL 5.659999847412109)
Descriptors: MW 311.34, ALogP 3.35, HBA 4, HBD 1, PSA 66.84, QED 0.69
Compound (SMILES): O=C(N[C@H]1CCC[C@@H]1Nc1ncc(C(F)(F)F)nc1C1CC1)c1ncccc1-c1ncccn1
InChIKey: HMBQAXPSSHQUTP-HOTGVXAUSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Fluorescent Imaging Plate Reader (FLIPR) Assay: Orexin antagonist activity was determined by measuring changes in intracellular calcium levels using a Ca2+ sensitive fluorescent dye. The changes in fluorescent signal were monitored by Fluorescent Imaging Plate Reader (FLIPR®) technology available from Molecular Devices, LLC, U.S.A. Orexin mediated increases in intracellular Ca2+ concentration were readily detected upon activation with orexin-A. Twenty-four hours prior to the assay, RBL-2H3 cells stably expressing either human orexin receptor 1 or human orexin receptor 2 were seeded in cell culture medium in black, clear-bottom 384-well plates (commercially available from Corning Inc., U.S.A.) and grown overnight at 37 °C., 5% CO2. On the day of the assay, cell culture media was removed and cells were loaded with Calcium 5 Dye (commercially sold by Molecular Devices, LLC, U.S.A.) for 1 hour at 37 °C., 5% CO2.
Measurement: IC50 = 49 nM (pChEMBL 7.309999942779541)
Descriptors: MW 469.47, ALogP 3.99, HBA 7, HBD 2, PSA 105.58, QED 0.56
Compound (SMILES): C[C@H](Nc1nc(Cl)cc(N2C(=O)OCC2[C@@H](C)O)n1)c1cn(-c2ccc(Cl)cc2)cn1
InChIKey: OYVVZWSDLVZFTI-QXNREYOVSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Fluorescence Biochemical Assay: The IDH1 (R132H) mutant catalyzes the reduced form of NADP+ (NADPH) and α-ketoglutarate (α-KG) to form nicotinamide adenine dinucleotide phosphate (NADP+) and R (−)-2-hydroxyglutarate (2HG). The reaction can be monitored kinetically by following the oxidation of NADPH to NADP+ which is measured using fluorescence, excitation at 355 nm and emission at 530 nm. Reactions were monitored using the Perkin-Elmer Envision, Model 2101. More specifically, the biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Cat. No. 781076) using a final reaction volume of 20 μL and the following assay buffer conditions: 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.02% BSA, 0.02% Tween-20, 10 μM NADPH and 100 μM α-KG. The final reaction mixture contained 2.5% DMSO and test compounds with concentrations ranging 0.0000008-25 μM. The IDH1 (R132H) enzyme was used at a final concentration of 10 nM.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 463.33, ALogP 3.85, HBA 8, HBD 2, PSA 105.4, QED 0.54
Compound (SMILES): Cc1cc(-c2n[nH]c3ccc(C(=O)NC4CCCC(Cn5cnc6cccnc65)C4)cc23)ccn1
InChIKey: LNBFSJHSRZITEV-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Mus musculus
UniProt: P63085
Assay: Active ERK2 (aERK) Assay: Activated ERK2 activity was also determined in the IMAP assay format using the procedure outlined above. 1 μl of 25× compound was added to 14 μl of kinase buffer containing 0.25 ng fully phosphorylated, active Mouse ERK2 protein. Following a 30 minute incubation, the reactions were initiated by addition of 10 μl of kinase buffer containing 1 μM ERK2 IMAP substrate peptide (0.9 μM unlabeled IPTTPITTTYFFFK-CONH2 and 100 nM IPTTPITTTYFFFK(5-carboxyfluorescein)-CONH2) and 30 μM ATP. Reactions proceeded for 30 minutes before termination by addition of 60 μl IMAP detection beads in binding buffer. Plates were read as above after 30 minute binding equilibration. Active compounds were reconfirmed in an independent assay.
Measurement: IC50 = 60.8 nM (pChEMBL 7.21999979019165)
Descriptors: MW 465.56, ALogP 4.67, HBA 6, HBD 2, PSA 101.38, QED 0.39
Compound (SMILES): CCN(C(=O)Oc1ccc(F)cc1)[C@@H]1CN(C(=O)C2CCN(S(C)(=O)=O)CC2)C[C@H]1c1ccc(Cl)c(F)c1
InChIKey: WYAWFWHOVHCJQS-XUZZJYLKSA-N
Target: Neuromedin-K receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P29371
Assay: [3H]SR142801 Competition Binding Assay: hNK3 receptor binding experiment were performed using [3H]SR142801 (Catalog No. TRK1035, specific activity: 74.0 Ci/mmol, Amersham, GE Healthcare UK limited, Buckinghamshire, UK) and membrane isolated from HEK293 cells transiently expressing recombinant human NK3 receptor. After thawing, the membrane homogenates were centrifuged at 48,000×g for 10 mM at 4° C., the pellets were resuspended in the 50 mM Tris-HCl, 4 mM MnCl2, 1 μM phosphoramidon, 0.1% BSA binding buffer at pH 7.4 to a final assay concentration of 5 μg protein/well. For inhibition experiments, membranes were incubated with [3H]SR142801 at a concentration equal to KD value of radioligand and 10 concentrations of the inhibitory compound (0.0003-10 μM) (in a total reaction volume of 500 μl) for 75 min at room temperature (RT). At the end of the incubation, membranes were filtered onto unitfilter (96-well white microplate with bonded GF/C filter preincubated 1 h in 0.3% PEI+0.3% BSA, Packard BioScience, Meriden, Conn.) with a Filtermate 196 harvester (Packard BioScience) and washed 4 times with ice-cold 50 mM Tris-HCl, pH 7.4 buffer. Nonspecific binding was measured in the presence of 10 μM SB222200 for both radioligands. The radioactivity on the filter was counted (5 min) on a Packard Top-count microplate scintillation counter with quenching correction after addition of 45 μl of microscint 40 (Canberra Packard S.A., Zürich, Switzerland) and shaking for 1 h.
Measurement: Ki = 29 nM (pChEMBL 7.539999961853027)
Descriptors: MW 570.06, ALogP 4.11, HBA 5, HBD 0, PSA 87.23, QED 0.52