lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1cc(-c2ccc(F)c(C)c2)c(F)cc1-n1c(=O)ccc2cc(S(=O)(=O)Nc3ncc(F)cn3)ccc21
InChIKey: HTOQYVCXZSLFFN-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Nav1.7 In Vitro PX Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to -10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes.
Measurement: IC50 = 31 nM (pChEMBL 7.510000228881836)
Descriptors: MW 552.53, ALogP 4.98, HBA 7, HBD 1, PSA 103.18, QED 0.32
Compound (SMILES): Cn1cc2c(nc(NC(=O)CCCc3cn(CCCCCCNC(=O)c4ccc(-c5c6ccc(=N)c(S(=O)(=O)O)c-6oc6c(S(=O)(=O)O)c(N)ccc56)c(C(=O)O)c4)nn3)n3nc(-c4ccco4)nc23)n1
InChIKey: XEABBZPOOUCNPW-UHFFFAOYSA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: Radioligand Binding Assays: Radioligand binding assays at hA1, hA2A, and hA3ARs were performed according to the procedures described in Gao, Z. G., et al., Biochem Pharmacol. 2003; 65: 1675-84, using the radiolabeled agonists [3H]R-PIA from Moravek Biochemicals (Brea, Calif.) or [3H]CGS21680 and [125I]I-AB-MECA from PerkinElmer (Waltham, Mass.) for the hA1, hA2AAR, and hA3AR assays, respectively.
Measurement: Ki = 90 nM (pChEMBL 7.050000190734863)
Descriptors: MW 1008.02
Compound (SMILES): O=C(Nc1cc(C(=O)N2CCCC2CO)nn1-c1ccccc1)c1cc(-c2ccccn2)c(F)cc1Cl
InChIKey: DZDIYCUEJAQXKR-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Cell Based Assay: TRKA co-expressed with p75: The assays are based upon DiscoveRx's proprietary Enzyme Fragment Complementation (EFC) technology. In the case of the TRK cell lines, the enzyme acceptor (EA) protein is fused to a SH2 protein and the TRK receptor of interest has been tagged with a Prolink tag. Cryopreserved PathHunter cells are used from either in-house produced batches or bulk batches bought directly from DiscoveRx. Cryopreserved cells are resuscitated, spun 1000 rpm for 4 min to remove freezing media, and resuspended in MEM+0.5% horse serum (both Invitrogen) to 5e^5 cells/ml. The cells are then plated using a Multidrop into Greiner white tissue culture treated plates at 20 μl/well and incubated for 24 h at 37 °C., 5% CO2, high humidity. On the day of the assay, the cell plates are allowed to cool to room temperature for 30 min prior to the assay. 4 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using the compound of Example 135, WO2005/116035 at a top concentration of 150 μM is also prepared on each test plate. High percentage effect (HPE) is defined by 150 μM of the compound of Example 135, WO2005/116035 and 0% effect (ZPE) is defined by 100% DMSO. Plates containing 1 μl of serially diluted compound, standard and HPE/ZPE are diluted 1/66 in assay buffer (PBS minus Ca2+, minus Mg2+ with 0.05% pluronic F127) using a Wellmate. Using a Platemate Plus, 5 μl of 1/66 diluted test compounds is then transferred to the cell plate and allowed to reach equilibrium by incubating for 30 min at room temperature before addition of agonist stimulus: 10 μl/well of 2 nM (0.571 nM FAC) of the cognate neurotrophin (Peprotech) diluted in agonist buffer (HBSS with 0.25% BSA). Final assay concentration of the test compounds is 8.66 μM, (the compound of Example 135, WO2005/116035 FAC is 0.325 μM). The plates are left at room temperature for a further 2 hours before addition of 10 μl of the DiscoveRx PathHunter detection reagent (made up by adding 1 part Galacton Star, 5 parts Emerald II and 19 parts Cell Assay Buffer as per the manufacturer's instructions). After reagent addition, plates are covered and incubated at room temperature for 60 minutes. Luminescence signal is read using an Envision.
Measurement: IC50 = 26.3 nM (pChEMBL 7.579999923706055)
Descriptors: MW 519.96, ALogP 4.58, HBA 6, HBD 2, PSA 100.35, QED 0.39
Compound (SMILES): COCC(C)NC(=O)c1ccc(NC(=O)N2Cc3ccc(F)cc3C2)cc1
InChIKey: YKBKBANREBCUSV-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 2:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1287128.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 128 nM (pChEMBL 6.889999866485596)
Descriptors: MW 371.41, ALogP 3.14, HBA 3, HBD 2, PSA 70.67, QED 0.85
Compound (SMILES): Cc1nn(C)cc1-c1c[nH]c2ncnc(N3CCOCC3)c12
InChIKey: RGWYNOKIZOFIMS-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Lantha Screen Technology Assay: LRRK2 kinase activity was measured using Lantha Screen technology from Invitrogen. GST-tagged truncated LRRK2 from Invitrogen (Cat # PV4874) was incubated with a fluorescein-labeled peptide substrate based upon ezrin/radixin/moesin (ERM), also known as LRRKtide (Invitrogen cat #PR8976A), in the presence of a dose response of compound. Upon completion, the assay was stopped and detected with a terbium labeled anti-phospho-ERM antibody (Invitrogen, cat #PR8975A). The assay was carried out under the following protocol: 3 uL of a working solution of substrate (233 nM LRRKtide, 117 uM ATP) prepared in assay buffer (50 mM HEEPES, pH 7.5, 3 mM MgCl2, with 2 mM DTT and 0.01% Brij35 added fresh) was added to a low volume Greiner 384-well plate. The compound dose response was prepared by diluting compound to a top concentration of 3.16 mM in 100% DMSO and serial diluted by half-log in DMSO 11 times. Aliquots (3.5 uL) of the 100% DMSO dose response were mixed with 46.5 uL water.
Measurement: IC50 = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 298.35, ALogP 1.5, HBA 6, HBD 1, PSA 71.86, QED 0.78
Compound (SMILES): O=C(/C=C/c1cccnc1)Nc1ccc(S(=O)(=O)c2ccc(C(F)(F)F)cc2)cc1
InChIKey: LGNYNDABFIHOLK-KGVSQERTSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Biochemical Inhibition Assay: The NAMPT enzymatic reactions were carried out in Buffer A (50 mM Hepes pH 7.5, 50 mM NaCl, 5 mM MgCl2, and 1 mM THP) in 96-well V-bottom plates. The compound titrations were performed in a separate dilution plate by serially diluting the compounds in DMSO to make a 100x stock. Buffer A (89 uL) containing 33 nM of NAMPT protein was added to 1 uL of 100x compound plate containing controls (e.g. DMSO or blank). The compound and enzyme mix was incubated for 15 minutes at room temperature, then 10 uL of 10x substrate and co-factors in Buffer A were added to the test well to make a final concentration of 1 uM NAM, 100 uM 5-Phospho-D-ribose 1-diphosphate (PRPP), and 2.5 mM Adenosine 5'-triphosphate (ATP). The reaction was allowed to proceed for 30 minutes at room temperature, then was quenched with the addition of 11 uL of a solution of formic acid and L-Cystathionine to make a final concentration of 1% formic acid and 10 uM L-Cystathionine.
Measurement: IC50 = 56.5 nM (pChEMBL 7.25)
Descriptors: MW 432.42, ALogP 4.59, HBA 4, HBD 1, PSA 76.13, QED 0.6
Compound (SMILES): CC(C)N(Cc1n[nH]c2ncccc12)C(=O)Nc1ccc(SC(F)(F)F)cc1
InChIKey: VGKVDYWUTPIEQT-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Kinase Activity Assay: TrkA kinase activity was measured as the ability of the enzyme to phosphorylate a fluorescently labeled peptide substrate. Buffer salts, reagents, and fluorescently labeled peptides were purchased from Carna Biosciences and were of the highest quality available (QSS Assist TRKA_MSA Kit). Enzyme was purchased from Cell Signaling, and was used without further purification. 384-well round bottom assay plates were prepared by the addition of 200 nl of a DMSO solution of compound at various concentrations to final inhibitor concentrations ranging from 100 μM to 0.2 μM. Next, assay buffer (10 μl) containing substrate and ATP were added, followed by addition of 10 μl of enzyme in assay buffer. Final assays concentrations were: [E]=0.37 nM, [S]=1 μM, [ATP]=2 mM. The reactions were incubated at room temperature for 3 hours resulting in approximately 15% substrate phosphorylation and were terminated by the addition of 5 μl of stop buffer.
Measurement: EC50 = 24.9 nM (pChEMBL 7.599999904632568)
Descriptors: MW 409.44, ALogP 5.01, HBA 4, HBD 2, PSA 73.91, QED 0.58
Compound (SMILES): C=CC(=O)N1CCC(c2cn3nc(-c4ccc(Oc5ccccc5)cc4)c(C(N)=O)c3[nH]2)C1
InChIKey: KYMRHZMEIPRWGR-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Time-Resolved Fluorescence Resonance Energy Transfer Assay: Compounds disclosed herein were tested for inhibition of Btk kinase activity in an assay based on time-resolved fluorescence resonance energy transfer methodology. Recombinant Btk was pre-incubated with the compounds disclosed herein at room temperature for 1 hour in an assay buffer containing 50 mM Tris pH7.4, 10 mM MgCl2, 2 mM MnCl2, 0.1 mM EDTA, 1 mM DT, 20 nM SEB, 0.1% BSA, 0.005% tween-20. The reactions were initiated by the addition of ATP (at the concentration of ATP Km) and peptide substrate (Biotin-AVLESEEELYSSARQ-NH2). After incubating at room temperature for 1 h, an equal volume of stop solution containing 50 mM HEPES pH7.0, 800 mM KF, 20 mM EDTA, 0.1% BSA, Eu cryptate-conjugated p-Tyr66 antibody and streptavidin-labeled XL665 was added to stop the reaction. Plates were further incubated at room temperature for 1 hour, and then the TR-FRET signals (ex337 nm, em 620 nm/665 nm) were read on BMG PHERAstar FS instrument. The residual enzyme activity in presence of increasing concentrations of compounds was calculated based on the ratio of fluorescence at 615 nm to that at 665 nm. The IC50 for each compound was derived from fitting the data to the four-parameter logistic equation by Graphpad Prism software.
Measurement: IC50 = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 441.49, ALogP 3.72, HBA 5, HBD 2, PSA 105.72, QED 0.44
Compound (SMILES): Clc1ccc(CCNC[C@H]2CC[C@H](c3nnn4cnc5[nH]ccc5c34)CC2)cc1
InChIKey: HJSZIUDMNAXXLP-QAQDUYKDSA-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.
Measurement: IC50 = 2.6 nM (pChEMBL 8.59000015258789)
Descriptors: MW 408.94, ALogP 4.37, HBA 5, HBD 2, PSA 70.9, QED 0.46
Compound (SMILES): COC(=O)C[C@@H]1N=C(c2ccc(Nc3c(F)cc(F)cc3F)cc2)c2c(sc(CO)c2C)-n2c(C)nnc21
InChIKey: HBYLVKAXSCOSII-IBGZPJMESA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: An expression vector containing cDNA of BRD2, 3 and 4 added with Flag-tag was transfected to CHO cell, and a cell lysate was prepared 24 hr later. Binding of acetylated histone H4 and BRD was confirmed by a Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) method. To a 384-well white plate (manufactured by Coaster) were added 50 nmol/L biotin-labeled acetylated histone H4 peptide (manufactured by Upstate) and a serially diluted test compound. Furthermore, CHO cell lysate transfected with BRD expression vector, a europium-labeled anti-Flag antibody (manufactured by Cisbio), and XL-665-labeled avidin (manufactured by Cisbio) were added, and the mixture was reacted at room temperature for 30 min to 2 hr. The fluorescence by FRET was measured by EnVision 2103 Multilabel Reader (manufactured by Perkin Elmer).
Measurement: IC50 = 77.2 nM (pChEMBL 7.110000133514404)
Descriptors: MW 541.56, ALogP 5.05, HBA 9, HBD 2, PSA 101.63, QED 0.33
Compound (SMILES): CCOc1ncc(C(=O)Nc2ccc(OC(F)(F)F)cc2)cc1-c1cncnc1
InChIKey: QDFOKEWPQMANBZ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Radiometric Filter-Binding Assay: For determination of ABL kinase activity, the radiometric filter-binding assay was used. The assay was performed by mixing 10 μL of the compound pre-diluted with 10 μL of ATP (20 M ATP with 0.1 μCi [γ-33P]-ATP) with the phospho-acceptor peptide poly[Ala6Glu2LysHBr5Tyr1]=polyAEKY) in 20 mM Tris/HCl pH 7.5, 1 mM DTT, 10 mM MgCl2, 0.01 mM Na3VO4, 50 mM NaCl. 10 μL of enzyme (ranging between 5 nM to 20 nM) was added to initiate the reaction. Pre-incubation of enzyme with compounds (when stated) was performed by exposing the enzyme to compounds prior to addition of the substrate mixture (ATP and/or peptide substrate). After 15 min at room temperature, the reaction was stopped by the addition of 50 μL 125 mM EDTA, and the peptide-bound 33P separated on filter-plates (PVDF or MAIP; Millipore, Volketswil, Switzerland) prepared according to the manufacturer's instructions. Filter-plates were washed 3× with 0.5% H3PO4, followed by addition of 30 μL scintillation cocktail (Microscint, Perkin Elmer) per well and then analysed in a TopCount NXT scintillation counter (Perkin Elmer).
Measurement: IC50 = 49 nM (pChEMBL 7.309999942779541)
Descriptors: MW 404.35, ALogP 4.09, HBA 6, HBD 1, PSA 86.23, QED 0.67
Compound (SMILES): C[C@@H]1CN(c2sc(C(F)(F)F)nc2-c2nc3ccccc3[nH]2)CCN1C(=O)Cn1cc(C2CCN(C)CC2)cn1
InChIKey: MDOLKXWWPFVDTC-QGZVFWFLSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: FLIPR Assay: The bioactivity of compounds is tested in a fluorometric imaging plate reader (FLIPR: Molecular Devices) using engineered CHO-K1 cells expressing the human CXCR3A coupled to a G protein (Galpha(16)). Cells are plated the day prior to bioassay in F12 medium supplemented with 10% FBS and G418 and hygromycin antibiotics to maintain recombinant selection. At the day of bioassay, cells are washed and dye loaded for one hour with Fluo-4-AM (Invitrogen) in Hanks Balanced Salt Solution (Invitrogen), buffered with 20 mM Hepes at pH 7.4 and sodium bicarbonate (0.015%), containing 5 mM probenecid. This buffer, but lacking the dye and containing probenecid at a concentration of 2.5 nM, is also is used for washing steps (wash buffer); or lacking both dye and probenecid but supplemented with 0.1% BSA for compound dilution steps (dilution buffer). Cells are washed free of excess dye and 60 microliter of wash buffer is added. Stock solutions of test compounds are made up at a concentration of 10 mM in DMSO, and serially diluted in dilution buffer to concentrations required for inhibition dose response curves. After a 10 minute incubation period at 37° C., 10 microliters of each compound dilution are transferred from a compound plate to the plate containing the recombinant cells in the FLIPR instrument according to the manufacturer's instructions. Following basal readings, 10 microliter CXCL10 agonist at a concentration of 20 nM (from Peprotech) is added, again using the FLIPR instrument. Changes in fluorescence are monitored before and after addition of the test compounds.
Measurement: IC50 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 572.66, ALogP 4.45, HBA 8, HBD 1, PSA 86.18, QED 0.38
Compound (SMILES): CC(C)CCNC(=O)c1ccc(NC(=O)N2Cc3ccc(F)cc3C2)cc1
InChIKey: GBBURVMXNCSFRE-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 1:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1251667.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 369.44, ALogP 4.15, HBA 2, HBD 2, PSA 61.44, QED 0.83
Compound (SMILES): CC(C)(COc1ccc(-c2ccc(-c3ncc(C4CCC(F)(F)CC4)[nH]3)cn2)cc1)C(=O)O
InChIKey: WXPPVKSNEHSEBC-UHFFFAOYSA-N
Target: Diacylglycerol O-acyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75907
Assay: DGAT1 Inhibitory Assay: As a buffer to be used in the enzymatic reaction of DGAT1, 100 mM Tris-HCl (pH 7.4), 200 mM Sucrose, 20 mM MgCl2, 0.125% Bovine Serum Albumin (BSA) were used. To the buffer were added Test compound with a predetermined concentration as well as 15 μM dioleoylglycerol, 5 μM [14C]-palmitoyl-CoA, 100 μg protein/nth DGAT1 highly expressed-expresSF+® microsome, 0.75% acetone, and 1% dimethylsulfoxide, and triglyceride (TG) synthetic reaction was carried out at 30 °C. for 20 minutes with a volume of 100 μL. 90 μL of the reaction solution was added to 810 μL of methanol to stop the reaction. The reaction solution was added to Oasie® μElution plate (available from Waters Corporation), and eluted with 150 μL of a mixed solution of acetonitrile: isopropanol (=2:3). To elute was added 150 μL of MicroScinti™-40 (available from PerkinElmer Inc.), and after thoroughly stirring the mixture, a [14C]-TG amount formed by the reaction was quantitated by measuring the same using TopCount™-NXT (available from PerkinElmer Inc.).
Measurement: IC50 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 455.51, ALogP 5.92, HBA 4, HBD 2, PSA 88.1, QED 0.46
Compound (SMILES): Cn1nc(-c2ccc(F)cc2)c(-c2ccc3nc(NC(=O)c4ccnc(F)c4)cn3n2)c1N1CCN(Cc2ccon2)CC1
InChIKey: YLZRLYXUAQSEHW-UHFFFAOYSA-N
Target: Casein kinase I isoform epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: P49674
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 ul prepared from 15 ul additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours and terminated by adding 45 ul of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIPÂ®3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the unphosphorylated substrate and phosphorylated product Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 uM ATP, 1.5 uM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 3.9 nM (pChEMBL 8.40999984741211)
Descriptors: MW 596.6, ALogP 4.03, HBA 11, HBD 1, PSA 122.51, QED 0.27
Compound (SMILES): COc1cc2[nH]ncc2cc1NC(=O)c1cnn2cccnc12
InChIKey: GLVJMFTUFFHUHM-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: In vitro IRAK-1 and IRAK-4 Assay: Purified recombinant IRAK-4 protein was incubated with 250 uM synthetic peptide(KKARFSRFAGSSPSQSSMVAR) in 30 ul of kinase buffer including (20 mM MOPS pH7.2, 25 mM beta glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM DTT, 50 uM ATP, 20 mM MgCl2, 10 uCi gamma-33P, 0.1% BSA) for the indicated time. For purified recombinant IRAK-1 protein kinase assay, 50 uM ATP was used. A 25 ul aliquot of the reaction mixture was transferred on to p81 phosphocellulose squares (Upstate Biotechnology, Lake Placid, N.Y.). The assay squares were washed three times with 0.75% phosphoric acid and once with acetone. Enzyme activity was measured by determining the bound radioactivity by liquid scintillation counting.
Measurement: IC50 = 231 nM (pChEMBL 6.639999866485596)
Descriptors: MW 308.3, ALogP 1.87, HBA 6, HBD 2, PSA 97.2, QED 0.6
Compound (SMILES): CC(C)(O)c1c(N(Cc2ccc(OC(F)(F)F)cc2)S(=O)(=O)c2ccc(Br)cc2)sc2ccccc12
InChIKey: BGAAQUYIODZIJT-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Functional Assay: The functional activity of compounds of the formula (I) was determined by measuring changes in intracellular calcium concentration using a Ca2+-sensitive fluorescent dye. The changes in fluorescent signal were monitored by a fluorescence plate reader, either a FLIPR (Molecular Devices) or FDSS (Hamamatsu). Increases in intracellular Ca2+ concentration were readily detected upon activation with icilin.At 24 hrs prior to assay, HEK293 cells stably expressing canine TRPM8 were seeded in culture medium in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences, NJ, USA) and grown overnight in 5% CO2 at 37° C. On assay day, growth media was removed and cells were loaded with Calcium 3 Dye (Molecular Devices) for 35 min at 37° C., under 5% CO2 and then for 25 min at room temperature and atmosphere. Subsequently, cells were tested for agonist-induced increases in intracellular Ca2+ levels using FLIPR or FDSS. Cells were challenged with a compound of the Formula (I) (at varying concentrations) and intracellular Ca2+ was measured for 5 min prior to the addition of icilin to all wells to achieve a final concentration that produces approximately an 80% maximal response. EC50 or IC50 values for compounds of the present invention were determined from eight-point dose-response studies.
Measurement: IC50 = 26.8 nM (pChEMBL 7.570000171661377)
Descriptors: MW 600.48, ALogP 7.19, HBA 5, HBD 1, PSA 66.84, QED 0.24
Compound (SMILES): O=CNC(CNC(=O)Cn1nc(-c2ccc(Cl)cc2)n(C[C@H](O)C(F)(F)F)c1=O)c1cccc(C(F)(F)F)c1
InChIKey: YBICINNGDZZTIZ-ZVAWYAOSSA-N
Target: Vasopressin V1a receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P37288
Assay: Cellular in Vitro Assay: On the day before the assay, the cells are plated out in culture medium (DMEM, 10% FCS, 2 mM glutamine, 10 mM HEPES) in 384-well microtitre plates and kept in a cell incubator (96% humidity, 5% v/v CO2, 37° C.). On the day of the assay, the culture medium is replaced by a Tyrode solution (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 20 mM glucose, 20 mM HEPES), which additionally contains the cofactor coelenterazine (50 μM), and the microtitre plate is then incubated for a further 3-4 hours. The test substances in various concentrations are placed for 10 to 20 minutes in the wells of the microtitre plate before the agonist [Arg8]-vasopressin is added, and the resulting light signal is measured immediately in the luminometer.
Measurement: IC50 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 579.89, ALogP 2.91, HBA 7, HBD 3, PSA 118.25, QED 0.25
Compound (SMILES): COc1cc2ncnc(N3CCN(C(=S)Nc4ccc(S(=O)(=O)Nc5ncccn5)cc4)CC3)c2cc1OC
InChIKey: MUQXLPQDKAJNMQ-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Inhibition of c-kit gain of function mutant in human GIST882 cells after 96 hrs by Cell-Titer Glo luciferase assay
Measurement: IC50 = 50000 nM (pChEMBL 4.300000190734863)
Descriptors: MW 566.67, ALogP 2.76, HBA 10, HBD 2, PSA 134.7, QED 0.32
Compound (SMILES): Cc1cc(Nc2nccc(C(F)(F)F)n2)cc(-c2ccc(OCc3ccncc3)c(C(=O)O)c2)c1
InChIKey: LVNIQLJKFXAIOY-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Homogeneous Time-Resolved Fluorescence (HTRF) Assay: A recombinant GST-hSYK fusion protein was used to measure potency of compounds to inhibit human Syk activity. The recombinant human GST-SYK (Carna Biosciences #08-176) (5 μM final concentration) was incubated with various concentrations of the inhibitor diluted in DMSO (0.1% final concentration) for 10 minutes at room temperature in 15 mM Tris-HCl (pH 7.5), 0.01% tween 20, 2 mM DTT in 384 well plate format. To initiate the reaction the biotinylated substrate peptide (250 nM final concentration) that contains the phosphorylation site for Syk was added with magnesium (5 mM final concentration) and ATP (25 μM final concentration). Final volume of the reaction was 10 μL. Phosphorylation of the peptide was allowed to proceed for 45' at room temperature. To quench the reaction and detect the phosphorylated product, 2 nM of a Europium-anti-phosphotyrosine antibody (Perkin Elmer #AD0161) and 70 nM SA-APC (Perkin-Elmer #CR130-100) were added together in 15 mM Tris pH 7.5, 40 mM EDTA, 0.01% tween 20. Final volume of the quenching solution was 10 μL. The resulting HTRF signal was measured after 30 minutes on an EnVision (Perkin-Elmer) reader using a time-resolved fluorescence protocol.
Measurement: IC50 = 16.3 nM (pChEMBL 7.789999961853027)
Descriptors: MW 480.45, ALogP 5.89, HBA 6, HBD 2, PSA 97.23, QED 0.34
Compound (SMILES): NC(=O)c1c(-c2ccc(Oc3ccccc3)cc2)nn2c1[nH]c(=O)c1cc(N)ccc12
InChIKey: JEHLFDYRJAKFNT-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Time-Resolved Fluorescence Resonance Energy Transfer Assay: Compounds disclosed herein were tested for inhibition of Btk kinase activity in an assay based on time-resolved fluorescence resonance energy transfer methodology. Recombinant Btk was pre-incubated with the compounds disclosed herein at room temperature for 1 hour in an assay buffer containing 50 mM Tris pH7.4, 10 mM MgCl2, 2 mM MnCl2, 0.1 mM EDTA, 1 mM DT, 20 nM SEB, 0.1% BSA, 0.005% tween-20. The reactions were initiated by the addition of ATP (at the concentration of ATP Km) and peptide substrate (Biotin-AVLESEEELYSSARQ-NH2). After incubating at room temperature for 1 h, an equal volume of stop solution containing 50 mM HEPES pH7.0, 800 mM KF, 20 mM EDTA, 0.1% BSA, Eu cryptate-conjugated p-Tyr66 antibody and streptavidin-labeled XL665 was added to stop the reaction. Plates were further incubated at room temperature for 1 hour, and then the TR-FRET signals (ex337 nm, em 620 nm/665 nm) were read on BMG PHERAstar FS instrument. The residual enzyme activity in presence of increasing concentrations of compounds was calculated based on the ratio of fluorescence at 615 nm to that at 665 nm. The IC50 for each compound was derived from fitting the data to the four-parameter logistic equation by Graphpad Prism software.
Measurement: IC50 = 480 nM (pChEMBL 6.320000171661377)
Descriptors: MW 411.42, ALogP 3.32, HBA 6, HBD 3, PSA 128.5, QED 0.39
Compound (SMILES): Cn1ccc2c(-c3ccc(OCC4(C)CCNCC4)c(C(F)(F)F)c3)nc(C#N)nc21
InChIKey: NEOUOOCYIYRPCD-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 = 3.6 nM (pChEMBL 8.4399995803833)
Descriptors: MW 429.45, ALogP 4.29, HBA 6, HBD 1, PSA 75.76, QED 0.67
Compound (SMILES): CN1CCN(CCNc2ccc(S(=O)(=O)NC(=O)c3ccc(N4CCN(CC5=C(c6ccc(Cl)cc6)CC(C)(C)CC5)CC4)cc3Oc3ccc4[nH]ccc4c3)cc2[N+](=O)[O-])CC1
InChIKey: OZZNCUWTNDYZJX-UHFFFAOYSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Mus musculus
UniProt: P10417
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The inhibition constant (Ki) is the dissociation constant of an enzyme-inhibitor complex or a protein/small molecule complex, wherein the small molecule is inhibiting binding of one protein to another protein or peptide. Where the Ki for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-XL) is greater than the limits of detection of the assay used. Where the binding selectivity ratio for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the selectivity of a particular compound for Bcl-2 over Bcl-XL is at least as great as the number indicated. Where the Ki for a compound is represented as < (less than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-2) is lower than the limit of detection of the assay used. Inhibition constants were determined using Wang's equation (Wang Z-X).
Measurement: Ki = 0.06 nM (pChEMBL 10.220000267028809)
Descriptors: MW 895.53, ALogP 8.09, HBA 11, HBD 3, PSA 156.39, QED 0.07
Compound (SMILES): COc1cc(C2CCCC2)c(F)cc1-n1c(=O)ccc2cc(S(=O)(=O)Nc3ccon3)ccc21
InChIKey: ZHFAQBFVVGHYSV-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Nav1.7 In Vitro PX Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to -10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes.
Measurement: IC50 = 88 nM (pChEMBL 7.059999942779541)
Descriptors: MW 483.52, ALogP 4.58, HBA 7, HBD 1, PSA 103.43, QED 0.43
Compound (SMILES): Cc1ccc(-c2noc(C3(NC(=O)OC(C)(C)C)CC3)n2)cc1NC(=O)c1cnc2ccccn12
InChIKey: NSRLVENBQNMIER-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Cell Proliferation Assay: The compounds was tested for inhibition of SCF dependent proliferation using human Mo7e cells which endogenously express c-kit in a 384 well format. Three-fold serially diluted test compounds (Cmax=10 mM) were evaluated for their antiproliferative activity of Mo7e cells stimulated with human recombinant SCF. After 48 hours of incubation at 37° C., cell viability was measured by adding 25 uL of CellTiter Glo (Promega) to the cells and the luminescence was measured by a CLIPR CCD camera (Molecular Devices).
Measurement: IC50 = 89 nM (pChEMBL 7.050000190734863)
Descriptors: MW 474.52, ALogP 4.46, HBA 8, HBD 2, PSA 123.65, QED 0.44
Compound (SMILES): Cc1c(N2C(=O)O[C@@H](C)[C@@H]2[C@H](O)C(F)(F)F)ccc(C#N)c1C(F)(F)F
InChIKey: CDCCUYIGQVCZGD-BPTDKIDVSA-N
Target: Androgen receptor (SINGLE PROTEIN), Rattus norvegicus
UniProt: P15207
Assay: Biological Assay: In order to demonstrate the utility of the compounds of this invention, an androgen receptor binding assay was performed wherein many of the compounds of this invention are shown to demonstrate significant affinity for the androgen receptor. The assay was performed as specified by the manufacturer (Invitrogen, Madison, Wis.). Briefly, 1 ul of 10 mM compound was added to 500 ul of AR screening buffer in a 1.5 ml eppendorf tube to make a 2x10E-5M stock. 10-fold serial dilutions of the test compounds were prepared ranging in concentration from 10E-5M to 10E-12M. Each dilution was added in triplicate to a black 384-microtiter plate. The test compounds will be diluted 2-fold in the final reaction. 2x AR-Fluormone complex was prepared with 2 nM Flourmone AL Green and 30 nM AR. 25 ul of 2x complex was aliquoted to each reaction well, such that the final reaction volume was 50 ul per well.
Measurement: IC50 = 33 nM (pChEMBL 7.480000019073486)
Descriptors: MW 382.26, ALogP 3.52, HBA 4, HBD 1, PSA 73.56, QED 0.79
Compound (SMILES): Cc1c(Cc2ccccc2)nnc(N2CCN(c3cnc(C(=O)NCC(C)O)cn3)[C@H](C)C2)c1C
InChIKey: RGTBFZDODFBZGR-QNSVNVJESA-N
Target: Protein smoothened (SINGLE PROTEIN), Mus musculus
UniProt: P56726
Assay: Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 475.6, ALogP 2.3, HBA 8, HBD 2, PSA 107.37, QED 0.54
Compound (SMILES): O=C1c2ccccc2S(=O)(=O)N1CCN1CCN(Cc2ccccc2)CC1
InChIKey: WEQPEFFNYGRQOJ-UHFFFAOYSA-N
Target: Acetylcholinesterase (SINGLE PROTEIN), Electrophorus electricus
UniProt: O42275
Assay: Inhibition of Electric eel AChE using acetylthiocholine iodide as substrate preincubated for 5 mins followed by substrate addition measured after 5 mins by Ellman's method
Measurement: IC50 = 830 nM (pChEMBL 6.079999923706055)
Descriptors: MW 385.49, ALogP 1.65, HBA 5, HBD 0, PSA 60.93, QED 0.78
Compound (SMILES): CCC(C(=O)Nc1ccc(C2CCNC2)cc1)c1ccccc1
InChIKey: OYEZXPCVBHKJLG-UHFFFAOYSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q923Y8
Assay: Radioligand Binding Assay: HEK-293 cells stably expressing mouse TAAR1 were maintained at 37° C. and 5% CO2 in DMEM high glucose medium, containing fetal calf serum (10%, heat inactivated for 30 min at 56° C.), penicillin/streptomycin (1%), and 375 ug/ml geneticin (Gibco). Cells were released from culture flasks using trypsin/EDTA, harvested, washed twice with ice-cold PBS (without Ca2+ and Mg2+), pelleted at 1,000 rpm for 5 min at 4° C., frozen and stored at -80° C. Frozen pellets were suspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 10 mM EDTA and homogenized with a Polytron (PT 6000, Kinematica) at 14,000 rpm for 20 s. The homogenate was centrifuged at 48,000Ã¿g for 30 min at 4° C. Subsequently, the supernatant was removed and discarded, and the pellet resuspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 0.1 mM EDTA using the Polytron (20 s at 14,000 rpm). This procedure was repeated and the final pellet resuspended in HEPES-NaOH containing 0.1 mM EDTA and homogenized using the Polytron. Typically, aliquots of 2 ml membrane portions were stored at -80° C. With each new membrane batch the dissociation constant (Kd) was determined via a saturation curve. The TAAR1 radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine (described in WO 2008/098857) was used at a concentration equal to the calculated Kd value, that was usually around 2.3 nM, resulting in the binding of approximately 0.2% of the radioligand and a specific binding representing approximately 85% of the total binding. Nonspecific binding was defined as the amount of 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine bound in the presence of 10 uM unlabeled ligand. All compounds were tested at a broad range of concentrations (10 pM to 10 uM) in duplicates. The test compounds (20 ul/well) were transferred into a 96 deep well plate (TreffLab), and 180 ul of HEPES-NaOH (20 mM, pH 7.4) containing MgCl2 (10 mM) and CaCl2 (2 mM) (binding buffer), 300 ul of the radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine at a concentration of 3.3xKd in nM and 500 ul of the membranes (resuspended at 50 ug protein per ml) added. The 96 deep well plates were incubated for 1 hr at 4° C. Incubations were terminated by rapid filtration through Unifilter-96 plates (Packard Instrument Company) and glass filters GF/C (Perkin Elmer) presoaked for 1 hr in polyethylenimine (0.3%) and washed 3 times with 1 ml of cold binding buffer. After addition of 45 ul of Microscint 40 (PerkinElmer) the Unifilter-96 plate was sealed and after 1 hr the ratioactivity counted using a TopCount Microplate Scintillation Counter (Packard Instrument Company).
Measurement: Ki = 40.7 nM (pChEMBL 7.389999866485596)
Descriptors: MW 308.42, ALogP 3.9, HBA 2, HBD 2, PSA 41.13, QED 0.88
Compound (SMILES): Cc1ncoc1-c1nnc(SCCCN2CC[C@@]3(C[C@H]3c3ccc(F)cc3F)C2)n1C
InChIKey: XIHOJUYAGODVFH-HTAPYJJXSA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Displacement of [3H]-spiperone from human dopamine D3 receptor expressed in CHO-K1 cell membranes after 90 mins by liquid scintillation counting
Measurement: Ki = 0.8128 nM (pChEMBL 9.09000015258789)
Descriptors: MW 445.54, ALogP 4.42, HBA 7, HBD 0, PSA 59.98, QED 0.4
Compound (SMILES): Cc1ccc2ncccc2c1-c1cc(-c2c(C)noc2C)cc2nc(C3CC3)[nH]c12
InChIKey: KHQQNXFBTALTQF-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Homogeneous Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Binding of the two tandem bromodomains, BRD4-1 and BRD4-2, to an acetylated histone H4 peptide was measured using a homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay. The synthetic peptide containing amino acids 1-18 of histone H4 was acetylated at lysine 5, 8, 12, 16 and conjugated to biotin (SGRGACKGGACKGLGACKGGAACKRH-GSGSK-biotin[SEQ ID NO:1]) was purchased from Millipore. BRD4-1 and BRD4-2 were expressed and purified from Escherichia coli as N-terminal His6-tagged proteins. An XL665 labeled anti-His antibody (Cisbio) was used to specifically bind BRD4 and a cryptate labeled streptavidin protein was used because it specifically recognized the biotinylated H4 peptide. Binding of BRD4 to the peptide resulted in an increase in FRET signal whereas disruption of this protein-peptide interaction with a small molecule inhibitor resulted in a decrease in FRET signal. Assays were performed in 50 mM Hepes (pH 7.5), 150 mM NaCl, 0.1 mg/ml BSA, 0.01% (v/v) Brij, 0.5% (v/v) DMSO and 200 nM H4 peptide at the following concentrations for each BRD4 isoform: 60 nM BRD4-1 and 120 nM BRD4-2. After an assay reaction time of 60 minutes at 25° C., binding was measured with 2 nM crytptate labeled streptavidin and 10 nM anti-His-XL665 antibody. TR-FRET signal was detected on an Envision plate reader (Ex: 320 nm; Em: 615/665 nm; 100 As delay and 200 Î¼s read window). Data were normalized based on a positive (2 Î¼M I-BET) and negative (DMSO) controls and IC50values were 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 = 87.1 nM (pChEMBL 7.059999942779541)
Descriptors: MW 394.48, ALogP 6.24, HBA 4, HBD 1, PSA 67.6, QED 0.39
Compound (SMILES): Cn1nc(C2CCN(C(=O)CC(=O)O)CC2)nc1-c1cnc(N)c(-c2nnc(C(C)(C)C)o2)n1
InChIKey: LLGDRAUZZHUOQT-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: In Vitro Enzyme Inhibition Assay: The inhibition of PI3K-beta, PI3K-alpha, PI3K-gamma and PI3K-delta was evaluated in a Kinase Glo based enzyme activity assay using human recombinant enzymes. Compounds in 100% DMSO were added to assay plates by acoustic dispensing. PI3K enzyme was added in a Tris buffer (50 mM Tris pH7.4, 0.05% CHAPS, 2.1 mM DTT, and 10 mM magnesium chloride) and allowed to preincubate with compound for 20 minutes prior to addition of substrate solution containing PIP2 and ATP. The enzyme reaction was stopped after 80 minutes by the addition of Kinase Glo detection solution containing Lucferin and Luciferase (from Kinase Glo® Plus Luminecent Kinase Assay kit (Promega #V3772). Plates were left for 30 minutes at room temperature then read on a Pherastar Instrument with a standard Luminescence filter block. The final concentration of DMSO, ATP and PIP2 in the assay were, 1%, 8 uM, and 80 uM respectively.
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 469.51, ALogP 1.38, HBA 11, HBD 2, PSA 179.04, QED 0.52
Compound (SMILES): Cc1cc(F)cc(CN2CC[C@H](CNC(=O)c3ccc4ccccc4c3)N[C@H](CCCN)C2=O)c1
InChIKey: PXFWVGFTDAQGCB-CLJLJLNGSA-N
Target: Melanocortin receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P33032
Assay: Radioligand Binding Assay: Assessments of compound binding to human MC5R (hMC5R)) by displacement of an 125I-labeled NDP-MSH receptor ligand peptide were performed essentially as described in the data sheets produced by Perkin Elmer to accompany their frozen hMC5R membranes (Perkin Elmer catalog number RBXMC5M400UA). [125I] NDP-MSH: Radiolabeled in House and Purified by HPLC: Na125I (0.5 mCi, 17.4 Ci/mg) was added to 50 μL sodium phosphate (50 mM, pH 7.4) in an eppendorf tube precoated with IODOGEN. After incubation for 10 mins the phosphate buffer containing the iodine was added to NDP-MSH (10 μl at 1 mg/mL) in a separate eppendorf tube. This was incubated for a further 10 mins. The iodinated NDP-MSH was purified by HPLC on a Zorbax SB 300 column using solvent A: 0.05% TFA and solvent B: 90% acetonitrile 0.045% TFA with a linear gradient, 0-67% B over 60 mins. The 125I NDP-MSH eluted at 52 mins after the unlabeled starting material (48 min) and was counted and stored in the freezer. It was used within 48 hrs, as radioactive decay and ligand decomposition resulted in greatly reduced specific binding observed after 72 hrs. Incubation buffer: 25 mM HEPES-KOH (pH 7.0), 1.5 mM CaCl2, 1 mM MgSO4, 0.1 M NaCl, 1 mM 1,10-phenanthroline, and 1 Complete™ protease inhibitor tablet/100 mL (Roche, catalog number 1873580). Perkin Elmer frozen hMC5 membranes: catalog number RBXMC5M400UA, 0.4 mL/vial; 400 microassays/vial, 0.78 mg/mL protein concentration. Vials of frozen membranes were thawed rapidly immediately before use, diluted with binding buffer and vortexed. Resuspended membranes were kept on ice until they were added to the wells of the plate. Assays were performed in 96 well polypropylene plates. Membranes (0.78 μg 40 μL of a 1:40 dilution in incubation buffer) were added to [125I] NDP-MSH (0.84 nM; 2200 Ci/mmol) and test compounds in a total volume of 140 μL. This was incubated for 1 hr at 37° C. Non-specific binding was determined with 3 mM NDP-MSH. Plates were filtered using a Tomtec cell harvester with GF/A filters (Wallac) (presoaked in 0.6% polyethylenimine) and washed three times with 1.0 mL ice-cold wash buffer (the above incubation buffer without 1,10-phenanthroline and Complete™ protease inhibitor tablet). The filters were dried in a 37° C. oven, placed in a sample bag and 5 mL Betaplatescint (Wallac) was added. Prepared filters were counted in cassettes in a Microbeta Trilux (Wallac) for 1 min.
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 476.6, ALogP 3.52, HBA 4, HBD 3, PSA 87.46, QED 0.46
Compound (SMILES): COc1cc(/C=N/NC(=O)CCn2cc(C)c3c(C)cccc32)cc2cccnc12
InChIKey: CDIDCUYYRZSZGW-VULFUBBASA-N
Target: Transient receptor potential cation channel subfamily M member 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZQ8
Assay: Inhibition of human TRPM5 transfected in HEK293 cells assessed as inhibition of ATP-mediated calcium mobilization by Calcium3 dye-based FLIPR assay
Measurement: IC50 = 1.4 nM (pChEMBL 8.850000381469727)
Descriptors: MW 400.48, ALogP 4.36, HBA 5, HBD 1, PSA 68.51, QED 0.39
Compound (SMILES): NS(=O)(=O)OC[C@H]1O[C@@H](n2cnc3c(C#Cc4ccccc4Br)ncnc32)[C@H](O)[C@@H]1O
InChIKey: KYJCDEJSZMDHOV-GFOCRRMGSA-N
Target: NEDD8-activating enzyme E1 catalytic subunit (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TBC4
Assay: Inhibition of recombinant human untagged NAEalpha/N-terminal His-tagged NAEbeta expressed in Bac-to-Bac expression system using FLAG-tagged NEDD8 as substrate after 90 mins in presence of GST-tagged Ubc12 by HTRF assay
Measurement: IC50 <= 100 nM (pChEMBL 7)
Descriptors: MW 510.33, ALogP -0.17, HBA 10, HBD 3, PSA 162.68, QED 0.4
Compound (SMILES): COCc1ccc(C(C)=O)c2sc(C(=O)Nc3cc(CN4CC[C@H](O)C4)c4ccccc4n3)c(C)c12
InChIKey: UXOLHRWLSKHKNL-FQEVSTJZSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Activity Assay: Inhibitory activity-1 assay: To a solution containing 20 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 100 μM EDTA, 330 μg/ml bovine serum albumin, 50 kU/ml 5′-nucleotidase, 0.1 μCi 3H-cAMP (64 nM cAMP), and PDE10A (H-PDE10A2, Human Phosphodiesterase 10A2, Scottish Biomedical), a test compound was added, and the mixture was reacted at 25° C. for 2 hours. To the reaction solution, QAE-Sephadex (17-0190-01, GE Healthcare Japan Corp.) suspended in 10 mM HEPES-Na (pH 7.0) (hereinafter, also referred to as a “QAE-Sephadex suspension”) was added, and the mixture was shaken for 1 minute and left standing for 5 minutes to obtain a supernatant. To the supernatant, a QAE-Sephadex suspension was further added, and the mixture was shaken for 1 minute and left standing for 5 minutes. Then, the obtained supernatant was transferred to LumaPlate (PerkinElmer, Inc.) and assayed using a radiation counter (TopCount NXT, PerkinElmer, Inc.).
Measurement: IC50 = 0.42 nM (pChEMBL 9.380000114440918)
Descriptors: MW 503.62, ALogP 4.93, HBA 7, HBD 2, PSA 91.76, QED 0.35
Compound (SMILES): COc1cccc([S+]([O-])N2CCC(c3cc(NC(=O)c4cnn5cccnc45)n(-c4ccc(C)cc4)n3)CC2)c1
InChIKey: AUOVRDJVIJGKNE-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Kinase Assay: The kinase activity of IRAK-4 is determined by its ability to catalyze the phosphorylation of a fluorescent polypeptide substrate. The extent of phosphorylation is measured using the IMAP technology (Molecular Devices) where the phosphorylated fluorescent substrate binds to the large M(III)-based nanoparticles which reduces the rotational speed of the substrate and thus increases its fluorescent polarization (FP). 20 μl reaction mixture contains 10 mM TriHCl, pH 7.2, 0.5 nM GST tagged IRAK-4 (SignalChem), 100 nM fluorescent peptide substrate (RP7030, Molecular Devices), 100 μM ATP, 1 mM DTT, 1 mM MgCl2, and 0.01% Tween 20. The reaction is initiated by the addition of ATP. After incubation for 30 minutes at 25° C., 60 μl of Progressive IMAP Reagent (Molecular Devices) is added to stop the reaction. Change in RP7030's FP is determined by a FP reader (Analyst HT, LH, BioSystems).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 555.66, ALogP 4.39, HBA 9, HBD 1, PSA 112.64, QED 0.3