lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(C)=Cc1ccc2[nH]nc(-c3cc(N4CCN(S(C)(=O)=O)CC4)ncn3)c2c1
InChIKey: DSONQBLFICMRBH-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LanthaScreen™ Assay (5 mM ATP): a) 400 nl of a 1:2.15 serial dilution of test compound(98 μM top assay concentration) is spotted via Labcyte Echo to certain wells in a 384 well black, untreated plate. Control wells contain 400 nl of either DMSO or 400 nl of a known inhibitor in DMSO. b) 10 μl of a 2.5 nM LRRK2(G2019S mutation, GST-LRRK2(amino acids 970-2527)) enzyme solution in 1× assay buffer(50 mM Tris pH 8.5, 10 mM MgCl2, 0.01% Brij-35, 1.0 mM EGTA, 2 mM DTT, 0.05 mM NaVO4) is added to all wells. c) A 30 minute room temperature incubation is followed by addition of 10 μl of 800 nM fluorescein labeled LRRKtide peptide substrate and 10 mM ATP solution in 1× assay buffer to all wells. d) After a 35 minute room temperature incubation, 20 μl of TR-FRET Dilution Buffer(Invitrogen PV3756B) containing 4 nM Tb-labeled anti-phospho LRRKtide antibody and 20 mM EDTA is added to all wells. e) Plates are incubated at room temperature for 1 hour and read on an Envision™ multi-mode plate reader with LanthaScreen™ settings. Results are analysed using Assay Data Analyzer.
Measurement: IC50 = 28.8 nM (pChEMBL 7.539999961853027)
Descriptors: MW 412.52, ALogP 2.52, HBA 6, HBD 1, PSA 95.08, QED 0.71
Compound (SMILES): CNc1nc(Nc2ccc(C(=O)O)cc2OC)ncc1Cl
InChIKey: FDJDDPNOJDABJG-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 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 308.73, ALogP 2.62, HBA 6, HBD 3, PSA 96.37, QED 0.78
Compound (SMILES): O=C1/C(=C/c2ccc(=O)n(O)c2)Oc2cc(O)ccc21
InChIKey: SFGCAZJFHWIMTD-XGICHPGQSA-N
Target: Tyrosinase (SINGLE PROTEIN), Homo sapiens
UniProt: P14679
Assay: Inhibition of tyrosinase (unknown origin)
Measurement: IC50 = 1500 nM (pChEMBL 5.820000171661377)
Descriptors: MW 271.23, ALogP 1.41, HBA 6, HBD 2, PSA 88.76, QED 0.6
Compound (SMILES): C[C@H](O)CNC(=O)c1cc(NC(=O)c2cc(-c3ncccc3F)c(F)cc2Cl)n(-c2ccccc2)n1
InChIKey: XWBPKJRHDXVFDJ-AWEZNQCLSA-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 = 17.76 nM (pChEMBL 7.75)
Descriptors: MW 511.92, ALogP 4.23, HBA 6, HBD 3, PSA 109.14, QED 0.34
Compound (SMILES): CC(C)c1cccc(NC(=O)c2cccc(N3CCc4c(cnc5ccnn45)C3)c2)c1
InChIKey: ZLDDEUAUHUMPTJ-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: VEGFR2 Kinase Assay: Biochemical KDR kinase assays were performed in 96 well microtiter plates that were coated overnight with 75 μg/well of poly-Glu-Tyr (4:1) in 10 mM Phosphate Buffered Saline (PBS), pH 7.4. The coated plates were washed with 2 mls per well PBS+0.05% Tween-20 (PBS-T), blocked by incubation with PBS containing 1% BSA, then washed with 2 mls per well PBS-T prior to starting the reaction. Reactions were carried out in 100 μL reaction volumes containing 2.7 μM ATP in kinase buffer (50 mM Hepes buffer pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2 and 0.2 mM Na3VO4). Test compounds were reconstituted in 100% DMSO and added to the reaction to give a final DMSO concentration of 5%. Reactions were initiated by the addition 20 μl per well of kinase buffer containing 200-300 ng purified cytoplasmic domain KDR protein (BPS Bioscience, San Diego, Calif.). Following a 15 minute incubation at 30° C., the reactions were washed 2 mls per well PBS-T. 100 μl of a monoclonal anti-phosphotyrosine antibody-peroxidase conjugate diluted 1:10,000 in PBS-T was added to the wells for 30 minutes. Following a 2 mis per well wash with PBS-Tween-20, 100 μl of O-Phenylenediamine Dihydrochloride in phosphate-citrate buffer, containing urea hydrogen peroxide, was added to the wells for 7-10 minutes as a colorimetric substrate for the peroxidase. The reaction was terminated by the addition of 100 μl of 2.5N H2SO4 to each well and read using a microplate ELISA reader set at 492 nm.
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 411.51, ALogP 4.67, HBA 5, HBD 1, PSA 62.53, QED 0.53
Compound (SMILES): C[C@H]1CN(C(=O)C2CC2)CCN1c1nc2cc(-c3nc(=O)o[nH]3)nc(-c3cncc(Cl)c3)c2n1C[C@H]1CC[C@H](C)CC1
InChIKey: IYPCCQZCPSITRD-FHWLQOOXSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: FRET Assay: Methods: An HDM2 FRET assay was developed to assess the compounds' inhibitory activity towards binding of p53 protein. A truncated version of HDM2 with residues 17 to 125 (containing p53 binding surface, Science (1994) 265, 346-355), with N-terminal His and Thioredoxin tag was generated in pET32a expression vector and expressed in E. coli strain BL21(DE3)Rosetta. Protein was purified using Ni-affinity chromatography, followed by size exclusion chromatography using Superdex 75 26/60 column. To assess inhibition of p53 binding to HDM2, a FITC labeled 8-mer peptide (SEQ ID NO:1: Ac-Phe-Arg-Dpr-Ac6c-(6-Br)Trp-Glu-Glu-Leu-NH2; Anal Biochem. 2004 Aug. 1; 331(1):138-46) with strong affinity towards the p53 binding pocket of HDM2 was used. The HDM2 assay buffer contained 1x Phosphate Buffered Saline (Invitrogen, Cat#14190), 0.01% BSA (Jackson ImmunoResearch, Cat#001-000-162), 0.01% Tween-20. In the 1x assay buffer recombinant HDM2 protein, peptide and Lumi-4-Tb Cryptate-conjugate mouse.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 591.12, ALogP 4.76, HBA 9, HBD 1, PSA 126.04, QED 0.34
Compound (SMILES): COc1ccc2cc1[C@@H](C)CN(C)C(=O)Nc1ccc(S(=O)(=O)C3CC3)c(c1)CN(C)C(=O)[C@@H]2Nc1ccc2c(N)ncc(F)c2c1
InChIKey: DRLAOAUSFABNAU-QPHCHLROSA-N
Target: Coagulation factor VII/tissue factor (PROTEIN COMPLEX), Homo sapiens
UniProt: P13726
Assay: Inhibition of recombinant human factor-7a/TF using S2288 as substrate measured after 60 mins at 25 degC
Measurement: Ki < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 660.77, ALogP 5.29, HBA 8, HBD 3, PSA 146.96, QED 0.26
Compound (SMILES): CCC1OC(=O)NN=C1N1CCc2nc(-c3ccccc3)oc2C1
InChIKey: HTCXRVMFMCUFKZ-UHFFFAOYSA-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 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 326.36, ALogP 2.53, HBA 6, HBD 1, PSA 79.96, QED 0.92
Compound (SMILES): Cc1ccc(CNc2nc(NC3CC3)n3ncc(/C=C4\NC(=O)NC4=O)c3n2)o1
InChIKey: DRZPXLBQDPRBDV-MLPAPPSSSA-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 > 1000 nM (pChEMBL 6)
Descriptors: MW 394.4, ALogP 1.39, HBA 9, HBD 4, PSA 138.48, QED 0.36
Compound (SMILES): C[C@H]1CCCN1c1cccc(Nc2cc(-c3cccc(Cl)c3)nn3ccnc23)n1
InChIKey: YABLVULUVFKKSF-HNNXBMFYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Inhibition Assay: Assay plates: 96-well MultiScreen 0.65 um filter plates (Millipore Cat. No.: MADVNOB10)Streptavidin coated beads: Streptavidin Sepharose, suspension 5.0 mL, in 50 mM EDTA/PBS diluted (1:100), (Amersham, Cat. No.: 17-5113-01)Compounds: 10 mM in 100% dimethylsulfoxide (DMSO), final conc.: compound 0.003-100 uM in 10% DMSOEnzyme: SYK RPA purified, truncated construct of Spleen Tyrosine Kinase aa 360-635, stock solution 1 mg/mL, MW: 31.2 KDa, final conc.:0.0005 uM. Peptide 1: biotinylated peptide is derived from a naturally occurring phosphor-acceptor consensus sequence (Biotin-EPEGDYEEVLE), special order from QCB, stock solution 20 mM, final conc.: 5.0 uM.ATP: Adenosine-5'-triphosphate 20 mM, (ROCHE Cat. No.: 93202720), final concentration: 20 uM Buffer: HEPES: 2-Hydroxyethyl piperazine-2-ethanesulfonic acid (Sigma, Cat. No.: H-3375)final concentration: 50 mM HEPES pH7.5BSA: Bovine Serum Albumin Fraction V, fatty acid free (Roche Diagnostics GmbH, Cat. No. 9100221).
Measurement: IC50 = 246.05 nM (pChEMBL 6.610000133514404)
Descriptors: MW 404.91, ALogP 5.18, HBA 6, HBD 1, PSA 58.35, QED 0.51
Compound (SMILES): Cc1cc(CN(C)C(c2ccc(Cl)cc2)C(C)C(=O)O)ccc1OCCN1C(=O)CCC1=O
InChIKey: QAYGSYUPTPNMJG-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 = 5520 nM (pChEMBL 5.260000228881836)
Descriptors: MW 472.97, ALogP 4.07, HBA 5, HBD 1, PSA 87.15, QED 0.52
Compound (SMILES): O=C(NCCc1c[nH]c2ccc(Cl)cc12)c1cccc(Cc2ccc(F)cc2)c1
InChIKey: JBNRDPPJWNKOFR-UHFFFAOYSA-N
Target: Alpha-synuclein (SINGLE PROTEIN), Homo sapiens
UniProt: P37840
Assay: In Vitro Inhibition Assay: α-Synucleinopathies are characterised by aggregation of α-synuclein in neurons. Aggregation of purified α-synuclein is performed essentially as described by Gerard et al. FASEB. 20(3):524-6 (2006). 20-100 μg purified α-synuclein (Sigma; S7820) at a concentration of about 2.5 μg/mL is incubated in the presence of spermin (250 μM) or paraquat (32 mM) or 6-hydroxydopamine (400 μM) or vehicle in a 384 well plate. Spermin, paraquat and 6-hydroxydopamine promote the α-synuclein aggregation process. Aggregation kinetics is determined by measuring turbidity at 340 nm, every 1-15 minutes for at least one hour.
Measurement: EC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 406.89, ALogP 5.52, HBA 1, HBD 2, PSA 44.89, QED 0.43
Compound (SMILES): CC1(C)CCC(CN2CCN(c3ccc(C(=O)NS(=O)(=O)c4cnc(OCC5(F)CCC(C)(O)CC5)c(Cl)c4)c(Oc4cnc5[nH]ccc5c4)c3)CC2)=C(c2ccc(Cl)cc2)C1
InChIKey: PXMGHKWJLJTPIN-UHFFFAOYSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Representative compounds were serially diluted in dimethyl sulfoxide (DMSO) starting at 50 uM (2x starting concentration; 10% DMSO) and 10 uL were transferred into a 384-well plate. Then 10 uL of a protein/probe/antibody mix was added to each well at final concentrations listed in TABLE 1. Protein:GST-Bcl-2, Probe: F-Bak Peptide Probe Acetyl-GQVGRQLAIIGDK(6-FAM)INR-amide(SEQ ID NO: 1), Protein(nM): 1, Probe (nM): 100, Antibody: Tb-anit-GST, Antibody (nm): 1.  The samples are then mixed on a shaker for 1 minute and incubated for an additional 3 hours at room temperature. For each assay, the probe/antibody and protein/probe/antibody were included on each assay plate as negative and positive controls, respectively. Fluorescence was measured on the ENVISION plate reader (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak peptide) and 495/510 nm (Tb-labeled anti-Histidine antibody) emission filters.
Measurement: Ki = 0.014 nM (pChEMBL 10.850000381469727)
Descriptors: MW 905.92, ALogP 9.37, HBA 10, HBD 3, PSA 149.98, QED 0.11
Compound (SMILES): CCCNC(=O)c1ccc(NC(=O)N2Cc3ccc(Br)cc3C2)cc1
InChIKey: BCAUBLUVWFUWHK-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 = 33.3 nM (pChEMBL 7.480000019073486)
Descriptors: MW 402.29, ALogP 4.14, HBA 2, HBD 2, PSA 61.44, QED 0.81
Compound (SMILES): O=c1[nH]nc2c3c(cccc13)NC1=C2CCNC1
InChIKey: XQOHUNRPGOTXDR-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: HT F Homogeneous PARP Inhibition Assay: PARP1 enzymatic assay was conducted using a method modified from HT F Homogeneous PARP Inhibition Assay Kit (Trevigen). 8.8 nM PARP1 was pre-incubated with different concentrations of compounds in a buffer containing 100 mM Tris-HCl pH 8.0, 100 mM NaCl, 20 mM MgCl2, and 1% DMSO for 30 min at RT. The auto-PARsylation reaction was initiated by addition of 500 nM NAD and 20 ng/ul activated DNA (Sigma) and incubated at RT for 40 min. The remaining NAD was detected by incubation with cycling assay solution containing 1% ethanol, 0.30 U/ml alcohol dehydrogenase, 25 uM resazurin, and 0.25 U/ml diaphorase for 50 min at RT. The concentration of NAD is proportional to the fluorescence signal at Ex540 nm/Em 590 nm.
Measurement: IC50 = 7.6 nM (pChEMBL 8.119999885559082)
Descriptors: MW 240.27, ALogP 1.05, HBA 4, HBD 3, PSA 69.81, QED 0.64
Compound (SMILES): CN1CC(Nc2ccc(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=O
InChIKey: LIUGYXVBDBKFBW-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 = 0.076 nM (pChEMBL 10.119999885559082)
Descriptors: MW 866.44, ALogP 8.07, HBA 10, HBD 3, PSA 170.22, QED 0.08
Compound (SMILES): CC(C)CC(C(=O)Nc1ccc(C(=O)O)cc1)n1cc(Cl)c(-c2cc(Cl)ccc2C#N)cc1=O
InChIKey: AMIQZUOXODCISO-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Test substances are dissolved in dimethyl sulphoxide and serially diluted in dimethyl sulphoxide (3000 μM to 0.0078 μM; resulting final concentrations in the test: 50 μM to 0.00013 μM). In each case 1 μl of the diluted substance solutions are placed into the wells of white microtitre plates from Greiner (384 wells). 20 μl of assay buffer (50 mM of Tris/HCl pH 7.4; 100 mM of sodium chloride solution; 5 mM of calcium chloride solution; 0.1% of bovine serum albumin) and 20 μl of factor XIa from Kordia (0.45 nM in assay buffer) are then added successively. After 15 min of incubation, the enzyme reaction is started by addition of 20 μl of the factor XIa substrate Boc-Glu(OBzl)-Ala-Arg-AMC dissolved in assay buffer (10 μM in assay buffer) from Bachem, the mixture is incubated at room temperature (22° C.) for 30 min and fluorescence is then measured (excitation: 360 nm, emission: 460 nm).
Measurement: IC50 = 49 nM (pChEMBL 7.309999942779541)
Descriptors: MW 498.37, ALogP 5.62, HBA 5, HBD 2, PSA 112.19, QED 0.44
Compound (SMILES): C[C@H](NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](C)NC(=O)C1CC1)C(N)=O
InChIKey: TTWCYSGAPRELIP-QXEWZRGKSA-N
Target: WD repeat-containing protein 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P61964
Assay: Binding Assay: All the FP based experiments were performed in MICROFLUOR 2 Black, "U" Bottom, 96-well Microtiter Plates (ThermoSci.) and FP was measured as mP units in a microplate reader (Tecan Ultra) with excitation at 485 nm and emission at 530 nm. To dilutions of WDR5Δ23 (2.2-0 uM) in 100 ul assay buffer (0.1M Phosphate, 25 mM KCl, 0.01% Triton, pH 6.5) 20 ul of a fixed concentration of the tracer in the assay buffer was added, followed by an addition of 5 ul DMSO to give 125 ul of total volume. Each assay had two controls: blank (without protein and tracer) and tracer only (without protein). The plates were incubated on a shaker at room temperature to reach equilibrium and mP values were measured at the 3 hour time point.
Measurement: IC50 < 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 383.45, ALogP -2.36, HBA 5, HBD 7, PSA 192.29, QED 0.12
Compound (SMILES): O=C(N[C@H]1CCC[C@@H]1Nc1cnc(C(F)(F)F)cn1)c1cc(F)ccc1-n1cccn1
InChIKey: IJOPDICBJZUTHP-GJZGRUSLSA-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 = 445 nM (pChEMBL 6.349999904632568)
Descriptors: MW 434.4, ALogP 3.58, HBA 6, HBD 2, PSA 84.73, QED 0.6
Compound (SMILES): CSCC[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc1cnc[nH]1)NC(=O)CNC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](Cc1c[nH]c2ccccc12)NC(=O)[C@H](CCC(N)=O)NC(=O)CC[C@@H](C)[C@H]1CC[C@H]2[C@@H]3CC[C@@H]4C[C@@H](NC(=O)CNC(=O)CN5CCN(CC(=O)O)CCN(CC(=O)O)CCN(CC(=O)O)CC5)CC[C@]4(C)[C@H]3CC(=O)[C@]12C)C(C)C)C(N)=O
InChIKey: DCTUWJWPBYIYMK-KXOAHDAXSA-N
Target: Gastrin-releasing peptide receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P30550
Assay: Displacement of [125I]-BBN from GRPR in human PC3 cells after 1 hr by compuGamma counting or microplate scintillation counting method
Measurement: IC50 = 4.5 nM (pChEMBL 8.350000381469727)
Descriptors: MW 1755.16
Compound (SMILES): Cc1ccc(CN2CCC(C(=O)NC(C)(C)C)CC2)cc1NC(=O)c1cccc(C(F)(F)F)n1
InChIKey: CATVBXAOUQSPII-UHFFFAOYSA-N
Target: Atypical chemokine receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P25106
Assay: In Vitro Assay: CHO-K1 CXCR7 b-arrestin cells are detached from culture dishes with a cell dissociation buffer (Invitrogen, #13151-014) and collected in growing medium (F12 HAMS 90% (v/v)/FCS 10% (v/v), Penicilin/streptomycin 1% (v/v)). 5000 cells per well (in 20 μl) are seeded in a 384 well plate (white-walled, clear bottom; BD Falcon #353274). The plate is incubated at 37° C./5% CO2 for 24 hours. Medium is then replaced by 20 μl OPTIMEM (Invitrogen #31985) for 3 to 4 hours. Test compounds are dissolved at 10 mM in DMSO and serially diluted in DMSO to 200× of the final concentration for dose response curves. Compounds are then diluted 1:33.3 in HBSS1×. 5 μl/well of HBSS1×/20 mM HEPES/0.2% BSA are added to the assay plate followed by addition of 5 μl/well of diluted compounds. CXCL12 (Peprotech #300-28A) may be used as a reference agonist. The plate is incubated for 90 minutes at 37° C. 12 μl of detection reagent (Path Hunter Detection Kit, DiscoveRx, #93-0001) is transferred to the assay plate and to the plate is incubated for 1 hour at room temperature. Luminescent signal is read in a microplate reader (FLUOstar Optima, bmg).
Measurement: EC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 476.54, ALogP 4.79, HBA 4, HBD 2, PSA 74.33, QED 0.65
Compound (SMILES): COc1ccc(F)cc1NC(=O)c1cnn2ccccc12
InChIKey: IMGSGWSHRWJHOL-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 = 4302 nM (pChEMBL 5.369999885559082)
Descriptors: MW 285.28, ALogP 2.73, HBA 4, HBD 1, PSA 55.63, QED 0.8
Compound (SMILES): COC(=O)Nc1ccc2c(c1)NC(C(F)(F)F)CC/C=C\C[C@H](NC(=O)/C=C/c1cc(Cl)ccc1-n1cnnn1)c1ncc-2[nH]1
InChIKey: HSIMPIIZQSDVFI-QXYIAPGXSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: In Vitro Assays: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 2171 nM (pChEMBL 5.659999847412109)
Descriptors: MW 642.04, ALogP 5.84, HBA 9, HBD 4, PSA 151.74, QED 0.16
Compound (SMILES): CN1CC(=O)N(Cc2ccc3c(-c4ccc(F)cc4)cc(C#N)nc3c2)C1=O
InChIKey: GBDJXOQJHMYYCQ-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: Biological Assay: The utility of the compounds in accordance with the present invention as antagonists of metabotropic glutamate receptor activity, in particular mGluR2 activity, may be demonstrated by methodology known in the art. Antagonist constants are determined as follows. The compounds of the present invention were tested in a fluorescence laser imaging plate reader based assay. This assay is a common functional assay to monitor Ca2+ mobilization in whole cells expressing recombinant receptor coupled with a promiscuous G-protein. CHO dhfr- cells stably expressing recombinant human mGluR2 and Gα16 loaded with Fluo-4 AM (Invitrogen, Carlsbad Calif.) are treated with various concentrations of antagonists of compounds and the Ca2+ response is monitored on a FLIPR384 (Molecular Devices, Sunnydale Calif.) for agonist activity.
Measurement: IC50 = 588 nM (pChEMBL 6.230000019073486)
Descriptors: MW 374.38, ALogP 3.31, HBA 4, HBD 0, PSA 77.3, QED 0.66
Compound (SMILES): COC(=O)[C@@H]1CNC[C@H](N(CC(C)C)C(=O)c2cnc(C(C)(C)C)nc2NCc2ccco2)C1
InChIKey: XWKPQIHEQDPCKK-ZWKOTPCHSA-N
Target: Renin (SINGLE PROTEIN), Homo sapiens
UniProt: P00797
Assay: Inhibition of renin in human plasma by radioimmunoassay
Measurement: IC50 > 10 nM (pChEMBL 8)
Descriptors: MW 471.6, ALogP 3.23, HBA 8, HBD 2, PSA 109.59, QED 0.56
Compound (SMILES): O=C(NN1CCCCC1)c1cccc(C2CCCC2)n1
InChIKey: HVLCSBIIKZAQSW-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1.times.HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated.
Measurement: EC50 = 129.4 nM (pChEMBL 6.889999866485596)
Descriptors: MW 273.38, ALogP 2.87, HBA 3, HBD 1, PSA 45.23, QED 0.92
Compound (SMILES): CC(C)(C)OC(=O)N1CCC(CCOc2ccc(-c3nc4cc(C(N)=O)ccc4[nH]3)cc2)CC1
InChIKey: KVWJJYNQHAPJPL-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase Chk2 (SINGLE PROTEIN), Homo sapiens
UniProt: O96017
Assay: Inhibition of human CDS1 using N-terminal biotin labeled SGLYRSPSMPENLNRPR as substrate in presence of [gamma-33P]-ATP after 3 hrs by scintillation counting method
Measurement: IC50 = 134 nM (pChEMBL 6.869999885559082)
Descriptors: MW 464.57, ALogP 4.74, HBA 5, HBD 2, PSA 110.54, QED 0.55
Compound (SMILES): COc1cc(C#N)ccc1C1C2=C(CCC2=O)N(c2cccc(C(F)(F)F)c2)C(=O)N1C
InChIKey: RTPRAIIYHYRDDS-UHFFFAOYSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: 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 = 9.5 nM (pChEMBL 8.020000457763672)
Descriptors: MW 441.41, ALogP 4.82, HBA 4, HBD 0, PSA 73.64, QED 0.68
Compound (SMILES): Cc1ccc(-n2nc(C(C)(C)C)cc2NC(=O)Nc2ccc(Oc3ccnc(Nc4cccc5cn[nH]c45)n3)c3ccccc23)cc1
InChIKey: QEXZVQWGGYXQDO-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16539
Assay: p38 MAPKα Enzyme Inhibition Assay: The inhibitory activities of test compounds against the p38 MAPKα isoform (MAPK14: Invitrogen), are evaluated indirectly by determining the level of activation/phosphorylation of the down-stream molecule, MAPKAP-K2. The p38 MAPK&dlpha; protein (80 ng/mL, 2.5 uL) is mixed with the test compound (2.5 uL of either 4 ug/mL, 0.4 ug/mL, 0.04 ug/mL or 0.004 ug/mL) for 2 hr at RT. The mix solution (2.5 uL) of the p38&dlpha; inactive target MAPKAP-K2 (Invitrogen, 600 ng/mL) and FRET peptide (8 uM; a phosphorylation target for MAPKAP-K2) is then added and the kinase reaction is initiated by adding ATP (40 uM, 2.5 uL). The mixture is incubated for 1 hr at RT. Development reagent (protease, 5 uL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan Flash, ThermoFisher Scientific).
Measurement: IC50 = 523 nM (pChEMBL 6.28000020980835)
Descriptors: MW 623.72, ALogP 8.48, HBA 8, HBD 4, PSA 134.67, QED 0.14
Compound (SMILES): Cn1cc(-c2cc(C(N)=O)nc3cc(CN4CCC(C(F)(F)F)CC4)ccc23)cn1
InChIKey: VZDSEFUNSQKPKU-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: Biological Assay: The utility of the compounds in accordance with the present invention as antagonists of metabotropic glutamate receptor activity, in particular mGluR2 activity, may be demonstrated by methodology known in the art. Antagonist constants are determined as follows. The compounds of the present invention were tested in a fluorescence laser imaging plate reader based assay. This assay is a common functional assay to monitor Ca2+ mobilization in whole cells expressing recombinant receptor coupled with a promiscuous G-protein. CHO dhfr- cells stably expressing recombinant human mGluR2 and Gα16 loaded with Fluo-4 AM (Invitrogen, Carlsbad Calif.) are treated with various concentrations of antagonists of compounds and the Ca2+ response is monitored on a FLIPR384 (Molecular Devices, Sunnydale Calif.) for agonist activity.
Measurement: IC50 = 114 nM (pChEMBL 6.940000057220459)
Descriptors: MW 417.44, ALogP 3.51, HBA 5, HBD 1, PSA 77.04, QED 0.7
Compound (SMILES): COc1cccc(C(=O)N2CCC(F)(F)C(Oc3ccc4ccccc4n3)C2)c1
InChIKey: QZXSVMSMRJTFPZ-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Displacement of (2S)-N-(2-pyrrol-1-ylphenyl)-1-[2-[1-(tritritiomethyl)benzimidazol-2-yl]sulfanylacetyl]pyrrolidine-2-carboxamide from human OX2 receptor expressed on CHO cell membrane measured after 3 hrs by TopCount method
Measurement: Ki > 4379 nM (pChEMBL 5.359000205993652)
Descriptors: MW 398.41, ALogP 4.17, HBA 4, HBD 0, PSA 51.66, QED 0.66
Compound (SMILES): Nc1n[nH]c2cc(NC(=O)NC3CCCC3c3ccccc3)ncc12
InChIKey: AUXOOYVTNXXNQO-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 uM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 uL) was dispensed, followed by the addition of 15 uL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 uL kinase buffer containing 2.45 uM ERK2 IMAP substrate peptides (2.25 uM-unlabeled IPTTPITTTYFFFK-COOH and 200 nM-labeled IPTTPITTTYFFFK-SFAM (5-carboxyfluorescein)-COOH), and 75 uM ATP. The final reaction in each well of 25 uL consists of 0.5 nM hERK2, 900 nM unlabeled peptide, 80 nM labeled-peptide, and 30 uM ATP.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 336.4, ALogP 3, HBA 4, HBD 4, PSA 108.72, QED 0.59
Compound (SMILES): CS(=O)(=O)c1cc(C#N)ccc1[C@@H]1C2=C(CCC2=O)N(c2cccc(C(F)(F)F)c2)C(=O)N1C(=O)N[C@H]1CC[C@H](O)C1
InChIKey: GROIZKGWAOMLQO-FXJWSJNKSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: 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 602.59, ALogP 4.2, HBA 7, HBD 2, PSA 147.88, QED 0.53
Compound (SMILES): O=C(c1cn(CC(F)(F)F)c2ccc(Nc3nccc(-n4cc(CN5C[C@H](O)CO5)cn4)n3)cc12)C1CC1
InChIKey: YBDFABRBIIRYCZ-SFHVURJKSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Inhibition Assay: Compounds of the invention were initially diluted to 10 mM in 100% DMSO (CALBIOCHEM) for storage and made into kinase buffer solution to create a compound concentration ranging from 1 uM and 10 uM. Serial dilutions of compounds of the invention were dispensed into a 96-well plate (GREINER BIOSCIENCES) at 6 uL each. Purified full-length human SYK (CARNA BIOSCIENCES) were diluted in kinase buffer and added to the compound solutions and pre-incubated for 30 minutes at room temperature. Next, ATP (TEKNOVA) of Km (15 uM) and substrate solution (suggested manufacture substrates of PerkinElmer, Ulight-TK peptide for SYK) was added (12 uL each) to the wells containing the compound solution and enzyme. The reaction mixture was incubated for 1 hour. Following the incubation, the stop solution made with EDTA, water, and Lance detection buffer (PERKINELMER) was added (12 uL each) to stop phosphorylation. Following the addition of the stop solution and 5 minutes of shaking.
Measurement: IC50 = 5.8 nM (pChEMBL 8.239999771118164)
Descriptors: MW 527.51, ALogP 3.62, HBA 10, HBD 2, PSA 110.33, QED 0.33
Compound (SMILES): O=C(O)C[C@H]1C[C@H](c2cccc(Cl)c2)[C@@H](c2ccc(Cl)cc2)N(CC2CCCC2)C1=O
InChIKey: UQGIWBFVKAEKAL-ZFJSRUIDSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: Homogenous Time-Resolved Fluorescence Assay (HTRF1 Assay): The standard assay conditions for the in vitro HTRF assay consisted of a 50 ul total reaction volume in black 384-well Costar polypropylene plates in 1xPBS buffer pH 7.4, 1 mM DTT, 0.1% BSA, 2.5 nM GST-hMDM2 (aa 1-188), 5 nM biotinylated-p53 (aa 1-83), 1.8 nM SA-XLent (Cisbio; Bedford, Mass.), 0.6 nM anti-GST cryptate monoclonal antibody (Cisbio; Bedford, Mass.) and 200 mM KF. Amino acid residues 1-188 of human MDM2 were expressed as an amino-terminal glutathione-S-transferase (GST) fusion protein (GST-hMDM2) in Escherichia coli. Residues 1-83 of human p53 were expressed as an amino-terminal AviTag-TrxA-6-His fusion protein (biotinylated p53) in E. coli. Each protein was purified from cell paste by affinity chromatography.Specifically, 10 uL of GST-hMDM2 was incubated with 10 ul of diluted compound (various concentrations, serially diluted) in 10% DMSO for 20 minutes at room temperature. 20 uL of biotinylated-p53 was added to the GST-hMDM2+compound mixture, and then incubated at room temperature for 60 min. 10 uL of detection buffer consisting of SA-XLent, anti-GST cryptate antibody and KF was added to GST-hMDM2, biotinylated-p53 and compound reaction and left at room temperature to reach equilibrium for >4 hrs. The final concentration of DMSO in the reaction was 2%. Time-resolved fluorescence readings were measured on a microplate multilabel reader. Percentage of inhibition was calculated relative to nutlin-3.
Measurement: IC50 = 190 nM (pChEMBL 6.71999979019165)
Descriptors: MW 460.4, ALogP 6.33, HBA 2, HBD 1, PSA 57.61, QED 0.55
Compound (SMILES): CC1(C)CC(c2ccc(Cl)cc2)=C(CN2CCN(c3ccc(C(=O)NS(=O)(=O)c4ccc(NC5CCC(N6CCOCC6)CC5)c(S(=O)(=O)C(F)(F)F)c4)c(Oc4cnc5[nH]ccc5c4)c3)CC2)CO1
InChIKey: XBANGVMCIRMPAC-UHFFFAOYSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Representative compounds were serially diluted in dimethyl sulfoxide (DMSO) starting at 50 uM (2x starting concentration; 10% DMSO) and 10 uL were transferred into a 384-well plate. Then 10 uL of a protein/probe/antibody mix was added to each well at final concentrations listed in TABLE 1. Protein:GST-Bcl-2, Probe: F-Bak Peptide Probe Acetyl-GQVGRQLAIIGDK(6-FAM)INR-amide(SEQ ID NO: 1), Protein(nM): 1, Probe (nM): 100, Antibody: Tb-anit-GST, Antibody (nm): 1.  The samples are then mixed on a shaker for 1 minute and incubated for an additional 3 hours at room temperature. For each assay, the probe/antibody and protein/probe/antibody were included on each assay plate as negative and positive controls, respectively. Fluorescence was measured on the ENVISION plate reader (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak peptide) and 495/510 nm (Tb-labeled anti-Histidine antibody) emission filters.
Measurement: Ki < 0.01 nM (pChEMBL 11)
Descriptors: MW 1026.6
Compound (SMILES): CC(C)(C)[C@@H]1OC(=O)[C@H]1NC(=O)OCc1ccc(-c2ccccc2)cc1
InChIKey: ZAIPGFQJHPUGIG-ZWKOTPCHSA-N
Target: N-acylethanolamine-hydrolyzing acid amidase (SINGLE PROTEIN), Homo sapiens
UniProt: Q02083
Assay: Inhibition of recombinant human NAAA expressed in HEK293 cells preincubated for 10 mins followed by PAMCA substrate addition after 30 mins by fluorescence assay
Measurement: IC50 = 507 nM (pChEMBL 6.289999961853027)
Descriptors: MW 353.42, ALogP 3.92, HBA 4, HBD 1, PSA 64.63, QED 0.85
Compound (SMILES): CC(C)(C)OC(=O)N1[C@@H]2CC[C@@H]1CC(Nc1ncnc(Nc3ccc(C#N)cc3Cl)c1[N+](=O)[O-])C2
InChIKey: IEEALEILWWFWFN-HZPDHXFCSA-N
Target: Glucose-dependent insulinotropic receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TDV5
Assay: Agonist activity at human GPR119 expressed in HEK293 cells assessed as stimulation of cAMP level measured after 60 mins by HTRF assay
Measurement: EC50 = 205 nM (pChEMBL 6.690000057220459)
Descriptors: MW 499.96, ALogP 5, HBA 9, HBD 2, PSA 146.31, QED 0.43
Compound (SMILES): Cc1cnc(-c2ccc(N3CCN(S(=O)(=O)c4ccc(OC(C)C)cc4)CC3)cc2OCC(=O)O)o1
InChIKey: NDORPLODVISQRV-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q13258
Assay: Antagonist activity at PGD2 receptor in human platelets assessed as inhibition of PGD2-mediated cAMP accumulation preincubated for 5 mins followed by PGD2 addition measured after 2 mins by HTRF assay
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 515.59, ALogP 3.41, HBA 8, HBD 1, PSA 122.41, QED 0.46
Compound (SMILES): Cc1cccc(-c2ccc(C(=O)c3ccccc3)cc2O)c1
InChIKey: ZVNWAOBWZTZWDN-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: CB1 Receptor Binding Protocol: 8 ug of membranes (20 ul of 1:8 dilution in incubation buffer) was incubated with 5 ul of drug solution (10E-4M to 10E-12M) and 5ul of 5.4 nM [3H]CP55,940 in a total volume of 200 ul for 90 mins at 30 C. Non-specific binding was determined using 10 uM WIN55,212-2 (Ki=4.4 nM). The membranes were filtered and the filters washed 7x with 0.2 ml ice-cold incubation buffer and allowed to air dry under vacuum.
Measurement: Ki > 1000 nM (pChEMBL 6)
Descriptors: MW 288.35, ALogP 4.6, HBA 2, HBD 1, PSA 37.3, QED 0.72
Compound (SMILES): COc1cc(-c2ccncc2)c(F)cc1-n1c(=O)ccc2cc(S(=O)(=O)Nc3ccon3)ccc21
InChIKey: KWBLCJVEGVXGLC-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 = 1800 nM (pChEMBL 5.75)
Descriptors: MW 492.49, ALogP 3.99, HBA 8, HBD 1, PSA 116.32, QED 0.38
Compound (SMILES): Cc1cc(Nc2cc(C(F)(F)F)ccn2)nc(-c2ccc(NC3CCC(C(=O)O)C(C)(C)C3)nc2)c1
InChIKey: ALNPMABMDQCLCT-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 pM 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 uM final concentration). Final volume of the reaction was 10 uL. Phosphorylation of the peptide was allowed to proceed for 45x 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.
Measurement: IC50 < 0.5 nM (pChEMBL 9.300999641418457)
Descriptors: MW 499.54, ALogP 6.3, HBA 6, HBD 3, PSA 100.03, QED 0.37