lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C(C1CCS(=O)(=O)CC1)N1CCN(c2cc(C(F)(F)F)on2)CC1c1cccc(F)c1F
InChIKey: YTLUKWXDWZGNJB-UHFFFAOYSA-N
Target: Sodium- and chloride-dependent glycine transporter 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P48067
Assay: Cytostar-T Assay: In detail, SK-N-MC cells are seeded into 96-well Cytostar-T assay plates at a density of 200,000 cells/well and grown for 16-18 hours to confluence in growth medium as recommended by ATCC. Before starting the assay, cells are washed once with HBSS (Hank's buffered salt solution; Sigma, H8264) cont. 5 mM alanine (referred in here as HBSS/Ala) and afterwards the following reagents are added: 1.80 ul/well HBSS/A1a 2. 20 ul/well of HBSS/A1a containing 6x the concentration of compound in 6% DMSO 3. approx. 5-10 min incubation 4. 20 ul/well 3 uM glycine (3H-glycine (Perkin Elmer, NET004001MC, specific activity: 52 Ci/mmol; diluted 1:1 with unlabelled glycine) in HBSS/A1a. In the final assay, glycine concentration is 500 nM (250 nM derived from the 3H-glycine Perkin Elmer, 250 nM unlabelled glycine), DMSO concentration is 1%. The assay plate is immediately after addition of the 3H-glycine placed into a Micro-Beta Counter (Perkin Elmer) and the signal is recorded over 60 min.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 493.45, ALogP 3.19, HBA 6, HBD 0, PSA 83.72, QED 0.61
Compound (SMILES): CC(C)N1CCC(Cc2ccc3c(c2)C(=O)c2c([nH]c4cc(C#N)ccc24)C3(C)C)CC1
InChIKey: LDOAYQLHGUNOOO-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Inhibition Assay: ALK-inhibiting activity was measured by following an activity of inhibiting phosphorylation by biotinylated peptide (EGPWLEEEEEAYGWMDF). For the detection of phosphorylation of the biotinylated peptide, time-resolved fluorescence measurement was performed using an anti-phosphorylated tyrosine antibody labeled with europium cryptate and streptavidin conjugated to XL665, i.e., an allophycocyanin derivative.
Measurement: IC50 = 11.73 nM (pChEMBL 7.929999828338623)
Descriptors: MW 425.58, ALogP 5.57, HBA 3, HBD 1, PSA 59.89, QED 0.6
Compound (SMILES): CC[C@@H](N[C@@H](C)CC(N)=O)c1ccc(Cl)c(C(=O)c2cccc(O)c2)c1F
InChIKey: RQUXHMOLNCHSSI-MEDUHNTESA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: FRET-Based Assay: The HCV NS3 protease functions have been extensively studied and are considered as potential targets for antiviral therapy: see for example the many references listed in the introductory section of this application. Therefore, the activity of the compounds of the invention as anti-HCV agents was assessed using a full length HCV NS3 protease.The protease activity of the full length NS3/4a was measured using a FRET-based assay utilizing a peptide substrate derived from the NS4A/B cleavage site (Anaspec) and labelled at one end with a quencher (QXL520) and at the other with a fluorophore (5-FAMsp). NS3/4a (produced in-house by literature methods) was incubated with test compounds and peptide substrate in 50 mM Tris pH8, 20 mM DTT, 1% CHAPS, 10% glycerol and 5% DMSO. The reaction was followed by monitoring the change in fluorescence on a Molecular Devices Gemini plate reader for 30 minutes at room temperature.
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 392.86, ALogP 3.72, HBA 4, HBD 3, PSA 92.42, QED 0.6
Compound (SMILES): O=C(N1C[C@H](O)C=C[C@@H]1Cc1ccccc1)n1ncc(C(O)(c2ccc(F)cc2)c2ccc(F)cc2)n1
InChIKey: WTPKQSZKNFVAOG-JWQCQUIFSA-N
Target: Monoacylglycerol lipase ABHD6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BV23
Assay: Inhibition of human ABHD6 using 2-AG as natural substrate by fluorescence based assay
Measurement: IC50 = 39.81 nM (pChEMBL 7.400000095367432)
Descriptors: MW 502.52, ALogP 3.65, HBA 6, HBD 2, PSA 91.48, QED 0.41
Compound (SMILES): CC#CCC1CNC(=O)c2cc(-c3cccc4nc(C)c(NC(C)(C)C)nc34)[nH]c21
InChIKey: UTNCOKVXILEJCJ-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Homogeneous Time Resolved Fluorescence Assay (HTRF): The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 384-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction. Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 22-point dosing curve having a high dose of 1 μM. A reference compound was included on each assay plate [Costar 3658] in order to validate that plate; on one plate of every assay run, two additional reference compounds were included. The Reaction Buffer consisted of 45 mM Hepes, pH 7.0, 15 mM NaCl, and 1 mM MgCl. The quench/detection buffer consisted of 50 mM Tris, 100 mM NaCl, 0.05% BSA, 0.1% Tween and 3 mM EDTA. Biotinylated BAD peptide (Biopeptide), 10 mM ATP (Sigma), Labeled p-BAD (S112) mAb (Cell Signalling and Perkin Elmer) [with 0.05% BSA and 2 mM DTT added] streptavidin:Allophycocyanin [Perkin Elmer]. Final concentrations either Pim-1 enzyme [5 pM], or Pim-2 enzyme [0.5 pM], DMSO [1%], BLC BAD (S112) [0.5 μM], ATP [1.5 μM], streptavidin:Allophycocyanin [0.002 mg/mL] and biotinylated-BAD (S112) mAb [100 pM]. Initial incubations were carried out at RT (22° C.) for 30 min for both Pim-1 and for Pim-2. Pim enzyme is added to compound in buffer, and plates are incubated of 30 min. Biotinylated BAD and ATP are added and plates are incubated for 1 h. A mixture of labeled p-BAD (S112) mAb and quench/detection buffer are added and incubated for 2 h. Fluorescence was measured by an HTRF® Envision microplate reader.
Measurement: IC50 = 3.1 nM (pChEMBL 8.510000228881836)
Descriptors: MW 401.51, ALogP 4.38, HBA 4, HBD 3, PSA 82.7, QED 0.57
Compound (SMILES): OC[C@@]12C[C@@H]1[C@@H](n1cnc3c(N[C@@H](C4CCC4)C4CC4)nc(Cl)nc31)[C@H](O)[C@@H]2O
InChIKey: YLEQGEOHZFVIAR-OHTVDBCQSA-N
Target: 5-hydroxytryptamine receptor 2A (SINGLE PROTEIN), Homo sapiens
UniProt: P28223
Assay: Displacement of [3H]ketanserin from human 5-HT2A receptor expressed in HEK-T cell membranes incubated for 90 mins under dark condition by microbeta scintillation counting method
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 419.91, ALogP 1.75, HBA 8, HBD 4, PSA 116.32, QED 0.53
Compound (SMILES): O=S(=O)(c1ccccc1)N(Cc1ccc(OC(F)(F)F)cc1)c1sc2ccccc2c1Br
InChIKey: JAQJKLKOLXUWJA-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 = 2.5 nM (pChEMBL 8.600000381469727)
Descriptors: MW 542.4, ALogP 6.96, HBA 4, HBD 0, PSA 46.61, QED 0.26
Compound (SMILES): Cc1ncc(-c2cc(C(=O)Nc3ccc(OC(F)(F)F)cc3)cnc2N2CC[C@H](CO)C2)cc1F
InChIKey: FDGBVSBVELNCPQ-HNNXBMFYSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Radio ABL1 (64-515) Assay: For determination of ABL kinase activity, the radiometric filter-binding assay was used. The assay was performed by mixing 10 uL of the compound pre-diluted with 10 uL of ATP (20 uM ATP with 0.1 uCi [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 uL 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 mM at room temperature, the reaction was stopped by the addition of 50 uL 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 3x with 0.5% H3PO4, followed by addition of 30 uL scintillation cocktail (Microscint, Perkin Elmer) per well and then analysed in a TopCount NXT scintillation counter (Perkin Elmer).
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 490.46, ALogP 4.56, HBA 6, HBD 2, PSA 87.58, QED 0.49
Compound (SMILES): COCCOC(=O)N[C@H]1CC[C@H](Nc2cc(NC3CC3)n3ncc(/C=C4\NC(=O)NC4=O)c3n2)CC1
InChIKey: SICWEILFSXFMPV-HKBWDPEPSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 75 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and the resid
Measurement: IC50 > 2500 nM (pChEMBL 5.6020002365112305)
Descriptors: MW 498.54, ALogP 1.58, HBA 10, HBD 5, PSA 160.01, QED 0.2
Compound (SMILES): CCCOc1cccc(CN2CCN(C(=O)Nc3ccccc3)CC2)c1
InChIKey: BEWFBOJQQKJVLJ-UHFFFAOYSA-N
Target: Fatty-acid amide hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O00519
Assay: Biological Assay: Assay Method 1: T84 frozen pellets (contents of 1-4x15 cm culture dishes) were homogenized in 300 mL of FAAH assay buffer (125 mM Tris, 1 mM EDTA, 0.2% glycerol, 0.02% Triton X-100, 0.4 mM Hepes, pH 9). The assay mixture was prepared from 50 uL of the cell homogenate, 10 uL of the test compound, and 40 uL of anandamide [1-3H-ethanolamine] (3H-AEA; Perkin-Elmer, 10.3 Ci/mmol), which was added last, for a final tracer concentration of 200 nM. The reaction mixture was incubated at rt for 1 hour (h). During the incubation, 96-well Multiscreen filter plates (catalog number MAFCNOB50; Millipore, Bedford, Mass., USA) were loaded with 25 uL of activated charcoal (Multiscreen column loader, catalog number MACL09625, Millipore) and washed once with 100 uL of MeOH. Also during the incubation, 96-well DYNEX MicroLite plates (catalog number NL510410) were loaded with 100 uL of MicroScint40 (catalog number 6013641, Packard Bioscience, Meriden, Conn., USA).
Measurement: IC50 = 170 nM (pChEMBL 6.769999980926514)
Descriptors: MW 353.47, ALogP 3.83, HBA 3, HBD 1, PSA 44.81, QED 0.86
Compound (SMILES): Cc1cc(OCCC(C)O)cc(C)c1-c1ccc(F)c(COc2cc3c(cn2)[C@@H]2C(C(=O)O)[C@@H]2C3)c1
InChIKey: MZGKJBGXGMABJL-ZINOHTFKSA-N
Target: Free fatty acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14842
Assay: Agonist activity at human GPR40 expressed in CHO cells coexpressing NFAT BLA assessed as increase in intracellular Ca2+ flux by fluo-4-AM dye based FLIPR assay
Measurement: EC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 491.56, ALogP 5.2, HBA 5, HBD 2, PSA 88.88, QED 0.43
Compound (SMILES): O=C(Nc1ccc2c(c1)OCO2)N(CCN1[C@@H]2CC[C@H]1C[C@@H](c1cccc(O)c1)C2)CC1CCCCC1
InChIKey: UFWSTWFZMFARFW-YGTOKIDASA-N
Target: Delta-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41143
Assay: Displacement of [3H]-diprenorphine from human delta opioid receptor expressed in cell membranes
Measurement: Ki = 100 nM (pChEMBL 7)
Descriptors: MW 505.66, ALogP 5.95, HBA 5, HBD 2, PSA 74.27, QED 0.49
Compound (SMILES): COC(=O)[C@@H]1C[C@H](c2cnc(N)c(-c3ccc(C(=O)N[C@H](CO)c4cccc(Cl)c4)c(F)c3)c2)CN1
InChIKey: MTRVVVZWNCZPGY-PDIWNELESA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Kinase Assay (New): Compound potency against activated ERK2 is determined using a kinase assay that measures ERK2-catalyzed phosphorylation of biotinylated ERKtide peptide substrate ([Biotin]-AHA-K-R-E-L-V-E-P-L-T-P-S-G-E-A-P-N-Q-A-L-L-R-[NH2], the peptide sequence derived from EGF receptor: SEQ ID NO:1). The assay is carried out in 50 mM HEPES [pH 7.5], 5 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 0.05% BSA using 0.25 nM ERK2, 200 nM ERKtide peptide and 35 uM ATP (all concentrations are final in the reaction) in a total volume of 10.25 uL. A 16-point, half-log dilution series of compounds at 41x final concentration is used for generating IC50 curves. Compound dilution series are prepared in 100% DMSO. ERK2 is preincubated with compounds for 30 minutes at ambient temperature. Reaction is initiated by addition of a substrate cocktail of ERKtide peptide and ATP and is allowed to proceed for 2-3 hours at ambient temperature. Reaction is terminated by addition of 10 uL of a 2x stop buffer consisting of 100 mM Tris-Cl [pH 7.5], 25 mM EDTA, 0.01% Tween 20, 10 ug/mL of AlphaScreen Protein A Acceptor Beads, 10 ug/mL of Streptavidin Donor Beads (PerkinElmer, Waltham, Mass.), and 1.4 ug/mL phospho-EGF Receptor (Thr669) antibody (Cat #3056, Cell Signaling Technology, Danvers, Mass.). Terminated reactions are read, after overnight incubation in the dark, on an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, Mass.), with excitation and emission wavelengths set to 680 nm and 570 nm, respectively. IC50 values are determined using a four-parameter fit.
Measurement: IC50 = 24.8 nM (pChEMBL 7.610000133514404)
Descriptors: MW 512.97, ALogP 3.21, HBA 7, HBD 4, PSA 126.57, QED 0.36
Compound (SMILES): CCCN1Cc2nc(NC(=O)[C@H](C)NC)ccc2N(c2ccccc2)C1=O
InChIKey: CGXRIGREXGFZPP-AWEZNQCLSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: In Vitro Competitive Displacement Binding Assay: Modified SMAC peptides and compounds were tested for their ability to displace the fluorescent tracer from either XIAP, cIAP-1 or cIAP-2. BIR3 domains of cIAP-1, cIAP-2 and XIAP were incubated with test compounds or SMAC based peptides and their respective peptide probes (Peptide Protein Research) in assay buffer (50 mM Hepes pH 7.5, 0.025% Tween-20, 0.01% BSA, and 1 mM DTT). Positive controls consisted of BIR3 proteins and tracer (no inhibition) and negative controls contained tracer only (100% inhibition). The samples were incubated at room temperature for 1 hr (XIAP and cIAP-2) or 3 hrs (cIAP-1) prior to being read in the BMG Pherastar in Fluorescence Polarization mode (FP 485 nm, 520 nm, 520 nm).
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 367.45, ALogP 3.11, HBA 4, HBD 2, PSA 77.57, QED 0.82
Compound (SMILES): O=C(NCc1cncnc1)c1cc(NC(=O)c2cc(-c3ncccc3F)c(Cl)cc2Cl)n(-c2ccccc2)n1
InChIKey: XYCBAYLIRXHFPB-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 = 2.02 nM (pChEMBL 8.699999809265137)
Descriptors: MW 562.39, ALogP 5.35, HBA 7, HBD 2, PSA 114.69, QED 0.28
Compound (SMILES): CC(O)CN1CCN(CC[S+]([O-])C(c2ccc(F)cc2)c2ccc(F)cc2)CC1
InChIKey: PHGOEMMHIGVMIN-UHFFFAOYSA-N
Target: Sodium-dependent dopamine transporter (SINGLE PROTEIN), Rattus norvegicus
UniProt: P23977
Assay: Displacement of [3H]WIN35,428 from DAT in Sprague-Dawley rat brain membranes incubated for 120 mins by radioligand binding assay
Measurement: Ki = 289 nM (pChEMBL 6.539999961853027)
Descriptors: MW 422.54, ALogP 2.8, HBA 4, HBD 1, PSA 49.77, QED 0.67
Compound (SMILES): Cn1cc(-c2cc(CC#N)ccc2OCC2CC2)c2cc[nH]c2c1=O
InChIKey: MDFOZSDQSGLITB-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Bromodomain Domain Binding Assay: A time-resolved fluorescence resonance energy transfer (TR-FRET) assay was used to determine the affinities of compounds of the Examples listed in Table 1 for each bromodomain of human BRD4. His-tagged first (BD1: amino acids K57-E168) and second (BD2: amino acids E352-E168) bromodomains of human BRD4 were expressed and purified. An Alexa647-labeled BET-inhibitor was used as the fluorescent probe in the assay. Compound dilution series were prepared in DMSO via a 3-fold serial dilution from 2.5 mM to 42 nM. Compounds were then diluted 6:100 in assay buffer (20 mM Sodium Phosphate, pH 6.0, 50 mM NaCl, 1 mM Ethylenediaminetetraacetic acid disodium salt dihydrate, 0.01% Triton X-100, 1 mM DL-Dithiothreitol) to yield 3× working solutions. Six microliters (L) of the working solution was then transferred to white, low-volume assay plates (Costar #3673). A 1.5× assay mixture containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and the Alexa-647-conjugated probe molecule was also prepared. Twelve L of this solution were added to the assay plate to reach a final volume of 18 L. The final concentration of 1× assay buffer contains 2% DMSO, 50 M-0.85 nM compound, 8 nM His-tagged bromodomain, 1 nM Europium-conjugated anti-His-tag antibody and 100 nM or 30 nM probe (for BDI or BDII, respectively). After a one-hour incubation at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 76.2 nM (pChEMBL 7.119999885559082)
Descriptors: MW 333.39, ALogP 3.39, HBA 4, HBD 1, PSA 70.81, QED 0.78
Compound (SMILES): Cc1c(C(=O)Nc2cccc(C(F)(F)F)c2)cc(C(C)(C)C)n1CC(C)(C)CN1CCNCC1
InChIKey: BJTCMWHIXBIYQG-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBA0
Assay: Inhibition Assay: Test Example 1: (1) The TRPV4 inhibitory activity was measured using CHO-K1 cells stably expressing human TRPV4 (hTRPV4/CHO cells). (2) In the day before the experimental day, frozen cell were thawed and washed with the culture medium (MEM-α, 10% FBS, 2 mmol/L GlutaMax, 50 unit Penicillin, 50 μg/mL Streptomycin). Then cells were suspended in the culture medium. (3) The 384-well plates which hTRPV4/CHO cells were seeded at densities of 4000 cells/well in the culture medium and cultured in a CO2 incubator in the presence of 5% CO2 at 37° C. for overnight was used as assay plates. (4) The assay plate was washed with assay buffer (Hanks, 20 mmol/L HEPES, 2.5 mmol/L probenecid, pH7.4), and the buffer were remained at 20 μL/well. (5) 10 μl of dye loading buffer (9 μmol/L Fluo 4-AM, 0.09% Pluronic F-127/assay buffer) was added to each well, then the assay plate was incubated in a CO2 incubator in the presence of 5% CO2 at 37° C. for one hour. (final 3 μmol/L Fluo4-AM). (6) The assay plate was washed with the assay buffer, and the buffers were remained at 20 μL/well. Then the assay plate was incubated for 10 min at room temperature. (7) Diluted compound solution was dispensed to each well in the compound plate, and mixed with built-in Pipette and Mixer of the Fluorescence analysis system FLIPR TETRA (Molecular devices). (8) After incubation for 5 min, 20 μL of 4α-PDD solution were applied to each well of assay plate and mixed with the FLIPR TETRA. (final 1 Î¼mol/L 4α-PDD). (9) The fluorescent intensity was measured with FLIPR TETRA system for 10 min from the point of time addition the compound solution, at Ex 470-495 nm, Em 515-575 nm wavelength.
Measurement: Ki = 450 nM (pChEMBL 6.349999904632568)
Descriptors: MW 478.6, ALogP 5.3, HBA 4, HBD 2, PSA 49.3, QED 0.59
Compound (SMILES): CC[C@H](NC[C@H](C)C(N)=O)c1ccc(Cl)c(C(=O)c2cccnc2)c1F
InChIKey: RAEPFGFONTWXGZ-NHYWBVRUSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: FRET-Based Assay: The HCV NS3 protease functions have been extensively studied and are considered as potential targets for antiviral therapy: see for example the many references listed in the introductory section of this application. Therefore, the activity of the compounds of the invention as anti-HCV agents was assessed using a full length HCV NS3 protease.The protease activity of the full length NS3/4a was measured using a FRET-based assay utilizing a peptide substrate derived from the NS4A/B cleavage site (Anaspec) and labelled at one end with a quencher (QXL520) and at the other with a fluorophore (5-FAMsp). NS3/4a (produced in-house by literature methods) was incubated with test compounds and peptide substrate in 50 mM Tris pH8, 20 mM DTT, 1% CHAPS, 10% glycerol and 5% DMSO. The reaction was followed by monitoring the change in fluorescence on a Molecular Devices Gemini plate reader for 30 minutes at room temperature.
Measurement: IC50 = 1100 nM (pChEMBL 5.960000038146973)
Descriptors: MW 377.85, ALogP 3.27, HBA 4, HBD 2, PSA 85.08, QED 0.69
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccc(F)cc3)n2)CCC(Nc2ccccn2)CC1
InChIKey: IOIBKVPYKBKQAC-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 433.49, ALogP 3.92, HBA 7, HBD 3, PSA 121.65, QED 0.52
Compound (SMILES): O=C(NCCc1c[nH]nn1)c1cc(NC(=O)c2cc(-c3ncccc3F)ccc2Cl)n(-c2ccccc2)n1
InChIKey: NJGSOIZRAUSYIZ-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 = 68.63 nM (pChEMBL 7.159999847412109)
Descriptors: MW 530.95, ALogP 4.07, HBA 7, HBD 3, PSA 130.48, QED 0.28
Compound (SMILES): COCCCN1CCN(c2cncc(-c3cn(-c4cc(NC(=O)c5ccc(C(C)(C)C)s5)ccc4C)nn3)c2)CC1
InChIKey: SGNZPOHNKGXXOZ-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: B-RAF (V600E) Kinase Assay: In a dilution series 10 uL/well of test substance solution are placed in a multiwell plate. The dilution series is selected so that generally a range of concentrations of 2 uM to 0.119 nM or 0.017 nM is covered. If necessary the initial concentration of 2 uM is changed to 50 uM, 10 uM or 0.4 uM or 0.2857 uM and further dilution is carried out accordingly. The final concentration of DMSO is 5%. 10 uL/well of the B-Raf (V600E)-kinase solution are pipetted in (containing 0.5 ng B-Raf (V600E)-kinase (e.g. from Upstate) in 20 mM Tris-HCl pH 7.5, 0.1 mM EDTA, 0.1 mM EGTA, 0.286 mM sodium orthovanadate, 10% glycerol, 1 mg/mL bovine serum albumin, 1 mM dithiothreitol) and the mixture is incubated for 1 h at RT under with shaking. The kinase reaction is started by the addition of 20 uL/well ATP solution [final concentration: 250 uM ATP, 30 mM Tris-HCl pH 7.5, 0.02% Brij, 0.2 mM sodium orthovanadate, 10 mM magnesium acetate, 0.1 mM EGTA, phosphatase cocktail (Sigma, #P2850, dilution recommended by the manufacturer)] and 10 uL/well MEK1 solution [containing 50 ng biotinylated MEK1 (prepared from purified MEK1 according to standard procedure, e.g. with EZ-Link Sulpho-NHS-LC-Biotin reagent, Pierce, #21335)] and carried out for 60 min at RT with constant shaking. The reaction is stopped by the addition of 12 uL/well of a 100 mM EDTA solution and incubation is continued for a further 5 min. 55 uL/well of the reaction solution are transferred into a streptavidin-coated plate (e.g. Streptawell HighBond, Roche, #11989685001) and shaken gently for 1 h at RT, in order to bind biotinylated MEK1 to the plate. After elimination of the liquid the plate is washed five times with 200 uL/well of 1Ã¿PBS and 100 uL/well solution of primary antibody plus europium-labelled secondary antibody [Anti Phospho-MEK (Ser217/221), Cell Signaling, #9121 and Eu-N1 labelled goat-anti-rabbit antibody, Perkin Elmer, #AD0105] is added, the primary antibody is diluted 1:2000 and the secondary antibody is diluted to 0.4-0.5 ug/mL in Delfia Assay Buffer (Perkin Elmer, #1244-111). After 1 h shaking at RT the solution is poured away and washed five times with 200 uL/well Delfia Wash Buffer (Perkin Elmer, #4010-0010/#1244-114). After the addition of 200 uL/well Enhancement Solution (Perkin Elmer, #4001-0010/#1244-105) the mixture is shaken for 10 min at RT and then measured in a Wallac Victor using the program "Delfia Time Resolved Fluorescence (Europium)". IC50 values are obtained from these dosage-activity curves using a software program (GraphPadPrizm).
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 573.77, ALogP 5.41, HBA 9, HBD 1, PSA 88.41, QED 0.27
Compound (SMILES): O=C(c1cccc(C(F)(F)F)c1Cl)N1C=Cc2c(nnn2-c2cnccn2)C1c1ccccc1
InChIKey: QGKRIXUZLHLZOB-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Radioligand Binding Assay: human or rat P2X7-1321 N1 cells were collected and frozen @−80° C. On the day of the experiment, cell membrane preparations were made according to standard published methods. The total assay volume was 100 μl: 10 μl compound (10×)+(b) 40 μl tracer (2.5×)+50 μl membrane (2×). The tracer used for the assay was tritiated A-804598. The compound can be prepared as described in the literature. (Donnelly-Roberts, D. Neuropharmacology 2009, 56 (1), 223-229.) Compounds, tracer and membranes were incubated for 1 hour @ 4° C. The assay was terminated by filtration (GF/B filters pre-soaked with 0.3% PEI) and washed with washing buffer (Tris-HCl 50 mM).
Measurement: Ki = 363.1 nM (pChEMBL 6.440000057220459)
Descriptors: MW 482.85, ALogP 4.95, HBA 6, HBD 0, PSA 76.8, QED 0.41
Compound (SMILES): COc1cc2ccn(-c3ccc(C(N)=O)c(Nc4ccccc4)c3)c2cc1OC
InChIKey: VGLUYNGPQJIQPN-UHFFFAOYSA-N
Target: Heat shock protein HSP 90-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P07900
Assay: AlphaScreen Competitive Assay: The HSP90-binding activity was measured by an AlphaScreen competitive assay system. The purified HSP90 solution was diluted with a binding buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Triton-X100, 1 mM DTT, 0.1% BSA) and added to a 384-well plate (No. 3673, Corning Incorporated) containing test substances. After reaction at room temperature for 2 hours, biotin-labeled geldanamycin was added to each reaction solution in an amount of 40 nM, followed by reaction for further 1 hour. Detection mix (20 mM HEPES-KOH (pH 7.5), 0.5% BSA, 0.04 mg/mL Nickel Chelate Acceptor beads, 0.04 mg/mL Streptavidin-coated Donor beads) (No. 6760619C, Perkin Elmer, Inc.) was added to each well in the same amount as that of the reaction solution. After reaction in a dark place at room temperature for 1 hour, the fluorescence intensity in each well was measured with a multilabel plate reader, EnVision (Perkin Elmer, Inc.).
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 387.44, ALogP 4.49, HBA 5, HBD 2, PSA 78.51, QED 0.51
Compound (SMILES): CC1=C[C@](C)(O)[C@H](/C(C)=C/C=C/C=C/C=C/[C@]2(C)[C@H]3OC(=O)[C@]2(C)C(=O)[C@@H]3C)O[C@H]1C
InChIKey: KSCZOENBBUGNJI-OIPKEOGYSA-N
Target: Fumarate reductase (SINGLE PROTEIN), Ascaris suum
UniProt: F1KRD8
Assay: Inhibition of NADH-Fumarate reductase in Ascaris suum muscle sub-mitochondrial particles using disodium fumarate as substrate in presence of NADH after 3 mins measured every 15 sec for 10 mins under anaerobic condition
Measurement: IC50 = 850 nM (pChEMBL 6.070000171661377)
Descriptors: MW 426.55, ALogP 4.24, HBA 5, HBD 1, PSA 72.83, QED 0.31
Compound (SMILES): [Na+].[O-]c1c(-n2ccnn2)cnn1-c1cc(N2CCOCC2)ncn1
InChIKey: VYRQLKYGGSWDNH-UHFFFAOYSA-M
Target: Egl nine homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZT9
Assay: Inhibition of recombinant human HIF-PHD2 expressed in insect Sf9 cells using biotinyl-DLDLEMLAPYIPMDDDFQL as substrate after 60 mins by DELFIA
Measurement: IC50 = 280 nM (pChEMBL 6.550000190734863)
Descriptors: MW 314.31, ALogP -0.63, HBA 9, HBD 1, PSA 106.75, QED 0.69
Compound (SMILES): Cc1ncc(C(CNC(=O)c2c(Cl)ccc(F)c2Cl)N2CCC(F)(F)CC2)cn1
InChIKey: AXGMSNUKRWJXFR-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Fluorescent Imaging Plate Reader (FLIPR) Assay: Briefly, 293-human or mouse P2X7 stable cells were incubated in sucrose buffer, pH 7.4 [KCl (5 mM), NaH2PO42H2O (9.6 mM), HEPES (25 mM), sucrose (280 mM), glucose (5 mM), CaCl2 (0.5 mM), and probenecid (0.1425 g in 3 mL 1N NaOH was added for 500 mL solution)] in 384-well plates. 93-rat P2X7 stable cells were incubated in HHPB (pH 7.4) [consisting of Hank's BSS (1X); HEPES (pH 7.4) (20 mM) (Sigma); probenecid (0.710 g/5 mL 1N NaOH) (Sigma); and BSA (0.05%) (Roche) which was added after the pH had been adjusted] in 384-well plates. Fluo-4 NW dye mix (Molecular Probes, Inc., Eugene, Oreg., USA) was prepared in buffer (see manufacturer's instructions). Cell plates were removed from the 37° C. incubator, the media discarded and then 30 μL of dye was added to each well. Plates were placed in the 37° C., non-CO2 incubator for 30 minutes and then room temperature for 30 minutes. Two sets of drug plates were prepared: A) Mixtures of compound plus agonist were prepared as follows, in order to determine close response: BzATP: 11 point ½ log, diluted in buffer, starting from 1 mM. Testing compounds: 11 point ½ log, diluted in 2% DMSO buffer starting from 10 μM. B) Agonist only mixture was prepared with BzATP at a single concentration in buffer (concentration determined by dose response). Compound mixtures (A) were added to assay plates containing cells and placed at room temperature for 30 minutes, then BzATP (B) was added. Fluorescence was read using the Tetra FLIPR® (Molecular Devices, Inc., Sunnyvale, Calif., USA) and IC50 values were calculated by standard methods to determine antagonist activity.
Measurement: IC50 = 37 nM (pChEMBL 7.429999828338623)
Descriptors: MW 447.29, ALogP 4.43, HBA 4, HBD 1, PSA 58.12, QED 0.69
Compound (SMILES): Cc1ccc(CN2CCC(N3CCC(c4ccc(O)cc4)C(F)(F)C3)C2=O)cc1
InChIKey: HKJQOALDDUBRBV-UHFFFAOYSA-N
Target: Glutamate receptor ionotropic, NMDA 2B (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q00960
Assay: Radioligand Binding Assay: To perform the competition binding assay, thawed membrane homogenate was added to each well of a 96-well plate (20 ug/well). The experimental compounds were serially diluted in 100% DMSO and added to each row of the assay plate to achieve desired compound concentrations, keeping the DMSO concentration in the assay plate at 1.33% of the final reaction volume. Next, 3H Ro 25-6981 (4 nM) was added to the assay plate. After incubation for 1 hr at room temperature, the membrane bound radioligand was harvested on to GF/B filter plates (treated with 0.5% PEI for 1 hr at room temperature). The filter plates were dried at 50° C. for 20 mins, incubated with microscint 20 for 10 minutes and finally, the counts were read on TopCount (Perkin Elmer). Non-specific binding was determined using MK-0657 (the preparation of this compound is described as example 1 in WO 2004 108705 (40 uM).
Measurement: Ki = 660 nM (pChEMBL 6.179999828338623)
Descriptors: MW 400.47, ALogP 3.93, HBA 3, HBD 1, PSA 43.78, QED 0.85
Compound (SMILES): CC(C)Oc1ccc(-c2nc(-c3cccc4c3CC[C@@H]4NCCS(C)(=O)=O)no2)cc1C#N
InChIKey: RPDHHUCVIGBRNS-NRFANRHFSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: Agonist activity at human S1P1 expressed in CRE-bla CHOK1 cells assessed as inhibition of forskolin-induced cAMP formation preincubated for 4 hrs followed by FRET-based beta-lactamase fluorescent substrate addition measured after 2 hrs
Measurement: EC50 = 0.05 nM (pChEMBL 10.300000190734863)
Descriptors: MW 466.56, ALogP 3.68, HBA 8, HBD 1, PSA 118.11, QED 0.53
Compound (SMILES): O=C(O)[C@H]1CC[C@H](C(=O)N2CC[C@](c3ccc(C(OCc4c(F)cccc4F)(C(F)(F)F)C(F)(F)F)cc3)(S(=O)(=O)c3ccc(F)cc3)C2)CC1
InChIKey: BFZCOFNHRDYFJK-IHPVIADISA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Scintillation Proximity Assay (SPA): The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 ug/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 uM to 0.28 nM for 10 min at room temperature in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% Glycerol (Sigma Cat#G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat #RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce [3H] 25-hydroxycholesterol specific binding by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 85 nM (pChEMBL 7.070000171661377)
Descriptors: MW 751.66, ALogP 7.43, HBA 5, HBD 1, PSA 100.98, QED 0.18
Compound (SMILES): O=S(=O)(Nc1ccc(S(=O)(=O)N(CCC2CC2)c2sc3ccccc3c2Cl)cc1)C1CC1
InChIKey: JGVVDJZNLXBIBW-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 = 39 nM (pChEMBL 7.409999847412109)
Descriptors: MW 511.09, ALogP 5.45, HBA 5, HBD 1, PSA 83.55, QED 0.41
Compound (SMILES): COc1ccc(F)cc1-c1c[nH]c2ncnc(N3CCOC(c4noc(C)n4)C3)c12
InChIKey: SURQEPAOBKWDSC-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 = 44 nM (pChEMBL 7.360000133514404)
Descriptors: MW 410.41, ALogP 3.04, HBA 8, HBD 1, PSA 102.19, QED 0.55
Compound (SMILES): COc1cc(OC)cc(N(CCN2CCCC(C(N)=O)C2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: VGCISMLLKUTUAO-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 50.12 nM (pChEMBL 7.300000190734863)
Descriptors: MW 515.62, ALogP 3.38, HBA 9, HBD 1, PSA 111.63, QED 0.36
Compound (SMILES): CN(CCC1CCN(C(=O)OCc2cc(Cl)cc(Cl)c2)CC1)C(=O)c1c[nH]c(=O)o1
InChIKey: BYNGXNVERRUMKS-UHFFFAOYSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Inhibition Assay: ATX activity was determined by measurement of released choline in reactions containing ATX (10 nM), choline oxidase (0.1 U/ml), HRP (100 U/ml), amplex red (50 μM) and LPC 18:1 (10 μM). Compounds of the invention were prepared as 10 point serial dilutions from 1 μM in duplicate and pre-incubated with ATX at 37° C. for 20 minutes prior to the addition of remaining reagents. The liberated choline was measured from changes in fluorescence intensity (λex 530 nm, λem 590 nm) of the product resurofin at 37° C. every 2 minutes over a 40-minute period. ATX activity was measured as a slope of the linear portion of the progress curve, typically between 14 to 24 minutes.
Measurement: IC50 = 68 nM (pChEMBL 7.170000076293945)
Descriptors: MW 456.33, ALogP 3.79, HBA 5, HBD 1, PSA 95.85, QED 0.71
Compound (SMILES): CC1=C2Cn3c(c(C4CCCCC4)c4ccc(cc43)C(=O)NS(=O)(=O)N(C)CCOCCN(C)C2=O)-c2ccc(F)cc21
InChIKey: VKPJSPACCVYHEI-UHFFFAOYSA-N
Target: RNA-directed RNA polymerase (SINGLE PROTEIN), Hepacivirus hominis
UniProt: O39930
Assay: Primer-Dependent Transcription Assay: IC50 con1b were determined according to the method as described previously (Pauwels et al, 2007, J Virol 81:6909-19) using a primer-dependent transcription assay. Following a 10 minute preincubation with the inhibitor, 20 nM of purified Con1b NS5B enzyme was incubated for 10 min. with 150 nM 5′-biotinylated oligo (rG13) primer, 15 nM poly(rC) template, 19 mM Tris-HCl, 5 mM MgCl2, 17 mM NaCl, 21 mM KCl, and 2.5 mM DTT. 600 nM GTP and 0.13 μCi of [3H]GTP was then added to initiate the 40-μl reaction mixture, which was then incubated at room temperature for 2 h before the reaction was stopped by addition of 40-μl streptavidin-coated SPA beads.
Measurement: IC50 = 38 nM (pChEMBL 7.420000076293945)
Descriptors: MW 608.74, ALogP 4.68, HBA 6, HBD 1, PSA 100.95, QED 0.43
Compound (SMILES): O=C(CC1CCCCC1)NC[C@H](O)CN1CCc2ccccc2C1
InChIKey: KMVRFQZNMXCWOF-IBGZPJMESA-N
Target: PRMT5/MEP50 complex (PROTEIN COMPLEX), Homo sapiens
UniProt: Q9BQA1
Assay: Inhibition of recombinant human fetal brain FLAG-tagged PRMT5/full length human testis 6His-tagged MEP50 expressed in baculovirus infected insect Sf9 cells using human histone H4 (1 to 15 residues) as substrate preincubated for 30 mins followed by [3H]-SAM addition
Measurement: IC50 = 1001 nM (pChEMBL 6)
Descriptors: MW 330.47, ALogP 2.49, HBA 3, HBD 2, PSA 52.57, QED 0.84
Compound (SMILES): CC1(C)OCC(CNC(=O)COc2ccc3c(c2)C(C)(C)c2[nH]c4cc(C#N)ccc4c2C3=O)O1
InChIKey: ZHSVGGLZOMQPJF-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Inhibition Assay: ALK-inhibiting activity was measured by following an activity of inhibiting phosphorylation by biotinylated peptide (EGPWLEEEEEAYGWMDF). For the detection of phosphorylation of the biotinylated peptide, time-resolved fluorescence measurement was performed using an anti-phosphorylated tyrosine antibody labeled with europium cryptate and streptavidin conjugated to XL665, i.e., an allophycocyanin derivative.
Measurement: IC50 = 51.12 nM (pChEMBL 7.289999961853027)
Descriptors: MW 473.53, ALogP 3.56, HBA 6, HBD 2, PSA 113.44, QED 0.59