lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C(NCc1ccccc1)N[C@H]1CC[C@@H](CCN2CCN(c3nsc4ccccc34)CC2)CC1
InChIKey: HTHHKXUTDJTRGL-DKXQDJALSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Receptor Binding Test: 1. Experimental Materials:(1) D2 receptor Cell Transfection:This experiment utilizes the plasmid vector containing the gene of D2 receptor protein for transfecting HEK293 cells by calcium phosphate transfection method. By culturing the transfected cells in culture medium containing G418 and selecting cell clones followed by radioligand binding test, a stable cell strain capable of expressing the D2 receptor protein stably is finally obtained.(2) Materials for the Receptor Binding Test:Isotopic ligand [3H] Spiperone (113.0 Ci/mmol) purchased from Sigma Corporation; (+) spiperone purchased from RBI Corporation; GF/B glassfiber filter purchased from Whatman Corporation; imported and subpackaged Tris; PPO and POPOP purchased from Shanghai No. 1 Reagent Factory; and fat-soluble scintillation fluid. Beckman LS-6500 multifunction liquid scintillation counter.2. Experimental Methods:    (1) Cells:HEK-293 cells are infected with the recombinant viruses containing various genes. 48-72 h later, receptor proteins are massively expressed on the membrane. The cells are centrifuged at 1000 rpm for 5 min, then the culture supernatant is discarded and the cell pellets are collected, preserved in refrigerator at −20° C., and resuspended with Tris-HCl reaction buffer (pH=7.5) before use.(2) Competitive Receptor Binding Test:The compound to be tested, the radioligand (20 μL for each) and the receptor protein (160 μL) are added to a reaction tube, wherein the compound to be tested and the positive drug both have the final concentration of 10 μmol/L.After being incubated in water bath at 30° C. for 50 min, the tube is immediately transferred to ice bath to stop the reaction. The mixture is subjected to rapid suction filtration through GF/C glassfiber filter on Millipore cell sample collector, washed with eluent (50 mM Tris-HCl, pH 7.5) (3 mL×3) and dried under microwave for 5-6 min. The filter is transferred to a centrifuge tube (0.5 mL), added with 500 μL fat-soluble scintillation fluid, left in the dark for over min, and counted to determine the radioactivity intensity. The percentage inhibition ratio for each compound to inhibit the isotopic ligand binding is calculated with the following equation:inhibition ratio (1%)=(total binding tube CPM−compound CPM)/(total binding tube CPM−non-specific binding tube CPM)×100%Each compound is tested in duplicate, and the tests are carried out independently twice.Compounds with an inhibition ratio of over 95% are subjected to the receptor binding test at a series of concentrations so as to determine half maximal inhibitory concentration (IC50, the concentration of the compound required for inhibiting 50% binding of [3H]-Spiperone to D2 receptor). Each concentration is tested in duplicate, and tests are carried out independently twice for each compound.
Measurement: Ki = 1.02 nM (pChEMBL 8.989999771118164)
Descriptors: MW 477.68, ALogP 4.87, HBA 5, HBD 2, PSA 60.5, QED 0.51
Compound (SMILES): COc1cc(OCc2cccc(-c3ccccc3)c2C)cc(OC)c1CNCCN1CCN(C)CC1
InChIKey: UAKMDFGBYWWAOW-UHFFFAOYSA-N
Target: Programmed cell death 1 ligand 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZQ7
Assay: Homogenous Time-Resolved Fluorescence (HTRF) Binding Assay: All binding studies were performed in an HTRF assay buffer consisting of dPBS supplemented with 0.1% (withv) bovine serum albumin and 0.05% (v/v) Tween-20. For the PD-1-Ig/PD-L1-His binding assay, inhibitors were pre-incubated with PD-L1-His (10 nM final) for 15 m in 4 μl of assay buffer, followed by addition of PD-1-Ig (20 nM final) in 1 μl of assay buffer and further incubation for 15 m. PD-L1 from either human, cyno, or mouse were used. HTRF detection was achieved using europium crypate-labeled anti-Ig (1 nM final) and allophycocyanin (APC) labeled anti-His (20 nM final). Antibodies were diluted in HTRF detection buffer and 5 μl was dispensed on top of binding reaction. The reaction mixture was allowed to equilibrate for 30 minutes and signal (665 nm/620 nm ratio) was obtained using an EnVision fluorometer. Additional binding assays were established between PD-1-Ig/PD-L2-His (20 & 5 nM, respectively), CD80-His/PD-L1-Ig (100 & 10 nM, respectively) and CD80-His/CTLA4-Ig (10 & 5 nM, respectively). Competition studies between biotinylated SEQ ID NO:71 and human PD-L1-His were performed as follows. Inhibitors were pre-incubated with PD-L1-His (10 nM final) for 60 m in 4 μl of assay buffer followed by addition of biotinylated SEQ ID NO:71 (0.5 nM final) in 1 μl of assay buffer. Binding was allowed to equilibrate for 30 m followed by addition of europium crypated labeled Strepatavidin (2.5 pM final) and APC-labeled anti-His (20 nM final) in 5 μl of HTRF buffer. The reaction was allowed to equilibrate for 30 m and signal (665 nm/620 nm ratio) was obtained using an EnVision fluorometer.
Measurement: IC50 = 555 nM (pChEMBL 6.260000228881836)
Descriptors: MW 489.66, ALogP 4.6, HBA 6, HBD 1, PSA 46.2, QED 0.4
Compound (SMILES): CCC(c1cn(-c2ccc(F)c(F)c2)nn1)c1c(C#N)c(-c2cnc(C(F)(F)F)nc2)c2c(N)ncnn12
InChIKey: WWOJLYKLBDUQQC-UHFFFAOYSA-N
Target: Cystic fibrosis transmembrane conductance regulator (SINGLE PROTEIN), Homo sapiens
UniProt: P13569
Assay: Chamber Electrophysiology Assay of CFTR Potentiation in CF Bronchial Epithelial Cells: Primary cystic fibrosis human bronchial epithelial (CF hBE) cells were expanded and cultured according to published methods (Neuberger et al., Ch. 4 of Cystic Fibrosis, Methods in Molecular Biology vol. 741, pp. 39-54 (2011)). Well-differentiated cells (>30 days at air/liquid interface) on Snapwell filters (Corning Costar, cat. no. 3801) were mounted in Ussing chambers (Physiologic Instruments, Inc., San Diego, Calif.). F508del/F508del cultures were assayed at 27° C. and G551D/F508del cells were assayed at 35° C. HEPES buffered physiological saline (composition (in mM): 137 NaCl, 4 KCl, 1 MgCl2, 1.8 CaCl2, 10 HEPES Na) was used in both apical and basolateral chambers. Chambers were bubbled with air to promote mixing and the voltage was clamped to zero. Amiloride (30 uM), forskolin (10 uM), test compound (4 increasing concentrations), and CFTRinh-172 (20 uM) were added sequentially with 20-25 minutes between additions. Short-circuit currents were acquired and analyzed using LabScribe2. Test compound responses were scaled relative to responses for DMSO (0%) and the maximal response of a positive control potentiator (100%).
Measurement: EC50 = 28.4 nM (pChEMBL 7.550000190734863)
Descriptors: MW 526.43, ALogP 4.06, HBA 10, HBD 1, PSA 136.49, QED 0.34
Compound (SMILES): CC(=O)N1CCCN(c2ccc3ncn(CC(=O)N(C)Cc4ccc(Cl)c(C#N)c4)c(=O)c3c2)CC1
InChIKey: MCNSCCICKMJYQM-UHFFFAOYSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Human ATX Enzyme Inhibition Assay: ATX inhibition was measured by a fluorescence quenching assay using a specifically labeled substrate analogue (MR121 substrate). To obtain this MR121 substrate, BOC and TBS protected 6-amino-hexanoic acid (R)-3-({2-[3-(2-{2-[2-(2-amino-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propionylamino]-ethoxy}-hydroxy-phosphoryloxy)-2-hydroxy-propyl ester (Ferguson et al., Org. Lett. 2006, 8, 2023) was labeled with MR121 fluorophore [CAS RN 185308-24-1], 1-(3-carboxypropyl)-11-ethyl-1,2,3,4,8,9,10,11-octahydro-dipyrido[3,2-b:2′,3′-i]phenoxazin-13-ium) on the free amine of the ethanolamine side and then, after deprotection, subsequently with tryptophan on the side of the aminohexanoic acid.Assay working solutions were made as follows:Assay buffer (50 mM Tris-HCl, 140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 0.01% Triton-X-100, pH 8.0;ATX solution: ATX (human His-tagged) stock solution (1.08 mg/mL in 20 mM bicine, pH 8.5, 0.15 M NaCl, 10% glycerol, 0.3% CHAPS, 0.02% NaN3), diluted to 1.4-2.5× final concentration in assay buffer;MR121 substrate solution: MR121 substrate stock solution (800 μM MR121 substrate in DMSO), diluted to 2-5× final concentration in assay buffer.Test compounds (10 mM stock in DMSO, 8 μL) were obtained in 384 well sample plates (Corning Costar #3655) and diluted with 8 μL DMSO. Row-wise serial dilutions were made by transferring 8 μL cpd solution to the next row up to row O. The compound and control solutions were mixed five times and 2 μL were transferred to 384 well assay plates (Corning Costar #3702). Then, 15 μL of 41.7 nM ATX solution was added (30 nM final concentration), mixed five times and then incubated for 15 minutes at 30° C. 10 μL of MR121 substrate solution was added (1 μM final concentration), mixed 30 times and then incubated for 15 minutes at 30° C. Fluorescence was then measured every 2 min for 1 h (Perkin Elmer plate: vision multimode reader); light intensity: 2.5%; exp. time: 1.4 s, Filter: Fluo_630/690 nm) and IC50 values were calculated from these readouts.
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 506.99, ALogP 2.64, HBA 7, HBD 0, PSA 102.54, QED 0.53
Compound (SMILES): COCC(C)(C)NC(=O)CN1[C@@H]2CC[C@H]1C[C@H](CNC(=O)c1cc(F)cc(Cl)c1)C2
InChIKey: YFLGMWJIOZROLO-QIDUDWOJSA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1H (SINGLE PROTEIN), Homo sapiens
UniProt: O95180
Assay: Activity Assay: The HEK293 cells having human Cav3.2 stably expressed therein were cultured at 37° C. in Alpha-MEM, to which 10% (v/v) fetal bovine serum (FBS), penicillin (100 U/mL), streptomycin (100 μg/mL), and G418 (250 g/mL) had been added. The cells were suspended in the culture liquid and were inoculated onto a 96-well plate. Subsequently, the cells were cultured for 48 hours. The culture liquid was removed, and the liquid was changed to S-MEM, to which 5% (v/v) FBS, calcium chloride (0.5 mmol/L), L-glutamine (2 mmol/L), L-alanine (8.9 ng/mL), L-asparagine (13.2 ng/mL), L-aspartic acid (1.33 ng/mL), L-glutamic acid (14.7 ng/mL), glycine (7.5 ng/mL), L-proline (11.5 ng/mL, L-serine (10.5 ng/mL), penicillin (100 U/mL), and streptomycin (100 μg/mL) had been added. The cells were cultured for another 24 hours. The culture liquid was removed again, and the cells were washed with an assay buffer (140 mmol/L sodium chloride, 5 mmol/L potassium chloride, 0.5 mmol/L magnesium chloride, 0.5 mmol/L calcium chloride, 10 mmol/L glucose, 0.4 mmol/L magnesium sulfate, 10 mmol/L HEPES, and 250 mol/L sulfinpyrazone, pH 7.4) that had been kept warm at 37° C. Subsequently, an assay buffer prepared by dissolving Fura2-AM, which is a fluorescent Ca2+ indicator, to a concentration of 5 μM, was added to the cells, and the system was incubated for 30 minutes at 37° C. The assay buffer having Fura2 dissolved therein was removed, and the cells were washed with the assay buffer. Subsequently, an assay buffer prepared by adding a test compound was added to the cells, and the system was incubated for 15 minutes. The plate was mounted on a fluorescence analyzer (FLEX STATION II, Molecular Devices, LLC), and the baseline was measured for 20 seconds. Subsequently, any change in the intracellular calcium concentration that was induced when an assay buffer prepared by adding 100 mmol/L calcium chloride was added to the cells, was measured (excited at 340 nm or 380 nm, and detected at 510 nm).
Measurement: IC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 439.96, ALogP 2.99, HBA 4, HBD 2, PSA 70.67, QED 0.65
Compound (SMILES): CC(C)c1ccnc(-c2nc3nc(C(=O)O)nc(OC(C)C4CCC4)c3n2C[C@H]2CC[C@H](C)CC2)c1
InChIKey: RTRXLBZTEBOHLE-DDAAKYIKSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: FRET Assay: 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 (sequence: 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 p53 binding pocket of HDM2 was used. The HDM2 assay buffer contained 1× Phosphate Buffered Saline (Invitrogen, Cat#14190), 0.01% BSA (Jackson ImmunoResearch, Cat#001-000-162), 0.01% Tween-20. In the 1× assay buffer recombinant HDM2 protein, peptide and Lumi4-Tb Cryptate-conjugate mouse anti-6×His antibody (cisbio, Cat# Tb61HISTLB) were added and transferred to ProxiPlate PLUS (PerkinElmer, Cat#6008269), containing compounds so that final DMSO concentration is 0.1%. Final concentrations of reagents in the assay wells are 0.5 nM HDM2, 0.25 nM anti HIS (Tb label) antibody and 3 nM peptide. After two hour incubation at room temperature in a humidified chamber plates were read on EnVision plate reader with the following settings: excitation: UV, 340 nM, two emission filters: 520 nm and 495 nm respectively. Ratio of em520/em495 was used to calculate % inhibition and to obtain IC50 with 4-parameter logistic equation.
Measurement: IC50 = 10.28 nM (pChEMBL 7.989999771118164)
Descriptors: MW 491.64, ALogP 6.1, HBA 7, HBD 1, PSA 103.02, QED 0.41
Compound (SMILES): NCC(c1ccc(C(F)(F)F)cc1)N1CCN(c2ncnc3c2CCCN3)CC1
InChIKey: CUBQBLLPFKWXRJ-UHFFFAOYSA-N
Target: Proline-rich AKT1 substrate 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96B36
Assay: AKT Enzyme Assay: A TTP Mosquito liquid handling instrument was used to place 125 nl of the appropriate concentration of inhibitor in 100% DMSO (for a dose response curve calculation) into each well of a 384-well plate. To this reaction components were added to a final volume of 12.5 μl:0.1 ng/μl His-AKT (Full Length), (Invitrogen, Part # P2999, Lot #641228C).160 uM ATP (Fluka, 02055)1 mM DTT (Sigma, D0632)1 mM MgCl2 (Sigma, M1028)1 μM substrate peptide (sequence FITC-AHA-GRPRTSSFAEG-NH2),synthesized by Tufts Peptide Synthesis service.100 mM HEPES pH 7.5 (Calbiochem, 391338)0.015% Brij-35 (Sigma, B4184)The reaction was incubated for 90 min at 25° C., and then stopped by the addition of 70 μl of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij-35, 10 mM EDTA (Sigma, E7889)).The plate was read on a Caliper LC 3000 in an Off-Chip mobility shift assay format, using the following parameters for a 12-sipper chip: screening pressure −2.3 psi, upstream voltage −500, and downstream voltage −3000. These conditions caused unphosphorylated substrate and phosphorylated product peptide to resolve as separate peaks allowing direct measurement of percentage of conversion of substrate to product. The percent conversion was plotted against concentration of inhibitor to produce a sigmoidal dose response curve, from which an IC50 was calculated.
Measurement: IC50 = 62 nM (pChEMBL 7.210000038146973)
Descriptors: MW 406.46, ALogP 2.68, HBA 6, HBD 2, PSA 70.31, QED 0.81
Compound (SMILES): C=CC(=O)N1CC[C@H](F)[C@@H](Oc2nc(Nc3cnn(CC(F)F)c3)nc3[nH]ccc23)C1
InChIKey: SGJIYRCDJHFJIF-KBPBESRZSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Inhibitory Activity Against JAK 3 and BTK Enzymes: Specifically, the inhibitory activities against JAK3 and BTK kinase were measured using a JAK3 kinase assay kit (Promega, V9441) and a BTK kinase assay kit (Promega, V9071) which were purchased from Promega. Recombinant purified human JAK3 and BTK were diluted with 1×kinase reaction buffer (JAK3: 40 mM Tris-Cl, pH 7.5, 20 mM MgCl2, 0.1 mg/mL BSA and 50 uM DTT/BTK: 40 mM Tris-Cl, pH 7.5, 20 mM MgCl2, 0.1 mg/mL BSA, 2 mM MnCl2 and 50 uM DTT) and added to 96 well plates (JAK3: final concentration of 4 ng per reaction/BTK: final concentration of 8 ng per reaction). The compounds prepared in the previous Examples were treated so as to be finally a 1% DMSO aqueous solution, and a substrate cocktail containing ATP (JAK3: final concentration of 5 uM/BTK: final concentration of 10 uM) and 0.2 ug/uL of Poly(Glu4, Tyr1)peptide (JAK3 and BTK final concentration) in the total 25 uL reactants was added to 96-well plates to initiate enzymatic reaction. After incubation (30° C.) for 1 hour, equivalent volume (25 uL per reaction) of ADP Glo was added and incubated (30° C.) for 40 minutes at room temperature. Then, a kinase detection reagent (50 uL per reaction) was added and incubated (30° C.) for 30 minutes at room temperature.
Measurement: IC50 = 0.3 nM (pChEMBL 9.520000457763672)
Descriptors: MW 435.41, ALogP 2.67, HBA 7, HBD 2, PSA 100.96, QED 0.55
Compound (SMILES): N#Cc1ccc(-n2ncc(C(=O)Nc3ccnc(C(F)(F)F)c3)c2C(F)(F)F)c2ccnn12
InChIKey: DRGRPQJKWCPPOY-UHFFFAOYSA-N
Target: Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UDY8
Assay: Biochemical Protease Assay: MALT1 protease activity was assessed in an in vitro assay using a tetrapeptide as substrate and full-length MALT1 protein (Strep-MALT1(1-824)-His) purified from baculovirus-infected insect cells. The tetrapeptide LRSR is coupled to AMC (7-amino-4-methylcoumarin) and provides a quenched, fluorescent substrate for the MALT1 protease (SM Biochemicals). Cleavage of AMC from the Arginine residue results in an increase in coumarin fluorescence measured at 460 nm (excitation 355 nm). The final assay buffer consisted of 10 nM FL MALT1 protein, 200 μM Ac-LRSR-AMC, 50 mM Tris pH 7.5, 0.6 M Citrate, 1 mM DTT, 1 mM EDTA, 0.05% BSA and 1.5% DMSO. Test compounds were spotted at 50 nL in 100% DMSO per well of a black 384-Proxiplate (Perkin Elmer). Test compound concentrations ranged from 30 μM to 0.5 nM using 11 dilution steps (1:3). Background signal was measured from control wells containing assay buffer without enzyme which functions as low control (LC). High control (HC) values were generated using the reaction with enzyme but no compound treatment. Compounds were pre-incubated with MALT1 enzyme for 50 minutes at RT. Substrate was added subsequently and fluorescence was measured in Labsystems fluoroskan at excitation 355 nm and emission 460 nm to determine time 0. The reaction was subsequently incubated for 4 h at RT and fluorescence was measured. For ICso calculations, timepoint 0 was subtracted from the 4 h timepoint to correct for any potential autofluorescence of the compounds. The enzyme reaction was linear during the 4 h incubation period. Characterization of the substrate Ac-LRSR-AMC determined the Michaelis constant KM at 200 μM.
Measurement: IC50 = 89 nM (pChEMBL 7.050000190734863)
Descriptors: MW 465.32, ALogP 4.08, HBA 7, HBD 1, PSA 100.9, QED 0.46
Compound (SMILES): Cn1cc(-c2cc(F)c3[nH]nc(C(=O)NC4CCC(F)(F)CC4)c3c2)cn1
InChIKey: UZYANKODRVJYDI-UHFFFAOYSA-N
Target: Glycogen synthase kinase-3 beta (SINGLE PROTEIN), Homo sapiens
UniProt: P49841
Assay: Kinase Assay: GSK3β: Each compound is dissolved in DMSO as a 10 mM stock and used to prepare compound source plates. Serial dilution (1:3, 11-point dose-response curves from 10 M to 0.0003 M) and compound transfer was performed using the ECHO 550 (Labcyte, Sunnyvale, Calif.) into 1536-well black-walled round bottom plates (Corning).The GSK3β kinase assay is run using the Ser/Thr 09 peptide Z-lyte assay kit according to manufacturer's instructions (Life Technologiesa Division of Thermo-Fisher). This is a non-radioactive assay using fluorescence resonance energy transfer (FRET) between coumarin and fluorescein to detect kinase activity which is represented as ratio of coumarin emission/fluorescein emission.Briefly, recombinant GSK3β kinase, ATP and Ser/Thr peptide 09 are prepared in 1× Kinase buffer to final concentrations of 0.04 μg/mL, 46 μM, and 4 μM respectively. The mixture is allowed to incubate with the representative compounds for one hour at room temperature. All reactions were performed in duplicate. Unphosphorylated (0% Control) and phosphorylated (100% control) forms of Ser/Thr 18 serve as control reactions.After incubation, diluted Development Buffer is added to the reaction and allowed to further incubate for one hour at room temperature. The plate is read at Ex 400 Em 455 to detect the coumarin signal and Ex 400 Em 520 to measure the signal (EnVision Multilabel Plate Reader, PerkinElmer).
Measurement: EC50 = 41 nM (pChEMBL 7.389999866485596)
Descriptors: MW 377.37, ALogP 3.41, HBA 4, HBD 2, PSA 75.6, QED 0.73
Compound (SMILES): COc1ccccc1-n1cnc(C(=O)N2[C@H]3CCC[C@@H]2c2nn(C)c(-c4cc(F)c(F)c(F)c4)c2C3)n1
InChIKey: XPKYEGDQLHBJPD-MGPUTAFESA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: In Vitro Activity Assay: The assay used to measure the in vitro activity of MGL is adapted from the assay used for another serine hydrolase (FAAH) described in Wilson et al., 2003 (A high-throughput-compatible assay for determining the activity of fatty acid amide hydrolase. Wilson S J, Lovenberg T W, Barbier A J. Anal Biochem. 2003 Jul. 15; 318(2):270-5). The assay consists of combining endogenously expressed MGL from HeLa cells with test compounds, adding [glycerol-1,3-3H]-oleoyl glycerol, incubating for one hour, and then measuring the amount of cleaved [1,3-3H]-glycerol that passes through an activated carbon filter. The amount of cleaved, tritiated glycerol passing through the carbon filter is proportional to the activity of the MGL enzyme in a particular well/test condition. Standard conditions for this assay combine 300 nM [Glycerol-1,3-3H]-oleoyl glycerol with human MGL from HeLa cells and test compounds for one hour, after which the reaction is filtered through activated carbon and tritium is measured in the flow through. The test compound concentration in screening mode is 10 uM, while the highest compound concentration in IC50 assays is determined empirically.
Measurement: IC50 = 18.1 nM (pChEMBL 7.739999771118164)
Descriptors: MW 508.5, ALogP 4.39, HBA 7, HBD 0, PSA 78.07, QED 0.38
Compound (SMILES): C=CC(=O)Nc1cc(F)ccc1-c1n[nH]c2c1CCC(c1c(Cl)c(OC)cc(OC)c1Cl)=C2
InChIKey: QLBJHBHQFMWBQW-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: FGFR4 Activity Inhibition Test: A HTRF KinEASE-TK 20000 tests kinase assay kit from CISBIO Corp. was used. The kit provided a biotin-labeled substrate, an EU-labeled phosphorylation site-specific antibody, and XL665-labeled avidin and a related buffer. FGFR4 phosphorylated the substrate, Eu-Ab identified the phosphorylated substrate, and XL665-SA binded to the biotin on the substrate, making Eu get closer to XL665, thus generating the HTRF signal. Changes in the activity of kinase was detected by reading the intensity of the HTRF signal. In the kinase assay experiment, there were mainly two steps of reaction: kinase reaction and assay reaction, respectively. In the kinase reaction, the kinase consumed ATP to phosphorylate the substrate, and at the same time produced a substrate containing a phosphate group. In the assay reaction, an assay reagent was added to terminate the kinase reaction. At the same time, the specific antibody and the XL665-labeled avidin in the assay reagent were binded to the phosphate group on the substrate and biotin respectively to generate HTRF signals. The signal intensity was directly proportional to the phosphorylation level of the substrate, and thereby the activity of the kinase FGFR4 could be quantitatively assayed.
Measurement: IC50 = 118 nM (pChEMBL 6.929999828338623)
Descriptors: MW 488.35, ALogP 6.15, HBA 4, HBD 2, PSA 76.24, QED 0.41
Compound (SMILES): CP(C)(=O)c1c(F)cccc1-c1ccc(N2CC[C@@H](NC(=O)Nc3ccc(C(F)(F)F)cc3F)C2=O)c(F)c1Cl
InChIKey: DOCSSCRGZGFMBT-LJQANCHMSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: FPR2 and FPR1 Cyclic Adenosine Monophosphate (cAMP) Assays: A mixture of forskolin (5 μM final for FPR2 or 10 μM final for FPR1) and IBMX (200 μM final) were added to 384-well Proxiplates (Perkin-Elmer) pre-dotted with test compounds in DMSO (1% final) at final concentrations in the range of 0.020 nM to 100 μM. Chinese Hamster Ovary cells (CHO) overexpressing human FPR1 or human FPR2 receptors were cultured in F-12 (Ham's) medium supplemented with 10% qualified FBS, 250 μg/ml zeocin and 300 μg/ml hygromycin (Life Technologies). Reactions were initiated by adding 2,000 human FPR2 cells per well or 4,000 human FPR1 cells per well in Dulbecco's PBS (with calcium and magnesium) (Life Technologies) supplemented with 0.1% BSA (Perkin-Elmer). The reaction mixtures were incubated for 30 min at room temperature. The level of intracellular cAMP was determined using the HTRF HiRange cAMP assay reagent kit (Cisbio) according to manufacturer's instruction. Solutions of cryptate conjugated anti-cAMP and d2 fluorophore-labelled cAMP were made in a supplied lysis buffer separately. Upon completion of the reaction, the cells were lysed with equal volume of the d2-cAMP solution and anti-cAMP solution. After a 1-h room temperature incubation, time-resolved fluorescence intensity was measured using the Envision (Perkin-Elmer) at 400 nm excitation and dual emission at 590 nm and 665 nm. A calibration curve was constructed with an external cAMP standard at concentrations ranging from 1 μM to 0.1 pM by plotting the fluorescent intensity ratio from 665 nm emission to the intensity from the 590 nm emission against cAMP concentrations. The potency and activity of a compound to inhibit cAMP production was then determined by fitting to a 4-parametric logistic equation from a plot of cAMP level versus compound concentrations.
Measurement: EC50 = 0.85 nM (pChEMBL 9.069999694824219)
Descriptors: MW 603.89, ALogP 6.62, HBA 3, HBD 2, PSA 78.51, QED 0.25
Compound (SMILES): CNCCC(Oc1cccc2ncccc12)c1cccnc1
InChIKey: PFSZSDBPCYENAG-UHFFFAOYSA-N
Target: Voltage-dependent calcium channel subunit alpha-2/delta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P54289
Assay: Binding Assay: Human α2δ-1 enriched membranes (2.5 μg) were incubated with 15 nM of radiolabeled [3H]-Gabapentin in assay buffer containing Hepes-KOH 10 mM, pH 7.4.NSB (non specific binding) was measured by adding 10 μM pregabalin. The binding of the test compound was measured at five different concentrations. After 60 min incubation at 27° C., binding reaction was terminated by filtering through Multiscreen GF/C (Millipore) presoaked in 0.5% polyethyleneimine in Vacuum Manifold Station, followed by 3 washes with ice-cold filtration buffer containing 50 mM Tris-HCl, pH 7.4. Filter plates were dried at 60° C. for 1 h and 30 μl of scintillation cocktail were added to each well before radioactivity reading.Readings were performed in a Trilux 1450 Microbeta radioactive counter (Perkin Elmer).
Measurement: Ki > 3000 nM (pChEMBL 5.5229997634887695)
Descriptors: MW 293.37, ALogP 3.36, HBA 4, HBD 1, PSA 47.04, QED 0.76
Compound (SMILES): COc1ccc(-c2cc(C(=O)NCc3c(C)cc(C)[nH]c3=O)cc3c2OC2(CCCC2)C3)cn1
InChIKey: BSPSVCZCZLQCET-UHFFFAOYSA-N
Target: Histone-lysine N-methyltransferase EZH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15910
Assay: Biological Assay: EZH2 Histone Methyl Transferase Assay: The effectiveness of compounds of the present invention as inhibitors of histone methyl transferases can be readily tested by assays known to those skilled in the art. For example, in vitro histone methyl transferase assays may be conducted with a relevant purified histone methyl transferase and an appropriate synthetic substrate to determine the inhibitory activity of the compounds. Assays for inhibition of EZH2 by the instant compounds were performed in 384-well plates with reaction mixtures containing 350 nM of histone peptide substrate (ATKAAR-K(Me2)-SAPATGGVKKPHRYRPG-GK(Biotin), 500 nM S-[methyl-3H]adenosyl-L-methionine (55-85 Ci/mmol), 50 mM Tris-HCl (pH 9.0), 50 mM NaCl, 1 mM dithiothreitol, Tween-20 at 0.01% and fatty-acid free bovine serum albumin at 0.01%, and recombinant EZH2-641F complex (EZH2 Y641F/EED/SUZ12/RbAp48/AEBP2) at 5 nM (>98% purity, BPS Bioscience) or 15 nM (50% purity, in-house). Reaction mixtures were incubated at room temperature for 3 hours, and the reactions were terminated by 0.005% poly-L-lysine solution in 20 mM Tris-HCl (pH 7.5) and 150 mM NaCl. Reaction products were captured by binding to strepavidin-conjugated imaging beads. Incorporation of radioactive methyl group into the histone peptide substrate was determined in Leadseeker (GE Healthcare) by means of scintillation proximity assay.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 459.55, ALogP 4.24, HBA 5, HBD 2, PSA 93.31, QED 0.6
Compound (SMILES): Cn1ccnc1CN1CN(c2ccccc2)C2(CCN(C(=O)c3ccc4[nH]ncc4c3)CC2)C1=O
InChIKey: XYTHPBVXOPRPDS-UHFFFAOYSA-N
Target: Epithelial discoidin domain-containing receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q08345
Assay: In Vitro Binding Assay for DDR1: This assay is based on the intracellular domain of the DDR1 protein which contains the kinase active site. The recombinant protein additionally carries a GST-tag that can be recognized by an Eu-labeled anti-GST antibody. A tracer compound binding to the active site is labeled with a dye so that a FRET donor acceptor pair can be formed. Excitation energy absorbed by the Europium complex (350 nm flash light or pulsed laser) is transferred to a suitable fluorescent dye, if it is in close proximity. Compounds binding competitively with the tracer molecule will displace the bound tracer molecules and reduce the FRET signal in a dose dependent manner. Due to the long lifetime of the Eu excited state, the emission of the donor and the acceptor can be measured in time-gated mode such that most of the intrinsic fluorescence contributions have already decayed. This results in high sensitivity, excellent reproducibility and high data quality. This sensitive detection method enables protein concentrations below 20 nM.Protein, tracer and labeled antibody were obtained from commercial sources. The assay was performed in 384 low volume microtiter-plates with a final volume of 15 μl. Dose response curves were generated from 16 compound dilutions in DMSO as solvent compound dilutions, a solution containing protein and labeled antibody, and a solution containing the tracer which is added in the last step. The fluorescence of donor and acceptor were then measured after one hour incubation at room temperature. Every assay run was quality-controlled with dose response curves for two reference compounds.
Measurement: IC50 = 897 nM (pChEMBL 6.050000190734863)
Descriptors: MW 469.55, ALogP 2.78, HBA 6, HBD 1, PSA 90.36, QED 0.5
Compound (SMILES): COCC1(COC)C(=N)N[C@](C)(c2sc(-c3cccc(C#N)c3)cc2Cl)CS1(=O)=O
InChIKey: JINCEVSAACSHAP-IBGZPJMESA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE1 HTRF FRET Assay: A homogeneous time-resolved FRET assay can be used to determine IC50 values for inhibitors of the soluble human BACE1 catalytic domain. This assay monitors the increase of 620 nm fluorescence that resulted from BACE1 cleavage of an APPswedish APPswe mutant peptide FRET substrate (QSY7-EISEVNLDAEFC-Europium-amide). This substrate contains an N-terminal QSY7 moiety that serves as a quencher of the C-terminal Europium fluorophore (620 nm Em). In the absence of enzyme activity, 620 nm fluorescence is low in the assay and increased linearly over 3 hours in the presence of uninhibited BACE1 enzyme. Inhibition of BACE1 cleavage of the QSY7-APPswe-Eu substrate by inhibitors is manifested as a suppression of 620 nm fluorescence.Varying concentrations of inhibitors at 3Ã¿ the final desired concentration in a volume of 10 ul are preincubated with purified human BACE1 catalytic domain (3 nM in 10 ul) for 30 minutes at 30° C. in reaction buffer containing 20 mM Na-Acetate pH 5.0, 10% glycerol, 0.1% Brij-35 and 7.5% DSMO. Reactions are initiated by addition of 10 ul of 600 nM QSY7-APPswe-Eu substrate (200 nM final) to give a final reaction volume of 30 ul in a 384 well Nunc HTRF plate. The reactions are incubated at 30° C. for 1.5 hours. The 620 nm fluorescence is then read on a Rubystar HTRF plate reader (BMG Labtechnologies) using a 50 millisecond delay followed by a 400 millisecond acquisition time window. Inhibitor IC50 values are derived from non-linear regression analysis of concentration response curves. Ki values are then calculated from IC50 values using the Cheng-Prusoff equation using a previously determined um value of 8 uM for the QSY7-APPswe-Eu substrate at BACE1. The compounds of Examples 1, 2, and 3 were measured in this assay and each exhibited a Ki value of less than about 5 nM.
Measurement: Ki = 135 nM (pChEMBL 6.869999885559082)
Descriptors: MW 468, ALogP 3.18, HBA 7, HBD 2, PSA 112.27, QED 0.67
Compound (SMILES): CS(=O)(=O)Nc1ccc(CC(Nc2ncc(-c3nnc(C(F)F)o3)cn2)c2ccc(F)cc2)cc1
InChIKey: GTVQQMOFNROWOL-UHFFFAOYSA-N
Target: Protein deacetylase HDAC6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBN7
Assay: Enzymatic Activity Assay: All enzymatic reactions were conducted in duplicate at room temperature for 17 hours in a 50 μl mixture containing HDAC assay buffer (50 mM Tris-HCl, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.05% Tween 20, 5 μg BSA), an HDAC substrate, an HDAC enzyme, and a test compound. Compound dilution was prepared ten-fold higher than the final concentration of the compounds with 10% DMSO in HDAC assay buffer and 5 μl of the dilution was added to a 50 μl reaction so that the final concentration of DMSO is 1% in all of reactions. After enzymatic reactions, 50 μl of HDAC Developer was added to each well and the plate was incubated at room temperature for an additional 20 minutes. Fluorescence intensity was measured at an excitation of 360 nm and an emission of 460 nm using a Tecan Infinite M1000 or Biotek Synergy microplate reader.The fluorescent intensity data were analyzed using the computer software, Graphpad Prism. In the absence of the compound, the fluorescent intensity (Ft) in each data set was defined as 100% activity. In the absence of HDAC, the fluorescent intensity (Fb) in each data set was defined as 0% activity. The percent activity in the presence of each compound was calculated according to the following equation: % activity=(F−Fb)/(Ft−Fb), where F=the fluorescent intensity in the presence of the compound.
Measurement: IC50 = 330 nM (pChEMBL 6.480000019073486)
Descriptors: MW 504.49, ALogP 4.37, HBA 8, HBD 2, PSA 122.9, QED 0.34
Compound (SMILES): COCCN1C[C@@H](NC(=O)Nc2cc(-c3cccc(F)c3)nn2C)[C@H](c2ccc(F)c(F)c2)C1
InChIKey: KGAKVAIYSCRCLS-PGRDOPGGSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: TrkA Omnia Assay: Trk enzymatic selectivity was assessed using Omnia Kinase Assay reagents from Invitrogen Corp. Enzyme (TrkA from Invitrogen Corp.) and test compound (various concentrations) were incubated for 10 minutes at ambient temperature in a 384-well white polypropylene plate (Nunc catalog#267462). Omnia Tyr Peptide #4, as well as ATP, were then added to the plate. Final concentrations were as follows: 20 nM enzyme, 500 μM of ATP, 10 μM peptide substrate. The assay buffer consisted of 25 mM MOPS pH 7.5, 0.005% (v/v) Triton X-100 and 5 mM MgCl2. The production of phosphorylated peptide was monitored continuously for 70 minutes using a Molecular Devices FlexStation II384 microplate reader (excitation=360 nm; emission=485 nm). Initial rates were calculated from the progress curves. IC50 values were calculated from these rates using either a 4 or 5-parameter logistic curve fit.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 473.5, ALogP 3.74, HBA 5, HBD 2, PSA 71.42, QED 0.55
Compound (SMILES): Cn1ccnc1CN1CCC2(CC1)COC1c3cc(Cl)ccc3OC(C)(C)C1C2
InChIKey: NOZAHGUXMSQBKL-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Test for Evaluating hERG Inhibition: To a cultured CHO cell strain which stably expresses hERG was added each test compound to adjust the final concentration of DMSO to 0.0135 to 0.5%. The hERG current was measured with QPatch HT (Sophion Inc.), and the concentration at which 50% of the hERG current was inhibited by each test compound (IC50 value; μM) was calculated.
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 415.97, ALogP 4.6, HBA 5, HBD 0, PSA 39.52, QED 0.72
Compound (SMILES): COc1ccc(-c2c(-c3ccc(C#N)c(F)c3)nc(N3CCC(N)CC3)c(=O)n2CCCC(N)=O)cc1
InChIKey: OEJLBMDVHPTFDF-UHFFFAOYSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: In Vitro Enzyme Inhibition Assay: The enzymatic assay of LSD1 activity is based on Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) detection. The inhibitory properties of compounds to LSD1 were determined in 384-well plate format under the following reaction conditions: 0.1-0.5 nM LSD1, 50 nM H3K4me1-biotin labeled peptide (Anaspec cat #64355), 2 uM FAD in assay buffer of 50 mM HEPES, pH7.3, 10 mM NaCl, 0.005% Brij35, 0.5 mM TCEP, 0.2 mg/ml BSA. Reaction product was determined quantitatively by TR-FRET after the addition of detection reagent Phycolink Streptavidin-allophycocyanin (Prozyme) and Europium-anti-unmodified histone H3 lysine 4 (H3K4) antibody (PerkinElmer) in the presence of LSD1 inhibitor such as 1.8 mM of Tranylcypromine hydrochloride (2-PCPA) in LANCE detection buffer (PerkinElmer) to final concentration of 12.5 nM and 0.25 nM respectively. The assay reaction was performed according to the following procedure: 2 uL of the mixture of 150 nM H3K4me1-biotin labeled peptide with 2 uL of 11-point serial diluted test compound in 3% DMSO were added to each well of plate, followed by the addition of 2 uL of 0.3 nM LSD1 and 6 LM of FAD to initiate the reaction. The reaction mixture was then incubated at room temperature for one hour, and terminated by the addition of 6 uL of 1.8 mM 2-PCPA in LANCE detection buffer containing 25 nM Phycolink Streptavidin-allophycocyanin and 0.5 nM Europium-anti-unmodified H3K4 antibody. Enzymatic reaction is terminated within 15 minutes if 0.5 LSD1 enzyme is used in the plate. Plates were read by EnVision Multilabel Reader in TR-FRET mode (excitation at 320 nm, emission at 615 nm and 665 nm) after 1 hour incubation at room temperature.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 504.57, ALogP 2.79, HBA 8, HBD 2, PSA 140.26, QED 0.48
Compound (SMILES): Cc1cc(N)nc(C)c1CNC(=O)c1ccc2c(c1)C1CC2C=C1c1ccccn1
InChIKey: NLSKYUYHLSRTNU-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Plasma Kallikrein Inhibition Assay: The chromogenic substrate D-Pro-Phe-Arg-pNa, 2HCl (BIOPHEN CS-31(02) from Hyphen BioMed, Neuville-Sur-oise, France) was dissolved in 5 mL deionized water and stored at 4° C. Concentration was determined in the spectrophotometer at 342 nm using an extinction coefficient of 8270. All other chemicals were of analytical grade.Human plasma kallikrein was purchased from Enzyme Research Labs (South Bend, Ind., USA, batch HPKa 2830). A stock solution of 7 μM in 50% glycerol was stored at −20° C.Enzyme reactions were conducted in assay buffer comprised of 20 mM HEPES at pH 7.4, 150 mM NaCl, 0.1% PEG-8000 and 0.01% Triton X-100.Both enzyme and substrate were diluted in assay buffer.The compound solutions as well as the enzyme and the substrate solutions were transferred to 96-well plates (Clear, UV-Star, Flat-bottom, Half-Area plates; cat. No. 675801 Greiner Bio-one, purchased from VWR International, Arlington Heights, Ill., USA) using a Rainin LTS 96-channel pipettor (Rainin, Columbus, Ohio, USA). Plate measurements were conducted using a SPECTROStar Nano reader (BMG Labtech, San Francisco, Calif., USA). The SPECTROStar Nano is a spectrophotometer and absorbance was measured at 405 nm. We used discrete wavelength, precise, kinetic reads of 15 cycles with a 60 sec cycle time.Determination of IC50 valuesFor the determination of IC50 values, the assays were performed at room temperature in 96-well plates with a total assay volume of 85 μL per well. The test compound was dissolved in 100% DMSO. The compounds were serially diluted in DMSO in a 7 point dose response. For the assays, 66.5 μL of protease solution (protease in assay buffer) was added per well followed by the addition of 8.5 μL of compound in 100% DMSO. The final assay concentration of the human plasma kallikrein was 250 μM. After 30 min. incubation at room temperature on an orbital shaker, the reactions were started by the addition of 10 μL substrate solution (in assay buffer, final assay concentration was 600 uM). After the addition of the substrate solution the final DMSO concentration was 10%. The plate was placed again on the shaker for 5 sec, spun at 2000 rpm for 5 sec and read on the spectrophotometer. The effect of the compound on the enzymatic activity was obtained from the linear part of the progress curves and determined after 15 minutes.
Measurement: IC50 = 14454 nM (pChEMBL 4.840000152587891)
Descriptors: MW 396.49, ALogP 4.27, HBA 4, HBD 2, PSA 80.9, QED 0.69
Compound (SMILES): CC[C@H]1[C@@H](O)[C@@H]2[C@H](CC[C@]3(C)[C@@H]([C@H](C)CCNC(=O)NS(=O)(=O)c4ccc(C5CCCCC5)cc4)CC[C@@H]23)[C@@]2(C)CC[C@@H](O)C[C@@H]12
InChIKey: NVGBQEPMFILTKP-ILSHJBTGSA-N
Target: Bile acid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q96RI1
Assay: Human FXR (NR1H4) Assay: The potency and efficacy of the compounds of the invention on TGR5 receptor was evaluated using in vitro assays which carried out using the express kit from DiscoverX (cAMP HUNTER eXpress GPBAR1 CHO-K1 GPCR Assay; Cataloguer number: 95-0049E2CP2S)GPBAR1 (G protein-coupled bile acid receptor 1) encodes a member of the G protein-coupled receptor (GPCR) superfamily. GPBAR1 activation following ligand binding initiates a series of second messenger cascades that result in a cellular response. Treatment of CHO cells expressing GPBAR1 with bile acids induces the production of intracellular cAMP and internalization of the receptor. The potency and efficacy of compound for GPBAR1 activation by measuring cyclic adenosine monophosphate (cyclic AMP or cAMP) levels in live cells using a competitive immunoassay based on Enzyme Fragment Complementation (EFC).In briefly, following seeding the cells into the white, 96 well microplate, place it in a 37° C., 5% CO2 in a humidified incubator for 18-24 hours prior to testing. On second day, proceed to the appropriate cAMP HUNTER eXpress Protocol according to the manufacturer's instructions. Dissolve agonist compound in DMSO at the desired stock concentration, and prepare 3-fold serial dilutions of agonist compound in Cell Assay Buffer. The concentration of each dilution should be prepared at 4× of the final screening concentration (i.e. 15 μL compound+45 μL Cell Assay Buffer/cAMP Antibody Reagent). For each dilution, the final concentration of solvent should remain constant. Transfer 15 μL diluted compound the assay plate and incubate the plate for 30 minutes at 37° C. Following agonist incubation, add 60 μL of working cAMP detection reagents/cAMP Solution mixture (cAMP Lysis Buffer, Substrate Reagent 1, cAMP Solution D) to the appropriate wells. Incubate for 1 hour at room temperature (23° C.), protected from light. Add 60 μl of cAMP Solution A to the appropriate wells. Incubate for 3 hours at room temperature (23° C.), protected from light. Read samples on Envision standard luminescence plate reader. Calculate of average EC50 after logarithm transformation.
Measurement: EC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 656.97, ALogP 7.38, HBA 5, HBD 4, PSA 115.73, QED 0.23
Compound (SMILES): N#CN1Cc2[nH]nc(-c3ccc(OCc4ccccc4)cc3)c2C1
InChIKey: JBGQYSLZJROGNG-UHFFFAOYSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q93009
Assay: Biochemical Assay: Dilution plates were prepared at 21 times the final concentration (2100 μM for a final concentration of 100 μM) in 50% DMSO in a 96-well polypropylene V-bottom plate (Greiner #651201). A typical 8-point dilution series would be 100, 30, 10, 3, 1, 0.3, 0.1, 0.03 μM final. Reactions were performed in duplicate in black 384 well plates (small volume, Greiner 784076) in a final reaction volume of 21 μl. Either 1 μl of 50% DMSO or diluted compound was added to the plate. USP7 was diluted in reaction buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 0.05% Tween 20, 0.5 mg/ml BSA, 5 mMbeta mercaptoethanol) to the equivalent of 0.0025 μl/well and 10 μl of diluted USP7 was added to the compound. Enzyme and compound were incubated for 30 min at room temp. Reactions were initiated by the addition of 50 nM of TAMRA labelled peptide linked to ubiquitin via an iso-peptide bond as fluorescence polarisation substrate. Reactions were read immediately after addition of substrate and following a 2 hr incubation at room temperature. Readings were performed on a Pherastar Plus (BMG Labtech). λ Excitation 540 nm; λ Emission 590 nm.
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 316.36, ALogP 3.45, HBA 4, HBD 1, PSA 64.94, QED 0.75
Compound (SMILES): COc1ccc(-n2nc(C)c3c(Cl)c(C(=O)Nc4ccc(C(F)(F)F)cc4)cnc32)cc1
InChIKey: RBEJZYKVSZZSJM-UHFFFAOYSA-N
Target: Glucocorticoid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04150
Assay: Biological Evaluation: The assay was performed using H4 neuroglioma cells and using indirect immunofluorescence detection of the endogenous GR receptor. Fixative: 4% PFA. Permeabilization buffer: 0.3% Triton X-100 in PBS. Primary antibody: 1:200 in 5% BSA block. Secondary antibody: 1:800 in 5% BSA block. HCS Cell Mask: 1:6000 in PBS. DAPI: 100 ng/ml.
Measurement: IC50 = 275 nM (pChEMBL 6.559999942779541)
Descriptors: MW 460.84, ALogP 5.66, HBA 5, HBD 1, PSA 69.04, QED 0.43
Compound (SMILES): O=C1CCC(N2Cc3cc(N[C@H]4CCCC[C@@H]4NCC4CCCCC4)ccc3C2=O)C(=O)N1
InChIKey: OCJMRBGAANJKCG-OJSMNCEXSA-N
Target: Zinc finger protein Helios (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UKS7
Assay: Prolabel Quantification of IKZF1, IKZF2, or GSPT1 Protein Levels in 293GT Cells: The Prolabel system from DiscoverX was used to develop high-throughput and quantitative assays to measure changes in IKZF1, IKZF2, and GSPT1 protein levels in response to compounds. The prolabel tag is derived from the alpha fragment of beta galactosidase and has the following protein sequence: mssnslavvlqrrdwenpgvtqlnrlaahppfaswrnseeartdrpsqqlrslnge. The complementary fragment of beta-galactosidase (from DiscoverX), is added to the prolabel tag to form an active beta galactosidase enzyme whose activity can be precisely measured. In this way, the levels of a fusion protein with the prolabel tag can be quantified in cell lysates.Lentiviral vectors, based on the Invitrogen pLenti6.2/V5 DEST backbone, were constructed that placed the prolabel tag upstream of IKZF1, IKZF2, or GSPT1 and expressed the fusion protein from a CMV promoter.To ensure moderate and consistent expression of the prolabel fusion proteins across all cells in the population, stable cell lines were constructed from cells expressing a single copy of the construct. Lentivirus packaged with the constructs was made using the Virapower kit from Invitrogen. Strongly adherent 293GT cell, GripTite 293 MSR cells from Thermo Fisher Scientific (Catalog number: R79507), were infected with the virus at low multiplicity of infection and selected by 5 μg/mL blasticidin for 2 weeks.
Measurement: EC50 = 36 nM (pChEMBL 7.440000057220459)
Descriptors: MW 452.6, ALogP 3.34, HBA 5, HBD 3, PSA 90.54, QED 0.58