lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): C[C@]1(O)CCC[C@@H]1n1c(=O)c(Cl)cc2cnc(NC3CCN(S(N)(=O)=O)CC3)nc21
InChIKey: AQSKGIKUFDLXKA-KSSFIOAISA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24941
Assay: CDK2/Cyclin E1 Mobility Shift Assay: The purpose of the CDK2/Cyclin E1 assay is to evaluate the inhibition (% inhibition, Kiapp and Ki values) of small molecule inhibitors by using a fluorescence-based microfluidic mobility shift assay. CDK2/Cyclin E1 catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide FL-Peptide-18 (5-FAM-QSPKKG-CONH2) (SEQ ID NO:1). (CPC Scientific, Sunnyvale, Calif.). The mobility shift assay electrophoretically separates the fluorescently labeled peptides (substrate and phosphorylated product) following the kinase reaction. Both substrate and product are measured and the ratio of these values is used to generate % conversion of substrate to product by the LabChip EZ Reader. Wild-type full length CDK2/wild-type full length Cyclin E1 enzyme complex was produced in-house (baculoviral expression, LJIC-2080/LJIC-2103) and phosphorylated by CDK7/Cyclin H1/Mat1 enzyme complex with CDK2:CDK7 ratio of 50:1 (concentration mg/mL) in the presence of 10 mM MgCl2 and 5 mM ATP at room temperature for one hour. Typical reaction solutions (50 μL final reaction volume) contained 2% DMSO (± inhibitor), 4 mM MgCl2, 1 mM DTT, 150 μM ATP (ATP Km=67.4 μM), 0.005% Tween-20, 3 μM FL-Peptide-18, and 0.36 nM (catalytically competent active site) phosphorylated wild-type full length CDK2/Cyclin E1 enzyme complex in 25 mM HEPES buffer at pH 7.15. The assay was initiated with the addition of ATP, following a fifteen minutes pre-incubation of enzyme and inhibitor at room temperature in the reaction mixture. The reaction was stopped after 45 minutes at room temperature by the addition of 50 μL of 80 mM EDTA, pH 7.5. The Ki value was determined from the fit of the data to the Morrison tight-binding competitive inhibition equation with the enzyme concentration as a variable.
Measurement: Ki = 1.47 nM (pChEMBL 8.829999923706055)
Descriptors: MW 456.96, ALogP 1, HBA 8, HBD 3, PSA 143.44, QED 0.62
Compound (SMILES): CC(C)n1c(CCl)nc2c(F)cc(-c3nc(N[C@@H]4CCOC[C@H]4O)ncc3Cl)cc21
InChIKey: LTJSYFAVWOFESN-GDBMZVCRSA-N
Target: Cyclin-dependent kinase 4/cyclin D1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P11802
Assay: Biological Assay: CDK4/Cyclin D1: The purpose CDK4/Cyclin D1 assay is to evaluate the inhibition (% inhibition, Kiapp and Ki values) in the presence of small molecule inhibitors by using a fluorescence based microfluidic mobility shift assay. CDK4/Cyclin D1 catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide 5-FAM-Dyrktide (5-FAM-RRRFRPASPLRGPPK) (SEQ ID NO:1). The mobility shift assay electrophoretically separates the fluorescently labeled peptides (substrate and phosphorylated product) following the kinase reaction. Both substrate and product are measured and the ratio of these values is used to generate % Conversion of substrate to product by the LabChip EZ Reader. Typical reaction solutions contained 2% DMSO (±inhibitor), 10 mM MgCl2, 1 mM DTT, 3.5 mM ATP, 0.005% TW-20, 3 μM 5-FAM-Dyrktide, 3 nM (active sites) activated CDK4/Cyclin D1 in 40 mM HEPES buffer at pH 7.5.
Measurement: Ki = 2.4 nM (pChEMBL 8.619999885559082)
Descriptors: MW 454.33, ALogP 4.17, HBA 7, HBD 2, PSA 85.09, QED 0.56
Compound (SMILES): COc1ccc(F)cc1-c1ccnc2[nH]c(C3=CCN(C(=O)NCCNCc4cccc(C(F)(F)F)c4)CC3)cc12
InChIKey: KQKRNCWYCGHHSU-UHFFFAOYSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), Homo sapiens
UniProt: O60563
Assay: LANCE ULight TR-FRET Kinase Assay: CDK9 enzyme activities were measured using LANCE ULight TR-FRET kinase assay reagents (PerkinElmer, Waltham, Mass.). Compounds were directly added in 100% DMSO to white low volume assay plates (Perkin Elmer Proxiplate 6008289) using a Labcyte Echo acoustic dispenser. Assay reagents in serine/threonine kinase assay buffer containing 20 mM HEPES, 10 mM MgCl2, 100 mM Na3VO4, and 0.0075% Triton X-100. were added for final reaction mixture concentrations of 1000 μM ATP, 100 nM U-light MBP peptide (Perkin Elmer TRF0109M) and reaction initiated with 4 nM CDK9/Cyclin T1 (Carna Biosciences 04-110). The kinase reaction was carried out for 30 minutes before addition of stopping buffer to a final of 20 mM EDTA and 0.5 nM of LANCE Ultra Europium anti-phospho-MBP antibody (PerkinElmer TRF0201M) in LANCE detection buffer (PerkinElmer CR97-100). The reaction was equilibrated for 1 hour and the signal read in the Perkin Elmer Envision in TR-FRET mode (excitation at 320 nm and emission at 615/665 nm).
Measurement: IC50 = 180 nM (pChEMBL 6.75)
Descriptors: MW 567.59, ALogP 5.98, HBA 4, HBD 3, PSA 82.28, QED 0.18
Compound (SMILES): O=C(CCC(=O)c1ncccn1)c1ccc2c(c1)CCCC2
InChIKey: NVNGARWVDDHUHA-UHFFFAOYSA-N
Target: Catenin beta-1/Protein Wnt-3a (PROTEIN COMPLEX), Homo sapiens
UniProt: P35222
Assay: Evaluation of Biological Activity: Compounds that enhance the Wnt activity, or Activators, were assayed as follows. Reporter cell lines were generated by stably transducing cells of colon cancer cell lines (with a lentiviral construct that included a Wnt-responsive promoter driving expression of the firefly luciferase gene).Lentiviral constructs were made in which the SP5 promoter, a promoter having eight TCF/LEF binding sites derived from the SP5 promoter, was linked upstream of the firefly luciferase gene. The lentiviral constructs also included a hygromycin resistance gene as a selectable marker. The SP5 promoter construct was used to transduce SW480 cells, a colon cancer cell line having a mutated APC gene that generates a truncated APC protein, leading to de-regulated accumulation of β-catenin.Cultured SW480 cells bearing a reporter construct were distributed at approximately 10,000 cells per well into 384- or 96-well multiwell plates. Compounds from a small molecule compound library were then added to the wells in half-log dilutions using a maximum concentration of three or ten micromolar. A series of control wells for each cell type received only buffer and compound solvent DMSO. Twenty-four hours after the addition of compound, reporter activity for luciferase was assayed, for example, by addition of the BrightGlo luminescence reagent (Promega) and the Victor3 plate reader (Perkin Elmer). Readings were normalized to DMSO only treated cells, and any activities above DMSO were considered activation. Compounds were considered activators if reporter activities were 2× fold or greater than DMSO. EC50 is the concentration at half maximal activation.
Measurement: EC50 = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 294.35, ALogP 3.2, HBA 4, HBD 0, PSA 59.92, QED 0.79
Compound (SMILES): CC(=O)c1c(C)c2cnc(N[C@@H]3CCOC[C@H]3O)nc2n([C@H]2C[C@H](O)C2)c1=O
InChIKey: VTEOJXOIAQEYFO-QHSBEEBCSA-N
Target: Cyclin-dependent kinase 4/cyclin D1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P11802
Assay: Chelation-Enhanced Fluorescence (CHEF) Assay: CDK4/Cyclin D1 and CDK6/Cylcin D3: The Chelation-Enhanced Fluorescence (CHEF) monitors the phosphorylation state in real time where the level of fluorescence is directly proportional to the amount of phosphorylated substrate. CHEF exploits a synthetic α-amino acid with a side chain bearing an 8-hydroxyquinoline derivative (sulfonamido-oxine, Sox) which, upon coordination to Mg(II), relays information on the phosphorylation state of a proximal serine, threonine or tyrosine residue in peptide-based kinase substrates. Phosphorylation of a specific peptide (Catalog # AQT0258) from AssayQuant Technologies results in an increase in fluorescence at the excitation and emission wavelengths of 360 nm Ex/485 nm Em.Examples along with DMSO (negative) and Palbociclib (positive) controls were added to 384-well plates at 100-fold their final concentrations followed by the addition of 10 nM CDK4/Cyclin D1 (LJIC-2007F1) or 10 nM CDK6/Cyclin D3 (LJIC-2009H2) for a 20 minute pre-incubation in assay buffer containing 40 mM HEPES, 1 mM Dithiothreito (DTT, 10 mM MgCl2, 1% Glycerol, 0.1% BSA. Enzyme reactions were initiated by the addition of AssayQuant Technologies peptide and ATP substrates (10 μM CHEF peptide (Catalog # AQT0258), 2 mM ATP) and allowed to proceed for 2 hours followed by fluorescence read of the reaction. Ki determinations were made from a plot of the fractional velocity as a function of inhibitor concentration fit to the Morrison equation with the enzyme concentration as a variable.
Measurement: Ki = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 388.42, ALogP 0.56, HBA 9, HBD 3, PSA 126.57, QED 0.65
Compound (SMILES): CC(F)(F)c1cc(-n2cc(COc3nccc(N4CCCC4)n3)nn2)ccc1Cl
InChIKey: XQHJKFUMKCVZTG-UHFFFAOYSA-N
Target: Glutamate NMDA receptor; GRIN1/GRIN2B (PROTEIN COMPLEX), Homo sapiens
UniProt: Q13224
Assay: NMDA receptor FLIPR assay: NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 149 mM NaCl, 4 mM KCl, 2 mM CaCl2, and 1.5 mM MgCl2, 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (149 mM NaCl (standard assay) or 150 mM (HTS assay), 4 mM KCl (standard assay) or 3 mM (HTS assay), 2 mM CaCl2, 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to EC80 (standard assay) or EC40 (HTS assay) is used to maximize the assay's signal window or the ability to detect NMDA receptor antagonists and negative allosteric modulators, respectively. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 43 nM (pChEMBL 7.369999885559082)
Descriptors: MW 420.85, ALogP 4, HBA 7, HBD 0, PSA 68.96, QED 0.6
Compound (SMILES): CN1CCN(c2ccc(Nc3ncc4cc5n(c4n3)[C@@H](c3ccccc3)CNC5=O)nc2)CC1
InChIKey: PBMNQMWMHILVLQ-OAQYLSRUSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24941
Assay: Kinase Enzymatic Assay: Kinase enzymatic reactions were performed in 384-well microplates using a 12-channel Caliper LabChip instrument as a detection device. The enzymatic phosphorylation of a peptide results in a change in net charge, enabling electrophoretic separation of product from substrate. As substrate and product are separated, two peaks of fluorescence are observed. Change in the relative fluorescence intensity of the substrate and product peaks is the parameter measured, reflecting enzyme activity. In the presence of an inhibitor, the ratio between product and substrate is altered. The signal of the product decreases, while the signal of the substrate increases.For the measurement of CDK2/cyclinE activity, enzyme (0.22 nM) was incubated with 100 mM ATP and the phosphoacceptor substrate peptide (1 mM) for one hour. For the measurement of CDK4/CyclinD activity, enzyme (0.85 nM) was incubated with 200 mM ATP and the phosphoacceptor substrate peptide (1 mM) for three hours. Potential inhibitor compounds (as HCl salts) were tested using 12-point dose response curves in single point at the Km for ATP.
Measurement: IC50 = 50500 nM (pChEMBL 4.300000190734863)
Descriptors: MW 454.54, ALogP 2.65, HBA 8, HBD 2, PSA 91.21, QED 0.49
Compound (SMILES): C=CC(=O)N1CCC(C(=O)Nc2cccc(CNc3nc(OCCOC)nc4c(C(C)C)cnn34)c2)C1
InChIKey: IZLKPZFDMWBZLM-UHFFFAOYSA-N
Target: CDK7/Cyclin H/MNAT1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P50613
Assay: Biochemical Assay for CDK7: The ability of compounds to inhibit CDK7 kinase activity was tested in a TR-FRET assay using 5 nM of CDK7/CycH/MNAT1 obtained from Invitrogen, USA. Test compounds were pre-incubated with the kinase at room temperature for 60 min. After incubation, substrate mix [100 nM Ultra-light MBP (Perkin Elmer, USA) and 1 mM ATP (Sigma)] was added. The above reaction was stopped by the addition of 40 mM EDTA after 60 minutes of kinase reaction. 1 nM Eu-labelled antiphospho-MBP antibody [Perkin Elmer, USA] was added, mixed well and the fluorescence emission at 615 nm and 665 nm [excitation at 340 nm] was measured. The final DMSO concentration in the assay was 1%. For IC50 determination, appropriate concentrations were made by ⅓rd serial dilutions of 10 mM DMSO stock solution of test compound. All the fluorescence measurements were made in a Victor 3 Multilabel Counter [Perkin Elmer, USA]. The IC50 was determined by fitting the dose response data to sigmoidal curve fitting equation using GraphPad Prism software V5. To identify compounds that inhibit CDK7 irreversibly, time depended inhibition studies were carried by pre-incubating compound with the enzyme at three time points (20, 60 and 180 min) and carrying out assay as described above.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 507.6, ALogP 2.86, HBA 9, HBD 2, PSA 122.98, QED 0.3
Compound (SMILES): C=CC(=O)Nc1cn(C)nc1C(=O)Nc1cccc(CNc2nc(NC3CCOCC3)nc3c(C(C)C)cnn23)c1
InChIKey: VSIOELSCXPKQDT-UHFFFAOYSA-N
Target: CDK7/Cyclin H/MNAT1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P50613
Assay: Biochemical Assay for CDK7: The ability of compounds to inhibit CDK7 kinase activity was tested in a TR-FRET assay using 5 nM of CDK7/CycH/MNAT1 obtained from Invitrogen, USA. Test compounds were pre-incubated with the kinase at room temperature for 60 min. After incubation, substrate mix [100 nM Ultra-light MBP (Perkin Elmer, USA) and 1 mM ATP (Sigma)] was added. The above reaction was stopped by the addition of 40 mM EDTA after 60 minutes of kinase reaction. 1 nM Eu-labelled antiphospho-MBP antibody [Perkin Elmer, USA] was added, mixed well and the fluorescence emission at 615 nm and 665 nm [excitation at 340 nm] was measured. The final DMSO concentration in the assay was 1%. For IC50 determination, appropriate concentrations were made by ⅓rd serial dilutions of 10 mM DMSO stock solution of test compound. All the fluorescence measurements were made in a Victor 3 Multilabel Counter [Perkin Elmer, USA]. The IC50 was determined by fitting the dose response data to sigmoidal curve fitting equation using GraphPad Prism software V5. To identify compounds that inhibit CDK7 irreversibly, time depended inhibition studies were carried by pre-incubating compound with the enzyme at three time points (20, 60 and 180 min) and carrying out assay as described above.
Measurement: IC50 < 300 nM (pChEMBL 6.5229997634887695)
Descriptors: MW 558.65, ALogP 3.56, HBA 11, HBD 4, PSA 152.39, QED 0.21
Compound (SMILES): N#Cc1ccc(OC2CN(Cc3ccc(-c4cccc5nc(NC(=O)C6CC6)nn45)cc3)C2)cc1
InChIKey: YMKCUJXCCBORDH-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK2 Inhibition Assay: Recombinant human JAK2 catalytic domain (amino acids 808-1132; catalog number PV4210) was purchased from Invitrogen. 0.025 mU of JAK2 was incubated with 2.5 μg polyGT substrate (Sigma catalog number P0275) in kinase reaction buffer (5 mM MOPS pH 7.5, 9 mM MgAc, 0.3 mM EDTA, 0.06% Brij and 0.6 mM DTT, 1 μM non-radioactive ATP, 0.25 μCi 33P-gamma-ATP (GE Healthcare, catalog number AH9968) final concentrations) with or without 5 μL containing test compound or vehicle (DMSO, 1% final concentration), in a total volume of 25 μL, in a polypropylene 96-well plate (Greiner, V-bottom). After 90 min at 30° C., reactions were stopped by adding of 25 μL/well of 150 mM phosphoric acid. All of the terminated kinase reaction was transferred to prewashed (75 mM phosphoric acid) 96 well filter plates (Perkin Elmer catalog number 6005177) using a cell harvester (Perkin Elmer). Plates were washed 6 times with 300 μL per well of a 75 mM phosphoric acid solution and the bottom of the plates was sealed. 40 μL/well of Microscint-20 was added, the top of the plates was sealed and readout was performed using the Topcount (Perkin Elmer). Kinase activity was calculated by subtracting counts per minute (cpm) obtained in the presence of a positive control inhibitor (10 μM staurosporine) from cpm obtained in the presence of vehicle. The ability of a test compound to inhibit this activity was determined as:Percentage inhibition=((cpm determined for sample with test compound present−cpm determined for sample with positive control inhibitor) divided by (cpm determined in the presence of vehicle−cpm determined for sample with positive control inhibitor))*100%.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 464.53, ALogP 3.88, HBA 7, HBD 1, PSA 95.55, QED 0.45
Compound (SMILES): CC(C)(C)OC(=O)n1nnnc1CCl
InChIKey: JFRJOVIKCHLSMA-UHFFFAOYSA-N
Target: Fatty-acid amide hydrolase 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P97612
Assay: FAAH Assay: All reagents were purchased from Sigma-Aldrich unless specified. Human and Rat Fatty Acid Amide Hydrolase (FAAH) genes used in assay have been described by Patricelli et al. (Biochemistry. 1998, 37(43), 15177-87). The transmembrane domain-deleted Fatty Acid Amide Hydrolase (FAAH) genes were cloned into pET15b (Novagen, #69661) (human FAAH)/pET 28a (Novagen, #69864-3) (rat FAAH gene) plasmids and expressed in E coli BL21 DE3. Chaperone protein groEL-groES in pGRO7 plasmid (Takara Bio Inc, Japan) was co-expressed with Fatty Acid Amide Hydrolase (FAAH) to improve solubility of the protein expressed in E coli. The protein was expressed and enriched as described in Mileni et al. (Proc Natl Acad Sci USA. 2008, 105(35),12820-4). Briefly, the bacterial cultures in Luria Broth (2 L) were induced with arabinose (2 mM) and Isopropyl (β-D-1-thiogalactopyranoside, IPTG (1 mM), for 20 h at room temperature. The cultures were centrifuged at 1200×g for 10 min and the cell pellet was resuspended in 100 mL buffer containing of 20 mM NaPi (pH 7.4), 100 mM NaCl, Benzonase (500u), Aprotinin (1 μg/mL) and Leupeptin (1 μg/mL). Cells were lyzed by sonication (Amp 20%, Pulse 15 s×15, on ice) and the cell debris removed by centrifugation at 5000×g for 20 min. The supernatant was enriched via ultracentrifugation at 100,000×g for 1 h and the cell pellet was resuspended in 16 mL buffer containing 20 mM NaPi (pH 7.8), 500 mM NaCl, 1% Triton X-100. The resuspended cell extract was subjected to ultra-centrifugation at 100,000×g for 1 h and the enriched supernatant was used in the in vitro assay. All protein extraction steps were carried out on ice or at 4° C.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 218.64, ALogP 1.2, HBA 6, HBD 0, PSA 69.9, QED 0.52
Compound (SMILES): CO[C@@H](C)CN1CCN(c2ccc(-c3cc(-c4cnn(C)c4)cn4ncc(C#N)c34)cn2)CC1
InChIKey: NZEKNDOXTKUGJH-SFHVURJKSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of General Formula I were screened for their ability to inhibit wild type and V804M mutant RET kinase using CisBio's HTRF® KinEASE -TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Cat. No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Cat. No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Cat. No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 158 nM (pChEMBL 6.800000190734863)
Descriptors: MW 456.55, ALogP 2.83, HBA 9, HBD 0, PSA 87.51, QED 0.44
Compound (SMILES): CC1(C(=O)N2CCC(O)(Cn3cnc4c(cc(Cl)n4-c4ccc5c(c4)OCO5)c3=O)CC2)CC1
InChIKey: HQHQDIGQORKBKP-UHFFFAOYSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q93009
Assay: Fluorescence Polarisation (FP) Homogeneous Assay: USP7 activity was monitored in a fluorescence polarisation (FP) homogeneous assay using the isopeptide ubiquitin-Lys-TAMRA substrate (U-558, Boston Biochem). Full-length USP7 was purchased from Boston Biochem (His6-USP7FL, E-519). Unless otherwise stated, all other reagents were purchased from Sigma-Aldrich. Enzymatic reactions were conducted in black flat-bottom polystyrene 384-well plates (Nunc) and 15 μL total volume. USP7 (2.5 nM, 5 μL) was incubated in assay buffer (50 mM HEPES (pH 7.2), 150 mM NaCl, 0.5 mM EDTA, 5 mM DTT, 0.05% BSA (w/v), 0.05% CHAPS) in the presence or absence of inhibitor (5 μl). Inhibitors were stored as 10 mM DMSO stocks in an inert environment (low humidity, dark, low oxygen, room temperature) using a Storage Pod System and serial dilutions were prepared in buffer just prior to the assay (from 200 to 0.1 μM, 8 dp curve). Following incubation at room temperature for 30 min, the enzymatic reactions were initiated by dispensing the Ub substrate (250 nM, 5 μL). FP was measured every 15 min over a period of 1.5 h (within the linear range of the assay) using a Synergy 4 plate reader (BioTek) exciting at 530 nm and measuring the amount of parallel and perpendicular light at 575 nm.
Measurement: IC50 < 250 nM (pChEMBL 6.6020002365112305)
Descriptors: MW 484.94, ALogP 2.72, HBA 8, HBD 1, PSA 98.82, QED 0.61
Compound (SMILES): NCC1(c2cccc(-c3ncnc4[nH]ccc34)c2)Cc2ccc(CN3CC[C@H](C(N)=O)C3)cc2C1
InChIKey: XXFPJRQVCHAZQC-QDLLFRBVSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: TAM Kinase Assay: The kinase assay buffer contained 50 mM HEPES, pH7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% NP-40 and 2 mM DTT. 0.1 ul test compounds dissolved in DMSO were transferred from compound plates to white 384-well assay plates (Greiner LUMITRAC plates). The final concentration of DMSO was 1.25%. Enzyme solutions of 5.1 nM phosphor-Axl (see Axl autophosphorylation assay above), or 0.0625 nM c-Mer (Carna Biosciences, 08-108), or 0.366 nM Tyro3 (Life Technologies, PR7480A) were prepared in assay buffer. A 1 mM stock solution of peptide substrate Biotin-EQEDEPEGDYFEWLE-amide SEQ ID NO: 1 (Quality Controlled Biochemicals, MA) dissolved in DMSO was diluted to 1 uM in assay buffer containing 2000 uM ATP. 4 ul enzyme solution (or assay buffer for the enzyme blank) was added to the appropriate wells in each plate, and then 4 ul/well substrate solution was added to initiate the reaction. The plate was protected from light and incubated at room temperature for 60 min. The reaction was stopped by adding 4 ul detection solution containing 50 mM Tris-HCl, pH7.8, 150 mM NaCl, 0.05% BSA, 45 mM EDTA, 180 nM SA-APC (Perkin Elmer, CR130-100) and 3 nM Eu¿W1024 anti-phosphotyrosine PY20 (Perkin Elmer, AD0067). The plate was incubated for 1 h at room temperature, and HTRF (homogenous time resolved fluorescence) signal was measured on a PHERAstar FS plate reader (BMG labtech). Percentage of inhibition was calculated for each concentration and IC50 value was generated from curve fitting with GraphPad Prism software.
Measurement: IC50 = 2000 nM (pChEMBL 5.699999809265137)
Descriptors: MW 466.59, ALogP 2.93, HBA 5, HBD 3, PSA 113.92, QED 0.4
Compound (SMILES): CCNc1ncc2c(n1)N(C)CCN(c1cccc(COCC(NC)c3ccccc3)c1)C2=O
InChIKey: PWQZZRUUAPCYPI-UHFFFAOYSA-N
Target: Voltage-dependent calcium channel subunit alpha-2/delta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P54289
Assay: Binding Assay: α2δ-1: Binding Assay to Human α2δ-1 Subunit of Cav2.2 Calcium Channel.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. 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 hour 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 = 1750 nM (pChEMBL 5.760000228881836)
Descriptors: MW 460.58, ALogP 3.48, HBA 7, HBD 2, PSA 82.62, QED 0.51
Compound (SMILES): Cc1nc(C(=O)N[C@]2(c3ccc(C(F)(F)F)cc3)C[C@@H]2C)cc(=O)[nH]1
InChIKey: UGRBWVGOUDRIQP-XXFAHNHDSA-N
Target: cGMP-dependent 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O00408
Assay: Fluorescence Polarization (FP) Assay: In a typical experiment the PDE2 inhibitory activity of the compounds of the present invention was determined in accordance with the following experimental method. Rhesus PDE2A3 was amplified from rhesus macaque brain cDNA (Biochain Institute, Hayward, Calif.) using primers based on human PDE2A sequence (accession NM_002599.3) where the forward primer containing a Kozak consensus was 5′-gccaccatggggcaggcatgtggc-3′ and the reverse primer was 5′-tcactcagcatcaaggctgca-3′. Amplification with Easy-A High-Fidelity PCR cloning enzyme (Stratagene, La Jolla, Calif.) was 95° C. for 2 minutes followed by thirty three cycles of 95° C. for 40 seconds, 52° C. for 30 seconds, and 72° C. for 2 minutes 48 seconds. Final extension was 72° C. for 7 minutes. The PCR product was TA cloned into pcDNA3.3-TOPO (Invitrogen, Carlsbad, Calif.) according to standard protocol. A consensus sequence was developed from multiple clones and then deposited into GenBank (EU812167). AD293 cells (Stratagene, La Jolla, Calif.) with 70-80% confluency were transiently transfected with rhesus PDE2A3/pcDNA3.3-TOPO using Lipofectamine 2000 according to manufacturer specifications (Invitrogen, Carlsbad, Calif.).
Measurement: Ki = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 351.33, ALogP 2.76, HBA 3, HBD 2, PSA 74.85, QED 0.89
Compound (SMILES): Cc1ccc(C(=O)N2CCC[C@H]2c2noc(-c3cccc(F)c3C)n2)c(-n2nccn2)c1
InChIKey: CXZXWKINGRPWER-IBGZPJMESA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: In Vitro Assay: Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES. On the day of the assay, 50 uL of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/L, 5 mM probenecid (Sigma) and 3 uM of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at rt for 30 min before measurement.Within the Fluorescent Imaging Plate Reader (FLIPR Tetra, Molecular Devices), antagonists are added to the plate in a volume of 10 uL/well, incubated for 120 min and finally 10 uL/well of agonist is added. Fluorescence is measured for each well at 1 second intervals, and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 3 nM orexin-A with vehicle in place of antagonist. The IC50 value (the concentration of compound needed to inhibit 50% of the agonistic response) is determined and may be normalized using the obtained IC50 value of an on-plate reference compound. Optimized conditions were achieved by adjustment of pipetting speed and cell splitting regime. The calculated IC50 values may fluctuate depending on the daily cellular assay performance. Fluctuations of this kind are known to those skilled in the art. In the case where IC50 values have been determined several times for the same compound, the geometric mean has been given.
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 432.46, ALogP 4.05, HBA 7, HBD 0, PSA 89.94, QED 0.48
Compound (SMILES): COC[C@@H](COc1cn[nH]c(=O)c1C(F)(F)F)OCCC(=O)N1CCN(c2ncc(Cl)cn2)CC1
InChIKey: UJLVBHGBPNSFJF-AWEZNQCLSA-N
Target: Protein mono-ADP-ribosyltransferase TIPARP (SINGLE PROTEIN), Homo sapiens
UniProt: Q7Z3E1
Assay: Enzymatic Assay for Inhibition of PARP7: Displacement of Probe A, a biotinylated probe binding to the TIPARP active site, was measured using a time-resolved fluorescence energy transfer (TR-FRET) assay. 20 nL of a dose response curve of each test compound was spotted in black 384-well polystyrene proxiplates (Perkin Elmer) using a Mosquito (TTP Labtech). Reactions were performed in a 8 μL volume by adding 6 μL of TIPARP and Probe A in assay buffer (20 mM HEPES pH=8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002% Tween20), incubating with test compound at 25° C. for 30 min, then adding 2 μL of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin (Perkin Elmer). The final concentrations of TIPARP and Probe A were 6 nM and 2 nM, respectively. The final concentration of ULight-anti 6xHis and LANCE Eu-W1024 labeled streptavidin were 4 nM and 0.25 nM, respectively. Reactions were incubated at 25° C. for an additional 30 min, then read on an Envision platereader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 320 nm and emission of 615 nm and 665 nM with a 90 μs delay. The ratio of the 665/615 nm emission were calculated for each well to determine the amount of complex of TIPARP and Probe A in each well. Control wells containing a negative control of 0.25% DMSO vehicle or a positive control of 100 μM Example 190 were used to calculate the % inhibition as described below:% ⁢ ⁢ inhibition = 100 × TRF cmpd - TRF min TRF max - TRF minwhere TRFcmpd is the TR-FRET ratio from the compound treated well, TRFmin is the TR-FRET ratio from the Example 190-treated positive control well and TRFmax is the TR-FRET ratio from the DMSO-treated negative control well.The % inhibition values were plotted as a function of compound concentration and the following 4-parameter fit was applied to derive the IC50 the values:Y = Bottom + ( Top - Bottom ) ( 1 + ( X IC 50 ) Hill ⁢ ⁢ Coefficientwhere top and bottom are normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit. The Hill Coefficient is normally allowed to float but may also be fixed at 1 in a 3-parameter fit. Y is the % inhibition and x is the compound concentration.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 520.9, ALogP 1.38, HBA 9, HBD 1, PSA 122.77, QED 0.49
Compound (SMILES): N#CCC1(N2CCC(N[C@@H]3CC3c3ccccc3)CC2)CN(C(=O)c2nccs2)C1
InChIKey: DEVUVKQKULAJNV-GFOWMXPYSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: Biochemical Assay: LANCE LSD1/KDM1A demethylase assay 10 μL of 1 nM LSD-1 enzyme (ENZO BML-SE544-0050) in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 25 mM NaCl, 5 mM DTT) were preincubated for 1 hour at 25° C. with 0.8 μL compound/DMSO dotted in black 384 well polystyrene plates. Reactions were started by addition of 10 μL of assay buffer containing 0.4 μM Biotin-labeled Histone H3 peptide substrate: ART-K(Me1)-QTARKSTGGKAPRKQLA-GGK(Biotin) SEQ ID NO:1 (AnaSpec 64355) and incubated for 1 hour at 25° C. Reactions were stopped by addition of 10 μL 1×LANCE Detection Buffer (PerkinElmer CR97-100) supplemented with 1.5 nM Eu-anti-unmodified H3K4 Antibody (PerkinElmer TRF0404), and 225 nM LANCE Ultra Streptavidin (PerkinElmer TRF102) along with 0.9 mM Tranylcypromine-HCl (Millipore 616431). After stopping the reactions plates were incubated for 30 minutes and read on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 421.57, ALogP 2.86, HBA 6, HBD 1, PSA 72.26, QED 0.78
Compound (SMILES): C=CC(=O)Nc1ccccc1Nc1ncc2c(n1)N(Cc1ccnc(N)c1)C(=O)N(c1cc(OC)cc(OC)c1)C2
InChIKey: WUVOYOFXWAPEOG-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: Omnia Assay Protocol for Potency Assessment Against FGFR 4 Enzyme: A 10× stock solution of FGFR4-WT (PR4380C or P3054), (from Invitrogen, Carlsbad, Calif.) corresponding to method a in Table 10, was prepared as described below. Alternatively, a 10× stock solution of FGFR4-WT (F01-11G), (SignalChem, Richmond, BC) corresponding to method b in Table 10, was prepared as described below. A solution of 1.4×ATP (AS001A) and 5× Tyr-Sox conjugated peptide substrate (KNZ3101) were prepared in 1× kinase reaction buffer consisting of 20 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 5 mM P3-glycerophosphate, 5% glycerol (10× stock, KB002A) and 0.2 mM DTT (DS001A). 5 μL of FGFR4 was pipetted into a Corning (#3574) 384-well, white, non-binding surface microtiter plate (Corning, N.Y.), containing a 0.5 μL volume of 100% DMSO. The serially diluted compounds were prepared on a Tecan EVO100. A second addition of 10 μl of Tyr-Sox FGFR4 substrate was added to each well and the kinase reactions were started with the addition of 35 μL of 1.4×ATP. The reactions were monitored every 71 seconds for 240 minutes at λex360/λem485 in a Synergy plate reader from BioTek (Winooski, Vt.). At the conclusion of each assay, progress curves from each well were examined for linear reaction kinetics and fit statistics (R2, 95% confidence interval, absolute sum of squares). Initial velocity (0 minutes to −60 minutes) from each reaction was determined from the slope of a plot of relative fluorescence units vs time (seconds) and then plotted against inhibitor concentration to estimate IC50 from log[Inhibitor] vs Response (Variable Slope model in GraphPad Prism from GraphPad Software (San Diego, Calif.)).
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 552.6, ALogP 4.49, HBA 9, HBD 3, PSA 147.83, QED 0.26
Compound (SMILES): Cc1cc(Nc2cc3ncccc3c(N[C@@H]3C[C@H]4CC[C@@H](C3)N4S(=O)(=O)C3CNC3)n2)n[nH]1
InChIKey: FNXXIMPCAHLECD-PHZGNYQRSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biochemical JAK Assay: A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 and Tyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH 7.5, 0.01% Brij-35, 10 mM MgCl2, and 1 mM EGTA). Recombinant GST-tagged JAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained from Life Technologies.Serially diluted compounds were pre-incubated with each of the four JAK enzymes and the substrate in white 384-well microplates (Corning) at ambient temperature for lh. ATP was subsequently added to initiate the kinase reactions in 10 μL total volume, with 1% DMSO. The final enzyme concentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and 0.25 nM respectively; the corresponding Km ATP concentrations used are 25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200 nM for all four assays. Kinase reactions were allowed to proceed for 1 hour at ambient temperature before a 10 μL preparation of EDTA (10 mM final concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (Life Technologies, 2 nM final concentration) in TR-FRET dilution buffer (Life Technologies) was added. The plates were allowed to incubate at ambient temperature for lh before being read on the EnVision reader (Perkin Elmer). Emission ratio signals (520 nm/495 nm) were recorded and utilized to calculate the percent inhibition values based on DMSO and background controls.For dose-response analysis, percent inhibition data were plotted vs. compound concentrations, and IC50 values were determined from a 4-parameter robust fit model with the Prism software (GraphPad Software). Results were expressed as pIC50 (negative logarithm of IC50) and subsequently converted to pKi (negative logarithm of dissociation constant, Ki) using the Cheng-Prusoff equation.Test compounds having a higher pKi value in each of the four JAK assays show greater inhibition of JAK activity. Compounds of the invention tested in this assay typically exhibited pKi values between about 7 and about 10.3
Measurement: Ki = 0.55 nM (pChEMBL 9.260000228881836)
Descriptors: MW 468.59, ALogP 2.11, HBA 8, HBD 4, PSA 127.93, QED 0.43
Compound (SMILES): COc1cc(-c2[nH]c3ccc(CCC4CN(Cc5c[nH]nn5)C4)nc3c2C(C)C)cn2ncnc12
InChIKey: LOBIZHCPZHVQCY-UHFFFAOYSA-N
Target: Toll-like receptor 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR96
Assay: TLR7/8/9 Inhibition Reporter Assays: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 177 nM (pChEMBL 6.75)
Descriptors: MW 471.57, ALogP 3.59, HBA 8, HBD 2, PSA 112.91, QED 0.36
Compound (SMILES): O=C(Nc1cnn(Cc2cccc(F)c2)c1)c1cc(C2(O)CCC2)on1
InChIKey: BQOYGEFWXGYNCU-UHFFFAOYSA-N
Target: Stearoyl-CoA desaturase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86SK9
Assay: Compound Profiling Methods: Strains expressing SCD1 or SCD5 as the sole desaturase, the human SCD1 and SCD5 genes were used to evaluate inhibition of SCD1/SCD5 using reduced growth as a surrogate for SCD inhibition. These yeast strains express human SCD1 or SCD5 from a plasmid harbored in a strain in which the yeast OLE1 gene is deleted.All compound profiling experiments were performed using the same basic protocol. Yeast were cultured using standard techniques in complete synthetic media lacking uracil and containing yeast nitrogen base supplemented with 2% (w/v) glucose (SD-Ura) Starter cultures were inoculated in 3 mL SD-Ura media containing 0.01% tween and 0.2 mM palmitoleic and oleic acid. Cultures were incubated overnight in a 30° C. shaker incubator (225 rpm). Saturated morning cultures were centrifuged, washed in SD-Ura media lacking TWEEN-20 and fatty acids, and then diluted 1:20 in fresh SD-Ura media also lacking TWEEN-20 and fatty acids. Cells were grown for 6 h to an  (optical density) of 0.4-0.8 at 30° C. with shaking.Compound stocks (10 mM in 100% DMSO) were arrayed into 384-round well, v-bottom polypropylene plates and diluted according to indicated dilution factors. Compound administration was performed in two separate steps. First, 15 μL of SD-Ura was dispensed into clear 384-well assay plates using a MULTIDROP Combi reagent dispenser. The diluted compound stock plates were then applied to the assay plates using an automated workstation (Perkin Elmer JANUS) outfitted with a 384-pin tool containing slotted pins that deliver 100 nL of compound. The cultures described above were centrifuged and washed with media lacking TWEEN-20 or oleic and palmitoleic acids. Cultures were then resuspended at a 2-fold concentrated  of 0.02 (final  of 0.0.01) in SD-Ura. 15 μL of diluted culture was then dispensed into the pinned assay plate to achieve 30 μL of the 1× culture (0.01) and a top drug concentration of 33.3 μM.After yeast delivery, assay plates were incubated under humidified conditions at 30° C. for 40 h. Yeast growth was monitored by reading the  of each well using a microplate reader (Perkin Elmer EnVision). Data were analyzed as follows. Raw data were processed by background subtracting and converting values to a percent of the nontreated condition for that strain [(EXP-0.035)/(DMSO-0.035)×100%].
Measurement: IC50 = 20000 nM (pChEMBL 4.699999809265137)
Descriptors: MW 356.36, ALogP 2.68, HBA 6, HBD 2, PSA 93.18, QED 0.73
Compound (SMILES): CCOc1c(F)cccc1-c1noc([C@@H]2CCCN2C(=O)c2cc(OC)c(C)cc2-n2nccn2)n1
InChIKey: LYLJJUAAANDBTD-IBGZPJMESA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: In Vitro Assay: Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES. On the day of the assay, 50 uL of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/L, 5 mM probenecid (Sigma) and 3 uM of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at rt for 30 min before measurement.Within the Fluorescent Imaging Plate Reader (FLIPR Tetra, Molecular Devices), antagonists are added to the plate in a volume of 10 uL/well, incubated for 120 min and finally 10 uL/well of agonist is added. Fluorescence is measured for each well at 1 second intervals, and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 3 nM orexin-A with vehicle in place of antagonist. The IC50 value (the concentration of compound needed to inhibit 50% of the agonistic response) is determined and may be normalized using the obtained IC50 value of an on-plate reference compound. Optimized conditions were achieved by adjustment of pipetting speed and cell splitting regime. The calculated IC50 values may fluctuate depending on the daily cellular assay performance. Fluctuations of this kind are known to those skilled in the art. In the case where IC50 values have been determined several times for the same compound, the geometric mean has been given.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 492.51, ALogP 4.15, HBA 9, HBD 0, PSA 108.4, QED 0.38
Compound (SMILES): CCN1CC2(CCN(Cc3ccc(OC)cc3)CC2)OC(C)(C)C1=O
InChIKey: ARYMTVNDCSUXCA-UHFFFAOYSA-N
Target: Sigma non-opioid intracellular receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99720
Assay: Radioligand Assay: To investigate binding properties of test compounds to human σ1 receptor, transfected HEK-293 membranes and [3H](+) -pentazocine (Perkin Elmer, NET-1056), as the radioligand, were used. The assay was carried out with 7 μg of membrane suspension, 5 nM of [3H](+)-pentazocine in either absence or presence of either buffer or 10 μM Haloperidol for total and non-specific binding, respectively. Binding buffer contained Tris-HCl 50 mM at pH 8. Plates were incubated at 37° C. for 120 minutes. After the incubation period, the reaction mix was then transferred to Multiscreen HTS, FC plates (Millipore), filtered and plates were washed 3 times with ice-cold 10 mM Tris-HCL (pH 7.4). Filters were dried and counted at approximately 40% efficiency in a MicroBeta scintillation counter (Perkin-Elmer) using EcoScint liquid scintillation cocktail Results: As this invention is aimed at providing a compound or a chemically related series of compounds which act as ligands of the σ1 receptor it is a very preferred embodiment in which the compounds are selected which act as ligands of the σ1 receptor and especially compounds which have a binding expressed as Ki which is preferably <1000 nM, more preferably <500 nM, even more preferably <100 nM.
Measurement: Ki < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 346.47, ALogP 2.69, HBA 4, HBD 0, PSA 42.01, QED 0.84
Compound (SMILES): CC[C@H](NS(=O)(=O)c1ccc(-c2sc(-c3nnc(CC(C)(C)C(=O)O)o3)nc2COC(C)C)c2ccccc12)C(F)(F)F
InChIKey: ZGMWSUYIZOUFIP-NRFANRHFSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: RORgammat (full-length human) Reporter Assay A: Cells used in this assay were transiently co-transfected with three different plasmids, one expressing the GAL4-DNA binding domain (DBD)-RORγt fusion protein under control of a CMV promoter (NH2-Gal4-DBD:RORC¿COOH in pCMV-BD, Stratagene #211342), and two reporter plasmids¿the firefly luciferase reporter under control of a GAL4 promoter (pFR-Luc 2× GAL4) and Renilla luciferase reporter under control of CMV promoter (pRL-CMV, Promega #E2261). The full-length coding sequence was used for human RORγt, i.e., nucleotides 142-1635 of human RORγt, transcript variant 2, NCBI Accession: NM_001001523.1 (SEQ ID NO: 1). HEK293T cells were plated at 35000 per well in 96-well plate in medium of MEM with 8.6% FBS. After 18-22 hours incubation, the transfection was carried out by using a PEI solution with 170.5 ng total DNA/well (50 ng pCMV-BD-ROR plus 20 ng of pFR-Luc reporter and 0.5 ng of pRL-CMV reporter plus 100 ng Carrier DNA (Clontech #630440) for each well). 4-6 hours after transfection, cells were treated with compounds for overnight in the medium with final concentration of FBS 1.1% and DMSO 0.1%. After overnight (16 to 20 hours) incubation, media were removed and cells were lysed with 20 μL 1× Passive Lysis Buffer (Promega) for 10-15 minutes. Luminescence was measured using a BMG LUMIstar OPTIMA plate reader, after addition of 75 μL/well firefly luciferase buffer, followed by 75 μL/well Renilla luciferase buffer. To calculate the effect of compounds on RORγt activity, firefly values were normalized against values of DMSO only and values of reference compound at saturating concentration, then further normalized against Renilla signals. IC50s were generated by plotting final Renilla normalized data against compound concentration and percent inhibition was calculated against DMSO control.
Measurement: IC50 = 39 nM (pChEMBL 7.409999847412109)
Descriptors: MW 640.71, ALogP 6.21, HBA 9, HBD 2, PSA 144.51, QED 0.18