lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1nc(OCc2cn(-c3ccc(F)c(C(F)F)c3)nn2)ncc1Cl
InChIKey: RXLRURYBQIBHOJ-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 = 1 nM (pChEMBL 9)
Descriptors: MW 369.73, ALogP 3.67, HBA 6, HBD 0, PSA 65.72, QED 0.69
Compound (SMILES): CC(C)n1c(CC(C)(C)N)nc2c(F)cc(-c3nc(N[C@@H]4CCOC[C@H]4O)ncc3Cl)cc21
InChIKey: PSILLYORWULWQE-SJLPKXTDSA-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 = 1 nM (pChEMBL 9)
Descriptors: MW 476.98, ALogP 3.71, HBA 8, HBD 3, PSA 111.11, QED 0.5
Compound (SMILES): CS(=O)(=O)N1CCC(Nc2ncc(C(F)(F)F)c(-c3cn(-c4ccc(CN5CC(C#N)C5)cc4Cl)cn3)n2)CC1
InChIKey: OLIFRVDPEYJVIN-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24941
Assay: HTRF Enzyme Activity Assay: CDK2/Cyclin E1 enzyme activity assays utilize full-length human CDK2 co-expressed as N-terminal GST-tagged protein with FLAG-Cyclin E1 in a baculovirus expression system (Carna Product Number 04-165). Assays were conducted in white 384-well polystyrene plates in a final reaction volume of 8 μL. CDK2/Cyclin E1 (0.25 nM) was incubated with the compounds of the Examples (40 nL serially diluted in DMSO) in the presence of ATP (50 μM or 1 mM) and 50 nM ULight-labeled eIF4E-binding protein 1 (THR37/46) peptide (PerkinElmer) in assay buffer (containing 50 mM HEPES pH 7.5, 1 mM EGTA, 10 mM MgCl2, 2 mM DTT, 0.05 mg/mL BSA, and 0.01% Tween 20) for 60 minutes at room temperature. The reactions were stopped by the addition of EDTA and Europium-labeled anti-phospho-4E-BP1 antibody (PerkinElmer), for a final concentration of 15 mM and 1.5 nM, respectively. HTRF signals were read after 1 hour at room temperature on a PHERAstar FS plate reader (BMG Labtech). Data was analyzed with IDBS XLFit and GraphPad Prism 5.0 software using a three or four parameter dose response curve to determine IC50 for each compound.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 595.05, ALogP 3.79, HBA 9, HBD 1, PSA 120.04, QED 0.44
Compound (SMILES): COCCN1CCC(c2cc3c(-c4cc(F)ccc4OC)ccnc3[nH]2)CC1
InChIKey: YXJQLUNLSQAWKV-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 = 66 nM (pChEMBL 7.179999828338623)
Descriptors: MW 383.47, ALogP 4.2, HBA 4, HBD 1, PSA 50.38, QED 0.69
Compound (SMILES): CC1(C)OCCn2nc3c(F)cc(-c4nc(N[C@@H]5CCOC[C@H]5O)ncc4Cl)cc3c21
InChIKey: OYBGABJZRRNVII-HZPDHXFCSA-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 = 1 nM (pChEMBL 9)
Descriptors: MW 447.9, ALogP 3.11, HBA 8, HBD 2, PSA 94.32, QED 0.64
Compound (SMILES): OCC1(CO)CCN(Cc2ccc(-c3ccc(-c4cc5nc(O[C@@H]6CO[C@H]7[C@@H]6OC[C@H]7O)[nH]c5cc4Cl)cc3)cc2)CC1
InChIKey: DVRFRHQBFWVLQS-OMRVPHBLSA-N
Target: AMP-activated protein kinase (AMPK) alpha-1/beta-1/gamma-1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P54619
Assay: In Vitro pAMPK1 Kinase Activation Assay: Compound effect on AMPK enzyme activation was determined in a cell-free format with a 12-point concentration curve. The ADP-Glo detection system was used to determine phosphorylation of a SAMS peptide substrate. Recombinant AMPK α1/β1/γ1 complex was pre-activated by phosphorylation with CAMKK2 followed by incubated with compound for 15 minutes prior to the SAMS phosphorylation reaction. Activity curves and EC50 values were fitted by interpolation to an ATP:ADP standard curve as indicated by the ADP-Glo manufacturer using Prism software.
Measurement: EC50 = 0.85 nM (pChEMBL 9.069999694824219)
Descriptors: MW 606.12, ALogP 4.02, HBA 8, HBD 4, PSA 120.3, QED 0.24
Compound (SMILES): COc1ccc(F)cc1-c1ccnc2[nH]c(C3CCN(C4CNC4)CC3)cc12
InChIKey: CZKVVJGRIZBXFO-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 = 190 nM (pChEMBL 6.71999979019165)
Descriptors: MW 380.47, ALogP 3.53, HBA 4, HBD 2, PSA 53.18, QED 0.73
Compound (SMILES): O=C(CNC(=O)c1cccc(Nc2ccccn2)c1)NC[C@H](NC(=O)[C@@H]1CCCN1S(=O)(=O)c1ccccc1)C(=O)O
InChIKey: MDJYIURRUWWBLA-GOTSBHOMSA-N
Target: Integrin alpha-V/beta-1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P05556
Assay: Cell adhesion assay to SW480: The blockade of integrin alpha 5 beta 1 provides a novel target for the treatment of asthma. As shown in FIGS. 1A and 1B, delivery of ATN-161, a low affinity yet specific inhibitor of alpha5beta1, reduces the magnitude of airway narrowing in vivo in mice sensitized and challenged with ovalbumin, a widely used model of allergic asthma. In the same model, it was found that mice with a specific deletion of this integrin in smooth muscle also have reduced airway narrowing. The intracellular actin-myosin cross-bridging pathway serves as a useful target. Modulating the interactions of the cell and the extracellular matrix impairs the ability of the smooth muscle to transmit tension effectively. The blockade of integrin alpha 5 beta 1 was tested in vitro with human cell lines as well as in vivo with a mouse model of airway hyperresponsiveness. Novel compounds as described herein, and identified in Table 1, have been synthesized and tested in vitro for their ability to inhibit cell adhesion mediated by alpha5beta1 (adhesion of the colon carcinoma cell line SW480 to fibronectin, a response that is entirely dependent on binding of alpha5beta1 to fibronectin).
Measurement: IC50 = 5050 nM (pChEMBL 5.300000190734863)
Descriptors: MW 594.65, ALogP 1.09, HBA 8, HBD 5, PSA 186.9, QED 0.2
Compound (SMILES): CC(C(=O)Nc1cc(C2CC2)[nH]n1)c1cccc(-c2ccc(NC(=O)/C=C/CN3CCC[C@H]3CC#N)nc2)c1
InChIKey: KDCKKNHYXJQMRS-LGDADKFGSA-N
Target: CDK7/Cyclin H/MNAT1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P51946
Assay: Biochemical Assay: 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 the 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 = 62.5 nM (pChEMBL 7.199999809265137)
Descriptors: MW 523.64, ALogP 4.96, HBA 6, HBD 3, PSA 126.8, QED 0.32
Compound (SMILES): O=C(NCCOCCO)c1ccc(-c2ccc(-c3cc4nc(O[C@@H]5CO[C@H]6[C@@H]5OC[C@H]6O)[nH]c4cc3Cl)cc2)c(O)c1
InChIKey: YLKRXJXHAQUJIP-BIYDSLDMSA-N
Target: AMP-activated protein kinase (AMPK) alpha-1/beta-1/gamma-1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P54619
Assay: In Vitro pAMPK1 Kinase Activation Assay: Compound effect on AMPK enzyme activation was determined in a cell-free format with a 12-point concentration curve. The ADP-Glo detection system was used to determine phosphorylation of a SAMS peptide substrate. Recombinant AMPK α1/β1/γ1 complex was pre-activated by phosphorylation with CAMKK2 followed by incubated with compound for 15 minutes prior to the SAMS phosphorylation reaction. Activity curves and EC50 values were fitted by interpolation to an ATP:ADP standard curve as indicated by the ADP-Glo manufacturer using Prism software.
Measurement: EC50 = 0.37 nM (pChEMBL 9.430000305175781)
Descriptors: MW 596.04, ALogP 2.9, HBA 9, HBD 5, PSA 155.39, QED 0.17
Compound (SMILES): Cc1ncccc1O[C@H]1C[C@@H]2CN(CC(O)c3ccc(O)cc3)C[C@@H]2C1
InChIKey: NYFNWILXAIGWGL-HRARKVMDSA-N
Target: Glutamate NMDA receptor; GRIN1/GRIN2B (PROTEIN COMPLEX), Homo sapiens
UniProt: Q13224
Assay: NR2B NAM Assay: Cell Culture and plating: HEK293 cells expressing NR1/NR2B (Chantest, Cleveland, Ohio) were grown to 70-80% confluency as an adherent monolayer in standard tissue culture flasks at 37° C., 5% CO2 per supplier's instructions. NR2B expression was induced by incubation with 0.3-0.4 μg/ml tetracycline in the presence of 4 mM ARL-15896 for 18-24 hours under the same growth conditions, then transferred to 30° C. for another 3-5 hours.After induction, cell culture medium was removed and cells were rinsed once with Ca2+ and Mg2+-free Dulbecco's phosphate buffered saline. Cells were then removed from the flask using TrypLE Express (Life Technologies) according to the manufacturer's instructions and collected to 50 ml centrifuge tubes. Following two washes in Ca2+/Mg2+-free HBSS with 20 mM HEPES (HHnoCa), cells were counted and viability assessed using trypan blue. To load cells with Ca2+-sensitive dye, they were resuspended in fluo-8 plus Component B (AAT Bioquest Products) diluted in HHnoCa and incubated 15 minutes at 37° C., followed by 30 minutes at room temp (in dark). Cells were then washed and resuspended in HHnoCa to remove extracellular dye and plated in 384-well plates (Falcon, uncoated) at 20,000-30,000 cells/well in a final volume of 25 μl/well.
Measurement: IC50 = 188 nM (pChEMBL 6.730000019073486)
Descriptors: MW 354.45, ALogP 2.92, HBA 5, HBD 2, PSA 65.82, QED 0.86
Compound (SMILES): CS(=O)(=O)N1CC[C@@H](Nc2ncc(Cl)c(-c3cc(F)c4ncn(C5CC5)c4c3)n2)[C@H](O)C1
InChIKey: UVGRSOFHEUUOQF-NVXWUHKLSA-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 = 0.3 nM (pChEMBL 9.520000457763672)
Descriptors: MW 480.95, ALogP 2.43, HBA 8, HBD 2, PSA 113.24, QED 0.58
Compound (SMILES): CCS(=O)(=O)N1CCC(Nc2ncc(C(F)(F)F)c(-c3cn(-c4ccc(C)nc4C#N)cn3)n2)CC1
InChIKey: MRPJCTDDXNSMCJ-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24941
Assay: HTRF Enzyme Activity Assay: CDK2/Cyclin E1 enzyme activity assays utilize full-length human CDK2 co-expressed as N-terminal GST-tagged protein with FLAG-Cyclin E1 in a baculovirus expression system (Carna Product Number 04-165). Assays were conducted in white 384-well polystyrene plates in a final reaction volume of 8 μL. CDK2/Cyclin E1 (0.25 nM) was incubated with the compounds of the Examples (40 nL serially diluted in DMSO) in the presence of ATP (50 μM or 1 mM) and 50 nM ULight-labeled eIF4E-binding protein 1 (THR37/46) peptide (PerkinElmer) in assay buffer (containing 50 mM HEPES pH 7.5, 1 mM EGTA, 10 mM MgCl2, 2 mM DTT, 0.05 mg/mL BSA, and 0.01% Tween 20) for 60 minutes at room temperature. The reactions were stopped by the addition of EDTA and Europium-labeled anti-phospho-4E-BP1 antibody (PerkinElmer), for a final concentration of 15 mM and 1.5 nM, respectively. HTRF signals were read after 1 hour at room temperature on a PHERAstar FS plate reader (BMG Labtech). Data was analyzed with IDBS XLFit and GraphPad Prism 5.0 software using a three or four parameter dose response curve to determine IC50 for each compound.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 520.54, ALogP 3.15, HBA 9, HBD 1, PSA 129.69, QED 0.52
Compound (SMILES): COc1cnc(OCc2cn(-c3ccc(F)cc3C)nn2)nc1
InChIKey: BLOKUKDWVMHARP-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 = 2650 nM (pChEMBL 5.579999923706055)
Descriptors: MW 315.31, ALogP 2.09, HBA 7, HBD 0, PSA 74.95, QED 0.72
Compound (SMILES): Fc1cccc(CNc2cc(-c3ccnc4[nH]c(C5=CCNCC5)cc34)c(F)cc2F)c1
InChIKey: UXZMBRBBGQVJFU-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 = 35 nM (pChEMBL 7.460000038146973)
Descriptors: MW 434.47, ALogP 5.64, HBA 3, HBD 3, PSA 52.74, QED 0.38
Compound (SMILES): CC(C(=O)Nc1cc(C2CC2)[nH]n1)c1cccc(-c2ccc(NC(=O)/C=C/CN3CCOCC3)nc2F)c1
InChIKey: KVOMOZYLJKQEDA-ZZXKWVIFSA-N
Target: CDK7/Cyclin H/MNAT1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P50613
Assay: Biochemical Assay: 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 the 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 = 62.5 nM (pChEMBL 7.199999809265137)
Descriptors: MW 518.59, ALogP 4.06, HBA 6, HBD 3, PSA 112.24, QED 0.29
Compound (SMILES): OCC1(CO)CCN(Cc2ccc(-c3ccc(-c4cc5nc(O[C@@H]6CO[C@H]7[C@@H]6OC[C@H]7O)[nH]c5cc4Cl)cc3)cc2)CC1
InChIKey: DVRFRHQBFWVLQS-OMRVPHBLSA-N
Target: AMP-activated protein kinase (AMPK) alpha-1/beta-1/gamma-1 (PROTEIN COMPLEX), Homo sapiens
UniProt: Q9Y478
Assay: In Vitro pAMPK1 Kinase Activation Assay: Compound effect on AMPK enzyme activation was determined in a cell-free format with a 12-point concentration curve. The ADP-Glo detection system was used to determine phosphorylation of a SAMS peptide substrate. Recombinant AMPK α1/β1/γ1 complex was pre-activated by phosphorylation with CAMKK2 followed by incubated with compound for 15 minutes prior to the SAMS phosphorylation reaction. Activity curves and EC50 values were fitted by interpolation to an ATP:ADP standard curve as indicated by the ADP-Glo manufacturer using Prism software.
Measurement: EC50 = 0.85 nM (pChEMBL 9.069999694824219)
Descriptors: MW 606.12, ALogP 4.02, HBA 8, HBD 4, PSA 120.3, QED 0.24
Compound (SMILES): CC(C(=O)Nc1cc(C2CC2)[nH]n1)c1cccc(-c2ccc(NC(=O)/C=C/CN3CCC[C@H]3CC#N)nc2)c1
InChIKey: KDCKKNHYXJQMRS-LGDADKFGSA-N
Target: CDK7/Cyclin H/MNAT1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P50613
Assay: Biochemical Assay: 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 the 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 = 62.5 nM (pChEMBL 7.199999809265137)
Descriptors: MW 523.64, ALogP 4.96, HBA 6, HBD 3, PSA 126.8, QED 0.32
Compound (SMILES): O=C(NCCOCCO)c1ccc(-c2ccc(-c3cc4nc(O[C@@H]5CO[C@H]6[C@@H]5OC[C@H]6O)[nH]c4cc3Cl)cc2)c(O)c1
InChIKey: YLKRXJXHAQUJIP-BIYDSLDMSA-N
Target: AMP-activated protein kinase (AMPK) alpha-1/beta-1/gamma-1 (PROTEIN COMPLEX), Homo sapiens
UniProt: Q9Y478
Assay: In Vitro pAMPK1 Kinase Activation Assay: Compound effect on AMPK enzyme activation was determined in a cell-free format with a 12-point concentration curve. The ADP-Glo detection system was used to determine phosphorylation of a SAMS peptide substrate. Recombinant AMPK α1/β1/γ1 complex was pre-activated by phosphorylation with CAMKK2 followed by incubated with compound for 15 minutes prior to the SAMS phosphorylation reaction. Activity curves and EC50 values were fitted by interpolation to an ATP:ADP standard curve as indicated by the ADP-Glo manufacturer using Prism software.
Measurement: EC50 = 0.37 nM (pChEMBL 9.430000305175781)
Descriptors: MW 596.04, ALogP 2.9, HBA 9, HBD 5, PSA 155.39, QED 0.17
Compound (SMILES): COc1cc(Nc2cc(Oc3ccc(NC(=O)Nc4cc(C(C)(C)C)cc(CC(N)=O)c4OC)c4ccccc34)ccn2)ccc1C(=O)NCCN1CCOCC1
InChIKey: ACNBWIILAUOAHP-UHFFFAOYSA-N
Target: Glycogen synthase kinase-3 alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P49840
Assay: Enzyme Inhibition Assay: Method 1: The inhibitory activities of compounds of the invention against the GSK 3α enzyme isoform (Invitrogen), are evaluated by determining the level of activation/phosphorylation of the target peptide. The GSK3-α protein (500 ng/mL, 2.5 μL) is mixed with the test compound (2.5 μL at either 4 μg/mL, 0.4 μg/mL, 0.04 μg/mL, or 0.004 μg/mL) for 2 hr at RT. The FRET peptide (8 μM, 2.5 μL), which is a phosphorylation target for GSK3α, and ATP (40 μM, 2.5 μL) are then added to the enzyme/compound mixture and the resulting mixture incubated for 1 hr. Development reagent (protease, 5 μL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan® Flash, ThermoFisher Scientific).
Measurement: IC50 = 2281 nM (pChEMBL 5.639999866485596)
Descriptors: MW 775.91, ALogP 6.82, HBA 10, HBD 5, PSA 178.4, QED 0.08
Compound (SMILES): COc1cc(F)c2[nH]c(-c3nn(C(C)C)c4ncnc(N)c34)cc2c1
InChIKey: VYRPJOQRNZMEEG-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Inhibition Assay: Class I PI3-Ks can be either purchased (p110α/p85α, p110β/p85α, p110Î´/p85α from Upstate, and p110γ from Sigma) or expressed as previously described (Knight et al., 2004). IC50 values are measured using either a standard TLC assay for lipid kinase activity (described below) or a high-throughput membrane capture assay. Kinase reactions are performed by preparing a reaction mixture containing kinase, inhibitor (2% DMSO final concentration), buffer (25 mM HEPES, pH 7.4, 10 mM MgCl2), and freshly sonicated phosphatidylinositol (100 ug/ml). Reactions are initiated by the addition of ATP containing 10 uCi of γ-32P-ATP to a final concentration 10 or 100 uM and allowed to proceed for 5 minutes at room temperature. For TLC analysis, reactions are then terminated by the addition of 105 ul 1N HCl followed by 160 ul CHCl3:MeOH (1:1). The biphasic mixture is vortexed, briefly centrifuged, and the organic phase is transferred to a new tube using a gel loading pipette tip precoated with CHCl3. This extract is spotted on TLC plates and developed for 3-4 hours in a 65:35 solution of n-propanol:1M acetic acid. The TLC plates are then dried, exposed to a phosphorimager screen (Storm, Amersham), and quantitated. For each compound, kinase activity is measured at 10-12 inhibitor concentrations representing two-fold dilutions from the highest concentration tested (typically, 200 uM).
Measurement: IC50 > 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 340.36, ALogP 3.29, HBA 6, HBD 2, PSA 94.64, QED 0.6
Compound (SMILES): Cc1ccc(NC[C@@H]2[C@H](C)CCCN2C(=O)c2nc(C)sc2-c2ccc(F)cc2)nc1
InChIKey: VULABSKJDXVOCQ-OXQOHEQNSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Cell-based Functional Assay: Measurement of [Ca2+]i using a FLIPR: CHO-OX1 or CHO-OX2 cells are seeded into black-walled clear-base 384-well plates (Costar) at a density of 20,000 cells per well in F12-K medium supplemented with 10% FBS and selection antibiotic (500 ug/ml G418 or 15 ug/ml Blasticidin) and cultured overnight. The cells are incubated with equal volume of calcium4 loading buffer (Molecular Devices, Inc.) containing 2.5 mM probenecid at 37° C. for 30 min, followed by putative OX1 or OX2 receptor antagonists (dose-range 0.1 nM-10 μM) for another 30 min.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 438.57, ALogP 5.31, HBA 5, HBD 1, PSA 58.12, QED 0.59
Compound (SMILES): Cc1ccc(-n2nc(C(C)(C)CO)cc2NC(=O)Nc2ccc(Oc3ccncc3)cc2)cc1
InChIKey: WQGFCOHRFHVPPO-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16539
Assay: MAPK13 and MAPK14 enzyme assays: ach of these analogs were checked for physical chemical characteristics based on molecular weight, Lipinski's Rule of 5, partition coefficient (log P), and topological polar surface area (tPSA). Acceptable analogs were assessed for IC50 in MAPK13 and MAPK14 enzyme assays, using inhibition of the closely related MAPK14 as a surrogate for specificity against the broader kinome.
Measurement: IC50 = 94 nM (pChEMBL 7.03000020980835)
Descriptors: MW 457.53, ALogP 5.28, HBA 6, HBD 3, PSA 101.3, QED 0.35
Compound (SMILES): COc1ccc2c(=O)[nH]cc(-c3ccc(NC(=O)N4CCc5ccccc54)cc3)c2c1
InChIKey: SSFWFVCXZGWPHN-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50; i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 411.46, ALogP 4.8, HBA 3, HBD 2, PSA 74.43, QED 0.51
Compound (SMILES): Cc1ccc(F)c(NC(=O)c2cccc(C#Cc3cc(C(=O)N=S4(=O)CCOCC4)cnc3N)c2)c1
InChIKey: LFNAERHMPRGPKC-UHFFFAOYSA-N
Target: Platelet-derived growth factor receptor beta (SINGLE PROTEIN), Homo sapiens
UniProt: P09619
Assay: PDGFRβ Kinase Assay: Biochemical PDGFRβ kinase assays were performed in 96 well microtiter plates that were coated overnight with 75 μg of poly-Glu-Tyr (4:1) in 10 mM Phosphate Buffered Saline (PBS), pH 7.4. The coated plates were washed with 2 mls per well PBS+0.05% Tween-20 (PBS-T), blocked by incubation with PBS containing 1% BSA, then washed with 2 mls per well PBS-T prior to starting the reaction. Reactions were carried out in 100 μL reaction volumes containing 36 μM ATP in kinase buffer (50 mM Hepes buffer pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2 and 0.2 mM Na3VO4). Test compounds were reconstituted in 100% DMSO and added to the reaction to give a final DMSO concentration of 5%. Reactions were initiated by the addition 20 ul per well of kinase buffer containing 200-300 ng purified cytoplasmic domain PDGFR-b protein (Millipore). Following a 60 minute incubation at 30° C., the reactions were washed 2 mls per well PBS-T. 100 μl of a monoclonal anti-phosphotyrosine antibody-peroxidase conjugate diluted 1:10,000 in PBS-T was added to the wells for 30 minutes. Following a 2 mls per well wash with PBS-Tween-20, 100 μl of O-Phenylenediamine Dihydrochloride in phosphate-citrate buffer, containing urea hydrogen peroxide, was added to the wells for 7-10 minutes as a colorimetric substrate for the peroxidase. The reaction was terminated by the addition of 100 μl of 2.5N H2SO4 to each well and read using a microplate ELISA reader set at 492 nm.
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 506.56, ALogP 3.4, HBA 6, HBD 2, PSA 123.74, QED 0.53
Compound (SMILES): Fc1ccc(-c2ncccc2-c2ccc3[nH]ncc3c2)nc1
InChIKey: QWIQXNOJDBEHSZ-UHFFFAOYSA-N
Target: TGF-beta receptor type-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P37173
Assay: Biological Assay: Assays for the compounds reported below were conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGFβR1 T204D or HIS-TGFβR2 WT, anti-HIS detection antibody, a labeled small molecule probe (Kd=<100 nM; koff=<0.001 s−1.) and test compounds in assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij35, 4 mM DTT, and 0.05 mg/ml BSA). The reaction is incubated for 1 hour at room temperature and the HTRF signal was measured on an Envision plate reader (Ex: 340 nm; Em: 520 nm/495 nm). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay are 1 nM HIS-TGFβR1 T204D or HIS-TGFβR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule probe (at Kd) and 0.5% DMSO. Dose response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations.
Measurement: IC50 > 15000 nM (pChEMBL 4.823999881744385)
Descriptors: MW 290.3, ALogP 3.83, HBA 3, HBD 1, PSA 54.46, QED 0.61
Compound (SMILES): C[C@@H]1Cc2c(nnn2-c2cc[nH]n2)CN1C(=O)c1ccnc(C(F)(F)F)c1F
InChIKey: FKTFTNYSZYYCFD-MRVPVSSYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q64663
Assay: Ca2+ flux assay: 1321N1 cells expressing the recombinant human or rat P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 μl volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250× the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 μL of the compound into 300 μL of assay buffer. A further 3× dilution occurred when transferring 50 μL/well of the compound plate to 100 μL/well in the cell plate. Cells were incubated with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 μL/well of BzATP (final concentration is 250 μM BzATP (human and rat)). The fluorescence change was measured 180 seconds after adding the agonist. Peak fluorescence was plotted as a function of BzATP concentration using Origin 7 software.
Measurement: IC50 = 5321 nM (pChEMBL 5.269999980926514)
Descriptors: MW 395.32, ALogP 2.13, HBA 6, HBD 1, PSA 92.59, QED 0.67
Compound (SMILES): C[C@@H]1CC[C@@H](c2nc([C@@H](O)C(F)F)cs2)CN1C(=O)c1ccccc1-n1nccn1
InChIKey: HDQTZRAZBKTWTQ-PBFPGSCMSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: FLIPR Ca2+ Flux Assay: Briefly, for intracellular calcium measurements, Chinese hamster ovary (CHO) cells expressing the orexin-1 receptor (e.g., rat or human) or the orexin-2 receptor (e.g., rat or human), are grown in Iscove's modified DMEM containing 2 mM L-glutamine, 0.5 g/ml G418, 1% hypoxanthine-thymidine supplement, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat-inactivated fetal calf serum (FCS). The cells are seeded at approximately 20,000 cells per well into 384-well clear bottom sterile plates coated with poly-D-lysine. The seeded plates are incubated overnight at 37° C. and 5% CO2. Human ala-6,12 orexin-A can be used as the agonist and prepared as a 1 mM stock solution in 1% bovine serum albumin (BSA) and diluted in assay buffer (HBSS containing 20 mM HEPES, 0.1% BSA and 2.5 mM probenecid, pH7.4) for use in the assay at a final concentration of 70 pM. Test compounds are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates, first in DMSO, then in assay buffer. On the day of the assay, cells are washed 3 times with 100 μl assay buffer and then incubated for 60 minutes (37° C., 5% CO2) in 60 μl assay buffer containing 1 μM Fluo-4 AM ester, 0.02% pluronic acid, and 1% BSA. The dye loading solution is then aspirated and cells are washed 3 times with 100 μl assay buffer. 30 μl of that same buffer is left in each well. Within the Fluorescent Imaging Plate Reader (FLIPR, Molecular Devices), test compounds are added to the plate in a volume of 25 μl, incubated for 5 minutes, and then 25 μl of agonist is added. Fluorescence is measured for each well at 1 second intervals for 5 minutes, and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 70 pM of agonist ala-6,12 orexin-A with buffer in place of test compound.
Measurement: IC50 = 520 nM (pChEMBL 6.28000020980835)
Descriptors: MW 433.48, ALogP 3.43, HBA 7, HBD 1, PSA 84.14, QED 0.67
Compound (SMILES): COc1cccc(C(CO)NC(=O)c2cc(-c3ccc4[nH]ncc4c3)ccc2F)c1
InChIKey: LDNRNYZSQGXYFE-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Evaluation of the Inhibitory Activity Against ROCK Protein Kinase: Assay buffer solution: 20 mM 4-hydroxyethylpiperazine ethanesulfonic acid (pH 7.5), 10 mM magnesium chloride, 1 mM ethylene glycol-bis-(2-aminoethyl)tetraacetic acid, 0.02% polyethylene glycol monododecyl ether, 0.02 mg/mL bovine serum albumin, 0.1 mM sodium vanadate, 2 mM dithiothreitol, 1% DMSO.Experimental Operation:The freshly prepared buffer solution was added to ROCK protein kinase substrate (Long S6 Kinase substrate peptide), at a concentration of 20 μM, then 1 nM ROCK protein kinase was added thereto and stirred evenly. Echo550 was used to add a series of DMSO dilutions containing the test compound (starting from 10 μM, serially diluted by 3 times), the solution was pre-incubated at room temperature for 20 minutes, then 33P-ATP (radiation intensity 10 μCi/μL) was added to initiate the reaction, and the reaction was performed at room temperature for two hours. Then the solution was filtered by P81 ion exchange paper (Whatman #3698-915) and washed with 0.75% phosphoric acid. The radiation intensity was determined by Filter-Binding method.
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 405.43, ALogP 3.84, HBA 4, HBD 3, PSA 87.24, QED 0.45
Compound (SMILES): CC(C)(C)c1nc(N2CCN(CCCCNC(=O)N3CCN(c4ccnc5cc(Cl)ccc45)CC3)CC2)cc(C(F)(F)F)n1
InChIKey: RNKUEZGSYUCLKY-UHFFFAOYSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Binding Assay: The affinities of the target compounds were determined using radioligand binding experiments. All radioligand binding experiments were performed according to previously described methods (Luedtke R. R. et al. Synapse 2000, 38, 438-449; Chu, W. et al. Bioorg. Med. Chem. 2005, 13, 77-87; Taylor, M. et al. Synapse 2010, 64, 251-266). Stably transfected HEK cells expressing the human D2-long and the D3 dopamine receptor were developed using the pIRESneo2 bicistronic expression vector (Clontech, Palo Alto, Calif.). Levels of expression of human D2 and D3 dopamine receptors in HEK cells were 57,941±14,686 fmol/mg protein and 4202±1516 fmol/mg protein, respectively. Competition curves were performed using 125I-IABN. Membrane homogenates (50 μL) were suspended in 50 mM Tris-HCl/150 mM NaCl/10 mM EDTA buffer, pH 7.5 and incubated with 50 μL of 125I-IABN at 37° C. for 60 min. Nonspecific binding was defined using 4 μM (+)-butaclamol. The radioligand concentration used was approximately equal to the Kd value, and the concentration of the competitive inhibitor ranged over five orders of magnitude. Binding was terminated by the addition of cold wash buffer (10 mM Tris-HCl/150 mM NaCl, pH 7.4) and filtration over a glass-fiber filter (Schleicher and Schuell No. 32). Filters were washed with 10 mL of cold buffer, and the radioactivity was quantitated. A Packard Cobra gamma counter was used for 125I-labeled radioligands (efficiency=75%). The protein concentration was determined using a BCA reagent (Pierce) with bovine serum albumin as the protein standard. Data from competitive inhibition experiments was modeled using nonlinear regression analysis to determine the concentration of inhibitor that inhibits 50% of the specific binding of the radioligand (IC50 value). Competition curves were modeled for a single site. Data from competition dose-response curves were analyzed using Tablecurve program (Jandel). IC50 values were converted to equilibrium dissociation constants (Ki values) using the Cheng and Prusoff correction.
Measurement: Ki = 359 nM (pChEMBL 6.449999809265137)
Descriptors: MW 633.16, ALogP 5.43, HBA 7, HBD 1, PSA 80.73, QED 0.35
Compound (SMILES): O=C1NN=C(c2ccc(-c3cccc(F)c3F)c(F)c2)CO1
InChIKey: YDEBOSSOBPCHNV-UHFFFAOYSA-N
Target: cGMP-inhibited 3',5'-cyclic phosphodiesterase 3B (SINGLE PROTEIN), Homo sapiens
UniProt: Q13370
Assay: PDE3B Enzyme Inhibition: The commercially available 3H-cAMP Scintillation Proximity Assay (SPA, Perkin Elmer) system was used for enzyme inhibition studies. For the determination of the in vitro effect of example compounds on the PDE3B reactions 2 μl of the respective example compound solution in DMSO (serial dilutions) were placed in wells of microtiter plates (Isoplate-96/200W; Perkin Elmer). 50 μl of a dilution of PDE3B cell extract from Sf9 cells overexpressing human full length PDE3B (SB Drug Discovery, UK) in buffer A (50 mM Tris/HCl pH 7.5, 8.3 mM MgCl2, 1.7 mM EDTA, 0.2% BSA) was added. The dilution of the PDE3B cell extract was chosen such that the reaction kinetics was linear and less than 70% of the substrate was consumed (typical dilution 1:6000). The reaction was started by addition of 50 μl (0.025 μCi) of 1:2000 in buffer A w/o BSA diluted substrate [8-3H]adenosine 3′, 5′-cyclic phosphate (1 μCi/μl; Perkin Elmer). After incubation at room temperature for 60 min, the reaction was stopped by addition of 25 μl of a suspension containing 18 mg/ml yttrium scintillation proximity beads (Perkin Elmer) in water. The microtiter plates were sealed and measured in a Microbeta scintillation counter (PerkinElmer Wallac). IC50 values were determined from sigmoidal curves by plotting percentage PDE3B activity vs log compound concentration.
Measurement: IC50 = 32 nM (pChEMBL 7.5)
Descriptors: MW 306.24, ALogP 3.21, HBA 3, HBD 1, PSA 50.69, QED 0.93
Compound (SMILES): CNc1cc(C(C)(C)C)cc(NC(=O)Nc2ccc(Oc3ccnc(Nc4cc(OC)cc(OCCOCCOCCOC)c4)c3)c3ccccc23)c1OC
InChIKey: CKVWFKWUVXFXPC-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase Src (SINGLE PROTEIN), Homo sapiens
UniProt: P12931
Assay: Enzyme Inhibition: The inhibitory activities of compounds of the invention against c-Src and Syk enzymes (Invitrogen), are evaluated in a similar fashion to that described hereinabove. The relevant enzyme (3000 ng/mL or 2000 ng/mL respectively, 2.5 μL) is incubated with the test compound (either 1 μg/mL, 0.1 μg/mL, 0.01 μg/mL, or 0.001 μg/mL, 2.5 μL each) for 2 hr at RT. The FRET peptides (8 μM 2.5 μL), and appropriate ATP solutions (2.5 μL, 800 μM for c-Src, and 60 μM ATP for Syk) are then added to the enzymes/compound mixtures and the mixture incubated for 1 hr. Development reagent (protease, 5 μL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan® Flash, ThermoFisher Scientific).
Measurement: IC50 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 753.9, ALogP 8.83, HBA 11, HBD 4, PSA 142.69, QED 0.06
Compound (SMILES): Cc1nc(C(=O)N[C@@H](c2ccc(C(F)(F)F)cc2)C2CC2)cc(=O)[nH]1
InChIKey: SEIUPVJTHVYQEP-OAHLLOKOSA-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 = 18 nM (pChEMBL 7.75)
Descriptors: MW 351.33, ALogP 2.98, HBA 3, HBD 2, PSA 74.85, QED 0.89