lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(=O)c1nn(CC(=O)N2[C@@H]3C[C@@H]3C[C@H]2C(=O)NC2CC(C)(C)C2)c2ccc(OCc3ncccn3)cc12
InChIKey: GEGJXBVUZUOIQE-ZQMYSKGWSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Human Complement Factor D Assay: Method 1: Recombinant human factor D (expressed in E. coli and purified using standard methods) at 10 nM concentration is incubated with test compound at various concentrations for 1 hour at room temperature in 0.1 M Hepes buffer, pH 7.5, containing 1 mM MgCl2, 1 M NaCl and 0.05% CHAPS. A synthetic substrate Z-Lys-thiobenzyl and 2,4-dinitrobenzenesulfonyl-fluoresceine are added to final concentrations of 200 μM and 25 μM, respectively. The increase in fluorescence is recorded at excitation of 485 nm and emission at 535 nm in a microplate spectrofluorimeter. IC50 values are calculated from percentage of inhibition of complement factor D-activity as a function of test compound concentration.
Measurement: IC50 = 36 nM (pChEMBL 7.440000057220459)
Descriptors: MW 516.6, ALogP 2.9, HBA 8, HBD 1, PSA 119.31, QED 0.46
Compound (SMILES): O=c1[nH]c(CSC2CCOCC2)nc2cc(OCC3CCOCC3)c(F)c(F)c12
InChIKey: UFTXVCMHPHSYSJ-UHFFFAOYSA-N
Target: Protein mono-ADP-ribosyltransferase PARP14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q460N5
Assay: Enzymatic Inhibition Assay: Enzymatic inhibition of PARP14 was measured using a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) monitoring the auto-modification of PARP14 by biotinylated nicotinamide adenine dinucleotide (biotin-NAD). 1 μL of a dose response curve of each test compound was spotted in 384-well nickel-coated white microplates (Thermo) using a Mosquito (TTP Labtech). Reactions were performed in a 50 μL volume by adding 40 μL of PARP14 in assay buffer (20 mM HEPES pH==8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002%0 Tween20), incubating with test compound at 25° C. for 30 min, then adding 10 μL of biotin-NAD (Biolog). The final concentrations of PARP14 and biotin-NAD are 50 nM and 3 μM, respectively. Reactions proceeded at 25° C. for 3 h, then were quenched with 5 μL of 10 mM unmodified nicotinamide adenine dinucleotide (Sigma-Aldrich). The quenched reactions were washed 3 times with 100 μL of TBST wash buffer (50 mM Tris-HCl, 150 mM NaCl and 0.1% Tween20). Next, to the washed and dried plate was added 25 μL of DELFIA Europium-N1 streptavidin (Perkin Elmer) diluted in DELFIA assay buffer (Perkin Elmer). After a 30 min incubation at 25° C., the plate was washed 5 times with TBST wash buffer. Finally, 25 μL of DELFIA enhancement solution was added. After a 5 min incubation the plate was read on an Envision platereader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 340 nm and emission of 615 nm to measure the amount of Europium present in each well, informing on the amount of biotin-NAD that was transferred in the automodification reaction.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 426.49, ALogP 3.42, HBA 6, HBD 1, PSA 73.44, QED 0.76
Compound (SMILES): COc1cccc(NC2CCN(c3ccc(Br)c(OC)n3)C2=O)c1C
InChIKey: BFAYVIGGIHFOKM-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assays: 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 (SEQ ID NO: 1)). 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 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 406.28, ALogP 3.39, HBA 5, HBD 1, PSA 63.69, QED 0.83
Compound (SMILES): CC(=O)NC1CN(C(=O)Nc2cc(C3=CCCCC3)ccc2N)C1
InChIKey: VGGCQWTYZDEBLJ-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: Inhibition of Histone Deacetylase Enzymatic Activity Assay: HDAC1: The following trypsin-coupled protocol and Caliper protocol described Herein was used to assay the compounds of the invention. Trypsin Coupled Assay: HDAC1-Kinetic IC50 3Hr PreIncubation. Working Stocks: 1xHDAC Assay Buffer: 50 mM HEPES (GIBCO 15630-114), 100mM KCl, 0.001% Tweem-20 (Zymed 00-3005), 0.05% BSA (Invitrogen, P2489), pH 7.4, 1.5xHDAC+1.5xTrypsin: 225nM Trypsin(Worthinton)+0.18 ug/ml HDAC1 (BPS Inc) in assay buffer 300x Trypsin stock is made up in HDAC Assay Buffer, aliquoted and stored at -80C. 3xSubstrate: 18uM Substrate in assay buffer BPS Enzyme and Substrate amounts (per well).
Measurement: IC50 = 3050 nM (pChEMBL 5.519999980926514)
Descriptors: MW 328.42, ALogP 2.58, HBA 3, HBD 3, PSA 87.46, QED 0.75
Compound (SMILES): COc1cc(CO)c(-c2cn3ccc(N4CC[C@H](NCc5ccncc5)C4)cc3n2)cc1Cl
InChIKey: FMJZNPOJPUYKBS-IBGZPJMESA-N
Target: Histone-lysine N-methyltransferase SUV39H2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H5I1
Assay: Suv 39H2 Assay: Assay buffer (4 μL) was added into all the wells, including test compound and control wells (except for Sinefungin control wells), using the Multidrop Combi. The plate was centrifuged at 1000 rpm for 1 min. Substrate mix (8 μL) containing radiolabelled 3H-SAM (final conc: 100 nM) and H3 Histone Peptide (final conc: 350 nM) was added to all wells using Multidrop Combi and centrifuged at 1000 rpm for 1 min. SUV39H2 (8 μL, final conc: optimized for each lot of the enzyme based on specific activity) was added by using the Multidrop Combi. The assay plate was centrifuged at 1000 rpm for 1 min. Assay buffer was used as background control, and incubated at room temperature for 3 hours. The reaction was stopped with 20 μL of 2.5 mg/mL Streptavidin SPA beads (final conc. 50 μg/well) in buffer using the Multidrop Combi and centrifuged for 1 min. The plate was loaded into the Trilux-Microbeta counter and a delayed read of 10 hours was made. The radioactive signal was measured at 1 min/well. Sinefungin and the internal standard compound were used as tool compounds to determine assay performance.
Measurement: IC50 = 6.49 nM (pChEMBL 8.1899995803833)
Descriptors: MW 463.97, ALogP 3.92, HBA 7, HBD 2, PSA 74.92, QED 0.43
Compound (SMILES): Cn1nccc1Nc1nccc(-c2cc3n4c(nnc4c2)[C@@H](Oc2ccc(F)cc2)CCO3)n1
InChIKey: CTWHMTCAEZFXMK-SFHVURJKSA-N
Target: Mitogen-activated protein kinase 6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16659
Assay: Enzymatic Assay: Compounds were tested in an enzymatic assay using human ERK-2 (Mitogen Activated Kinase 1), recombinantly expressed as an n-terminal 6-His fusion protein in E. coli and corresponding to aa 8-360. The substrate used was the fluorescent Omnia peptide S/T17 (Invitrogen of Carlsbad, Calif.; Cat. KNZ1171C). Test compounds were diluted in DMSO in 3-fold serial dilutions at 100× final concentrations. In addition to compound, the assay contained 50 mM HEPES [pH 7.3], 10 mM MgCl2, 2 mM DTT, 0.005% Triton-X100, 5 nM ERK-2 enzyme, 6.25 μM S/T17 peptide substrate and 25 μM ATP (corresponding to the observed Km) for a total reaction volume of 25 μL. The assay was run at ambient temperature in a white 384-well polypropylene plate (Nunc, Inc of Naperville, Ill.; Cat. 267462) collecting data every 50 seconds for approximately 30 minutes on an Envision plate reader (PerkinElmer, Inc. of Waltham, Mass.); Excitation 340 nm/Emission 495 nm. The data collected from each well was fit to a straight line, and the resulting rates were used to calculate percent of control. Percent of control was plotted against compound concentration, and IC50 values were determined using a four-parameter fit.
Measurement: IC50 = 0.02 nM (pChEMBL 10.699999809265137)
Descriptors: MW 458.46, ALogP 3.71, HBA 10, HBD 1, PSA 104.28, QED 0.43
Compound (SMILES): CC1(C)[C@H]2CC[C@]1(c1ccnc(-c3ccc(CS(C)(=O)=O)nc3)n1)c1nnc(-c3c(F)cccc3F)cc12
InChIKey: YZAQTKDLVVXBNL-VKGTZQKMSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: In Vitro Ligand Binding Assay: 50 mM HEPES buffer (pH 7.4); 150 mM NaCl; 1 mM DTT; 5 mM MgCl2; 0.01% BSA; 5% DMSO; 0.6 ug/mL RORc receptor; 6 nM 25-[3H]hydroxycholesterol. For NSB wells, 1 uM 25-hydroxycholesterol was also present. Assay plates were 96-well polypropylene V-bottom plates. 10 uL of 5× compound in 25% DMSO/75% Assay Buffer was added to Test wells. 10 uL of 25% DMSO/75% Assay Buffer was added to Total Binding or No Receptor wells. 10 uL of 5 uM 25-hydroxycholesterol in 25% DMSO/75% Assay Buffer was added to NSB wells. 20 uL of 15 nM 25-[3H]hydroxycholesterol prepared in Assay Buffer was added to all wells. 20 uL of 1.5 ug/mL RORc receptor was added to wells (or 40 uL Assay Buffer to No R wells). Following addition to the wells, the plates were incubated 3 h at 25° C. Filtration
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 533.6, ALogP 5.02, HBA 7, HBD 0, PSA 98.59, QED 0.36
Compound (SMILES): Cn1cc(-c2cn3nccc3c(-c3cnn(C4(CC#N)CN(S(C)(=O)=O)C4)c3)n2)cn1
InChIKey: NBHWKOZWTUTPEQ-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: JAK Caliper Enzyme Assay at 1 mM ATP: Compounds were added to a 384-well plate. Reaction mixtures contained 10 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% BSA, 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20% to 30% phosphorylation. The assays were stopped with 15 μL of 180 mM HEPES, pH 7.4, 20 mM EDTA, and 0.2% Coating Reagent 3. The assay plates were placed on a Caliper Life Science LC3000 instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 437.49, ALogP 0.88, HBA 10, HBD 0, PSA 127, QED 0.45
Compound (SMILES): O=C(Nc1cc2c(cc1N1CCOCC1)OCCN2)c1cnn2cccnc12
InChIKey: PIWBBIIWOWZKFI-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Biological Assay: Kinase activities were assayed using the Transcreener-Fluorecescence polarization platform (BelBrook Labs, Madison, Wis., USA) that measures amounts of the reaction product, ADP. The IRAK4 reaction conditions were optimized using an IRAK1-derived peptide (sequence H-KKARFSRFAGSSPSQSSMVAR) to provide a linear reaction rate over the course of a 90 min incubation, which resulted in 10-12% conversion of the starting ATP to ADP. Final IRAK4 assay conditions were 1.25 nM IRAK4; 125 uM ATP; 10 uM MgCl2; 125 uM peptide in reaction buffer (25 mM HEPES (pH7.4); 2 mM Dithiothreitol; 0.015% Brij-35; and 0.5% dimethyl sulfoxide. The IRAK1 activity was optimized similarly, yielding final assay conditions of 3 mM IRAK1; 62.5 uM ATP; 5 uM MgCl2, and 62.5 uM IRAK1 peptide in reaction buffer for 60 min.Assays of compounds for kinase inhibition were performed using inhibitors serially-diluted in dimethyl sulfoxide, which was accomplished with a LabCyte Echo 555 liquid dispenser. 384 well assay plates spotted with compound received 4 ul of a 2× substrate (ATP+peptide) mix in reaction buffer, followed by 4 ul of 2× enzyme diluted in reaction buffer. Reactions were halted at 60 (IRAK1) or 90 (IRAK4) min by addition of 6 ul of detection buffer, containing EDTA (40 nM final concentration), 0.95 ug of the ADP-binding antibody ADP2, ADP tracer (3 nM final concentration), and 25 uM HEPES. Following a 1 hr incubation, fluorescence polarization of the ADP2-antibody TRACER complex was read on a Tecan M1000 plate reader using a 635/20 excitation filter in combination with a 670/20 emission filter.
Measurement: Ki = 98 nM (pChEMBL 7.010000228881836)
Descriptors: MW 380.41, ALogP 1.62, HBA 8, HBD 2, PSA 93.02, QED 0.71
Compound (SMILES): C=C(C)[C@H]1CCC(C)=CC1c1c(O)cc(CCCCC)c(C(=O)OCCO)c1O
InChIKey: IINCKHXDYQYLTO-MRTLOADZSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: In Vitro Binding to CB1 and CB2 Receptors: Membranes from human CB1 or CB2 receptor-transfected cells (RBHCB1M400UA and RBXCB2M400UA, respectively) were supplied by Perkin-Elmer Life and Analytical Sciences (Boston, Mass.). The values of Bmax and Kd for the CB1 or CB2 receptor membranes are variable. The used batch showed the following Bmax and Kd values, 1.9 pmol/mg membrane protein and 0.16 nM, respectively, for CB1 receptor membranes, and 5.2 pmol/mg membrane protein and 0.18 nM, respectively, for CB2 receptor membranes. The protein concentration for the CB1 receptor membranes was 8.0 mg/ml, whereas for the CB2 receptor membranes 4.0 mg/ml. The commercial membranes were diluted (1:20) with the binding buffer (50 mM TrisCl, 5 mM MgCl2.H2O, 2.5 mM EDTA, 0.5 mg/mL BSA and pH=7.4 for CB1 binding buffer; 50 mM TrisCl, 5 mM MgCl2.H2O, 2.5 mM EGTA, 1 mg/mL BSA and pH=7.5 for CB2 binding buffer). The radioligand was [3H]-CP55940 (144 Ci/mmol; PerkinElmer) used at a concentration of 0.10 nM with a final volume of 200 μl for CB1 binding and at a concentration of 0.15 nM with a final volume of 600 μl for CB2 binding.
Measurement: Ki = 359 nM (pChEMBL 6.449999809265137)
Descriptors: MW 402.53, ALogP 5, HBA 5, HBD 3, PSA 86.99, QED 0.31
Compound (SMILES): CN1C(=O)C(NC(=O)c2cc(Oc3ccccc3)ccn2)COc2ccc(C#CC(C)(C)O)cc21
InChIKey: UIKLFGXQZRQKMF-UHFFFAOYSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: biochemical assay using the ADP-Glo technology: ADP-Glo (Promega, Madison, Wis., USA) reagents were thawed at ambient temperature. Kinase Detection Reagent was prepared by mixing kinase detection buffer with the lyophilized kinase detection substrate.A 500 ml stock volume of 5× Reaction Kinase Buffer was made by mixing 1000 μl of 1M MgCl2, 500 μl of 1M Tris-HCL pH7.4, 0.5 mg/ml (25 mg) of BSA, and 3475 μl of distilled H2O. A 3 ml 2× working stock volume of Reaction Kinase Buffer was made containing a final concentration of 100 μM DTT and 4 mM MnCl2.Components of RIPK1 enzyme (Rigel Pharmaceuticals, South San Francisco, Calif., USA) were thawed on ice. Diluted RIPK1 was prepared in 1× Kinase Reaction Buffer (diluted from 2× buffer) to 31 ng/well. A 166 M working stock ATP assay solution was prepared in 1× Kinase Reaction Buffer (diluted from 2× buffer).Compounds were serially diluted in DMSO from 250 uM in 4-fold dilutions then diluted 1:5 in 2× Reaction Buffer in a 96 well plate. 1.0 ul of diluted compound was added to a 384 well plate in duplicate. 2 μl of diluted Active RIPK1 was added to 384 well plate (do not add to column1) add 2× rxn buffer to column 1. AKT (Anaspec, Fremont, Calif., USA) at 150 nM was combined with ATP working stock at equal volume and 2 ul/well were added to the 384 well plate. The final reaction volume was 5.0 μl. The plate was quickly centrifuged and the reaction was incubated at 30° C. for 30 minutes. Adding 5 μl of ADP-Glo terminated the reaction. The plate was quickly centrifuged and the reaction was incubated at room temperature for 40 minutes. Kinase Detection Reagent was then added and incubated at room temperature for 30 minutes. The relative light unit (RLU) of kinase reaction was determined by luminescent (Luminescence 0.1 s) using a Wallac Victor2 Luminometer (PerkinElmer, Waltham, Mass., USA).
Measurement: IC50 = 22.2 nM (pChEMBL 7.650000095367432)
Descriptors: MW 471.51, ALogP 3.15, HBA 6, HBD 2, PSA 100.99, QED 0.57
Compound (SMILES): COc1nccc(Sc2cnc(N3CCC4(CC3)CO[C@@H](C)[C@H]4N)c([S+](C)[O-])n2)c1Cl
InChIKey: KMIPGWLKADTXHS-IXORFKRPSA-N
Target: Tyrosine-protein phosphatase non-receptor type 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06124
Assay: SHP2 Allosteric Inhibition Assay: SHP2 possesses two N-terminal Src homology 2 (SH2) domains, a central protein-tyrosine phosphatase (PTP) domain, and C-terminal tail. At the basal state, SHP2 is auto-inhibited and access of substrates to the catalytic site is blocked by the intermolecular interactions between the SH2 domains and the PTP domain. When bis-tyrosyl-phosphorylated peptides bind to SH2 domain of SHP2, the PTP domain becomes available for substrate recognition and reaction catalysis and SHP2 is allosterically activated. SHP2 catalytic activity can be measured using a fluorogenic artificial substrate DiFMUP.The phosphatase reactions were carried out at room temperature in 384-well black polystyrene plates (Greiner Bio-One, Cat #784076) using assay buffers containing 60 mM HEPES, pH 7.2, 75 mM NaCl, 75 mM KCl, 1 mM EDTA, 0.05% P-20, and 5 mM DTT.0.33 nM of STIP2 was co-incubated with of 0.5 μM of bisphos-IRS 1 peptide (sequence: H2N-LN(pY)IDLDLV(dPEG8)LST(pY)ASINFQK-amide) and various concentrations of compounds for 30-60 min at room temperature. Then the reaction was initiated by addition of the surrogate substrate DiFMUP (Invitrogen, Cat #D6567, 100 uM final).The real-time conversion of DiFMUP to DiFMU (6,8-difluoro-7-hydroxyl-4-methyl-coumarin) was measured every 5 min for 30 min using a microplate reader (CLARIOstar, BMG Labtech) with excitation and emission wavelengths of 340 nm and 450 nm, respectively. Initial reaction rates were determined by linear fitting of the data and the inhibitor dose response curves were analyzed using normalized IC50 regression curve fitting with control based normalization.
Measurement: IC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 484.05, ALogP 2.75, HBA 9, HBD 1, PSA 109.45, QED 0.64
Compound (SMILES): O=C(/C=C/c1ccc(O)cc1)C(C(=O)/C=C/c1ccc(O)cc1)C(=O)CO
InChIKey: INOCVMCIBUBQTR-YDWXAUTNSA-N
Target: Matrix metalloproteinase-9 (SINGLE PROTEIN), Homo sapiens
UniProt: P14780
Assay: Inhibition of MMPs: It has been observed that 50 and 100 μM concentrations of curcumin decreased TNFα production by endotoxin-stimulated human monocytes (HMs) in culture by 80-90% (lower concentrations of curcumin, 10 and 20 μM, had no effect). However, this inhibitory effect was associated with some precipitation of the curcumin in cell culture and with significant cytotoxicity. It was hypothesized that increasing the solubility of curcumin will: (i) enhance its efficacy as an inhibitor of cytokine expression, (ii) reduce its cytotoxicity, and (iii) preserve (perhaps even enhance; see below) its potency, as an MMP inhibitor (MMPI) compound, which was found to be similar to that of the Zn++ chelating compound, 1,10-O-phenanthroline (FIG. 1). However, it should be noted that excessive inhibition of MMP activity may not be desirable therapeutically because a minimal, or basal, level of MMPs may be necessary for optimal defense of the host.
Measurement: IC50 = 6000 nM (pChEMBL 5.21999979019165)
Descriptors: MW 366.37, ALogP 2.14, HBA 6, HBD 3, PSA 111.9, QED 0.49
Compound (SMILES): Cc1noc(-c2cc(-c3cc(S(=O)(=O)NCCC(F)(F)F)ccc3C)cnc2N)n1
InChIKey: YMHFHEFMQSMIQU-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42338
Assay: Kinase Glo Luminescent Kinase Assay (Kglo) for PI 3-Kinase Alpha (A), PI 3-Kinase Beta (B), Vps34 (C), PI 4-Kinase Beta (D): The luminescence-based ATP detection reagent KinaseGlo was obtained from Promega, (Cat. No. V6714, Lot No. 236161) through Catalys, Wallisellen, Switzerland. L-alpha-phosphatidylinositol (PI, liver, bovine) was obtained from Avanti Polar Lipid (Cat. No. 840042C, Lot#LPI-274), phosphatidylinositol-4,5-bisphosphate (PIP(4,5)2) was obtained from Avanti Polar Lipid (Cat. No. 840046X). L-α-phosphatidylserine (PS) was obtained from Avanti Polar Lipid (Cat. No. 840032C), n-octylglucoside from Avanti Polar Lipid (Cat. No. 10634425001). Luminescence is a well established readout to determine ATP concentrations and can thus be used to follow the activity of many kinases regardless of their substrate. The Kinase Glo Luminescent Kinase Assay (Promega, Madison, Wis., USA) is a homogeneous HTS method of measuring kinase activity by quantifying the amount of ATP remaining in solution following a kinase reaction.50 nL of compound dilutions were dispensed onto black 384-well low volume Non Binding Styrene (NBS) plates (Costar Cat. No. NBS#3676). L-α-phosphatidylinositol (PI), provided as 10 mg/ml solution in methanol, was transferred into a glass tube and dried under a nitrogen beam. It was then resuspended in 3% OctylGlucoside (1-O-n-octyl-beta-D-glucopyranoside) by vortexing and stored at 4° C. 5 μl of a mix of PI/OctylGlucoside with the PI 3-kinase alpha and PI 3-kinase beta subtypes, or Vps34 or PI 4-kinase beta were added. Kinase reactions were started by the addition of 5 μl of an ATP-mix containing in a final volume 10 μl 10 mM TRIS-HCl pH 7.5, 3 mM MgCl2, 50 mM NaCl, 0.05% CHAPS, 1 mM DTT and 1 μM ATP at room temperature. Reactions were stopped with 10 μl of KinaseGlo and plates were read 10 mins later in a Synergy2 reader using an integration time of 0.1 seconds per well. 2.5 μM of NVP-BGT226 (1-(3-(trifluoromethyl)-4-(piperazin-1-yl)phenyl)-8-(6-methoxypyridin-3-yl)-3-methyl-1H-imidazo[4,5-c]quinolin-2(3H)-one) was added to the assay plates to generate the 100% inhibition of the kinase reaction, and the 0% inhibition was given by the solvent vehicle (90% DMSO in water). (1-(3-(trifluoromethyl)-4-(piperazin-1-yl)phenyl)-8-(6-methoxypyridin-3-yl)-3-methyl-1H-imidazo[4,5-c]quinolin-2(3H)-one) was used as a reference compound and included in all assay plates in the form of 16 dilution points in duplicate.IC50 values of the percentage inhibition of each compound at 8 concentrations (10, 3.0, 1.0, 0.3, 0.1, 0.030, 0.010 and 0.003 μM) n=2 were derived by fitting a sigmoidal dose-response curve to a plot of assay readout over inhibitor concentration as described. All fits were performed with the program XLfit4 (ID Business Solutions, Guildford, UK).
Measurement: IC50 = 5100 nM (pChEMBL 5.289999961853027)
Descriptors: MW 441.44, ALogP 3.23, HBA 7, HBD 2, PSA 124, QED 0.6
Compound (SMILES): Cn1ncnc1C(=O)N[C@H]1CC[C@H](N)CC1
InChIKey: OPNFFEBTPPTLEU-ZKCHVHJHSA-N
Target: Histone-lysine N-methyltransferase SMYD3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H7B4
Assay: SMYD3 Biochemical Assay: The assays were all performed in a buffer consisting of 25 mM Tris-Cl pH 8.0, 1 mM TCEP, 0.005% BSG, and 0.005% Tween 20, prepared on the day of use. Compounds in 100% DMSO (1 ul) were spotted into a 384-well white opaque OptiPlate using a Bravo automated liquid handling platform outfitted with a 384-channel head (Agilent Technologies). DMSO (1 ul) was added to Columns 11, 12, 23, 24, rows A-H for the maximum signal control and 1 ul of SAH, a known product and inhibitor of SMYD3, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control. A cocktail (40 ul) containing the SMYD3 enzyme was added by Multidrop Combi (Thermo-Fisher). The compounds were allowed to incubate with SMYD3 for 30 min at room temperature, then a cocktail (10 ul) containing SAM and MEKK2 was added to initiate the reaction (final volume=51 ul). The final concentrations of the components were as follows: SMYD3 was 0.4 nM, 3H-SAM was 8 nM, MEKK2 was 12 nM, SAH in the minimum signal control wells was 1 mM, and the DMSO concentration was 2%. The assays were stopped by the addition of non-radiolabeled SAM (10 ul) to a final concentration of 100 uM, which dilutes the 3H-SAM to a level where its incorporation into MEKK2 is no longer detectable. Radiolabeled MEKK2 was detected using a scintillation proximity assay (SPA). 10 uL of a 10 mg/mL solution of SPA beads in 0.5 M citric acid was added and the plates centrifuged at 600 rpm for 1 min to precipitate the radiolabeled MEKK2 onto the SPA beads. The plates were then read in a PerkinElmer TopCount plate reader to measure the quantity of 3H-labeled MEKK2 as disintegrations per minute (dpm) or alternatively, referred to as counts per minute (cpm).
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 223.28, ALogP -0.19, HBA 5, HBD 2, PSA 85.83, QED 0.73
Compound (SMILES): O=c1[nH]c2ncc(-c3cccc(C(F)(F)F)c3)cc2n1Cc1cccnc1
InChIKey: NUOJESOUQWSUMZ-UHFFFAOYSA-N
Target: Glutamate receptor ionotropic, NMDA 2B (SINGLE PROTEIN), Homo sapiens
UniProt: Q13224
Assay: Standard cell-based calcium flux 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 137 mM NaCl, 4 mM KCl, 2 mM CaCl2, 0.5 mM MgCl2 (standard assay) or 1.5 mM MgCl2 (HTS assay), 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 (137 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 EC40 (standard assay) is used to maximize the assay's signal window and ability to detect NMDA receptor antagonists and negative allosteric modulators. 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 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 370.33, ALogP 3.85, HBA 4, HBD 1, PSA 63.57, QED 0.6
Compound (SMILES): CCOc1cc(-c2ccc(N3CC[C@@]4(CCCN(C(C)=O)C4)C3)nc2)c2c(C#N)cnn2c1
InChIKey: BNKXTTSSKXPWRK-RUZDIDTESA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of Formula I were screened for their ability to inhibit wildtype 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; Catalog No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Catalog No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog 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 = 1415 nM (pChEMBL 5.849999904632568)
Descriptors: MW 444.54, ALogP 3.51, HBA 7, HBD 0, PSA 86.76, QED 0.61
Compound (SMILES): CCc1nc2ccc(C3=CCN(S(=O)(=O)CCCOC(C)=O)CC3)cn2c1N(C)c1nc(-c2ccc(F)cc2)cs1
InChIKey: RZEZTSNMQNPOFZ-UHFFFAOYSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Human ENPP2 (hENPP2) Assay: Compound IC50 values are determined in a hENPP2 (UniProtKB/SwissProt Sequence ref Q13822) biochemical assay using LPC as substrate. 5 μL of a dilution series of compound, starting from 20 μM highest concentration, 1/5 dilution, is added to the wells. hENPP2 is used at a final concentration of 1 μg/mL or 3 μg/mL (it will be appreciated by the skilled person that the potency read out is independent of the enzyme concentration). The enzyme is diluted in 50 mM Tris-HCl pH 8.5, 500 mM NaCl, 5 mM KCl, 10 mM CaCl2, 0.1% fatty acid free BSA in a total volume of 10 μL. the reaction is started by the addition of 10 μL of 150 μM LPC (palmitoyl 16:0) diluted in the same buffer as described above and the mixture is incubated at 37° C. for 30 min. The reaction is terminated and choline quantified by the addition of a 25 μL of a mixture containing 0.6 U/mL of choline oxidase, 0.6 U/mL of peroxydase, 1.8 mM TOOS, 1.2 mM amino-antipyrine, 20 mM EGTA (stop-developer solution) diluted in the buffer described above. Luminescence is read on the Envision after an incubation of 30 min at room temperature (Excitation 555 nm, excitation light=70%).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 597.74, ALogP 5.3, HBA 9, HBD 0, PSA 97.11, QED 0.18
Compound (SMILES): Cc1ccc(C(O)(C(N)=O)C(F)(F)F)cc1-c1cnc(N)c(-c2ccnn2C)n1
InChIKey: ZPSFLAJYOZXKOV-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: PI3K-gamma Scintillation Proximity Assay: Materials: [γ-33P]ATP (10 mCi/mL) and Wheat Germ Agglutinin (WGA) YSi SPA Scintillation Beads was purchased from Perkin Elmer (Waltham, Mass.). Lipid kinase substrate, D-myo-Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)D (+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho linked (PIP2), CAS 204858-53-7, was purchased from Echelon Biosciences (Salt Lake City, Utah). PI3Kγ (p110γ) Recombinant Human Protein was purchased from Life technology (Grand Island, N.Y.). ATP, MgCl2, DTT, EDTA, MOPS and CHAPS were purchased from Sigma Aldrich (St. Louis, Mo.).The kinase reaction was conducted in polystyrene 384-well Greiner Bio-one white plate from Thermo Fisher Scientific in a final volume of 25 μL. Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 2%. The PI3Kγ assay was carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 μM PIP2, 2 μM ATP, 0.5 μCi [γ-33P] ATP, 13 nM PI3Kγ. Reactions were incubated for 120 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 163 mM potassium phosphate pH 7.8, 20% glycerol, 25 mM EDTA. The final concentration of SPA beads is 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1500 rpm for 10 min, the radioactivity of the product was determined by scintillation counting on Topcount (Perkin-Elmer). IC50 determination was performed by fitting the curve of percent of the solvent control activity versus the log of the inhibitor concentration using the GraphPad Prism 6.0 software.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 406.37, ALogP 1.67, HBA 7, HBD 3, PSA 132.94, QED 0.6
Compound (SMILES): Cc1cc(-c2n[nH]c3cc4c(cc23)CN(C2CCNCC2)C(=O)N4)ccn1
InChIKey: NCLDKPUNZJAPMU-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Z′-LYTE Kinase Assay: The inhibition of ERK activity by the compounds disclosed herein was determined using the Z′-LYTE kinase assay kit (Life Technologies) with a Ser/Thr 3 peptide substrate (Life Technologies) according to manufacturers' instructions. The assay was run with an ERK2 enzyme (Life Technologies) concentration of 0.47 ng/μL at 100 μM ATP (approximately the ATP Km for ERK2). The IC50 values for the compounds were determined with 3-fold serial dilutions in duplicate. The compounds were first diluted in 1:3 dilutions in 100% DMSO at 100× the desired concentration, and then further diluted (1:25) in 20 mM HEPES buffer (Invitrogen) to make 4× solutions prior to adding to the enzyme solution. The final DMSO concentration in the assay was 1%. Final reaction volume was 20 μL/well in 384-well plates. Kinase reactions were conducted or 1 hour followed by the assay development reaction (1 hour) in a 20 ul/well in a 384 well plate format.
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 362.44, ALogP 3.03, HBA 4, HBD 3, PSA 85.94, QED 0.65
Compound (SMILES): CCCOc1cnccc1-c1[nH]c2c(c1Nc1ccccc1)C(=O)N(C)CC2
InChIKey: HZPXRJQBQGCGHX-UHFFFAOYSA-N
Target: Mitotic checkpoint serine/threonine-protein kinase BUB1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43683
Assay: Kinase Assay: In a typical assay 11 different concentrations of each compound (0.1 nM, 0.33 nM, 1.1 nM, 3.8 nM, 13 nM, 44 nM, 0.15 μM, 0.51 μM, 1.7 μM, 5.9 μM and 20 μM) were tested in duplicate within the same microtiter plate. To this end, 100-fold concentrated compound solutions (in DMSO) were previously prepared by serial dilution (1:3.4) of 2 mM stocks in a clear low volume 384-well source microtiter plate (Greiner Bio-One, Frickenhausen, Germany), from which 50 nl of compounds were transferred into a black low volume test microtiter plate from the same supplier. Subsequently, 2 μl of Bub1 (the final concentration of Bub1 was adjusted depending on the activity of the enzyme lot in order to be within the linear dynamic range of the assay: typically 200 ng/ml were used) in aqueous assay buffer [50 mM Tris/HCl pH 7.5, 10 mM magnesium chloride (MgCl2), 200 mM potassium chloride (KCl), 1.0 mM dithiothreitol (DTT), 0.1 mM sodium ortho-vanadate, 1% (v/v) glycerol, 0.01% (w/v) bovine serum albumine (BSA), 0.005% (v/v) Trition X-100 (Sigma), 1× Complete EDTA-free protease inhibitor mixture (Roche)] were added to the compounds in the test plate and the mixture was incubated for 15 min at 22° C. to allow pre-equilibration of the putative enzyme-inhibitor complexes before the start of the kinase reaction, which was initiated by the addition of 3 μl 1.67-fold concentrated solution (in assay buffer) of adenosine-tri-phosphate (ATP, 10 μM final concentration) and peptide substrate (1 μM final concentration). The resulting mixture (5 μl final volume) was incubated at 22° C. during 60 min., and the reaction was stopped by the addition of 5 μl of an aqueous EDTA-solution (50 mM EDTA, in 100 mM HEPES pH 7.5 and 0.2% (w/v) bovine serum albumin) which also contained the TR-FRET detection reagents (0.2 μM streptavidin-XL665 [Cisbio Bioassays, Codolet, France] and 1 nM anti-phosho-Serine antibody [Merck Millipore, cat. #35-002] and 0.4 nM LANCE EU-W1024 labeled anti-mouse IgG antibody [Perkin-Elmer, product no. AD0077, alternatively a Terbium-cryptate-labeled anti-mouse IgG antibody from Cisbio Bioassays can be used]).
Measurement: IC50 = 51.6 nM (pChEMBL 7.289999961853027)
Descriptors: MW 376.46, ALogP 4.24, HBA 4, HBD 2, PSA 70.25, QED 0.68
Compound (SMILES): O=C(O)CNC(=O)C1=C(O)C2(CCCC2)n2cccc2C1=O
InChIKey: WVXGAQQJNSAMDM-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZT9
Assay: Effect of Compounds on Activities of HIF Prolyl Hydroxylase-2: Activities of HIF prolyl hydroxylase was determined according to the method as described in Anal Biochem, 2004, 330: 74-80, which was slightly modified. A 96-well plate was pretreated with blocker casein and 1 mM biotin for 30 minutes, and then biotin-linked HIF-1α556-574 (biotinyl-DLDLEMLAPYIPMDDDFQL) was immobilized on the 96-well plate. The 96-well plate was then filled with an appropriate amount of HIF-PHD2-containing buffer (20 mM Tris (pH 7.5), 5 mM KCl, 1.5 mM MgCl2, 20 mM 2-oxoglutarate, 10 mM FeSO4, 2 mM ascorbic acid, 4% EDTA-free protease inhibitor) and was incubated for 1 to 60 minutes at RT. The reaction mixture also contained different concentrations of HIF prolyl hydroxylase inhibitors to be tested. The reaction was stopped by rinsing the 96-well plate three times with washing buffer. In 100 μl binding buffer (50 mM tris(hydroxymethyl)-aminomethane, pH 7.5, 120 mM NaCl), hydroxylated HIF-1α556-574 was reacted with EuVBC protein in the binding buffer at RT for 60 minutes. The reaction solution was aspirated, and the unbound EuVBC protein was washed away by washing 3 times with an elution buffer. Subsequently, 10 μl of rabbit anti-EuVBC polyclonal antibody was added. After further 30 minutes, 10 μl of anti-rabbit polyclonal antibody immunoglobulin coupled to horseradish peroxidase was added to the binding buffer. To determine the amount of bound EuVBC protein, it was incubated with TMB for 15 minutes. The color reaction was terminated by addition of 100 μl of 1 M sulfuric acid. The amount of bound EuVBC protein was determined by measuring optical density at 450 nm, which was proportional to the amount of hydroxylated proline in the peptide substrate.
Measurement: IC50 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 304.3, ALogP 0.97, HBA 5, HBD 3, PSA 108.63, QED 0.72
Compound (SMILES): CC(C)c1ccc(-c2nc3ncccn3c2CN2CC3COCC(C2)N3C(=O)c2ccccc2F)cc1
InChIKey: BKNDMWLGGMBYTD-UHFFFAOYSA-N
Target: Potassium channel subfamily K member 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O14649
Assay: In Vitro Electrophysiological Assay: Table 1: In Vitro Electrophysiological Analysis of the Human TASK-1 and TASK-3 Channels Via Two-Electrode Voltage Clamp Technique in Xenopus laevis Oocytes. Xenopus laevis oocytes were selected as described elsewhere by way of illustration [Decher et al., FEBS Lett. 492, 84-89 (2001)]. Subsequently, the oocytes were injected with 0.5-5 ng of a cRNA solution coding for TASK-1 or TASK-3. For the electrophysiological analysis of the channel proteins expressed in the oocytes, the two-electrode voltage clamp technique [Stühmer, Methods Enzymol. 207, 319-339 (1992)] was used. The measurements were conducted as described [Decher et al., FEBS Lett. 492, 84-89 (2001)] at room temperature (21-22° C.) using a Turbo TEC 10CD amplifier (NPI), recorded at 2 kHz and filtered with 0.4 kHz. Substance administration was performed using a gravitation-driven perfusion system. Here, the oocyte is located in a measuring chamber and exposed to the solution stream of 10 ml/min. The level in the measuring chamber is monitored and regulated by sucking off the solution using a peristaltic pump.
Measurement: IC50 = 9400 nM (pChEMBL 5.03000020980835)
Descriptors: MW 499.59, ALogP 4.38, HBA 6, HBD 0, PSA 62.97, QED 0.41
Compound (SMILES): CCOc1cc(-c2ccc(N3CCC(CN4CCCC4)(C(=O)NCC(C)C)CC3)nc2)c2c(C#N)cnn2c1
InChIKey: VSNILXFTYNBLPP-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of Formula I were screened for their ability to inhibit wildtype 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; Catalog No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Catalog No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog 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 = 8.4 nM (pChEMBL 8.079999923706055)
Descriptors: MW 529.69, ALogP 4.12, HBA 8, HBD 1, PSA 98.79, QED 0.45
Compound (SMILES): COc1nc(N2CCC(N3CCC(F)(F)C3)C2=O)ccc1-c1cn[nH]c1
InChIKey: WBEJCXNUSDQXMC-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assays: 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 (SEQ ID NO: 1)). 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 = 1150 nM (pChEMBL 5.940000057220459)
Descriptors: MW 363.37, ALogP 1.93, HBA 5, HBD 1, PSA 74.35, QED 0.9
Compound (SMILES): CC1(C)[C@H]2CC[C@]1(c1cccc(-c3ccn(C[C@@H](O)CO)n3)n1)c1nnc(-c3c(F)cccc3F)cc12
InChIKey: YYWSVNUKCZZVJJ-AUPSIVQCSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: In Vitro Ligand Binding Assay: 50 mM HEPES buffer (pH 7.4); 150 mM NaCl; 1 mM DTT; 5 mM MgCl2; 0.01% BSA; 5% DMSO; 0.6 ug/mL RORc receptor; 6 nM 25-[3H]hydroxycholesterol. For NSB wells, 1 uM 25-hydroxycholesterol was also present. Assay plates were 96-well polypropylene V-bottom plates. 10 uL of 5× compound in 25% DMSO/75% Assay Buffer was added to Test wells. 10 uL of 25% DMSO/75% Assay Buffer was added to Total Binding or No Receptor wells. 10 uL of 5 uM 25-hydroxycholesterol in 25% DMSO/75% Assay Buffer was added to NSB wells. 20 uL of 15 nM 25-[3H]hydroxycholesterol prepared in Assay Buffer was added to all wells. 20 uL of 1.5 ug/mL RORc receptor was added to wells (or 40 uL Assay Buffer to No R wells). Following addition to the wells, the plates were incubated 3 h at 25° C. Filtration
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 503.55, ALogP 4.24, HBA 7, HBD 2, PSA 96.95, QED 0.41
Compound (SMILES): Cn1cc(-c2ccc3c(=O)n(C)cc(-c4nc(CS(C)(=O)=O)ncc4OCC4CC4)c3c2)cn1
InChIKey: DYFKVBCVUNGUHG-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: In Vitro Enzyme Inhibition Assay: Determination of the IC50 for the heterocyclic derivative BRD4 inhibitors disclosed herein was performed as follows. His-tagged BRD4 was cloned, expressed and purified to homogeneity. Filipakopoulos et al., Nature 468:1067 (2010). BRD4 binding and inhibition was assessed by monitoring the interaction of biotinylated H4-tetraacetyl peptide (AnaSpec, H4K5/8/12/16(Ac), biotin-labeled) with the target using the AlphaScreen technology (Life Technologies). In a 384-well ProxiPlate BRD4(BD1) (2 nM final) was combined with peptide (15 nM final) in 50 mM HEPES (pH 7.3), 10 mM NaCl, 0.25 mM TCEP, 0.1% (w/v) BSA, and 0.005% (w/v) Brij-35 either in the presence of DMSO (final 0.4% DMSO) or compound dilution series in DMSO. After 20 min incubation at room temp, Alpha streptavidin donor beads and Nickel Chelate acceptor beads were added to a final concentration of 5 μg/mL. After 2 hr of equilibration, plates were read on an Envision instrument and the IC50 was calculated using a four parameter non-linear curve fit. Chemistry Example 1 (2-methyl-4-phenylisoquinolin-1-one) had an IC50 of 2.782 μM in this assay format.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 479.56, ALogP 2.73, HBA 9, HBD 0, PSA 108.97, QED 0.4
Compound (SMILES): CC1C2CC(Oc3cnc(C(F)(F)F)cn3)C(C2)N1C(=O)c1cccc(F)c1-n1nccn1
InChIKey: QTBFUIIFOKACFK-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P56718
Assay: Receptor Radioligand Binding Assay: Human Embryonic Kidney 293 cells (HEK293) stably expressing rat orexin 1 receptor (Genebank accession number NM_001525) or Chinese ovary cells (CHO) stably expressing human orexin 1 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1× Pen/Strep, 1× sodium pyruvate, 10 mM HEPES, 600 μg/mL G418 and DMEM/F12 (Gibco, Cat #11039), 10% FBS, 1× Pen/Strep, 600 μg/mL G418 media, respectively on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1× with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2K×G, 5 min at 4° C.), the supernatant was aspirated and the pellets frozen and stored at −80° C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and homogenized for 45 sec prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H] (1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone) (Moraveck Corporation, specific activity=35.3 Ci/mmol), diluted to a 10 nM concentration in PBS (4 nM final). Compounds were solubilized in 100% DMSO (Acros Organics, Cat. #61042-1000) and tested over a range of 7 concentrations (from 0.1 nM to 10 μM). The final concentration of DMSO in the reactions is equal to or less than 0.1%. Total and nonspecific binding was determined in the absence and presence of 10 μM almorexant. The total volume of each reaction is 200 μL (20 μL of diluted compounds, 80 μL of [3H]¿ (1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone) diluted in PBS and 100 μL of the cell suspension). Reactions were run for 60 min at room temperature and terminated by filtration through GF/C filter plates (PerkinElmer, Cat. #6005174) presoaked in 0.3% polyethylenimine using the cell harvester (PerkinElmer Filtermate).
Measurement: Ki = 18 nM (pChEMBL 7.75)
Descriptors: MW 462.41, ALogP 3.29, HBA 7, HBD 0, PSA 86.03, QED 0.55