lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC#Cc1ccc2sc3ccc([C@]4(C)CS(=O)(=O)C(C)(C)C(=N)N4)cc3c2c1
InChIKey: TYFDPTNOAQWDOO-QFIPXVFZSA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: Activity Assay: A BACE2 activity assay kit from AnaSpec (Fremont, Calif.) was used to determine the potency of the selected compounds by AnaSpec BACE2 fluorescent assay. To each well of a black 96-well micro plate, 50 μl of the β-secretase substrate (Hylite Fluor 488) diluted 1:100 using assay buffer was first added.The compounds were serially diluted (1:2) into DMSO from their 10 mM stock concentrations in DMSO. The serially diluted inhibitors were diluted 1:100 into 1× assay buffer in a 96-well polypropylene plate (assay dilution plate). The final concentration of these compounds tested was 300 nM, 150 nM, 75 nM, 37.5 nM, 18.75 nM, 9.375 nM, 4.688 nM, 2.344 nM, 1.172 nM, 0.586 nM, and 0.293 nM.A volume of 10 μl of the inhibitor from the assay dilution plate, a control inhibitor LY2886721 or inhibitor vehicle was then added to each well of the black 96-well micro plate.The recombinant BACE2 was diluted in an assay buffer and the final BACE2 was 10.6 nM. The enzyme was added in a volume of 40 μl to each well of a black 96-well micro plate.The reagents were mixed gently and the assay plate was placed into a POLARstar fluorescent plate reader with excitation wavelength set at 485 nm and the emission wavelength set at 520 nm. The plate reader was set to take readings every 5 minutes for up to 30 minutes or a 30 minute endpoint reading was used.
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 410.56, ALogP 4.41, HBA 4, HBD 2, PSA 70.02, QED 0.58
Compound (SMILES): O=C(NCc1ccncc1)C1CCN(c2ncnc3[nH]ccc23)CC12CC2
InChIKey: YQSILSKDYCDGTQ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK Kinase Assays: Human baculovirus-expressed JAK1, 2, 3 and TYK2 were purchased from Carna Biosciences, Inc. All four purified enzymes contain only the catalytic domain. JAK1 (aa 850-1154) and TYK2 (aa 871-1187) are expressed with an N-terminally fused GST-tag, and JAK2 and JAK3 with an N-terminally fused His-tag.Inhibition of phosphorylation of a synthetic peptide was measured in an HTRF-based assay using the TK substrate-Biotin from the Cisbio HTRFKinEASE TK kit. First, 2 μl of TK solution (TK substrate-biotin in kinase buffer [1× enzymatic buffer from HTRFKinEASE TK kit, 1 mM DTT]) is added to a plate containing 1 μl prediluted compound (final assay concentration DMSO: 0.75%). Then, 5 μl kinase-ATP mix (prepared in kinase buffer) is added to the wells and the plates are incubated at RT for 20-30 min. For all four kinases a concentration of ATP that corresponded to the Km for ATP was used. The final concentrations of buffers, substrate, kinase and ATP were: JAK1: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 10 mM MgCl2, 1 mM DTT, 7 μM ATP, 50 nM SEB, 1 μM TK Substrate-Biotin and 5 ng JAK1; JAK2: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 4 μM ATP, 1 μM TK Substrate-Biotin and 0.1 ng JAK2; JAK3: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 2 μM ATP, 1 μM TK Substrate-Biotin and 0.3 ng JAK3; TYK2: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 13 μM ATP, 50 nM SEB, 1 μM TK Substrate-Biotin and 0.8 ng TYK2. Thereafter, the kinase reaction is stopped by adding 4 μl detection mix (final concentrations: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 0.8 M KF, 20 mM EDTA, 42 nM Streptavidin-XL665 and 1:400 STK Ab Cryptate) and the plates are incubated overnight in the dark. The HTRF signal is read using an Envision plate reader.
Measurement: EC50 = 28.7 nM (pChEMBL 7.539999961853027)
Descriptors: MW 362.44, ALogP 2.28, HBA 5, HBD 2, PSA 86.8, QED 0.74
Compound (SMILES): CCOc1ncccc1C(=O)N1C[C@H](c2cc(C(C)(C)O)no2)CC[C@H]1C
InChIKey: ZUSUKYYEFVQQPM-ZIAGYGMSSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: FLIPR Ca2+ Flux Assay: n a typical experiment the OX1 and OX2 receptor antagonistic activity of the compounds of the present invention was determined in accordance with the following experimental method. For intracellular calcium measurements, Chinese hamster ovary (CHO) cells expressing the rat orexin-1 receptor or the human orexin-2 receptor, 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 20,000 cells/well into Becton-Dickinson black 384-well clear bottom sterile plates coated with poly-D-lysine. All reagents are from GIBCO-Invitrogen Corp. The seeded plates are incubated overnight at 37° C. and 5% CO2. Ala-6,12 human orexin-A as the agonist is 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 assay buffer. On the day of the assay, cells are washed 3 times with 100 μl assay buffer and then incubated for 60 min (37° C., 5% CO2) in 60 μl assay buffer containing 1 μM Fluo-4AM 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 min and finally 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 Ala-6,12 orexin-A with buffer in place of antagonist.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 373.45, ALogP 3.1, HBA 6, HBD 1, PSA 88.69, QED 0.87
Compound (SMILES): CS(=O)(=O)N1CCC(n2nc(-c3cc4ccccc4[nH]3)c3c(N)ncnc32)CC1
InChIKey: JRIKXAWUIRWPQH-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Time-Resolved Fluorescence Transfer (FRET) Assay: RET and KDR: Kinase activity was detected using CisBio HTRF kinEASE kit based on time-resolved fluorescence transfer (FRET). The assay was performed in 384-well white plates (Corning #3574) in a reaction volume of 10 μL containing 1× CisBio enzymatic buffer supplemented with a final concentration of 5 mM MgCl2, 1 mM DTT, 10 nM SEB and 0.01% Triton X100 for RET. The same buffer was used for the KDR biochemical assay with the addition of 2 mM MnCl2.Inhibitors were pre-incubated in the plate for 15 mins with 5 μL kinase and assay buffer at the following concentrations; 13 pM RET (Carna Biosciences; 08-159) and 150 pM KDR (Millipore; 14-630). The reaction was initiated by the addition of 5 μL ATP and substrate at 2× final reaction concentrations. For RET, this was 18 pM and 2 μM; for KDR, this was 16 μM and 1 μM, respectively. Reactions were performed at ATP Km for each target. The assay was allowed to proceed at room temperature for 20 mins before terminating with the addition of 10 μL HTRF detection buffer containing EDTA supplemented with TK-antibody labelled with Eu3+-Cryptate (1:100 dilution) and streptavidin-XL665 (128 nM). Following incubation at room temperature for 1 hour, FRET signal was measured using the Pherastar FS Microplate Reader.
Measurement: IC50 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 411.49, ALogP 2.15, HBA 7, HBD 2, PSA 122.79, QED 0.53
Compound (SMILES): CC(=O)N(C)c1cnn(C(=O)N2CC3CN(Cc4cc(Cl)cc(N5CCN(C6CC6)CC5)c4)CC3C2)c1
InChIKey: YIEAXGXEDWGMHB-UHFFFAOYSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Mus musculus
UniProt: O35678
Assay: In Vitro Competitive Activity-Based Protein Profiling: Proteomes (mouse brain membrane fraction or cell lysates for mouse assays; human prefrontal cortex or cell membrane fractions for human assays) (50 μL, 1.0 mg/mL total protein concentration) were preincubated with varying concentrations of inhibitors at 37° C. After 30 min, FPRh or HT-01 (1.0 μL, 50 μM in DMSO) was added and the mixture was incubated for another 30 min at 37° C. Reactions were quenched with SDS loading buffer (15 μL-4×) and run on SDS-PAGE. Following gel imaging, serine hydrolase activity was determined by measuring fluorescent intensity of gel bands corresponding to MAGL and FAAH using ImageJ 1.43u software.Preparation of Mouse Brain Proteomes from Inhibitor Treated Mice.Inhibitors were administered to wild-type C57Bl/6J by oral gavage in a vehicle of polyethylene glycol. Each animal was sacrificed 4 h following administration and brain proteomes were prepared and analyzed according to previously established methods (See Niphakis, M. J., et al. (2011) ACS Chem. Neurosci. and Long, J. Z., et al. Nat. Chem. Biol. 5:37-44).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 526.09, ALogP 2.84, HBA 7, HBD 0, PSA 68.16, QED 0.6
Compound (SMILES): C=CC(=O)N1CCC(Oc2cc3c(Nc4ccc(OC(F)(F)F)c(Cl)c4)ncnc3cc2OC)C1
InChIKey: ISCZCVDYFSPLOP-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Cell Viability Assay and IC50 Estimation: Cell viability was determined using the Cell Titer Glo assay (Promega) as previously described. Briefly, 2000-3000 cells per well were plated in 384-well plates (Greiner Bio-One) in technical triplicate. Cells were treated with seven different concentrations of tyrosine kinase inhibitors or vehicle alone at a final volume of 40 μL per well. After 3 days hours, 11 μL of Cell Titer Glo was added to each well. Plates were shaken for 15 minutes, and bioluminescence was determined using a FLUOstar OPTIMA multi-mode micro-plate reader (BMG LABTECH). Bioluminescence values were normalized to DMSO treated cells, and normalized values were plotted in GraphPad Prism using non-linear regression fit to normalized data with a variable slope. IC50 values were calculated by GraphPad Prism at 50% inhibition.
Measurement: IC50 = 510 nM (pChEMBL 6.289999961853027)
Descriptors: MW 508.88, ALogP 5.1, HBA 7, HBD 1, PSA 85.81, QED 0.44
Compound (SMILES): CN(CCCOc1cc2nccc(Oc3ccc(NC(=O)Nc4ccc(Cl)c(C(F)(F)F)c4)cc3)c2c2c1OCCO2)C1CCC1
InChIKey: VPCGFHNKNCLGPN-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Assay of Small Molecular Compounds for Inhibiting the Activity of VEGFR-2 Kinase: The assay is based on the LANCE TR-FRET technology of Perkin Elmer Inc., and the assay method is as follows:1. Dilution of compounds: a total of 11 concentrations were obtained using a 3-fold gradient dilution from the highest concentration of 2500 nM (the maximum final concentration of the drug used in this assay was 2500 nM, and the minimum final concentration was 0.042 nM).2. 2.5 μL of the gradient-diluted compounds were taken with a transfer pipette to a 384-well plate.3. Addition of enzyme: 5 μL of 2× VEGFR2 kinase solution (concentration was 0.5 nM) was taken with a transfer pipette to the corresponding reaction well of the 384-well plate, mixed well and pre-reacted at room temperature for 30 minutes.4. 2.5 μL 4× Ultra ULight-JAK-1 (Tyr1023) Peptide (concentration was 200 nM)/ATP (concentration was 40 μMM) mixture was taken with a transfer pipette to the corresponding reaction well of the 384-well plate.5. Negative control: 2.5 μL/well 4× substrate/ATP mixture and 7.5 μL 1× Kinase Assay Buffer were added to the wells of the 384-well plate.6. Positive control: 2.5 μL/well 4× substrate/ATP mixture, 2.5 μL/well 1× Kinase Assay Buffer containing 16% DMSO, and 5 μL/well 2× VEGFR2 kinase solution were added to the 384-well plate. The final concentration of DMSO in the reaction system was 4%.7. The mixture was mixed well and then centrifuged and reacted at room temperature in dark for 60 min.8. Termination of the enzymatic reaction: 5 μL of 4× stop solution was taken with a transfer pipette to the wells of the 384-well plate, mixed and then centrifuged, and reacted at room temperature for 5 min.9. Development of the reaction: 5 μL of 4× detection solution was taken with a transfer pipette to the wells of the 384-well plate for color development, and the mixture was mixed and then centrifuged and reacted at room temperature for 60 min.10. The 384-well plate was placed into the Envision plate reader and the signal was detected using the appropriate program.11. Analysis and processing of the raw data:The drug concentrations and the corresponding inhibition rates were input into GraphPad Prism5 for calculation, and the inhibition rate of the compounds were calculated as follows: inhibition rate (%)=(reading of positive well−reading of experimental well)/(reading of positive control well−reading of negative control well)×100%. Processing with GraphPad Prism5 software yielded the corresponding IC50 values (the concentration of the compound at which 50% of the highest inhibition of the enzyme is achieved).
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 657.09, ALogP 8.37, HBA 7, HBD 2, PSA 94.18, QED 0.17
Compound (SMILES): Cc1cc(Cl)cc2c1nc(N)n1nc(C3CCCN(c4cnn(C)c4)C3)nc21
InChIKey: AVOBSDIPGJUDLP-UHFFFAOYSA-N
Target: Adenosine receptor A2b (SINGLE PROTEIN), Homo sapiens
UniProt: P29275
Assay: Measurement of A2b Binding Affinity: The reported affinity of the compounds of the invention for the human A2b adenosine receptor was determined experimentally using a radioligand filtration binding assay. This assay measures the amount of binding of a tritiated proprietary A2b receptor antagonist, in the presence and absence of a compound of the invention, to membranes made from HEK293 cells recombinantly expressing the human A2b adenosine receptor (Perkin Elmer, Cat. No. ES-013-C).To perform the assay, compounds of the invention to be tested were first solubilized in 100% DMSO and further diluted in 100% DMSO to generate, typically, a 10-point titration at half-log intervals such that the final assay concentrations did not exceed 10 μM of compound or 1% DMSO. 148 μL (135 μg/mL) membranes and 2 μL test compounds were transferred to individual wells of a 96-well polypropylene assay plate and incubated for 15 to 30 minutes at room temperature with agitation. Tritiated radioligand was diluted to a concentration of 14 nM in assay buffer (phosphate buffered saline without Magnesium and Calcium, pH 7.4; GE Healthcare Life Sciences, Cat. No. SH30256.01) and then 50 μL of the solution were transferred to each well of the assay plate. To define total and non-specific binding, wells containing 1% DMSO and 20 μM N-ethylcarboxamidoadenosine (Tocris Bioscience, Cat. No. 1691) respectively, were also included. The wells of the assay plate were incubated at room temperature for 60 minutes with agitation, then filtered using a FilterMate Harvester® (Perkin Elmer) or similar equipment through a UniFilter-96® PEI coated plate (Perkin Elmer Cat. No. 6005274 or 6005277). Filtering was achieved by aspirating the contents of the assay plate for 5 seconds, then washing and aspirating the contents three times with ice-cooled wash buffer (assay buffer supplemented with 0.0025% Brij58) and allowing the vacuum manifold to dry the plate for 30 seconds. The filter plate was incubated for at least 1 hour at 55° C. and allowed to dry. The bottom of the filter plate was then sealed with backing tape. 40 μL Ultima Gold (Perkin Elmer, Cat. No. 6013329) was added to each well of the filter plate and the top of the plate was sealed with TopSeal-A PLUS® clear plate seal (Perkin Elmer, Cat. No. 6050185). The plates were then incubated for at least 20 minutes, and then the amount of radioactivity remaining in each well was determined using a TopCount® (Perkin Elmer) scintillation counter. After normalization to total and non-specific binding, the percent effect at each compound concentration was calculated. The plot of percent effect versus the log of compound concentration was analyzed electronically using a 4-parameter logistic fit based on the Levenberg-Marquardt algorithm to generate IC50 values.
Measurement: IC50 = 743 nM (pChEMBL 6.130000114440918)
Descriptors: MW 396.89, ALogP 2.94, HBA 8, HBD 1, PSA 90.16, QED 0.56
Compound (SMILES): O=C(O)c1cccc(CN(C(=O)c2cc(-c3cc4c(cc3C(=O)N3Cc5ccccc5C[C@H]3CN3CCOCC3)OCO4)n3c2CCCC3)c2ccc(O)cc2)c1
InChIKey: DYQGOQWRBZLCMH-UMSFTDKQSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07817
Assay: BCL-XL TR-FRET Assay: The following assay concentrations and times were used: 10.5 ng BCL-XL, 5 μl of 1:100 anti-His Tb-labeled donor, 5 μl of 1:100 Dye-labeled acceptor, 5 μl of 1:80 BCL-XL Peptide Ligand, and 2 μl of test compound, with 60 min incubation time. The results of the assay were read using a Clariostar (BMG Labtech) plate reader with the following parameters: TR FRET, 340ex/620 and 665em; 60 μsec Delay; and 500 μsec integration.
Measurement: IC50 = 3000 nM (pChEMBL 5.519999980926514)
Descriptors: MW 768.87, ALogP 6.37, HBA 9, HBD 2, PSA 134.01, QED 0.18
Compound (SMILES): Cc1ccc(N)cc1-c1ccc2nc(NC(=O)[C@@H]3C[C@@H]3F)cn2c1
InChIKey: MAFRULWHJSGRCY-CABCVRRESA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Kinase Assay: The LRRK2 and LRRK2 G2019S kinase assays were performed using the Adapta technology in ThermoFisher Scientific. This experiment was carried out according to the supplier's protocol. Briefly, assay conditions were as follows. The mixture of substrate (LRRKtide) and each kinase was prepared in 50 mM Tris pH 8.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA, 0.02% NaN3. Assays were performed in the presence of 70 μM ATP and 100 μM ATP (KmATP) in LRRK2 and LRRK2 G2019S, respectively. The reaction was progressed at room temperature for 1 hour. Upon completion of kinase reaction, 5 μL of Detection Mix was added and after 60 min incubation time, the emission ratio of 665/615 nm was calculated.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 324.36, ALogP 3.19, HBA 4, HBD 2, PSA 72.42, QED 0.73
Compound (SMILES): Cc1ccc(C(O)(C(N)=O)C(F)(F)F)cc1-c1cnc2c(N)nc(C(=O)NC3(C)CCOC3C)cn12
InChIKey: ZIMYTKSWIRPFNE-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Scintillation Proximity Assay: PI3Kδ: [γ-33P]ATP (10 mCi/mL) and Wheat Germ Agglutinin (WGA) YSi SPA Scintillation Beads was purchased from PerkinElmer (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). PI3Kδ (p110δ/p85α) Recombinant Human Protein was purchased from Eurofins. ATP, MgCl2, DTT, EDTA, MOPS and CHAPS were purchased from SigmaAldrich.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 PI3Kδ 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, 3.4 nM PI3Kδ. 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.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 506.49, ALogP 1.82, HBA 8, HBD 4, PSA 157.86, QED 0.41
Compound (SMILES): N#Cc1cccnc1N1CCN(Cc2ccc3c4c(c(=O)[nH]c3c2)CCCN4)CC1
InChIKey: PITKHMJQSBHPED-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TNK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13470
Assay: Inhibition Assay: The tankyrase-1 or tankyrase-2 enzyme inhibitory activities of the compounds of the present invention were assayed in the following manner by use of a kit (cat. 80573 or 80578) purchased from BPS Bioscience.The 96-well plate provided in BPS Bioscience kit was coated with histone and incubated at 4° C. for 16 hours. Then, the plate was washed four times with PBST (7.5 mM Na2HPO4, 2.5 mM NaH2PO4, 145 mM NaCl, 0.05% Tween 20, pH 7.4), and blocking buffer (provided in BPS Bioscience kit) was added thereto in order to block nonspecific reaction, and was then incubated at 25° C. for 1 hour. After incubation for 1 hour, the plate was washed four times with PBST, and varying concentrations of each of the compounds of the Examples were added to a reaction solution containing tankyrase-1 enzyme (40 ng/well) or tankyrase-2 enzyme (15 ng/well) and an assay mixture, and allowed to react at 25° C. for 1 hour. After 1 hour, each well was washed four times with PBST, and in order to measure the level of ribosylation by PARP enzyme, streptavidin-linked peroxidase (Strep-HRP, 1:50 dilution) was added and allowed to react at 25° C. for 30 minutes. The plate was washed four times with PBST, and then finally an HRP chemiluminescent substrate was added and allowed to react. The level of histone ribosylation formed by each enzyme was quantified using Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Inc., USA).
Measurement: IC50 = 10.4 nM (pChEMBL 7.980000019073486)
Descriptors: MW 400.49, ALogP 2.48, HBA 6, HBD 2, PSA 88.05, QED 0.7
Compound (SMILES): CN1C(=O)Cc2cc(-c3cncc(-c4ccc(=O)n(C5CCCC5)c4)c3)ccc21
InChIKey: AFIAWFRELDLTGM-UHFFFAOYSA-N
Target: Bone morphogenetic protein receptor type-1A (SINGLE PROTEIN), Homo sapiens
UniProt: P36894
Assay: Luminescence-Based ADPGlo Kinase Activity Assay: Example 53: The assays were performed in 384-well, low volume microtiter assay plates in a final reaction volume of 6 ul. Dose-response curves were generated by incubating 10 nM of each kinase in 50 mM Hepes pH 7.5, 0.02% Tween 20, 0.02% BSA, 1 mM DTT, 10 uM Na3VO4, 10 mM ß-Glycerolphosphate, 1 mM MgCl2, 12 mM MnCl2 and 15 uM ATP for 60 min at 32° C. in the presence or absence of compound diluted in DMS. The amount of generated ADP is a measure of kinase activity and is quantified using the ADP-Glo Kinase Assay (Promega) according to manufacturer's instructions. ADP is converted to ATP by adding 3 ul of ADP-Glo Reagent and incubation at 32° C. for 60 min. ATP is subsequently converted into a bioluminescent signal by adding 6 ul luciferase assay reagents (Kinase detection buffer+Kinase Detection Substrate (Promega)) and further incubation at 32° C. for 60 min. For the measurement of luminescence a PHERAstar Multilabel Reader was used at a measurement interval time of 0.1 second (optical module for luminescence measurements in the 230 nm to 750 nm wavelength range). The luminescent signal positively correlates with kinase activity.
Measurement: IC50 = 2400 nM (pChEMBL 5.619999885559082)
Descriptors: MW 385.47, ALogP 4.21, HBA 4, HBD 0, PSA 55.2, QED 0.68
Compound (SMILES): Fc1cnc(Nc2ccc(CN3CCN(C4CCCCCC4)CC3)cn2)nc1-c1ccc2c(c1)c(C1CC1)cn2C1CCC1
InChIKey: ZMPGSRVISHYRKW-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00534
Assay: Caliper Mobility Shift Assay: CDK6: The test compound was dissolved in DMSO and diluted with a kinase buffer solution (20 mM HEPES-pH 7.5, 0.01% Triton X-100, 10 mM MgCl2, 2 mM DTT) and 5 μL of compound at 5-fold final concentration of reaction dissolved in 10% DMSO was added in a 384-well plate. The compound-free control well was 5 μL of 10% DMSO, and the no-activity control well was 5 μL of kinase buffer. 10 μL of a 2.5-fold diluted CDK6 enzyme solution (GST-CDK6(1-326 end)) was added and incubated at room temperature for 10 min, and then 10 μL of the 2.5-fold diluted substrate solution Peptide FAM-P8 was added. The reaction was stopped by adding 25 μL of stop solution after incubation at 28° C. for 3 h, and the conversion rate data was read on a Caliper EZ Reader II (Caliper Life Sciences) and the conversion rate was converted to inhibition rate data according to above method.
Measurement: IC50 < 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 593.8, ALogP 7.82, HBA 7, HBD 1, PSA 62.11, QED 0.21
Compound (SMILES): COC(=O)c1c(C#N)ccnc1O[C@@H]1CC[C@@H](C)N(C(=O)c2ccc(C)cc2-n2nccn2)C1
InChIKey: NPLVYXLBAUOJGN-SJLPKXTDSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: FLIPR Ca2+ Flux Assay: . In a typical experiment the OX1 and OX2 receptor antagonistic activity of the compounds of the present invention was determined in accordance with the following experimental method. For intracellular calcium measurements, Chinese hamster ovary (CHO) cells expressing the rat orexin-1 receptor or the human orexin-2 receptor, 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 20,000 cells/well into Becton-Dickinson black 384-well clear bottom sterile plates coated with poly-D-lysine. All reagents were from GIBCO-Invitrogen Corp. The seeded plates are incubated overnight at 37° C. and 5% CO2. Ala-6,12 human orexin-A as the agonist is 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 assay buffer. On the day of the assay, cells are washed 3 times with 100 μl assay buffer and then incubated for 60 min (37° C., 5% CO2) in 60 μl assay buffer containing 1 μM Fluo-4AM 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 min and finally 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 Ala-6,12 orexin-A with buffer in place of antagonist.
Measurement: IC50 = 24.3 nM (pChEMBL 7.610000133514404)
Descriptors: MW 460.49, ALogP 2.7, HBA 9, HBD 0, PSA 123.23, QED 0.53
Compound (SMILES): CCc1cnc(Nc2ccc3c(c2)CCN2CCNC[C@@H]32)nc1
InChIKey: CPDXPOVYDVDNGM-KRWDZBQOSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q923Y8
Assay: Radioligand Binding Assay: HEK-293 cells stably expressing mouse TAAR1 were maintained at 37° C. and 5% CO2 in DMEM high glucose medium, containing fetal calf serum (10%, heat inactivated for 30 min at 56° C.), penicillin/streptomycin (1%), and 375 μg/ml geneticin (Gibco). Cells were released from culture flasks using trypsin/EDTA, harvested, washed twice with ice-cold PBS (without Ca2+ and Mg2+), pelleted at 1,000 rpm for 5 min at 4° C., frozen and stored at −80° C. Frozen pellets were suspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 10 mM EDTA and homogenized with a Polytron (PT 6000, Kinematica) at 14,000 rpm for 20 s. The homogenate was centrifuged at 48,000×g for 30 min at 4° C. Subsequently, the supernatant was removed and discarded, and the pellet resuspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 0.1 mM EDTA using the Polytron (20 s at 14,000 rpm). This procedure was repeated and the final pellet resuspended in HEPES-NaOH containing 0.1 mM EDTA and homogenized using the Polytron. Typically, aliquots of 2 ml membrane portions were stored at −80° C. With each new membrane batch the dissociation constant (Kd) was determined via a saturation curve.
Measurement: Ki = 43.8 nM (pChEMBL 7.360000133514404)
Descriptors: MW 309.42, ALogP 2.29, HBA 5, HBD 2, PSA 53.08, QED 0.91
Compound (SMILES): COc1cc(OC)c(F)c(N2Cc3cnc(-c4cc(N)cnc4C)cc3C3(CC3)C2=O)c1F
InChIKey: NQSGEAWINZPUMZ-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P21802
Assay: FGFR Enzymatic Assay 1: FGFR1, FGFR2, and FGFR4 are measured under equivalent conditions with the following changes in enzyme and ATP concentrations: FGFR1, 0.02 nM and 210 uM respectively, FGFR2, 0.01 nM and 100 uM, respectively, and FGFR4, 0.04 nM and 600 uM respectively. The enzymes were purchased from Millipore or Invitrogen. GraphPad prism3 was used to analyze the data. The IC50 values were derived by fitting the data to the equation for a sigmoidal dose-response with a variable slope. Y=Bottom+(Top−Bottom)/(1+10^((Log IC50−X)*HillSlope)) where X is the logarithm of concentration and Y is the response. Compounds having an IC50 of 1 μM or less are considered active. The compounds of the invention were found to be selective inhibitors of FGFR3 and/or FGFR4 according to the FGFR Enzymatic Assays. Compounds of Formula (I′) and (I) and all the compounds as described herein have been tested and exhibit an IC50 of less than 1 μM. Table 1 provides IC50 data for compounds of the invention assayed in the FGFR Enzymatic Assay after dilution in assay buffer, added to the plate and pre-incubated for 4 hours.
Measurement: IC50 > 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 452.46, ALogP 3.91, HBA 6, HBD 1, PSA 90.57, QED 0.65
Compound (SMILES): COc1cc(-c2cnc(N)c(-c3ccc(NC(=O)c4cn(CC5CCOCC5)cc(-c5ccc(C)cc5)c4=O)nn3)c2)ccc1OCCN1CCOCC1
InChIKey: XYELEXOQONCGOW-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: Cell-Free Mer Kinase Inhibitory Activity: A kinase dilution solution containing 20 ng/ml Mer (the 528th to 999th amino acids of the intracellular domain of human MER expressed as a fusion protein with glutathione transferase in a baculovirus expression system and purified by glutathione Sepharose chromatography and ion-exchange chromatography; Carna Biosciences, Inc., catalog No. 08-108) was prepared using a kinase reaction buffer solution (100 mM HEPES (pH 7.4), 0.003% Brij-35, 0.004% Tween-20, 1 mM DTT, and 10 mM MgCl2) and added at 19 μl/well to a 384-well plate.Next, each test compound was diluted with DMSO, and this dilution solution was added at 1 μl/well to the plate.After preincubation at room temperature for 20 minutes, a solution containing a substrate peptide (FL-Peptide 27 (5FAM-EFPIYDFLPAKKK-CONH2), Caliper Life Sciences, catalog No. 760424) and ATP at 5 mM was prepared and added at 5 μl/well to the plate to start kinase reaction. The plate was incubated at 28° C. for 45 minutes, and the reaction was terminated by the addition of a termination buffer solution (100 mM HEPES (pH 7.4), 0.015% Brij-35, 40 mM EDTA, and 0.1% Coating Reagent 3) at 40 μl/well.The substrate peptide and the phosphorylated peptide in the reaction mixture were separated and quantified using EZ Reader II (Caliper Life Sciences).The kinase reaction was evaluated on the basis of a product ratio (P/(P+S)) calculated from the peak height (S) of the substrate peptide and the peak height (P) of the phosphorylated peptide.The rate of inhibition (Inhibition) was determined according to the following expression (automatically calculated using the software of the EZ Reader II system).
Measurement: IC50 > 4000 nM (pChEMBL 5.3979997634887695)
Descriptors: MW 731.85, ALogP 5.32, HBA 12, HBD 2, PSA 155.95, QED 0.17
Compound (SMILES): CC(=O)N1CC(N2Cc3c(F)c(Cl)cc([C@H](C)n4nc(C)c5c(N)ncnc54)c3O[C@H](C)C2)C1
InChIKey: PHXYAQRIODZOPM-YPMHNXCESA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Scintillation Proximity Assay: The kinase reaction was conducted in polystyrene 384-well matrix 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 0.5%. The PI3K assays were 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 M PIP2, 20 M ATP, 0.2 μCi [γ-33P]ATP, 4 nM PI3Kδ. Reactions were incubated for 210 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 150 mM potassium phosphate pH 8.0, 20% glycerol. 25 mM EDTA, 400 M ATP. The final concentration of SPA beads was 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1800 rpm for 10 minutes, the radioactivity of the product was determined by scintillation counting on Topcount (Perkin-Elmer). IC50 determination was performed by fitting the curve of percent control activity versus the log of the inhibitor concentration using the GraphPad Prism 3.0 software.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 487.97, ALogP 2.93, HBA 8, HBD 1, PSA 102.4, QED 0.61
Compound (SMILES): Cc1cc(Nc2cc(C)[nH]n2)nc(N2CCC(CC#N)(C(=O)N[C@@H](C)c3ccc(-n4cccn4)nc3)CC2)n1
InChIKey: MBNNDXIBMURPRW-FQEVSTJZSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET V804L Gatekeeper Mutant Assay at KM: In each well of a 384-well plate, 7.5 nM-10 nM of mutant RET (ProQinase 1096-0000-1) was incubated in a total of 12.5 μL of buffer (100 mM HEPES pH 7.5, 0.015% BriJ 35, 10 mM MgCl2, 1 mM DTT) with 1 μM CSKtide (FITC-AHA-KKKKDDIYFFFG-NH2) (SEQ ID NO: 5) and 10 μM ATP at 25° C. for 120 minutes in the presence or absence of a dosed concentration series of compound (1% DMSO final concentration). The reaction was stopped by the addition of 70 μL of Stop buffer (100 mM HEPES pH 7.5, 0.015% BriJ 35, 35 mM EDTA and 0.2% of Coating Reagent 3 (Caliper Lifesciences)). The plate was then read on a Caliper EZReader 2 (protocol settings: −1.7 psi, upstream voltage −500, downstream voltage −3000, post sample sip 35 s). Data was normalized to 0% and 100% inhibition controls and the IC50 calculated using a 4-parameter fit in the CORE LIMS.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 525.62, ALogP 3.52, HBA 10, HBD 3, PSA 153.33, QED 0.31
Compound (SMILES): CC#CC(=O)N1CC[C@]2(CCN([C@H](C(=O)N[C@H]3Cc4nc(cs4)-c4ccc5c(c4)c(c(-c4cccnc4[C@H](C)OC)n5CC)CC(C)(C)COC(=O)[C@@H]4CCCN(N4)C3=O)C(C)C)C2=O)C1
InChIKey: NFAKFDVIGJUJAH-LLXFPOQXSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Biochemical Assay: A variety of Ras proteins may be inhibited by compounds of the present invention (e.g., K-Ras, N-Ras, H-Ras, and mutants thereof at positions 12, 13 and 61, such as G12C, G12D, G12V, G12S, G13C, G13D, and Q61L, and others described herein). The purpose of this biochemical assay is to measure the ability of test compounds to covalently label nucleotide-loaded K-Ras isoforms. In assay buffer containing 12.5 mM HEPES pH 7.4, 75 mM NaCl, 1 mM MgCl2, 1 mM BME, 5 uM Cyclophilin A and 2 uM test compound, a 5 uM stock of GMP-PNP-loaded K-Ras (1-169) G12C is diluted 10-fold to yield a final concentration of 0.5 uM; with final sample volume being 100 uL. The sample is incubated at 25�� C. for a time period of up to 24 hours prior to quenching by the addition of 10 uL of 5% Formic Acid. Quenched samples are centrifuged at 15000 rpm for 15 minutes in a benchtop centrifuge before injecting a 10 uL aliquot onto a reverse phase C4 column and eluting into the mass spectrometer with an increasing acetonitrile gradient in the mobile phase. Analysis of raw data may be carried out using Waters MassLynx MS software, with % bound calculated from the deconvoluted protein peaks for labeled and unlabeled K-Ras.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 919.16, ALogP 5.7, HBA 12, HBD 2, PSA 168.3, QED 0.17
Compound (SMILES): O=C(NC1=CC2C(=C1)C=CC=C2c1ccc(CNC(=O)N2CCOCC2)cc1)C1CC1
InChIKey: RJRNYCOPKQAFRA-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK2 Inhibition Assay: Recombinant human JAK2 catalytic domain (amino acids 808-1132; catalog number PV4210) was purchased from Invitrogen. 0.025 mU of JAK2 was incubated with 2.5 μg polyGT substrate (Sigma catalog number P0275) in kinase reaction buffer (5 mM MOPS pH 7.5, 9 mM MgAc, 0.3 mM EDTA, 0.06% Brij and 0.6 mM DTT, 1 μM non-radioactive ATP, 0.25 μCi 33P-gamma-ATP (GE Healthcare, catalog number AH9968) final concentrations) with or without 5 μL containing test compound or vehicle (DMSO, 1% final concentration), in a total volume of 25 μL, in a polypropylene 96-well plate (Greiner, V-bottom). After 90 min at 30° C., reactions were stopped by adding of 25 μL/well of 150 mM phosphoric acid. All of the terminated kinase reaction was transferred to prewashed (75 mM phosphoric acid) 96 well filter plates (Perkin Elmer catalog number 6005177) using a cell harvester (Perkin Elmer). Plates were washed 6 times with 300 μL per well of a 75 mM phosphoric acid solution and the bottom of the plates was sealed. 40 μL/well of Microscint-20 was added, the top of the plates was sealed and readout was performed using the Topcount (Perkin Elmer). Kinase activity was calculated by subtracting counts per minute (cpm) obtained in the presence of a positive control inhibitor (10 μM staurosporine) from cpm obtained in the presence of vehicle. The ability of a test compound to inhibit this activity was determined as:Percentage inhibition=((cpm determined for sample with test compound present−cpm determined for sample with positive control inhibitor) divided by (cpm determined in the presence of vehicle−cpm determined for sample with positive control inhibitor))*100%.
Measurement: IC50 = 750 nM (pChEMBL 6.119999885559082)
Descriptors: MW 417.51, ALogP 3.15, HBA 3, HBD 2, PSA 70.67, QED 0.77
Compound (SMILES): N#CC[C@@H](C1CC1)n1nc(Nc2ccc3nccnc3c2)c2c(=O)[nH]ccc21
InChIKey: QPIDJZFQSBXWQZ-INIZCTEOSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Jak Biochemical HTRF Assay Protocol: The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer #PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at room temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading. After normalization to untreated controls, the percent inhibition of the HTRF signal at each compound concentration was calculated.
Measurement: IC50 = 5570 nM (pChEMBL 5.25)
Descriptors: MW 371.4, ALogP 3.28, HBA 7, HBD 2, PSA 112.28, QED 0.56
Compound (SMILES): Cn1cc(-c2nn(C3CCC(Oc4cn(C)nc4C(F)(F)F)CC3)c3cc(Nc4ccccc4)ncc23)cn1
InChIKey: RRBNREHEKNKYJD-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: Enzyme Assay: MER: Compounds of Formula I were screened for their ability to inhibit MER kinase using Invitrogen's LanthaScreen Eu Kinase Binding technology. His-tagged recombinant human MER cytoplasmic domain (5 nM) was incubated with 20 nM Alexa-Fluor® Tracer 236 (PR9078A), 2 nM biotinylated anti-His (Cat. No. M4408), and 2 nM europium-labeled Streptavidin (Cat. No. PV5899) 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. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-minute incubation at 22° C., the reaction was measured using a PerkinElmer EnVision multimode plate reader via TR-FRET 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 a concentration of control compound that completely inhibits the enzyme. The POC values are fit to a 4 parameter logistic curve and the IC50 value is point where the curve crosses 50 POC.
Measurement: IC50 = 110 nM (pChEMBL 6.960000038146973)
Descriptors: MW 536.56, ALogP 5.89, HBA 9, HBD 1, PSA 87.61, QED 0.29
Compound (SMILES): C=CC(=O)N1CCC[C@@H](Nc2ncnc3[nH]cc(-c4ccc5c(cnn5C)c4)c23)C1
InChIKey: MPAMGEMLJRTOKJ-MRXNPFEDSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK Caliper Enzyme Assay: Test article was solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. An 11-point half log dilution series was created in DMSO with a top concentration of 600 μM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 resulting in a top final assay compound concentration of 10 μM and a 2% DMSO concentration.Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition 1 nM JAK3 enzyme and were incubated at room temperature 75 minutes for JAK3. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 4822 nM (pChEMBL 5.320000171661377)
Descriptors: MW 401.47, ALogP 3.1, HBA 6, HBD 2, PSA 91.73, QED 0.51
Compound (SMILES): Fc1cnccc1Nc1nc(-c2ccccn2)nn2ccc(CNC3CNC3)c12
InChIKey: UCSKPHSZKIKARD-UHFFFAOYSA-N
Target: TGF-beta receptor type-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P37173
Assay: Biological Assays: Assays are 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. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 14000 nM (pChEMBL 4.849999904632568)
Descriptors: MW 390.43, ALogP 2.13, HBA 8, HBD 3, PSA 92.06, QED 0.46
Compound (SMILES): Cc1cc(S(=O)(=O)Nc2cscn2)c(F)cc1N(C)[C@H]1CCN(Cc2cccc(F)c2F)C1
InChIKey: RVAUTCOLGIFJMK-INIZCTEOSA-N
Target: Sodium channel protein type 1 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P35498
Assay: Sodium Influx Assay (In Vitro Assay): This sodium influx assay employs the use of the cell permeable, sodium sensitive dye ANG2 to quantify sodium ion influx through sodium channels which are maintained in an open state by use of sodium channel modulators. This high throughput sodium influx assay allows for rapid profiling and characterization of sodium channel blockers.In general, Trex HEK293 cells were stably transfected with an inducible expression vector containing the full-length cDNA coding for the desired human sodium channel α-subunit and with an expression vector containing full length cDNA coding for the β1-subunit. Sodium channel expressing cell lines were induced with tetracycline (1 μg/mL) and plated on 384-well PDL-coated plates at a density of 25K-30K cells/well in culture media (DMEM, containing 10% FBS and 1% L-glutamine). After overnight incubation (37° C., 5% CO2), culture media was removed and cells were loaded with 5 uM ANG2 dye for 1-1.5 h in Buffer 1 (155 mM NMDG, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, adjusted with Tris to pH 7.4). Access dye was removed and cells were incubated with test compounds for 1 hr in buffer 1 containing sodium channel modulator(s) at room temperature. Hamamatsu FDSS ρCell was used to perform a 1:1 addition of Na/K challenge buffer (140 mM NaCl, 20 mM HEPES, 1 mM CaCl2, 15 mM KCl, 1 mM MgCl2, 10 mM glucose, adjusted with Tris to pH 7.4) and simultaneously read plates at excitation wavelength of 530 nm and emission wavelength set at 558 nm. Percent inhibition of sodium ion influx was calculated for each test compound at each test concentration to determine the IC50 values.
Measurement: IC50 = 7798 nM (pChEMBL 5.110000133514404)
Descriptors: MW 496.58, ALogP 4.38, HBA 6, HBD 1, PSA 65.54, QED 0.53