lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1cc(C(=O)O)ccc1OCC12CCC(/C=C/c3c(-c4c(Cl)cncc4Cl)noc3C3CC3)(CC1)CC2
InChIKey: JGWDGYNZEHNIKF-VOTSOKGWSA-N
Target: Bile acid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q96RI1
Assay: transient human FXR/Gal4-luciferase reporter assay: A Gal4-hFXR fusion construct reporter system was used as the primary assay to characterize compound activity. A construct including 5 copies of the Gal4 promoter response element upstream of a firefly luciferase reporter cDNA was stably expressed in HEK293 cells. This reporter cell line was maintained in Dulbecco's Modified Eagle's medium (DMEM; Gibco) supplemented with 1% penicillin-streptomycin (P/S) solution, 500 μg/ml Zeocin and 10% charcoal/dextran-treated fetal bovine serum (cs-FBS) at 37° C. in a humidified 5% CO2 atmosphere. Another plasmid was constructed in which the human cytomegalovirus promoter in the pcDNA3.1 vector directs the expression of the cDNA encoding a fusion protein comprised of the DNA binding domain from the Gal4 transcription factor fused to the ligand binding domain from human FXR.The day prior to transfection, the reporter cells in culture are detached from the plate with trypsin and plated into a T75 flask at a sufficient density to achieve approximately 90% confluence the next morning. The transfection reagents are prepared by separately diluting 25 μg of the pcDNA3.1-Gal4-FXR plasmid into 1.87 mL of Opti-MEM (Thermo-Fisher), and 40 μL of Lipofectamine 2000 (Thermo-Fisher) into 1.87 mL of Opti-MEM, and then adding the diluted DNA solution into the diluted Lipofectamine 2000 solution and incubating at room temperature for 15-20 minutes. The mixture is further diluted with 10 mL of a solution comprised of DMEM, 10% cs-FBS, and 1% P/S immediately prior to transferring to the cells. The maintenance culture media is aspirated from the cells and the final transfection mixture is added before the cells are incubated overnight at 37° C. in a humidified 5% CO2 atmosphere. This protocol can be scaled up, and the transiently transfected cells can be cryopreserved in an assay-ready format.For compound testing, 100 nL of the compounds (serial dilutions in DMSO) are dispensed with an Echo acoustic dispenser (Labcyte) into the wells of a Corning/Costar clear bottom 384-well white plate. The transfected cells are harvested, counted, and diluted such that 10-25,000 cells in 25 μL are plated into each well of the 384-well compound assay plate. The compound-treated cells are incubated overnight at 37° C. in a humidified 5% CO2 atmosphere. The next morning 25 μL of Steady-Glo (Promega) are added to each well of the plate, the mixture is incubated for 15 min. with shaking, and luminescence is measured on an Envision (Perkin Elmer) plate reader. Background counts from cells treated with DMSO alone are subtracted from all raw counts, and the corrected values are converted to a percentage of the control response attained with 8 μM GW-4064. These data are fit to a 4-parameter log agonist-response equation to calculate an EC50 value.
Measurement: EC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 569.49, ALogP 8.06, HBA 6, HBD 1, PSA 94.68, QED 0.28
Compound (SMILES): Nc1c(C(=O)c2cc3cc(C(=O)N4CCOCC4)ccc3[nH]2)cnn1-c1ccc(Oc2ccccc2)cc1Cl
InChIKey: XRMDUUNRWNFGCZ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: TR-FRET (Time Resolved FRET) Assay: This BTK competition assay measures compound potency (IC50) for the inactivated state of Bruton's Tyrosine Kinase using FRET (Förster/Fluorescence Resonance Energy Transfer) technology. The BTK-Eu complex was incubated on ice one hour prior to use at a starting concentration of 50 nM BTK-Bioease: 10 nM Eu-streptavidin (Perkin-Elmer Catalog#AD0062). The assay buffer consisted of 20 mM HEPES (pH 7.15), 0.1 mM DTT, 10 mM MgCl2, 0.5 mg/ml BSA with 3% Kinase Stabilizer (Fremont Biosolutions, Catalog #STB-K02). After 1 h, the reaction mixture from above was diluted 10 fold in assay buffer to make 5 nM BTK: 1 nM Eu-Streptavidin complex (donor fluorophore). 18 μl of a mixture of 0.11 nM BTK-Eu and 0.11 nM Kinase Tracer 178 (Invitrogen, Catalog #PV5593,) with BTK-Eu alone as no negative control, was then dispensed into 384-well flat bottom plates (Greiner, 784076). Compounds to be tested in assay were prepared as 10× concentrations and serial dilution in half-log increments was performed in DMSO so as to generate 10 point curves. To initiate the FRET reaction, compounds prepared as 10× stock in DMSO was added to the plates and the plates were incubated 18-24 h at 14° C.After the incubation the plates were read on a BMG Pherastar Fluorescent plate reader (or equivalent) and used to measure the emission energy from the europium donor fluorophore (620 nm emission) and the FRET (665 nm emission).
Measurement: IC50 = 0.46 nM (pChEMBL 9.34000015258789)
Descriptors: MW 542, ALogP 5.08, HBA 7, HBD 2, PSA 115.47, QED 0.29
Compound (SMILES): Cn1c(=O)ccc2ncn(Cc3nn([C@H]4C[C@H](c5ccc(Cl)cc5)C4)c(=O)o3)c(=O)c21
InChIKey: JOVMFRRSFHVPLP-CTYIDZIISA-N
Target: Transient receptor potential cation channel subfamily A member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75762
Assay: FLIPR Assay: The IC50 (effective concentration) of compounds on the human and rat TRPA1 channels were determined using a FLIPR Tetra instrument. CHO cells expressing TRPA1 were plated into 384-well plates, incubated overnight at 37° C., and loaded with BD calcium indicator dye for 1 hr at 37° C. followed by 15 minutes at room temperature. The assay buffer was Hank's Balanced Salt Solution (HBSS) containing 20 mM HEPES (pH readjusted to 7.4) along with 0.02% BSA.Following dye load and plate cool down, compounds were added to the cells using FLIPR Tetra. Plates were then incubated with compounds for 20 minutes at room temperature prior to adding agonist. Following this incubation, about an EC50 concentration of cinnamaldehyde (75 uM for human TRPA1 and 45 uM for rat TRPA1) was added to active the channels and block of cinnamaldehyde induced calcium influx was measured.The IC50 results were fit with a standard Hill function, keeping the Hill coefficient (n) fixed to 1.5. Fixing the Hill coefficient will generally reduce variability of the IC50 determination. The IC50 results were individually examined to make sure the MIN and MAX points were set correctly prior to validation of the results.
Measurement: IC50 = 15.2 nM (pChEMBL 7.820000171661377)
Descriptors: MW 439.86, ALogP 2.07, HBA 9, HBD 0, PSA 104.92, QED 0.48
Compound (SMILES): CC1(c2ccc(NC(=O)c3ccc(Cl)cc3)cc2)CCN(Cc2ccccc2)C1
InChIKey: OKJPXMKIVRWLDB-UHFFFAOYSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q923Y9
Assay: Radioligand Binding Assay: Rat: The TAAR1 radioligand 3[H]-(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine (described in WO 2008/098857) was used at a concentration equal to the calculated Kd value, that was usually around 2.3 nM, resulting in the binding of approximately 0.2% of the radioligand and a specific binding representing approximately 85% of the total binding. Nonspecific binding was defined as the amount of 3[H]-(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine bound in the presence of 10 μM unlabeled ligand. All compounds were tested at a broad range of concentrations (10 pM to 10 μM) in duplicates. The test compounds (20□ μl/well) were transferred into a 96 deep well plate (TreffLab), and 180 μl of HEPES-NaOH (20 mM, pH 7.4) containing MgCl2 (10 mM) and CaCl2 (2 mM) (binding buffer), 300 μl of the radioligand 3[H]-(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine at a concentration of 3.3×Kd in nM and 500 μl of the membranes (resuspended at 50 g protein per ml) added.
Measurement: Ki = 522 nM (pChEMBL 6.28000020980835)
Descriptors: MW 404.94, ALogP 5.76, HBA 2, HBD 1, PSA 32.34, QED 0.59
Compound (SMILES): O=C(Nc1cnn(Cc2cnccc2C(F)(F)F)c1)c1cc(-c2ccccc2)on1
InChIKey: OXGGKSGJMQDQMY-UHFFFAOYSA-N
Target: Stearoyl-CoA desaturase (SINGLE PROTEIN), Homo sapiens
UniProt: O00767
Assay: Compound Profiling Methods: Strains expressing SCD1 or SCD5 as the sole desaturase, the human SCD1 and SCD5 genes were used to evaluate inhibition of SCD1/SCD5 using reduced growth as a surrogate for SCD inhibition. These yeast strains express human SCD1 or SCD5 from a plasmid harbored in a strain in which the yeast OLE1 gene is deleted.All compound profiling experiments were performed using the same basic protocol. Yeast were cultured using standard techniques in complete synthetic media lacking uracil and containing yeast nitrogen base supplemented with 2% (w/v) glucose (SD-Ura) Starter cultures were inoculated in 3 mL SD-Ura media containing 0.01% tween and 0.2 mM palmitoleic and oleic acid. Cultures were incubated overnight in a 30° C. shaker incubator (225 rpm). Saturated morning cultures were centrifuged, washed in SD-Ura media lacking TWEEN-20 and fatty acids, and then diluted 1:20 in fresh SD-Ura media also lacking TWEEN-20 and fatty acids. Cells were grown for 6 h to an  (optical density) of 0.4-0.8 at 30° C. with shaking.Compound stocks (10 mM in 100% DMSO) were arrayed into 384-round well, v-bottom polypropylene plates and diluted according to indicated dilution factors. Compound administration was performed in two separate steps. First, 15 μL of SD-Ura was dispensed into clear 384-well assay plates using a MULTIDROP Combi reagent dispenser. The diluted compound stock plates were then applied to the assay plates using an automated workstation (Perkin Elmer JANUS) outfitted with a 384-pin tool containing slotted pins that deliver 100 nL of compound. The cultures described above were centrifuged and washed with media lacking TWEEN-20 or oleic and palmitoleic acids. Cultures were then resuspended at a 2-fold concentrated  of 0.02 (final  of 0.0.01) in SD-Ura. 15 μL of diluted culture was then dispensed into the pinned assay plate to achieve 30 μL of the 1× culture (0.01) and a top drug concentration of 33.3 μM.After yeast delivery, assay plates were incubated under humidified conditions at 30° C. for 40 h. Yeast growth was monitored by reading the  of each well using a microplate reader (Perkin Elmer EnVision). Data were analyzed as follows. Raw data were processed by background subtracting and converting values to a percent of the nontreated condition for that strain [(EXP-0.035)/(DMSO-0.035)×100%].
Measurement: IC50 > 45000 nM (pChEMBL 4.3470001220703125)
Descriptors: MW 413.36, ALogP 4.25, HBA 6, HBD 1, PSA 85.84, QED 0.53
Compound (SMILES): COc1ccc(-c2cc(=O)n(C)cc2-c2cnn(-c3ccccc3F)c2)cn1
InChIKey: BTLLORLAUZFBCR-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: In Vitro CBP Inhibition: The CBP-inhibitory activity of the compounds described herein was determined by calculating the IC50. More specifically, CBP inhibitor activity was assayed as follows: CBP was cloned and expressed in E. coli as His-tag protein and purified by Nickel affinity and gel-filtration chromatography. The protein was further characterized as a single band with the correct molecular weight by SDS-PAGE. CBP binding and inhibition is 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 (Perkin Elmer). In a 384-well ProxiPlate CBP (50 nM final) was combined with peptide (20 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 calculated using a four parameter non-linear curve fit.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 376.39, ALogP 3.45, HBA 6, HBD 0, PSA 61.94, QED 0.55
Compound (SMILES): CCN1Cc2c(ccnc2N[C@@H](C)c2ccc(C(=O)Nc3ccccc3)cc2)C1=O
InChIKey: WFLLHOBMFMCACG-INIZCTEOSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Biochemical Assay for IDH Inhibition: Solution A was prepared by combining 1 M Tris-HCl pH7.5 (300 μL), 5 M NaCl (450 μL), 1 M MgCl2 (150 μL), 1 M DTT (15 μL), 20 mg/mL BSA (37.5 μL), 20 mM NADPH (20 μL), and 0.5 mM α-KG (40 μL), followed by adding deionized water to a final volume of 15 mL.Reaction buffer was prepared by combining 1 M Tris-HCl pH 7.5 (200 μL), 5 M NaCl (300 μL), 1 M MgCl2 (100 μL), 1 M DTT (10 μL) and 20 mg/mL BSA (25 μL), followed by adding deionized water to a final volume of 10 mL.Solution B was prepared by combining the reaction buffer (5 mL) and 192 M IDH1-R132H (5.21 μL) or 107 μM IDH1-R132C (4.67 μL).148 μL Solution A was added into each sample well of a 96-well plate P1, and then 2 μL of the test compound solution at each concentration and 2 μL of DMSO (negative control/blank) were added into individual sample well respectively. 50 μL of Solution B was added into each of the sample wells having test compounds and one sample well having DMSO only, and 50 μL of reaction buffer was added into another sample well having DMSO only, respectively. P1 was put into BioTek Synergy H4 Microplate reader (BioTek Instruments Inc., Winooski, U.S.), and the program was set as follows: 37° C., shake plate 5 seconds, run kinetic mode, total detection time 20 min, detection intervals 40 seconds, detection mode fluorescence (at Excitation 340 nm, Emission 460 nm). The data were exported, the relative enzyme activity and the half-maximal inhibitory concentration (IC50) for the test compound were calculated. Relative enzyme activity was calculated as the absolute value of the slope of the Emission 460 reads of each well plotted against time (which represents the conversion rate of ca-KG to 2-HG through IDH1 modulated utilization of NADPH). The Dose-response curve was drawn and the IC50 was calculated via fitting. The IDH inhibition and selectivity of each test compound can be evaluated according to the effect of the compounds on the IDH enzyme activity.
Measurement: IC50 = 12500 nM (pChEMBL 4.900000095367432)
Descriptors: MW 400.48, ALogP 4.48, HBA 4, HBD 2, PSA 74.33, QED 0.64
Compound (SMILES): CCOc1c([C@@H](C)n2nc(C)c3c(N)ncnc32)cc(Cl)c(F)c1[C@@H]1COC(=O)N1
InChIKey: UCYDWQYLQRHHCX-SKDRFNHKSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: PI3Kδ 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 μM PIP2, 20 μM 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).
Measurement: IC50 > 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 434.86, ALogP 3.3, HBA 8, HBD 2, PSA 117.18, QED 0.63
Compound (SMILES): CC(=O)Nc1cc(-c2cncc(S(=O)(=O)NC(C)(C)C)c2C)ccn1
InChIKey: DFEHGJHPOVRYBQ-UHFFFAOYSA-N
Target: Phosphatidylinositol 3-kinase catalytic subunit type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NEB9
Assay: Enzyme Assay : 100 nL compounds in DMSO are added to wells of a 384 well microtitre plate (Greiner 780076). At room temperature: 5 ul VPS34 reaction buffer (Invitrogen Assay Buffer Q (diluted 1 in 5 with nanopure water) plus 2 mM DTT and 2 mM MnCl2) containing ATP (20 uM, Promega) and 200 uM PI-PS substrate (Invitrogen PV5122) is added followed immediately by 5 ul VPS34 reaction buffer (as above) containing VPS34 (5 nM, Millennium Protein Sciences Group) and the mixture is incubated with shaking at room temperature for 1 hour. Then 5 ul VPS34 stop-detect mix (as per Invitrogen Adapta Assay kit (PV5009) instructions (contains kinase quench buffer, TR-FRET buffer, Adapta Eu anti-ADP antibody and Alexa Fluor 647 ADP tracer)) is added to quench the reaction. The plates are then incubated for 30 minutes at room temperature with shaking and then read on a BMG PheraStar Plus reader.For the assay methods described above, test compound percent inhibition, at various concentrations, is calculated relative to control (DMSO and EDTA) treated samples. Compound concentration versus percent inhibition curves are fitted to generate IC50 values. One skilled in the art will appreciate that the values generated either as percentage inhibition at a single concentration or IC50 values are subject to experimental variation.
Measurement: IC50 > 100 nM (pChEMBL 7)
Descriptors: MW 362.46, ALogP 2.49, HBA 5, HBD 2, PSA 101.05, QED 0.87
Compound (SMILES): C(#CC12CC1CN(c1ccc3[nH]ccc3n1)C2)c1ccccn1
InChIKey: YMKCZBJXPRPWHM-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 5 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P31424
Assay: Functional Calcium Flux Assay: For functional assays, HEK293 cells stably expressing recombinant rat mGluR5 were seeded in 384-well plates and dye loaded using Fluo-8. Cells were then washed to remove the un-incorporated dye. Antagonist evaluation was performed following a 15 min incubation of the test compound followed by the addition of submaximal concentration of glutamate. Intracellular calcium ([Ca2+]i) measurements were performed using a fluorometric imaging plate reader (FLIPR, Molecular Devices). The glutamate-evoked increase in [Ca2+]i in the presence of the test compounds was compared to the response to glutamate alone (the positive control). Antagonist inhibition curves were fitted with a 4-parameter logistic equation giving IC50 values, and Hill coefficients using an iterative nonlinear curve fitting algorithm.
Measurement: IC50 = 3000 nM (pChEMBL 5.519999980926514)
Descriptors: MW 300.37, ALogP 2.84, HBA 3, HBD 1, PSA 44.81, QED 0.7
Compound (SMILES): COC(=O)[C@@H](C)Cn1c(=O)[nH]/c(=N\c2ccc(Oc3ccc(F)cn3)cc2)n(CC2CCC3(CC2)CC3)c1=O
InChIKey: DKJIKGQVXZFJLP-SFHVURJKSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 cell transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 384-well PDL-coated microtiter plate at a concentration of 3000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, and 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.5% BSA, and 0.04% Pluronic P-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), and each well was added with 20 μL of the washing buffer. The plate was placed in High-Throughput Screening System FLIPR 384 (Molecular Device Co.). Measurement of fluorescence intensity by FLIPR 884 was started, and 20 μL of DMSO solutions containing different concentrations of the test compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 150 nM ATP solution (25 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 4 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement.
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 537.59, ALogP 3.68, HBA 9, HBD 1, PSA 120.57, QED 0.44
Compound (SMILES): Cc1cc(Nc2nn([C@@H](CC#N)C3CC3)c3cc[nH]c(=O)c23)ccc1C(=O)N1CCS(=O)(=O)CC1
InChIKey: GFMASRNLUNUSTO-IBGZPJMESA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
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 = 410 nM (pChEMBL 6.389999866485596)
Descriptors: MW 494.58, ALogP 2.51, HBA 8, HBD 2, PSA 140.95, QED 0.54
Compound (SMILES): COc1ccc(Nc2cc(-c3ccccc3OC)ncn2)cc1CO
InChIKey: MTXAAMINIIKRFL-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P50750
Assay: Fluorescent Polarization Assay: The activity of the compounds described in the present invention can be determined by measuring the phosphorylation of a fluorescently-labeled peptide by human CDK9/CyclinT kinase complex by fluorescent polarization using a commercially available IMAP Screening Express Assay Kit (Molecular devices).Test compounds were diluted in 100% DMSO to 5 mM stock concentration, then further dilutions were made in H2O or 100% DMSO to desirable concentrations. Each reaction consisted of 5 nM enzyme: CDK9/CyclinT (Proqinase cat#0371-0345-1), 400 nM TAMRA-Rbtide (synthetic 15-mer peptide derived from human retinoblastoma tumor suppressor protein labeled with TAMRA dye, Genecust Europe), 12 μM ATP (=Kmapp, Sigma-Aldrich) and kinase buffer: 20 mM MOPS pH 7 (Sigma-Aldrich), 1 mM DTT (Sigma-Aldrich), 10 mM MgCl2 (Sigma-Aldrich), 0.01% Tween 20 (Sigma-Aldrich).For each reaction, 4 or 6 μl containing TAMRA-Rbtide, ATP and kianse buffer were combined with 2 μl diluted compound in H2O or 0.028 μl compound in 100% DMSO. The kinase reaction was started by the addition of 2 μl diluted enzyme.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 337.38, ALogP 3.4, HBA 6, HBD 2, PSA 76.5, QED 0.72
Compound (SMILES): Cc1cc(F)c(C(=O)Nc2cccc(-c3nncn3C(C)C)n2)cc1C1CC1C(=O)N1CCC(F)(F)C1
InChIKey: KZRJQVKEPZZHTN-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99683
Assay: HTRF KinEASE Assay: ASK1 was purchased from Thermofisher (Catalogue #PV4011), ATP was purchased from Sigma (Catalogue #A7699), HTRF® KinEASE Assay System was obtained from Cisbio (Bedford, Mass.). ½ Area plate was purchased from Perkin Elmer (Catalogue # #6005560). HTRF® KinEASE-STK is a generic method for measuring serine/threonine kinase activities using a time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay. The IC50 value for each compound was determined in the presence of compound (various concentration from 0 to 10 μM) and a fixed amount of ATP and peptide substrates. The test compound, 1 uM STK3 peptide substrate, and 5 nM of ASK1 kinase are incubated with kinase reaction buffer containing 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, and 1 mM EGTA for 30 minutes. 100 uM ATP is added to start kinase reaction and incubated for 3 hours. The STK3-antibody labeled with Eu3+-Cryptate and 125 nM streptavidin-XL665 are mixed in a single addition with stop reagents provided by the Cisbio kit used to stop the kinase reaction. Fluorescence is detected using an Envision Multilabeled 2014 reader from PerkinElmer. The Fluorescence is measured at 615 nm (Cryptate) and 665 nm (XL665) and a ratio of 665 nm/615 nm is calculated for each well. The resulting TR-FRET is proportional to the phosphorylation level. Staurosporine was used as the positive control.
Measurement: IC50 > 10 nM (pChEMBL 8)
Descriptors: MW 512.54, ALogP 4.59, HBA 6, HBD 1, PSA 93.01, QED 0.52
Compound (SMILES): [C-]#[N+]c1c(Cl)cc(C(C)n2nc(C)c3c(N)ncnc32)c(OC)c1C1CN(S(C)(=O)=O)C1
InChIKey: WGQRYIKNZFHQQR-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: PI3K delta Scintillation Proximity Assay: [γ-33P]ATP (10 mCi/mL) 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). PI3Kδ (p110δ/p85α) was purchased from Millipore (Bedford, Mass.). ATP, MgCl2, DTT, EDTA, MOPS and CHAPS were purchased from Sigma-Aldrich (St. Louis, Mo.). Wheat Germ Agglutinin (WGA) YSi SPA Scintillation Beads was purchased from GE healthcare life sciences (Piscataway, N.J.). 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 μM PIP2, 20 μM 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 μM 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 < 10 nM (pChEMBL 8)
Descriptors: MW 475.96, ALogP 2.9, HBA 8, HBD 1, PSA 120.59, QED 0.56
Compound (SMILES): Cc1nnc(-c2cc(N3CCOCC3)cc3c2nc(C)n3-c2ccnc3c(F)cccc23)[nH]1
InChIKey: XQKMOFHRANHWSC-UHFFFAOYSA-N
Target: Phosphatidylinositol 3-kinase regulatory subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O00459
Assay: TR-FRET PI3K Beta Assay: Class I PI3K isoforms were expressed and purified as heterodimeric recombinant proteins. All assay reagents and buffers for the TR-FRET assay were purchased from Millipore. PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2× Km ATP (75-500 μM), 2 μM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, and 1% (v/v) DMSO at the following concentrations for each isoform: PI3Kα, PI3Kβ, and PI3Kδ between 25 and 50 μM, and PI3Kγ at 2 nM. The compounds were added to the assay solution and incubated for 30 minutes at 25° C. The reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 500 μs read window).The results were normalized based on positive (1 μM wortmanin) and negative (DMSO) controls, and the IC50 values for PI3K α, β, δ, and γ were calculated from the fit of the dose-response curves to a four-parameter equation. These assays generally produced results within 3-fold of the reported mean. As shown in Table 1, the compounds provided herein are inhibitors of PI3Kβ.
Measurement: IC50 = 87 nM (pChEMBL 7.059999942779541)
Descriptors: MW 443.49, ALogP 3.95, HBA 7, HBD 1, PSA 84.75, QED 0.45
Compound (SMILES): CN1CC[C@@H](n2c(C3CCC(F)C3)nc3cnc4ccc(F)cc4c32)C1
InChIKey: KLSMZNHKHHARTE-SSDMNJCBSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LRRK2 Assay: LRRK2 kinase activity was measured using Lantha Screen technology from Invitrogen. GST-tagged truncated LRRK2 from Invitrogen (Cat #PV4874) was incubated with a fluorescein-labeled peptide substrate based upon ezrin/radixin/moesin (ERM), also known as LRRKtide (Invitrogen cat #PR8976A), in the presence of a dose response of compound. Upon completion, the assay was stopped and detected with a terbium labeled anti-phospho-ERM antibody (Invitrogen, cat #PR8975A). The assay was carried out under the following protocol: The compound dose response was prepared by diluting compound to a top concentration of 0.3 mM in 100% DMSO and serial diluted by half-log in DMSO to give an 11 point curve, 100× final assay concentration. Using Echo acoustic dispensing, 60 nL of compound was transferred to a low volume Corning 384-well assay plate. 3 μL of a working solution of substrate (200 nM LRRKtide, 2 mM ATP) prepared in assay buffer (50 mM HEPES, pH 7.5, 3 mM MgCl2, with 2 mM DTT and 0.01% Brij35 added fresh) was added to the 60 nL compound assay plate. The kinase reaction was started with 3 μL of a working solution of LRRK2 enzyme at a concentration of 4 μg/mL. The final reaction concentrations were 100 nM LRRKtide, 1 mM ATP, 2 μg/mL LRRK2 enzyme and a compound dose response with a top dose of 3 μM. The reaction was allowed to progress at room temperature for 30 minutes and then stopped with the addition of 6 μL of detection buffer (20 mM Tris pH 7.6, 0.01% NP-40, 6 mM EDTA with 2 nM terbium labeled anti-phospho-ERM). After an incubation of 1 hour at room temperature, the plate was read on an Envision with an excitation wavelength of 340 nm and a reading emission at both 520 nm and 495 nm. The ratio of the 520 nm and 495 nm emission was used to analyze the data. Inhibition of mutant G2019S LRRK2 (Invitrogen cat #PV4881) was measured in the exact same method. All final concentrations of substrate ATP and enzyme were the same.
Measurement: IC50 = 59.6 nM (pChEMBL 7.21999979019165)
Descriptors: MW 356.42, ALogP 4.21, HBA 4, HBD 0, PSA 33.95, QED 0.69
Compound (SMILES): Cc1cc(Nc2ncnc3sc(C(=O)N4CC(N)C4)cc23)c(=O)n2c1C(=O)NC21CCCCC1
InChIKey: XFEFHVZCXJMXGZ-UHFFFAOYSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BUB5
Assay: MKNK1 Kinase High ATP Assay: MKNK1-inhibitory activity at high ATP of compounds of the present invention after their preincubation with MKNK1 was quantified employing the TR-FRET-based MKNK1 high ATP assay as described in the following paragraphs.A recombinant fusion protein of Glutathione-S-Transferase (GST, N-terminally) and human full-length MKNK1 (amino acids 1-424 and T344D of accession number BAA 19885.1), expressed in insect cells using baculovirus expression system and purified via glutathione sepharose affinity chromatography, was purchased from Carna Biosciences (product no 02-145) and used as enzyme. As substrate for the kinase reaction the biotinylated peptide biotin-Ahx-IKKRKLTRRKSLKG (SEQ ID NO: 1) (C-terminus in amide form) was used, which can be purchased e.g. from the company Biosyntan (Berlin-Buch, Germany)For the assay 50 nL of a 100 fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 μL of a solution of MKNK1 in aqueous assay buffer [50 mM HEPES pH 7.5, 5 mM MgCl2, 1.0 mM dithiothreitol, 0.005% (v/v) Nonidet-P40 (Sigma)] was added and the mixture was incubated for 15 mM at 22° C. to allow pre-binding of the test compounds to the enzyme before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 μL of a solution of adenosine-tri-phosphate (ATP, 3.3 mM=>final conc. in the 5 μL assay volume is 2 mM) and substrate (0.1 μM=>final conc. in the 5 μL assay volume is 0.06 μM) in assay buffer and the resulting mixture was incubated for a reaction time of 30 min at 22° C. The concentration of MKNK1 was adjusted depending of the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical concentrations were in the range of 0.003 μg/mL. The reaction was stopped by the addition of 5 μL of a solution of TR-FRET detection reagents (5 nM streptavidine-XL665 [Cisbio Bioassays, Codolet, France] and 1 nM anti-ribosomal protein S6 (pSer236)-antibody from Invitrogen [#44921G] and 1 nM LANCE EU-W1024 labeled ProteinG [Perkin-Elmer, product no. AD0071]) in an aqueous EDTA-solution (100 mM EDTA, 0.1% (w/v) bovine serum albumin in 50 mM HEPES pH 7.5).
Measurement: IC50 = 0.6 nM (pChEMBL 9.220000267028809)
Descriptors: MW 479.57, ALogP 2.05, HBA 9, HBD 3, PSA 135.24, QED 0.52
Compound (SMILES): CCOc1cc(O[C@H]2CCc3c(-c4cccc(OC)c4F)cccc32)c(Cl)cc1CN[C@@H](CO)C(=O)O
InChIKey: VNPYFUNZDMEACC-GOTSBHOMSA-N
Target: Programmed cell death 1 ligand 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZQ7
Assay: Enzyme-Linked Immunosorbent AssayâELISA: Plates were coated with 1 Î¼g/mL of human PD-L1 (obtained from R&D) in PBS overnight at 4Â° C. The wells were then blocked with PBS containing 2% BSA in PBS (W/V) with 0.05% TWEEN-20 for 1 hour at 37Â° C. The plates were washed 3 times with PBS/0.05% TWEEN-20 and the samples were diluted to 1:5 in dilution medium in the ELISA plates. Human PD-1 and biotin 0.3 Î¼g/mL (ACRO Biosystems) were added and incubated for 1 hour at 37Â° C. then washed 3 times with PBS/0.05% TWEEN-20. A second block was added with PBS containing 2% BSA in PBS (W/V)/0.05% TWEEN-20 for 10 min at 37Â° C. and was washed 3 times with PBS/0.05% TWEEN-20. Streptavidin-HRP was added for 1 hour at 37Â° C. then washed 3 times with PBS/0.05% TWEEN-20. TMB substrate was added and reacted for 20 min at 37Â° C. A stop solution (2 N aqueous H2SO4) was added. The absorbance was read at 450 nm using a micro-plate spectrophotometer.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 529.99, ALogP 5.15, HBA 6, HBD 3, PSA 97.25, QED 0.32
Compound (SMILES): Cc1nc(Cl)ccc1OCC(C)(C)C(=O)NC1CCN(C)CC1
InChIKey: QMWPFQAOZDYONM-UHFFFAOYSA-N
Target: Somatostatin receptor type 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P31391
Assay: Inhibition of Forskolin Stimulated cAMP in Cells Overexpressing SSTR4: This cell-based assay measures the ability of compounds to inhibit forskolin stimulated cAMP in CHO-K1 cells overexpressing SSTR4. CHO-K1 cells overexpressing SSTR4 (CHO-SSTR4) are purchased from DiscoveRx (product code 95-0059C2). The CHO-SSTR4 cells are maintained in F12K media with 10% Fetal Bovine Serum (Hyclone), 1% Pen/Strep (Life Technologies), and 800 μg/mL G418 (Life Technologies). To perform the assay, 3000 cells are plated per well in white 384-well plate (Corning 3570) in 50 μL complete media and the cells are allowed to attach for 16 hours in a 37° C., 5% CO2 incubator. The next day, the culture media is removed from the cells and the cells are washed (added then removed) with Kreb's Ringer Buffer (ZenBio, KRB-1000 mL). Test compounds are suspended in DMSO and diluted in stimulation buffer: Kreb's Ringer Buffer plus 0.5% BSA (Roche), 300 μM IBMX (Sigma), and 350 nM forskolin (Sigma). The cells are incubated in L compound/stimulation buffer for 30 minutes at room temperature. Cellular cAMP levels are detected with a HTRF LANCE Ultra cAMP kit (Perkin Elmer, catalog number TRF0264).The assay is performed in accordance with the manufacturer's instructions. Five μL of diluted Eu-W8044 labeled streptavidin (dilution: 1:50 in cAMP Detection Buffer) is added to each well. Then 5 μL of diluted biotin cAMP (dilution: 1:150 in cAMP Detection Buffer) is added to each well. The plates are covered and allowed to incubate for 60 minutes at room temperature on a shaker. HTRF (665 nm/615 nm) is read on a Perkin Elmer ENVISION plate reader. The pEC50 values are generated using Activity Base for Screening Data Management.
Measurement: EC50 = 3981 nM (pChEMBL 5.400000095367432)
Descriptors: MW 339.87, ALogP 2.66, HBA 4, HBD 1, PSA 54.46, QED 0.84
Compound (SMILES): CN1C(=O)[C@@H](NC(=O)c2n[nH]c(Cc3ccccc3)n2)COc2ccc(C#CC3(O)CCC3)cc21
InChIKey: JOPXYPLAXUBVQB-IBGZPJMESA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: Biochemical Assay: 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).
Measurement: IC50 = 31.7 nM (pChEMBL 7.5)
Descriptors: MW 471.52, ALogP 1.82, HBA 6, HBD 3, PSA 120.44, QED 0.5
Compound (SMILES): Cc1nnn(C)c1-c1cnc2c3ccnc(N4CCN(C)CC4)c3n(C(c3ccccc3)C3CCOCC3)c2c1
InChIKey: WRJIAXCPOMKVLN-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The binding of compounds to bromodomain BRD4 (44-168), BRD4 (333-460), and BRD4 (1-477 or 44-460) was assessed using a time resolved fluorescent resonance energy transfer binding assay (1), that measures the binding of a fluorescently labeled probe molecule to the bromodomain protein. The bromodomain protein, fluorescent probe molecule (either a biotinylated histone peptide or a fluorescently labeled small molecule), and dose-responded test compound are incubated together to reach thermodynamic equilibrium. In the absence of a test compound, the bromodomain and small molecule are bound, resulting in a high fluorescent signal. In the presence of a sufficient concentration of inhibitor, this intercation is disrupted resulting in a lost of fluorescent resonance energy transfer.All assay components were dissolved in buffer composition 20 mM Hepes pH 7.5, 150 mM NaCl, 5 mM DTT, 0.005% Tween 20, and 100 ug/ml BSA for BRD4 (1-477 and 44-460). The final concentrations of the bromodomain proteins are 1.6 nM BRD4(44-168), 1 nM BRD4(333-460), and 1 nM BRD4(1-477 or 44-460), and the fluorescent probe molecule is 100 nM, 50 nM, and 7.5 nM respectively. All proteins were biotinylated. A streptavidin labeled with terbium cryptate (Cisbio SA-Tb) was used as detection, and pre-mixed with the bromodomain protein at a final concentration of 0.2 nM. In some instances for BRD4 (44-460), anti-His terbium cryptate was used as a detection. 7.5 nl of dose-responded test compound or dmso vehicle (0.0375%) was pre-spotted in a black Corning 384 well plate and 10 ul each of bromodomain/detection reagent and fluorescent small molecule solution were added to the plate, and the reaction incubated for 60 min at room temperature. Plates were then read on EnVision plate reader, (λex=340 nm, acceptor λEm=520 nm, and donor λEm=615 nm, LANCE D400 mirror). Time resolved fluorescnce intensity measurements were made at both emissions, and the ratio of acceptor/donor was calculated and used for data analysis.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 536.68, ALogP 4.46, HBA 9, HBD 0, PSA 77.13, QED 0.33
Compound (SMILES): CC(C)(O)[C@H](F)CNC(=O)c1cnc(-c2cnc3cc(Cl)cnn23)cc1NC1CC(F)C1
InChIKey: JVNYKHCBJJIOTO-PWGQDJSOSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IRAK4 Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 μL prepared from 15 μL additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij 35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction mixture was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 464.9, ALogP 3.2, HBA 7, HBD 3, PSA 104.44, QED 0.5
Compound (SMILES): Cc1ccc(-c2cn(CC3CCOCC3)cc(C(=O)Nc3ccc(-c4cc(-c5ccncc5F)cnc4N)cc3)c2=O)cc1
InChIKey: XGAALYULZCHKHO-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: Cell-Free Axl Kinase Inhibitory Activity: A kinase dilution solution containing 170 ng/ml AXL (the 464th to 885th amino acids of the intracellular domain of human AXL expressed as a fusion protein with glutathione transferase in a baculovirus expression system and purified by glutathione Sepharose chromatography; Carna Biosciences, Inc., catalog No. 08-107) 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 30 minutes, a solution containing a substrate peptide (FL-Peptide 30 (5FAM-KKKKEEIYFFF-CONH2), Caliper Life Sciences, catalog No. 760430) and ATP at 1.5 μM and 10 μM, respectively, was prepared and added at 5 μl/well to the plate to start kinase reaction. The plate was incubated at 28° C. for 1.5 hours, 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 = 1 nM (pChEMBL 9)
Descriptors: MW 589.67, ALogP 6.35, HBA 7, HBD 2, PSA 112.13, QED 0.23
Compound (SMILES): Nc1nc(Cl)nc2c1ncn2[C@@H]1O[C@H](COC(Cc2cccnc2)(C(=O)O)c2nn[nH]n2)[C@@H](O)[C@H]1O
InChIKey: AOVZQHDMNFKFFE-REUWOWEOSA-N
Target: 5'-nucleotidase (SINGLE PROTEIN), Homo sapiens
UniProt: P21589
Assay: Inhibition Enzyme In Vitro Assay: For measurements of soluble CD73 enzyme activity, recombinant CD73 was obtained from R&D Systems, Cat. No. 5795-EN-010. Serial dilutions of test compounds were incubated with recombinant CD73 and AMP in reaction buffer (25 mM Tris HCl pH7.5, 5 mM MgCl2, 50 mM NaCl, 0.25 mM DTT, 0.005% Triton X-100). The final reaction volume was 25 μL and the final concentrations of recombinant CD73 and AMP were 0.5 nM and 50 μM, respectively. Reactions were allowed to proceed for 30 minutes at room temperature before the addition of 100 μL Malachite Green (Cell Signaling Technology, Cat. No. 12776). After 5 minutes at room temperature, absorbance at 630 nm was determined on a microplate spectrophotometer.
Measurement: IC50 = 1178 nM (pChEMBL 5.929999828338623)
Descriptors: MW 518.88, ALogP -1.17, HBA 14, HBD 5, PSA 233.19, QED 0.17
Compound (SMILES): C[C@@H]1CC[C@@H](n2ncc(C(C)(C)O)n2)CN1C(=O)c1ccccc1SC(F)(F)F
InChIKey: GMAADJYGYZXUKG-CHWSQXEVSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P56718
Assay: FLIPR Ca2+ Flux Assay: 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 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 428.48, ALogP 3.98, HBA 6, HBD 1, PSA 71.25, QED 0.75