lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1ccnc(NCCC#CCCNC(=O)c2cccn(Cc3ccc(Cl)c(Cl)c3)c2=O)n1
InChIKey: RPDMVKUBXUQLOY-UHFFFAOYSA-N
Target: 3-phosphoinositide-dependent protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O15530
Assay: Kinase Assay: A PDK1 kinase assay was performed as follows. PDK1 (amino acids 51-360) and AKT2 (amino acids 140-467 fused to PIFtide, amino acids EEQEMFRDFDYIADW) were expressed as N-terminally tagged GST fusion proteins in insect cells and purified to greater than 90% homogeneity. PDK1 protein was divided into two fractions, one of which was subsequently dephosphorylated. To generate dephosphorylated PDK1, the PDK1 was reacted with GST-tagged lambda-phosphatase in vitro. GST was subsequently cleaved proteolytically for both phosphorylated and dephosphorylated PDK1. Protein preparations were run on glutathione Sepharose columns to remove GST and GST-tagged lambda-phosphatase, if present. Phosphorylated PDK1 and dephosphorylated PDK1 were verified by mass-spectrometry. Enzyme activity was determined in a coupled PDK1/AKT/FAM-crosstide assay using either phosphorylated or unphosphorylated PDK1 and phosphorylation of FAM-crosstide was determined by standard IMAP protocol (Molecular Devices). For inhibition studies, compounds were titrated 3-fold in DMSO and diluted 40-fold into assay buffer (10 mM Tris HCl pH7.2; 10 mM MgCl2; 0.01% Triton X-100; 1 mM DTT) containing PDK1, AKT2, and FAM-crosstide. (final concentrations: 25 nM un-phosphorylated PDK1 or 0.5 nM phosphorylated PDK1, 30 nM unphosphorylated AKT2, and 100 nM crosstide substrate). The kinase reaction was initiated by adding ATP to a final concentration of 24 μM for both forms of PDK1 and incubated at 25° C. for 30 min. To detect assay product, the kinase reaction was combined with Progressive Binding Solution (1:600 Progressive Binding Reagent, 50% Buffer A, 50% Buffer B, Molecular Devices) in a 1:3 ratio. The mixture was incubated for 2 hours at 25° C. and the plate was scanned on an Analyst AD with excitation at 485 nm and emission at 530 nm.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 500.39, ALogP 3.63, HBA 7, HBD 2, PSA 98.14, QED 0.34
Compound (SMILES): O=C1CCc2cc(OCCCN3CCC(C4=NOC5C=C(F)C=CC45)CC3)ccc2N1
InChIKey: OCNUPGFDBUFRFR-UHFFFAOYSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Receptor Binding Assay: D2:Procedures(1) The prepared membrane was applied with appropriate amount of buffer, and homogenizer was used for evenly dispersing. 15 tubes were mixed into a 100 ml container, and appropriate amount of buffer was added to give 50 ml of membrane suspension, which was reserved for future use.(2) 100 μL of membrane preparation and 100 μL of buffer were added into each reaction tube.(3) 100 μL of buffer was added into the total binding tube (TB), 100 μL of Butaclamol (final concentration 10−5M) was added into the nonspecific binding tube (NB), 100 μL of the test compound (final concentration 10−5M) was added into the specific binding tube (SB) of each test compound.(4) 10 μL of radioactive ligand 3H-Spiperone was respectively added into each reaction tube (2 parallel tubes were used for each reaction tube, and each of them was placed on ice when adding sample).(5) Each of the reaction tubes was incubated at 37° C. for 20 min. After the reaction was completed, the bound ligands were rapidly filtered under reduced pressure, and sufficiently washed with ice-chilled assay buffer. The filter was taken out and put into a 3 ml scintillation vial, and 2 ml of toluene scintillation cocktail was added and blended.(6) The scintillation vials were put into Liquid Scintillation Counter for counting.Inhibition rate(I%)=(Total binding tube cpm−compound cpm)/(Total binding tube cpm−nonspecific binding tube cpm)×100% .
Measurement: Ki = 5.6 nM (pChEMBL 8.25)
Descriptors: MW 425.5, ALogP 3.85, HBA 5, HBD 1, PSA 63.16, QED 0.7
Compound (SMILES): COc1cn(Cc2cc(F)cc(F)c2)c(Nc2cccc(C)c2C)nc1=O
InChIKey: UIRDXCCONCAMLD-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Evaluation of a Human P2X7 Receptor Inhibitory Activity: Stably expressing cell line (1321N1 cell transfected with the human P2X7 receptor gene (GenBank accession number NM_002562.5 including T606C and G952A SNP)) was used. The cells were seeded in a 384-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (10% fetal bovine serum, 25 mM HEPES, 1% penicillin and streptomycin in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. After replacing with 20 μL of the HBSS buffer (20 mM HEPES, 55.6 mM D-glucose, 1×HBSS(−), pH7.4-7.5), 15 μL of 17.3 μM Yo-Pro solution in the HBSS buffer was added. The plate was placed in high-throughput cellular screening system FLIPR TETRA (Molecullar Devices, LLC.) and 15 μL of 130 μM BzATP solution in the HBSS buffer was added. Measurement of fluorescence intensity by FLIPR TETRA was started. After eight minutes, 15 μL of DMSO solutions containing different concentrations of the compound of the present invention as prepared by dilution with the HBSS buffer were dispensed to each well through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 20 minutes. The maximum fluorescence intensity without the compound of the present invention is calculated as 0% inhibition and the maximum fluorescence intensity when the reference compound was added is calculated as 100% inhibition. Changing values of fluorescence intensity by the compound of the present invention were calculated by difference between maximum and minimum fluorescence intensity for 20 minutes. IC50 was calculated using logistic approximation.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 371.39, ALogP 3.94, HBA 5, HBD 1, PSA 56.15, QED 0.74
Compound (SMILES): Cn1nc(C(C)(C)n2nccn2)cc1Nc1ncc(Br)c(OC2CC2)n1
InChIKey: TWKLMKZGLOLTJN-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Biochemical Assay: Materials:    LRRK2 G2019S enzyme    Substrate (LRRKtide)    ATP    TR-FRET dilution buffer    pLRRKtide antibody    384-well assay plate    DMSOEnzyme Reaction Conditions    50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Brij-35, 2 mM DTT    5 nM LRRK2    134 μM ATP    60 minute reaction time    23° C. reaction temperature    10 μL total reaction volumeDetection Reaction Conditions    1× TR-FRET dilution buffer    10 mM EDTA    2 nM antibody    23° C. reaction temperature    10 μL total reaction volumeCompounds were prepared by initially diluting to 1 mM with DMSO. 35 μL of reference compound solution, 35 μL of test compound solution, and 35 μL HPE were successively added to the source plate (384-well assay plate, Labcyte). The plates were centrifuged at 2500 rpm for 1 minute and sealed in foil. POD was used to perform a 3.162 fold serial dilution and 100 nL of reference compound solution, test compound solution, HPE and ZPE were transferred to assay plates. The assay plate was centrifuged at 2500 rpm for 1 minute, and sealed with foil.To perform the enzyme reaction, 5 μL of LRRKtide substrate and kinase mixture in assay buffer was added to all wells of the assay plate. The plate was centrifuged to concentrate the mixture at the bottom of the wells. The assay plate was incubated at 23° C. for 20 minutes. Following incubation, 5 μL of 2× ATP in assay buffer was added to each well, and plates were centrifuged to concentrate the mixture at the bottom of the wells. The plate was incubated at 23° C. for 60 minutes.To perform the detection of the reaction, EDTA completely mixed in TR-FRET dilution buffer was added to antibody reagent. 10 μL of detection reagent was added to all wells of each well of the assay plate and the plate was centrifuged to concentrate the mixture at the bottom of the wells. The plate was then incubated at 23° C. for 60 minutes. Plates were read on Perkin Elmer Envision 2104 instrument in TR-FRET mode using a 340 nm excitation filter, 520 nm fluorescence emission filter, and 490 or 495 nm terbium emission filter.
Measurement: IC50 < 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 419.29, ALogP 2.63, HBA 9, HBD 1, PSA 95.57, QED 0.66
Compound (SMILES): Cc1cc(Nc2cc3ncc(S(C)(=O)=O)cc3c(N[C@@H]3C[C@H]4CC[C@@H](C3)N4CCC(F)(F)F)n2)n[nH]1
InChIKey: LDWSNTGPPCCVRW-PHZGNYQRSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Biochemical JAK Assay: A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 and Tyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH 7.5, 0.01% Brij-35, 10 mM MgCl2, and 1 mM EGTA). Recombinant GST-tagged JAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained from Life Technologies.Serially diluted compounds were pre-incubated with each of the four JAK enzymes and the substrate in white 384-well microplates (Corning) at ambient temperature for lh. ATP was subsequently added to initiate the kinase reactions in 10 μL total volume, with 1% DMSO. The final enzyme concentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and 0.25 nM respectively; the corresponding Km ATP concentrations used are 25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200 nM for all four assays. Kinase reactions were allowed to proceed for 1 hour at ambient temperature before a 10 μL preparation of EDTA (10 mM final concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (Life Technologies, 2 nM final concentration) in TR-FRET dilution buffer (Life Technologies) was added. The plates were allowed to incubate at ambient temperature for lh before being read on the EnVision reader (Perkin Elmer). Emission ratio signals (520 nm/495 nm) were recorded and utilized to calculate the percent inhibition values based on DMSO and background controls.For dose-response analysis, percent inhibition data were plotted vs. compound concentrations, and IC50 values were determined from a 4-parameter robust fit model with the Prism software (GraphPad Software). Results were expressed as pIC50 (negative logarithm of IC50) and subsequently converted to pKi (negative logarithm of dissociation constant, Ki) using the Cheng-Prusoff equation.Test compounds having a higher pKi value in each of the four JAK assays show greater inhibition of JAK activity. Compounds of the invention tested in this assay typically exhibited pKi values between about 7 and about 10.3
Measurement: Ki = 0.55 nM (pChEMBL 9.260000228881836)
Descriptors: MW 523.59, ALogP 4.17, HBA 8, HBD 3, PSA 115.9, QED 0.42
Compound (SMILES): CC(F)(F)COc1nc2c(s1)CCN(CC[C@H]1CC[C@H](NC(=O)c3cnn4ccccc34)CC1)CC2
InChIKey: PJMKWDTWNMTUMU-WGSAOQKQSA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Binding Inhibition for Dopamine D3 Receptor: Buffer solution: 50 mM Tris-HCl (35409-45, Nacalai Tesque) (pH 7.4) containing 120 mM NaCl (31320-05, Nacalai Tesque), 1 mM MgCl.6HO (20909-55, Nacalai Tesque), 5 mM KCl (28514-75, Nacalai Tesque) and 2 mM CaCl) (067-31, NAKARAI CHEMICALS, LTD.) Radioligand: (final concentration) 2 nM [H]-Methylspiperone ([H���N-methyl-]-Methylspiperone, NET-856, 83.8 Ci/mmol, PerkinElmer)Non-specific ligand: (final concentration) 10 uM Butaclamol [(+)-Butaclamol Hydrochloride, D033, Sigma] SPA beads solution: SPA beads [WGA PVT SPA Scintillation Beads, RPNQ0001 (500 mg), RPNQ0060 (2 g), PerkinElmer] (0.2 mg/well) Incubation time and temperature: 120 min at 25° C.
Measurement: Ki = 0.43 nM (pChEMBL 9.369999885559082)
Descriptors: MW 517.65, ALogP 4.6, HBA 7, HBD 1, PSA 71.76, QED 0.47
Compound (SMILES): COc1cc(OC)c(F)c(N2Cc3cnc4[nH]c(CCN5CCOCC5)cc4c3N(c3ccc(F)cc3)C2=O)c1F
InChIKey: SJSQASZJOOONGL-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Enzymatic Assay: The inhibitor potency of the exemplified compounds was measured in an enzyme assay that measures peptide phosphorylation using FRET measurements to detect product formation. Inhibitors were serially diluted in DMSO and a volume of 0.5 μL was transferred to the wells of a 384-well plate. For FGFR3, a 10 μL volume of FGFR3 enzyme (Millipore) diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 5 mM DTT, pH 7.5) was added to the plate and pre-incubated for 5-10 minutes. Appropriate controls (enzyme blank and enzyme with no inhibitor) were included on the plate. The assay was initiated by the addition of a 10 μL solution containing biotinylated EQEDEPEGDYFEWLE peptide substrate (SEQ ID NO: 1) and ATP (final concentrations of 500 nM and 140 μM respectively) in assay buffer to the wells. The plate was incubated at 25° C. for 1 hr. The reactions were ended with the addition of 10 μL/well of quench solution (50 mM Tris, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.8; 30 mM EDTA with Perkin Elmer Lance Reagents at 3.75 nM Eu-antibody PY20 and 180 nM APC-Streptavidin). The plate was allowed to equilibrate for 1 hr before scanning the wells on a PheraStar plate reader (BMG Labtech).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 567.57, ALogP 5.15, HBA 6, HBD 1, PSA 83.16, QED 0.33
Compound (SMILES): CN1CCN(Cc2cccc(-c3nc4ccncc4n3-c3ccc(Cl)cc3)c2)CC1
InChIKey: OXOPNEMWNNOVGW-UHFFFAOYSA-N
Target: Amine oxidase [copper-containing] 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16853
Assay: Biological Assay: SSAO: All primary assays were performed at RT. with purified recombinantly expressed human SSAO. Enzyme was prepared essentially as described in Öhman et al. (Protein Expression and Purification 46 (2006) 321-331). In addition, secondary- and selectivity assays were performed using SSAO prepared from various tissues or purified rat recombinant SSAO. The enzyme activity was assayed with benzylamine as substrate by measuring either benzaldehyde production, using 14C-labeled substrate, or by utilizing the production of hydrogen peroxide in a horseradish peroxidase (HRP) coupled reaction. Briefly, test compounds were dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 mM. Dose-response measurements were assayed by either creating 1:10 serial dilutions in DMSO to produce a 7 point curve or by making 1:3 serial dilutions in DMSO to produce 11 point curves. The top concentrations were adjusted depending on the potency of the compounds and subsequent dilution in reaction buffer yielded a final DMSO concentration≤2%.
Measurement: IC50 = 700 nM (pChEMBL 6.159999847412109)
Descriptors: MW 417.94, ALogP 4.49, HBA 5, HBD 0, PSA 37.19, QED 0.49
Compound (SMILES): CC(C)n1ncc2nc(-c3ccc(N4CCN(C(=O)Nc5ccccc5Cl)CC4)cc3)[nH]c21
InChIKey: FWQJAHKUEHDJRR-UHFFFAOYSA-N
Target: Diacylglycerol O-acyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75907
Assay: FlashPlate™ Assay: DGAT activity in membrane preparations was assayed in 50 mM Tris-HCl (pH 7.4), 150 mM MgCl2, 1 mM EDTA and 0.2% BSA, containing 50 μM DAG, 32 μg/ml PC/PS and 8.4 μM [3H]-oleoylCoA (at a specific activity of 30 nCi/well) in a final volume of 50 μl in 384-well format using the red shifted Basic Image FlashPlate™ (Perkin Elmer Cat.No. SMP400). In detail, 10 μl enzyme mix and 10 μl substrate mix were added to 30 μl of assay buffer, optionally in the presence of 1 μl DMSO (blank and controls) or 1 μl of the compound to be tested. This reaction mixture was incubated for 120 minutes at 37° C. and the enzymatic reaction stopped by adding 20 μl of the stop mix. The plates were sealed and the vesicles allowed to settle overnight at room temperature. Plates were centrifuged for 5 minutes at 1500 rpm and measured in Leadseeker.
Measurement: IC50 = 85 nM (pChEMBL 7.070000171661377)
Descriptors: MW 463.97, ALogP 5.01, HBA 5, HBD 2, PSA 82.08, QED 0.45
Compound (SMILES): C[C@]1(O)CC[C@@H](Nc2ncnc3[nH]cc(C(=O)c4ccccc4Cl)c23)CC1
InChIKey: UKOAPNKZWOJGJD-KWUMSVIQSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Mobility Shift Assay (MSA) Type A: Full length unphosphorylated form of BTK expressed in Sf9 cells was employed to test inhibitory activity in the inactive BTK assay. The assay was measured in buffer solution containing 100 mM HEPES pH7.5, 0.01% Triton X-100, 0.1% BSA, 5 mM MgCl2, 1 mM DTT. The enzyme and increasing concentrations of inhibitor was incubated at room temperature for 30 minutes and the kinase reaction was initiated by the addition an activation mixture diluted in assay buffer containing Srctide peptide substrate, DOPS/DOPC, PtdIns(3,4,5)P3, and ATP for final concentrations of 1 μM Srctide, 5.5 μM DOPS, 5.5 μM DOPC, 0.5 μM PtdIns(3,4,5)P3, and 16 μM ATP. The plates were incubated for 60 minutes at room temperature, and then the reaction stopped with 100 mM HEPES buffer containing 0.01% Triton and 40 mM EDTA and read on Caliper Life Sciences Labchip EZ Reader II instrument.
Measurement: IC50 = 3.5 nM (pChEMBL 8.460000038146973)
Descriptors: MW 384.87, ALogP 3.95, HBA 5, HBD 3, PSA 90.9, QED 0.59
Compound (SMILES): Nc1nc2cc(-c3ccc4ncn(C5CC5)c(=O)c4c3)ccc2o1
InChIKey: WMZMCTLVCWIPLX-UHFFFAOYSA-N
Target: Phosphatidylinositol 3-kinase regulatory subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O00459
Assay: PI 3-Kinase HTRF Assay: Briefly, the assay is a time resolved FRET assay that indirectly measures PIP3 product formed by the activity of a PI3-K. The kinase reaction is performed in a microtitre plate (e.g., a 384 well microtitre plate). The total reaction volume is approximately 20 ul per well. In the first step, each well receives 2 ul of test compound in 20% dimethylsulphoxide resulting in a 2% DMSO final concentration. Next, approximately 14.5 ul of a kinase/PIP2 mixture (diluted in 1x reaction buffer) is added per well for a final concentration of 0.25-0.3 ug/ml kinase and 10 uM PIP2. The plate is sealed and incubated for 15 minutes at room temperature. To start the reaction, 3.5 ul of ATP (diluted in 1x reaction buffer) is added per well for a final concentration of 10 uM ATP. The plate is sealed and incubated for 1 hour at room temperature. The reaction is stopped by adding 5 ul of Stop Solution per well and then 5 ul of Detection Mix is added per well. The plate is sealed, incubated for 1 hour at room temperature, and then read on an appropriate plate reader.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 318.34, ALogP 3.12, HBA 6, HBD 1, PSA 86.94, QED 0.61
Compound (SMILES): COCCCN1C(=O)N(CC(=O)Nc2cccc(OC)n2)C[C@]12CC[C@](c1ccccc1)(N(C)C)CC2
InChIKey: MFYGIWUIQJLEPR-HNRBIFIRSA-N
Target: Nociceptin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41146
Assay: Human Nociceptin/Orphanin FQ Peptide (hNOP) Receptor Binding Assay: The hNOP receptor binding assay was performed as homogeneous SPA-assay (scintillation proximity assay) using the assay buffer 50 mM TRIS-HCl, 10 mM MgCl2, 1 mM EDTA (pH 7.4). The final assay volume (250 μl/well) included 0.5 nM of [leucyl-3H]nociceptin as ligand (PerkinElmer Life Sciences, Inc. Boston, Mass., USA), and either test compound in dilution series or 1 μM unlabelled nociceptin for determination of unspecific binding. The test compound was diluted with 25% DMSO in H2O to yield a final 0.5% DMSO concentration, which also served as a respective vehicle control. The assay was started by adding wheat germ agglutinin coated SPA beads (GE Healthcare UK Ltd., Buckinghamshire, UK) which had been preloaded with hMOP receptor membranes (PerkinElmer Life Sciences, Inc. Boston, Mass., USA). After incubation for 60 minutes at RT and centrifugation for 20 minutes at 500 rpm the signal rate was measured by means of a 1450 Microbeta Trilux ß-counter (PerkinElmer Life Sciences/Wallac, Turku, Finland). Half-maximal inhibitory concentration (IC50) values reflecting 50% displacement of [3H]nociceptin-specific receptor binding were calculated by nonlinear regression analysis and Ki values were calculated by using the Cheng-Prusoff equation, (Cheng and Prusoff, 1973).
Measurement: Ki = 9.1 nM (pChEMBL 8.039999961853027)
Descriptors: MW 509.65, ALogP 3.57, HBA 6, HBD 1, PSA 87.24, QED 0.49
Compound (SMILES): Cc1cc2c(cc1C#N)c(-c1cncc(N)c1Cl)cn2[C@H]1C[C@H](O)C1
InChIKey: ZQHCJZGVESBCID-JOCQHMNTSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: FL-LSD1 LC-MS Assay: Representative compounds of the present invention were serially and separately diluted 3-fold in DMSO to obtain a total of twelve concentrations. Then the test compounds at each concentration (100 nL of each) were transferred into a 384-well Perkin Elmer ProxiPlate 384 plus plates by Mosquito Solutions (5 μL) of 0.8 nM, the full-length LSD1 and 0.5 μM FAD in reaction buffer (40 mM Tris-HCl, 0.01% Triton-×100.10 mM KCl, 1 mM DTT) were added into the wells and then incubated with the test compound for 30 min. A 5 μL solution of 1 μM of the peptide substrate H3K4me1 (histone H3[1-21]-biotin) in reaction buffer was added to each initiate reaction. The final components in the reaction solution include 0.4 nM FL-LSD1, 0.25 μM FAD, and 0.5 μM H3K4me1 peptide with varying concentration of the compounds. A positive control consisted of the enzyme, 0.25 μM FAD and 0.5 μM substrate in the absence of the test compound, and a negative control consisted of 0.5 μM substrate only. Each reaction was incubated at room temperature for 60 min, and then stopped by the addition of 3 μL quench solution (2.5% TFA with 320 nM d4-SAH). The reaction mixture was centrifuged (Eppendorf centrifuge 5810, Rotor A-4-62) for 1 min at 2000 rpm and read on an API 4000 triple quadrupole mass spec with Turbulon Spray (Applied Biosystem) coupled with Prominence UFLC (Shimadzu).
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 352.83, ALogP 3.81, HBA 5, HBD 2, PSA 87.86, QED 0.73
Compound (SMILES): O=C(C[C@@H]1CCc2cc(C(O)(C(F)(F)F)C(F)(F)F)ccc2N1S(=O)(=O)c1ccc(F)cc1)N1CCCC1c1cccnc1
InChIKey: ZGJNQNWNNBOBMW-OWJIYDKWSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Spa Binding Assay: The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromotography. 50 nM RORγ (A262-S507) is incubated with test compound at varying concentrations for 15 min at room temperature in PBS buffer containing 0.5% fatty acid free BSA. 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 15 min. 4 mg/mL of Ysi Copper HIS-TAG SPA Beads (Perkin Elmer) are added, and the mixture is incubated for 30 min. The reaction is read on a MicroBeta Trilux scintillation plate reader (Perkin Elmer).
Measurement: IC50 = 47.4 nM (pChEMBL 7.320000171661377)
Descriptors: MW 645.6, ALogP 5.8, HBA 5, HBD 1, PSA 90.81, QED 0.34
Compound (SMILES): Cc1cc(N)nc(C)c1CNC(=O)c1nc(Cc2ccc3ncc(Cl)cc3c2)c2ccccn12
InChIKey: PMJMPOLQHRXHNS-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Enzyme Inhibition: A 10 mM solution of the test compound was made in DMSO. This solution was serially diluted 1:5 in DMSO to yield 2000, 400, 80, 16, 3.2, 0.64, 0.128, 0.0256 and 0.00512 μM compound test solutions. A control tube containing only DMSO is included. 16 μL of each compound test solution was combined with 384 μL of assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.01% Triton X-100) to yield a 4× test compound buffer stock.Separately, a 40 nM solution of human Plasma Kallikrein (Abcam) and a 93.6 μM solution Pro-Phe-Arg-AMC (Bachem) were made using assay buffer. These solutions are hereby referred to as 4×hPK and 2×PFR-AMC, respectively.60 μL of each 4× test compound buffer stock was combined with 60 μL of 4×hPK to yield 120 μL of 2× test compound buffer stock/2×hPK. 50 μL was removed from this mixture and placed into duplicate wells on a Microfluor 1Black U-bottom microtiter plate (Thermo Scientific). This plate was incubated for 5 minutes at 37° C. To each well, 50 μL of pre-warmed 2×PFR-AMC was added to start the enzymatic reaction. Cleavage of PFR-AMC was monitored in a Biotek Synergy H4 reader set at 37° C. Readings are taken every 43 seconds for 1 hour. The highest mean velocity over 20 reads (15 minutes) is used to calculate the IC50. The IC50 is calculated using the Gen5 (Biotek Instruments).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 470.96, ALogP 4.65, HBA 6, HBD 2, PSA 98.2, QED 0.39
Compound (SMILES): CNC(=O)c1cnc(Nc2nc3ccc(Cl)nc3s2)cc1NC(C)C
InChIKey: LGOMLXCQJHQUIC-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: 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% Brij35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction 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 = 18 nM (pChEMBL 7.75)
Descriptors: MW 376.87, ALogP 3.66, HBA 7, HBD 3, PSA 91.83, QED 0.59
Compound (SMILES): C[C@H](c1cccc2c1CCC2)[C@H](NS(=O)(=O)c1ccc(F)c(C(C)(C)O)c1F)c1n[nH]c(=O)o1
InChIKey: QDMNOCJCRKNVJN-ODXCJYRJSA-N
Target: Ribonucleotide reductase (PROTEIN COMPLEX), Homo sapiens
UniProt: P23921
Assay: Human RNR Inhibition Effect: The inhibitory activity against the ribonucleotide reduction reaction (hereinafter referred to as RNR reaction) of the test compound was determined by measuring the formation of deoxycytidine diphosphate (hereinafter referred to as dCDP) from cytidine diphosphate (hereinafter referred to as CDP) by the following method.Human M1 subunit and human M2 subunit (mutant lacking amino terminal 59 amino acids), which are fused a histidine tag at the amino terminus, are overexpressed in Escherichia coli and are solubilized after collection, and histidine tagged human M1 and M2 proteins were purified on a nickel chelate column.For measuring the inhibitory activity of the test compound against the RNR reaction, the method described in the document [CANCER RESEARCH 64, 1-6, 2004] was referred to.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 493.53, ALogP 3.18, HBA 6, HBD 3, PSA 125.29, QED 0.46
Compound (SMILES): COc1cc(-c2cc(F)cc(F)c2)ccc1-c1nccc2cc(S(=O)(=O)Nc3cnsn3)ccc12
InChIKey: HXZNGNARIZVEDE-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: In Vitro PX Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to â¿¿10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes.
Measurement: IC50 = 428 nM (pChEMBL 6.369999885559082)
Descriptors: MW 510.55, ALogP 5.51, HBA 7, HBD 1, PSA 94.07, QED 0.32
Compound (SMILES): COc1cc(-n2cnc(Nc3nc(-c4cccc(CO)c4)nc4sc(CO)cc34)c2)cc(OC)c1OC
InChIKey: YXTPTQDXIUGTOE-UHFFFAOYSA-N
Target: Activin receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q04771
Assay: Biochemical assay: Biochemical assays to measure the inhibitory effects of the compounds were performed by ThermoFisher Scientific (Life Technologies). ALK2 inhibition was tested using LanthaScreen Eu Kinase Binding Assay screening protocol.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 519.58, ALogP 4.3, HBA 11, HBD 3, PSA 123.78, QED 0.26
Compound (SMILES): C=CC(=O)N1CCC[C@H](Nc2nc(Nc3ccc(OC)cc3)ncc2F)C1
InChIKey: BSGQCGPLYCUUAR-AWEZNQCLSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Omnia Assay: Briefly, 10x stocks of EGFR-WT (PV3872) from Invitrogen and EGFR-T790M/L858R (40350) from BPS Bioscience, San Diego, Calif., 1.13×ATP (AS001A) and appropriate Tyr-Sox conjugated peptide substrates (KCZ1001) were prepared in 1× kinase reaction buffer consisting of 20 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 5 mM β-glycerophosphate, 5% glycerol (10× stock, KB002A) and 0.2 mM DTT (DS001A). 5 μL of each enzyme were pre-incubated in a Corning (#3574) 384-well, white, non-binding surface microtiter plate (Corning, N.Y.) for 30 min. at 27° C. with a 0.5 μL volume of 50% DMSO and serially diluted compounds prepared in 50% DMSO. Kinase reactions were started with the addition of 45 μL of the ATP/Tyr-Sox peptide substrate mix and monitored every 30-90 seconds for 60 minutes at λex360/λem485 in a Synergy4 plate reader from BioTek (Winooski, Vt.). At the conclusion of each assay, progress curves from each well were examined for linear reaction kinetics and fit statistics (R2, 95% confidence interval, absolute sum of squares). Initial velocity (0 minutes to 30 minutes) from each reaction was determined from the slope of a plot of relative fluorescence units vs time (minutes) and then plotted against inhibitor concentration to estimate IC50 from log[Inhibitor] vs Response, Variable Slope model in GraphPad Prism from GraphPad Software (San Diego, Calif.).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 371.42, ALogP 2.96, HBA 6, HBD 2, PSA 79.38, QED 0.76
Compound (SMILES): N#Cc1c(N)nn2ccc(N3C[C@@H](F)C[C@@H]3c3cc(F)ccc3F)nc12
InChIKey: ZXYSMZITRHMVCJ-IINYFYTJSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Inhibitory Activity Assay: TrkA, TrkB and TrkC:A testing platform for TrkA, TrkB and TrkC kinase activity was established based on Homogeneous Time-Resolved Fluorescence (HTRF) assay, and the activities of the compounds were tested using the platform. The compounds were subjected to three-fold gradient dilution with 100% DMSO with a starting concentration of 1 mM (11 concentrations in total). 4 μL of diluted sample for each concentration was added to 96 μL of reaction buffer (50 mM HEPES, pH7.4, 5 mM MgCl2, 1 mM NaVO3, 0.001% Tween-20, 0.01% BSA and 1 mM DTT) and mixed homogeneously to be used as a 4* compound. The reaction buffer was used to formulate 2* TrkA, TrkB and TrkC kinases (purchased from Carna Biosciences 08-186, 08-187, 08-197, and the final concentrations thereof were 0.5 nM, 0.1 nM, and 1 nM, respectively) and 4* substrate mixture (ATP+TK peptide) (wherein the final concentrations of ATP were 40 μM, 50 μM, and 20 μM, respectively; TK peptide, HTRF® KinEASE-TK, was purchased from Cisbio, and the final concentration thereof was 100 nM) for use. 2.5 μL of the 4* compound was added to a 384-well plate (OptiPlate-384, purchased from PerkinElmer), and then 5 μL of the 2* TrkA, TrkB and TrkC kinases were added, and mixed homogeneously by centrifugation. Then 2.5 μL of the 4* substrate mixture was added to initiate the reaction (the total reaction volume is 10 μL). The 384-well plate was placed in an incubator and incubated for 60 min at 23° C. Then the reaction was terminated by adding 5 μL of Eu3+ cryptate-labeled anti-phosphotyrosine antibody (HTRF® KinEASE-TK, purchased from Cisbio), and 5 μL of Streptavidin-XL-665 (HTRF® KinEASE-TK, purchased from Cisbio). After incubated for 1 hr in the incubator, the fluorescence values were read on Envision (purchased from PerkinElmer).
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 358.33, ALogP 2.75, HBA 6, HBD 1, PSA 83.24, QED 0.76
Compound (SMILES): COc1ccccc1-c1cc(C(=O)/N=c2\[nH]c3ccccc3n2[C@H]2CC[C@@H](O)CC2)ccn1
InChIKey: AMRMZQYHUGBUGI-KDURUIRLSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Biochemical EGFR Inhibition Assays (5 mM ATP): 5 μL of EGFR enzyme form in assay buffer (50 mM HEPES pH 7.3, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween 20, 2 mM DTT) are dispensed into columns 1-23, than 5 μL of ATP and ULight-poly-GT substrate (PerkinElmer; TRF0100-M) mix in assay buffer is added to all wells (final assay concentration of the ULight-poly-GT substrate 200 nM). Each of the different EGFR enzyme form assays is available at low ATP (final assay concentration 5 μM). After 90 minutes incubation at room temperature 5 μL EDTA (final assay concentration 50 mM) and LANCE Eu-anti-P-Tyr (PT66) antibody (PerkinElmer, AD0069) (final assay concentration 6 nM) mix are added to stop the reaction and start the binding of the antibody. After additional 60 minutes incubation at room temperature the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the TR-FRET LANCE Ultra specs of PerkinElmer (used wavelengths: excitation 320 nm, emission 1 665 nm, emission 2 615 nm). Each plate contains 16 wells of a negative control (diluted DMSO instead of test compound; w EGFR enzyme form; column 23) and 16 wells of a positive control (diluted DMSO instead of test compound; w/o EGFR enzyme form; column 24). Negative and positive control values are used for normalization and IC50 values are calculated and analysed using a 4 parametric logistic model.
Measurement: IC50 = 0.6 nM (pChEMBL 9.220000267028809)
Descriptors: MW 442.52, ALogP 4.26, HBA 5, HBD 2, PSA 92.5, QED 0.49
Compound (SMILES): COc1cccc(C2CCCC(NC(=O)c3ccc(Oc4cc5c(cc4Cl)C(C(=O)O)CCO5)cc3)C2)c1
InChIKey: ZRCAKGXNNCTYQI-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Binding Inhibition Assay (4% HSA): The coding sequence of human DP2 was introduced into the human Leukemic cell line K562 by electroporation and stable clones expressing DP2 were obtained by limiting dilution followed by cell surface staining with a rat monoclonal antibody specific for human DP2. Membranes were prepared from one of these DP2 expressing clones and used to determine the ability of compounds of the present invention to inhibit binding of prostaglandin D2 (PGD2) to its receptor DP2 in the presence of one or more of the following serum protein concentrations, 4% HSA, by the following procedure. Membranes (1.25 μg/well for 4% HSA) were mixed with 3H-labeled PGD2 and various concentrations of test compounds in 150 μL of binding buffer (50 mM Tris-HCl, pH 7.4, 40 mM MgCl2, 0.1% bovine serum albumin, 0.1% NaN3) in 96-well U-bottom polypropylene plates. After incubation for 60 minutes at room temperature, the assay was transferred to a filtration plate (#MAFB; Millipore Corporation, Bedford, Mass.), and washed three times with binding buffer. Radioactivity was measured by a scintillation counter (TopCount; PerkinElmer Life Sciences, Boston, Mass.). Nonspecific binding was determined by incubations in the presence of 1 μM unlabeled PGD2 or 5 μM of a known DP2 antagonist. EC50 values for inhibition of binding were determined for each compound tested from the inflexion point of a standard 4-parameter logistical curve fitted to the values obtained. Compounds of the invention had EC50 values less than 5 micromolar in one or more of the binding assays.
Measurement: EC50 = 509 nM (pChEMBL 6.289999961853027)
Descriptors: MW 536.02, ALogP 6.55, HBA 5, HBD 2, PSA 94.09, QED 0.36
Compound (SMILES): COCCN1CCC(n2cc(-c3cnc(N)c(-c4cc(C)c(=O)n(-c5c(Cl)c(OC)cc(OC)c5Cl)n4)n3)cn2)CC1
InChIKey: ULZYEHHPVPISAH-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Enzyme Assay: FGFR1, 2 and 3 kinase activity was measured by the Invitrogen LanthaScreen Assay technology which directly measures the amount of substrate phosphorylation by TR-FRET using a fluorescein-labeled peptide and Europium-labeled antibody.To measure FGFR1 kinase activity, 200 pM His-tagged recombinant human FGFR1 catalytic domain (amino acids 308-731) (Life Technologies Cat. No. PR4660A) was incubated with 100 nM Alexa Fluor® 647-Poly-GT Peptide Substrate (Life Technologies Cat. No. PV5836) and ATP in the presence of Mg++, along with test compound in a buffer consisting of 250 mM HEPES, 25 mM MgCl2, 0.05% TritonX-100, pH 7.5, 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 20 minutes incubation at 22° C., an equal volume of 2 nM LanthaScreen® Eu-PY20 Antibody (Life Technologies Cat. No. PV5691) and EDTA were added to quench the kinase reaction and start the detection reaction. After an additional 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) calculated using a ratiometric emission factor. 100 POC is determined using no test compound and 0 POC is determined using no enzyme. The POC values were fit to a 4 parameter logistic curve and the IC50 value is point where the curve crosses 50 POC.To measure FGFR2 kinase activity: 200 pM His-tagged recombinant human FGFR2 cytoplasmic domain (amino acids 403-822), (Life Technologies Cat. No. PR5332A); 20 minutes incubation at 22° C., 60 minute detection incubation at 22° C.To measure FGFR3 kinase activity: 750 pM N-terminal GST-HIS6 fusion protein with a 3C cleavage site recombinant human FGFR3 (amino acids R397-T806) (ProQinase Cat. No. 1068-0000-1); 10 minutes incubation at 22° C., 60 minute detection incubation at 22° C.
Measurement: IC50 = 5.6 nM (pChEMBL 8.25)
Descriptors: MW 615.52, ALogP 4.05, HBA 12, HBD 1, PSA 135.44, QED 0.29
Compound (SMILES): COc1ncc(CNC(=O)N2CCN(c3ncnc4[nH]c(-c5ccc(F)cc5)cc34)CC2)cc1F
InChIKey: CVAKFWMBBBJPCA-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Cell-Based Assays of c-Kit Mutant Kinase Activity: The c-Kit mutant D816V inhibitors were assessed using an engineered BaF3-FL KIT D816V or BaF3-FL KIT V560G/D816V cell line. The BaF3-FL KIT D816V cell lines were created by introduction of KIT mutant (D816V) full length constructs that render the cells dependent on the introduced kinase for growth. Inhibitors of c-Kit mutant D816V kinase reduce or eliminate the mediated c-kit mutant D816V kinase activation, resulting in reduced cell proliferation of the BaF3-FL Kit mutant D816V cells. This inhibition is measured by the effect of compound concentration on cell growth to assess IC50 values. BaF3-FL KIT D816V cells were seeded at 1×104 cells per well of a 96 well cell culture plate in 50 μl of cell culture medium of RPMI Medium 1× (Invitrogen #11875-093) supplemented with 10% FBS (Invitrogen #10438), 1% Non Essential Amino Acids (Invitrogen #11140), 1% Penicillin Streptomycin (Invitrogen #15140), 1% L-Glutamine (Invitrogen #25030-081). Compounds were dissolved in DMSO at a concentration of 5 mM and were serially diluted 1:3 for a total of eight points and added to the cells to a final maximum concentration of 10 μM in 100 μl cell culture medium (final concentration 0.2% DMSO). Cells were also treated with Dasatinib as a positive control. The cells were incubated at 37° C., 5% CO2 for three days. ATPlite Buffer (Perkin Elmer #6016739) and substrate were equilibrated to room temperature, and enzyme/substrate Recombinant Firefly Luciferase/D-Luciferin was reconstituted. The cell plates were equilibrated to room temperature for 30 minutes, then lysed by addition of 25 uL per well of the ATPlite Reagent. The plate was mixed for 5 minutes on a plate shaker to lyse the cells. The plates were read on a Tecan Safire using Luminescence protocol modified to read 0.1s per well. The luminescence reading assesses the ATP content, which correlates directly with cell number such that the reading as a function of compound concentration is used to determine the IC50 value.
Measurement: Ki = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 479.49, ALogP 3.34, HBA 6, HBD 2, PSA 99.27, QED 0.46
Compound (SMILES): C=CC(=O)Nc1cccc(-n2c(NC(=O)c3ccc(-c4cnco4)s3)nc3cc(CN[C@@H](C)C(C)(C)C)ccc32)c1
InChIKey: NURMJDRYQZYZDK-IBGZPJMESA-N
Target: Tyrosine-protein kinase ITK/TSK (SINGLE PROTEIN), Homo sapiens
UniProt: Q08881
Assay: Kinase Inhibition Assay: ITK: The in vitro kinase assays were performed at Nanosyn utilizing micro-fluidic detection technology. The test compounds were serially pre-diluted in DMSO and added, by the acoustic dispensing (Labcyte® 550), directly to 384 well assay plates into 10 uL of a buffer with enzyme comprising: 100 mM HEPES, pH7.5, 5 mM MgCl2, 0.1% bovine serum albumin, 1 mM DTT, 0.01% Triton X-100 and the enzyme. Final DMSO concentration was maintained at 1% in all samples, including the controls. The reactions were initiated by addition of ATP (to the specified concentration) and the fluorescently labeled peptide substrate to a final concentration of 1 uM, and incubated for 3 hours at 25° C. Following incubation, the reactions were quenched by addition of 40 μL of termination buffer (100 mM HEPES, pH7.5, 0.01% Triton X-100, 50 mM EDTA). Terminated plates were analyzed using Caliper LabChip® 3000 microfluidic electrophoresis instrument (Caliper Life Sciences/Perkin Elmer). The enzymatic modification of the peptide substrate (phosphorylation) results in a change of net charge enabling electrophoretic separation of product from substrate. As substrate and product are separated by electrophoresis, two peaks of fluorescence are observed. Change in the relative fluorescence intensity of the substrate and product peaks was the parameter measured, reflecting enzyme activity. In the presence of inhibitor, the ratio between product and substrate is altered: signal of the product decreases, while the signal of the substrate increases. Activity in each test sample was determined as the product to sum ratio (PSR):P/(S+P), where P is the peak height of the product and S is the peak height of the FAM-cAMP substrate. For each compound, enzyme activity was measured at 12 concentrations spaced by 3× dilution intervals.
Measurement: EC50 < 10 nM (pChEMBL 8)
Descriptors: MW 568.7, ALogP 6.64, HBA 8, HBD 3, PSA 114.08, QED 0.17
Compound (SMILES): CC#C[C@@H](CC(=O)O)c1ccc(OCc2cccc(N3CCN(c4ccc(OC)cc4)CC3)c2)cc1
InChIKey: NBXAESOOXILHLN-VWLOTQADSA-N
Target: Free fatty acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14842
Assay: Activity Assay: The GPR40 protein activity according to the novel 3-(4-(benzyloxy)phenyl)hex-4-inoic acid derivative of the present invention was measured by investigating the changes in intracellular calcium concentration affected by the GRP40 activation. First, HEK-293 cells were transfected with human GPR40 DNA (Origene, RC218370) by using Fugene HD (Promega, E2311). The transfected HEK-293 cells were distributed in a 96-well black clear bottom floor plate (Costar, 3603), followed by culture. 24 hours later, the cell culture medium was replaced with Dulbecco's Modified Eagle Medium (DMEM, 50 μl/well) supplemented with 1% fetal bovine serum (FBS). To measure the calcium concentration, 50 μl of Fluo-4 reagent (Invitrogen, F10471) was added to each well, followed by culture in a 37° C. incubator for 2 hours. During the culture, the compounds of Examples and the compounds of Comparative Examples 1 and 2 were diluted with 1×HBSS (Hank's Buffered Salt Solution) containing 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, resulting in the preparation of the samples for treating the cells. 2 hours after the culture began, those prepared samples were automatically injected to the cells by using Flexstation 3 (Molecular Devices). Then, intracellular calcium concentration was measured for 120 seconds by using SoftMax® Pro software. At this time, dimethylsulfoxide (DMSO) was injected to the cells for the non-treated group, followed by measuring the calcium concentration therein.
Measurement: EC50 = 250 nM (pChEMBL 6.599999904632568)
Descriptors: MW 484.6, ALogP 5.18, HBA 5, HBD 1, PSA 62.24, QED 0.42
Compound (SMILES): C=CC(=O)N1CC2(CCNc3c(C(N)=O)c(-c4ccc(Oc5ccccc5)cc4)nn32)C1
InChIKey: WCCNQMBCIGEKBN-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Assay: Compounds disclosed herein were tested for inhibition of Btk kinase activity in an assay based on time-resolved fluorescence resonance energy transfer methodology. Recombinant Btk was pre-incubated with the compounds disclosed herein at room temperature for 1 hour in an assay buffer containing 50 mM Tris pH7.4, 10 mM MgCl2, 2 mM MnCl2, 0.1 mM EDTA, 1 mM DTT, 20 nM SEB, 0.1% BSA, 0.005% tween-20. The reactions were initiated by the addition of ATP (at the concentration of ATP Km) and peptide substrate (Biotin-AVLESEEELYSSARQ-NH2). After incubating at room temperature for 1 h, an equal volume of stop solution containing 50 mM HEPES pH7.0, 800 mM KF, 20 mM EDTA, 0.1% BSA, Eu cryptate-conjugated p-Tyr66 antibody and streptavidin-labeled XL665 was added to stop the reaction. Plates were further incubated at room temperature for 1 hour, and then the TR-FRET signals (ex337 nm, em 620 nm/665 nm) were read on BMG PHERAstar FS instrument. The residual enzyme activity in presence of increasing concentrations of compounds was calculated based on the ratio of fluorescence at 615 nm to that at 665 nm.
Measurement: IC50 = 40 nM (pChEMBL 7.400000095367432)
Descriptors: MW 429.48, ALogP 2.98, HBA 6, HBD 2, PSA 102.48, QED 0.61
Compound (SMILES): CS(=O)(=O)Nc1ccccc1-c1ccc(N2CCC[C@@H](NC(=O)Nc3ccc(OC(F)F)nc3)C2=O)cc1
InChIKey: HVMILXXMZDTLGK-OAQYLSRUSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: FPR2 and FPR1 Cyclic Adenosine Monophosphate (cAMP) Assays: A mixture of forskolin (5 μM final for FPR2 or 10 μM final for FPR1) and IBMX (200 μM final) were added to 384-well Proxiplates (Perkin-Elmer) pre-dotted with test compounds in DMSO (1% final) at final concentrations in the range of 0.020 nM to 100 μM. Chinese Hamster Ovary cells (CHO) overexpressing human FPR1 or human FPR2 receptors were cultured in F-12 (Ham's) medium supplemented with 10% qualified FBS, 250 μg/ml zeocin and 300 μg/ml hygromycin (Life Technologies). Reactions were initiated by adding 2,000 human FPR2 cells per well or 4,000 human FPR1 cells per well in Dulbecco's PBS (with calcium and magnesium) (Life Technologies) supplemented with 0.1% BSA (Perkin-Elmer). The reaction mixtures were incubated for 30 min at room temperature. The level of intracellular cAMP was determined using the HTRF HiRange cAMP assay reagent kit (Cisbio) according to manufacturer's instruction. Solutions of cryptate conjugated anti-cAMP and d2 flurorophore-labelled cAMP were made in a supplied lysis buffer separately. Upon completion of the reaction, the cells were lysed with equal volume of the d2-cAMP solution and anti-cAMP solution. After a 1-h room temperature incubation, time-resolved fluorescence intensity was measured using the Envision (Perkin-Elmer) at 400 nm excitation and dual emission at 590 nm and 665 nm. A calibration curve was constructed with an external cAMP standard at concentrations ranging from 1 μM to 0.1 pM by plotting the fluorescent intensity ratio from 665 nm emission to the intensity from the 590 nm emission against cAMP concentrations. The potency and activity of a compound to inhibit cAMP production was then determined by fitting to a 4-parametric logistic equation from a plot of cAMP level versus compound concentrations.
Measurement: EC50 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 545.57, ALogP 4.04, HBA 6, HBD 3, PSA 129.73, QED 0.39