lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1ccc([C@@H](C)NC(=O)c2cc(OCC3CCOCC3)cc(-c3ncc(C)s3)c2)nn1
InChIKey: FNFCPAXHCILJLE-QGZVFWFLSA-N
Target: P2X2/P2X3 heterotrimeric receptor (PROTEIN COMPLEX), Homo sapiens
UniProt: P56373
Assay: Biological Assay: A fluorescent imaging plate reader (FILEX/FLIPR station; Molecular Devices) was used to monitor intracellular calcium levels using the calcium-chelating dye Rim-4 (Molecular Probes). The excitation and emission wavelengths used to monitor fluorescence were 470-495 nm and 515-575 nm, respectively. Cells expressing purinergic receptors P2X3 (human) or P2X2/3 (human) were plated at a density of 15,000 cells/well in collagen-coated 384-well plates approximately 20 hours before beginning the assay. On the day of the assay, 20 μl of loading buffer (Hank's balanced salt solution, 20 mM HEPES, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, 5 mM probenecid, 10 mM D-glucose monohydrate, 2 μM Fluo-4, and 5 units/mL, hexokinase, pH=7.4) was added and cells dye-loaded for 90 min at 37° C. The dye supernatant was removed and replaced with 45 μl probenecid buffer (Hank's balanced salt solution, 20 mM HEPES, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, 5 mM probenecid, 10 mM D-glucose monohydrate, pH=7.4). The test compound was added in a volume of 5 μl and allowed to incubate for 30 min at 37° C.
Measurement: IC50 = 27 nM (pChEMBL 7.570000171661377)
Descriptors: MW 452.58, ALogP 4.51, HBA 7, HBD 1, PSA 86.23, QED 0.57
Compound (SMILES): Cc1cnc(-c2cc(OC[C@H]3CN(C)C(=O)O3)cc(C(=O)N[C@H](C)c3cnc(C(F)(F)F)nc3)c2)s1
InChIKey: OAEQMLBAMDEFGR-FZKQIMNGSA-N
Target: P2X2/P2X3 heterotrimeric receptor (PROTEIN COMPLEX), Homo sapiens
UniProt: P56373
Assay: Biological Assay: A fluorescent imaging plate reader (FILEX/FLIPR station; Molecular Devices) was used to monitor intracellular calcium levels using the calcium-chelating dye Rim-4 (Molecular Probes). The excitation and emission wavelengths used to monitor fluorescence were 470-495 nm and 515-575 nm, respectively. Cells expressing purinergic receptors P2X3 (human) or P2X2/3 (human) were plated at a density of 15,000 cells/well in collagen-coated 384-well plates approximately 20 hours before beginning the assay. On the day of the assay, 20 μl of loading buffer (Hank's balanced salt solution, 20 mM HEPES, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, 5 mM probenecid, 10 mM D-glucose monohydrate, 2 μM Fluo-4, and 5 units/mL, hexokinase, pH=7.4) was added and cells dye-loaded for 90 min at 37° C. The dye supernatant was removed and replaced with 45 μl probenecid buffer (Hank's balanced salt solution, 20 mM HEPES, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, 5 mM probenecid, 10 mM D-glucose monohydrate, pH=7.4). The test compound was added in a volume of 5 μl and allowed to incubate for 30 min at 37° C.
Measurement: IC50 = 1795 nM (pChEMBL 5.75)
Descriptors: MW 521.52, ALogP 4.25, HBA 8, HBD 1, PSA 106.54, QED 0.5
Compound (SMILES): CCc1cnc(-c2cc(OC[C@H]3CCOC3)cc(C(=O)N[C@H](C)c3cnc(C)cn3)c2)s1
InChIKey: PZFOCXJBZDWRSU-SJORKVTESA-N
Target: P2X2/P2X3 heterotrimeric receptor (PROTEIN COMPLEX), Homo sapiens
UniProt: P56373
Assay: Biological Assay: A fluorescent imaging plate reader (FILEX/FLIPR station; Molecular Devices) was used to monitor intracellular calcium levels using the calcium-chelating dye Rim-4 (Molecular Probes). The excitation and emission wavelengths used to monitor fluorescence were 470-495 nm and 515-575 nm, respectively. Cells expressing purinergic receptors P2X3 (human) or P2X2/3 (human) were plated at a density of 15,000 cells/well in collagen-coated 384-well plates approximately 20 hours before beginning the assay. On the day of the assay, 20 μl of loading buffer (Hank's balanced salt solution, 20 mM HEPES, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, 5 mM probenecid, 10 mM D-glucose monohydrate, 2 μM Fluo-4, and 5 units/mL, hexokinase, pH=7.4) was added and cells dye-loaded for 90 min at 37° C. The dye supernatant was removed and replaced with 45 μl probenecid buffer (Hank's balanced salt solution, 20 mM HEPES, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, 5 mM probenecid, 10 mM D-glucose monohydrate, pH=7.4). The test compound was added in a volume of 5 μl and allowed to incubate for 30 min at 37° C.
Measurement: IC50 = 392 nM (pChEMBL 6.409999847412109)
Descriptors: MW 452.58, ALogP 4.38, HBA 7, HBD 1, PSA 86.23, QED 0.55
Compound (SMILES): CCc1cnc(-c2cc(OC[C@H]3CCOC3)cc(C(=O)N[C@H](C)c3cnc(C)cn3)c2)s1
InChIKey: PZFOCXJBZDWRSU-SJORKVTESA-N
Target: P2X2/P2X3 heterotrimeric receptor (PROTEIN COMPLEX), Homo sapiens
UniProt: P56373
Assay: Biological Assay: A fluorescent imaging plate reader (FILEX/FLIPR station; Molecular Devices) was used to monitor intracellular calcium levels using the calcium-chelating dye Rim-4 (Molecular Probes). The excitation and emission wavelengths used to monitor fluorescence were 470-495 nm and 515-575 nm, respectively. Cells expressing purinergic receptors P2X3 (human) or P2X2/3 (human) were plated at a density of 15,000 cells/well in collagen-coated 384-well plates approximately 20 hours before beginning the assay. On the day of the assay, 20 μl of loading buffer (Hank's balanced salt solution, 20 mM HEPES, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, 5 mM probenecid, 10 mM D-glucose monohydrate, 2 μM Fluo-4, and 5 units/mL, hexokinase, pH=7.4) was added and cells dye-loaded for 90 min at 37° C. The dye supernatant was removed and replaced with 45 μl probenecid buffer (Hank's balanced salt solution, 20 mM HEPES, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, 5 mM probenecid, 10 mM D-glucose monohydrate, pH=7.4). The test compound was added in a volume of 5 μl and allowed to incubate for 30 min at 37° C.
Measurement: IC50 = 96 nM (pChEMBL 7.019999980926514)
Descriptors: MW 452.58, ALogP 4.38, HBA 7, HBD 1, PSA 86.23, QED 0.55
Compound (SMILES): CC(C)O[C@H]1CC[C@H](C(=O)N2Cc3cccnc3Nc3ccc(C4COCCO4)cc32)CC1
InChIKey: VEIJZLITLDVJAC-XEMOTPFLSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Enzymatic Assay: Each test compound (10 mM stock in DMSO) is diluted in DMSO to make a 10-point, 3-fold dilution series. 125 nL of each dilution or DMSO alone is dispensed to a 384-well Greiner Lumitrac 200 assay plate using an Echo® Liquid Handler. To each well of the plate is added 20 uL of enzyme in assay buffer or assay buffer alone. Assay buffer consists of 50 mM sodium phosphate, pH 7.0, 50 mM magnesium chloride, 50 mM sodium chloride, and 0.01% (w/v) bovine serum albumin. When present, the R132H mutant IDH1 enzyme is at a working concentration of 1.875 nM (final concentration in assay of 1.5 nM). The assay plate is allowed to incubate for 30 minutes at room temperature and 5 uL of 5× substrate mixture (2.5 uM nicotinamide adenine dinucleotide phosphate, 100 uM adenosine diphosphate, 7.5 mM glyceraldehyde-3-phosphate, 7.5 ug/mL of spinach glyceraldehyde-3-phosphate dehydrogenase, 25 nM phosphoglycerate kinase, and 5 mM alpha-ketoglutarate in assay buffer) is added to all wells. The reaction plate is incubated for 60 minutes followed by addition of 25 uL of Promega Kinase-GLO reagent to all wells and 10-minute incubation.Luminescence is measured using a PerkinElmer Envision plate reader. The percent activity of each dilution is determined as the ratio of background corrected signal to the background corrected signal of wells receiving only DMSO. IC50 values are determined by fitting percent activity data to a four-parameter logistic dose response equation.
Measurement: IC50 = 39 nM (pChEMBL 7.409999847412109)
Descriptors: MW 451.57, ALogP 4.74, HBA 6, HBD 1, PSA 72.92, QED 0.72
Compound (SMILES): Cn1cnc(-c2cc(Cl)c(F)c(C(=O)NNS(=O)(=O)CC3CCN(C(=O)NCCO)C3)c2)c1
InChIKey: KUJOYUJMAPULDO-UHFFFAOYSA-N
Target: Histone acetyltransferase KAT6A (SINGLE PROTEIN), Homo sapiens
UniProt: Q92794
Assay: KAT-6A Enzyme Assay: KAT-6A. Enzyme assay buffer was 50 mM Tris pH 8.0, 0.002% Tween20, 0.005% bovine skin gelatin, and 1 mM dithiothreitol (DTT). For determination of IC50values, compounds were serially diluted with 2% (v/v) DMSO in the final reaction, pre-incubating each dilution of each compound with 40 μL of assay buffer containing KAT-6A enzyme (12.5 nM final concentration). 10 μL of assay buffer containing 1 μM peptide substrate and 1 μM acetyl coenzyme A (final concentrations) was added. Reactions (50 μL total) were then carried out at 25° C. for 90 minutes. Reactions were terminated by the addition of 0.5% formic acid (final concentration), and a sample of each reaction was analyzed by SAMDI Tech, Inc. (Chicago, Ill.) using self-assembled monolayer desorption/ionization time-of-flight mass spectrometry (Mrksich, M. (2008) Mass spectrometry of self-assembled monolayers: a new tool for molecular surface science. ACS Nano 2, 7-18).
Measurement: IC50 = 30000 nM (pChEMBL 4.519999980926514)
Descriptors: MW 502.96, ALogP 0.47, HBA 7, HBD 4, PSA 145.66, QED 0.39
Compound (SMILES): Cc1cc(OCCCc2c3n(c4c(-c5c(C)nn(C)c5C)c(Cl)ccc24)CCCN(c2cc(C(=O)O)c4c(Cl)cn(C)c4c2)C3=O)cc(C)c1Cl
InChIKey: VQAIMLFHIPYVBD-UHFFFAOYSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07820
Assay: Fluorescence Polarization Anisotropy Competition Assay: Fluorescence Polarization Anisotropy Competition Assay. Anisotropy measurements were carried out in 384-well, black, flat-bottom plates (Greiner Bio-one, Monroe, N.C., USA). The assay was run using either a fluorescein isothiocyanate-labeled BH3 peptide derived from Bak (FITC-AHx-GQVGRQLAIIGDDINR-NH2) or a fluorescein isothiocyanate-labeled BH3 peptide derived from Bim (FITC-AHx-EARIAQELRRIGDEFNETYTR-NH2)that were purchased from GenScript (Piscataway, N.J.) at >95% purity and used without further purification. 10 nM FITC-Bak peptide and 15 nM recombinant Mcl-1 (residues 172-327) were added to assay buffer (3 mM dithiothreitol, 50 mM NaCl, 20 mM Tris, pH 7.5). The Bim based assay was run with 1 nM FITC-Bim peptide and 1.5 nM recombinant Mcl-1 (residues 172-327) added to assay buffer (20 mM TRIS pH 7.5, 50 mM NaCl, 3 mM DTT, 0.01% CHAPS). For selectivity assays, 40 nM Bcl-2 (residues 1-207 A96T,G110R, Δ35-91, replaced with Bcl-xL35-50) or 4 nM Bcl-xL (residues 1-209, loop 45-86 deleted) were incubated with 10 nM FITC-Bak in assay buffer. Compounds are diluted in DMSO in a 10-point, 3-fold serial dilution scheme. For the FITC-BAK assay 2.5 uL compound is added to 47.5 μL of assay buffer containing FITC-Bak and protein, for a final DMSO concentration of 5% and a top concentration of 20 μM. A FITC-Bak peptide alone (100% inhibition) and peptide plus protein (0% inhibition) control is included on each assay plate. For the FITC-Bim assay, compound is added to 40 uL of assay buffer containing protein, 15 minutes prior to addition of 10 μL of the FITC-Bim peptide, for a final DMSO concentration of 0.165% and a top concentration of 200 nM. A FITC-Bim peptide alone (100% inhibition) and peptide plus protein (0% inhibition) control is included on each assay plate. The plate was mixed and incubated for 90 minutes at room temperature.
Measurement: Ki = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 747.12, ALogP 9.49, HBA 7, HBD 1, PSA 94.52, QED 0.16
Compound (SMILES): CCOc1cc(-c2ccc(N3CC4CC(C3)N4Cc3cncc(OC)c3)nc2)c2c(C#N)cnn2c1
InChIKey: LBIHHXYFWKSAJN-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of Formula I were screened for their ability to inhibit wildtype and V804M mutant RET kinase using CisBio's HTRF® KinEASE¿-TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Catalog No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Catalog No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 222 nM (pChEMBL 6.650000095367432)
Descriptors: MW 481.56, ALogP 3.53, HBA 9, HBD 0, PSA 91.81, QED 0.4
Compound (SMILES): Cc1cc(Nc2ncnc(-c3ccc(O[C@@H]4CCC(=O)NC4)c(C#N)c3)n2)ccc1N1CCN(C2COC2)CC1
InChIKey: USCFDHISMFVVSX-XMMPIXPASA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: hJAK2 Biological Assay: Enzymatic activity of hJAK2 was measured using a LANCE homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay that monitors enzyme dependent phosphorylation of a biotinylated serine/threonine peptide substrate (PTK). An increase in the amount of phosphorylated peptide results in an increase in TR-FRET signal. hJAK2 was expressed and purified as full length recombinant proteins. Detection reagents for the assay were purchased from PerkinElmer. hJAK2 enzyme was assayed under initial rate conditions in the presence of 2×Km ATP (30 μM) and 1 μM peptide, 50 mM Tris-Cl (pH 7.5), 10 mM MgCl2, 1 mM dithiothreitol, 0.01% BSA, 0.05% (v/v) DMSO at the following concentration of enzyme: hJAK2 at 0.3 nM. After an assay reaction time of 40 minutes at 25° C., reactions were terminated with EDTA and LANCE Detection Buffer.Amount of phosphorylated peptide was determined by the addition of 20 nM SA-APC and 1 nM europium cryptate labeled anti-phospho PTK monoclonal antibody PY66 and the resulting TR-FRET signal was recorded on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 200 μs read window). Data was normalized based on a positive (1 μM Staurosporine) and negative (DMSO) controls and IC50 values calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations.
Measurement: IC50 = 57000 nM (pChEMBL 4.239999771118164)
Descriptors: MW 540.63, ALogP 2.64, HBA 10, HBD 2, PSA 128.53, QED 0.46
Compound (SMILES): N#Cc1cccc(C(=O)NCc2ccc(-c3nn(C4CCCC4)c4ncnc(N)c34)cc2)c1
InChIKey: XEPZBYPPIOVUTE-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: EGFR Binding Affinity: EGFR binding affinity was determined using a time-resolved fluorescence resonance energy transfer (TR-FRET) methodology. 2.5 nM Recombinant EGFR, varying concentrations of inhibitor, 2 nM LanthaScreen Eu anti-GST Antibody and 3 nM Kinase Tracer 199 was incubated in 1× LanthaScreen Kinase Buffer A for 5 h. Recombinant EGFR and all LanthaScreen components were purchased from Invitrogen. Measurements were performed in a reaction volume of 30 μL using half-area 96-well assay plates. The TR-FRET signal was read on a plate reader with an excitation wavelength of 340 nm and detection wavelengths of 615 and 665 nm. Binding affinity was determined for each compound by measuring TR-FRET signal at various concentrations of compound and plotting the relative fluorescence units against the inhibitor concentration to estimate the IC50 from log [Inhibitor] vs response using the Variable Slope model in Graphpad prism from Graphpad software (SanDiego, Calif.).
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 437.51, ALogP 3.99, HBA 7, HBD 2, PSA 122.51, QED 0.49
Compound (SMILES): C[C@@H]1C[C@H](N)CN(c2ccncc2NC(=O)c2c(N)oc3cc(OC(F)F)cnc23)C1
InChIKey: MENUNSBFZDLLDG-MNOVXSKESA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Enzyme Assay: Pim-1 and Pim-3 kinase assays-20 μL reactions were run in white 384 well polystyrene plates dotted with 0.8 μL compound/DMSO in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM MgCl2, 0.01% BSA, 5 mM DTT), containing 0.05 μm Biotin-labeled BAD peptide substrate (AnaSpec 62269), 1 mM ATP, and 2.5 pM (Pim-1, Invitrogen PV3503) or 1.25 pM (Pim-3, Millipore 14-738) enzyme for 1 h at 25° C. Reactions were stopped by addition of 10 μL STOP Buffer (150 mM Tris, pH=7.5, 150 mM NaCl, 75 mM EDTA, 0.01% Tween-20, 0.3% BSA) supplemented with Phospho-Bad (Ser112) Antibody (Cell Signaling 9291) diluted 666-fold, and Streptavidin donor beads (PerkinElmer 6760002) along with Protein-A acceptor beads (PerkinElmer 6760137) at 15 μg/mL each. Supplementation of the STOP buffer with beads and stopping the reactions were done under reduced light. Prior to the stopping reactions STOP buffer with beads was pre-incubated for 1 h in the dark at room temperature. After stopping the reactions, plates were incubated for 1 h in the dark at room temperature before reading on a PHERAstar FS plate reader (BMG Labtech) under reduced light.Pim-2 kinase assay-20 μL reactions were run in white 384 well polystyrene plates dotted with 0.8 μL compound/DMSO in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM MgCl2, 0.01% BSA, 5 mM DTT), containing 0.05 μm Fluorescein-labeled CREBtide peptide substrate (Invitrogen PV3508), 1 mM ATP, and 1 nM enzyme (Invitrogen PV3649) for 2 h at 25° C. Reactions were stopped by addition of 10 μL TR-FRET Dilution Buffer (Invitrogen PV3574) with 30 mM EDTA and 1.5 nM LanthaScreen Tb-CREB pSer133 antibody (Invitrogen PV3566). After 30 min. incubation at room temperature, plates were read on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 432.43, ALogP 2.83, HBA 8, HBD 3, PSA 132.53, QED 0.56
Compound (SMILES): Cc1cc(Nc2cc(C3CCCO3)nc(N(C)[C@@H]3C[C@H]4CCC[C@@H](C3)N4CC(=O)N3CCCC3)n2)n[nH]1
InChIKey: LNCZXRDIYFEVAH-SUIYAGFRSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Biochemical JAK and Tyk2 Kinase Assays: 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 or discretely 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 1 h. 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 1 h 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.
Measurement: Ki = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 508.67, ALogP 3.55, HBA 8, HBD 2, PSA 102.51, QED 0.59
Compound (SMILES): CNC(=O)CN1C[C@]2(CC[C@](c3ccccc3)(N(C)CC(C)C)CC2)N(CC2CCC2)C1=O
InChIKey: OLKNEYLQFQEAAF-MKPDMIMOSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Human Mu-Opioid Peptide (hMOP) Receptor Binding Assay: The hMOP receptor binding assay was performed as homogeneous SPA-assay (scintillation proximity assay) using the assay buffer 50 mM TRIS-HCl (pH 7.4) supplemented with 0.052 mg/ml bovine serum albumin (Sigma-Aldrich Co., St. Louis, Mo.). The final assay volume (250 μl/well) included 1 nM of [N-allyl-2,3-3H]naloxone as ligand (PerkinElmer Life Sciences, Inc. Boston, Mass., USA), and either test compound in dilution series or 25 μM unlabelled naloxone 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 90 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]naloxone-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 = 1740 nM (pChEMBL 5.760000228881836)
Descriptors: MW 454.66, ALogP 4.07, HBA 3, HBD 1, PSA 55.89, QED 0.64
Compound (SMILES): CC(C)n1nc(N2CCN(CC3CCN(c4ccc5c(c4)C(=O)N(C4CCC(=O)NC4=O)C5=O)CC3)CC2)c2cnc(Nc3ccnc(-c4cnn(S(=O)(=O)C5CC5)c4)n3)cc21
InChIKey: OGRYXOIGKDXQED-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: TR-FRET Assay for ERBB Kinases: All compounds and PROTACs were serially diluted in three-fold increments using 100% DMSO, followed by an intermediate 10-fold dilution using Buffer A (50 mM HEPES, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 1 mM DTT, and 0.1% Pluronic F-68). Two microliters of serially diluted compound or PROTAC were then transferred to black 384-well Proxiplates (PerkinElmer, #6008260) using an Integra Viaflo96. Next, 10 uL of protein kinase in Buffer A was added to each well of the assay plate and pre-incubated with compound for 10 minutes. Kinase reactions were then initiated by addition of 5 uL substrate mix containing 3 mM ATP and 30 uM fluorescein-labeled Poly-GluTyr (Thermo Fisher, #PV3610) in Buffer A and allowed to proceed for 10 minutes at room temperature. Reactions were quenched by addition of a 5 uL mixture containing 5 nM LanthaScreen® Tb-pY20 Antibody (Thermo Fisher, #PV3552) and 40 mM EDTA in Buffer A. Assay plates were then read using a Synergy2 (Biotek Instruments, Winooski, Vt.) via excitation thru a 340/20 nm bandpass filter and emission collected thru 490/10 nm (donor) and 520/25 nm (acceptor) bandpass filters. The final kinase concentrations used for each 15 uL reaction were as follows: 0.2 nM EGFR Exon20NPG (SignalChem, #E10-132GG), 0.1 nM wild type EGFR (BPS Bioscience, #40187), 0.3 nM EGFR L858R/T790M/C797S (BPS Bioscience, #40351), 0.1 nM EGFR L858R (BPS Bioscience, #40189), 0.4 nM L858R/T790M (BPS Bioscience, #40350), InM EGFR Dell9 (SignalChem, #E10-122JG), 10 nM EGFR Dell9 T790M (SignalChem, #E10-122KG), 0.3 nM Her2 (BPS Bioscience, #40230), 15 nM Her2 InsYVMA (SignalChem, #E27-13BG).
Measurement: IC50 = 170 nM (pChEMBL 6.769999980926514)
Descriptors: MW 861.99, ALogP 3.19, HBA 17, HBD 2, PSA 213.75, QED 0.18
Compound (SMILES): CC1(C)Cc2cc(NC(=O)c3cnn4cccnc34)c(N3CCN(CC(F)F)CC3)cc2O1
InChIKey: WFQAMUFXBXJWTQ-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Biological Assay: Kinase activities were assayed using the Transcreener-Fluorecescence polarization platform (BelBrook Labs, Madison, Wis., USA) that measures amounts of the reaction product, ADP. The IRAK4 reaction conditions were optimized using an IRAK1-derived peptide (sequence H-KKARFSRFAGSSPSQSSMVAR) to provide a linear reaction rate over the course of a 90 min incubation, which resulted in 10-12% conversion of the starting ATP to ADP. Final IRAK4 assay conditions were 1.25 nM IRAK4; 125 uM ATP; 10 uM MgCl2; 125 uM peptide in reaction buffer (25 mM HEPES (pH7.4); 2 mM Dithiothreitol; 0.015% Brij-35; and 0.5% dimethyl sulfoxide. The IRAK1 activity was optimized similarly, yielding final assay conditions of 3 mM IRAK1; 62.5 uM ATP; 5 uM MgCl2, and 62.5 uM IRAK1 peptide in reaction buffer for 60 min.Assays of compounds for kinase inhibition were performed using inhibitors serially-diluted in dimethyl sulfoxide, which was accomplished with a LabCyte Echo 555 liquid dispenser. 384 well assay plates spotted with compound received 4 ul of a 2× substrate (ATP+peptide) mix in reaction buffer, followed by 4 ul of 2× enzyme diluted in reaction buffer. Reactions were halted at 60 (IRAK1) or 90 (IRAK4) min by addition of 6 ul of detection buffer, containing EDTA (40 nM final concentration), 0.95 ug of the ADP-binding antibody ADP2, ADP tracer (3 nM final concentration), and 25 uM HEPES. Following a 1 hr incubation, fluorescence polarization of the ADP2-antibody TRACER complex was read on a Tecan M1000 plate reader using a 635/20 excitation filter in combination with a 670/20 emission filter.
Measurement: Ki = 26 nM (pChEMBL 7.579999923706055)
Descriptors: MW 456.5, ALogP 3.08, HBA 7, HBD 1, PSA 75, QED 0.64
Compound (SMILES): O=c1c2c(O)cc(O)cc2c(-c2ccccc2)cn1-c1ccc(O)cc1
InChIKey: UQJVSWJIGSWDBK-UHFFFAOYSA-N
Target: Estrogen receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P03372
Assay: Binding Assay: Recombinant ER-α or ER-β ligand binding domain (LBD) was combined with [3H]E2 (PerkinElmer, Waltham, Mass.) in buffer A (10 mM Tris, pH 7.4, 1.5 mM disodium EDTA, 0.25 M sucrose, 10 mM sodium molybdate, 1 mM PMSF) to determine the equilibrium dissociation constant (Kd) of [3H]E2. Protein was incubated with increasing concentrations of [3H]E2 with and without a high concentration of unlabeled E2 at 4° C. for 18h in order to determine total and non-specific binding. Non-specific binding was then subtracted from total binding to determine specific binding. Ligand binding curves were analyzed by nonlinear regression with one site saturation to determine the Kd of E2 (ER-α: 0.65 nM; ER-β: 1.83 nM). In addition, the concentration of [3H]E2 required to saturate ER-α and ER-β LBD was determined to be 1-3 nM.Increasing concentrations of two β-SERMs (14m and 12u) (range: 10−11 to 10−6 M) were incubated with [3H]E2 (1-2 nM) and ER LBD using the conditions described above. Following incubation, plates were harvested with GF/B filters on the Unifilter-96 Harvester (PerkinElmer) and washed three times with ice-cold buffer B (50 mM Tris, pH 7.2). The filter plates were dried at room temperature, then Microscint-O cocktail was added to each well and the filter plates were sealed with TopSeal-A. Radioactivity was counted in a TopCount® NXT Microplate Scintillation Counter using the settings for [3H] in Microscint cocktail (PerkinElmer).The specific binding of [3H]E2 at each concentration of compound was determined by subtracting the nonspecific binding of [3H]E2 (determined by incubating with 10−6 M unlabeled E2) and expressing it as a percentage of the specific binding in the absence of compound. The concentration of compound that reduced the specific binding of [3H]E2 by 50% (IC50) was determined by computer-fitting the data with SigmaPlot and non-linear regression with the four parameter logistic curve. The equilibrium binding constant (Ki) of each compound was then calculated by: Ki=Kd×IC50/(Kd+L), where Kd is the equilibrium dissociation constant of [3H]E2, and L is the concentration of [3H]E2.
Measurement: Ki = 124 nM (pChEMBL 6.909999847412109)
Descriptors: MW 345.35, ALogP 3.77, HBA 5, HBD 3, PSA 82.69, QED 0.52
Compound (SMILES): COc1cc2ncnc(NCCCCNS(N)(=O)=O)c2cc1OC
InChIKey: LUIDVNMEIDIUAT-UHFFFAOYSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: ENPP2 Assay: Table 3: Assay Buffer: 50 mM Tris, 10 mM CaCl2, 5 mM MgCl2, 0.02% Brij-35 (v/v), pH 8.5Substrate: 20 mM Bis(p-Nitrophenyl) Phosphate Sodium Salt (BPNPP) (Sigma, Catalog #N3002) Enzyme: 0.01 ng/μL Recombinant Human ENPP-2/Autotaxin (rhENPP-2) (R&D Catalog #5255-EN) DMSO (Selleck S8218)>96-well clear assay plates Protocol: An eight point serial dilution of drugs was prepared in 10× in assay buffer with the final assay concentrations starting at 10 μM, 3 μM, 1 μM, 0.3 μM . . . 0 μM. A dilution of DMSO was included as a control. The assay plate was set up as follows with each well in duplicate: 81 μL assay buffer+10 μL ENPP1 inhibitor or DMSO+5 μL Substrate+5 μL Enzyme (0.05 ng). Both the enzyme and substrate were added to opposite sides of the well to ensure that there was no interaction until all wells had both components. The plate was then centrifuged gently for 10 seconds, followed by an incubation at 37° C. for 10 minutes. The reaction was quantified by measuring absorbance at 400 nm using the Envision. PF8380 was used as a positive control for the assay.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 355.42, ALogP 0.63, HBA 7, HBD 3, PSA 128.46, QED 0.56
Compound (SMILES): CC1C(=O)N(C)Cc2c(-c3cccc4nc(-c5cnn(C)c5)c(C#N)cc34)nc(C3CC3)n21
InChIKey: PSWWDDSOCOGNCT-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: AlphaScreen Assay: CBP: 5 nM GST-CBP(1081-1197) and 20 nM biotin-H4(1-21) Ac-K5/8/12/16 (AnaSpec. 64989) were incubated with varying concentrations of CBP inhibitors in 15 μL of buffer containing 50 mM HEPES 7.5, 100 nM NaCl, 1 mM TCEP, and 0.003% Tween-20. After 30 minutes incubation at room temperature, 15 μL of detection buffer (BPS Bio. 33006) containing 7 μg/mL of Glutathione AlphaLisa acceptor beads (Perkin Elmer AL109) and 14 μg/mL of Streptavidin donor beads (Perkin Elmer 676002) was then added to the previous mixture. The reaction was incubated for an additional 2 hours at at room temperature, and the AlphaScreen signal was quantified using the Envision Multilabel plate reader. As negative control, GST-CBP(1081-1197) was incubated with the non-acetylated biotin-H4(1-21) peptide(AnaSpec. 62555) and in presence of 0.25% of final DMSO concentration.
Measurement: IC50 = 25000 nM (pChEMBL 4.599999904632568)
Descriptors: MW 437.51, ALogP 3.78, HBA 7, HBD 0, PSA 92.63, QED 0.49
Compound (SMILES): CCCCCCCCc1cccc(-c2noc([C@@H]3CCCN3C(=N)N)n2)c1
InChIKey: ZMOZBJKDEONOQO-SFHVURJKSA-N
Target: Sphingosine kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NRA0
Assay: Scintillation Proximity Assay: SphK activity was measured by a scintillation proximity assay as previously described [1], the disclosure of which is incorporated herein. Briefly, recombinant SphK1 or SphK2 were expressed in Sf9 insect cells, crude homogenates were prepared and incubated in 96 well FlashPlates (Perkin-Elmer) in a buffer containing D-erythro-sphingosine and γ-[33P]ATP. The [33P]S1P product, which adheres to the plate wall, was quantified by scintillation counting. To assay ceramide kinase or diacylglycerol kinases, the recombinant proteins were incubated with γ-[32P]ATP and substrate (C6 ceramide or 1-Ohexadecyl-2-acetyl-sn-glycerol, respectively) and the lipid product, after recovery by organic extraction, was resolved by thin layer chromatography, detected by autoradiography, and quantified by liquid scintillation counting. These assays were performed with and without a fixed concentration of inhibitor and its effect on KM and Vmax determined.
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 369.51, ALogP 4.67, HBA 4, HBD 2, PSA 92.03, QED 0.38
Compound (SMILES): CCC(=O)Cn1c(=O)[nH]c2ncc(-c3ccc(F)c(C)c3F)cc21
InChIKey: JBGDMRZEUCZMPN-UHFFFAOYSA-N
Target: Glutamate NMDA receptor; GRIN1/GRIN2B (PROTEIN COMPLEX), Homo sapiens
UniProt: Q05586
Assay: Biological Assay (standard): NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 137 mM NaCl, 4 mM KCl, 2 mM CaCl2), 0.5 mM MgCl2, 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (137 mM NaCl, 4 mM KCl, 2 mM CaCl2), 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to ~EC40 used to maximize the assay's signal window and ability to detect NMDA receptor antagonists and negative allosteric modulators. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 331.32, ALogP 2.96, HBA 4, HBD 1, PSA 67.75, QED 0.8
Compound (SMILES): CC(C)(C)OC(=O)[C@@H]1CC[C@@](CC#N)(n2nc(Nc3ccccc3)c3c(=O)[nH]ccc32)CO1
InChIKey: KHOTXVKXPBBUFY-UUOWRZLLSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biochemical HTRF Assay : 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.
Measurement: IC50 = 1.62 nM (pChEMBL 8.789999961853027)
Descriptors: MW 449.51, ALogP 3.6, HBA 8, HBD 2, PSA 122.03, QED 0.57
Compound (SMILES): COc1cc2c(OC[C@H]3NC(=O)[C@]4(F)C[C@H]34)ccnc2cc1C(N)=O
InChIKey: VGYNVEKRCVCWQZ-HSOHIQCKSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IRAK4 Enzymatic DELFIA Assay, Protocol A: This is an in vitro assay to measure IRAK4 enzymatic activity utilizing the DELFIA (Dissociation-Enhanced Lanthanide Fluorescent Immunoassay, Perkin-Elmer) platform, with the human IRAK4 FL (Full Length) construct to characterize IRAK4 inhibitor and control compounds at 0.6 mM ATP (KM). The final amount of enzyme in the assay is 0.1 nM IRAK4 FL, final concentration of substrate is 50 nM, and final concentration of DMSO is 2.5%.The test compound was solubilized in DMSO to a stock concentration of 30 mM. The dose response plates were prepared with a 4 mM primary compound concentration, and then diluted in DMSO in a four-fold series for a total of 11 data points. Compounds were prepared as a 40-fold multiple of the final in-assay concentration.To begin the assay, 19 μL of reaction mixture containing 20 mM HEPES pH=7.5, 5 mM MgCl2, 0.0025% Brij-35, 600 μM ATP, 0.21 nM Full-length phosphorylated recombinant human IRAK4 (GenBank ID AF445802) were aliquoted into Ultra-Clear Polypropylene, 384-well, U-Bottom Plates (Corning Life Sciences). 1 μL of test compound from the dose-response plate was added to the reaction mixture and incubated for 20 minutes at room temperature. Then 20 μL of 20 mM HEPES pH=7.5, 5 mM MgCl2, 0.0025% Brij-35, 600 μM ATP, and 100 nM ERM-biotinylated peptide (AGAGRDKYKTLRQIR) was added to start the reaction. The reaction was incubated for 60 minutes at room temperature and stopped by the addition of 20 μL 0.3M EDTA.50 μL of the reaction mixture was transferred to a streptavidin coated detection plate (DELFIA streptavidin coated plates, 384-well, white plates, Perkin-Elmer Life Sciences) and incubated for 30 minutes at room temperature. The plates were washed 4× with 75 μL per well of PBS containing 0.05% Tween-20. Plates were then incubated with 50 μL per well of antibody cocktail of Anti-pERM antibody at 0.125 μg/mL (Cell Signaling Technology), plus Anti-Rabbit IgG EuN1 at 0.25 ug/ml (Perkin-Elmer Life Sciences) in a solution of 10 mM MOPS pH=7.5, 150 mM NaCl, 0.05% Tween-20, 0.02% NaN3, 1% BSA, 0.1% Gelatin for 45 minutes. The plates were washed 4× with 50 μL per well of PBS containing 0.05% Tween-20. Then 50 μL per well of DELFIA Enhancement Solution (Perkin-Elmer Life Sciences) were added to the plate and then read on an EnVision Model 2103 using a 340 nm excitation wavelength and a 665 nm emission wavelength for detection.
Measurement: IC50 = 0.4 nM (pChEMBL 9.399999618530273)
Descriptors: MW 345.33, ALogP 0.95, HBA 5, HBD 2, PSA 103.54, QED 0.84
Compound (SMILES): CCC(CC)n1cc(-c2nc(-c3cnn(C4CCN(C(=O)N(C)C)CC4)c3)cn3nccc23)cn1
InChIKey: LDDOGZGJPXCIRH-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK1 Inhibition Assay: Compounds of Formula I were screened for their ability to inhibit JAK1 using the general enzyme inhibition assay method, in which the assay mixture contained 1 mM ATP, 8 μM Omnia Y12 peptide (Catalog # IVGN KPZ3121C; Invitrogen Corporation, Carlsbad, Calif.) and 12.5 nM JAK1 in a total volume of 25 μL. JAK1 was purchased from Invitrogen Corporation, Carlsbad, Calif. (catalog # IVGN PV4775).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 475.6, ALogP 4.14, HBA 8, HBD 0, PSA 89.38, QED 0.42
Compound (SMILES): Cc1nc(N)nc(N[C@H](c2nc3cccc(Cl)c3c(=O)n2[C@H]2[C@@H]3CNC[C@@H]32)C2CC2)c1C#N
InChIKey: IJBKSHRETAMTRG-QABNGEJJSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: TR-FRET PIK3 Assay: TR-FRET monitored the formation of 3,4,5-inositol triphosphate molecule that competed with fluorescently labeled PIPS for binding to the GRP-1 pleckstrin homology domain protein. An increase in phosphatidylinositide 3-phosphate product resulted in a decrease in TR-FRET signal as the labeled fluorophore was displaced from the GRP-1 protein binding site.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 pM, and PI3Kγ at 2 nM. The compounds of Table 1 and Compound X ((S)-2,4-diamino-6-((1-(5-chloro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)ethyl)amino)pyrimidine-5-carbonitrile) 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-PIPS, 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 is delay and 500 is read window).The results were normalized based on positive (1 μM wortmanin) and negative (DMSO) controls, and the IC50 values for PI3Kα, 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.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 462.95, ALogP 2.56, HBA 9, HBD 3, PSA 134.54, QED 0.53
Compound (SMILES): O=c1n(Cc2cccc(Cl)c2)c2sc(Cc3ccccc3)cc2c2ncnn12
InChIKey: WIUKRYCFMGWGIT-UHFFFAOYSA-N
Target: Dual specificity calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1B (SINGLE PROTEIN), Homo sapiens
UniProt: Q01064
Assay: PDE1B Inhibitory Assay: PDE1B inhibition was determined by an IMAP TR-FRET assay. The IMAP TR-FRET PDE assay was optimized for concentration of enzyme, Calmodulin, cAMP or cGMP substrate, DMSO tolerance, and incubation time. Into each well of a solid white 1536 well plate (Corning) was dispensed 250 pg full-length recombinant NH-terminal GST tagged human PDE1B enzyme (BPS Bioscience Cat #60011, San Diego, Calif.) in 2.5 μL IMAP BSA reaction buffer (Molecular Devices, Sunnyvale, Calif.) containing 10 U/ml Calmodulin and 2.5 mM CaCl2 (Sigma Aldrich.) After a brief centrifugation, 30 nl compound was added by transfer from 1 mM stock in DMSO using a Kalypsys 1536 Pintool. Plates were incubated for 5 minutes at room temperature before dispensing 1.5 μL of 533 nM 5-carboxy fluorescein (FAM)-labeled cAMP (Molecular Devices, Sunnyvale, Calif.) for a final concentration of 200 nM. After a brief centrifugation, the plates were incubated for 30 minutes at room temperature. The assay was terminated by adding 5 μL IMAP binding reagent/Tb complex (Molecular Devices, Sunnyvale, Calif.) to each well. Plates were incubated 1 hour at room temperature and read on a Viewlux multimode plate reader (Perkin Elmer). The instrument was set to excite using the DUG11 filter and measure using 490/10 nm and 520/10 nm filters. Ratios of acceptor and donor were then calculated.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 406.9, ALogP 4.4, HBA 6, HBD 0, PSA 52.19, QED 0.44
Compound (SMILES): Fc1ccc2c(c1)CN(c1nccc(Nc3ccc4[nH]ncc4c3)n1)C2
InChIKey: PPRQSCUSEPEPNV-UHFFFAOYSA-N
Target: Rho-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13464
Assay: Rho-kinase inhibition assay for ROC2 and ROCK1: Dose response curves for Rho-kinase inhibition were derived from a Invitrogen Z′-LYTE Kinase Assay Kit (Invitrogen catalog number PV3793). Purified active ROCK1 and ROCK2 were obtained from Invitrogen (catalog numbers ROCK1, PV3691 and ROCK2, PV3759). The kit components include a coumarin and fluorescein labeled peptide based on myosin light chain 2 (KKRPQRRYSNVF), a proprietary protease containing development reagent and a proprietary Stop buffer used to terminate the development reaction. The inhibitory activities of compounds are measured according to the manufactures protocol. Briefly, decreasing concentrations of test compounds or the known ROCK inhibitor Y-27963, are added, from 10 uM to 2.56×10−5 uM to reaction buffer containing 50 mM HEPES pH 7.5, 10 mM MgCl2, 5 mM EGTA, and 0.05% Brij-35 and of ROCK1 at 0.18 ug/mL or ROCK2 at 0.8 ug/mL in assay dilution buffer. This mixture is overlayed into a white 96-well half area plate and the reaction is initiated with the addition of 5 uM ATP for ROCK1 or 12 uM ATP for ROCK2. The assay proceeds at room temperature for 1 hour followed by the addition of development reagent, and further incubation for 1 hour at room temperature. STOP reagent is then added and the reaction and immediately the coumarin and fluorescein emission signals are read on a Tecan Infinite M1000 fluorescence plate reader (excitation: 400 nm; emission 445 and 520 nm, respectively). By comparing the emission ratios of the test samples against control samples, percent phosphorylation values are calculated and the concentration of inhibitor that produces ½ inhibition of kinase activity (IC50) is determined using Prism. Table 1 provides IC50 concentrations for compounds of the above examples. Several of the compounds also demonstrated activity in a preliminary assay that measured inhibition of myosin light chain phosphorylation (pMLC). For compounds marked ND, activity was not determinable under the test conditions employed.
Measurement: IC50 = 2930 nM (pChEMBL 5.53000020980835)
Descriptors: MW 346.37, ALogP 3.76, HBA 5, HBD 2, PSA 69.73, QED 0.59
Compound (SMILES): CO[C@H]1CCN(c2cc(NC(=O)N3CCCc4cc(CN5CC[C@H](OC)C5=O)c(C=O)nc43)ncc2C#N)C1
InChIKey: LFTRUGBNIVGOGN-REWPJTCUSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Enzymologic Experiment of FGFR1: In this experiment, the inhibitory effect of the compounds on FGFR1 kinase activity was tested by a fluorescence resonance energy transfer (TR-FRET) method, and the half maximal inhibitory concentration (IC50) of the compounds on the FGFR1 kinase activity was determined. 1) 15 μL of FGFR1 enzymatic solution was added to a 384-well plate, and the final concentration of the enzyme was 0.15 nM. 2) 15 μL of diluted solution in gradient of the compound was added.3) 15 μL of a substrate mixture containing substrate polypeptide with a final concentration of 550 nM and ATP with a final concentration of 10200 μM was added. 4) The mixture was incubated for 0.5-3 hours at room temperature. 5) 10 μL of EDTA and a test solution comprising labeled antibody were added, and the plate was incubated for 1 hour at room temperature. 6) The fluorescence signal values of each plate were determined by a microplate reader at 665 nm. 7) The inhibition rates were calculated according to the fluorescence signal values.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 533.59, ALogP 2.12, HBA 9, HBD 1, PSA 140.99, QED 0.53