lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Nc1ncc(N2CCN(c3cccc(-c4cnc(N5CCC5)nc4)c3)CC2)c(N)n1
InChIKey: DYSYUHAHEWMCMM-UHFFFAOYSA-N
Target: Dihydrofolate reductase (SINGLE PROTEIN), Homo sapiens
UniProt: P00374
Assay: TBD: TBD
Measurement: IC50 = 955 nM (pChEMBL 6.019999980926514)
Descriptors: MW 403.49, ALogP 1.63, HBA 9, HBD 2, PSA 113.32, QED 0.67
Compound (SMILES): COc1cc2nc(NCCCCC(=O)NC(C)C)c3nnc(C)n3c2cc1OC
InChIKey: GGLOCXZVMKPMOK-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Evaluation of BRD4 binding inhibition ability: The following experiment was performed to evaluate the ability of [1,2,4]triazolo[4,3-a]quinoxaline derivative of the present invention to inhibit the interaction between BRD4 (BD1+BD2) bromodomain, one of BET protein family, and tetraacetylated histone H4 peptide.The compound was serially diluted at the ratio of 1:5 in assay buffer from 10 mM stock in DMSO (initial concentration: 100 μM) on white OptiPlate-384 (PerkinElmer). A mixture comprising 100 nM GST-BRD4 (BD1+BD2) and 100 nM biotinylated acetyl-histone H4 (Lys5,8,12,16) peptide was prepared in assay buffer (50 mM HEPES pH 7.4; 25 mM NaCl; 0.05% Tween 20; 0.1% bovine serum albumin (BSA); 10 mM dithiothreitol (DTT)). After adding 6 μl of the mixture to the diluent, 6 μl of the pre-mixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads (PerkinElmer, 10 μg/ml in assay buffer, respectively) was added thereto. The samples were incubated in the dark at room temperature for 30 minutes (shaking at 300 rpm). Then, the signals were measured with PerkinElmer Envision HTS Multilabel Reader using an alpha screen protocol of PerkinElmer. Each plate contained the negative control in which biotinylated acetyl-histone H4 peptide and GST-BRD4 (BD1+BD2) were replaced by assay buffer. In the case of using the software GraphPad Prism for calculation, the negative control point was input as a low standard value. The positive control (probe molecule l-BET762 containing protein/peptide mixture) proceeded to pipetting. IC50 value was determined by using GraphPad Prism 3.03 software (or an updated version thereof).
Measurement: IC50 = 29 nM (pChEMBL 7.539999961853027)
Descriptors: MW 400.48, ALogP 2.71, HBA 8, HBD 2, PSA 102.67, QED 0.53
Compound (SMILES): CN[C@H]1CC[C@@H](Nc2nc(Nc3cc(C)ns3)nc3ccsc23)CC1
InChIKey: IGGSSNMNNIFMQN-TXEJJXNPSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Kinase Activity Inhibition Assay: IRAK4 kinase (purchased from Life Technologies, Cat. No.: PR5612U) was diluted to 2 folds of the final concentration (the final concentration is 0.76 ng/μL) with reaction buffer (40 mM Tris-HCl, pH 7.5; 20 mM MgCl2; 0.1 mg/mL BSA; 1 mM DTT), and added to a 384-well plate at 5 μL/well. The test drugs were 10-fold serial diluted from 10 μM to set six concentration points and added to the test wells of the 384-well plate at 2.5 μL/well. After incubation for 10 min at 25° C., 10 μM of ATP and 0.1 μg/L of an enzyme reaction substrate, myelin basic protein MBP (purchased from SignalChem, Cat. No.: M42-51N) were added at 2.5 L/well, and reacted at 25° C. for 60 minutes. After the reaction was completed, the kinase activity assay was performed using the ADP-Glo kinase assay kit (purchased from Promega, Cat. No.: V9102) according to the manufacture's instruction, i.e., 10 μL of ADP-Glo reaction reagent was first added and reacted at 25° C. for 40 minutes, 10 μL of the solution to be tested was taken and mixed with 10 μL of ADP-Glo detection reagent, and reacted at 25° C. for 30 minutes.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 374.54, ALogP 4.14, HBA 8, HBD 3, PSA 74.76, QED 0.62
Compound (SMILES): N#Cc1cnn(C2CC(c3ccc(F)cc3)CC2N2CC(N)CC(F)C2)c1
InChIKey: JIQJJGRQXFKITC-UHFFFAOYSA-N
Target: Short transient receptor potential channel 6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y210
Assay: Electrophysiology Assay: For TRPC6 current recording, the intracellular solution contained (in mM): 120 CsOH, 120 Aspartate, 20 CsCl, 2 MgCl2, 0.4 CaCl2, 10 HEPES, 2 Na2ATP, 0.1 Na3GTP, 10 Glucose, 1 EGTA (pH 7.2-7.25, adjusted with CsOH). The extracellular solution contained (in mM): 145 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 10 Glucose (pH 7.4 adjusted with NaOH).For TRPC3 current recording, the intracellular solution contained (in mM): 130 CsCl, 5 HEPES, 5 EGTA, 5 Na2ATP, Na3GTP, 5.5 MgCl2 (pH 7.2-7.25, adjusted with CsOH). The extracellular solution contained (in mM): 140 NaCl, 4 KCl, 1 MgCl2, 10 HEPES, 0.2 CaCl2, 10 Glucose, 2 Na4EDTA (pH7.4 adjusted with NaOH).
Measurement: IC50 = 4.06 nM (pChEMBL 8.390000343322754)
Descriptors: MW 371.44, ALogP 2.75, HBA 5, HBD 1, PSA 70.87, QED 0.9
Compound (SMILES): CN(C)C(c1ccccc1)n1nc(COC(C)(C)C(=O)O)cc1-c1cccc(OC2CC2)c1
InChIKey: UHFITIMNKSXRTJ-UHFFFAOYSA-N
Target: Monocarboxylate transporter 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O15427
Assay: MCT4-Mediated Lactate Transport in MDA-MB-231 Breast Cancer Cells: MDA-MB-231 breast cancer cells may be used to measure MCT4 activity in cells with high native levels of MCT4 and low levels of MCT1 and are known to those with skill in the art. MCT4 activity may be assessed by monitoring the intracellular pH change that accompanies lactate/proton symport, using the pH-sensitive fluorescent dye 2′,7′-bis-(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), in a manner similar to that previously reported for MCT1 and MCT4. The following is an exemplary procedure for assaying MCT4 activity of the compounds of Formula (I).
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 449.55, ALogP 4.58, HBA 6, HBD 1, PSA 76.82, QED 0.49
Compound (SMILES): CC(=O)Nc1cnc(N2CCN(C(C)c3ccc4scnc4c3)CC2)nc1
InChIKey: GKHWFSHBAJDYQQ-UHFFFAOYSA-N
Target: Protein O-GlcNAcase (SINGLE PROTEIN), Homo sapiens
UniProt: O60502
Assay: Human O-GlcNAcase enzyme inhibition assay: 5 μl of the appropriate concentration of a solution of inhibitor in McIlvaine's Buffer (pH 6.5) in 2% DMSO (for a dose response curve calculation) is added into each well of a 384-well plate (Greiner, 781900). Then, 20 nM of His-Tagged hOGA and 10 μM of FL-GlcNAc (Fluorescein mono-beta-D-(2-deoxy-2-N-acetyl) glucopyranoside; Marker Gene Technologies Inc, M1485) were added to the 384-well plate for a final volume of 20 μl. After incubation for 60 min at room temperature, the reaction was terminated by the addition of 10 μL of stop buffer (200 mM glycine, pH 10.75). The level of fluorescence (λexc 485 nm; (λemm 520 nm) was read on a PHERAstar machine. The amount of fluorescence measured was plotted against the concentration of inhibitor to produce a sigmoidal dose response curve to calculate an IC50. All individual data was corrected by subtraction of the background (Thiamet 3 uM=100% inhibition) whilst 0.5% DMSO was considered as the control value (no inhibition).
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 382.49, ALogP 2.93, HBA 7, HBD 1, PSA 74.25, QED 0.75
Compound (SMILES): COC[C@@H]1CCCN1CCC(=O)N1CCC(c2ccc3[nH]c(-c4cn5ncnc5c(C)c4C)c(C(C)C)c3c2)CC1
InChIKey: WRSAFFXSXFMPMB-SANMLTNESA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 556.76, ALogP 5.82, HBA 6, HBD 1, PSA 78.76, QED 0.3
Compound (SMILES): Cn1cc(-c2ccc3cnc(NC(=O)c4cccc(N5CCNCC5)c4)cc3c2)cn1
InChIKey: KYSSLQKDASBZNF-UHFFFAOYSA-N
Target: Dual specificity tyrosine-phosphorylation-regulated kinase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: Q13627
Assay: The DYRK1A kinase assa: Briefly, recombinant DYRK1A kinase, ATP and Ser/Thr peptide 18 were prepared in 1× Kinase buffer to final concentrations of 0.19 μg/mL, 30 μM, and 4 μM respectively. The mixture was allowed to incubate with the representative compounds for one hour at room temperature. All reactions were performed in duplicate. Unphosphorylated (0% Control) and phosphorylated (100% control) forms of Ser/Thr 18 served as control reactions. Additionally, an 11-point dose-response curve of Staurosporine (1 uM top) was run to serve as a positive compound control.After incubation, Development Reagent A was diluted in Development Buffer then added to the reaction and allowed to further incubate for one hour at room temperature. The plate was read at Ex 400 Em 455 to detect the coumarin signal and Ex 400 Em 520 to measure the signal (EnVision Multilabel Plate Reader, PerkinElmer).
Measurement: EC50 = 1.3 nM (pChEMBL 8.890000343322754)
Descriptors: MW 412.5, ALogP 3.3, HBA 6, HBD 2, PSA 75.08, QED 0.54
Compound (SMILES): CCS(=O)(=O)c1ccc(Cl)cc1CNC(=O)c1cc(Cl)c(CN2CC[C@H](NC(C)C)C2)c(C(F)(F)F)c1
InChIKey: XBKYTUJHJNIECL-IBGZPJMESA-N
Target: Epithelial discoidin domain-containing receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q08345
Assay: LanthaScreen Eu Kinase Binding Assay: DDR1 binding activity was measured by the LanthaScreen (Registered trademark) Eu Kinase Binding Assay (manufactured by Life Technologies Corporation). A test compound and the Alexa Fluor (Registered trademark) 647-labeled Kinase Tracer 178 (manufactured by Life Technologies Corporation) were added to a mixture of DDR1 and the LanthaScreen (Registered trademark) Eu-anti-GST antibody. After reacting at room temperature for one hour, the fluorescence resonance energy transfer was measured. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate relative to the test compound-free control.
Measurement: IC50 = 39 nM (pChEMBL 7.409999847412109)
Descriptors: MW 580.5, ALogP 5.31, HBA 5, HBD 2, PSA 78.51, QED 0.42
Compound (SMILES): N#Cc1ccc(-c2cc(NCCN3CCCC3=O)nc3ncnn23)cc1
InChIKey: BUWDIWNTAXRQNN-UHFFFAOYSA-N
Target: Prolyl hydroxylase EGLN2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96KS0
Assay: PHD1 Enzyme Assay: The IC50 values for the PHD1 enzyme (residues 1-407) were determined by mixing increasing amounts of a compound of the invention with a fixed amount of the enzyme (20 nM final concentration) and peptide substrate (Asp-Leu-Asp-Leu-Glu-Ala-Leu-Ala-Pro-Tyr-Ile-Pro-Ala-Asp-Asp-Asp-Phe-Gln-Leu, 1 μM final concentration) and 2-oxoglutarate (0.5 M final concentration) in an assay buffer comprising 30 mM 2-(N-morpholino)ethanesulfonic acid pH 6.0, 2 mM sodium ascorbate, 100 μM dithiothreitol, 2 mg/ml bovine serum albumin, 60 μg/ml catalase enzyme and 1 μM iron (II) sulphate (FeSO4). The reaction was conducted by pre-incubating the PHD1 enzyme in the presence of a compound of the invention for 60 minutes at room temperature. The activity of the free enzyme was measured by adding the peptide, the 2-oxoglutarate and sodium ascorbate (see above for final concentrations). The assay was quenched by the addition of 30% v/v trichloroacetic acid (final concentration 5%). The amount of product released was measured using a UPLC-MS (Agilent 1290 with an ABSciex 4000qTrap Mass Spectrometer). Data were analysed using the classical isotherm equation for the determination of IC50.
Measurement: IC50 = 884 nM (pChEMBL 6.050000190734863)
Descriptors: MW 347.38, ALogP 1.7, HBA 7, HBD 1, PSA 99.21, QED 0.75
Compound (SMILES): CN(Cc1nc2cc(Cl)c3c(c2o1)C1(CCCCC1)NC(=O)N3)C1CCOCC1
InChIKey: IWDMWRFGBFCYQD-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 7B (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NP56
Assay: Enzymatic Assay: PDE7 inhibition was determined by an IMAP TR-FRET assay using PDE7B. The IMAP TR-FRET PDE assay was optimized for concentration of enzyme, FAM-cAMP substrate, reducing agent, DMSO tolerance, and incubation time. First, PDE7 inhibitor compounds in 10 mM DMSO stock were serially diluted in 100% DMSO. Next, into each well of a solid white 1536 well plate (Corning) was dispensed 12.8 pg of N-terminal truncated recombinant human PDE7B enzyme (91-450aa, prep #DBVC-D04614, made in-house by structural biology) in 2.5 μL IMAP BSA reaction buffer (Molecular Devices, Sunnyvale, Calif.) containing 1 mM DTT (Sigma Aldrich.) After a brief centrifugation, 30 nL of serially diluted compounds in DMSO were added by transfer from 1 mM stock using a Kalypsys 1536 Pintool. Plates were incubated for 5 minutes at room temperature before dispensing 1.5 μL of 134 nM 5-carboxy fluorescein (FAM)-labeled cAMP (Molecular Devices, Sunnyvale, Calif.) for a final concentration of 50 nM. After a brief centrifugation, the plates were incubated for 15 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).
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 418.93, ALogP 4.39, HBA 5, HBD 2, PSA 79.63, QED 0.77
Compound (SMILES): CCc1nn(-c2ccc(C(=O)Nc3c(F)cccc3F)c(O[C@@H](C)C(F)(F)F)c2)c(=O)n1C
InChIKey: VRLPFCYJYQMGPV-NSHDSACASA-N
Target: Dihydroorotate dehydrogenase (quinone), mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q02127
Assay: Enzymatic Assay: Assay 1: The enzymatic assay couples DHODH activity with bleaching of the dye 2,6-dichlorophenolindophenol (DCIP) (Knecht and Loffler, 1998; Miller et al., 1968). The assay was conducted in buffer containing 50 mM Tris, 0.1% Triton X-100, 150 mM potassium chloride, 2 nM DHODH, 1 mM dihydroorotate, 0.1 mM decylubiquinone, 0.06 mM DCIP, and 2% DMSO at pH 8.0 at 32 degree Celsius. The reaction was initiated by addition of substrates. Enzyme activity was monitored kinetically by the reduction in DCIP absorbance at 600 nm. Purified recombinant human DHODH enzyme was purchased from Novus (cat. no. NBP1-98916). Other chemicals were purchased from Sigma-Aldrich. Absorbance measurements were obtained using a BMG clarion star plate-reading spectrophotometer.
Measurement: IC50 = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 470.4, ALogP 3.99, HBA 6, HBD 1, PSA 78.15, QED 0.55
Compound (SMILES): CC(c1ccc2scnc2c1)N1CCN(c2ncc3c(n2)CCS(=N)(=O)C3)CC1
InChIKey: GYIVDPSXYVZVSW-UHFFFAOYSA-N
Target: Protein O-GlcNAcase (SINGLE PROTEIN), Homo sapiens
UniProt: O60502
Assay: Human O-GlcNAcase enzyme inhibition assay: 5 μl of the appropriate concentration of a solution of inhibitor in McIlvaine's Buffer (pH 6.5) in 2% DMSO (for a dose response curve calculation) is added into each well of a 384-well plate (Greiner, 781900). Then, 20 nM of His-Tagged hOGA and 10 μM of FL-GlcNAc (Fluorescein mono-beta-D-(2-deoxy-2-N-acetyl) glucopyranoside; Marker Gene Technologies Inc, M1485) were added to the 384-well plate for a final volume of 20 μl. After incubation for 60 min at room temperature, the reaction was terminated by the addition of 10 μL of stop buffer (200 mM glycine, pH 10.75). The level of fluorescence (λexc 485 nm; (Δemm 520 nm) was read on a PHERAstar machine. The amount of fluorescence measured was plotted against the concentration of inhibitor to produce a sigmoidal dose response curve to calculate an IC50. All individual data was corrected by subtraction of the background (Thiamet 3 uM=100% inhibition) whilst 0.5% DMSO was considered as the control value (no inhibition).
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 428.59, ALogP 3.07, HBA 8, HBD 1, PSA 86.07, QED 0.69
Compound (SMILES): CCOc1cc(-c2ccc(N3CCC(O)(c4ccccc4)CC3)nc2)c2c(C#N)cnn2c1
InChIKey: SCGLSIOKIDHBQJ-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 = 13.2 nM (pChEMBL 7.880000114440918)
Descriptors: MW 439.52, ALogP 4.15, HBA 7, HBD 1, PSA 86.68, QED 0.5
Compound (SMILES): Cc1ccc(C(=O)NC2(C)CCN(c3ccc(-c4cc(OCC(C)(C)O)cn5ncc(C#N)c45)cn3)CC2)nc1
InChIKey: ASBVJJJIOUBNPG-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 = 156 nM (pChEMBL 6.809999942779541)
Descriptors: MW 539.64, ALogP 3.91, HBA 9, HBD 2, PSA 128.67, QED 0.36
Compound (SMILES): Cc1cc(-c2cc(Cl)ccc2OCCn2c(C)nc3cc(C(F)(F)F)c(Br)c(C#N)c3c2=O)c2scc(C(=O)O)c2n1
InChIKey: QJBDNPFTBPMKGC-UHFFFAOYSA-N
Target: Eukaryotic translation initiation factor 4E-binding protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13541
Assay: Fluorescence Polarization Assay: eIF4E protein was pre-incubated with EDA-m7GDP-ATTO-550 in FPB at 2xconcentrations for 5 minutes prior to the addition of compounds. Compounds (100x) were prepared using 3-fold serial dilutions in 100% DMSO, and subsequently diluted 1:50 in FPB to produce 2x stocks. 50 ul of 2x eIF4E/EDA-m7GDP-ATTO-550 were transferred to the wells of a 96-well half-area black flat bottom polystyrene plate. 50 ul of 2x test compound were added, and binding reactions were allowed to equilibrate for 30 minutes at room temperature while protected from light. The fluorescent polarization signal was detected using a Victor 2 multi-label counter (Perkin Elmer) and the concentration necessary to achieve inhibition of eIF4E/EDA-m7GDP-ATTO-550 binding by 50% (IC50) was calculated using data from an 8-point compound dilution series.
Measurement: IC50 = 73.3 nM (pChEMBL 7.130000114440918)
Descriptors: MW 677.89, ALogP 7.37, HBA 8, HBD 1, PSA 118.1, QED 0.2
Compound (SMILES): O=C(NCc1ccc(Cl)cc1N1CCCC1)Nc1cccc2[nH]c(=O)[nH]c12
InChIKey: HZDOWKSLUNQEPU-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: O35433
Assay: calcium assay: Capsaicin (0.3 μM) caused an increase in [Ca2+] in the vast majority (95%) of dorsal root ganglia neurons, which were thereby identified as TRPV1 expressing neurons. All synthesized derivatives were tested and all were able to inhibit the calcium uptake and several compounds exhibited more than 80% inhibition, e.g. compounds of Examples 1, 3, 4, 5, 6, 10, 11, 12, 13, 16, 19, 23, 31, 32, 35, 36, 39, 41, 45, 46, 47, 48, 51, 53, 67, 68, 69, 70, 71, 74, 75, 78, 80, 81, 82, 86, 87 and 89. Among them, derivatives such as compounds of Examples 4, 5, 6, 13, 19, 31, 36, 39, 45, 46, 47, 48, 51, 52, 53, 67, 70, 71, 74, 75, 78, 80, 81, 86 and 87, appeared the most potent TRPV1 antagonists exhibiting a complete abolition of capsaicin response (around 100%) at 300 nM.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 385.86, ALogP 3.43, HBA 3, HBD 4, PSA 93.02, QED 0.55
Compound (SMILES): OC(CN1C[C@H]2C[C@@H](c3ccccc3)C[C@H]2C1)c1cccc(C(F)(F)F)c1
InChIKey: CSONBADFIUOLGA-QHUCILSMSA-N
Target: Glutamate receptor ionotropic, NMDA 2B (SINGLE PROTEIN), Homo sapiens
UniProt: Q13224
Assay: FDSS Assay: To each well of the plate, 10 μL test compound, control (MK801) or HHnoCa buffer was added to a final concentration of 10 μM with a final concentration of DMSO of 0.1%. Following 10 minutes pre-incubation in the dark, plates are loaded onto the Hamamatsu FDSS 6000. After collecting baseline fluorescence images, 3 μM glutamate, 3 glycine, and 1 mM Ca2+ in HHnoCa buffer is added to each well, and Ca2+ is recorded for 3 minutes. Data were processed by computing ratio of fluorescence at the end of data collection to baseline fluorescence to assess degree of Ca′ influx inhibition relative to that observed in MK801.
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 375.43, ALogP 4.86, HBA 2, HBD 1, PSA 23.47, QED 0.82
Compound (SMILES): Cc1nnc(C2CCN(C3CCN(C(=O)[C@@H](Cc4cc(C)c5[nH]ncc5c4)NC(=O)N4CCC(c5cc6ccccc6[nH]c5=O)CC4)CC3)CC2)[nH]1
InChIKey: FGDVWCLHQLZDDN-UUWRZZSWSA-N
Target: Calcitonin gene-related peptide 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P06881
Assay: Radioligand Binding Assay: Human CGRP receptors (consisting of CRLR and RAMP1) expressed in insect Sf21 cell membrane homogenates were re-suspended in the binding buffer (10 mM HEPES, pH 7.4, 5 mM MgCl2, 0.2% BSA) to a final assay concentration of 0.6 protein per well. Saturation isotherms were determined by the addition of various concentrations of 3H-telcagepant (Ho et al, The Lancet, 2008, 372, 2115) (in a total reaction volume of 250 μL) for 60 min at room temperature. At the end of the incubation, membranes were filtered onto a unifilter, a 96-well white microplate with bonded GF/B filter pre-incubated with 0.5% PEI, with a Tomtec cell harvester and washed 5 times with distilled water. Non-specific binding (NSB) was measured in the presence of 10 nM MK-3207 hydrochloride (CAS No. 957116-20-0). Radioactivity on the filter was counted (1 min) on a microbeta counter after addition of 50 μL of scintillation fluid. For inhibition experiments, membranes were incubated with 0.5 nM 3H-telcagepant and 10 concentrations of the inhibitory compound (0.001-10 IC50 values were derived from the inhibition curve and the affinity constant (Ki) values were calculated using the Cheng-Prussoff equation (Cheng et al, Biochem. Pharmacol. 1973, 22, 3099-3108).
Measurement: Ki = 0.1 nM (pChEMBL 10)
Descriptors: MW 704.88, ALogP 4.51, HBA 7, HBD 4, PSA 159, QED 0.19
Compound (SMILES): Cc1cnc2c(C(=O)Nc3cc4c(nc3N3CCN(CC(N)=O)CC3)O[C@@](C)(CO)C4)cnn2c1
InChIKey: JOHXLJCCDLLMLY-HSZRJFAPSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Enzymatic 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 MgC2; 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.
Measurement: Ki = 6.6 nM (pChEMBL 8.180000305175781)
Descriptors: MW 480.53, ALogP -0.02, HBA 10, HBD 3, PSA 151.21, QED 0.44
Compound (SMILES): O=C1CCC(N2C(=O)c3cccc(NCCOCCOCCN4CCN(Cc5ccc(-c6cc7c(NCc8ccccc8Cl)ncnc7[nH]6)cc5)CC4)c3C2=O)C(=O)N1
InChIKey: PAQSVWSJPJUALL-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 = 27 nM (pChEMBL 7.570000171661377)
Descriptors: MW 820.35, ALogP 4.55, HBA 12, HBD 4, PSA 174.12, QED 0.08
Compound (SMILES): COC(=O)c1c(C#N)ccnc1O[C@@H]1CC[C@@H](C)N(C(=O)c2cccc(F)c2-c2noc(C)n2)C1
InChIKey: BHEHAWNZFJGPDP-CZUORRHYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Radioligand Binding Assay: Described in Bergman et. al. Bioorg. Med. Chem. Lett. 2008, 18, 1425-1430.
Measurement: Ki = 128 nM (pChEMBL 6.889999866485596)
Descriptors: MW 479.47, ALogP 3.31, HBA 9, HBD 0, PSA 131.44, QED 0.51
Compound (SMILES): CCCC[C@@H](CC(=O)O)Nc1nc(N)nc(C)c1Cc1cc(C(=O)NCCCP(=O)(O)O)ccc1OC
InChIKey: BUCBKQGKMDVFBE-SFHVURJKSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: HEK TLR7 Assay: HEK-Blue TLR7 cells were purchased from Invivogen (San Diego, Calif.). The following description was taken from the product information sheet."HEK-Blue hTLR7 cells are designed for studying the stimulation of human TLR7 (hTLR7) by monitoring the activation of NF-kB. HEK-Blue hTLR7 cells were obtained by co-transfection of the hTLR7 gene and an optimized secreted embryonic alkaline phosphatase (SEAP) reporter gene into HEK293 cells. The SEAP reporter gene is placed under the control of the IFN-b minimal promoter fused to five NF-kB and AP-1-binding sites. Stimulation with a TLR7 ligand activates NF-kB and AP-1 which induce the production of SEAP, which is detected by the HEK-Blue Detection cell culture medium."A typical assay protocol involved the following steps:1. Cells were cultured according to the product information sheet.2. 10 mM compound stock in DMSO were first diluted to 3 mM and then 3-fold serially diluted using DMSO to afford a 10-pt dilution.3. 3 μl of the diluted DMSO were added to 57 μl HEK-Blue Detection media for a further 20-fold dilution.4. 10 μl of the diluted compound in assay media were added into 40 μl cell culture (in HEK-Blue Detection media) in 384-well plate. Final cell concentration=8,000 cells per well.5. The plates were incubated at 37° C. in 5% CO2 for 16 h. SEAP was determined using a spectrophotometer at 620-655 nm.
Measurement: EC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 537.55, ALogP 2.71, HBA 8, HBD 6, PSA 196.99, QED 0.14
Compound (SMILES): C=CC(=O)Nc1cccc(C)c1Nc1ncc2cc(-c3c(Cl)c(OC)cc(OC)c3Cl)cc(OC)c2n1
InChIKey: IQHHYGWMDMHKLJ-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: FGFR-4 wild type assay: In each well of a 384-well plate, 0.5 ng/ul of wild type FGFR-4 (Carna Biosciences, Inc.) was incubated in a total of 12.5 ul of buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 10 mM MgCl2, 1 mM DTT) with 1 uM CSKtide (5-FAM-KKKKEEIYFFFG-NH2) and 400 uM ATP at 25 C for 90 minutes in the presence or absence of a dosed concentration series of compound (1% DMSO final concentration). The reaction was stopped by the addition of 70 ul of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 35 mM EDTA and 0.2% of Coating Reagent 3 (Caliper Lifesciences)). The plate was then read on a Caliper LabChip® EZ Reader II (protocol settings: −1.9 psi, upstream voltage −700, downstream voltage −3000, post sample sip 35 s).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 539.42, ALogP 6.81, HBA 7, HBD 2, PSA 94.6, QED 0.24
Compound (SMILES): CC(=O)Nc1ccc(C(=O)N2CCC3(CC2)NC(=O)NC3=O)cc1
InChIKey: RLJBRGWDAVMMEZ-UHFFFAOYSA-N
Target: Protein cereblon (SINGLE PROTEIN), Homo sapiens
UniProt: Q96SW2
Assay: Fluorescence Direct Binding Asaay: Determination of the affinities of compounds to protein containing one or more tryptophan is measurable by monitoring the fluorescence emission in direct mode. The measurements, depending on the protein available amounts, were performed either manually in a cuvette on ISS-PCI photon counting spectrofluorometer or automatically in well plates on a fluorescence plate reader device. Fluorescence titrations were performed at 20° C. in the chosen binding assay buffer by using a defined constant protein concentration against ligand concentration variations. Small aliquots of known ligand concentration solubilized in DMSO were added and the fluorescence, excited at 280 nm, was recorded at 340 nm. The fluorescence intensity was corrected for protein dilution and for the filter effect (Birdsall, B., King, R. W., Wheeler, M. R., Lewis, C. A. Jr, Goode, S. R., Dunlap, R. B. & Roberts, G. C. (1983). Anal. Biochem.132, 353-361). The corrected fluorescence intensity was plotted against the ligand concentration and fitted using a four-parameter sigmoidal function from which the equilibrium dissociation constant Kd was computed using the law of mass action assuming a 1:1 proteinligand complex (Eftink, Methods Enzymol. 1997; 278:221-57). The process includes:Optimization of measurement parameters to minimize protein consumption and to minimize the dilution effect and the DMSO content. Titration measurements of the protein against ligand by at least 12 titration steps to obtain an s-curve fit. Repeat the same titration measurements with the ligand alone to enable correction. Check the stability of the protein once by titration against DMSO alone. Determination of the molar extinction coefficients of the ligand at 280 and 340 nm with help of an UV-spectrophotometer. Use Excel template for the correction of the measured raw data. Use GraphPad Prism software for the quadratic binding fit and the KD evaluation.
Measurement: Kd = 27 nM (pChEMBL 7.570000171661377)
Descriptors: MW 330.34, ALogP 0.46, HBA 4, HBD 3, PSA 107.61, QED 0.68
Compound (SMILES): Cc1ccc2c(c1)CN(C1CCc3ccc(C(c4ccc5c(nnn5C)c4C)C(C)(C)C(=O)O)cc31)C[C@@H](C)O2
InChIKey: SQRQXRPSWVMYOV-QNJQMVSBSA-N
Target: NAD(P)H dehydrogenase [quinone] 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P15559
Assay: BEAS-2B NQO1 MTT Assay: NAD(P)H:quinone oxidoreductase 1 (NQO1), also called DT diaphorase, is a homodimeric FAD-containing enzyme that catalyzes obligatory NAD(P)H-dependent two-electron reductions of quinones and protects cells against the toxic and neoplastic effects of free radicals and reactive oxygen species arising from one-electron reductions. The transcription of NQO1 is finely regulated by NRF2, and thus NQO1 activity is a good marker for NRF2 activation. On day one, frozen BEAS-2B cells (ATCC) are thawed in a water bath, counted, and re-suspended at a concentration of 250,000 cells/mL. Fifty microliters of cells are plated in 384 well black clear-bottomed plates. Plates are incubated at 37° C., 5% CO2 overnight. On day two, plates are centrifuged and 50 nL of compound or controls are added to the cells. Plates are then incubated at 37° C., 5% CO2 for 48 hours. On day four, medium is aspirated from the plate and crude cell lysates are made by adding 13 uL of 1× Cell Signaling Technologies lysis buffer with 1 Complete, Mini, EDTA-free Protease Inhibitor Tablet (Roche) for each 10 mL of lysis buffer. After lysis plates are incubated for 20 minutes at room temperature. Two microliters of lysate are removed for use in Cell Titer Glo assay (Promega) and MTT cocktail is prepared (Prochaska et. al. 1998) for measurement of NQO1 activity. Fifty microliters of MTT cocktail is added to each well, plate is centrifuged, and analyzed on an Envision plate reader (Perkin Elmer) using Absorbance 570 nm label for 30 minutes. Product formation is measured kinetically and the pEC50 of NQO1 specific activity induction is calculated by plotting the change in absorbance (Delta OD/min) versus the log of compound concentration followed by 3-parameter fitting.
Measurement: EC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 538.69, ALogP 6.1, HBA 6, HBD 1, PSA 80.48, QED 0.33
Compound (SMILES): CC(C)COc1nc2ccc(S(=O)(=O)NC3(C)CC3)cc2c(=O)n1C
InChIKey: HOWIEDWSBQTSQA-UHFFFAOYSA-N
Target: Poly(ADP-ribose) glycohydrolase (SINGLE PROTEIN), Homo sapiens
UniProt: Q86W56
Assay: PARG Assay: PARG In vitro assays were conducted in a total volume of 15 ul in a standard 384 well format. 5 ul of Human Full Length PARG (Produced internally by Astra Zeneca), used at a final reaction concentration of 80 pM, was added to 5 ul of Ribosylated PARP substrate (also produced internally by Astra Zeneca) at final reaction concentration of 4.5 nM in assay buffer (50 mM Tris pH7.4, 0.1 mg/ml BSA, 3 mM EDTA, 0.4 mM EGTA, 1 mM DTT, 0.01% Tween 20, 50 mM KCl). The reaction was incubated at room temperature for 10 minutes and then 5 ul detection reagent was added. Detection Reagent consists of 42 nM MAb Anti-6HIS XL665 (CisBio: 61HISXLB) and 2.25 nM Streptavidin Europium Cryptate (CisBio: 610SAKLB), both at 3× working stock concentrations (final concentrations of 14 nM and 0.75 nM respectively), in a detection buffer of 50 mM Tris pH7.4, BSA at 0.1 mg/ml and KF at 100 mM. Following incubation at room temperature for 60 minutes in the dark, TR-FRET signal was measured at Ex 340 and Em 665 and Em 620. A ratio was calculated as Em665/EM620×104 for each well and used to calculate percent inhibition for test compounds.
Measurement: IC50 = 3506 nM (pChEMBL 5.460000038146973)
Descriptors: MW 365.46, ALogP 1.8, HBA 6, HBD 1, PSA 90.29, QED 0.84
Compound (SMILES): CC(C)NC(=O)[C@H]1CN(c2ccc(-c3cc(-c4cnn(C)c4)cn4ncc(C#N)c34)cn2)CCO1
InChIKey: CLEHHSAVKWGWBO-JOCHJYFZSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of General Formula I were screened for their ability to inhibit wild type 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; Cat. No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Cat. No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Cat. 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 = 202 nM (pChEMBL 6.699999809265137)
Descriptors: MW 470.54, ALogP 2.4, HBA 9, HBD 1, PSA 113.37, QED 0.48
Compound (SMILES): COCC(C)(C)NC(=O)[C@@H]1CN(CC2CC2)CC[C@H]1NC(=O)c1cn(-c2ccc(F)cc2F)nn1
InChIKey: DZHIXHSGPVKOQN-IEBWSBKVSA-N
Target: Atypical chemokine receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P25106
Assay: In Vitro Assay: Tango CXCR7-bla U2OS cells are detached from culture dishes with 0.05% trypsin-EDTA and collected in growing medium (McCoy's 5A 90% (v/v), dialyzed FCS 10% (v/v), 0.1 mM NEAA, 25 mM HEPES (pH7.3), 1 mM sodium pyruvate, P/S 1% (v/v) 50 μg/ml Hygromycin, 100 μg/ml Geneticin, 200 μg/ml Zeocin), spinned down and resuspended in assay medium (McCoy's 5A 90% (v/v), dialyzed FCS 1% (v/v), 0.1 mM NEAA, 25 mM HEPES (pH7.3), P/S 1% (v/v)). 10′000 cells per well (in 30 μl) are seeded in a 384 well plate (black-walled, clear bottom). The plate is incubated at 37° C./5% CO2 for 24 hours. Test compounds are dissolved to 10 mM in DMSO and serially diluted in DMSO to 500× of the final concentration for dose response curves. Compounds are then diluted 1:100 in assay medium to 5× of the final concentration. 10 μl/well of diluted compounds are added to the assay plate and incubated for 15 minutes at 37° C. Thereafter CXCL12/SDF1-α is diluted in assay medium to 5× of the final concentration (its EC80 value for receptor activation) and 10 μl/well are added to the assay plate. The agonist leads to activation of the receptor and therefore to b-arrestin recruitment. Compounds acting as antagonists reduce this activation. The plate is incubated for 22 hrs at 37° C. 10 μl/well of detection reagent (LiveBLAzer-FRET BIG (CCF4-AM) substrate) is transferred to the assay plate and the plate is incubated for 2 hours at room temperature protected from light. Fluorescent counts are determined (Scan1: Ex 409/20 nm, Em 460/30 nm, Scan 2: Ex 409/20 nm, Em 530/30 nm).
Measurement: IC50 = 535 nM (pChEMBL 6.269999980926514)
Descriptors: MW 490.56, ALogP 1.92, HBA 7, HBD 2, PSA 101.38, QED 0.56
Compound (SMILES): CNC(=O)CN1CCN(C(=O)c2ccc(N/C(=C3\C(=O)Nc4cc(C(=O)OC)c(C)cc43)c3ccccc3)cc2)CC1
InChIKey: RJIWXRGRXHMIJN-ZIADKAODSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: FGFR1 Enzyme Inhibition: The inhibitory activities of compounds of the invention against FGFR1 (FGFR1 Kinase Enzyme System: Promega), were evaluated by mixing the FGFR1 protein (3.12 ng/mL, 2 μL), substrate (Poly (4:1 Glu4, Tyr1), 100 ng/mL, 2 μL) with the test compound (2 μL at either 3 μM, 0.67 μM, 0.15 μM, 0.033 μM, 0.0073 μM, 0.0016 μM, 0.0036 μM or 0.00008 μM) for 90 min at 25° C. The kinase reaction was then initiated by adding ATP (50 μM, 2 μL) and the mixture was incubated for 1 hr at 25° C. ADP-Glo reagent was added for 40 min (8 μL) then development reagent (16 μL) was added for 40 min prior to detection in a microplate reader (EnVision Multilabel Reader, Perkin Elmer).
Measurement: Ki = 23.9 nM (pChEMBL 7.619999885559082)
Descriptors: MW 567.65, ALogP 3.22, HBA 7, HBD 3, PSA 120.08, QED 0.3
Compound (SMILES): CC(C)n1cnc2cc(-c3ccc4c(c3)N(C3CC(N5CCCC(C)(C)C5)C3)C(=O)C43CCN(C(=O)C4CCO4)CC3)nc(Nc3ccncc3F)c21
InChIKey: QLAVOARKAZHNBH-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: Enzymatic Activity Assay: Recombinant HPK1 kinase domain produced via baculovirus infection of insect cells was obtained from Proteros (Proteros Biostructures #PR-0322) and was pre-activated in the presence of 2 mM ATP (Sigma-Aldrich, cat #GE27-2056-01) and 2 mM magnesium chloride for 16 hours at 4° C. The protein reaction mixture was then loaded to a desalting column (Thermo Fisher Scientific, Cat #89889) to remove excess ATP. HPK1 was eluted with buffer containing 20 mM Tris (2-Amino-2-(hydroxymethyl)propane-1,3-diol) pH 8.0, 150 mM NaCl, 2 mM dithiothreitol and 5% glycerol, and was frozen at −80° C. for later use. HPK1 dual phosphorylation was confirmed by mass spectrometry.Ten nanoliters of test compounds dissolved in DMSO at various concentrations were dispensed into a 384-well ProxiPlate (PerkinElmer #6008289). Five microliters of a solution of recombinant HPK1 diluted in HPK1 kinase assay buffer (50 mM BES [N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid], pH 7.0; 10 mM magnesium chloride; 0.01% Triton X-100; 1 mM dithiothreitol; 0.01% bovine serum albumin; 0.1 mM sodium orthovanadate) was added to the compound-containing plate and was incubated for 15 minutes at 25° C. Five microliters of a mixture of ATP (Sigma-Aldrich #A6559) and peptide substrate STK S1 (Cisbio #61ST1BLC) diluted in HPK1 kinase assay buffer was then added to start the reaction. Final concentrations were 0.15 nM for HPK1, 10 μM for ATP, and 1 μM for the STK S1 peptide substrate. The reaction mixture was incubated at 25° C. for 3 hours and was stopped with the addition of 10 μl of an EDTA (Ethylenediaminetetraacetic acid)-containing detection buffer (Cisbio #62SDBRDF) supplemented with Europium cryptate-labeled anti-phospho-serine/threonine antibodies (Cisbio #62ST1PEJ) and XL665-labeled streptavidin (Cisbio #610SAXLG). The mixture was incubated for 16 hours at room temperature and peptide phosphorylation was measured by time-resolved fluorescence energy transfer (665 nm/620 nm) on an Envision plate reader (PerkinElmer).
Measurement: IC50 = 0.2 nM (pChEMBL 9.699999809265137)
Descriptors: MW 720.89, ALogP 6.61, HBA 9, HBD 1, PSA 108.72, QED 0.23
Compound (SMILES): CN1C(=O)N(c2ccc(C(=O)O)cc2)Cc2c1cnc1[nH]c(-c3ccc(CN4CCC(S(C)(=O)=O)CC4)cc3)cc21
InChIKey: NNLYRJAWAIDKSB-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK2 LanthaScreen JH1 Binding Assay: JAK2 JH1 binding assay utilizes catalytic domain (JH1, amino acids 826-1132) of human JAK2 expressed as N-terminal FLAG-tagged, biotinylated protein in a baculovirus expression system (Carna Biosciences, Product #08-445-20N). The assay was conducted in black 384-well polystyrene plates in a final reaction volume of 20 μL. JAK2 JH1 (1.5 nM) was incubated with compounds (100 nL serially diluted in DMSO) in the presence of 50 nM Fluorescent JAK2-JH1 Tracer and 0.5 nM Streptavidin-Tb cryptate (Cisbio Part #610SATLB) in assay buffer (50 mM Tris, pH=7.5, 10 mM MgCl, 0.01% Brij-35, 0.1% BSA, 1 mM EGTA, 5% Glycerol and 5 mM DTT). Non-specific binding was accessed in the presence of 2 mM ATP. After incubation for 2 hours at 25° C., LanthaScreen signals were read on a PHERAstar FS plate reader (BMG LABTECH). Data was analyzed with IDBS XLfit and GraphPad Prism 5.0 software using a four parameter dose response curve to determine IC50 for each compound.
Measurement: IC50 = 1750 nM (pChEMBL 5.760000228881836)
Descriptors: MW 573.68, ALogP 4.51, HBA 6, HBD 2, PSA 126.91, QED 0.35
Compound (SMILES): Cc1cc(F)c(C(=O)Nc2cccc(-c3nnc4n3CCC[C@@H]4O)n2)cc1-n1cnc(C2CC2)c1
InChIKey: CLFVPXSMEDLWQW-NRFANRHFSA-N
Target: Mitogen-activated protein kinase kinase kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99683
Assay: Enzyme Activity: Primary reaction buffer solution: 20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSOTreatment of the Compound:The tested compounds were formulated into a 10 mM stock solution in DMSO, diluted in 3-fold gradient for a total of 10 concentrations, and placed in a 384-well plate (Cyclic Olefin Copolymer LDV Echo®).Kinase Name: ASK1/MAP3K5 (Invitrogen, Carlsbad, Calif.)Type: Recombinant Human Full Length Protein, GST-taggedFinal reaction concentration of the enzyme: 20 nMSubstrate: Myelin basic protein, MBP (Active Motif, Carlsbad, Calif.)Final reaction concentration of the substrate: 20 μMExperimental Procedures:1. The substrate was dissolved in a freshly prepared primary reaction buffer solution,2. The desired coenzyme factor was added to the above substrate solution,3. The kinase was added to the substrate solution and mix gently,4. The solution tested compound in DMSO was added to the kinase reaction solution and incubated at room temperature for 20 minutes.5. The reaction was initiated by adding 33P-ATP (specific activity 10 μCi/μL) to the reaction solution.6. Incubated at room temperature for 2 hours.7. A small portion of the reactants were placed onto the P-81 ion exchange filter paper.8. The filter paper was washed three times with 0.75% phosphate buffer to wash away unbound phosphate, and then dried.9. The radioactivity remaining on the filter paper was determined,10. The data for the kinase activity was expressed as the ratio of the kinase activity remaining in the test sample to the kinase activity in the vehicle (DMSO).
Measurement: IC50 = 12.5 nM (pChEMBL 7.900000095367432)
Descriptors: MW 473.51, ALogP 3.93, HBA 8, HBD 2, PSA 110.75, QED 0.45