lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(C)N1CCOc2c(F)cc(-c3nc(Nc4ccc5c(n4)CCNC5)ncc3F)cc21
InChIKey: PSWOMNFIVDHMDM-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Activity Assay: PI3Kδ: Briefly, this assay was conducted in buffer (Tris-HCl 40 mM (pH7.5), Orthovanadate 3 mM, MgCl2 20 mM, DTT 2 mM, CHAPS 0.05%, DMSO 1%). PI3Kδ kinase was added to the reaction solution and mixed gently. The test compounds in 100% DMSO (at specific solutions) were mixed with the kinase reaction mixture to achieve the final compounds at pre-defined concentrations (e.g., range—0.5 nM to 100 μM) by Acoustic technology (Echo550; nanoliter range). After incubating for 10 min at room temp, ATP was added into the reaction mixture to initiate the reaction followed by a 30-min incubation at 30° C. After quenching the reaction with ADP-Glo reagent, the plates were incubated for 40 min. The Detection Mixture was added, and the plate was incubated for an additional 30 min. At the end of incubation, luminescence was measured. For data analysis, the luminescence was converted into μM ADP production based on ADP standard curves.
Measurement: IC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 438.48, ALogP 3.81, HBA 7, HBD 2, PSA 75.2, QED 0.64
Compound (SMILES): CCc1ccc(Oc2ccccc2)c(O)c1
InChIKey: XZOPHRPAUNJURP-UHFFFAOYSA-N
Target: Enoyl-[acyl-carrier-protein] reductase [NADPH] FabI (SINGLE PROTEIN), Staphylococcus aureus
UniProt: Q6GI75
Assay: Tight-binding assay: Kinetics were performed on a Cary 100 spectrophotometer (Varian) at 20° C. Reaction velocities were measured by monitoring the oxidation of NAD(P)H to NAD(P)+ at 340 nm (Figure US10071965-20180911-P00001=6220 M−1 cm−1). For saFabI, the reaction mixture was identical to that described previously for progress curve experiments (Schiebel et al. (2012). Structure 20:802-13). For ecFabI, the final reaction mixture contained ecFabI (75 nM), trans-2-butenoyl-CoA (800 μM; Sigma and Advent Bio), NADH (300 μM; Sigma), NAD+ (400 μM; Sigma), and inhibitor (2 v/v % DMSO) in 50 mM potassium phosphate, pH 7.5, 150 mM NaCl, 8 v/v % glycerol. For InhA, the final reaction mixture contained InhA (100 nM), trans-2-octenoyl-CoA (200 μM), NADH (250 μM), NAD+ (200 μM), and inhibitor (2 v/v % DMSO) in 30 mM PIPES, pH 6.8, 150 mM NaCl, 1 mM EDTA, 8 v/v % glycerol. The resulting curves were fit to the Morrison and Walsh integrated rate equation (Equation 1) (Morrison et al.
Measurement: Ki = 32.2 nM (pChEMBL 7.489999771118164)
Descriptors: MW 214.26, ALogP 3.75, HBA 2, HBD 1, PSA 29.46, QED 0.84
Compound (SMILES): Cc1c(-c2ccc3oc(N)nc3c2)ccc2ncc(N3CCN[C@@H](C)C3)nc12
InChIKey: QIQZPJHDPGTRMV-LBPRGKRZSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42338
Assay: Inhibition Assay of PI3K: An exemplary system is PI 3-Kinase (human) HTRF Assay from Upstate. The assay can be carried out according to the procedures suggested by the manufacturer. Briefly, the assay is a time resolved FRET assay that indirectly measures PIP3 product formed by the activity of a PI3-K. The kinase reaction is performed in a microtitre plate (e.g., a 384 well microtitre plate). The total reaction volume is approximately 20 ul per well. In the first step, each well receives 2 ul of test compound in 20% dimethylsulphoxide resulting in a 2% DMSO final concentration. Next, approximately 14.5 ul of a kinase/PIP2 mixture (diluted in 1× reaction buffer) is added per well for a final concentration of 0.25-0.3 ug/ml kinase and 10 μM PIP2. The plate is sealed and incubated for 15 minutes at room temperature. To start the reaction, 3.5 ul of ATP (diluted in 1× reaction buffer) is added per well for a final concentration of 10 μM ATP. The plate is sealed and incubated for 1 hour at room temperature. The reaction is stopped by adding 5 ul of Stop Solution per well and then 5 ul of Detection Mix is added per well. The plate is sealed, incubated for 1 hour at room temperature, and then read on an appropriate plate reader.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 374.45, ALogP 3.13, HBA 7, HBD 2, PSA 93.1, QED 0.56
Compound (SMILES): CCOC(=O)Cn1nc(-c2cc3cc(O)ccc3[nH]2)c2c(N)ncnc21
InChIKey: KFACBXKHIBUBDZ-UHFFFAOYSA-N
Target: Phosphatidylinositol 3-kinase regulatory subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O00459
Assay: In vitro kinase assay: This assay is relatively simple, reasonably sensitive, and the peptide substrate can be adjusted both in terms of sequence and concentration to meet the assay requirements. Other exemplary kinase assays are detailed in U.S. Pat. No. 5,759,787 and U.S. application Ser. No. 12/728,926, both of which are incorporated herein by reference.
Measurement: IC50 > 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 352.35, ALogP 1.83, HBA 8, HBD 3, PSA 131.94, QED 0.48
Compound (SMILES): COC(=O)c1cc2c(cc1C)/C(=C(/Nc1ccc(C(=O)NCCN3CCC(F)(F)CC3)cc1)c1ccccc1)C(=O)N2
InChIKey: JNFPOOBKOALVEY-DQSJHHFOSA-N
Target: Vascular endothelial growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P17948
Assay: VEGFR1 Enzyme Inhibition: The inhibitory activities of compounds of the invention against VEGFR1 (VEGFR1 Kinase Enzyme System: Promega), were evaluated by mixing the VEGFR1 protein (12.5 ng/mL, 2 μL), substrate (IGFR1Rtide, 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 initiated by adding ATP (50 μM, 2 μL) and the mixture was incubated for 90 min 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 = 513 nM (pChEMBL 6.289999961853027)
Descriptors: MW 574.63, ALogP 5.18, HBA 6, HBD 3, PSA 99.77, QED 0.25
Compound (SMILES): CC(C)C(=O)N1CCC(c2cc(-c3ccc(NC(=O)c4cn(C5CC5)c(=O)n(-c5ccccc5)c4=O)cc3)c3c(N)ncnn23)CC1
InChIKey: INCLUHXJYYFKBR-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: TAM Enzymatic Assay: The kinase assay buffer contained 50 mM HEPES, pH7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% NP-40 and 2 mM DTT. 0.1 ul test compounds dissolved in DMSO were transferred from compound plates to white 384-well assay plates (Greiner LLIMITRAC plates). The final concentration of DMSO was 1.25%. Enzyme solutions of 5.1 nM phosphor-Axl, or 0.0625 nM c-Mer (Carna Biosciences, 08-108), or 0.366 nM Tyro3 (Life Technologies, PR7480A) were prepared in assay buffer. A 1 mM stock solution of peptide substrate Biotin-EQEDEPEGDYFEWLE-amide SEQ ID NO: 1 (Quality Controlled Biochemicals, MA) dissolved in DMSO was diluted to 1 uM in assay buffer containing 2000 uM ATP. 4 ul enzyme solution (or assay buffer for the enzyme blank) was added to the appropriate wells in each plate, and then 4 ul/well substrate solution was added to initiate the reaction. The plate was protected from light and incubated at room temperature for 60 min. The reaction was stopped by adding 4 ul detection solution containing 50 mM Tris-HCl, pH7.8, 150 mM NaCl, 0.05% BSA, 45 mM EDTA, 180 nM SA-APC (Perkin Elmer, CR130-100) and 3 nM Eu-W1024 anti-phosphotyrosine PY20 (Perkin Elmer, AD0067). The plate was incubated for 1 h at room temperature, and HTRF (homogenous time resolved fluorescence) signal was measured on a PHERAstar FS plate reader (BMG labtech). Percentage of inhibition was calculated for each concentration and IC50 value was generated from curve fitting with GraphPad Prism software.
Measurement: IC50 > 100 nM (pChEMBL 7)
Descriptors: MW 632.73, ALogP 4.24, HBA 10, HBD 2, PSA 149.62, QED 0.27
Compound (SMILES): NCC1(O)CCN(c2nc(Oc3cccc(F)c3)cc(C(F)(F)F)n2)CC1
InChIKey: HYEMQFSXSZXWCD-UHFFFAOYSA-N
Target: Sodium-dependent noradrenaline transporter (SINGLE PROTEIN), Homo sapiens
UniProt: P23975
Assay: Competition Binding Assay: The affinity of the compounds of the invention to the human DAT or NET or SERT transporters is assessed by using the [3H]WIN-35,428 or [3H]nisoxetine or [3H]citalopram binding assays in recombinant human DAT, NET and SERT membranes with the SPA technology. The final assay volume is 50 μL in 384 well plates.Briefly, 0.5 μL of test compound in neat DMSO or 0.5 μL of DMSO for total binding (TB) or 0.5 μL of indatraline 1 mM (10 μM final concentration) for non specific binding (NSB) are added to the assay plate. 50 μL of the SPA mixture is added to each well, containing: 30 μg/mL or 10 μg/mL or 25 μg/mL DAT, NET, SERT membranes, respectively; 5 nM [3H]WIN-35,428 or 5 nM [3H]nisoxetine or 1 nM [3H]citalopram, for DAT, NET, SERT assay, respectively; 2.5 mg/mL or 1 mg/mL or 4 mg/mL WGA-PVT SPA beads (PerkinElmer RPNQ0001, for DAT, NET, SERT assay, respectively. All components are added to Assay Buffer (20 mM HEPES pH 7.4, 145 mM NaCl, 5 mM KCl, 0.01% Pluronic F-127). 0.02% BSA was used for DAT binding only. Plates are sealed with Topseal A and centrifuged 1 min, 800 rpm. Plates are loaded into a 1450 Microbeta TriLux (Perkin-Elmer) plate reader and the radioactivity counted after at least 4 hrs or overnight incubation at room temperature. Curve fitting and IC50 estimations are performed using a four parameter model in XLfit (IDBS, Guilford, UK) for Microdoft Excel (Microsoft, Redmond, Wash.).
Measurement: IC50 = 145 nM (pChEMBL 6.840000152587891)
Descriptors: MW 386.35, ALogP 2.72, HBA 6, HBD 2, PSA 84.5, QED 0.79
Compound (SMILES): COCC1(CC(C)C(=O)N2CCN(c3ccc(F)c(Cl)c3)[C@@H](C)C2)NC(=O)NC1=O
InChIKey: MKGHITYLXXYNMZ-BTTAGBNVSA-N
Target: 72 kDa type IV collagenase (SINGLE PROTEIN), Homo sapiens
UniProt: P08253
Assay: In Vitro Assay: MMP2: Protocol 2: The basis for the assay is the cleavage of the substrate 390 MMP FRET substrate I (Anaspec, Catalog n#: AS-27076) by human MMP2 (R&D SYSTEMS INC., Cat #902-MP).For the dose response (10 point), 4 μL of a dilution series of compound (2 mM highest concentration, 1/5 dilution in DMSO further diluted 1 in 10 in water, corresponding to a final highest concentration of 20 μM), is transferred to 384 well Fluotrac 200 plate (Greiner, cat #781076) and incubated at room temperature for 30 min with a 26 μL buffer solution (45 mM Tris pH 7.5, 9 mM CaCl2, 135 mM NaCl, 0.045% Brij35) containing MMP2 (0.03 ng/L) (it will be appreciated by the skilled person that the potency read out is independent of the enzyme concentration).The reaction is initiated by adding to the assay plate 390 MMP FRET substrate I (10 μL, 2.5 μM, Anaspec) in the same buffer.Finally, the fluorescence is read on the Envision (Perkin Elmer) after an incubation of 30 min at room temperature (Excitation 485 nm, Emission 530).
Measurement: IC50 = 581 nM (pChEMBL 6.239999771118164)
Descriptors: MW 440.9, ALogP 1.77, HBA 5, HBD 2, PSA 90.98, QED 0.66
Compound (SMILES): C=CC(=O)Nc1cccc(Nc2nc(Nc3cccc(OCC4CCCN(C)C4)c3)ncc2F)c1
InChIKey: UGWZEBRWDXGJIN-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ITK/TSK (SINGLE PROTEIN), Homo sapiens
UniProt: Q08881
Assay: Omnia Assay: ITK: This example describes continuous-read kinase assays to measure inherent potency of compound against active forms of ITK enzymes as described in Example 251 above except that the modified ITK-optimized reagent conditions are:[ITK]=10 nM, [ATP]=25 [Y6-Sox]=10 μM (ATP KMapp=33 μM).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 476.56, ALogP 4.95, HBA 7, HBD 3, PSA 91.41, QED 0.38
Compound (SMILES): CC(C)(O)C1(Nc2n[nH]c3nccc(Oc4ccc(NC(=O)c5ccnn(-c6ccc(F)cc6)c5=O)cc4F)c23)CCCC1
InChIKey: UVRZBVULQRZPDL-UHFFFAOYSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Enzyme Assay: The affinity of compound binding to wild type and mutant human MET kinases is measured using Invitrogen's LanthaScreen Eu Kinase Binding technology. Briefly, GST-tagged recombinant human MET kinase domain from Signal Chem (see Table 8 below for concentration in assay) is incubated with 50 nM Alexa-Fluor® Tracer 236 (Invitrogen Cat No. PR9078A) and 2 nM Europium-anti-GST (Invitrogen Cat. No. A151 6) along with test compound in a buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, 1 mM DTT, and 2% DMSO. Compounds are typically prepared in a three-fold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-minute incubation at 22° C., the reaction is measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) calculated using a ratiometric emission factor. 100 POC is determined using no test compounds and 0 POC is determined using a concentration of control compound that completely inhibits the enzyme
Measurement: IC50 = 625 nM (pChEMBL 6.199999809265137)
Descriptors: MW 601.61, ALogP 5.32, HBA 9, HBD 4, PSA 147.05, QED 0.19
Compound (SMILES): O=C(c1ccc(F)c(Cl)c1)N1CCC(F)(CNCCOc2cccc(Cl)n2)CC1
InChIKey: VTBXTDAXQSIVGY-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 1A (SINGLE PROTEIN), Homo sapiens
UniProt: P08908
Assay: Agonist activity at human 5-HT1A receptor expressed in CHO-K1 cells co-expressing mitochondrially targeted aequorin assessed as calcium mobilization incubated for 1 hr by MicroBeta2 LumiJET plate counter analysis
Measurement: EC50 = 147.91 nM (pChEMBL 6.829999923706055)
Descriptors: MW 444.31, ALogP 4.14, HBA 4, HBD 1, PSA 54.46, QED 0.52
Compound (SMILES): Cn1ncc(-c2cc(-c3cnn(C4(CC#N)CC(C#N)C4)n3)c3cccnc3c2)c1N
InChIKey: GRZXUCDONIKIEF-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: JAK Caliper Enzyme Assay at 1mM ATP: Compounds were added to a 384-well plate. Reaction mixtures contained 10 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% BSA, 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20% to 30% phosphorylation. The assays were stopped with 15 μL of 180 mM HEPES, pH 7.4, 20 mM EDTA, and 0.2% Coating Reagent 3. The assay plates were placed on a Caliper Life Science LC3000 instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 115 nM (pChEMBL 6.940000057220459)
Descriptors: MW 409.46, ALogP 3.02, HBA 9, HBD 1, PSA 135.02, QED 0.55
Compound (SMILES): CN1CCN([C@@H]2CCCN(c3cnc(C(N)=O)c(Nc4ccccc4)n3)C2)C1=O
InChIKey: SYXRBODSNFMHRI-OAHLLOKOSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: HotSpot kinase Assay: The degree of inhibition of a panel of kinases is determined using the in vitro HotSpot kinase assay (purified enzymes, 33P-ATP, an appropriate substrate and 1 uM ATP).
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 395.47, ALogP 1.66, HBA 6, HBD 2, PSA 107.69, QED 0.8
Compound (SMILES): Cn1cc(-c2cnc3ccc(-c4ccc5oc(N)nc5c4)cc3n2)cn1
InChIKey: JXVKOQWQVWGTSM-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42338
Assay: Inhibition Assay of PI3K: An exemplary system is PI 3-Kinase (human) HTRF Assay from Upstate. The assay can be carried out according to the procedures suggested by the manufacturer. Briefly, the assay is a time resolved FRET assay that indirectly measures PIP3 product formed by the activity of a PI3-K. The kinase reaction is performed in a microtitre plate (e.g., a 384 well microtitre plate). The total reaction volume is approximately 20 ul per well. In the first step, each well receives 2 ul of test compound in 20% dimethylsulphoxide resulting in a 2% DMSO final concentration. Next, approximately 14.5 ul of a kinase/PIP2 mixture (diluted in 1× reaction buffer) is added per well for a final concentration of 0.25-0.3 ug/ml kinase and 10 μM PIP2. The plate is sealed and incubated for 15 minutes at room temperature. To start the reaction, 3.5 ul of ATP (diluted in 1× reaction buffer) is added per well for a final concentration of 10 μM ATP. The plate is sealed and incubated for 1 hour at room temperature. The reaction is stopped by adding 5 ul of Stop Solution per well and then 5 ul of Detection Mix is added per well. The plate is sealed, incubated for 1 hour at room temperature, and then read on an appropriate plate reader.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 342.36, ALogP 3.42, HBA 7, HBD 1, PSA 95.65, QED 0.53
Compound (SMILES): CC(C)(C)C(=O)NCCN1CCC(c2cccc(C(F)(F)F)n2)CC1
InChIKey: WRRJZZBXRRDBBD-UHFFFAOYSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Evaluation of Binding Activity to Human 5-HT1A Receptor, Human D4 Receptor, and Human D2 Receptor : The binding affinity of the present compounds to human 5-HT1A receptor, human D4 receptor, and human D2 receptor was measured in a manner mentioned below.CHO cell membrane fraction in which human 5-HT1A receptor, human D4 receptor, and human D2 receptor were expressed was purchased from PerkinElmer Co., Ltd. In the evaluation test of binding, the test compound dissolved in DMSO, each receptor membrane preparation diluted with buffer solution, and [3H] 8-OH-DPAT (for 5-HT1A receptor), [3H] dopamine (for D4 receptor), or [3H] spiperone (for D2 receptor) (all were obtained from PerkinElmer Co., Ltd.) were mixed, and each mixture was incubated at room temperature for 30 or 60 minutes. The nonspecific binding to each receptor was evaluated by a competition binding experiment in the presence of 10 μmol/L 8-OH-DPAT, 10 μmol/L dopamine, or 10 μmol/L spiperone, respectively. The radioactivity of each receptor-binding sample was measured with a liquid scintillation counter (PerkinElmer Co., Ltd.), the 50% inhibitory concentration was calculated, and Ki value was evaluated based on the dissociation constant and the substrate concentration calculated in the saturated bond test, which was used as binding affinity.
Measurement: IC50 = 2443 nM (pChEMBL 5.610000133514404)
Descriptors: MW 357.42, ALogP 3.44, HBA 3, HBD 1, PSA 45.23, QED 0.9
Compound (SMILES): COC/C=C/C(=O)N1CCCC[C@H]1c1nc(-c2ccc(C(=O)Nc3ncccn3)cc2)c2c(N)nccn12
InChIKey: MFCPWIVFRPCSJE-PDGUBNMISA-N
Target: Tyrosine-protein kinase Fyn (SINGLE PROTEIN), Homo sapiens
UniProt: P06241
Assay: FynT Enzyme Activity : FynT enzyme activity is measured using the IMAP (immobilized metal ion affinity-based fluorescence polarization) assay as outlined below.FynT enzyme (Biomol catalog # SE-287), is diluted to 0.5 μg/mL in KR buffer (10 mM Tris-HCl, 10 mM MgCl2, 0.01% Tween-20, 0.05% NaN3, 1 mM DTT, 2 mM MnCl2, pH 7.2).Serial dilution log 10 from 2 mM to 63.2 nM of test compounds are made in 100% DMSO. The dilutions in DMSO are then diluted 50-fold in KR-buffer of which 5 μl is used in the assay, leading to a final compound concentration range in the assay from 10 μM to 0.316 nM.5 μL/well of test compound in KR buffer (final DMSO concentration in the assay is 1%) is mixed with 5 μl/well of 0.5 μg/mL FynT enzyme (final concentration in the assay is 125 ng/mL). Test compounds and FynT enzyme are pre-incubated 60 minutes at room temperature, before adding 5 μL/well of 400 nM Fluorescin labeled substrate peptide (p34cdc2 substrate peptide, e.g. #R7157/#R7172, Molecular Devices) in KR-buffer. Final peptide substrate concentration in assay is 100 nM. The kinase assay is started by adding 5 μL/well of 0.8 μM ATP in KR-buffer (final ATP concentration is 0.2 μM ATP, Km ATP in FynT IMAP assay). Following incubation for 2 h at room temperature the enzyme reaction is stopped by adding 40 μL/well IMAP Progressive Binding Solution (according to suppliers (Molecular Devices) protocol using 75% 1× buffer A and 25% 1× buffer B with 1:600 Progressive Binding Solution). After 60 min incubation at room temperature in the dark the FP signal is read. Fluorescence at 535 nm is measured using parallel and perpendicular filters to determine differences in rotation due to binding of the phosphorylated substrate peptide to the beads. Values are calculated as percentage of the difference in readout (ΔmPi) of the controls with and without ATP. EC50 values are determined by curve fitting of the experimental results using Activity Base.
Measurement: EC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 512.57, ALogP 3.28, HBA 9, HBD 2, PSA 140.63, QED 0.36
Compound (SMILES): Cc1c(-c2[nH]c3ccc(C4CC5CCC(C4)N5CCS(C)(=O)=O)cc3c2C(C)C)cn2ncnc2c1C
InChIKey: AQZULFVPKDRLSW-UHFFFAOYSA-N
Target: Toll-like receptor 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR96
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 = 4832 nM (pChEMBL 5.320000171661377)
Descriptors: MW 519.72, ALogP 5.37, HBA 6, HBD 1, PSA 83.36, QED 0.37
Compound (SMILES): CNCc1cc(OCc2cc(Cl)ccc2F)n(CC2CCCC2)n1
InChIKey: HTSGWANQFVFCRX-UHFFFAOYSA-N
Target: Sodium-dependent serotonin transporter (SINGLE PROTEIN), Homo sapiens
UniProt: P31645
Assay: Sert Binding Assay: Specifically, a solution containing 50 μL of [3H]-citalopram (GE Healthcare) diluted with SERT buffer (50 mmol/L Tris-HCl (pH=7.4) containing 120 mmol/L NaCl and 5 mmol/L KCl) (the final concentration: about 2 nmol/L), 149 μL of h-SERT/CHO cell membrane sample (40 μg/well for the amount of protein), and 1 μL of a solution of a test compound in DMSO or a solvent (DMSO) was reacted at room temperature for 60 minutes, and then filtered promptly by aspiration under lower pressure with a glass fiber filter coated with a 0.05% aqueous polyethyleneimine solution. The filtered substance on the glass fiber filter was washed with 250 μL of SERT buffer twice, and then transferred to an ACS-II (Amersham) 4-mL-glass vial. The radioactivity of the filtered substance remained on the filter was measured by liquid scintillation counter. The radioactivity value measured by liquid scintillation counter was deemed to be a receptor binding activity, and the binding inhibition was calculated from the total binding (TB), non-specific binding (NSB), and specific binding (SB) of a test compound according to the following equations.
Measurement: Ki = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 351.85, ALogP 4.16, HBA 4, HBD 1, PSA 39.08, QED 0.81
Compound (SMILES): CC(C)n1cnc2ccc(-c3ccc4oc(N)nc4c3)cc2c1=O
InChIKey: CXDMXFLYRNQGIH-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Inhibition Assay of PI3K: An exemplary system is PI 3-Kinase (human) HTRF Assay from Upstate. The assay can be carried out according to the procedures suggested by the manufacturer. Briefly, the assay is a time resolved FRET assay that indirectly measures PIP3 product formed by the activity of a PI3-K. The kinase reaction is performed in a microtitre plate (e.g., a 384 well microtitre plate). The total reaction volume is approximately 20 ul per well. In the first step, each well receives 2 ul of test compound in 20% dimethylsulphoxide resulting in a 2% DMSO final concentration. Next, approximately 14.5 ul of a kinase/PIP2 mixture (diluted in 1× reaction buffer) is added per well for a final concentration of 0.25-0.3 ug/ml kinase and 10 μM PIP2. The plate is sealed and incubated for 15 minutes at room temperature. To start the reaction, 3.5 ul of ATP (diluted in 1× reaction buffer) is added per well for a final concentration of 10 μM ATP. The plate is sealed and incubated for 1 hour at room temperature. The reaction is stopped by adding 5 ul of Stop Solution per well and then 5 ul of Detection Mix is added per well. The plate is sealed, incubated for 1 hour at room temperature, and then read on an appropriate plate reader.
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 320.35, ALogP 3.37, HBA 6, HBD 1, PSA 86.94, QED 0.61
Compound (SMILES): COc1cccc(-c2cc(COC(C)(C)C(=O)O)nn2Cc2ccccc2Cl)c1
InChIKey: XWCDPGKAMJDMKE-UHFFFAOYSA-N
Target: Monocarboxylate transporter 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O15427
Assay: MCT4-Mediated Lactate Transport in NCI-H358 Lung Adenocarcinoma Cell: NCI-H358 lung adenocarcinoma 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. Preparation of BCECF-loaded cells and lactate transport activity may be determined as described for Assay 1.
Measurement: IC50 = 44 nM (pChEMBL 7.360000133514404)
Descriptors: MW 414.89, ALogP 4.64, HBA 5, HBD 1, PSA 73.58, QED 0.58
Compound (SMILES): C=CC(=O)N1CCC(Oc2cc3c(Nc4cccc(C5CC5)c4)ncnc3cc2OC)C1
InChIKey: DVBRORLRXBJHPY-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Cell Viability Assay and IC50 Estimation: Cell viability was determined using the Cell Titer Glo assay (Promega) as previously described. Briefly, 2000-3000 cells per well were plated in 384-well plates (Greiner Bio-One) in technical triplicate. Cells were treated with seven different concentrations of tyrosine kinase inhibitors or vehicle alone at a final volume of 40 μL per well. After 3 days hours, 11 μL of Cell Titer Glo was added to each well. Plates were shaken for 15 minutes, and bioluminescence was determined using a FLUOstar OPTIMA multi-mode micro-plate reader (BMG LABTECH). Bioluminescence values were normalized to DMSO treated cells, and normalized values were plotted in GraphPad Prism using non-linear regression fit to normalized data with a variable slope. IC50 values were calculated by GraphPad Prism at 50% inhibition.
Measurement: IC50 = 2500 nM (pChEMBL 5.599999904632568)
Descriptors: MW 430.51, ALogP 4.43, HBA 6, HBD 1, PSA 76.58, QED 0.56
Compound (SMILES): CC#CC(=O)N1CCCC[C@H]1c1nc(-c2ccc(C(=O)Nc3nccc(C)n3)cc2)c2c(N)nccn12
InChIKey: MBDCFLUQPOZNTA-FQEVSTJZSA-N
Target: Tyrosine-protein kinase Lyn (SINGLE PROTEIN), Homo sapiens
UniProt: P07948
Assay: Lyn Enzyme Activity: Lyn enzyme (Millipore catalog #14-510), is diluted to 250 mU/mL in KR buffer (10 mM Tris-HCl, 10 mM MgCl2, 0.01% Tween-20, 0.05% NaN3, 1 mM DTT, 2 mM MnCl2, pH 7.2).Serial dilution log 10 from 2 mM to 63.2 nM of test compounds are made in 100% DMSO. The dilutions in DMSO are then diluted 50-fold in KR-buffer of which 5 μl is used in the assay, leading to a final compound concentration range in the assay from 10 μM to 0.316 nM.5 μL/well of test compound in KR buffer (final DMSO concentration in the assay is 1%) is mixed with 5 μl/well of 250 mU/mL Lyn enzyme (final concentration in the assay is 62.5 mU/mL). Test compounds and Lyn enzyme are pre-incubated 60 minutes at room temperature, before adding 5 μL/well of 400 nM Fluorescin labeled substrate peptide (Blk/Lyntide substrate, e.g. #R7188/#R7233, Molecular Devices) in KR-buffer. Final peptide substrate concentration in assay is 100 nM. The kinase assay is started by adding 5 μL/well of 8 μM ATP in KR-buffer (final ATP concentration is 2 μM ATP, Km ATP in Lyn IMAP assay). Following incubation for 2 h at room temperature the enzyme reaction is stopped by adding 40 μL/well IMAP Progressive Binding Solution (according to suppliers (Molecular Devices) protocol using 75% 1× buffer A and 25% 1× buffer B with 1:600 Progressive Binding Solution). After 60 min incubation at room temperature in the dark the FP signal is read. Fluorescence at 535 nm is measured using parallel and perpendicular filters to determine differences in rotation due to binding of the phosphorylated substrate peptide to the beads. Values are calculated as percentage of the difference in readout (ΔmPi) of the controls with and without ATP. EC50 values are determined by curve fitting of the experimental results using Activity Base.
Measurement: EC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 494.56, ALogP 3.41, HBA 8, HBD 2, PSA 131.4, QED 0.42
Compound (SMILES): C[C@H](NC(=O)c1c(N)nn2cccnc12)c1cc2cccc(C#Cc3cnn(C)c3)c2c(=O)n1C1CC1
InChIKey: WMYOILMBEYWYEX-INIZCTEOSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42338
Assay: Inhibition Assays: Class I PI3-Ks can be either purchased (p110α/p85α, p110β/p85α, p110δ/p85α from Upstate, and p110γ from Sigma) or expressed as previously described (Knight et al., 2004). IC50 values are measured using either a standard TLC assay for lipid kinase activity (described below) or a high-throughput membrane capture assay. Kinase reactions are performed by preparing a reaction mixture containing kinase, inhibitor (2% DMSO final concentration), buffer (25 mM HEPES, pH 7.4, 10 mM MgCl2), and freshly sonicated phosphatidylinositol (100 μg/mL). Reactions are initiated by the addition of ATP containing 10 μCi of γ-32P-ATP to a final concentration of 10 or 100 μM and allowed to proceed for 5 minutes at room temperature. For TLC analysis, reactions are then terminated by the addition of 105 μL 1N HCl followed by 160 μL CHCl3:MeOH (1:1). The biphasic mixture is vortexed, briefly centrifuged, and the organic phase is transferred to a new tube using a gel loading pipette tip precoated with CHCl3. This extract is spotted on TLC plates and developed for 3-4 hours in a 65:35 solution of n-propanol:1M acetic acid.
Measurement: IC50 = 2000 nM (pChEMBL 5.699999809265137)
Descriptors: MW 492.54, ALogP 2.59, HBA 9, HBD 2, PSA 125.13, QED 0.37
Compound (SMILES): CN(C)[C@H]1CCCN(c2ccnc3[nH]cc(-c4cncnc4)c23)C1
InChIKey: ZILLQAUSVGQYOB-AWEZNQCLSA-N
Target: Rho-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13464
Assay: Kinase Assay: MRCKα, MRCKβ, ROCK1 and ROCK2 assays were performed using an IMAP fluorescence polarization assay format (Molecular Devices Inc.). 8-12 nM of each kinase (Life Technologies) was incubated for 60 min at room temperature with 100 nM FAM-S6-ribosomal protein derived peptide (synthesized by Alta Biosciences, University of Birmingham UK) in the presence of 1 μM ATP and 0.5 mM MgCl2 in 20 mM Tris buffer (pH 7.4) containing 0.01% Tween-20 and 1 mM DTT (MRCKα and β); or 1 μM ATP, 10 mM MgCl2 in 20 mM Tris buffer (pH 7.5) containing 0.25 mM EGTA 0.01% Triton X-100 and 1 mM DTT (ROCK1 and ROCK2). Typically, dose response analyses were performed over concentration ranges from 0.005-100 μM. Reactions were stopped by adding 2 assay volumes of 0.25% (v/v) IMAP binding reagent in 1×IMAP binding buffer A (Molecular Devices). After 30 min incubation to allow binding reagent to bind phosphorylated peptide, fluorescence polarization was measured on a Tecan Saphire2 plate reader at excitation (470 nm) and emission (530 nm) wavelengths. Inhibition was calculated using no inhibitor and no enzyme controls as 0 and 100% inhibition, respectively.
Measurement: Ki = 404 nM (pChEMBL 6.389999866485596)
Descriptors: MW 322.42, ALogP 2.55, HBA 5, HBD 1, PSA 60.94, QED 0.8
Compound (SMILES): COc1cc2ncc3c(c2cc1-c1cnn(C)c1)n(-c1c(F)cncc1OC)c(=O)n3C
InChIKey: VMNZFFHECMZKEV-UHFFFAOYSA-N
Target: PI3-kinase p110-delta/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: O00329
Assay: PI3K p110delta/p85alpha (Human), Non-radioactive Assay: PI3K p110δ/p85α (human) was incubated in an assay buffer comprising 10 μM phosphati-dylinositol 4,5-bisphosphate and MgATP (concentration as necessary). The reaction was initiated by addition of MgATP solution. After an incubation time of 30 min at room temperature, the reaction was stopped by addition of a solution consisting of EDTA and biotinylated phosphatidylinositol 3,4,5-trisphosphate. Finally, the detection buffer, consisting of a europium-labelled anti-GST monoclonal antibody, a GST-labelled GRP1 PH domain and streptavidin allophycocyanin, was added. The plate was read out via homogeneous time-resolved fluorescence (HTRF) and the corresponding signals were evaluated via the formula HTRF=10000×(Em665nm/Em620nm).
Measurement: IC50 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 434.43, ALogP 2.83, HBA 9, HBD 0, PSA 88.99, QED 0.43
Compound (SMILES): CN(C)[C@]1(c2ccccc2)CC[C@]2(CC1)CN(CC1CCN(c3cncnc3)CC1)C(=O)N2CC1CC1
InChIKey: FEADKIBOZKXXHU-RNPORBBMSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: 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 ul/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 uM 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 ??-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 = 985 nM (pChEMBL 6.010000228881836)
Descriptors: MW 502.71, ALogP 4.61, HBA 5, HBD 0, PSA 55.81, QED 0.55
Compound (SMILES): CC(C)COc1ncnc2[nH]cc(C(=O)c3c(F)ccc(NS(=O)(=O)c4ccc(C(F)(F)F)cc4)c3F)c12
InChIKey: RXVSJIUWPQDZHM-UHFFFAOYSA-N
Target: Cytochrome P450 2D6 (SINGLE PROTEIN), Homo sapiens
UniProt: P10635
Assay: Inhibition Assay: The inhibition of CYP enzyme activity (IC50) for each of CYP1A2, CYP2C19, CYP2C9, CYP2D6, CYP3A4(BFC) and CYP3A4(BQ) is determined for compounds, where inhibition of metabolism of a known substrate leads to a decrease in the fluorescence of the metabolized product. The fluorescence of the product is monitored as a function of compound concentration. Compounds are dissolved in dimethyl sulfoxide to a concentration of 100 mM. These are diluted 1 μL into 82 μL of acetonitrile. An 11 μL aliquot of this solution is then added to 204 μL of cofactor mix (1.3% NADPH Regeneration system Solution A, 1.04% NADPH Regeneration system Solution B from BD Biosciences, 5% acetonitrile and 0.05% dimethyl sulfoxide). These are then serially diluted 1:1 (160 μL to 160 μL co-factor mix) for a total of 10 points. A 10 μL aliquot of this final mixture is dispensed into 384 well assay plates and incubated for 10 minutes at 37° C. Enzyme and substrate mix (10 μL; 0.5 pmol CYP1A2/5 μM CEC; 1.0 pmol CYP2C9/75 μM MFC; 0.5 pmol CYP2C19/25 μM CEC; 1.5 pmol CYP2D6/1.5 μM AMMC; 1.0 pmol CYP3A4/50 μM BFC; or 1.0 pmol CYP3A4/40 μM BQ) is added to these assay plates. Assay plates are incubated at 37° C. (CYP1A2-15 min; CYP2C9-45 min; CYP2C19, 2D6 and 3A4-30 min) and read in a Tecan Safire 2 plate reader (CYP1A2, 2C19 and 3A4 409 ex/460 em; CYP2C9 and 2D6 409 ex/530 em).
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 554.5, ALogP 5.32, HBA 6, HBD 2, PSA 114.04, QED 0.23
Compound (SMILES): Cc1c(-c2[nH]c3ccc(C4CCC(NCC(=O)NC(C)(C)C)CC4)nc3c2C(C)C)cn2ncnc2c1C
InChIKey: QWEOHFZXZHCPMY-UHFFFAOYSA-N
Target: Toll-like receptor 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR96
Assay: TLR7/8/9 Inhibition Reporter Assays: 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 = 6674 nM (pChEMBL 5.179999828338623)
Descriptors: MW 515.71, ALogP 5.54, HBA 6, HBD 3, PSA 100, QED 0.31
Compound (SMILES): COc1cc(OC)c(F)c(N(CC2CCC(=O)N2)c2ccc3ncc(-c4cnn(C)c4)cc3n2)c1F
InChIKey: YJAREBGBTVFDIK-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: FGFR1 (Enzymatic Assay): In a final reaction volume of 30 μL, FGFR1 (h) (25 ng/ml) was incubated with 50 mM HEPES pH 7.5, 6 mM MnCl2, 1 mM DTT, 0.1 mM Na3VO4, 0.01% Triton-X-100, 500 nM Btn-Flt3 and 5 μM ATP in the presence of compound (1% DMSO final). After incubation for 60 minutes at room temperature the reaction was stopped with 2.27 nM EU-anti P-Tyr, 7 mM EDTA, 31.25 nM SA-XL-665 and 0.02% BSA which was present for 60 minutes at room temperature. Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) signal (ex340 nm. Em 620 nm, em 655 nm) was measured afterwards and results are expressed in RFU (Relative Fluorescence Units). In this assay, the inhibitory effect of different compound concentrations (range 10 μM to 0.1 nM) was determined and used to calculate an IC50 (M) and pIC50 (−log IC50) value.
Measurement: IC50 = 0.955 nM (pChEMBL 9.020000457763672)
Descriptors: MW 494.5, ALogP 3.74, HBA 8, HBD 1, PSA 94.4, QED 0.42
Compound (SMILES): Nc1ncc(-c2ccc([C@]34C[C@H]3CN(CCN3CCOCC3)C4)cc2)cc1C(=O)NC12CCC(O)(CC1)CC2
InChIKey: GAIBPGQSYNAFNN-BVPJRLQBSA-N
Target: Activin receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q04771
Assay: Caliper Assay: A kinase selectivity panel which measures substrate peptide phosphorylation was set-up for wild-type ALK2 (aa172-499), ALK2 FOP mutant (aa172-499 R206H), ALK1 (aa166-493), ALK5 (aa162-503) and ALK6 (aa168-495). The technology used for the described assay is based on the separation and quantification of substrate and product in an electrical field. In the course of the kinase reaction the peptide substrate is phosphorylated by a kinase. The transfer of a phosphate residue also causes the introduction of two additional negative charges and hence to a change in the net charge of the phospho-peptide compared to the unphosphorylated peptide. Due to this difference in charge the phosphorylated und unphosphorylated peptides migrate with different velocities in an electrical field.In the applied method, this separation takes place inside a chip that contains a complex capillary system for simultaneous analysis of 12 samples (12-sipper chip, Caliper Technologies Corp., Mountain View, USA). In order to allow the detection and quantification of the peptides in the capillary system, the peptides carry a fluorescent label (fluorescein). With this label the peptides can be quantified by fluorescence intensity through the instruments laser and detection system (LC3000, Caliper Life Sciences).The assays were performed in 384-well, low volume microtiter assay plates in a final reaction volume of 9 ul. Dose-response curves were generated by incubating 10 nM of each kinase together with 2 μm of the fluorescently labeled substrate peptide 5-Fluo-Ahx-KKYQAEEN-T-YDEYENKK-amid (10 mM stock solution in DMSO) in 50 mM Hepes pH 7.5, 0.02% Tween 20, 0.02% BSA, 1 mM DTT, 10 m Na3VO4, 10 mM If-Glycerolphosphate, 1 mM MgCl2, 12 mM MnCl2 (ALK1 and ALK6 7 mM) and 15 μm ATP for 60 min at 30° C. in the presence or absence of compound diluted in DMSO. Kinase reaction were terminated by adding 15 ul STOP buffer (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35.Plates with terminated kinase reactions were transferred to the Caliper LC3000 workstation (Caliper Technologies Corp., Mountain View, USA) for reading. The relative amount of phosphorylated peptide r, was calculated using the heights of the substrate peak, s, and the product peak, p: r=p/(p+s).
Measurement: IC50 = 49 nM (pChEMBL 7.309999942779541)
Descriptors: MW 531.7, ALogP 2.8, HBA 7, HBD 3, PSA 103.95, QED 0.5
Compound (SMILES): COC(=O)c1cc2c(cc1C)/C(=C(/Nc1ccc(C(=O)NCCN3CCC(F)(F)CC3)cc1)c1ccccc1)C(=O)N2
InChIKey: JNFPOOBKOALVEY-DQSJHHFOSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: EGFR2 Enzyme Inhibition: The inhibitory activities of compounds of the invention against VEGFR2 (VEGFR2 Kinase Enzyme System: Promega), were evaluated by mixing the VEGFR2 protein (1.56 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 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 = 154 nM (pChEMBL 6.809999942779541)
Descriptors: MW 574.63, ALogP 5.18, HBA 6, HBD 3, PSA 99.77, QED 0.25
Compound (SMILES): CC(C)C(=O)N1CCC(c2cc(-c3ccc(NC(=O)c4cn(C5CC5)c(=O)n(-c5ccccc5)c4=O)cc3)c3c(N)ncnn23)CC1
InChIKey: INCLUHXJYYFKBR-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor TYRO3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06418
Assay: TAM Enzymatic Assay: The kinase assay buffer contained 50 mM HEPES, pH7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% NP-40 and 2 mM DTT. 0.1 ul test compounds dissolved in DMSO were transferred from compound plates to white 384-well assay plates (Greiner LLIMITRAC plates). The final concentration of DMSO was 1.25%. Enzyme solutions of 5.1 nM phosphor-Axl, or 0.0625 nM c-Mer (Carna Biosciences, 08-108), or 0.366 nM Tyro3 (Life Technologies, PR7480A) were prepared in assay buffer. A 1 mM stock solution of peptide substrate Biotin-EQEDEPEGDYFEWLE-amide SEQ ID NO: 1 (Quality Controlled Biochemicals, MA) dissolved in DMSO was diluted to 1 uM in assay buffer containing 2000 uM ATP. 4 ul enzyme solution (or assay buffer for the enzyme blank) was added to the appropriate wells in each plate, and then 4 ul/well substrate solution was added to initiate the reaction. The plate was protected from light and incubated at room temperature for 60 min. The reaction was stopped by adding 4 ul detection solution containing 50 mM Tris-HCl, pH7.8, 150 mM NaCl, 0.05% BSA, 45 mM EDTA, 180 nM SA-APC (Perkin Elmer, CR130-100) and 3 nM Eu-W1024 anti-phosphotyrosine PY20 (Perkin Elmer, AD0067). The plate was incubated for 1 h at room temperature, and HTRF (homogenous time resolved fluorescence) signal was measured on a PHERAstar FS plate reader (BMG labtech). Percentage of inhibition was calculated for each concentration and IC50 value was generated from curve fitting with GraphPad Prism software.
Measurement: IC50 < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 632.73, ALogP 4.24, HBA 10, HBD 2, PSA 149.62, QED 0.27
Compound (SMILES): NCC1(O)CCN(c2nc(Oc3cccc(F)c3)cc(C(F)(F)F)n2)CC1
InChIKey: HYEMQFSXSZXWCD-UHFFFAOYSA-N
Target: Sodium-dependent serotonin transporter (SINGLE PROTEIN), Homo sapiens
UniProt: P31645
Assay: Competition Binding Assay: The affinity of the compounds of the invention to the human DAT or NET or SERT transporters is assessed by using the [3H]WIN-35,428 or [3H]nisoxetine or [3H]citalopram binding assays in recombinant human DAT, NET and SERT membranes with the SPA technology. The final assay volume is 50 μL in 384 well plates.Briefly, 0.5 μL of test compound in neat DMSO or 0.5 μL of DMSO for total binding (TB) or 0.5 μL of indatraline 1 mM (10 μM final concentration) for non specific binding (NSB) are added to the assay plate. 50 μL of the SPA mixture is added to each well, containing: 30 μg/mL or 10 μg/mL or 25 μg/mL DAT, NET, SERT membranes, respectively; 5 nM [3H]WIN-35,428 or 5 nM [3H]nisoxetine or 1 nM [3H]citalopram, for DAT, NET, SERT assay, respectively; 2.5 mg/mL or 1 mg/mL or 4 mg/mL WGA-PVT SPA beads (PerkinElmer RPNQ0001, for DAT, NET, SERT assay, respectively. All components are added to Assay Buffer (20 mM HEPES pH 7.4, 145 mM NaCl, 5 mM KCl, 0.01% Pluronic F-127). 0.02% BSA was used for DAT binding only. Plates are sealed with Topseal A and centrifuged 1 min, 800 rpm. Plates are loaded into a 1450 Microbeta TriLux (Perkin-Elmer) plate reader and the radioactivity counted after at least 4 hrs or overnight incubation at room temperature. Curve fitting and IC50 estimations are performed using a four parameter model in XLfit (IDBS, Guilford, UK) for Microdoft Excel (Microsoft, Redmond, Wash.).
Measurement: IC50 = 8710 nM (pChEMBL 5.059999942779541)
Descriptors: MW 386.35, ALogP 2.72, HBA 6, HBD 2, PSA 84.5, QED 0.79
Compound (SMILES): C=CC(=O)Nc1cccc(Nc2nc(Nc3cccc(OCC4CCCN(C)C4)c3)ncc2F)c1
InChIKey: UGWZEBRWDXGJIN-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Omnia Assay: EGFR (WT) and EGFR (T790M/L858R): The Omnia Assay Protocol for potency assessment against EGFR is performed as described in Example 251 above except that the EGFR-WT- and EGFR T790M/L858R-modified optimized reagent conditions are:[EGFR-WT]=5 nM, [ATP]=15 mM, [Y12-Sox]=5 mM (ATP KMapp˜12 mM); and [EGFR-T790M/L858R]=3 nM, [ATP]=50 mM, [Y12-Sox]=5 mM (ATP KMapp˜45 mM).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 476.56, ALogP 4.95, HBA 7, HBD 3, PSA 91.41, QED 0.38