lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COCc1nnc(NS(=O)(=O)[C@@H](C)[C@H](C)c2cnc(C)cn2)n1-c1c(OC)ncnc1OC
InChIKey: YFWNWWXQUBQHPV-STQMWFEESA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: [35S]GTPγS Binding: The human APJ receptor was cloned by polymerase chain reaction and the gene encoding the receptor was subcloned in pFLAG-CMV-3 expression vector (Sigma, Saint Louis, Mo. USA) in-house at Amgen. A GTPγS binding assay was performed on membranes prepared from CHO cells stably expressing human APJ receptor. The optimum experimental conditions for the concentrations of GDP, MgCl2, and NaCl in the assay buffer were initially determined. The assay was performed in 9 μL assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, 100 mM NaCl and 0.1% (w/v) BSA], 1 μL of diluted test compound (starting with 0.75 mM, 2-fold serial dilution with DMSO, total 22 points), 10 μL of 18 M GDP (final concentration of 3 μM GDP), 20 μL of 0.25 μg/mL membrane protein expressing human APJ receptor captured with WGA PS beads (final concentration of 5 μg per well), and 20 μL of 0.3 nM [35S]GTPγS (final concentration is 0.1 nM [35S]GTPγS)(Perkin Elmer Life and Analytical Sciences, Waltham USA). One column of the plate was 1 μL of DMSO as background and another column of the plate was 1 μL of 180 M Pyr-Apelin-13 which was used as control at a final concentration of 3 M. Incubation was at RT for 90 min and the microplate was read using a ViewLux ultra HTS Microplate Imager (PerkinElmer, Inc.).
Measurement: EC50 = 630 nM (pChEMBL 6.199999809265137)
Descriptors: MW 478.54, ALogP 1.25, HBA 12, HBD 1, PSA 156.13, QED 0.45
Compound (SMILES): COc1cc2c(-c3c(N)nn4cc(N5CCC(N6CCN(C)CC6)CC5)cnc34)ccnc2cc1F
InChIKey: XSVVDPXAICTBLZ-UHFFFAOYSA-N
Target: Activin receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q04771
Assay: Luciferase Reporter Gene Assay for ALK2 and TGFbeta Activity: In this reporter gene cell based ALK2 and TGFβ assay, the C2Cl2 cell line was employed for the measurement of ALK2 activity, using a BRE-Luc SMAD1/5/8 reporter and BMP6 as the agonist. The HEK293T cell line was employed to measure TGFβ activity, using a SBE-Luc SMAD2/3 reporter and TGFβ as the agonist. Luciferase reporter assays were read using Promega Steady-Glo Luciferase Assay System.Cells were plated in 96 well white clear bottom assay plates at 10 k cells/well for C2Cl2-BRE, 15 k cells/well for HEK293-SBE in DMEM containing 2% FBS 1% P/S. Cells were given a minimum of 4 hours in incubator at 37° C./5% CO2 to adhere prior to further treatment. Compounds were diluted in DMSO to a 10-point dilution curve and added to the plate to reach the following final concentrations: 10000, 3000, 1000, 300, 100, and 30 nM in the HEK293-SBE assay and 1000, 300, 100, 30, 10, 3, 1, 0.3 nM in the C2Cl2-BRE assay. Negative and positive control wells received 2 μL DMSO as vehicle treatment. Plates were returned to incubator for 45 minutes and then BMP6 and TGFB were added to a final concentration of 50 ng/mL and 5 ng/mL, respectively. Plates were returned to the incubator and left overnight. After a minimum of 18 hours post BMP6/TGFβ addition, the plates were read using Promega Steady-Glo Luciferase Assay System. A 1:1 mixture of prepared steady-glo and phenol free DMEM was prepared, and 50 μL/well was added to the assay plates whose media had been flicked off. Plates were given 10 minutes post steady-glo addition before luminescence was read on a Spectramax M5e microplate reader. Negative control wells were averaged and subtracted from all other wells on the plate. Inhibition was calculated as the percent of signal loss compared to the averaged positive control wells.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 490.59, ALogP 2.89, HBA 9, HBD 1, PSA 88.05, QED 0.47
Compound (SMILES): Cc1nc(-c2ccc(Cl)cc2)c(-c2ccc3nc(NC(=O)c4ccnc(F)c4)cn3n2)n1CC(F)(F)F
InChIKey: FEHSVEZOWNLHMK-UHFFFAOYSA-N
Target: Casein kinase I isoform epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: P49674
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours and terminated by adding 45 μl of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP®3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the unphosphorylated substrate and phosphorylated product. Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 μM ATP, 1.5 μM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations.
Measurement: IC50 = 5.95 nM (pChEMBL 8.220000267028809)
Descriptors: MW 529.89, ALogP 5.57, HBA 7, HBD 1, PSA 90, QED 0.24
Compound (SMILES): Cc1cc(C(F)(F)F)cc2c1nc(Cc1c(Cl)ccc(C(=O)N3CCC(N(C)C)CC3)c1Cl)n2C
InChIKey: CPDIYNBCDGHCMB-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: transient transactivation assay: The primary screen was performed by transient transactivation assays. These cell-based assays were carried out using Cos-7 cells transfected with a chimeric human RORγ-Gal4 receptor expression plasmid and a 5Gal4 pGL3 TK Luc reporter plasmid. Transfections were performed by a chemical agent (Jet PEI). Transfected cells were distributed in 384-wells plates and were allowed to recover for 24 h. The culture medium was then removed and fresh medium containing the compounds to be tested was added (final concentration ranging from 10−4 M to 3 10−10 M). After an overnight incubation, luciferase expression was measured by adding SteadyGlo according to the manufacturer's instructions (Promega). T0901317 at 10−5 M was used as reference. Results were expressed as fold induction compared to basal level or as percentage activity compared to references taken as 100% (basal) or 0% (T0901317). Dose-effect curves and IC50 were calculated using the software Assay Explorer (MDL).
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 527.42, ALogP 5.96, HBA 4, HBD 0, PSA 41.37, QED 0.42
Compound (SMILES): COc1ncc(-c2c(F)cc3c(N)ncnn23)cc1C(=O)NCCC(C)c1ccccc1
InChIKey: RGPABHMRJNNPGZ-UHFFFAOYSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: RIPK1 HTRF Binding Assay: A solution was prepared containing 0.2 nM Anti GST-Tb (Cisbio, 61GSTTLB), 90.6 nM probe and 1 nM His-GST-TVMV-hRIPK1(1-324) in FRET Buffer (20 mM HEPES, 10 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 0.05 mg/mL BSA). Using Formulatrix Tempest, the detection antibody/enzyme/probe solution (2 mL) was dispensed into wells of a 1536 plate (Black Low Binding Polystyrene 1536 Plate (Corning, 3724)) containing 10 nL of compounds of interest at appropriate concentration in DMSO. The plate was incubated at rt for 1 h. FRET was measured using the EnVision plate reader (Excitation: 340 nM, Emission: 520 nM/495 nM). Total signal (0% inhibition) was calculated from wells containing 10 nL DMSO only. Blank signal (100% inhibition) calculated from wells containing 10 nL of 15 nM staurosporine and internal controls.
Measurement: IC50 = 7200 nM (pChEMBL 5.139999866485596)
Descriptors: MW 434.48, ALogP 3.44, HBA 7, HBD 2, PSA 107.43, QED 0.46
Compound (SMILES): CN(C)C(=O)Cn1cc(NC(=O)CN2CCN(c3nncc(-c4ccc(Cl)cc4)n3)CC2)cn1
InChIKey: KNAWLKPMXWQFEH-UHFFFAOYSA-N
Target: Cytochrome P450 11B2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P19099
Assay: Inhibitory Effect on hCYP11B2: The pcDNA3.1-human CYP11B2 plasmid was transfected into a Chinese hamster lung fibroblast V79 cell line to produce a cell line stably expressing human CYP11B2 gene.The cells were cultured and grown in the Dulbecco's modified Eagle's/Ham's medium supplemented with 10% fetal bovine serum and 1% G418 disulfate solution under the environment of 37° C., 95% air, and 5% CO2, and the grown cells were harvested.Then, the cells were fractionated to obtain mitochondria by reference to a method described in Chabre et al. JCE 86 M 85 (11) 4060-68, 2000. In particular, the cells suspended in a 5 mmol/L Tris-HCl buffer (pH 7.4) containing 250 mmol/L sucrose were homogenized in a Teflon (Registered Trademark) Potter Elvehjem homogenizer, and then the suspension was centrifuged (800×g, 15 min.). The supernatant was separated and again centrifuged (10000×g, 15 min.) to obtain a pellet (mitochondrial fraction).The mitochondrial fraction diluted with a buffer containing 10 mmol/L KH2PO4, 10 mmol/L Tris, 20 mmol/L KCl, 25 mmol/L sucrose, 5 mmol/L MgCl2, and 0.05% bovine serum albumin was dispensed to a 96-well plate. 0.5 μmol/L Deoxycorticosterone, 150 μmol/L NADPH and a compound of each concentration were added to each well, and incubated for 1.5-2 hours at room temperature to produce aldosterone. An amount of the produced aldosterone in the incubated solution was determined by using HTRF (Homogeneous Time Resolved Fluorescence) method.IC 50 (nmol/L) was calculated by analyzing the aldosterone production inhibition rate (%) of each concentration of compounds by non-linear regression to a logistic curve.
Measurement: IC50 = 4.5 nM (pChEMBL 8.350000381469727)
Descriptors: MW 483.96, ALogP 1.24, HBA 9, HBD 1, PSA 112.38, QED 0.53
Compound (SMILES): FC(F)Oc1cc(-n2cc(Cn3ccnc3C3CC3)nn2)ccc1Cl
InChIKey: BHASKRVNSLNZGW-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: hERG receptor binding assay: TBD
Measurement: IC50 = 1.2 nM (pChEMBL 8.920000076293945)
Descriptors: MW 365.77, ALogP 3.64, HBA 6, HBD 0, PSA 57.76, QED 0.67
Compound (SMILES): CCc1nn(C)c2c1-c1c(F)ccc3c(CCCOc4cccc5ccccc45)c(C(=O)O)n(c13)C/C=C\COC2
InChIKey: AOFDJIUTQZRWFM-SREVYHEPSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07820
Assay: Biological Assay: MCL-1 Assay: In the assay 11 different concentrations of each compound (0.1 nM, 0.33 nM, 1.1 nM, 3.8 nM, 13 nM, 44 nM, 0.15 μM, 0.51 μM, 1.7 μM, 5.9 μM and 20 μM) were typically measured as duplicates in the same microtiter plate. For that, 100-fold concentrated DMSO solutions were prepared by serial dilutions (1:3.4) of a 2 mM stock solution in a clear, 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany). From there, 50 nl were transferred in a dark test plate (Greiner Bio-One, Frickenhausen, Germany). The assay was initiated by addition of 2 μl of a 2,5-fold concentrated MBP-MCL-1 solution (usually for a 1 nM end concentration in 5 μl reaction volume) in aqueous assay buffer [50 mM Tris/HCl pH 7, 100 mM sodium chloride (NaCl), 50 mM potassium fluoride (KF), 0.005% Tween-20, 2 mM DTT, 0.1% bovine gamma globulin (BGG)] to the compounds in the assay plate. This was followed by a 10-minute incubation step at 22° C. for pre-equilibration of the putative complex between MBP-MCL-1 and the compounds. After that, 3 μl of a 1.67-fold concentrated solution (in assay buffer) consisting of Noxa BH3-derived peptide (1 nM end concentration) and TR-FRET detection reagents [1.67 nM anti-MBP-Eu cryptate and 1.67 nM streptavidin-XL665 (both from Cisbio Bioassays, Codolet, France)], were added.
Measurement: IC50 = 2.2 nM (pChEMBL 8.65999984741211)
Descriptors: MW 553.63, ALogP 6.69, HBA 6, HBD 1, PSA 78.51, QED 0.18
Compound (SMILES): Cc1c(Cl)cccc1Nc1nc(=O)cc2n1C(c1ccccc1)CCO2
InChIKey: JGHGOFLCSRHBNY-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Inhibition Assay: Evaluation of a human P2X7 receptor inhibitory activity Stably expressing cell line (1321N1 cell transfected with the human P2X7 receptor gene (GenBank accession number NM_002562.5 including T606C and G952A SNP)) was used. The cells were seeded in a 384-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (10% fetal bovine serum, 25 mM HEPES, 1% penicillin and streptomycin in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. After replacing with 20 μL of the HBSS buffer (20 mM HEPES, 55.6 mM D-glucose, 1× HBSS(−), pH7.4-7.5), 15 μL of 17.3 μM Yo-Pro solution in the HBSS buffer was added. The plate was placed in high-throughput cellular screening system FLIPR TETRA (Molecullar Devices, LLC.) and 15 μL of 130 μM BzATP solution in the HBSS buffer was added. Measurement of fluorescence intensity by FLIPR TETRA was started. After eight minutes, 15 μL of DMSO solutions containing different concentrations of the compound of the present invention as prepared by dilution with the HBSS buffer were dispensed to each well through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 20 minutes. The maximum fluorescence intensity without the compound of the present invention is calculated as 0% inhibition and the maximum fluorescence intensity when the reference compound was added is calculated as 100% inhibition.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 367.84, ALogP 4.32, HBA 5, HBD 1, PSA 56.15, QED 0.75
Compound (SMILES): COc1cnc2c(Nc3ccc(F)c([C@]4(C)CN(C)S(=O)(=O)C(C)(C)C(=N)N4)c3)nccc2c1
InChIKey: RKYGTOUJCHJNJL-QHCPKHFHSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: HTRF FRET Assay: Reagents: Na+-Acetate pH 5.0; 1% Brij-35; Glycerol; Dimethyl Sulfoxide (DMSO); Recombinant human soluble BACE1 catalytic domain (>95% pure); APP Swedish mutant peptide substrate (QSY7-APPswe-Eu): QSY7-EISEVNLDAEFC-Europium-amide (SEQ ID 1).A homogeneous time-resolved FRET assay can be used to determine IC50 values for inhibitors of the soluble human BACE1 catalytic domain. This assay monitors the increase of 620 nm fluorescence that resulted from BACE1 cleavage of an APPswedish APPswe mutant peptide FRET substrate (QSY7-EISEVNLDAEFC-Europium-amide (SEQ ID 1)). This substrate contains an N-terminal QSY7 moiety that serves as a quencher of the C-terminal Europium fluorophore (620 nm Em). In the absence of enzyme activity, 620 nm fluorescence is low in the assay and increased linearly over 3 hours in the presence of uninhibited BACE1 enzyme. Inhibition of BACE1 cleavage of the QSY7-APPswe-Eu substrate by inhibitors is manifested as a suppression of 620 nm fluorescence.
Measurement: Ki = 209 nM (pChEMBL 6.679999828338623)
Descriptors: MW 486.57, ALogP 3.36, HBA 7, HBD 3, PSA 120.3, QED 0.52
Compound (SMILES): COc1cc(OC)c(F)c(CCc2cnc3[nH]c(-c4cnn(CC(C)(C)O)c4)cc3n2)c1F
InChIKey: AFKKRBAHOGSSHF-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Enzymatic Assay: The inhibitor potency of compounds was measured in an enzyme assay that measures peptide phosphorylation using FRET measurements to detect product formation. Inhibitors were serially diluted in DMSO and a volume of 0.5 μL was transferred to the wells of a 384-well plate. For FGFR3, a 10 μL volume of FGFR3 enzyme (Millipore) diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 5 mM DTT, pH 7.5) was added to the plate and pre-incubated for 5-10 minutes. Appropriate controls (enzyme blank and enzyme with no inhibitor) were included on the plate. The assay was initiated by the addition of a 10 μL solution containing biotinylated EQEDEPEGDYFEWLE peptide substrate (SEQ ID NO: 1) and ATP (final concentrations of 500 nM and 140 μM respectively) in assay buffer to the wells. The plate was incubated at 25° C. for 1 hr. The reactions were ended with the addition of 10 μL/well of quench solution (50 mM Tris, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.8; added fresh 30 mM EDTA and Perkin Elmer Lance Reagents for HTRF at 3.75 nM Eu-antibody PY20 and 180 nM APC-Streptavidin). The plate was allowed to equilibrate for 1 hr before scanning the wells on a PheraStar plate reader.FGFR1 and FGFR2 were measured under equivalent conditions with the following changes in enzyme and ATP concentrations: FGFR1, 0.02 nM and 210 μM, respectively and FGFR2, 0.01 nM and 100 μM, respectively.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 457.48, ALogP 3.67, HBA 7, HBD 2, PSA 98.08, QED 0.42
Compound (SMILES): COc1cncc(-n2cc(-c3cc(C(=O)NC4CC4)c(F)cc3Cl)cn2)c1
InChIKey: DUCKBLQRHHLBQD-UHFFFAOYSA-N
Target: Receptor-interacting serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43353
Assay: V9103X ADP-Glo Kinase Assay Custom: In a 384-well plate, test compound diluted in assay buffer (1% DMSO final) is mixed with 8His-RIPK2 FL enzyme (final concentration of 8 nM). After 15 minutes of pre-incubation at RT, ATP dissolved in assay buffer is added (final concentration 5 μM). The mixture is incubated for 60 minutes at 37° C. in a humidified incubator. Then, ADP Glo Reagent is added, followed by a 40 minute incubation at rt. Finally, Kinase Detection Reagent is added and the entire mixture is incubated for 40 min at RT. The luminescence signal is measured with an Envision reader to determine the amount of ADP produced. Assay buffer: 25 mM HEPES (4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid), 0.1% BSA (bovine serum albumin), 10 mM MgCl2, 5 mM MnCl2, 50 mM KCl, 0.01% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), 10 μM Na3VO4, 1 mM DTT (dithiothreitol), pH 7.5 All plates contain wells with vehicle controls instead of compound (1% DMSO) as reference for the high signal (100% CTL (100% of control), high signal), and wells without enzyme as reference for low signal (0% CTL, low signal). The luminescent signal generated is proportional to the ADP produced and is correlated with enzyme activity. The analysis of the data is performed by the calculation of the percentage of ADP production in the presence of the test compound and RIPK2 as compared to the ADP production in the presence of RIPK2 plus 50 μM Gefitinib.
Measurement: IC50 = 8.9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 386.81, ALogP 3.63, HBA 5, HBD 1, PSA 69.04, QED 0.73
Compound (SMILES): Cc1nc2ccc(S(=O)(=O)NC3(C)CC3)cc2c(=O)[nH]1
InChIKey: ZBXMCBZWWQFQHF-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 = 6959 nM (pChEMBL 5.159999847412109)
Descriptors: MW 293.35, ALogP 1.06, HBA 4, HBD 2, PSA 91.92, QED 0.88
Compound (SMILES): CC[C@H](OCCCCCCCOc1ccc(-c2ccn[nH]2)nc1)C(=O)O
InChIKey: MZZPPPJXAZQVIH-SFHVURJKSA-N
Target: Cytochrome P450 4F2 (SINGLE PROTEIN), Homo sapiens
UniProt: P78329
Assay: Inhibition Test for Each Compound of the Present Invention Against 20-HETE Producing Enzymes (CYP4F2 and CYP4A11): In the CYP4F2 inhibition test, the reaction solution containing each compound [final concentration of 50 mM, KPO4 (pH 7.4), 2.5 μM luciferine derivative, and 1 mM NADPH] was added to an Escherichia coli membrane fraction (100 μg/mL protein) in which human CYP4F2 had been expressed. In the CYP4A11 inhibition test, the reaction solution containing each compound [final concentration of 100 mM, Tris-HCl (pH 7.5), 60 μM luciferine derivative, 1.3 mM NADP+, 3.3 mM Glucose 6-Phosphate, 3.3 mM MgCl2, and 0.4 U/mL Glucose 6-Phosphate dehydrogenase] was added to an Escherichia coli membrane fraction (100 μg/mL protein) in which human CYP4A11 had been expressed. Following this, the membrane fraction was left to stand at room temperature for 60 minutes to perform an enzymatic reaction. After the reaction, a luciferine detection reagent was added, and the luminescence value was measured using a plate reader. By using that value, the percent inhibition of 20-HETE producing enzyme (%) was calculated according to the equation described below, and the 50% inhibitory concentration (IC50 value) for each compound was calculated.
Measurement: IC50 = 90 nM (pChEMBL 7.050000190734863)
Descriptors: MW 361.44, ALogP 3.68, HBA 5, HBD 2, PSA 97.33, QED 0.53
Compound (SMILES): Nc1cc(C(=O)Nc2ccccc2)n(Cc2csc(N3CCCCC3)n2)c1
InChIKey: DFVJZELOGPBZEY-UHFFFAOYSA-N
Target: Dynamin-1-like protein (SINGLE PROTEIN), Homo sapiens
UniProt: O00429
Assay: Drp1 Activity Screen: The GTPase activity of Drp1 was assayed using a colorimetric assay similar to as described previously by Leonard M. et al. Robust colorimetric assays for dynamin's basal and stimulated GTPase activities. Methods Enzymol. 2005, Vol. 404, pp. 490-503. Briefly, for GTPase assays of purified Drp1 inhibitor samples, 0.6 μM Drp1 preincubated with 0.1 mg/mL liposomes (consisting of 80% soybean lipids/20% cardiolipin) and inhibitor was added to 1 mM GTP in a microtiter plate for 20 min at 37° C. in 4 mM MgCl2, 10 mM Hepes (pH 7.0), 100 mM KCl, 1 mM DTT. Reactions were stopped by diluting with 0.5 mM EDTA. Samples were then incubated with malachite green reagent (1 mM malachite green and 50 mM ammonium molybdate in 1 N HCl), and the absorbance at 650 nm was measured. The reaction rates, IC50 values, and max % inhibition were determined using Excel/GraphPad Prism software.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 381.51, ALogP 3.82, HBA 6, HBD 2, PSA 76.18, QED 0.7
Compound (SMILES): CCCCNc1nc(N)nc(C)c1Cc1ccc(Cc2nn[nH]n2)cc1OC
InChIKey: LDPQAUYDAXCRQL-UHFFFAOYSA-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 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 382.47, ALogP 2.28, HBA 8, HBD 3, PSA 127.52, QED 0.48
Compound (SMILES): COc1ccc(CN2CCN(c3ccc(-c4cc(OCC(C)C)cn5ncc(C#N)c45)cn3)CC2)nc1
InChIKey: TZQKBLMLGITGFR-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 = 133 nM (pChEMBL 6.880000114440918)
Descriptors: MW 497.6, ALogP 4.03, HBA 9, HBD 0, PSA 91.81, QED 0.36
Compound (SMILES): O=C(N(CC12CCC(c3noc(C4CC4(F)F)n3)(CC1)CC2)c1cccc(-c2nc(C3CC3)no2)c1)C12CC(F)(C1)C2
InChIKey: ZVWBNIOGNYQXNO-UHFFFAOYSA-N
Target: Bile acid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q96RI1
Assay: transient human FXR/Gal4-luciferase reporter assay: A Gal4-hFXR fusion construct reporter system was used as the primary assay to characterize compound activity. A construct including 5 copies of the Gal4 promoter response element upstream of a firefly luciferase reporter cDNA was stably expressed in HEK293 cells. This reporter cell line was maintained in Dulbecco's Modified Eagle's medium (DMEM; Gibco) supplemented with 1% penicillin-streptomycin (P/S) solution, 500 μg/mL Zeocin and 10% charcoal/dextran-treated fetal bovine serum (cs-FBS) at 37° C. in a humidified 5% CO2 atmosphere. Another plasmid was constructed in which the human cytomegalovirus promoter in the pcDNA3.1 vector directs the expression of the cDNA encoding a fusion protein comprised of the DNA binding domain from the Gal4 transcription factor fused to the ligand binding domain from human FXR.The day prior to transfection, the reporter cells in culture are detached from the plate with trypsin and plated into a T75 flask at a sufficient density to achieve approximately 90% confluence the next morning. The transfection reagents are prepared by separately diluting 25 μg of the pcDNA3.1-Gal4-FXR plasmid into 1.87 mL of Opti-MEM (Thermo-Fisher), and 40 μL of Lipofectamine 2000 (Thermo-Fisher) into 1.87 mL of Opti-MEM, and then adding the diluted DNA solution into the diluted Lipofectamine 2000 solution and incubating at room temperature for 15-20 minutes. The mixture is further diluted with 10 mL of a solution comprised of DMEM, 10% cs-FBS, and 1% P/S immediately prior to transferring to the cells. The maintenance culture media is aspirated from the cells and the final transfection mixture is added before the cells are incubated overnight at 37° C. in a humidified 5% CO2 atmosphere. This protocol can be scaled up, and the transiently transfected cells can be cryopreserved in an assay-ready format.For compound testing, 100 nL of the compounds (serial dilutions in DMSO) are dispensed with an Echo acoustic dispenser (Labcyte) into the wells of a Corning/Costar clear bottom 384-well white plate. The transfected cells are harvested, counted, and diluted such that 10-25,000 cells in 25 μL are plated into each well of the 384-well compound assay plate. The compound-treated cells are incubated overnight at 37° C. in a humidified 5% CO2 atmosphere. The next morning 25 μL of Steady-Glo (Promega) are added to each well of the plate, the mixture is incubated for 15 min. with shaking, and luminescence is measured on an Envision (Perkin Elmer) plate reader. Background counts from cells treated with DMSO alone are subtracted from all raw counts, and the corrected values are converted to a percentage of the control response attained with 8 μM GW-4064. These data are fit to a 4-parameter log agonist-response equation to calculate an EC50 value.
Measurement: EC50 = 73 nM (pChEMBL 7.139999866485596)
Descriptors: MW 579.62, ALogP 6.64, HBA 7, HBD 0, PSA 98.15, QED 0.3
Compound (SMILES): Cc1ccc(C(CC(=O)O)c2ccc3c(c2)nnn3C)cc1CN1C[C@@H](C)Oc2ccc(C#N)cc2S1(=O)=O
InChIKey: WMOGSKRCIKIOJQ-FKSKYRLFSA-N
Target: Nuclear factor erythroid 2-related factor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16236
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 h. On day four, medium is aspirated from the plate and crude cell lysates are made by adding 13 μL 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 min 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 min. 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 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 545.62, ALogP 3.73, HBA 8, HBD 1, PSA 138.41, QED 0.39
Compound (SMILES): O=C(NCc1ccc(S(=O)(=O)N2CC3CC2CC3F)cc1)c1ccc2nccn2c1
InChIKey: KXXBMMUSCGODAD-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Enzymatic Reaction Assay: The NAMPT enzymatic reactions were carried out in Buffer A (50 mM Hepes pH 7.5, 50 mM NaCl, 5 mM MgCl2, and 1 mM THP) in 96-well V-bottom plates. The compound titrations were performed in a separate dilution plate by serially diluting the compounds in DMSO to make a 100× stock. Buffer A (89 μL) containing 33 nM of NAMPT protein was added to 1 μL of 100× compound plate containing controls (e.g. DMSO or blank). The compound and enzyme mixture was incubated for 15 min at rt, then 10 μL of 10× substrate and co-factors in Buffer A were added to the test well to make a final concentration of 1 μM NAM, 100 μM 5-Phospho-D-ribose 1-diphosphate (PRPP), and 2.5 mM Adenosine 5′-triphosphate (ATP). The reaction was allowed to proceed for 30 min at rt, then was quenched with the addition of 11 μL of a solution of formic acid and L-Cystathionine to make a final concentration of 1% formic acid and 10 μM L-Cystathionine. Background and signal strength was determined by addition (or non-addition) of a serial dilution of NMN to a pre-quenched enzyme and cofactor mix.
Measurement: IC50 = 8.1 nM (pChEMBL 8.09000015258789)
Descriptors: MW 428.49, ALogP 2.39, HBA 5, HBD 1, PSA 83.78, QED 0.68
Compound (SMILES): COc1cc(C(=O)N2CCC[C@@H]2c2cccc(C(=O)NCC(C)(C)O)c2)ccc1-c1cn[nH]c1
InChIKey: ARCLBIGSHYNAHO-JOCHJYFZSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro ROCK Assays: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50, i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 = 4.52 nM (pChEMBL 8.350000381469727)
Descriptors: MW 462.55, ALogP 3.56, HBA 5, HBD 3, PSA 107.55, QED 0.5
Compound (SMILES): Cc1c(F)c2cc(C#N)cc(C#N)c2c(=O)n1CCOc1ccc(Cl)cc1-c1ccnc2c(C(=O)O)csc12
InChIKey: JOARQTZZRQJXBU-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 = 30.2 nM (pChEMBL 7.519999980926514)
Descriptors: MW 558.98, ALogP 5.9, HBA 8, HBD 1, PSA 129, QED 0.27
Compound (SMILES): Cc1cc(F)c(C(=O)Nc2cccc(-n3nnnc3C(C)C)n2)cc1-c1cnn(C2CC2)c1
InChIKey: WSBGWDHXXSULMH-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99683
Assay: HTRF KinEASE Assay: The ability (IC50) of compounds to inhibit ASK1 kinase activity was determined by HTRF® KinEASE Assay System. ASK1 was purchased from Thermofisher (Catalogue #PV4011), ATP was purchased from Sigma (Catalogue #A7699), HTRF® KinEASE Assay System was obtained from Cisbio (Bedford, Mass.). ½ Area plate was purchased from Perkin Elmer (Catalogue ##6005560). HTRF® KinEASE-STK is a generic method for measuring serine/threonine kinase activities using a time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay. The IC50 value for each compound was determined in the presence of compound (various concentration from 0 to 10 μM) and a fixed amount of ATP and peptide substrates. The test compound, 1 uM STK3 peptide substrate, and 5 nM of ASK1 kinase are incubated with kinase reaction buffer containing 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, and 1 mM EGTA for 30 minutes. 100 uM ATP is added to start kinase reaction and incubated for 3 hours. The STK3-antibody labeled with Eu3+-Cryptate and 125 nM streptavidin-XL665 are mixed in a single addition with stop reagents provided by the Cisbio kit used to stop the kinase reaction. Fluorescence is detected using an Envision Multilabeled 2014 reader from PerkinElmer. The Fluorescence is measured at 615 nm (Cryptate) and 665 nm (XL665) and a ratio of 665 nm/615 nm is calculated for each well.
Measurement: IC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 446.49, ALogP 4.08, HBA 8, HBD 1, PSA 103.41, QED 0.48
Compound (SMILES): CC(=O)N1CCc2c1ccnc2N[C@@H](C)c1cc2cc(Cl)ccc2[nH]c1=O
InChIKey: OYBWNNMYHQWGDX-NSHDSACASA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Enzymatic Assay: Assays were performed in a 384-well black plate. An aliquot of 250 nL of compound was incubated with 10 μL of 30 nM IDH1-R132H or 10 nM IDH1-R132C recombinant protein in assay buffer (50 mM Tris pH=7.5, 150 mM NaCl, 5 mM MgCl2, 0.1% (w/v) Bovine Serum Albumin, and 0.01% Triton X-100) in each well at 25° C. for 15 minutes. After the plate was centrifuged briefly, an aliquot of 10 μL of 2 mM α-ketoglutarate and 20 μM NADPH solution prepared in assay buffer was then added to each well and the reaction was maintained at 25° C. for 45 minutes. An aliquot of 10 μL of diaphorase solution (0.15 U/mL diaphorase and 30 μM Resazurin in assay buffer) was added to each well. The plate was maintained at 25° C. for 15 minutes and then read on a plate reader with excitation and emission wavelengths at 535 nm and 590 nm, respectively. The IC50 of a given compound was calculated by fitting the dose response curve of inhibition of NADPH consumption at a given concentration with the four parameter logistic equation.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 382.85, ALogP 3.66, HBA 4, HBD 2, PSA 78.09, QED 0.72
Compound (SMILES): CN(C(=O)OC(C)(C)C)N1CCC2(CC1)NC(=O)c1c(Cl)cc(Nc3ccncn3)c(=O)n12
InChIKey: UWOMHLCMEWOFFY-UHFFFAOYSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBH9
Assay: Mnk Biochemical Enzymatic Assay: Compounds are screened for Mnk inhibition using the ADP-Glo kinase assay kit (Promega, catalogue No. V9101). All kinase reactions are performed in Reaction Buffer E (15 mM HEPES pH7.4, 20 mM NaCl, 1 mM EGTA, 10 mM MgCl2, 0.1 mg/ml BGG, and 0.02% Tween-20). Final Mnk1 reactions contained 10 nM recombinant Mnk1 (Life Technologies, PR9138A), 100 μM Mnk substrate peptide Ac-TATKSGSTTKNR-NH2 (American Peptide Company), 300 μM ATP, and varying concentrations of the inhibitory compound of interest. Final Mnk2 reactions contained 3 nM recombinant Mnk2 (Life Technologies, PV5607), 50 μM Mnk substrate peptide Ac-TATKSGSTTKNR-NH2 (American Peptide Company), 10 μM ATP, and varying concentrations of the inhibitory compound of interest. Final DMSO concentration in each reaction is 1%. Kinase reactions are carried out in 96-well half-area white flat-bottom polystyrene plates in a final volume of 25 μl. Mnk1/2 enzymes are pre-incubated with compound and peptide substrate for 5 minutes prior to the addition of ATP. After the addition of ATP, kinase reactions are incubated at room temperature for 40 minutes. Reactions are subsequently stopped by the addition of 25 μl of ADP-Glo Reagent and incubating for an additional 40 minutes. The final luminescent signal used for kinase activity readout is produced by the addition of 45 μl of Kinase Detection Reagent (ADP-Glo kit, Promega) and incubating for 40 minutes. The luminescent signal is detected using a Victor 2 multilabel counter (Perkin Elmer) and the concentration of compound necessary to achieve inhibition of enzyme activity by 50% (IC50) is calculated using signals from an 8-point compound dilution series.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 475.94, ALogP 2.31, HBA 9, HBD 2, PSA 121.69, QED 0.69
Compound (SMILES): CCOc1cc(-c2ccc(Cl)cc2)nc2cc(C(=O)NCCCc3nnc[nH]3)ccc12
InChIKey: HIBZWYQBKWWJJI-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: NAMPT Enzyme Assay: To measure the inhibition of NAMPT activity hNAMPT protein stock and anti 6His-Tb (Cisbio; Cat. No. 61HISTLB) is diluted to 3× final concentration with assay buffer (50 mM Tris-HCl (pH 7.5), 1 mM DTT, 100 mM NaCl, 10 mM MgCl2, 0.005% Tween 20). To this solution is added a test compound or vehicle control (DMSO) and BodiPY ligand (structure below). The plate is shaken for 1-2 min sealed and incubated for 1 h at rt in the dark. The TR-FRET signal is measured using BMG Pherastar (Lanthascreen protocol on BMG Pherastar). Excitation was carried out at 320 nm, and the ratio of emission of BodiPY (520 nm) to terbium (486 nm) was determined. Concentration response curves are generated by calculating the excitation increase in test compound-treated samples relative to DMSO-treated controls.For the assay method described above, test compound percent inhibition values at a single concentration are calculated relative to control (DMSO) treated samples. Compound concentration response curves are fitted to generate IC50 values from those curves. One skilled in the art will appreciate that these values generated either as percentage inhibition at a single concentration or IC50 values are subject to experimental variation.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 435.92, ALogP 4.43, HBA 5, HBD 2, PSA 92.79, QED 0.4
Compound (SMILES): O=C(Nc1cccc(-c2c(C#CCCO)sc(-c3ccc(F)cc3)c2-c2ccncc2)c1)c1ccccc1
InChIKey: JJBBQZUHVGUGBD-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16539
Assay: Fluorescence polarisation (FP) assay for inactive p38alpha MARK: FP signals were measured with a PHERAstar microplate reader (BMG Labtech) using black, low-binding half-area 96 well plates (Corning). Each binding data point was carried out in duplicate or triplicate. Data were recorded in millipolarisation (mP) units and measured at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. Tris(hydroxymethyl)aminomethane (Tris) assay buffer was used in all binding experiments and consisted of 10 mM Tris, 50 nM potassium chloride, and 3.5 mM 3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate (Chaps) at pH 8.0. The recombinant inactive His-tag p38α MAPK used in the ligand binding assays were prepared as described by Bukhtiyarova et al.
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 518.61, ALogP 7.27, HBA 4, HBD 2, PSA 62.22, QED 0.23
Compound (SMILES): COC(=O)c1cc(C)nc(C2CCN(C(=O)c3cc(C(C)C)n[nH]3)C2)c1
InChIKey: BQCMBTSQAJPEBX-UHFFFAOYSA-N
Target: Lysine-specific demethylase 5A (SINGLE PROTEIN), Homo sapiens
UniProt: P29375
Assay: KDM5 Enzyme Assay: Full length KDM5A enzyme was expressed and purified inhouse. Biotin-H3K4me3 peptide was purchased from New England Biolabs. HTRF reagents (containing Eu-labeled H3K4me1-2 antibody, and streptavidin-XL665) were purchased from Cis-Bio International. Plates were read on an Envision multi-label plate reader (Perkin Elmer).The HTRF assay mixture contained 2 nM full length KDM5A enzyme, 100 nM H3K4Me3 peptide substrate, 1 uM 2-OG, 100 uM Fe2+, 500 uM ascorbate. 50 mM HEPES pH 7.0 buffer, 0.01% Triton-X 100, 2 mM DTT, 0.25% DMSO at a final volume of 10 uL.The enzyme reaction was carried out at room temperature in black Proxiplate 384-Plus plate (Corning. Costar) within 30 minutes, in the presence of varying concentration of a test compound. At the end of enzyme reaction, 5 uL of 1 mM EDTA were added to quench the reaction and then the detection reagents (5 uL) were added to give final concentrations of 0.5 nM Eu-labeled H3K4me1-2 antibody, and 50 nM streptavidin-XL665. The plates were incubated at room temperature for 60 minutes and then read in the Envision plate reader. The readouts were transformed into % inhibition, and IC50 value of a test compounds was generated by using four parameters curve fitting (Model 205 in XLFIT5, iDBS).
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 356.43, ALogP 2.65, HBA 5, HBD 1, PSA 88.18, QED 0.85
Compound (SMILES): CNC(=O)c1cn(C[C@H](F)CCc2ccc(NC(=O)Cc3cc(OC4CC(OCC(F)(F)F)C4)ccn3)nn2)nn1
InChIKey: CBRUFHWLRPJRLK-APMMTJHUSA-N
Target: Glutaminase kidney isoform, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: O94925
Assay: Enzymatic Activity Assay: The inhibition of purified recombinant human GAC by varying concentrations of inhibitors is assessed via a dual-coupled enzymatic assay. The glutamate produced by the glutaminase reaction is used by glutamate oxidase to produce α-ketoglutarate, ammonia, and hydrogen peroxide, with this hydrogen peroxide subsequently being used by horseradish peroxidase to produce resorufin in the presence of Amplex UltraRed. The assay buffer consisted of 50 mM Hepes (pH 7.4), 0.25 mM EDTA and 0.1 mM Triton X-100. GAC was incubated with potassium phosphate (10 minutes at room temperature) prior to incubation with inhibitor (10 minutes at room temperature). The final reaction conditions were as follows: 2 nM GAC, 50 mM potassium phosphate, 100 mU/mL glutamate oxidase (Sigma), 1 mM glutamine (Sigma), 100 mU/mL horseradish peroxidase (Sigma), 75 μM Amplex UltraRed (Life Technologies), and 1% (v/v) DMSO. The production of resorufin was monitored on a Perkin Elmer Envision plate reader (excitation 530 nm, emission 590 nm) either in a kinetics or endpoint mode (at 20 minutes). IC50 values were calculated using a four-parameter logistic curve fit.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 580.54, ALogP 2.46, HBA 10, HBD 2, PSA 146.04, QED 0.29
Compound (SMILES): CC1(C)[C@H]2CC[C@]1(c1ccnc(-c3cn(CCS(C)(=O)=O)cn3)n1)c1nnc(-c3c(F)cccc3F)cc12
InChIKey: CVEUQTZOAYJFLY-SOKVYYICSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: In Vitro Ligand Binding Assay: 50 mM HEPES buffer (pH 7.4); 150 mM NaCl; 1 mM DTT; 5 mM MgCl2; 0.01% BSA; 5% DMSO; 0.6 ug/mL RORc receptor; 6 nM 25-[3H]hydroxycholesterol. For NSB wells, 1 uM 25-hydroxycholesterol was also present. Assay plates were 96-well polypropylene V-bottom plates. 10 uL of 5× compound in 25% DMSO/75% Assay Buffer was added to Test wells. 10 uL of 25% DMSO/75% Assay Buffer was added to Total Binding or No Receptor wells. 10 uL of 5 uM 25-hydroxycholesterol in 25% DMSO/75% Assay Buffer was added to NSB wells. 20 uL of 15 nM 25-[3H]hydroxycholesterol prepared in Assay Buffer was added to all wells. 20 uL of 1.5 ug/mL RORc receptor was added to wells (or 40 uL Assay Buffer to No R wells). Following addition to the wells, the plates were incubated 3 h at 25° C. Filtration
Measurement: IC50 = 56 nM (pChEMBL 7.25)
Descriptors: MW 536.61, ALogP 4.32, HBA 8, HBD 0, PSA 103.52, QED 0.36