lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1cc(OCCCc2c(C(=O)NS(=O)(=O)c3ccccc3)[nH]c3c(-c4ccnn4C(C)C)cccc23)cc(C)c1Cl
InChIKey: JLWBSFUSGSRMQJ-UHFFFAOYSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07820
Assay: Bak Peptide Binding Assay: Compound affinity was measured using a fluorescence polarization anisotropy competition assay. Anisotropy measurements were carried out in 384-well, black, flat-bottom plates (Greiner Bio-one, Monroe, N.C., USA). The assay was run using a fluorescein isothiocyanate-labeled BH3 peptide derived from Bak (FITC-AHx-GQVGRQLAIIGDDINR-NH2) that was purchased from GenScript (Piscataway, N.J.) at >95% purity and used without further purification. 10 nM FITC-Bak peptide and 14 nM recombinant Mcl-1 (residues 172-327) were added to assay buffer (3 mM dithiothreitol, 50 mM NaCl, 20 mM Tris, pH 7.5). For selectivity assays, 40 nM Bcl-2 (residues 1-207A96T,G110R, Δ35-91, replaced with Bcl-xL35-50) or 4 nM Bcl-xL (residues 1-209, loop 45-86 deleted) were incubated with 10 nM FITC-Bak in assay buffer. Compounds are diluted in DMSO in a 10-point, 3-fold serial dilution scheme. 2.5 uL compound is added to 47.5 uL of assay buffer containing FITC-Bak and protein, for a final DMSO concentration of 5% and a top concentration of 20 uM. A FITC-Bak peptide alone (100% inhibition) and peptide plus protein (0% inhibition) control is included on each assay plate. The plate was mixed and incubated for 90 minutes at room temperature. Anisotropy is measured at excitation wavelength 480 nm and emission wavelength 535 nm using an EnVision Multi-label plate reader (PerkinElmer, Wellesley, Mass., USA). Fluorescence anisotropy is plotted against compound concentration to generate an IC50 (inhibitor concentration at which 50% of bound peptide is displaced) by fitting the data to a 4-parameter logistic model using XLFit software (Guildford, Surrey, UK). IC50 is converted to a binding dissociation constant (Ki value) according to the formula of Wang Z. FEBS Lett (1996) 3, 245:K i =[I] 50/([L] 50 /K d +[P] 0 /K d+1)where [I]50 is the concentration of the free inhibitor at 50% inhibition, [L]50 is the concentration of the free labeled ligand at 50% inhibition, [P]0 is the concentration of the free protein at 0% inhibition, Kd represents the dissociation constant of the FITC peptide probe.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 605.16, ALogP 7.01, HBA 6, HBD 2, PSA 106.08, QED 0.17
Compound (SMILES): CN(Cc1ccc(F)cc1)C(=O)N1[C@H]2CC[C@@H]1[C@@H](C(=O)O)N(C(=O)N(c1ccccc1)c1ccccc1)C2
InChIKey: RJNOOUSDOXZAPP-NXCFDTQHSA-N
Target: Type-2 angiotensin II receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P50052
Assay: Biological Assay: Measurement of Inhibitory Activity on AT1 Receptor (AT4R)/AT2 Receptor (AT2R)Through the following steps, the inhibitory activity of the compound on AT1R/AT2R (IC50 value) was determined:1) An appropriate amount of 1×TLB (Tag-lite Buffer) was prepared and well mixed for use.2) The compound was diluted by 10 times with ddH2O or DMSO. The compound was then dilute to 4 times of the working concentration with 1×TLB and mixed well for use.3) 8600 nM Tag-lite angiotensin receptor red agonist was diluted to 12 nM (4×Kd) with 1×TLB.4) 5 ml 1×TLB was taken into a 15 ml centrifuge tube.5) After thawing 1 tube of Tb-labeled AT1R/AT2R cells in a 37° C. water bath, the cells were quickly transferred to the 1×TLB in step 4), mixed gently, and centrifuged at 1200 g for 5 minutes at room temperature.6) The supernatant was aspirated gently, and the cells were resuspended and mixed in 2.7 ml 1×TLB, and then placed at room temperature until use.7) 10 μl cells were added to all test wells, and 5 μl 4× working solution of the compound from step 2) was added to the corresponding test wells. 5 μl 4× Tag-lite angiotensin receptor red agonist well diluted in step 3) was added to all test wells.8) After leaving the reaction plate at room temperature for 1 h, data were measured and analyzed using Envision HTRF Reader, and the half inhibitory concentration (IC50) of the compound on AT1R/AT2R was calculated with the GraphPad Prism four-parameter equation.
Measurement: IC50 = 30.5 nM (pChEMBL 7.519999980926514)
Descriptors: MW 516.57, ALogP 4.94, HBA 3, HBD 1, PSA 84.4, QED 0.52
Compound (SMILES): Cc1c(NC(=O)c2cccc(C#N)c2)cnc2c1c(C1CCN(C(=O)C3CC4CC[C@@H]3C4)CC1)cn2C
InChIKey: ZROQDFAPXXPPAV-HWDGFJGFSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: TR-FRET: The activity of compound of the invention can be determined by a co-activator recruitment by TR-FRET (time-resolved fluorescence resonance energy transfer) assay. In general, the assay is based on the interaction between N-terminally Six-Histidine-tagged-RORC2 ligand binding domain (6-His-RORC2 LBD), expressed in E. coli and purified by affinity chromatography, and biotin-coactivator peptide SRC1-2 (biotin-aminohexanoic acid-CPSSHSSLTERHKILHRLLQEGSPS-NH2; SEQ ID NO: 1) containing the LXXLL (SEQ ID NO: 2) consensus domain which is responsible for receptor binding. This interaction is detected by addition of Europium labeled-anti-His antibody (Ex. 337 nm, Em. 620 nm, which binds to 6His) and Streptavidin-APC (Ex. 620 nm, Em. 665 nm, which binds to biotin). When receptor and coactivator are bound to each other, upon shining light at 337 nm on the sample, the Europium emits fluorescence that excites APC due to close proximity (FRET) and this signal is measured at 665 nm. Due to the long lasting fluorescence emission of Europium, the non-specific, short-lived fluorescence is time-resolved (TR) from the fluorescence of interest. Inhibitors of the interaction of receptor and coactivator peptide are detected by a decrease in TR-FRET signal.
Measurement: IC50 = 7.2 nM (pChEMBL 8.140000343322754)
Descriptors: MW 495.63, ALogP 5.15, HBA 5, HBD 1, PSA 91.02, QED 0.54
Compound (SMILES): C[C@@]1(CO)Cc2cc(NC(=O)c3cnn4cccnc34)c(N3CCC([C@@](C)(O)C(F)(F)F)CC3)cc2O1
InChIKey: SKHPIZDOPBOMIW-BJKOFHAPSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Biological Assay: Kinase activities were assayed using the Transcreener-Fluorecescence polarization platform (BelBrook Labs, Madison, Wis., USA) that measures amounts of the reaction product, ADP. The IRAK4 reaction conditions were optimized using an IRAK1-derived peptide (sequence H-KKARFSRFAGSSPSQSSMVAR) to provide a linear reaction rate over the course of a 90 min incubation, which resulted in 10-12% conversion of the starting ATP to ADP. Final IRAK4 assay conditions were 1.25 nM IRAK4; 125 uM ATP; 10 uM MgCl2; 125 uM peptide in reaction buffer (25 mM HEPES (pH7.4); 2 mM Dithiothreitol; 0.015% Brij-35; and 0.5% dimethyl sulfoxide. The IRAK1 activity was optimized similarly, yielding final assay conditions of 3 mM IRAK1; 62.5 uM ATP; 5 uM MgCl2, and 62.5 uM IRAK1 peptide in reaction buffer for 60 min.Assays of compounds for kinase inhibition were performed using inhibitors serially-diluted in dimethyl sulfoxide, which was accomplished with a LabCyte Echo 555 liquid dispenser. 384 well assay plates spotted with compound received 4 ul of a 2× substrate (ATP+peptide) mix in reaction buffer, followed by 4 ul of 2× enzyme diluted in reaction buffer. Reactions were halted at 60 (IRAK1) or 90 (IRAK4) min by addition of 6 ul of detection buffer, containing EDTA (40 nM final concentration), 0.95 ug of the ADP-binding antibody ADP2, ADP tracer (3 nM final concentration), and 25 uM HEPES. Following a 1 hr incubation, fluorescence polarization of the ADP2-antibody TRACER complex was read on a Tecan M1000 plate reader using a 635/20 excitation filter in combination with a 670/20 emission filter.
Measurement: Ki = 7.6 nM (pChEMBL 8.119999885559082)
Descriptors: MW 519.52, ALogP 3.2, HBA 8, HBD 3, PSA 112.22, QED 0.47
Compound (SMILES): Cc1ncoc1-c1nnc(SCCCN2C[C@H]3CCN(c4cccc(C#N)c4)[C@H]3C2)n1C
InChIKey: QCALWRVXYCHRSG-QUCCMNQESA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Inhibition Assay: 3H]-Spiperone Binding Assay at hD3 and hD4 recombinant receptors CHO cells transiently transfected with human dopamine type 3 or 4 receptors (CHO-hD3 or CHO-hD4, respectively), were re-suspended in 20 mM HEPES, 2 mM EDTA (pH 7.4), homogenised and centrifuged at 40,000 g (20 min, 4° C.). After re-suspension, homogenization and centrifugation as above, the final pellet was re-suspended in 20 mM HEPES, 100 mM NaCl, 10 mM MgCl2, 1 mM EDTA (pH 7.4) and aliquots were kept at −80° C. [3H]-Spiperone Binding experiments were performed in 96 deep-well polypropylene plates in 50 mM Tris/HCl, 120 mM NaCl, 5 mM KCl, 5 mM MgCl2 (pH 7.4). Compounds of invention were serially diluted in DMSO at 100 fold final concentrations in the assay (1% DMSO final in the assay). Displacement was performed in the presence of 0.3 nM [3H]-Spiperone. The reaction was initiated by the addition of membrane suspension (4 μg and 12 μg of protein for CHO-hD3- and CHO-hD4 membranes, respectively) and lasted for 90 or 100 min (for hD3 or hD4 membranes, respectively) at 23° C. in a final volume of 500 μl. Non specific binding (NSB) was determined in the presence of 1 μM Spiperone. The binding reaction was stopped by rapid filtration through GF/B filterplates pre-soaked in 0.5% polyetylenimmine (PEI) using a Packard cell harvester. After washing with ice-cold 0.9% NaCl, the plate was left to dry before the addition of Microscint 20 (50 μl/well, PerkinElmer). Radioactivity was counted with a TopCount (PerkinElmer). Data were analysed by non-linear regression analysis using GraphPad Prism 5.0 (GraphPad Software). Saturation binding experiments were performed similar to the competition binding experiments using a radioligand concentrations ranging from 0.015 to 4.0 nM. Ref: Mackenzie R. G. et al. (1994). Characterization of the human dopamine D3 receptor expressed in transfected cell lines. Eur. J. Pharmacol. 266: 79-85.
Measurement: Ki = 5.89 nM (pChEMBL 8.229999542236328)
Descriptors: MW 449.58, ALogP 3.34, HBA 9, HBD 0, PSA 87.01, QED 0.4
Compound (SMILES): NS(=O)(=O)c1cc(NC(=O)c2cc(C(F)(F)F)cnc2N2CCC(Cl)(Cl)CC2)ccn1
InChIKey: MYVBIHHBPDHRRM-UHFFFAOYSA-N
Target: Sodium channel protein type 10 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y9
Assay: Biological Assay: The following buffers were used for the Qube® recordings: External buffer for Nav1.8 Qube® recording: 150 NaCl, 2 CaCl2, 5 KCl, 1 Mg Cl2, 10 HEPES, 12 Dextrose; External buffer for Qube® Nav1.5 recording: 120 N-Methyl-D-Glucamine, 40 NaCl, 1 KCl, 2.7 CaCl2), 5 HEPES, 0.5 MgCl2; and Internal buffer for Qube® recording: 120 CsF, 30 CsCl, 10 EGTA, 5 HEPES, 5 NaF, 2 MgCl2.
Measurement: IC50 = 31 nM (pChEMBL 7.510000228881836)
Descriptors: MW 498.31, ALogP 3.17, HBA 6, HBD 2, PSA 118.28, QED 0.63
Compound (SMILES): COc1cccc(CN2C(=O)N(c3ccc(-c4cn[nH]c4)c(OC)n3)CC23CCN(C(=O)C(C)O)CC3)c1
InChIKey: PXWJXCLKPHMOIM-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: 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) (SEQ ID NO. 1). 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.
Measurement: IC50 < 3 nM (pChEMBL 8.52299976348877)
Descriptors: MW 520.59, ALogP 2.67, HBA 7, HBD 2, PSA 124.12, QED 0.49
Compound (SMILES): N=C(Nc1ccc(F)c(Br)c1)c1nonc1SCCn1cncn1
InChIKey: HQKVEMNAAIAZKK-UHFFFAOYSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: Enzyme Assay: HIS-tagged IDO1 protein was recombinantly expressed in Escherichia coli using ZYP5052 autoinduction media supplemented with 500 uM delta aminolevulinic acid for 48 h at 16° C. IDO1 protein was purified using Ni2+-affinity resin and size exclusion chromatography. Purified protein was then diluted in assay buffer (50 mM Tris pH 7.0, 1% glycerol, 20 uM methylene blue, 0.05% Tween-20, 20 mM sodium ascorbate, 100 units/mL catalase to obtain a final IDO1 concentration of 40 nM. IDO1 solution (30 uM) or buffer alone (30 uM) were dispensed to wells of the assay plate using a BioRAPTR liquid dispenser (Beckman Coulter). Assay plates containing compound and IDO1 enzyme were incubated at RT for 30 min. Afterwards, 10 uL of 400 uM tryptophan in assay buffer were added to each well of the assay plate using a BioRAPTR liquid dispenser. Plates were incubated at RT for 60 min and reactions were quenched by addition of 10 uL of 0.5 M methyl isonipecotate in dimethyl sulfoxide. Plates were sealed and incubated at 37° C. for 4 h or 50° C. for 2 h. The plates are allowed to cool and then centrifuged for 1 min at 1000 ug. The resulting fluorescence was measured in an Envision plate reader (Perkin Elmer) with a 400/25 nm excitation filter and an 510/20 nm emission filter.
Measurement: IC50 = 31.4 nM (pChEMBL 7.5)
Descriptors: MW 412.25, ALogP 2.79, HBA 8, HBD 2, PSA 105.51, QED 0.36
Compound (SMILES): CCS(=O)(=O)c1ccc(Cl)cc1CNC(=O)c1cc(Cl)c(CN2CCC[C@H](NC)C2)c(C(F)(F)F)c1
InChIKey: TWGWKQNKYRFPRW-SFHVURJKSA-N
Target: Epithelial discoidin domain-containing receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q08345
Assay: LanthaScreen Eu Kinase Binding Assay: DDR1 binding activity was measured by the LanthaScreen (Registered trademark) Eu Kinase Binding Assay (manufactured by Life Technologies Corporation). A test compound and the Alexa Fluor (Registered trademark) 647-labeled Kinase Tracer 178 (manufactured by Life Technologies Corporation) were added to a mixture of DDR1 and the LanthaScreen (Registered trademark) Eu-anti-GST antibody. After reacting at room temperature for one hour, the fluorescence resonance energy transfer was measured. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate relative to the test compound-free control.
Measurement: IC50 = 6.7 nM (pChEMBL 8.170000076293945)
Descriptors: MW 566.47, ALogP 4.92, HBA 5, HBD 2, PSA 78.51, QED 0.47
Compound (SMILES): C1=CC2NC=C(c3nnc(SCc4ccccc4)n3CCCc3c[nH]cn3)C2C=C1
InChIKey: KOHHPNPAOWZSBW-UHFFFAOYSA-N
Target: Somatostatin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30872
Assay: Radioligand Binding Assay: Competitive radioligand binding experiments were performed using MEMBRANE TARGET Systems (Perkin-Elmer, Boston, Mass.) for human somatostatin receptors: SSTR1 (ES-520-M400UA), SSTR2A (ES-521-M400UA), SSTR3 (ES523-M400UA), SSTR4 (ES-524-M400UA) and SSTR5 (ES-522-M400UA) receptors. (See Table 1). Respective membrane receptor preparations were prepared in assay buffer (25 mM HEPES, 10 mM MgCl2, 1 mM CaCl2, 0.5% BSA, pH=7.4) at a 1:150 dilution. Binding assays were performed using 1251-Tyr-SRIF 14 (Perkin-Elmer, NEX 3890) dissolved in 1 mM HCl. Binding assays were performed in triplicate for each concentration of ligand in a total volume of 200 μL radioligand (25 μL ligand, 25 μL radioligand, 150 μL receptors) and incubated at room temperature for 90 minutes using a shaking table. Binding was terminated by filtration through GF/B glass fiber filters that were presoaked in 0.5% polyethyleneimine for a minimum of 4 hours. Filters were washed 9× with 1000 μL ice cold wash buffer (50 mM Tris-HCl, pH=7.4, 0.2% BSA). Filters were scored, transferred into plastic test tubes and counted in a gamma counter (Wizard2, Perkin-Elmer). Determination of the Ki for each compound was performed using non-linear regression with GraphPad PRISM-5 software (GraphPad Software, Inc., La Jolla, Calif.).
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 416.55, ALogP 3.98, HBA 6, HBD 2, PSA 71.42, QED 0.54
Compound (SMILES): Cc1cc(N2C(=O)c3c(C4CC4)nn(C)c3[C@H]2c2ccc(Cl)cc2)cc2c1nnn2C
InChIKey: XMRBIQDRTZCVGG-OAQYLSRUSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: In-Vitro Binding Assay: All assays were performed in 384 well microtiter plates. Each assay plate contained 8-point serial dilutions for 40 test compounds, plus 16 high- and 16 low controls. Liquid handling and incubation steps were done on an Innovadyne Nanodrop Express equipped with a robotic arm (Thermo CatX, Perkin Elmer/Caliper Twister II) and an incubator (Liconic STX40, Thermo Cytomat 2C450). The assay plates were prepared by addition of 50 nl per well of compound solution in 90% DMSO HummingBird nanodispenser (Zinsser Analytic). The assay was started by stepwise addition of 4.5 μl per well of bromo domain protein (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 45 nM His-Brd2(60-472) or 45 nM His-Brd3(20-477) or 45 nM His-Brd4(44-477) all proteins produced in-house) and 4.5 μl per well of peptide solution (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 60 nM acetyl-histone H4 (AcK 5, 8, 12, 16) (Biosyntan GmbH)). Reactions were incubated at 30° C. for 35 minutes. Subsequently 4.5 μl per well detection mix (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 3 nM Eu-labeled anti-His6 antibody, 21 nM streptavidin-allophycocyanin) were added. After 35 minutes incubation at 30° C., plates were measured in a Perkin Elmer EnVision multilabel reader. Concentrations causing 50% inhibition (IC50 values) were determined from percent inhibition values at different compound concentrations by non-linear regression analysis.
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 432.92, ALogP 4.29, HBA 6, HBD 0, PSA 68.84, QED 0.48
Compound (SMILES): CC1(C)Cc2cc(NC(=O)c3cnn4cccnc34)c(N3CCN(Cc4ncc[nH]4)CC3)cc2O1
InChIKey: BFKLLWAMWWKEHI-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Biological Assay: Kinase activities were assayed using the Transcreener-Fluorecescence polarization platform (BelBrook Labs, Madison, Wis., USA) that measures amounts of the reaction product, ADP. The IRAK4 reaction conditions were optimized using an IRAK1-derived peptide (sequence H-KKARFSRFAGSSPSQSSMVAR) to provide a linear reaction rate over the course of a 90 min incubation, which resulted in 10-12% conversion of the starting ATP to ADP. Final IRAK4 assay conditions were 1.25 nM IRAK4; 125 uM ATP; 10 uM MgCl2; 125 uM peptide in reaction buffer (25 mM HEPES (pH7.4); 2 mM Dithiothreitol; 0.015% Brij-35; and 0.5% dimethyl sulfoxide. The IRAK1 activity was optimized similarly, yielding final assay conditions of 3 mM IRAK1; 62.5 uM ATP; 5 uM MgCl2, and 62.5 uM IRAK1 peptide in reaction buffer for 60 min.Assays of compounds for kinase inhibition were performed using inhibitors serially-diluted in dimethyl sulfoxide, which was accomplished with a LabCyte Echo 555 liquid dispenser. 384 well assay plates spotted with compound received 4 ul of a 2× substrate (ATP+peptide) mix in reaction buffer, followed by 4 ul of 2× enzyme diluted in reaction buffer. Reactions were halted at 60 (IRAK1) or 90 (IRAK4) min by addition of 6 ul of detection buffer, containing EDTA (40 nM final concentration), 0.95 ug of the ADP-binding antibody ADP2, ADP tracer (3 nM final concentration), and 25 uM HEPES. Following a 1 hr incubation, fluorescence polarization of the ADP2-antibody TRACER complex was read on a Tecan M1000 plate reader using a 635/20 excitation filter in combination with a 670/20 emission filter.
Measurement: Ki = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 472.55, ALogP 2.74, HBA 8, HBD 2, PSA 103.68, QED 0.46
Compound (SMILES): CCc1cc2c(cc1Nc1nc(=O)c(OC)cn1Cc1ccccc1)OCCO2
InChIKey: DDZIXXBOMYQDOG-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Evaluation of a Human P2X7 Receptor Inhibitory Activity: Stably expressing cell line (1321N1 cell transfected with the human P2X7 receptor gene (GenBank accession number NM_002562.5 including T606C and G952A SNP)) was used. The cells were seeded in a 384-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (10% fetal bovine serum, 25 mM HEPES, 1% penicillin and streptomycin in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. After replacing with 20 μL of the HBSS buffer (20 mM HEPES, 55.6 mM D-glucose, 1×HBSS(−), pH7.4-7.5), 15 μL of 17.3 μM Yo-Pro solution in the HBSS buffer was added. The plate was placed in high-throughput cellular screening system FLIPR TETRA (Molecullar Devices, LLC.) and 15 μL of 130 μM BzATP solution in the HBSS buffer was added. Measurement of fluorescence intensity by FLIPR TETRA was started. After eight minutes, 15 μL of DMSO solutions containing different concentrations of the compound of the present invention as prepared by dilution with the HBSS buffer were dispensed to each well through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 20 minutes. The maximum fluorescence intensity without the compound of the present invention is calculated as 0% inhibition and the maximum fluorescence intensity when the reference compound was added is calculated as 100% inhibition. Changing values of fluorescence intensity by the compound of the present invention were calculated by difference between maximum and minimum fluorescence intensity for 20 minutes. IC50 was calculated using logistic approximation.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 393.44, ALogP 3.38, HBA 7, HBD 1, PSA 74.61, QED 0.69
Compound (SMILES): CC(C)C(=O)NCc1ccc(Cl)c(-c2nc(-c3ccc(OCC4CCOCC4)nc3)cc(=O)[nH]2)c1
InChIKey: HSDFPKJRGQTCSF-UHFFFAOYSA-N
Target: Prostaglandin E synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O14684
Assay: Inhibitory Activity Assay: Microsomes were prepared from COS-1 cells transiently transfected with a plasmid containing human mPGES-1 cDNA, and used as mPGES-1 enzyme. The mPGES-1 enzyme was diluted with a sodium phosphate buffer (pH 7.2) containing reduced glutathione (2.5 mM) and EDTA (1 mM), DMSO or a DMSO solution of a test compound (final concentration of DMSO was 1%) was added to the enzyme, and the mixture was preincubated at 4° C. for 15 minutes. Then, PGH2 as the substrate was added at a final concentration of 1 μM to start the enzymatic reaction, and after incubation at 4° C. for 4 minutes, a solution of ferric chloride (25 mM) and citric acid (50 mM) was added to terminate the enzymatic reaction. Generated PGE2 was measured by using Prostaglandin E2 Express EIA Kit (Cayman Chemical).
Measurement: IC50 = 1.9 nM (pChEMBL 8.720000267028809)
Descriptors: MW 497, ALogP 4.23, HBA 6, HBD 2, PSA 106.2, QED 0.48
Compound (SMILES): COc1ccc(COc2ccc(Nc3ccc4ncc(N5CCC(COC(C)=O)CC5)nc4c3C#N)cc2OC)cc1
InChIKey: GFTZYPXBIQIQBO-UHFFFAOYSA-N
Target: Macrophage colony-stimulating factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P07333
Assay: Kinase Assay: CSF-1R: Inhibition of CSF-1R kinase activity by a test compound disclosed herein was estimated by AlphaScreen (PerkinElmer). Standard assay conditions were 1.25 ng of recombinant CSF-1R kinase (SignalChem) with 10 ng Biotin-conjugated Poly-(Glu 4:Tyr 1) (Cisbio) in the assay buffer (10 μM ATP, 10 mM MOPs, pH7.0, 0.21 mM EDTA, 0.5% glycerol, 1 mg/ml BSA, 0.01% 2-mercaptoethanol, 0.001% Brij35) in a final volume of 25 μL. Reactions were incubated at 30° C. for 45 min and stopped by adding 5 μL of 50 mM EDTA. Analysis of resulting product using an AlphaScreen kit, and counted with Enspire Alpha (PerkinElmer). The readout from control reaction (complete reaction mixture) was designated as 0% inhibition and the readout for the reaction without enzyme as 100% inhibition. The IC50 values of quinoxaline compounds of this invention against CSF-1R kinase were determined using the GraphPad Prism 5 software.
Measurement: IC50 = 27.5 nM (pChEMBL 7.559999942779541)
Descriptors: MW 567.65, ALogP 5.62, HBA 10, HBD 1, PSA 118.83, QED 0.24
Compound (SMILES): CC(C)c1ccc2oc3nc(N)ccc3c(=O)c2c1
InChIKey: MJHQRCUJDFRTLG-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: Kinase Assays: Kinase assays are commonly used for determining the ability of a small molecule to inhibit enzyme activity. By measuring the degree of phosphorylation of a substrate at varying concentrations of inhibitor, the concentration of inhibitor resulting in 50% enzymatic activity (IC50) can be obtained. The IC50 is defined as the inflection point of the sigmoidal graph obtained when plotting substrate phosphorylation vs. inhibitor concentration. Experiments were conducted during development of embodiments herein using radiolabeled ATP to detect substrate phosphorylation in the presence of amlexanox analogs to determine the potency (IC50) of said analogs.TBK1 (residues 1-657) and IKKε (residues 1-655) were purified from insect cells to ≥90% purity by coomassie staining. Reactions containing 50 nM TBK1 or IKKε, 7 μM myelin basic protein (MBP), and inhibitor in reaction buffer (50 mM HEPES pH 7.5, 10 mM NaCl, 10 mM MgCl2, 1 mM DTT) were initiated with 5 μM ATP spiked with [γ-32P]-ATP and allowed to proceed for 30 minutes at room temperature. Reactions were quenched with SDS gel loading dye, run on 4-15% SDS-PAGE gels, and imaged on phosphorimaging screens. Band intensities corresponding to phosphorylated MBP were quantified with ImageQuant and the data analyzed in Prism 6. Dose-response assay band intensities were normalized and fit to a sigmoidal dose-response equation with the Hill slope fixed at −1 and the top constrained to 100.
Measurement: IC50 = 24400 nM (pChEMBL 4.610000133514404)
Descriptors: MW 254.29, ALogP 3.05, HBA 4, HBD 1, PSA 69.12, QED 0.68
Compound (SMILES): Cc1cc(-c2cnn(C)c2)nc(C2CCN(C(=O)c3cc(C(C)C)n[nH]3)C2)n1
InChIKey: KZMBBRVEMMYESC-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 = 0.76 nM (pChEMBL 9.119999885559082)
Descriptors: MW 379.47, ALogP 2.66, HBA 6, HBD 1, PSA 92.59, QED 0.75
Compound (SMILES): CCS(=O)(=O)Nc1ccc(Oc2ccc(F)cc2F)c(-c2cn(C)c(=O)c3sc(-c4nc5ccccc5o4)cc23)c1
InChIKey: ODORIAOXNUUHFP-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Binding Assay (BRD4-1): The bromodomain binding assays were performed by Reaction Biology Corp., Malvern, Pa., USA (www.reactionbiology.com). The BET binding assays were conducted in 384 well microplates in assay buffer (50 mM HEPES-HCl, pH 7.5, 100 mM NaCl, 1 mg/ml BSA, 0.05% CHAPS, and 0.5% DMSO) with compounds added as DMSO stocks at a single concentration or with 10-point dose response titrations. BET protein (20 nM for BRD4) or assay buffer were delivered to the appropriate wells of the microplate. Test compound (20 nM) was then delivered by acoustic technology via a Labcyte Echo550 liquid handler. The microplate was centrifuged for 5 min and pre-incubated for 30 min at RT with gentle shaking. The ligand (histone H4 peptide (1-21) K5/8/12/16Ac-biotin) was delivered and the microplate was again centrifuged for 5 min and allowed to incubate for 30 min at RT with gentle shaking. Donor beads were then added in the absence of light and the microplate was centrifuged and gently shaken. After 5 min, acceptor beads were added in the absence of light and the microplate was centrifuged and gently shaken in the dark for 60 min. The microplate was read using a Perkin Elmer EnSpire Alpha plate reader (λ Ex/λ Em=680/520-620 nm). Percent inhibition was calculated relative to positive and negative controls on a per plate basis.
Measurement: IC50 = 103 nM (pChEMBL 6.989999771118164)
Descriptors: MW 593.63, ALogP 6.91, HBA 8, HBD 1, PSA 103.43, QED 0.22
Compound (SMILES): CC(=O)c1csc2c(-c3cc(Cl)ccc3C#CCn3c(C)nc4cnc(N(C)[C@H]5C[C@H]6CC[C@@H](C5)N6CC(F)F)c(C#N)c4c3=O)ccnc12
InChIKey: JLQUJFWUIHVVQQ-GPOLMCQNSA-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 = 26.6 nM (pChEMBL 7.579999923706055)
Descriptors: MW 712.23, ALogP 6.85, HBA 10, HBD 0, PSA 108.01, QED 0.13
Compound (SMILES): CC(CC(C)(C)c1ccc(Cl)cc1)N1CCC(O)CC1
InChIKey: BWYUKDNDSYRADV-UHFFFAOYSA-N
Target: Sigma non-opioid intracellular receptor 1 (SINGLE PROTEIN), Cavia porcellus
UniProt: Q60492
Assay: Competitive Radioligand Binding Assay 2: The affinity of test compounds at sigma-1 and sigma-2 receptors was also determined by displacement of different known labeled sigma-2 or sigma-1 ligands. Filtration assays were conducted according the previously published procedure (Xu, et al., 2005). Test compounds were dissolved in N,N-Dimethylformamide (DMF), dimethyl sulfoxide (DMSO) or ethanol and then diluted in 50 mM Tris-HCl pH 7.4 buffer containing 150 mM NaCl and 100 mM EDTA. Membrane homogenates were made from guinea pig brain for sigma-1 binding assay and rat liver for sigma-2 binding assay. Membrane homogenates were diluted with 50 mM Tris-HCl buffer, pH 8.0 and incubated at 25° C. in a total volume of 150 uL in 96 well plates with the radioligand and test compounds with concentrations ranging from 0.1 nM to 10 uM. After incubation was completed, the reactions were terminated by the addition of 150 uL of ice-cold wash buffer (10 mM Tris HCl, 150 mM NaCl, pH 7.4) using a 96 channel transfer pipette (Fisher Scientific, Pittsburgh, Pa.) and the samples harvested and filtered rapidly through 96 well fiber glass filter plate (Millipore, Billerica, Mass.) that had been presoaked with 100 uL of 50 mM Tris-HCl buffer. Each filter was washed four times with 200 uL of ice-cold wash buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.4). A Wallac 1450 MicroBeta liquid scintillation counter (Perkin Elmer, Boston, Mass.) was used to quantitate the bound radioactivity.
Measurement: Ki = 1 nM (pChEMBL 9)
Descriptors: MW 295.85, ALogP 3.85, HBA 2, HBD 1, PSA 23.47, QED 0.91
Compound (SMILES): NC(=O)c1ccc(-n2cccc2-c2ccncc2)cc1NC1CCC(O)CC1
InChIKey: PFWJCVVCWPZZGP-UHFFFAOYSA-N
Target: Heat shock protein HSP 90-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P07900
Assay: Fluorescence Polarization: Assay buffer (25 μL, 20 mM HEPES pH 7.3, 50 mM KCl, 5 mM MgCl2, 1 mM DTT, 20 mM Na2MoO4, 0.01% NP-40, and 0.5 mg/mL BGG) was added to 96-well plate (black well, black bottom) followed by the desired compound at the indicated final concentrations in DMSO (1% DMSO final concentration). Recombinant cGrp94 (10 nM for compounds 2-27, 30 nM for compounds 28-48) and FITC-GDA were then added (6 nM). Plates were incubated with rocking for 5 h at 4° C. Fluorescence was determined using excitation and emission filters of 485 and 528 nm, respectively. Percent FITC-GDA bound was determined by using the DMSO millipolarization unit (mP) as the 100% bound value and the 0% for FITC-GDA. Kd values were calculated from separate experiments performed in triplicate using GraphPad Prism.
Measurement: Kd > 100000 nM (pChEMBL 4)
Descriptors: MW 376.46, ALogP 3.35, HBA 5, HBD 3, PSA 93.17, QED 0.64
Compound (SMILES): Cn1cc(NC(=O)c2ccc3ccc(N4C[C@H]5CC[C@@H]4CN5)nn23)c(Br)n1.O=C(O)C(F)(F)F
InChIKey: CPMYBXAAKPUKDR-LOCPCMAASA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IRAK4 Kinase Assay: The kinase activity of IRAK4 is determined by its ability to catalyze the phosphorylation of a fluorescent polypeptide substrate. The extent of phosphorylation is measured using the IMAP technology (Molecular Devices) where the phosphorylated fluorescent substrate binds to the large M (III)-based nanoparticles which reduces the rotational speed of the substrate and thus increases its fluorescent polarization (FP).20 μL reaction mixture contains 10 mM TriHCl, pH 7.2, 0.5 nM GST tagged IRAK4 (SignalChem), 100 nM fluorescent peptide substrate (RP7030, Molecular Devices), 100 μM ATP, 1 mM DDT, 1 mM MgCl2, and 0.01% Tween 20. The reaction is initiated by the addition of ATP. After incubation for 30 minutes at 25° C., 60 μL of Progressive IMAP Reagent (Molecular Devices) is added to stop the reaction. Change in RP7030's FP is determined by a FP reader (Analyst HT, LJL BioSystems).
Measurement: IC50 = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 430.31, ALogP 2.02, HBA 7, HBD 2, PSA 79.49, QED 0.67
Compound (SMILES): O=C(NCc1cccc(-c2cnc3n2CCNC3)c1)c1ccc2c(c1)OCc1cnccc1-2
InChIKey: JNTWFPLDVSBXRN-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: 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 = 370 nM (pChEMBL 6.429999828338623)
Descriptors: MW 437.5, ALogP 3.54, HBA 6, HBD 2, PSA 81.07, QED 0.51
Compound (SMILES): COC[C@H]1C[C@H]2C(=O)N(C)C(=N)N[C@@]2(c2cc(Oc3cc(C#N)ccn3)c(F)cc2F)CO1
InChIKey: AHGJFIJKACDGHF-QOAZOABKSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE1 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. 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). 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. Varying concentrations of inhibitors at 3× the final desired concentration in a volume of 10 ul are preincubated with purified human BACE1 catalytic domain (3 nM in 10 μl) for 30 minutes at 30° C. in reaction buffer containing 20 mM Na-Acetate pH 5.0, 10% glycerol, 0.1% Brij-35 and 7.5% DSMO. Reactions are initiated by addition of 10 μl of 600 nM QSY7-APPswe-Eu substrate (200 nM final) to give a final reaction volume of 30 μl in a 384 well Nunc HTRF plate. The reactions are incubated at 30° C. for 1.5 hours. The 620 nm fluorescence is then read on a Rubystar HTRF plate reader (BMG Labtechnologies) using a 50 millisecond delay followed by a 400 millisecond acquisition time window. Inhibitor IC50 values are derived from non-linear regression analysis of concentration response curves. Ki values are then calculated from IC50 values using the Cheng-Prusoff equation using a previously determined m value of 8 μM for the QSY7-APPswe-Eu substrate at BACE1.
Measurement: Ki = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 457.44, ALogP 2.27, HBA 7, HBD 2, PSA 120.56, QED 0.71
Compound (SMILES): Cc1nc(F)ccc1[C@H](Nc1cc(Cl)c2ncc(C#N)c(N[C@H](CCC#N)c3ccccc3)c2c1)C1=CN(C2CC2)NN1
InChIKey: KJADTWFOIBCKRK-ZUKKLESISA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: High Throughput Biochemical Assay: EGFR activity was measured using KinEASE (Cisbio), a time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay. In this assay, EGFR-catalyzes the phosphorylation of a universal Tyrosine kinase peptide substrate labeled with XL665. Europium conjugated phosphor-tyrosine specific antibody binds the resulting phosphorylated peptide. Formation of phosphorylated peptide is quantified by TR-FRET with Europium as the donor and XL665 the acceptor. The assay was performed in two main steps. The first step is the kinase reaction step and the second step is the detection step with TR-FRET reagents. In brief, test compounds 1:3 serially diluted in DMSO were delivered into Corning white, low volume, non-binding 384 well plates using the Echo 550 acoustic liquid dispenser (Labcyte®). EGFR enzyme (Human EGFR, cytoplasmic domain [669-1210] from Carna Biosciences Cat. No. 08-115) and substrates TK substrate-biotin (included in Cisbio HTRF KinEASE-TK kit Cat. No. 62TK0PEJ) were dispensed into assay plates using a Multi-Flo (Bio-Tek Instruments). The standard 10 μL reaction mixture contained 6 μM ATP (1×Km) or 12 μM ATP (2×Km), 1 μM biotinylated peptide, 0.3 nM EGFR (for 1×Km ATP) or 0.1 nM EGFR (for 2×Km ATP) in reaction buffer (10 mM MOPs, pH 7.0, 1.5% Glycerol, 0.5 mg/ml BSA, 10 mM Mg-Acetate, 1 mM DTT, 0.025% NP-40). After 60 min of incubation at room temperature, 10 μL of Stop and Detect Solution (1:400 Europium Cryptate labeled anti-phosphorylated peptide antibody solution and 125 nM strepavidin-XL665 Tracer in a 50 mM Hepes pH 7.0 detection buffer containing sufficient EDTA) was added. The plate was then further incubated for over 60 minutes at room temperature and read using an Envision 2103 Multilabeled reader (PerkinElmer) with excitation/emission/FRET emission at 340 nm/615 nm/665 nm, respectively.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 594.1, ALogP 6.54, HBA 9, HBD 4, PSA 124.72, QED 0.15
Compound (SMILES): Cc1nn(C2CCS(=O)(=O)CC2)c2ncc(C(=O)Nc3ccc(Cl)cc3)c(Cl)c12
InChIKey: SLJKFGHINGDLIZ-UHFFFAOYSA-N
Target: Glucocorticoid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04150
Assay: Biological Evaluation: The assay was performed using H4 neuroglioma cells and using indirect immunofluorescence detection of the endogenous GR receptor. Fixative: 4% PFA. Permeabilization buffer: 0.3% Triton X-100 in PBS. Primary antibody: 1:200 in 5% BSA block. Secondary antibody: 1:800 in 5% BSA block. HCS Cell Mask: 1:6000 in PBS. DAPI: 100 ng/ml.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 453.35, ALogP 4.05, HBA 6, HBD 1, PSA 93.95, QED 0.65
Compound (SMILES): O=COCOn1nnc2ccc(C(=O)N[C@H](Cc3ccc(-c4ccccc4Cl)cc3Cl)C[C@@H](O)C(=O)O)cc21
InChIKey: OWIPYCVUBCHUHS-HOYKHHGWSA-N
Target: Neprilysin (SINGLE PROTEIN), Homo sapiens
UniProt: P08473
Assay: Enzyme Inhibition Assay: The assays were performed in 384-well white opaque plates at 37° C. using the fluorogenic peptide substrates at a concentration of 10 μM in Assay Buffer (NEP: 50 mM HEPES, pH 7.5, 100 mM NaCl, 0.01% polyethylene glycol sorbitan monolaurate (Tween-20), 10 μM ZnSO4; ACE: 50 mM HEPES, pH 7.5, 100 mM NaCl, 0.01% Tween-20, 1 μM ZnSO4). The respective enzymes were used at concentrations that resulted in quantitative proteolysis of 1 μM of substrate after 20 minutes at 37° C.Test compounds were assayed over the range of concentrations from 10 μM to pM. Test compounds were added to the enzymes and incubated for 30 minute at 37° C. prior to initiating the reaction by the addition of substrate. Reactions were terminated after 20 minutes of incubation at 37° C. by the addition of glacial acetic acid to a final concentration of 3.6% (v/v).
Measurement: Ki < 1 nM (pChEMBL 9)
Descriptors: MW 573.39, ALogP 3.14, HBA 9, HBD 3, PSA 152.87, QED 0.13
Compound (SMILES): C=C(C)c1cn(-c2ccccc2C)c(=O)c2ccc(-n3nc(CO)n(CC)c3=O)cc12
InChIKey: YYMNDXDCONMGAF-UHFFFAOYSA-N
Target: Dihydroorotate dehydrogenase (quinone), mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q02127
Assay: In Vitro Assay: DHODH Enzymatic Assay: The following assay is referred to herein as the DHODH Enzymatic Assay. To detect DHODH enzyme activities, dichloroindophenol (DCIP) is added as the final electron acceptor in the assay. DCIP can accept electrons from the reduced coenzyme Q generated in the assay, or from dihydroorotate (DHO) via FMN by binding presumably to the ubiquinone pocket. DCIP solutions are blue, with an intense absorbance around 600 nm, but becomes colorless upon reduction (J. Biol. Chem. (1986) 261, 11386). The assay buffer contained 50 nM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, and 0.1% Triton X-100 in MilliQ water. Substrate consisting of 20 mM DHO, 5 mM CoQ6, and 1 mM DCIP in assay buffer, initiates the reaction. The assay is run in end-point mode by quenching the reaction with the potent DHODH inhibitor brequinar. Absorbance measurements were obtained using the BMG Phera Star plate-reading spectrophotomer. Purified human DHODH was purchased from Proteros (cat. No. PR-0044). Chemicals were purchased from Sigma-Aldrich, Teknova, and Avanti Polar Lipids. Liquid handling was performed using Labcyte Echo and Formulatrix Tempest.
Measurement: IC50 = 0.634 nM (pChEMBL 9.199999809265137)
Descriptors: MW 416.48, ALogP 3.19, HBA 7, HBD 1, PSA 82.05, QED 0.54
Compound (SMILES): COC1CN(c2nc3ccc(C)cc3cc2C(=O)OC(C)C)CCC1NC1CCCCC1
InChIKey: CLRXGWFAQYJWRY-UHFFFAOYSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: OPRK1 antagonist assay: The cell line for the OPRK1 antagonist assay stably expresses the following elements. The carboxy terminus of the OPRK1 receptor has a 7 amino acid linker, followed by the TEV protease cleavage site and a GAL4-VP16 fusion protein. The cell line also expresses a b-arrestin-2-TEV protease fusion protein and contains a reporter construct consisting of the UAS response element and the b-lactamase (bla) reporter gene. Upon activation of the receptor, g-protein receptor kinase (GRK) phosphorylates specific intracellular residues of OPKR1 and this induces recruitment of B-arrestin2-TEV protease fusion protein. The TEV protease recognizes and cleaves the TEV site, releasing the GAL4-VP16 fusion protein, which then translocates to the nucleus. The GAL4-V16 binds to the UAS element, driving expressing of the b-lactamase gene. B-lactamase expression is detected with the cell permeable, fluorescent substrate, CCF4-AM. This substrate consists of coumarin tethered to fluoroscein via a b-lactam ring. In the absence of b-lactamase, excitation of the dye with 405 nm light results in FRET from the coumarin to fluoroscein and emission of green (525 nm maximum) light. B-lactamase cleavage of the substrate separates the courmarin fluorophore from the fluorscein, and 405 nm excitation results in blue (460 nm maximum) emission. The assay is monitored by the blue/green emission ratio. The antagonist assay is performed by seeding the cells into 384 well plates and incubating them 16-24 hours at 37° C. Test antagonist compounds are added and incubated for 30 minutes at 37° C. Next an EC80 challenge of U-50488 (OKRK1 agonist) is added and the samples are incubated for 4 hours at 37° C., followed by addition of CCF4-AM substrate. The plates are then incubated 2 hours at room temperature in the dark and the blue/green ratio determined on a fluorescent plate reader. See J Biomol Screen April 2009, vol. 14 no. 4, pp 381-394.
Measurement: IC50 = 38 nM (pChEMBL 7.420000076293945)
Descriptors: MW 439.6, ALogP 4.62, HBA 6, HBD 1, PSA 63.69, QED 0.66
Compound (SMILES): COc1cc(OC(F)(F)F)cc(N2CCC(C(=O)NCCC3CCN(Cc4ccccc4)CC3)CC2)c1
InChIKey: WHNHWZWPVHPTPF-UHFFFAOYSA-N
Target: Muscarinic acetylcholine receptor M4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08173
Assay: Binding Assay: Binding affinity (Ki) of compounds was measured by inhibition of radioligand binding to membranes from CHO cells expressing human M1, M2, M3, M4 and M5 receptors. Membranes were prepared by nitrogen cavitation and differential centrifugation as previously described (Hoare et al., Mol. Pharmacol. 2003 March; 63(3): 751-65). The radioligand employed was tritiated N-methylscopolamine, used at a concentration of 1.5 nM. A dose-response of twelve concentrations of compound was used, ranging from 10 Î¼M to 32 Î¼M. The assay buffer was 50 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 1 mM ethylenediaminetetraacetic acid, pH-adjusted to pH 7.4.
Measurement: Ki = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 519.61, ALogP 5.23, HBA 5, HBD 1, PSA 54.04, QED 0.49
Compound (SMILES): COc1cc(C(=O)NS(C)(=O)=O)c(F)cc1-c1cnc(Oc2cc(Cl)ccc2F)c(Cl)c1
InChIKey: ZWLFMOMRVSTWIB-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Nav 1.7 In Vitro Patch Express (PX) Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to −10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes. Peak sodium current was measured at the end of the compound addition period to determine percent inhibition. Three to five cells were tested per concentration, and IC50 curves were fitted to percent inhibition as a function of concentration.
Measurement: IC50 = 83.6 nM (pChEMBL 7.079999923706055)
Descriptors: MW 503.31, ALogP 4.82, HBA 6, HBD 1, PSA 94.59, QED 0.52