lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COC[C@@H]1CCCC(=O)N1C[C@@H](C)S(=O)(=O)Nc1nnc(-c2ccc(C)o2)n1-c1c(OC)cccc1OC
InChIKey: IDHUFWPLGLIDRW-MSOLQXFVSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: 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 assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, and 0.1% (w/v) BSA with 200 mM NaCl, 3 μM GDP] and membranes expressing human APJ receptor/well along with WGA PS beads. The reaction was initiated by addition of 0.2 nM [35S]GTPγS in the absence or presence of various ligands and incubated at RT for 90 min. Nonspecific binding was determined in the presence of 100 μM GTPγS and was always less than 0.2% of total binding.
Measurement: EC50 = 0.24 nM (pChEMBL 9.619999885559082)
Descriptors: MW 547.63, ALogP 3.01, HBA 10, HBD 1, PSA 138.02, QED 0.38
Compound (SMILES): CC(C)(Cc1nnc(-c2nc(CN3CCCC(F)(F)C3)c(-c3ccc(C(O)(C(F)(F)F)C(F)(F)F)c(Cl)c3Cl)s2)o1)C(=O)O
InChIKey: OGUNKYJNRXHJAN-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: RORgammat (full-length human) Reporter Assay A: Cells used in this assay were transiently co-transfected with three different plasmids, one expressing the GAL4-DNA binding domain (DBD)-RORγt fusion protein under control of a CMV promoter (NH2-Gal4-DBD:RORC¿COOH in pCMV-BD, Stratagene #211342), and two reporter plasmids¿the firefly luciferase reporter under control of a GAL4 promoter (pFR-Luc 2× GAL4) and Renilla luciferase reporter under control of CMV promoter (pRL-CMV, Promega #E2261). The full-length coding sequence was used for human RORγt, i.e., nucleotides 142-1635 of human RORγt, transcript variant 2, NCBI Accession: NM_001001523.1 (SEQ ID NO: 1). HEK293T cells were plated at 35000 per well in 96-well plate in medium of MEM with 8.6% FBS. After 18-22 hours incubation, the transfection was carried out by using a PEI solution with 170.5 ng total DNA/well (50 ng pCMV-BD-ROR plus 20 ng of pFR-Luc reporter and 0.5 ng of pRL-CMV reporter plus 100 ng Carrier DNA (Clontech #630440) for each well). 4-6 hours after transfection, cells were treated with compounds for overnight in the medium with final concentration of FBS 1.1% and DMSO 0.1%. After overnight (16 to 20 hours) incubation, media were removed and cells were lysed with 20 μL 1× Passive Lysis Buffer (Promega) for 10-15 minutes. Luminescence was measured using a BMG LUMIstar OPTIMA plate reader, after addition of 75 μL/well firefly luciferase buffer, followed by 75 μL/well Renilla luciferase buffer. To calculate the effect of compounds on RORγt activity, firefly values were normalized against values of DMSO only and values of reference compound at saturating concentration, then further normalized against Renilla signals. IC50s were generated by plotting final Renilla normalized data against compound concentration and percent inhibition was calculated against DMSO control.
Measurement: IC50 = 37 nM (pChEMBL 7.429999828338623)
Descriptors: MW 697.43, ALogP 7.36, HBA 8, HBD 2, PSA 112.58, QED 0.24
Compound (SMILES): CNCC1(c2ccc(Cl)c(Cl)c2)CCC(F)CC1
InChIKey: FRFZOFGUQVIKSJ-UHFFFAOYSA-N
Target: Sodium-dependent dopamine transporter (SINGLE PROTEIN), Homo sapiens
UniProt: Q01959
Assay: Functional Uptake Assay (hDAT): Inhibition of human dopamine reuptake transporter was assayed using the recombinant human dopamine transporter expressed in either CHO-K1 or HEK293 cells using a published method (Pristupa, Z. B. et al., Mol. Pharmacol. 45: 125-135, 1994). Either CHO-K1 or HEK293 cells expressing human recombinant dopamine transporter were plated before the assay. Test compound and/or vehicle was preincubated with cells in modified HEPES buffer pH 7.1 or pH 7.4 for 20 minutes at 18 to 25° C. and 50 nM [3H]dopamine was then added for an additional timed incubation period (10 to 30 minutes). After washing the cells to remove [3H]dopamine not internalized, the cells were lysed, and the amount of tritium in the lysate was measured using a liquid scintillation counter to determine [3H]dopamine uptake. Non-specific binding of tritium was measured in a control reaction containing 10 uM nomifensine, and was subtracted from the counts for assays to correct for non-specific binding of tritium. Reduction of [3H]dopamine uptake by 50 percent or more (50%) relative to an uninhibited control reaction indicates significant inhibitory activity. Compounds were screened at 10, 1, 0.1, 0.01 and 0.001 uM. The reference compound for the assay was nomifensine, for which the IC50 value of 11 nM was obtained in a typical experiment.
Measurement: IC50 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 290.21, ALogP 4.36, HBA 1, HBD 1, PSA 12.03, QED 0.87
Compound (SMILES): Fc1ccc(-n2cc(COc3ncc(N4CCC(F)(F)C4)cn3)nn2)cc1C(F)F
InChIKey: XYFMTQICDRKSTF-UHFFFAOYSA-N
Target: Retinoic acid receptor RXR-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P19793
Assay: NR2B standard assay: NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 149 mM NaCl, 4 mM KCl, 2 mM CaCl2, and 1.5 mM MgCl2, 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (149 mM NaCl (standard assay) or 150 mM (HTS assay), 4 mM KCl (standard assay) or 3 mM (HTS assay), 2 mM CaCl2, 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to ¿EC80 (standard assay) or EC40 (HTS assay) is used to maximize the assay's signal window or the ability to detect NMDA receptor antagonists and negative allosteric modulators, respectively. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 68 nM (pChEMBL 7.170000076293945)
Descriptors: MW 426.35, ALogP 3.56, HBA 7, HBD 0, PSA 68.96, QED 0.56
Compound (SMILES): COc1cc2c(cc1Nc1ncc(Cl)c(Nc3ccccc3S(=O)(=O)C(C)C)n1)CCNC2(C)C
InChIKey: XBJYPECXRASMSS-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Inhibitory Activity Against Anaplastic Lymphoma Kinase (ALK): A proliferation inhibitory activity against the ALK of the compound represented by Chemical Formula 1 according to the present invention at an enzyme level, the following experiment was performed.In order to measure the inhibitory activity against the ALK, the compounds (2 μl) prepared in Examples 1 to 106 were added to Greiner 96 well round bottom plates, respectively, and the ALK enzyme (1 μl) and biotin-adhered peptide substrate (2 μl) were mixed for 15 minutes, and then, the plates were cultured. An ATP solution (5 μl) was added thereto, and then, a kinase reaction was performed at room temperature for 30 minutes. Streptavidin-adhered XL 665 (5 μl) and europium (Eu3+)-adhered anti-phosphotyrosine antibody (5 μl) dissolved in ethylenediaminetetraacetic acid solution was added to the reaction solution to stop the reaction, and cultured for 1 hour, and analyzed by using homogeneous time-resolved fluorescence (HTRF, Cisbio) The compounds were read in a wavelength range of 615/665 nm using a Wallac Envision 2103 instrument. The IC50 of each of the test compounds subjected to the above experiment was implemented using prism (version 5.01, GraphPad) software.Accordingly, IC50 of each of the compounds that reduce an ALK enzyme activity and a cell activity of L1196M (a non-small cell lung cancer cell including an ALK enzyme) to 50% was calculated, and as a result, it was found that all of the compounds prepared in Examples 1 to 106 according to the present invention had IC50 values of 0.01 μM or less.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 516.07, ALogP 5.19, HBA 8, HBD 3, PSA 105.24, QED 0.39
Compound (SMILES): O=C(Oc1cc2c(Cl)cccc2[nH]c1=O)c1cc(CCc2ccccc2)c[nH]1
InChIKey: HGXQHHQRKVUEAA-UHFFFAOYSA-N
Target: D-amino-acid oxidase (SINGLE PROTEIN), Sus scrofa
UniProt: P00371
Assay: In Vitro Measurements of porcine D-Amino Acid Oxidase (DAAO) Activities: The pkDAAO (porcine kidney DAAO) activity was measured by using D-Proine as a substrate to produce hydrogen peroxide (H2O2). The produced H2O2 would be oxidized by peroxidase, and the produced free radicals would further react with 1,2-Phenylenediamine (OPD) reagent. The reaction product had an absorbance on 450 nm. The OD450 would be measured to represent the activity of pkDAAO. All compounds were dissolved in DMSO. Each compound was diluted with DMSO in 3 or 4-fold serial dilution to create a 9-point dose response curve. Each sample was added in triplicate, 10 μL/well, into 96-well assay microplate. Positive control wells were added with 10 μL of DMSO. The diluted compounds were incubated with pkDAAO in dark for 10 minutes and then reacted with D-Proline. The final reaction mixture was composed of 0.01 U/mL pkDAAO, 0.03% OPD, 25 U/mL HRP and 40 mM D-Proline in PBS. The reaction plates were then incubated in the dark at room temperature. The OD450 absorbance readout was detected at 0 and 20 minute by Molecular Device Spectra Max Plus reader. The percentage of inhibition values for each well were calculated with the following equation:The percentage of inhibition=(OD450sample, 20 min−OD450sample, 0 min)/(OD450DMSO, 20 min−OD450DMSO, 0 min)×100%The nonlinear curve fitting model in GraphPad Prism 5 was used to calculate IC50 value for each compound.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 392.84, ALogP 4.51, HBA 3, HBD 2, PSA 74.95, QED 0.49
Compound (SMILES): COc1ccc(Nc2nccc(Oc3ccc(NC(=O)Nc4cc(C(C)(C)C)nn4-c4ccc(C)cc4)c4ccccc34)n2)cc1OC
InChIKey: KNRZDHNOISHOGX-UHFFFAOYSA-N
Target: Glycogen synthase kinase-3 alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P49840
Assay: GSK 3α Enzyme Inhibition: The GSK3-α protein (500 ng/mL, 2.5 μL) was mixed with the test compound (2.5 μL at either 4 μg/mL, 0.4 μg/mL, 0.04 μg/mL, or 0.004 μg/mL) for 2 hr at RT. The FRET peptide (8 μM, 2.5 μL), which is a phosphorylation target for GSK3α, and ATP (40 μM, 2.5 μL) were then added to the enzyme/compound mixture and the resulting mixture incubated for 1 hr. Development reagent (protease, 5 μL) was added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan® Flash, ThermoFisher Scientific).
Measurement: IC50 = 2108 nM (pChEMBL 5.679999828338623)
Descriptors: MW 643.75, ALogP 8.62, HBA 9, HBD 3, PSA 124.45, QED 0.14
Compound (SMILES): O=S(=O)(Nc1cscn1)c1cc(C(F)F)c(NCc2c(F)cccc2CN2CCCC2)cn1
InChIKey: XPDGKOOOSKSIAM-UHFFFAOYSA-N
Target: Sodium channel protein type 8 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UQD0
Assay: Sodium Influx Assay (In Vitro Assay): This sodium influx assay employs the use of the cell permeable, sodium sensitive dye ANG2 to quantify sodium ion influx through sodium channels which are maintained in an open state by use of sodium channel modulators. This high throughput sodium influx assay allows for rapid profiling and characterization of sodium channel blockers.In general, Trex HEK293 cells were stably transfected with an inducible expression vector containing the full-length cDNA coding for the desired human sodium channel α-subunit and with an expression vector containing full length cDNA coding for the β1-subunit. Sodium channel expressing cell lines were induced with tetracycline (1 μg/mL) and plated on 384-well PDL-coated plates at a density of 25K-30K cells/well in culture media (DMEM, containing 10% FBS and 1% L-glutamine). After overnight incubation (37° C., 5% CO2), culture media was removed and cells were loaded with 5 uM ANG2 dye for 1-1.5h in Buffer 1 (155 mM NMDG, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, adjusted with Tris to pH 7.4). Access dye was removed and cells were incubated with test compounds for 1 hr in buffer 1 containing sodium channel modulator(s) at room temperature. Hamamatsu FDSS μCell was used to perform a 1:1 addition of Na/K challenge buffer (140 mM NaCl, 20 mM HEPES, 1 mM CaC2, 15 mM KCl, 1 mM MgCl2, 10 mM glucose, adjusted with Tris to pH 7.4) and simultaneously read plates at excitation wavelength of 530 nm and emission wavelength set at 558 nm. Percent inhibition of sodium ion influx was calculated for each test compound at each test concentration to determine the IC50 values.
Measurement: IC50 = 1460 nM (pChEMBL 5.840000152587891)
Descriptors: MW 497.57, ALogP 4.62, HBA 7, HBD 2, PSA 87.22, QED 0.45
Compound (SMILES): Cc1cc(-c2[nH]c3ccc(C4CCN(C(=O)CN(C)C5CCOC5)CC4)cc3c2C(C)C)cc(C)n1
InChIKey: GDURWVZLWYYDOE-UHFFFAOYSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
Assay: Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications.
Measurement: IC50 = 61.1 nM (pChEMBL 7.210000038146973)
Descriptors: MW 488.68, ALogP 5.4, HBA 4, HBD 1, PSA 61.46, QED 0.5
Compound (SMILES): C=CC(=O)N1CCC(c2cc(-c3ccc(Oc4cc(C(F)(F)F)ccn4)cc3)c(N)c3cncnc23)C1
InChIKey: OBGIDMAZWFQCPQ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Inhibition assay for BTK: To determine IC50 of a compound against a kinase, a series of concentrations of the compound were tested for the inhibition. IC50 was calculated by plotting the concentration of compound vs the percentage of inhibition in treated wells using GraphPad Prism 5.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 505.5, ALogP 5.59, HBA 6, HBD 1, PSA 94.23, QED 0.28
Compound (SMILES): COCc1ccc(NC(=O)c2c(Br)n(C3(C)CC3)c3ncnc(N)c23)cc1
InChIKey: QLHJQWFGVCHOJR-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: Inhibitory Activity Against Resistance Mutation of RET (In Vitro): Regarding the conditions for measurement of in vitro inhibitory activity of compounds against RET (V804L) (i.e., RET with V804L mutation) kinase activity, the website of AnaSpec states that Srctide (GEEPLYWSFPAKKK) corresponds to the substrate peptide for reaction to measure RET kinase activity. Thus, the amino acid sequence was partly modified and biotinylated to prepare biotinylated peptides (biotin-EEPLYWSFPAKKK). The purified recombinant human RET (V804L) protein used in the test was purchased from Eurofins.To measure the inhibitory activity, first, the test compounds were individually diluted with dimethyl sulfoxide (DMSO) stepwise. Subsequently, RET (V804L) protein, the substrate peptides (the final concentration: 250 nM), magnesium chloride (the final concentration: 10 mM), ATP (the final concentration: 10 μM), and a solution of a test compound in DMSO (the final concentration of DMSO: 5%) were added to a buffer for kinase reaction (13.5 mM Tris, pH of 7.5, 2 mM dithiothreitol, 0.009% Tween-20). Each of the mixtures was incubated at 25° C. for 120 minutes to perform a kinase reaction. EDTA was then added thereto to give a final concentration of 40 mM so that the reaction was terminated. A detection solution containing Eu-labeled antiphosphotyrosine antibody PT66 (PerkinElmer) and SureLight APC-SA (PerkinElmer) was added thereto, and each mixture was allowed to stand at room temperature for 2 hours or more. Finally, the intensity of fluorescence under excitation light with a wavelength of 337 nm was measured with a PHERAstar FS (BMG Labtech) at the two wavelengths of 620 nm and 665 nm. The phosphorylation level was calculated from the ratio of the fluorescence intensity at the two wavelengths, and the compound concentration at which phosphorylation was inhibited by 50% was defined as the IC50 value (nM).
Measurement: IC50 = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 430.31, ALogP 3.68, HBA 6, HBD 2, PSA 95.06, QED 0.64
Compound (SMILES): Cc1nc2c(F)cc(-c3nc(Nc4ncc5c(n4)CCNC5)ncc3F)cc2n1C(C)C
InChIKey: GUPLVXQEYAKBCK-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00534
Assay: Inhibitory Activity Assay: 1. Take 10 mM stock solution of the test compound, in 96-well compound plate, DMSO was used to dilute the compound to an initial concentration of 100×, then this concentration was used as the first concentration, 3-fold diluted to make 10 serial concentrations; 1 μL each serial dilution was then added to 19 μL 1×reaction buffer to prepare 5×compound for use; 2 μL 5×compound was transferred from 96-well plate to 384-well plate; compound-free control well was added with 2 μL the following liquid: 1×reaction buffer with the addition of 1 μL DMSO; 2 μL 250 mM EDTA was added to the Min control well.2.1× reaction buffer was used to formulate the kinase, substrate and ATP into a 2.5×enzyme/substrate mixture and 2.5×ATP solution respectively. In the experiment, the final concentration of CDK4/CycD3 kinase is 0.76 ng/μL, the final concentration of ATP is 80 μM; the final concentration of CDK6/CycD3 kinase is 0.5 ng/μL, the final concentration of ATP is 50 μM; the final concentration of CDK2/CycA2 kinase is 0.86 ng/μL, the final concentration of ATP is 15 μM; the final concentration of CDK2/CycE1 kinase is 1.016 ng/μL, the final concentration of ATP is 20 μM; 2.5×enzyme/substrate mixture was added to a 384-well plate, incubated at room temperature for 5 minutes; then added with 2.5×ATP solution, reacted at room temperature for 30 minutes.3. LANCE® Detection Buffer was used, 1× to prepare 2×LANCE® Ultra Europium-anti-phospho-eIF4E-binding protein 1 (Thr37/46) for use. After the enzymatic reaction was continued for 30 minutes, 10 mM EDTA was added to 384-well plate and the mixture was reacted at room temperature for 5 minutes. Then LANCE® Ultra Europium-anti-phospho-eIF4E-binding protein 1 (Thr37/46) was added, reacted at room temperature for 1 hour.4. The 384-well plate was placed in HERAEUS Multifuge X1R centrifuge, centrifuged at 2000 rpm for 2 minutes; data were measured on EnVision, 337 nM wavelength laser was used as the excitation light, test at RFU665 nM and RFU615 nM, and RFU665 nM/RFU615 nM×10000 was used as the final data for analysis.
Measurement: IC50 = 327 nM (pChEMBL 6.489999771118164)
Descriptors: MW 436.47, ALogP 3.84, HBA 8, HBD 2, PSA 93.44, QED 0.5
Compound (SMILES): COc1ccc(NC(=O)c2n[nH]c3ccc(-c4cncc(N5CC(N)C5)n4)cc23)cn1
InChIKey: OKDAXNJHNXANQC-UHFFFAOYSA-N
Target: Dual specificity tyrosine-phosphorylation-regulated kinase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: Q13627
Assay: DYRK1A activity assay: SH-SY5Y cells are cultured in DMEM/F-12 medium supplemented with 15% FBS, Non-essential Amino Acid and Penicillin/Streptamycin. Two days before treatment, cells are seeded onto 96 well plates at 20e5 cells/well.DMSO-resuspended compounds are dispensed to 8 wells as a serial titration from 10 μM to 4.6 nM final and cells are exposed overnight (16-18 h) before harvest. Wells are visualized checked for cell death or change in morphology and supernatants are tested for cytotoxicity by measurement of lactate dehydrogenase release (LDH, CytoToxOne kit, Progema) if necessary. As controls, commercially available DYRK1A inhibitors, Harmine and Indy were shown to have good DYRK1A inhibition in the kinase assay with no CDK1 activity (EC50 18 and 53n M respectively, 6 μM for CDK1) but weak EC50 in the Tau assay: about 10 μM for Harmine and 30 μM for Indy.Cells are lyzed with RIPA buffer complemented with phosphatase and protease inhibitors (Thermo Scientific) then lysates are sonicated and spun down at 12,000 g for 10 min to remove any cellular debris. Lysates are then either directly tested for pSer396 by ELISA (Life Technology, Kit KHB7031) or loaded on NuPage Bis-Tris gels for western blot analysis. Colorimetric detection of ELISA signal is performed by Cytation3 plate reader (Biotek) and the chemoluminecence signal for HRP-linked antibodies used in western blotting is detected using a Carestream Image Station. The same pSer396 antibody is used for detection of pTau in both assays.Blot densitometry for pSer396 and beta-actin are analyzed using ImageJ (NIH) and pSer396/Total Tau ELISA signal was used to plot, draw the curve fitting, and determine each compounds EC50 in Prism (GraphPad).
Measurement: EC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 416.45, ALogP 1.82, HBA 8, HBD 3, PSA 134.94, QED 0.45
Compound (SMILES): CN(C)c1cccnc1CNC(=O)c1nc(-c2ccc(=O)n(C)c2)c(-c2ncco2)nc1N
InChIKey: GYSSUJQTLZXOER-UHFFFAOYSA-N
Target: Adenosine receptor A1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30542
Assay: Binding Affinity Assay: The compounds at different concentrations were incubate with hA1 membrane (from PerkinElmer) and [3H]-8-Cyclopentyl-1,3-dipropylxanthine (DPCPX) for 50 min at 25° C., meanwhile 100 μL 0.5% PEI solution was added into UNFILTER-96 GF/B filter plate for 60 min at 4° C., then UNIFILTER-96 GF/B filter plate was washed twice with 50 ml wash buffer, the membrane mix was transferred into UNIFILTER-96 GF/B filter plate, and the filter plate was washed 4 times before incubated at 55° C. for 10 min. At last, 40 μL ULTIMA GOLD was added into each well, and CPM was read by TopCount. The compounds at different concentrations were incubate with hA2a membrane (from PerkinElmer) and [3H]-CGS21680 for 90 min at 25° C., meanwhile 100 μL 0.5% PEI solution was added into UNFILTER-96 GF/B filter plate for 60 min at 4° C., then UNIFILTER-96 GF/B filter plate was washed twice with 50 ml wash buffer, the membrane mix was transferred into UNIFILTER-96 GF/B filter plate, and the filter plate was washed 4 times before incubated at 55° C. for 10 min. At last, 40 μL ULTIMA GOLD was added into each well, and CPM was read by TopCount. The compounds at different concentrations were incubate with hA2b membrane (from PerkinElmer) and [3H]-DPCPX for 60 min at 27° C., and the binding reactions were stopped by rapid filtration through 0.5% BSA coated UNIFILTER-96 GF/C plates using cell harvester. The filter plates were then washed three times with ice cold wash buffer, and dried at 37° C. for 120 min. At last, 50 μL of scintillation cocktail was added into each well, and CPM was read by TopCount. The compounds at different concentrations were incubate with hA3 membrane (from PerkinElmer) and [125I]-AB-MECA for 60 min at 27° C., the binding reactions were stopped by rapid filtration through 0.5% BSA coated UNIFILTER-96 GF/C plates using cell harvester. The filter plates were then washed three times with ice cold wash buffer, and dried at 37° C. for 120 min. At last, 50 μL of scintillation cocktail was added into each well, and CPM was read by TopCount.
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 446.47, ALogP 1.47, HBA 10, HBD 2, PSA 145.06, QED 0.45
Compound (SMILES): Cc1nc(C(=O)N2C3CC(C3)[C@H](C)C2COc2ccc(F)cn2)c(-c2ccccn2)s1
InChIKey: DKEGHGZGKXBHGG-YHHJOVQNSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Scintillation Proximity Assay (SPA) Binding Assay: CHO cells stably transfected with human Orexin type 1 receptors (CHO-hOX1) or HEK-293 cells transiently transfected with human Orexin type 2 receptors (HEK-hOX2) were collected after 16 h induction with 5 mM sodium butyrate. The cell pellets were re-suspended, homogenized in 15 mM Tris/HCl pH=7.5, 1 mM EGTA, 0.3 mM EDTA, 2 mM MgCl2, protease inhibitors and centrifuged at 40,000 g (20 min, 4° C.). After re-suspension, homogenization and centrifugation as above, the final pellets were re-suspended in 75 mM Tris/HCl pH=7.5, 1 mM EGTA, 0.3 mM EDTA, 12.5 mM MgCl2, 250 mM Sucrose, protease inhibitors, divided into aliquots and frozen down at −80° C.Compounds of invention were serially diluted in neat DMSO at 100-fold concentrations (1% DMSO final in the assay) and 2 μl/well were plated into 96-well Isoplates (Perkin Elmer).
Measurement: Ki = 3388 nM (pChEMBL 5.46999979019165)
Descriptors: MW 438.53, ALogP 4.37, HBA 6, HBD 0, PSA 68.21, QED 0.59
Compound (SMILES): CC(C)c1ccnc(OCc2cn(-c3ccc(Cl)c(C(F)F)c3)nn2)n1
InChIKey: DFALROLCFWWLDX-UHFFFAOYSA-N
Target: Retinoic acid receptor RXR-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P19793
Assay: NR2B standard assay: NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 149 mM NaCl, 4 mM KCl, 2 mM CaCl2, and 1.5 mM MgCl2, 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (149 mM NaCl (standard assay) or 150 mM (HTS assay), 4 mM KCl (standard assay) or 3 mM (HTS assay), 2 mM CaCl2, 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to ¿EC80 (standard assay) or EC40 (HTS assay) is used to maximize the assay's signal window or the ability to detect NMDA receptor antagonists and negative allosteric modulators, respectively. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 129 nM (pChEMBL 6.889999866485596)
Descriptors: MW 379.8, ALogP 4.35, HBA 6, HBD 0, PSA 65.72, QED 0.64
Compound (SMILES): CCCCOC(=O)c1ccc2nc(-c3ccc(F)cc3)c(N3CCCCC3)nc2c1
InChIKey: LNTSWYUOHQDHBB-UHFFFAOYSA-N
Target: PAS domain-containing serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: Q96RG2
Assay: FRET Assay: Compound screening was done via the following method. 10 mM stock solution of test compound in DMSO was prepared by dissolving test compound in DMSO at RT for 1 hour, and then sonicating at 100% output for 8 minutes. If compound is not soluble under this condition, it was diluted to 3 mM. Kinase reaction buffer was prepared containing 10 mM MgCl2, 50 mM HEPES, 1 mM EGTA, 0.01% TWEEN-20, 2 mM DTT. Serial dilutions of the test compounds were prepared at 4× final assay concentrations using Freedom EVO200® dispensing system as follows: 12×10−5 M, 4×10−5 M, 1.33×10−5 M, 4.44×10−6 M, 1.48×10−6 M, 4.92×10−7M, 1.65×10−7 M, 5.48×10−7 M, 1.82×10−8 M, 6.09×10−9, 2.03×10−9 M. Test compounds (2.5 μl at 4× the final assay concentration) was added to wells using Freedom EVO200 dispensing system. As a positive control, 2.5 μl of positive compound was added to assay wells, and 2.5 μl of DMSO to assay wells as vehicle control. Kinase solution was prepared in reaction buffer at 2× final assay concentration. Kinase solution (5 μl) was added to each well of the assay plate. The substrate and ATP solution was prepared in kinase reaction buffer at 4× final assay concentration. The kinase reaction was started by adding 2.5 μl of substrate+ATP mix solution to each well of the assay plate. The plate is mixed on a plate shaker; then covered and allowed to react for 2 hours in the dark at 25° C. without shaking. The reaction was stopped by adding 5 μl of stop solution to each test well followed by mixing and incubation at RT for 10 minutes in the dark. 5 μl of detection mix (detection antibody diluted in detection buffer) was added; the contents of the plate were mixed and then incubated in the dark for 1 hour at RT. The signal was recorded at TR-FR ET mode (665 nm/615 nm).
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 407.49, ALogP 5.38, HBA 5, HBD 0, PSA 55.32, QED 0.41
Compound (SMILES): COc1ccc2c(c1)[C@]1(CC1c1ccc3c(/C=C/c4csc(CN5CCOCC5)c4)n[nH]c3c1)C(=O)N2
InChIKey: GDNLKCHOVHUYTG-XDUDPQOESA-N
Target: Aurora kinase B (SINGLE PROTEIN), Homo sapiens
UniProt: Q96GD4
Assay: Inhibition Assay: Aurora B inhibition was determined using the Z-Lyte assay kit from Invitrogen. The assay was performed using the recommended manufacturer's instructions with 128 μM ATP and 28 nM Aurora B (Invitrogen cat #PV3970). The % inhibition values were determined according to the manufacturer's directions and IC50 values were obtained using a non-linear 4 point logistic curve fit (XLfit4, IDBS)
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 512.64, ALogP 5.01, HBA 6, HBD 2, PSA 79.48, QED 0.38
Compound (SMILES): CN1CC2(CC(C(=O)Nc3cc4cc(-c5cncn5C)ccc4cn3)C2)C1
InChIKey: LAJDZYPFDBOUJG-UHFFFAOYSA-N
Target: Glycogen synthase kinase-3 beta (SINGLE PROTEIN), Homo sapiens
UniProt: P49841
Assay: The screening for GSK3beta kinase activity: Each compound is dissolved in DMSO as a 10 mM stock and used to prepare compound source plates. Serial dilution (1:3, 11-point dose-response curves from 10 μM to 0.0003 μM) and compound transfer was performed using the ECHO 550 (Labcyte, Sunnyvale, Calif.) into 1536-well black-walled round bottom plates (Corning). The GSK3β kinase assay is run using the Ser/Thr 09 peptide Z-lyte assay kit according to manufacturer's instructions (Life Technologiesa Division of Thermo-Fisher). This is a non-radioactive assay using fluorescence resonance energy transfer (FRET) between coumarin and fluorescein to detect kinase activity which is represented as ratio of coumarin emission/fluorescein emission. Briefly, recombinant GSK3β kinase, ATP and Ser/Thr peptide 09 are prepared in 1× Kinase buffer to final concentrations of 0.04 μg/mL, 46 μM, and 4 μM respectively. The mixture is allowed to incubate with the representative compounds for one hour at room temperature. All reactions were performed in duplicate. Unphosphorylated (0% Control) and phosphorylated (100% control) forms of Ser/Thr 18 serve as control reactions. After incubation, diluted Development Buffer is added to the reaction and allowed to further incubate for one hour at room temperature. The plate is read at Ex 400 Em 455 to detect the coumarin signal and Ex 400 Em 520 to measure the signal (EnVision Multilabel Plate Reader, PerkinElmer). The Emission ratio (Em) is calculated as a ratio of the coumarin (C) emission signal (at 445 nm)/Fluorescein (F) emission signal (at 520 nm). The percent phosphorylation is then calculated using the following formula: [1−((Em ratio×F100%)−C100%)/((C0%−C100%)+(Em ratio×(F100%−F0%)))]. Dose-response curves are generated and inhibitory concentration (IC50) values are calculated using non-linear regression curve fit in the Dotmatics' Studies Software (Bishops Stortford, UK).
Measurement: EC50 = 42 nM (pChEMBL 7.380000114440918)
Descriptors: MW 361.45, ALogP 2.92, HBA 5, HBD 1, PSA 63.05, QED 0.78
Compound (SMILES): Cn1cc(-c2cnc(N)c(C(=O)N[C@H]3CCC[C@@H]3OCc3ccc(-c4ccccc4)cc3)c2)cn1
InChIKey: LPJSSPDAFKCJDK-UIOOFZCWSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: Inhibition of N-terminal His-tagged recombinant human Axl (473 to end residues) expressed in baculovirus-infected Sf9 cells using biotin-labeled TK and ATP as substrate preincubated with enzyme for 30 mins followed by substrate addition and measured after 1 hr by HTRF assay
Measurement: IC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 467.57, ALogP 4.6, HBA 6, HBD 2, PSA 95.06, QED 0.41
Compound (SMILES): Cc1ncsc1C(CNC(=O)c1c(F)cccc1Cl)N1CCC(Oc2cccc(F)c2)CC1
InChIKey: YPANXZDGNFFRJU-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q64663
Assay: Screen Quest Fluo-8 No Wash Calcium Assay Kit: Ca2+ influx was measured in HEK-293 cells stably transfected with the receptor using Screen Quest Fluo-8 No Wash Calcium Assay Kit (AAt Bioquest®). Briefly, once inside the cells, the lipophilic blocking groups of Fluo-8 are cleaved by non-specific cell esterases, resulting in a negatively-charged fluorescent-dye that stays inside cells. Its fluorescence increases upon binding to calcium. When HEK-293/P2X7 cells were stimulated with BzATP, Ca2+ entered the cells and the fluorescence of Fluo-8 NW increaseed. The dye absorption spectrum was compatible with excitation at 488 nm by argon laser sources and its emission wavelength was in the range of 515-575 nm.To routinely test the compounds, HEK-293 cells stably transfected with rat P2X7R were seeded overnight in growth medium at 10000, 15000 or 20000 cells/well in 384-well plate, according to the level of response after thawing. 24 hours later, the medium was removed and the cells were pre-loaded at RT for 1 hour with 20 μL/w of Fluo-8 NW prepared in Tyrode 0.3 mM Ca2+/Mg2+-free. Compounds of the invention were tested at 8 concentrations (4 replicates for each concentration): 10-3.16-1-0.316-0.1-0.0316-0.01 and 0.00316 μM, in the same plate.Compounds were tested at FLIPRTETRA according to the following method:    first injection at FLIPRTETRA of 10 μL of 3× test compound (in Tyrode's buffer 0.3 mM Ca2+/Mg2+-free+DMSO 0.5% final concentration)    5′ incubation    second injection at FLIPRTETRA of 15 □L of 3× BzATP at ECK) (in Tyrode's buffer 0.3 mM Ca2+/Mg2+-free+BSA 0.0003% final concentration)    Fluorescence recording for 3′Between one plate and the following, tips were extensively washed with water, then with 100% DMSO and finally with water to avoid carry-over inside the tips.The effect of the test compounds was measured as percent inhibition vs a reference antagonist and IC50 values were calculated accordingly.
Measurement: IC50 = 402 nM (pChEMBL 6.400000095367432)
Descriptors: MW 491.99, ALogP 5.4, HBA 5, HBD 1, PSA 54.46, QED 0.48
Compound (SMILES): COc1cc(-c2cc(N[C@@H]3CCOC3)c3c(cnn3C(C)C)n2)c(OC)cn1
InChIKey: FUMQGQGPKVHDSV-CYBMUJFWSA-N
Target: Dual specificity calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1B (SINGLE PROTEIN), Homo sapiens
UniProt: Q01064
Assay: Inhibition Assay: PDE1A, PDE1B and PDE1C assays were performed as follows: the assays were performed in 60 μL samples containing a fixed amount of the PDE1 enzyme (sufficient to convert 20-25% of the cyclic nucleotide substrate), a buffer (50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) pH 7.6; 10 mM MgCl2; 0.02% Tween20), 0.1 mg/ml BSA (bovine serum albumin), 15 nM tritium labelled cAMP and varying amounts of inhibitors. Reactions were initiated by addition of the cyclic nucleotide substrate, and reactions were allowed to proceed for 1 hr at room temperature before being terminated through mixing with 20 μL (0.2 mg) yttrium silicate SPA beads (PerkinElmer). The beads were allowed to settle for 1 hour in the dark before the plates were counted in a Wallac 1450 Microbeta counter. The measured signals were converted to activity relative to an uninhibited control (100%) and IC50 values were calculated using XIFit (model 205, IDBS).
Measurement: IC50 = 308 nM (pChEMBL 6.510000228881836)
Descriptors: MW 383.45, ALogP 3.29, HBA 8, HBD 1, PSA 83.32, QED 0.7
Compound (SMILES): O=C(c1cccc(C2CC2NCC2CC2)c1)N1Cc2ccccc2C1
InChIKey: BWLNQMDJHGJPOP-UHFFFAOYSA-N
Target: Amine oxidase [flavin-containing] A (SINGLE PROTEIN), Homo sapiens
UniProt: P21397
Assay: MAO-A Inhibition Assay: A test compound dissolved in 4% DMSO was added by 12.5 μL to 25 μL reaction solution (100 mM HEPES (pH 7.5), 5% glycerol) containing 400 ng of MAO-A enzyme (Sigma-Aldrich Co. LLC.), and the mixture was reacted at room temperature for 10 min. MAO substrate (Promega KK) (160 μM) was added by 12.5 μL to start the reaction. After reaction at room temperature for 60 min, Luciferine detection reagent (Promega KK) (50 μL) was added to terminate the reaction. After reaction at room temperature for min with stirring, the luminescence was measured by Envision (PerkinElmer). The MAO-A inhibitory rate (%) of the test compound was calculated by the following formula.inhibitory rate(%)=(1−(test compound count−blank)÷(control−blank))×100
Measurement: IC50 = 2900 nM (pChEMBL 5.539999961853027)
Descriptors: MW 332.45, ALogP 3.7, HBA 2, HBD 1, PSA 32.34, QED 0.91
Compound (SMILES): C=CC(=O)N1CC[C@H](F)[C@@H](Oc2nc(Nc3cnn(C4CC4)c3)nc3[nH]ccc23)C1
InChIKey: HBAFQIAOOXMYKV-HOTGVXAUSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Inhibitory Activity Against JAK 3 and BTK Enzymes: Specifically, the inhibitory activities against JAK3 and BTK kinase were measured using a JAK3 kinase assay kit (Promega, V9441) and a BTK kinase assay kit (Promega, V9071) which were purchased from Promega. Recombinant purified human JAK3 and BTK were diluted with 1×kinase reaction buffer (JAK3: 40 mM Tris-Cl, pH 7.5, 20 mM MgCl2, 0.1 mg/mL BSA and 50 uM DTT/BTK: 40 mM Tris-Cl, pH 7.5, 20 mM MgCl2, 0.1 mg/mL BSA, 2 mM MnCl2 and 50 uM DTT) and added to 96 well plates (JAK3: final concentration of 4 ng per reaction/BTK: final concentration of 8 ng per reaction). The compounds prepared in the previous Examples were treated so as to be finally a 1% DMSO aqueous solution, and a substrate cocktail containing ATP (JAK3: final concentration of 5 uM/BTK: final concentration of 10 uM) and 0.2 ug/uL of Poly(Glu4, Tyr1)peptide (JAK3 and BTK final concentration) in the total 25 uL reactants was added to 96-well plates to initiate enzymatic reaction. After incubation (30° C.) for 1 hour, equivalent volume (25 uL per reaction) of ADP Glo was added and incubated (30° C.) for 40 minutes at room temperature. Then, a kinase detection reagent (50 uL per reaction) was added and incubated (30° C.) for 30 minutes at room temperature.
Measurement: IC50 = 2.5 nM (pChEMBL 8.600000381469727)
Descriptors: MW 411.44, ALogP 2.74, HBA 7, HBD 2, PSA 100.96, QED 0.61
Compound (SMILES): COc1ccnc2cc(Nc3cc(C)[nH]n3)nc(N[C@@H]3C[C@H]4CC[C@@H](C3)N4CCC#N)c12
InChIKey: VSSIFCUCNODKKS-OSYLJGHBSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biochemical JAK Assay: A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 and Tyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH 7.5, 0.01% Brij-35, 10 mM MgCl2, and 1 mM EGTA). Recombinant GST-tagged JAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained from Life Technologies.Serially diluted compounds were pre-incubated with each of the four JAK enzymes and the substrate in white 384-well microplates (Corning) at ambient temperature for lh. ATP was subsequently added to initiate the kinase reactions in 10 μL total volume, with 1% DMSO. The final enzyme concentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and 0.25 nM respectively; the corresponding Km ATP concentrations used are 25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200 nM for all four assays. Kinase reactions were allowed to proceed for 1 hour at ambient temperature before a 10 μL preparation of EDTA (10 mM final concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (Life Technologies, 2 nM final concentration) in TR-FRET dilution buffer (Life Technologies) was added. The plates were allowed to incubate at ambient temperature for lh before being read on the EnVision reader (Perkin Elmer). Emission ratio signals (520 nm/495 nm) were recorded and utilized to calculate the percent inhibition values based on DMSO and background controls.For dose-response analysis, percent inhibition data were plotted vs. compound concentrations, and IC50 values were determined from a 4-parameter robust fit model with the Prism software (GraphPad Software). Results were expressed as pIC50 (negative logarithm of IC50) and subsequently converted to pKi (negative logarithm of dissociation constant, Ki) using the Cheng-Prusoff equation.Test compounds having a higher pKi value in each of the four JAK assays show greater inhibition of JAK activity. Compounds of the invention tested in this assay typically exhibited pKi values between about 7 and about 10.3
Measurement: Ki = 0.55 nM (pChEMBL 9.260000228881836)
Descriptors: MW 432.53, ALogP 3.73, HBA 8, HBD 3, PSA 114.78, QED 0.52
Compound (SMILES): COC(=O)c1ccc(C#Cc2ccc(NC(=O)C(=O)O)cc2)cc1
InChIKey: JMZYPZQWJABIMF-UHFFFAOYSA-N
Target: Tyrosine-protein phosphatase non-receptor type 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06124
Assay: Inhibition Assay: Compounds were tested against mPTPB using the p-nitrophenyl phosphate (pNPP) assay system in a Cary 100 UV-Vis spectrophotometer by monitoring the increase in absorbance of product formed, p-nitrophenol (pNP) at 405 nm. The reaction was started by the addition of mPTPB into a final volume of 200 μL master mix containing pH 7.0 3,3-Dimethyl glutarate buffer (50.0 mM, 1 mM EDTA, 0.15M NaCl), 3.0 mM pNPP. IC50 values for each compound were determined by varying the concentration of the inhibitor at fixed concentrations of the enzyme and by fitting the dose-response data into the four-parameter logistic curve (eq 1) model of GraphPad prism 7.02, as follows. A I /A 0 =IC 50/(IC 50+[I]).The inhibition assay for these PTPs were performed under the same conditions as mPTPB except using a different pNPP concentration corresponding to the Km of the FTP studied.
Measurement: IC50 = 52600 nM (pChEMBL 4.28000020980835)
Descriptors: MW 323.3, ALogP 1.9, HBA 4, HBD 2, PSA 92.7, QED 0.5
Compound (SMILES): CNC[C@@H](c1ccc2ccccc2c1)[C@@H](O)c1cncc(OC)c1
InChIKey: ZYQBVZPSYAIUDW-PMACEKPBSA-N
Target: Sodium-dependent dopamine transporter (SINGLE PROTEIN), Homo sapiens
UniProt: Q01959
Assay: Uptake Assay Protocol: DAT: This protocol was designed to measure inhibition of uptake by the human dopamine transporter. The reagents were human DAT (HEK293F) cells, GBR 12909 (Sigma), nomifensine, neurotransmitter transporter uptake assay kit (Molecular Devices), freestyle 293 expression medium (Invitrogen), 10× Hank's Balanced Salt Solution (HBSS; Invitrogen), 1 M HEPES (Mediatech), Biocoat poly-D-lysine 96-well, black, clear plates (Becton, Dickinson), and 500 pt polypropylene U-bottom 96-well plates (Fisher). The Assay Buffer (AB) was 1×HBSS and 0.02 M HEPES.The HEK293F cells were transfected with the human dopamine transporter and frozen in 1 mL aliquots at about 1E+07 cells/mL. On the day of the experiment, the cells were removed from −80° C. or liquid nitrogen and thawed in a room temperature water bath. The cells were dilute to about 1-2E+06 with Freestyle medium. A 1 mL sample (1:2 dilution) was prepared, and the cells were counted. The cells were spun at 1100 rpm for 5 minutes, and the medium was aspirated off. The cells were resuspend in medium at 1.5E+06 cells/mL for about 60,000 cells per well. 40 μL of cells were dispensed per well in the Biocoat plates. The plates were spun at 1100 rpm for 1 minute to improve homogeneity of the cell layer and were incubated at 37° C. for a minimum of 3 hours.12 μL of test compound (10 mM) in DMSO was added to the wells, and nomifensine was used as a control. GBR 12909 (final assay concentration of 10 μM) was used for background signal. The neurotransmitter transporter dye was prepared in AB prior to use.
Measurement: Ki = 49.6 nM (pChEMBL 7.300000190734863)
Descriptors: MW 322.41, ALogP 3.28, HBA 4, HBD 2, PSA 54.38, QED 0.73
Compound (SMILES): COc1ncc(CN(C)C2CCC(c3ccc4[nH]c(-c5cn6ncnc6c(C)c5C)c(C(C)C)c4n3)CC2)cn1
InChIKey: CGHFWMXFRIZLJA-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assays: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 3.6 nM (pChEMBL 8.4399995803833)
Descriptors: MW 538.7, ALogP 5.97, HBA 8, HBD 1, PSA 97.12, QED 0.27