Simple Search Results

ChEMBL_610945 (CHEMBL1069664) Displacement of [125I]echistatin from alphaVbeta3 receptor in human EA-Hy926 cells by gamma-counting
ChEMBL_1665457 (CHEMBL4015253) Agonist activity at human PK-tagged GPR84 expressed in CHOK1 cells assessed as EA-tagged beta-arrestin recruitment by beta-galactosidase complementation assay
ChEBML_1681651 Modulation of ProLink-fused human CXCR7 expressed in CHOK1 cell membranes assessed as induction of EA-tagged beta-arrestin recruitment after 30 mins by chemiluminescence method
ChEMBL_1714532 (CHEMBL4124581) Agonist activity at human PK-tagged OXER1 expressed in CHOK1 cells assessed as EA-tagged beta-arrestin recruitment by beta-galactosidase enzyme fragment complementation assay
ChEMBL_1681651 (CHEMBL4031928) Modulation of ProLink-fused human CXCR7 expressed in CHOK1 cell membranes assessed as induction of EA-tagged beta-arrestin recruitment after 30 mins by chemiluminescence method
ChEMBL_1755145 (CHEMBL4189905) Antagonist activity at C-terminal PK-fused human full length TrkA assessed as inhibition of betaNGF-induced protein binding to Shc1-EA by beta-galactosidase reporter assay
ChEMBL_2231871 (CHEMBL5145643) Inhibition of human TRPC5 channel expressed in HEK293 cells assessed as inhibition of EA-evoked channel current measured at +80 mV by whole-cell voltage clamp assay
ChEMBL_856119 (CHEMBL2162507) Agonist activity at dopamine D3 receptor in human U2OS-D3 cells assessed as EA-tagged beta-arrestin translocation after 1.5 hrs by luminescence based beta-galactosidase assay
ChEMBL_1666020 (CHEMBL4015816) Partial agonist activity at human D2SR expressed in HEK293T cells co-expressing (EA)beta-arrestin2 assessed as induction of beta-arrestin2 recruitment after 5 hrs by chemiluminescence assay
ChEMBL_819785 (CHEMBL2034297) Agonist activity at recombinant kappa opioid receptor expressed in human U2OS cells coexpressing beta arrestin/EA complex assessed as beta arrestin recruitment after 60 mins by luminescence spectrophotometry
ChEMBL_1613916 (CHEMBL3855716) Transactivation of ProLink tagged human PPARdelta transfected in CHO-K1 cells assessed as interaction with EA labelled SRCP after 3 to 16 hrs by beta galactosidase reporter gene assay
ChEMBL_1972416 (CHEMBL4605234) Agonist activity at muscarinic M3 receptor (unknown origin) expressed in HEK293 cells coexpressing EA tagged beta-arrestin2 assessed as beta-arrestin2 recruitment incubated for 90 mins by PathHunter assay
ChEMBL_2050542 (CHEMBL4705241) Agonist activity at EA-fragment beta-arrestin-tagged human MOR expressed in human U2OS cell membranes assessed as stimulation beta-arrestin recruitment after 60 mins by Pathhunter chemiluminescence assay
ChEMBL_2050544 (CHEMBL4705243) Agonist activity at EA-fragment beta-arrestin-tagged human DOR expressed in human U2OS cell membranes assessed as stimulation beta-arrestin recruitment after 60 mins by Pathhunter chemiluminescence assay
ChEMBL_792739 (CHEMBL1930654) Activation of rat TAAR1 expressed in Syrian hamster AV12-664 cells assessed as accumulation of cAMP after 30 mins using cAMP XS EA/CL as substrate by luminescence method
ChEMBL_1666025 (CHEMBL4015821) Partial agonist activity at human D2SR expressed in HEK293T cells co-expressing (EA)beta-arrestin2 and GRK2 assessed as induction of beta-arrestin2 recruitment after 5 hrs by chemiluminescence assay
ChEMBL_2080513 (CHEMBL4736304) Agonist activity at human Gi/G0-coupled mu opioid receptor expressed in human U2OS cells co-expressing EA-tagged beta-arrestin2 assessed as beta-arrestin 2 recruitment by PathHunter assay
ChEMBL_1578429 (CHEMBL3808246) Agonist activity at PK2-tagged human EP2 receptor expressed in HEK293 cells assessed as induction of EA-tagged beta-arrestin recruitment incubated for 90 mins by beta-galactosidase reporter gene assay
ChEMBL_2231866 (CHEMBL5145638) Inhibition of TRPC5 channel (unknown origin) expressed in HEK293 cells assessed as inhibition of EA-evoked Ca2+ entry by measuring reduction in intracellular Ca2+ level by Fluo-4 dye based FLIPR assay
ChEMBL_2231868 (CHEMBL5145640) Inhibition of TRPC4 channel (unknown origin) expressed in HEK293 cells assessed as inhibition of EA-evoked Ca2+ entry by measuring reduction in intracellular Ca2+ level by Fluo-4 dye based FLIPR assay
ChEMBL_2050539 (CHEMBL4705238) Agonist activity at EA-fragment beta-arrestin-tagged human MOR expressed in human U2OS cell membranes assessed as stimulation of [35S]-GTPgammaS binding after 60 mins by beta-galactosidase based scintillation counting method
ChEMBL_2050543 (CHEMBL4705242) Agonist activity at EA-fragment beta-arrestin-tagged human DOR expressed in human U2OS cell membranes assessed as stimulation of [35S]-GTPgammaS binding after 60 mins by beta-galactosidase based scintillation counting method
ChEBML_1671641 Inhibition of PK-tagged human TrkC expressed in human U2OS cells assessed as reduction in NT3-induced EA-tagged SHC1 recruitment pretreated for 1 hr followed by NT3 stimulation after 3 hrs by PathHunter assay
ChEMBL_1677401 (CHEMBL4027544) Agonist activity at human PK-tagged GPR40 expressed in HEK293 cells assessed as EA-tagged beta-arrestin recruitment after 90 mins in presence of 1 % heat inactivated FBS by beta-galactosidase reporter gene assay
ChEMBL_1719141 (CHEMBL4134141) Agonist activity at PK-tagged human GPR120 long isoform expressed in CHOK1 cells co-expressing EA-tagged beta-arrestin assessed as beta-gal enzyme complex formation after 90 mins by enzyme fragment complementation assay
ChEMBL_1496603 (CHEMBL3578969) Agonist activity at ARMS2-PK2-tagged human dopamine D2S receptor expressed in HEK293 cells co-expressing (EA)-tagged beta arrestin fusion protein assessed as recruitment of beta-arrestin-2 after 5 hrs by chemiluminescence assay
ChEMBL_1660569 (CHEMBL4010181) Agonist activity at ARMS2-PK2 tagged D2S receptor (unknown origin) expressed in HEK293 cells assessed as induction of EA-tagged beta-arrestin-2 recruitment incubated for 5 hrs measured after 60 mins by PathHunter assay
ChEMBL_1671641 (CHEMBL4021670) Inhibition of PK-tagged human TrkC expressed in human U2OS cells assessed as reduction in NT3-induced EA-tagged SHC1 recruitment pretreated for 1 hr followed by NT3 stimulation after 3 hrs by PathHunter assay
ChEMBL_1677402 (CHEMBL4027545) Agonist activity at rat PK-tagged GPR40 expressed in human U2OS cells assessed as EA-tagged beta-arrestin recruitment after 90 mins in presence of 1 % heat inactivated FBS by beta-galactosidase reporter gene assay
ChEMBL_1754033 (CHEMBL4188793) Agonist activity at recombinant human PK-tagged GPR120 long isoform expressed in CHOK1 cells co-expressing EA-tagged beta-arrestin assessed as beta-gal enzyme complex formation after 90 mins by enzyme fragment complementation assay
ChEMBL_2085012 (CHEMBL4766275) Agonist activity at Prolink-tagged mouse GPR40 fused with EA-tagged beta-arrestin expressed in human U2OS cells assessed as induction of beta-arrestin recruitment measured after 90 mins by beta-galactosidase based PathHunter assay
ChEMBL_2085014 (CHEMBL4766277) Agonist activity at Prolink-tagged rat GPR40 fused with EA-tagged beta-arrestin expressed in human U2OS cells assessed as induction of beta-arrestin recruitment measured after 90 mins by beta-galactosidase based PathHunter assay
ChEMBL_2216154 (CHEMBL5129286) Inhibition of human TRPC5 channel expressed in HEK293 cells assessed as inhibition of EA-evoked Calcium influx by measuring reduction in intracellular Ca2+ level and measured upto 360 sec by Fluo-4/AM dye based FLIPR assay
ChEMBL_2216155 (CHEMBL5129287) Inhibition of human TRPC4 channel expressed in HEK293 cells assessed as inhibition of EA-evoked Calcium influx by measuring reduction in intracellular Ca2+ level and measured upto 360 sec by Fluo-4/AM dye based FLIPR assay
ChEMBL_1671640 (CHEMBL4021669) Inhibition of PK-tagged human Trkalpha expressed in human U2OS cells assessed as reduction in beta NGF-induced EA-tagged SHC1 recruitment pretreated for 1 hr followed by beta NGF stimulation measured after 3 hrs by PathHunter assay
ChEMBL_2085010 (CHEMBL4766273) Agonist activity at human recombinant full-length Prolink-tagged GPR40 fused with EA-tagged beta-arrestin expressed in human HEK293 cells assessed as induction of beta-arrestin recruitment measured after 90 mins by beta-galactosidase based PathHunter assay
ChEMBL_792743 (CHEMBL1930658) Activation of human TAAR1 expressed in Syrian hamster AV12-664 cells co-expressing GalphaS protein assessed as accumulation of cAMP after 30 mins using cAMP XS EA/CL as substrate by luminescence method in presence of RX821002 and alprenolol
ChEMBL_1849192 (CHEMBL4349733) Antagonist activity at prolink-tagged full length human TrkA expressed in U2OS cells assessed as inhibition of NGF-induced receptor phosphorylation by measuring reduction in EA-tagged SH2 protein recruitment preincubated for 30 mins followed by NGF addition and measured by chemiluminescence method
ChEMBL_1972417 (CHEMBL4605235) Antagonist activity at muscarinic M3 receptor (unknown origin) expressed in HEK293 cells coexpressing EA tagged beta-arrestin2 assessed as inhibition of carbachol-induced beta-arrestin2 recruitment preincubated for 30 mins followed by carbachol addition and measured after 90 mins by PathHunter assay
beta-Arrestin Assay The DISCOVERX PATHHUNTER β-Arrestin assay is used to measure β-arrestin-2 activity. The technology is based on Enzyme Fragment Complementation (EFC) with β-galactosidase (β-Gal) as the reporter. The enzyme is split into two inactive complementary portions (EA for Enzyme Acceptor, and ED for Enzyme Donor) expressed as fusion proteins in the cell. EA is fused to β-arrestin-2, and ED is fused to the C-terminus of the GPCR of interest. When a ligand binds and the GPCR is activated, β-arrestin-2 is recruited to the receptor resulting in an ED/EA complementation, restoring β-Gal activity, which is measured using chemiluminescence. For this assay, the PathHunter human OPRM1 (μ, MOR) Arrestin CHO-K1 cell line was used. The EMAX values of the chemiluminescent signal were all normalized to DAMGO, which is defined as 100%.
ChEMBL_1760792 (CHEMBL4195800) Antagonist activity at prolink-tagged TrkB in human U2OS cells assessed as inhibition of BDNF-induced receptor phosphorylation by measuring reduction in EA-tagged SH2 protein recruitment preincubated for 30 mins followed by BDNF stimulation measured after 2 hrs by PathHunter enzyme complementation assay
ChEMBL_1760793 (CHEMBL4195801) Antagonist activity at prolink-tagged TrkC in human U2OS cells assessed as inhibition of NT3-induced receptor phosphorylation by measuring reduction in EA-tagged SH2 protein recruitment preincubated for 30 mins followed by NT3 stimulation measured after 2 hrs by PathHunter enzyme complementation assay
ChEMBL_1809731 (CHEMBL4309191) Antagonist activity at prolink-tagged TrkA (unknown origin) expressed in cells assessed as inhibition of NGF-induced receptor phosphorylation by measuring reduction in EA-tagged SH2 protein recruitment preincubated for 30 mins followed by NGF addition measured after 2 hrs by PathHunter enzyme complementation assay
ChEMBL_1809732 (CHEMBL4309192) Antagonist activity at prolink-tagged TrkB (unknown origin) expressed in cells assessed as inhibition of BDNF-induced receptor phosphorylation by measuring reduction in EA-tagged SH2 protein recruitment preincubated for 30 mins followed by BDNF addition measured after 2 hrs by PathHunter enzyme complementation assay
ChEMBL_1809733 (CHEMBL4309193) Antagonist activity at prolink-tagged TrkC (unknown origin) expressed in cells assessed as inhibition of NT3-induced receptor phosphorylation by measuring reduction in EA-tagged SH2 protein recruitment preincubated for 30 mins followed by NT3 addition measured after 2 hrs by PathHunter enzyme complementation assay
ChEMBL_1760791 (CHEMBL4195799) Antagonist activity at prolink-tagged TrkA in human U2OS cells assessed as inhibition of beta-NGF-induced receptor phosphorylation by measuring reduction in EA-tagged SH2 protein recruitment preincubated for 30 mins followed by beta-NGF stimulation measured after 2 hrs by PathHunter enzyme complementation assay
Biological Assay TrkA functional activity was measured using a DiscoverX PathHunter assay. In this assay, U2OS cells express the human TrkA receptor as a fusion with the weakly complementing fragment of B-galactosidase, which DiscoverX calls Prolink (PK) ; additionally, Shc1 is fused with a larger fragment, which is called Enzyme Acceptor (EA) . Activation of the TrkA receptor, upon NGF addition, results in the kinase domain being phosphorylated, resulting in subsequent recruitment of Shc1-EA protein. That recruitment results in an active B-galactosidase enzyme that is detected by addition of a chemiluminescent substrate. The human p75NTR protein was also expressed as a co-receptor for NGF.
Biological Assay TrkA functional activity was measured using a DiscoverX PathHunter assay. In this assay, U2OS cells express the human TrkA receptor as a fusion with the weakly complementing fragment of B-galactosidase, which DiscoverX calls Prolink (PK); additionally, Shc1 is fused with a larger fragment, which is called Enzyme Acceptor (EA). Activation of the TrkA receptor, upon NGF addition, results in the kinase domain being phosphorylated, resulting in subsequent recruitment of Shc1-EA protein. That recruitment results in an active B-galactosidase enzyme that is detected by addition of a chemiluminescent substrate. The human p75NTR protein was also expressed as a co-receptor for NGF.
Functional Activity Assay TrkA functional activity was measured using a DiscoverX PathHunter assay. In this assay, U2OS cells express the human TrkA receptor as a fusion with the weakly complementing fragment of B-galactosidase, which DiscoverX calls Prolink (PK) ; additionally, Shcl is fused with a larger fragment, which is called Enzyme Acceptor (EA) . Activation of the TrkA receptor, upon NGF addition, results in the kinase domain being phosphorylated, resulting in subsequent recruitment of Shcl-EA protein. That recruitment results in an active B-galactosidase enzyme that is detected by addition of a chemiluminescent substrate. The human p75NTR protein was also expressed as a co-receptor for NGF.All reagents were purchased from DiscoverX, except for the receptor agonists (NGF, BDNF, NT3) which were purchased from Peprotech. Cells were expanded and frozen into cryovials, and stored in the vapor phase of liquid nitrogen, and thawed immediately before use. Thawed cells were added to a 384-well plate at 7500 cells/well, and allowed to incubate overnight. Compound at various concentrations was added the following morning and allowed to incubate on cells for 1 h. Then, NGF was added at a concentration sufficient to elicit 80% of a maximal response and allowed to incubate for 3 h at ambient temperature.
PathHunter Beta-Arrestin Assay Compound 1 was screened for agonist activity against the human CB2 receptor using the DiscoveRx PathHunter β-arrestin assay which measures the β-arrestin binding to the CB2 receptor upon its activation. CB2 was cloned into the pCMV-PK vector (DiscoveRx, Fremont, Calif.; catalog #93-0167) and transfected into the CHO-K1 EA-Arrestin parental cell line (DiscoveRx, Fremont, Calif.; catalog #93-0164). CHO-K1 positive clones stably expressing the CB2-ProLink fusion protein were identified by their responses to the CB2 agonist CP55,940. Clone #61 was chosen for its big agonist window and homogenous expression as detected by anti-HA flow cytometry.
DiscoverX PathHunter Assay In this assay, U2OS cells express the human TrkA receptor as a fusion with the weakly complementing fragment of B-galactosidase, which DiscoverX calls Prolink (PK) ; additionally, Shc1 is fused with a larger fragment, which is called Enzyme Acceptor (EA) . Activation of the TrkA receptor, upon NGF addition, results in the kinase domain being phosphorylated, resulting in subsequent recruitment of Shc1-EA protein. That recruitment results in an active B-galactosidase enzyme that is detected by addition of a chemiluminescent substrate. The human p75NTR protein was also expressed as a co-receptor for NGF.All reagents were purchased from DiscoverX, except for the receptor agonists (NGF, BDNF, NT3) which were purchased from Peprotech. Cells were expanded and frozen into cryovials, and stored in the vapor phase of liquid nitrogen, and thawed immediately before use. Thawed cells were added to a 384-well plate at 7500 cells/well, and allowed to incubate overnight. Compound at various concentrations was added the following morning and allowed to incubate on cells for 1 h. Then, NGF was added at a concentration sufficient to elicit 80% of a maximal response and allowed to incubate for 3 h at ambient temperature. DiscoverX PathHunter detection reagent was then added and the plate was further incubated for 1 h in the dark. The plate was then read via luminescence on the Perkin Elmer Envision.
TrkA Receptor TrkA functional activity was measured using a DiscoverX PathHunter assay. In this assay, U2OS cells are express TrkA receptor as a fusion with the weakly complementing fragment of B-galactosidase, which DiscoverX calls Prolink (PK) ; additionally, Shc1 is fused with a larger fragment, which is called Enzyme Acceptor (EA) . Activation of the TrkA receptor, upon NGF addition, results in the kinase domain being phosphorylated, resulting in subsequent recruitment of Shc1-EA protein. That recruitment results in an active B-galactosidase enzyme that is detected by addition of a chemiluminescent substrate.All reagents were purchased from DiscoverX, except for the receptor agonists (NGF, BDNF, NT3) which were purchased from Peprotech. Cells were expanded and frozen into cryovials (TrkA p75 @ passage 13, 1.5×10^7 cells/vial; TrkC p75 @ passage 11, 1.5×10^7 cells/vial in InVitrogen Recovery Cell Freezing media), and stored in the vapor phase of liquid nitrogen, and thawed immediately before use. Thawed cells were added to a 384-well plate (BD Falcon 384-well white/clear, TC surface 120 uL assay plates (353963), 20 uL of 0.375e6 cells/mL=>7500 cells/well), and allowed to incubate overnight. Compound (10 mM starting dose: 202.5 nL compound yielding 0.81% DMSO final in total assay volume and starting doses 81 uM) was added the following morning and allowed to incubate on cells for 1 hour. Then, 5 uL EC80 of agonist (NGF for TrkA; BDNF for TrkB; NT3 for TrkC) was added and allowed to incubate for 3 hours at room temperature. DiscoverX PathHunter detection reagent is added (12 uL to plate and components diluted in parts as per kit instructions) and the plate is further incubated for 1 hour in the dark. The plate is read via luminescence on the Perkin Elmer Envision.
In Vitro Assay-LXRβ PathHunter NHR CHO cells stably transfected with tagged full-length human LXRβ and a nuclear fusion protein containing Steroid Receptor Co-activator Peptide (SRCP). The full-length human LXRβ was tagged with the ProLink component of the DiscoverX Enzyme Fragment Complementation (EFC) system and the SRCP domain is fused to the enzyme acceptor component (EA) expressed in the nucleus. Cells were seeded in a total volume of 20 μL into white walled, 384-well microplates and incubated at 37 C. overnight prior to testing. Assay media contained charcoal-dextran filtered serum to reduce the level of hormones present. Final assay vehicle concentration was 1%. Assay signal was generated through a single addition of 12.5 or 15 μL (50% v/v) of PathHunter Detection reagent cocktail, followed by a one-hour incubation at room temperature. Microplates were read following signal generation with a PerkinElmer Envision instrument for chemiluminescent signal detection.
In Vitro Assay The assay is using the PathHunter CHO-K1 CXCR7 β-arrestin cell line from DiscoverX. The system is based on the Enzyme Fragment Complementation Technology. Two complementing fragments of the β-galactosidase enzyme are expressed within stably transfected cells. The larger portion of β-gal, termed EA for Enzyme Acceptor, is fused to the C-terminus of b-arrestin 2. The smaller fragment, termed ProLink tag, is fused to CXCR7 at the C-terminus. Upon activation, b-arrestin is recruited which forces the interaction of ProLink and EA, allowing complementation of the two fragments of b-gal and the formation of a functional enzyme which is capable of hydrolysing the substrate and generating a chemiluminescent signal. CHO-K1 CXCR7 β-arrestin cells are detached from culture dishes with a cell dissociation buffer (Invitrogen, #13151-014) and collected in growing medium (F12 HAMS 90% (v/v)/FCS 10% (v/v), Penicillin/streptomycin 1% (v/v)). 5000 cells per well (in 20 μl) are seeded in a 384 well plate (white-walled, clear bottom; BD Falcon #353274). The plate is incubated at 37° C./5% CO2 for 24 hours. Medium is then replaced by 20 μl OPTIMEM (Invitrogen #31985) for 3 to 4 hours. Test compounds are dissolved at 10 mM in DMSO and serially diluted in DMSO to 200× of the final concentration for dose response curves. Compounds are then diluted 1:33.3 in HBSS1X. 5 μl/well of HBSS1X/20 mM HEPES/0.2% BSA are added to the assay plate followed by addition of 5 μl/well of diluted compounds. CXCL12 (Peprotech #300-28A) may be used as a reference agonist. The plate is incubated for 90 minutes at 37° C. 12 μl of detection reagent (Path Hunter Detection Kit, DiscoveRx, #93-0001) is transferred to the assay plate and to the plate is incubated for 1 hour at room temperature. Luminescent signal is read in a microplate reader (FLUOstar Optima, bmg). The calculated EC50 values may fluctuate depending on the daily cellular assay performance. Fluctuations of this kind are known to those skilled in the art. Average EC50 values from several measurements are given as geometric mean values.
Beta-Arrestin2 Recruitment Assay Agonist-promoted β-arrestin2 recruitment to the sphingosine-1-phosphate 1 receptor was determined using the β-arrestin PathHunter ) Detection kit (DiscoverX Corporation, Cat #: 93-0001). In this system, β-arrestin2 is fused to an N-terminal deletion mutant of β-galactosidase (termed the enzyme acceptor or EA) and the C-terminus of the GPCR of interest is fused to a smaller (42 amino acids), weakly-complementing fragment 15 termed ProLink . In cells that stably express these fusion proteins, stimulation with a cognate agonist results in the interaction of 0-arrestin2 and the Prolink -tagged GPCR. This allows the complementation of the two β-galactosidase fragments and results in the formation of a functional enzyme with β-galactosidase activity. AssayComplete Cell Plating 11 Reagent was removed and replaced with Ham's F-12 containing IBMX (500 μM), and NKH-477 (1.5 μM) along with test or control compounds at the desired concentrations. Following a 60 minute incubation at 37 C. and 5% CO2 in a humidified CO2 and temperature-controlled incubator, the components of the β-arrestin PathHunter Detection kit were added as per the manufacturer's instructions. After an hour incubation at room temperature, plates were analyzed by a BMG PheraStar microplate reader.
TAAR1 Biodata EC50 (the effective concentration of an agonist that produces half of the maximal effect) and Emax (the maximal cAMP level generated by the biding of a ligand) can be used as a measure of potency. In some embodiments, the TAAR1 agonists or partial agonists of the present disclosure have EC50≤10 μm, or EC50≤1 μm. An in vitro cAMP assay (agonist activity assay) is described below.cAMP Hunter cell lines were expanded from freezer stocks according to standard procedures. Cells were seeded in a total volume of 20 μL into white walled, 384-well microplates and incubated at 37° C. overnight prior to testing. cAMP modulation was determined using the HitHunter cAMP XS+ assay (DiscoverX, Fremont, Calif.).On the day of test, media was aspirated from cells and replaced with 15 μL HBSS/10 mM HEPES. A plate centrifuge was used for the media exchange, and the plate centrifuge was immediately stopped once its speed hit 270 RPM.5 μL of 4× compound (prepared in HBSS/10 mM HEPES/4% DMSO) was added to cells and incubated at 37° C. for 30 minutes. Final assay vehicle concentration was 1%.5 μL of cAMP XS+ Antibody reagent was then added, followed by 20 μL of ED/CL lysis mix. The mixture was incubated at room temperature for 60 minutes. 20 μL of EA reagent was added, and the mixture was incubated at room temperature for 120 minutes.Microplates were read following signal generation with a PerkinElmer Envision instrument (Waltham, Mass.) for chemiluminescent signal detection. Compound activity was analyzed using CBIS Data Analysis Suite (ChemInnovation, CA).
FGFR Enzyme Binding Assay Table EA: The potency of compounds inhibiting human isoforms of FGFR kinase was determined using Life Technologies' Homogeneous Time Resolved Fluorescence (HTRF)-based binding assay technology. An incubation was conducted with either 5 nM dephosphorylated FGFR1 (Array Biopharma, p1702; SEQ ID NO: 1, amino acids 458 to 765, dephosphorylated by co-expression with PTP1b (protein tyrosine phosphatase 1B)), 5 nM dephosphorylated FGFR2 (Life Technologies, Cat. No. PV4106 that had been dephosphorylated with Lambda protein phosphatase (New England Biolabs, cat #P0753)) or 5 nM phosphorylated FGFR3 (Array Biopharma, p1836; SEQ ID NO: 5, amino acids 449 to 759), 50 nM Kinase Tracer 236 (Life Technologies Cat. No. PR9078A), 2 nM Biotin-anti-6HIS (Life Technologies Cat. No. PV6090) and 2 nM Europium-Streptavidin (Life Technologies Cat. No. PV6025) along with test compound in a buffer consisting of 50 mM HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.5), 5 mM MgCl2, 0.005% Triton X-100, 1 mM DTT, 1 mM NaVO4 and 2% DMSO in a final volume of 12 μL Compounds were typically prepared as a 3-fold or 4-fold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60 minute incubation at 22° C., the extent of tracer displacement was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. One hundred POC was determined using no test compound, and 0 POC was determined in the presence of 1 μM of an appropriate control inhibitor. A 4-parameter logistic curve was fit to the POC values as a function of the concentration of compound, and the IC50 value was the point where the best-fit curve crossed 50 POC.
Biological Activity Test Assay TRPC5 is a type of non-selective cation channel with permeability to calcium ions. Therefore, TRPC5 agonist Englerin A (EA) and TRPC5 inhibitor Pico145 were used as positive controls in this experiment. Fluo-4 AM fluorescence dye was used to indirectly reflect the effect of the compound on TRPC5 channel by detecting intracellular Ca21 in TRPC5-HEK 293 cells.1. Cell culture1.1 Revival of TRPC5-HEK 293 cellsRevival solution: 100% high glucose DMEMSelection medium: high glucose DMEM+10% FBS+1% P/S+1% HEPES+Blasticidin (5 μg/mL)+Hygromycine (50 μg/mL)Induction medium: high glucose DMEM+10% FBS+1% P/S+1% HEPES+Doxycycline (1 μg/mL).Revival process: A cryopreservation solution containing TRPC5-HEK 293 cells was taken out from the liquid nitrogen tank, and transferred from the ice box to the water bath, and then dissolved in circles to a small piece of ice, and the cryopreservation solution was transferred to the revival solution, and centrifuged at 1000 rpm for 5 minutes, and then the supernatant was discarded, then the residue was transferred to the selection medium, and expanded in a CO2 incubator (5% CO2, humidity of 95%, 37° C.).1.2 Seeding TRPC5-HEK 293 cells in plates14 hours before the real-time fluorescence experiment, cells were washed with PBS and digested with trypsin-EDTA (TE). Cells were diluted to 200,000 cells/mL with induction medium and seeded onto a 96-well black wall bottom transparent plate coated with poly-D-lysine (PDL) (stock solution of 10 mg/mL, final concentration of 10 μg/mL) at 100 μL/well, i.e., 20,000 cells/well.2. Preparation of buffer500 mL of external solution for detecting TRPC5 calcium signals: 4.0908 g of NaCl, 0.1864 g of KCl, 0.111 g of CaCl2, 0.0476 g of MgCl2, 0.9 g of glucose, 1.1915 g of HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid).Calcium fluorescent dye: an external solution for detecting TRPC5 calcium signals containing Fluo-4 with a final concentration of 4 μM (containing 0.5% BSA)
Cell Based Assay TRKA co-expressed with p75: The assays are based upon DiscoveRx's proprietary Enzyme Fragment Complementation (EFC) technology. In the case of the TRK cell lines, the enzyme acceptor (EA) protein is fused to a SH2 protein and the TRK receptor of interest has been tagged with a Prolink tag. Cryopreserved PathHunter cells are used from either in-house produced batches or bulk batches bought directly from DiscoveRx. Cryopreserved cells are resuscitated, spun 1000 rpm for 4 min to remove freezing media, and resuspended in MEM+0.5% horse serum (both Invitrogen) to 5e^5 cells/ml. The cells are then plated using a Multidrop into Greiner white tissue culture treated plates at 20 μl/well and incubated for 24 h at 37 °C., 5% CO2, high humidity. On the day of the assay, the cell plates are allowed to cool to room temperature for 30 min prior to the assay. 4 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using the compound of Example 135, WO2005/116035 at a top concentration of 150 μM is also prepared on each test plate. High percentage effect (HPE) is defined by 150 μM of the compound of Example 135, WO2005/116035 and 0% effect (ZPE) is defined by 100% DMSO. Plates containing 1 μl of serially diluted compound, standard and HPE/ZPE are diluted 1/66 in assay buffer (PBS minus Ca2+, minus Mg2+ with 0.05% pluronic F127) using a Wellmate. Using a Platemate Plus, 5 μl of 1/66 diluted test compounds is then transferred to the cell plate and allowed to reach equilibrium by incubating for 30 min at room temperature before addition of agonist stimulus: 10 μl/well of 2 nM (0.571 nM FAC) of the cognate neurotrophin (Peprotech) diluted in agonist buffer (HBSS with 0.25% BSA). Final assay concentration of the test compounds is 8.66 μM, (the compound of Example 135, WO2005/116035 FAC is 0.325 μM). The plates are left at room temperature for a further 2 hours before addition of 10 μl of the DiscoveRx PathHunter detection reagent (made up by adding 1 part Galacton Star, 5 parts Emerald II and 19 parts Cell Assay Buffer as per the manufacturer's instructions). After reagent addition, plates are covered and incubated at room temperature for 60 minutes. Luminescence signal is read using an Envision.

Query String: Electroacupuncture EA