lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Oc1ccc2c(c1F)CCCC(c1ccc(Cl)cc1F)=C2c1ccc(O[C@H]2CCN(CCCF)C2)cc1
InChIKey: WGTZQBMGWDDRRV-QFIPXVFZSA-N
Target: Estrogen receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P03372
Assay: Estrogen Receptor Coactivator Assay: It is a competition assay, where binding of a test compound to a complex comprised of (i) His6-ERα298-554 protein representing ERα ligand-binding domain, (ii) Tb-labeled His6 antibody, (iii) a fluorescein-labeled PGC1α coactivator peptide (EAEEPSLLKKLLLAPANTQ), and (iv) estradiol, results in a decrease of the TR-FRET signal due to dissociation of the coactivator peptide. His6-ERα298-554 proteins were expressed as WT or D538G or Y537S mutants in E. coli and purified by affinity chromatography. The assay works in a homogeneous mix-and-read format. In a typical experiment, a 4 μL mixture of 0.5 nM His6-ERα298-554, 0.5 nM Tb-labeled His6 antibody, 250 nM PGC1a peptide, and 3 nM estradiol in 100 mM potassium phosphate, pH 7.4, 0.01% Tween-20, 0.02% NaN3, 5 mM DTT, was added to 40 nL test compound in DMSO and incubated overnight at room temperature. The TR-FRET 520:495 nm emission ratio was calculated and used to determine the IC50 value from a dose response curve fit to the 4-parameter logistic equation. The antagonist activity with respect to estrogen receptors in this test is given by the concentration which inhibits 50% of the estrogen receptor activity (or IC50) in nM.
Measurement: IC50 = 56 nM (pChEMBL 7.25)
Descriptors: MW 528.01, ALogP 7.43, HBA 3, HBD 1, PSA 32.7, QED 0.35
Compound (SMILES): CCNc1cc2c(cn1)c(-c1cnn(C)c1)nn2C1CCC(Oc2nccc(N(C)C)c2F)CC1
InChIKey: NEFYXQZFBKFSBT-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor TYRO3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06418
Assay: Enzyme Assay: TYRO3: Compounds of Formula I were screened for their ability to inhibit TYRO3 kinase using Invitrogen's LanthaScreen Eu Kinase Binding technology. GST-tagged recombinant human TYRO3 kinase domain from Carna (5 nM; Cat. No. PR7480A) was incubated with 20 nM Alexa-Fluor® Tracer 236 (PR9078A) and 2 nM Europium-anti-GST (Cat. No. A15116) along with test compound in a buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO. Compounds are typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-minute incubation at 22° C., the reaction was measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using a concentration of control compound that completely inhibits the enzyme. The POC values were fit to a 4 parameter logistic curve and the IC50 value is point where the curve crosses 50 POC.
Measurement: IC50 = 350 nM (pChEMBL 6.460000038146973)
Descriptors: MW 478.58, ALogP 4.43, HBA 9, HBD 1, PSA 85.92, QED 0.42
Compound (SMILES): CC1(O)COCC[C@@]1(NC(=O)c1cnn2cc(C3CC3)cnc12)c1ccc(C(F)(F)F)cc1F
InChIKey: FNEHCEZBZVEEDS-FOIFJWKZSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Biological Assay: Assay Principle: The PDE reaction cleaves cAMP to AMP. The IMAP system (Molecular Device) using fluorescence polarization (FP) as detection principle was used to measure enzyme activity. A fluorescent labeled cAMP was used as substrate for the reaction, generating a labeled AMP. The fluorescent AMP binds specifically to the large M(III)-based nano-particles which reduces the rotational speed of the substrate and thus increases its polarization.</p><p id="h-0016" num="0000">Detailed Method:The inhibition of PDE 2A or 10 enzyme activity was assessed using IMAP-Phosphodiesterase-cAMP fluorescence labeled substrate (Molecular Devices, Order No. R7506), IMAP TR-FRET screening express (Molecular Devices, Order No. R8160, the TR-FRET component will not be used) and PDE 2A or PDE10 protein expressed upon baculovirus infection in SF9 cells. The cells were incubated after infection for 3 days and protein production was confirmed by Western Blot. The cells were collected by centrifugation and the pellet frozen in liquid nitrogen before it was resuspended in PBS containing 1% Triton X-100 and protease inhibitors. After 45 min incubation on ice, the cell debris was removed by centrifugation (13.000 rpm, 30 min). Since SF 9 cells do not express cAMP hydrolyzing enzymes to a high extent, no further purification of the protein was needed. All reactions were performed in 384 well plates, Perkin Elmer black optiplates and IMAP reaction buffer with 0.1% Tween20 (kit component). Compounds were serial diluted in DMSO. With an intermediate dilution step with reaction buffer DMSO concentration was reduced to achieve 1% DMSO in the assay reaction. Setup of the assay started with 10 μl enzyme (10 ng/well, depending on prep. batch), 5 μl compound, reaction was started by addition of 5 μl labeled cAMP (30 nM, final concentration), immediately mixed for 15 seconds on a Eppendorf mixmate (2000 rpm) followed by an incubation at room temperature for 90 minutes in the dark. Reaction is stopped by adding of 60 μl binding buffer for FP/cAMP (kit component). After at least 90 min of further incubation (room temperature, dark) the assay was measured at 485 nm excitation/525 nm emission in an Envision multilabel reader (PerkinElmer).Each assay plate contained wells with vehicle controls (1% DMSO) for the measurement of non-inhibited reaction (=100% control) and wells without enzyme as 0% controls.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 478.45, ALogP 3.56, HBA 6, HBD 2, PSA 88.75, QED 0.56
Compound (SMILES): COc1ncc(-c2cc(NC(=O)c3ccnc(C4(C#N)CC4)c3)cnc2C)nc1N1CCOCC1
InChIKey: MKSMMQHYPGOHAM-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: In Vitro Raf Activity: Raf and biotinylated Mek, kinase dead, were combined at 2× final concentrations in assay buffer (50 mM Tris, pH 7.5, 15 mM MgCl2, 0.01% BSA and 1 mM DTT) and dispensed 5 ml per well in assay plates (Greiner white 384 well assay plates #781207) containing 0.25 ml of 40× of a Raf kinase inhibitor test compound diluted in 100% DMSO. The plate was incubated for 60 minutes at room temperature. The Raf kinase activity reaction was started by the addition of 5 mL per well of 2×ATP diluted in assay buffer. After 3 hours (b-Raf(V600E)) or 1 hour (c-Raf). The reactions were stopped and the phosphorylated product was measured using a rabbit anti-p-MEK (Cell Signaling, #9121) antibody and the Alpha Screen IgG (ProteinA) detection Kit (PerkinElmer #6760617R), by the addition of 10 mL to the well of a mixture of the antibody (1:2000 dilution) and detection beads (1:2000 dilution of both beads) in Stop/bead buffer (25 mM EDTA, 50 mM Tris, pH 7.5, 0.01% Tween20). The additions were carried out under dark conditions to protect the detection beads from light. A lid was placed on top of the plate and incubated for 1 hour at room temperature, then the luminescence was read on a PerkinElmer Envision instrument. The concentration of each compound for 50% inhibition (IC50) was calculated by non-linear regression using XL Fit data analysis software.
Measurement: IC50 = 2.9 nM (pChEMBL 8.539999961853027)
Descriptors: MW 471.52, ALogP 2.89, HBA 9, HBD 1, PSA 126.15, QED 0.58
Compound (SMILES): C=CC(=O)Nc1cnn(C[C@H]2CCOC2)c1-c1cc2c(cn1)CN(c1c(F)c(OC)cc(OC)c1F)C(=O)C21CC1
InChIKey: ADWOHKVEGSFDTQ-MRXNPFEDSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Enzymatic Assay: The inhibitor potency of the exemplified compounds was measured in an enzyme assay that measures peptide phosphorylation using FRET measurements to detect product formation. Inhibitors were serially diluted in DMSO and a volume of 0.5 μL was transferred to the wells of a 384-well plate. For FGFR3, a 10 μL volume of FGFR3 enzyme (Millipore) diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 5 mM DTT, pH 7.5) was added to the plate and pre-incubated for a time between 5-10 minutes and up to 4 hours. Appropriate controls (enzyme blank and enzyme with no inhibitor) were included on the plate. The assay was initiated by the addition of a 10 μL solution containing biotinylated EQEDEPEGDYFEWLE peptide substrate (SEQ ID NO: 1) and ATP (final concentrations of 500 nM and 140 μM respectively) in assay buffer to the wells. The plate was incubated at 25° C. for 1 hr. The reactions were ended with the addition of 10 μL/well of quench solution (50 mM Tris, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.8; 30 mM EDTA with Perkin Elmer Lance Reagents at 3.75 nM Eu-antibody PY20 and 180 nM APC-Streptavidin). The plate was allowed to equilibrate for 1 hr before scanning the wells on a PheraStar plate reader (BMG Labtech).FGFR1, FGFR2, and FGFR4 are measured under equivalent conditions with the following changes in enzyme and ATP concentrations: FGFR1, 0.02 nM and 210 uM respectively, FGFR2, 0.01 nM and 100 uM, respectively, and FGFR4, 0.04 nM and 600 uM respectively. The enzymes were purchased from Millipore or Invitrogen.
Measurement: IC50 > 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 565.58, ALogP 3.98, HBA 8, HBD 1, PSA 107.81, QED 0.41
Compound (SMILES): C[C@@H]1CN(c2cc(F)c(F)c(Cl)c2)C[C@H](C)N1C(=O)NCCC1CCN(Cc2ccccc2)CC1
InChIKey: PFQNHVGTLFNXFJ-BGYRXZFFSA-N
Target: Muscarinic acetylcholine receptor M2 (SINGLE PROTEIN), Homo sapiens
UniProt: P08172
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 505.05, ALogP 5.53, HBA 3, HBD 1, PSA 38.82, QED 0.53
Compound (SMILES): COc1cc(OC)cc(C(CCO)c2cc3cc(-c4cnn(C)c4)cnc3cn2)c1
InChIKey: NTIBKJMEIQFZET-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: FGFR3 (Enzymatic Assay): In a final reaction volume of 30 μL, FGFR3 (h) (40 ng/ml) was incubated with 50 mM HEPES pH 7.5, 6 mM MnCl2, 1 mM DTT, 0.1 mM Na3VO4, 0.01% Triton-X-100, 500 nM Btn-Flt3 and 25 μM ATP in the presence of compound (1% DMSO final). After incubation for 60 minutes at room temperature the reaction was stopped with 2.27 nM EU-anti P-Tyr, 7 mM EDTA, 31.25 nM SA-XL-665 and 0.02% BSA which was present for 60 minutes at room temperature. Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) signal (ex340 nm. Em 620 nm, em 655 nm) was measured afterwards and results are expressed in RFU (Relative Fluorescence Units). In this assay, the inhibitory effect of different compound concentrations (range 10 μM to 0.1 nM) was determined and used to calculate an IC50 (M) and pIC50 (−log IC50) value.
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 404.47, ALogP 3.56, HBA 7, HBD 1, PSA 82.29, QED 0.51
Compound (SMILES): O=S(=O)(Nc1cscn1)c1c(F)cc(NCc2c(F)cccc2CN2CC3(CCC3)C2)cc1F
InChIKey: ZZDVFOAEYNZLRR-UHFFFAOYSA-N
Target: Sodium channel protein type 1 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P35498
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 1-1.5 h 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 CaCl2, 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 = 9965 nM (pChEMBL 5)
Descriptors: MW 508.59, ALogP 4.96, HBA 6, HBD 2, PSA 74.33, QED 0.45
Compound (SMILES): Cn1cnc(-c2cc(Cl)c(F)c(C(=O)NNS(=O)(=O)C[C@H]3CCC[C@H](NC(=O)OC(C)(C)C)C3)c2)c1
InChIKey: WGFOUPFCONPGEL-HOCLYGCPSA-N
Target: Histone acetyltransferase KAT7 (SINGLE PROTEIN), Homo sapiens
UniProt: O95251
Assay: KAT-7 Enzyme Assay: Enzyme assay buffer was 50 mM Tris pH 8.0, 0.002% Tween20, 0.005% bovine skin gelatin, and 1 mM dithiothreitol (DTT). For determination of IC50values, compounds were serially diluted with 2% (v/v) DMSO in the final reaction, pre-incubating each dilution of each compound with 40 μL of assay buffer containing KAT-7 enzyme (6 nM final concentration). 10 μL of assay buffer containing 1 μM peptide substrate and 2 μM acetyl coenzyme A (final concentrations) was added. Reactions (50 μL total) were then carried out at 25° C. for 120 minutes. Reactions were terminated by the addition of 0.5% formic acid (final concentration), and a sample of each reaction was analyzed by SAMDI Tech, Inc. (Chicago, Ill.) using self-assembled monolayer desorption/ionization time-of-flight mass spectrometry (Mrksich, M. (2008) Mass spectrometry of self-assembled monolayers: a new tool for molecular surface science. ACS Nano 2, 7-18).
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 544.05, ALogP 3.53, HBA 7, HBD 3, PSA 131.42, QED 0.46
Compound (SMILES): Cc1cc(-c2[nH]c3ccc(C4CCN(C(=O)CNCCO)CC4)cc3c2C(C)C)cc(C)n1
InChIKey: JYKLBSVHZJEFIM-UHFFFAOYSA-N
Target: Toll-like receptor 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR96
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 = 272 nM (pChEMBL 6.570000171661377)
Descriptors: MW 448.61, ALogP 4.26, HBA 4, HBD 3, PSA 81.25, QED 0.47
Compound (SMILES): Cc1cccc2cc(Cn3nc(I)c4c(N)ncnc43)c(N3CCCC3)nc12
InChIKey: GNQJYDPUBFERML-UHFFFAOYSA-N
Target: Phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O00750
Assay: Inhibition Assay: Class I PI3-Ks can be either purchased (p110α/p85α, p110β/p85α, p110δ/p85α from Upstate, and p110γ from Sigma) or expressed as previously described (Knight et al., 2004). IC50 values are measured using either a standard TLC assay for lipid kinase activity (described below) or a high-throughput membrane capture assay. Kinase reactions are performed by preparing a reaction mixture containing kinase, inhibitor (2% DMSO final concentration), buffer (25 mM HEPES, pH 7.4, 10 mM MgCl2), and freshly sonicated phosphatidylinositol (100 μg/ml). Reactions are initiated by the addition of ATP containing 10 μCi of γ-32P-ATP to a final concentration 10 or 100 μM and allowed to proceed for 5 minutes at room temperature. For TLC analysis, reactions are then terminated by the addition of 105 l 1N HCl followed by 160 μl CHCl3:MeOH (1:1). The biphasic mixture is vortexed, briefly centrifuged, and the organic phase is transferred to a new tube using a gel loading pipette tip precoated with CHCl3. This extract is spotted on TLC plates and developed for 3-4 hours in a 65:35 solution of n-propanol:1M acetic acid. The TLC plates are then dried, exposed to a phosphorimager screen (Storm, Amersham), and quantitated. For each compound, kinase activity is measured at 10-12 inhibitor concentrations representing two-fold dilutions from the highest concentration tested (typically, 200 μM). For compounds showing significant activity, IC50 determinations are repeated two to four times, and the reported value is the average of these independent measurements.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 485.33, ALogP 3.52, HBA 7, HBD 1, PSA 85.75, QED 0.45
Compound (SMILES): C=CC(=O)N1CC[C@]2(C1)C[C@@H](n1c(NC(=O)c3ccc(-c4cnco4)s3)nc3ccccc31)C2
InChIKey: FINZRTUQZUBKBD-NDQYQSCMSA-N
Target: Tyrosine-protein kinase Tec (SINGLE PROTEIN), Homo sapiens
UniProt: P42680
Assay: Kinase Inhibition Assay: The in vitro kinase assays were performed at Nanosyn (Santa Clara, Calif.) utilizing microfluidic detection technology. The test compounds were serially pre-diluted in DMSO and added, by the acoustic dispensing (Labcyte 550), directly to 384 well assay plates into 10 uL of a buffer with enzyme (ITK, TXK, BTK or TEC) comprising: 100 mM HEPES, pH7.5, 5 mM MgCl2, 0.1% bovine serum albumin, 1 mM DTT, 0.01% Triton X-100 and the enzyme. Final DMSO concentration was maintained at 1% in all samples, including the controls. The reactions were initiated by addition of ATP (1 mM final concentration) and the fluorescently labeled peptide substrate to a final concentration of 1 uM, and incubated for 3 hours at 25° C. Following incubation, the reactions were quenched by addition of 40 μL of termination buffer (100 mM HEPES, pH7.5, 0.01% Triton X-100, 50 mM EDTA). Terminated plates were analyzed using Caliper LabChip 3000 microfluidic electrophoresis instrument (Caliper Life Sciences/Perkin Elmer). The enzymatic modification of the peptide substrate (phosphorylation) results in a change of net charge enabling electrophoretic separation of product from substrate. As substrate and product are separated by electrophoresis, two peaks of fluorescence are observed. Change in the relative fluorescence intensity of the substrate and product peaks was the parameter measured, reflecting enzyme activity. In the presence of inhibitor, the ratio between product and substrate is altered: signal of the product decreases, while the signal of the substrate increases. Activity in each test sample was determined as the product to sum ratio (PSR): P/(S+P), where P is the peak height of the product and S is the peak height of the FAM-cAMP substrate. For each compound, enzyme activity was measured at 12 concentrations spaced by 3× dilution intervals. Negative control samples (0%-inhibition in the absence of inhibitor, DMSO only) and positive control samples (100%-inhibition, in the absence of enzyme or in the presence of control inhibitor) were assembled in replicates of four and were used to calculate %-inhibition values in the presence of compounds. Percent inhibition (Pinh) was determined using the following equation: Pinh=(PSR0%−PSRinh)/(PSR0%−PSR100%)*100, where PSRinh is the product sum ratio in the presence of inhibitor, PSR0% is the product sum ratio in the absence of inhibitor and PSR100% is the product sum ratio in 100%-inhibition control samples. To determine IC50 values, the inhibition curves (Pinh versus inhibitor concentration) were fitted by 4 parameter sigmoid dose-response model using XLfit software (IDBS).
Measurement: IC50 = 5.15 nM (pChEMBL 8.289999961853027)
Descriptors: MW 473.56, ALogP 4.74, HBA 7, HBD 1, PSA 93.26, QED 0.42
Compound (SMILES): O=C1Cc2cc(CCN3CCN(c4cccc5sccc45)CC3)ccc2N1
InChIKey: YCNGRVYOTGGUSQ-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 2A (SINGLE PROTEIN), Homo sapiens
UniProt: P28223
Assay: 5-HT2A Receptor Antagonism Activity Test: The 5-HT2A receptor antagonism activity test (The antagonism activity of test compounds on 5-HT2A receptor expressing human recombinant 5-HT2A receptor in CHO-K1 cells) was performed using FLIPR Calcium 5 Assay Kit (Product of Molecular Devices USA Inc.) according to the method documented in the kit instructions. The concentration of the test compounds was 0.1 nM-10000 nM and risperidone was used as a positive control. Test method is as follows: On the first day the seed cells are placed in T-175 flask containing 25 ml growth medium (F-12 nutrient mixture+10% FBS+1% penicillin/streptomycin+1.2% 50 mg/ml Geneticin) at a density of 14 million per bottle, cells were cultured under 37° C./5% CO2/humidified conditions for 24 hours; On the next day the seed cells were inoculated to the 384-well cell culture plate (each well containing 20,000 cells), the growth medium was replaced by 50 μL detecting medium (F-12 nutrient mixture+1.5% activated carbon-treated FBS), the cells were cultured under 37° C./5% CO2/humidified conditions for 16 hours; On the third day the medium was removed, each well of the cells plates were added with 24 μL freshly prepared loading dye solution (formulated according to the instructions), the plates were placed in an incubator and incubated under 37° C./5% CO2/humidified conditions for 120 minutes; transfer 6 μL test compound solution to the assay plate and gently shaking for one minute, incubated under 37° C./5% CO2/humidified conditions for 30 minutes; each well of the assay plate was added with 10 μL freshly prepared α-methyl-5-hydroxytryptamine solution (1.2 M, the final concentration of α-methyl-5-hydroxytryptamine is 300 nM), data were detected and analyzed by FLIPR (Product of US Molecular Devices Corporation). Inhibition rate of the compounds at different concentrations was calculated, IC50 value was calculated by the Excelfit software
Measurement: IC50 = 6.36 nM (pChEMBL 8.199999809265137)
Descriptors: MW 377.51, ALogP 3.76, HBA 4, HBD 1, PSA 35.58, QED 0.75
Compound (SMILES): Cc1c(C)n2c(C3(C4CC4)CC3)nnc2c(=O)n1C[C@@H]1CCCCO1
InChIKey: NLIMXPGAJBMBAG-HNNXBMFYSA-N
Target: Dual specificity calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1C (SINGLE PROTEIN), Homo sapiens
UniProt: Q14123
Assay: Phosphodiesterase Enzyme Assay: TABLE 5: All 3′, 5′ cyclic nucleotide PDE enzyme activities are measured with a radiometric enzyme assay based on SPA detection system. Compounds to be tested are diluted in pure DMSO using ten point concentration response curves. Maximal compound concentration in the reaction mixture is either 10 or 100 μM. Compounds at the appropriate concentration are pre-incubated with either of the PDE enzymes for 30 minutes before the reaction is started by the addition of substrate. Reactions are allowed to proceed for 60 minutes at room temperature. Next, reactions are stopped by addition of SPA beads. Samples are read 12 hours later in a MICROBETA TRILUX© Counter. IC50 values are calculated by plotting the normalized data vs. log [compound] and fitting the data using a four parameter logistic equation.PDE1B, PDE1A, and PDE1C are cloned and purified following standard protein generation procedures. The assay buffer is prepared to give a final concentration in the assay of 50 mM Tris-HCl, 50 mM MgCl2, 4 mM CaCl2), 0.1% BSA and 6 U/mL Calmodulin in water, at pH 7.5. The final enzyme concentration is 0.25, 0.074 and 0.0012 nM, for PDE1A, PDE1B and PDE1C respectively. The reactions are started by addition of the substrate, [3H]cAMP, to give a final concentration of 47 nM.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 342.44, ALogP 2.52, HBA 6, HBD 0, PSA 61.42, QED 0.86
Compound (SMILES): COc1cc2c(Nc3c(F)cc(F)cc3Br)ncnc2cc1O[C@@H]1CO[C@@H]2[C@H]1OC[C@H]2F
InChIKey: HJOMYFVOXGNASK-PTLICHBZSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: AlphaScreen Assay: In AlphaScreen, when the donor and acceptor beads are close in proximity, a series of photochemical events will give rise to a fluorescent signal upon light activation. Here we use biotinylated poly-(Glu, Tyr) 4:1 as the kinase substrate, streptavidin-coated donor beads and anti-phosphortyrosine antibody PY100-coated acceptor beads. Upon phosphorylation, the peptide substrate can bind to both donor and acceptor beads, thus gives rise to fluorescence. Compounds, ATP, biotinylated poly-(Glu, Tyr) and kinases are mixed in a volume of 20 μL for 1 hr at ambient temperature using Greiner 384-well white clear bottom medium binding plates. Then 10 uL solution containing 15-30 mg/mL AlphaScreen beads, 75 mM Hepes, pH 7.4, 300 mM NaCl, 120 mM EDTA, 0.3% BSA and 0.03% Tween-20 is added to each well. After 2-16 hr incubation of the beads, plates are read in a Perkin Elmer AlphaQuest reader (available from PerkinElmer Life and Analytical Sciences, Inc., of Boston Mass.).
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 512.28, ALogP 4.31, HBA 7, HBD 1, PSA 74.73, QED 0.55
Compound (SMILES): Cc1c(Br)cc(C(=O)Nc2ccc(-c3cc(C4CCN(C(=O)C(C)C)CC4)n4ncnc(N)c34)cc2)c(=O)n1-c1ccccn1
InChIKey: LQWBLHVQPPKLHS-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor TYRO3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06418
Assay: Enzymatic Assay: Tyro3, and Mer:The kinase assay buffer contained 50 mM HEPES, pH7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% NP-40 and 2 mM DTT. 0.1 ul test compounds dissolved in DMSO were transferred from compound plates to white 384-well assay plates (Greiner LUMITRAC plates). The final concentration of DMSO was 1.25%. Enzyme solutions of 5.1 nM phosphor-Axl, or 0.0625 nM c-Mer (Carna Biosciences, 08-108), or 0.366 nM Tyro3 (Life Technologies, PR7480A) were prepared in assay buffer. A 1 mM stock solution of peptide substrate Biotin-EQEDEPEGDYFEWLE-amide SEQ ID NO: 1 (Quality Controlled Biochemicals, MA) dissolved in DMSO was diluted to 1 uM in assay buffer containing 2000 uM ATP. 4 ul enzyme solution (or assay buffer for the enzyme blank) was added to the appropriate wells in each plate, and then 4 ul/well substrate solution was added to initiate the reaction. The plate was protected from light and incubated at room temperature for 60 min. The reaction was stopped by adding 4 ul detection solution containing 50 mM Tris-HCl, pH7.8, 150 mM NaCl, 0.05% BSA, 45 mM EDTA, 180 nM SA-APC (Perkin Elmer, CR130-100) and 3 nM Eu-W1024 anti-phosphotyrosine PY20 (Perkin Elmer, AD0067). The plate was incubated for 1 h at room temperature, and HTRF (homogenous time resolved fluorescence) signal was measured on a PHERAstar FS plate reader (BMG labtech).
Measurement: IC50 > 100 nM (pChEMBL 7)
Descriptors: MW 669.58, ALogP 5.21, HBA 9, HBD 2, PSA 140.51, QED 0.25
Compound (SMILES): CC1(C)CN(c2ccc(C#N)nn2)CC[C@H]1Nc1c(C(N)=O)cnc2cc(-c3ccc(N)nc3)nn12
InChIKey: JXSIEYYMYPREPS-GOSISDBHSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: In Vitro JAK Kinase Inhibition Assay: Tested compounds were dissolved in 100% DMSO, and obtained stock solutions were serially diluted in the reaction buffer (50 mM Tris pH 7.5, 10 mM MgCl2, 0.25 mM EGTA, 0.1 mM Na3VO4, 0.01% Triton X-100, 2.5 mM DTT). Recombinant kinases JAK1 (ProQinase), JAK2, or JAK3 (Carna Biosciences) were diluted in the dilution buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 0.05% Triton X-100, 1 mM DTT) to the final concentration 3 ng/μL (JAK1), 0.1 ng/μL (JAK2), or 0.2 ng/μL (JAK3). 5 μL of obtained solution of the compounds and 5 μL of the solution of respective kinases were added to the well of a 96-well plate. To initiate interaction between tested compounds and an enzyme, the plate was incubated for 10 minutes at 25° C. in a Plate-Thermo-Shaker with orbital stirring at 400 rpm. Wells of a negative control contained all reagents as mentioned above with the exception of tested compounds and kinase, and wells of a positive controls contained all reagents as mentioned above with the exception of tested compounds. Enzymatic reaction was initiated by the addition of 15 μL of the following solution: 5× concentrated reaction buffer (50 mM Tris pH 7.5, 10 mM MgCl2, 0.25 mM EGTA, 0.1 mM Na3VO4, 0.01% Triton X-100, 2.5 mM DTT), water, 30 μM ATP and for particular kinases: JAK160 μM peptide IRS-1 (Enzo), JAK2 or JAK310 μM peptide IGF-1Rtide (Lipopharm). Then the plate was incubated for 1 hour at 25° C. in a Plate-Thermo-Shaker, with orbital stirring at 400 rpm. Detection of ADP formed in the enzymatic reaction was then performed using ADP-Glo Kinase Assay kit (Promega). For this purpose, to the well of the 96-well plate 25 μL of ADP-Glo Reagent were added, and the plate was incubated for 40 minutes at 25° C. in a Plate-Thermo-Shaker with orbital stirring at 400 rpm. Then to the well of the 96-well plate 50 μL of Kinase Detection Reagent were added and the plate was incubated for 30 minutes at 25° C. in a Plate-Thermo-Shaker with orbital stirring at 400 rpm. After incubation, luminescence intensity was measured using Victor×Light luminometer (Perkin Elmer, Inc.).
Measurement: IC50 = 0.16 nM (pChEMBL 9.800000190734863)
Descriptors: MW 483.54, ALogP 1.85, HBA 11, HBD 3, PSA 177.03, QED 0.38
Compound (SMILES): Cc1noc(-c2cc(F)ccc2C(=O)N2C3CCC2C(Nc2cnc(C(F)(F)F)cn2)C3)n1
InChIKey: LILLACVMWMBBKH-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Human Orexin 2 Receptor Radioligand Binding Assay: HEK293 stably expressing human orexin 2 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM (Hyclone, cat #SH30022), 10% FBS, 1× Pen/Strep, 1× NaPyruvate, 10 mM HEPES, 600 ug/ml G418 media on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1× with Calcium and Magnesium, Cat #SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2K×G, 5 min at 40° C.), the supernatant was aspirated and the pellets frozen and stored at −800° C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and homogenized for 45 sec just prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H]-EMPA (Moraveck Corporation, specific activity=29.6 Ci/mmol), diluted to a 5 nM concentration in PBS (2 nM final concentration). Compounds were solubilized in 100% DMSO (Acros Organics, Cat. #61042-1000) and tested over a range of 7 concentration (from 0.1 nM to 10 μM). The final concentration of DMSO in the reactions is equal to or less than 0.1%. Total and nonspecific binding was determined in the absence and presence of 10 μM almorexant. The total volume of each reaction is 200 μL (20 μL of diluted compounds, 80 μL of [3H]-EMPA diluted in PBS and 100 μL of the cell suspension). Reactions were run for 60 min at room temperature and terminated by filtration through GF/C filter plates (PerkinElmer, Cat. #6005174) presoaked in 0.3% polyethylenimine using the cell harvester (PerkinElmer Filtermate). The plates were washed 3 times by aspirating 30 ml PBS through the plates. Plates were dried in 55° C. oven for 60 min, scintillation fluid was added, and the radioactivity was counted on a Topcount (Packard).
Measurement: Ki = 4800 nM (pChEMBL 5.320000171661377)
Descriptors: MW 462.41, ALogP 3.85, HBA 7, HBD 1, PSA 97.04, QED 0.59
Compound (SMILES): COc1cc(OC)cc(N2Cc3cnc4[nH]ccc4c3N(Cc3ccccc3)C2=O)c1
InChIKey: PMJXPDNRNBOAKW-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Enzymatic Assay: The inhibitor potency of the exemplified compounds was measured in an enzyme assay that measures peptide phosphorylation using FRET measurements to detect product formation. Inhibitors were serially diluted in DMSO and a volume of 0.5 μL was transferred to the wells of a 384-well plate. For FGFR3, a 10 μL volume of FGFR3 enzyme (Millipore) diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 5 mM DTT, pH 7.5) was added to the plate and pre-incubated for 5-10 minutes. Appropriate controls (enzyme blank and enzyme with no inhibitor) were included on the plate. The assay was initiated by the addition of a 10 μL solution containing biotinylated EQEDEPEGDYFEWLE peptide substrate (SEQ ID NO: 1) and ATP (final concentrations of 500 nM and 140 μM respectively) in assay buffer to the wells. The plate was incubated at 25° C. for 1 hr. The reactions were ended with the addition of 10 μL/well of quench solution (50 mM Tris, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.8; 30 mM EDTA with Perkin Elmer Lance Reagents at 3.75 nM Eu-antibody PY20 and 180 nM APC-Streptavidin). The plate was allowed to equilibrate for 1 hr before scanning the wells on a PheraStar plate reader (BMG Labtech).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 414.47, ALogP 4.73, HBA 4, HBD 1, PSA 70.69, QED 0.51
Compound (SMILES): C[C@H]1CN(c2ccncc2NC(=O)c2ccc3sc(-c4c(F)cccc4F)cc3n2)C[C@@H](N)[C@@H]1O
InChIKey: YEBIBPUTVNACIB-CNAJZPKWSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Enzyme Assay: Pim-1 and Pim-3 kinase assays 20 μL reactions were run in white 384 well polystyrene plates dotted with 0.8 μL compound/DMSO in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM MgCl2, 0.01% BSA, 5 mM DTT), containing 0.05 μM Biotin-labeled BAD peptide substrate (AnaSpec 62269), 1 mM ATP, and 2.5 μM (Pim-1, Invitrogen PV3503) or 1.25 μM (Pim-3, Millipore 14-738) enzyme for 1 h at 25° C. Reactions were stopped by addition of 10 μL STOP Buffer (150 mM Tris, pH=7.5, 150 mM NaCl, 75 mM EDTA, 0.01% Tween-20, 0.3% BSA) supplemented with Phospho-Bad (Ser112) Antibody (Cell Signaling 9291) diluted 666-fold, and Streptavidin donor beads (PerkinElmer 6760002) along with Protein-A acceptor beads (PerkinElmer 6760137) at 15 μg/mL each. Supplementation of the STOP buffer with beads and stopping the reactions were done under reduced light. Prior to the stopping reactions STOP buffer with beads was preincubated for 1 h in the dark at room temperature. After stopping the reactions, plates were incubated for 1 h in the dark at room temperature before reading on a PHERAstar FS plate reader (BMG Labtech) under reduced light.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 495.56, ALogP 4.03, HBA 7, HBD 3, PSA 104.37, QED 0.39
Compound (SMILES): CCn1cc(Nc2ncc(C(N)=O)c(NCCC3CC3)n2)cn1
InChIKey: MTDSIDOSVRHPSI-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Kinase Activity Assay: JAK3 (the final concentration was 0.36 nM); ATP (the final concentration was 10 μM); ULight-labeled JAK-1 (Tyr1023) Peptide (the final concentration was 50 nM); the enzymatic reaction time was 1 hour. The maximum final concentration of compounds was 2.5 μM. After a 3-fold gradient dilution, 11 concentrations were obtained, with the minimum final concentration of 0.042 nM. The final DMSO concentration was 1%.
Measurement: IC50 = 1.09 nM (pChEMBL 8.960000038146973)
Descriptors: MW 315.38, ALogP 1.75, HBA 7, HBD 3, PSA 110.75, QED 0.68
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3F)ncnc2cc1OC[C@@H]1C[C@@H]2CN(CC(C)C)C[C@@H]2C1
InChIKey: RFKWUHOHGOAWTQ-NNZMDNLPSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: AlphaScreen Assay: In AlphaScreen, when the donor and acceptor beads are close in proximity, a series of photochemical events will give rise to a fluorescent signal upon light activation. Here we use biotinylated poly-(Glu, Tyr) 4:1 as the kinase substrate, streptavidin-coated donor beads and anti-phosphortyrosine antibody PY100-coated acceptor beads. Upon phosphorylation, the peptide substrate can bind to both donor and acceptor beads, thus gives rise to fluorescence. Compounds, ATP, biotinylated poly-(Glu, Tyr) and kinases are mixed in a volume of 20 μL for 1 hr at ambient temperature using Greiner 384-well white clear bottom medium binding plates. Then 10 uL solution containing 15-30 mg/mL AlphaScreen beads, 75 mM Hepes, pH 7.4, 300 mM NaCl, 120 mM EDTA, 0.3% BSA and 0.03% Tween-20 is added to each well. After 2-16 hr incubation of the beads, plates are read in a Perkin Elmer AlphaQuest reader (available from PerkinElmer Life and Analytical Sciences, Inc., of Boston Mass.).
Measurement: IC50 = 525 nM (pChEMBL 6.28000020980835)
Descriptors: MW 533.48, ALogP 6.82, HBA 6, HBD 1, PSA 59.51, QED 0.32
Compound (SMILES): C[C@@H](O)c1nc2cnc3ccsc3c2n1[C@H]1CC[C@H](O)CC1
InChIKey: DHTRFHYYKUCARY-GMTAPVOTSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: In Vitro JAK Kinase Assay: Compounds herein were tested for inhibitory activity of JAK targets according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (a.a. 837-1142), JAK2 (a.a. 828-1132) and JAK3 (a.a. 781-1124) were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 μL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hour and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a PHERA star plate reader (BMG, Cary, N.C.). The Example compounds were each tested in the Example A assay (see Table 1 for data for the compounds of the examples as tested by the assay of Example A at 1 mM ATP).
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 317.41, ALogP 3.18, HBA 6, HBD 2, PSA 71.17, QED 0.76
Compound (SMILES): CC1CCCN1S(=O)(=O)c1ccc(Nc2nn([C@@H](CC#N)C3CC3)c3cc[nH]c(=O)c23)cc1
InChIKey: NBPCQTQLAKHQTH-FUBQLUNQSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Jak Biochemical HTRF Assay Protocol: The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer #PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at room temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading. After normalization to untreated controls, the percent inhibition of the HTRF signal at each compound concentration was calculated.
Measurement: IC50 = 340 nM (pChEMBL 6.46999979019165)
Descriptors: MW 466.57, ALogP 3.51, HBA 7, HBD 2, PSA 123.88, QED 0.55
Compound (SMILES): N#CC[C@H]1CC[C@H](n2c(Cn3c(-c4ccccc4)noc3=O)nc3cnc4[nH]ccc4c32)CC1
InChIKey: SNSGNQJXZJKMAR-SAABIXHNSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzymatic Inhibition Assay: 11-point dosing series were made for each compound by serially diluting 1:3 or 1:4 in DMSO, with point 12 being a DMSO control. From the serial dilution plates, sample was transferred to a 384 wells assay plate (#781280, Greiner, Monroe, N.C.) using Labcyte Echo (Sunnyvale, Calif.), or Biosero ATS (San Diego, Calif.). The compounds were tested in duplicate. Column 12 was used for positive controls, and column 24 contained negative controls with no enzyme added. A compound from our internal collection, with inhibitory activity for JAK isoforms, was used as a reference compound. The final concentration of DMSO was ≤0.25% in a 20 μL reaction. Assay conditions for each of the proteins are summarized in Table 3. The enzyme reaction was initiated by the addition of 10 μL of 2× enzyme and ATP mixture to 10 μL of 2× substrate solution prepared in reaction buffer (50 mM MOPS pH 7.5, 10 mM MgCl2, 1 mM EDTA, 2 mM DTT, 0.002% Tween-20). The Tyk2 enzyme was pre-incubated with 2 mM ATP for 30 min prior to the reaction initiation. Immediately after the addition of the enzyme to the reaction mixture, the plate was centrifuged at 1000 rpm for 1 minute and incubated at 25° C. for 45 minutes for JAK 3 and 90 minutes for JAK1, JAK2 and Tyk2. The reaction was quenched by the addition of 20 μL of 0.5% TFA containing 0.15 μM of internal standard peptide using Multidrop Combi reagent dispenser (Thermo Scientific, Waltham, Mass.). Several wells in column 24 were typically used for the product standard curve. After the quench, the assay plate was centrifuged at 3000 rpm for 3 minutes and sealed with pierceable aluminum foil (Cat #06644-001, Agilent) using a PlateLoc (Agilent Technologies, Santa Clara, Calif.). The plates then were transferred on to the RapidFire for the MS analysis. Compound inhibition was assessed by a decrease of the phosphorylated product levels in sample wells compared to the non-inhibited enzyme reaction. The assay conditions for the above assays are shown in Table 3 and the results of Ex. 1-209 as tested in these assays are shown in Table 4.TABLE 3Assay conditions for JAK family enzyme assays* [p], [ATP], [Substrate], [IS],Enzyme nM μM μM nMJAK1-JH1JH2 8.0 12.5 200 100JAK2-JH1JH2 7.0 or 3.6 30 40 100JAK3-JH1JH2 2.0 150 40 100Tyk2-JH1JH2   25 or 14.7 50 200 100*Reaction buffer: 50 mM MOPS, pH 7.5 10 mM MgCl2, 1 mM EDTA, 2 mM DTT, 0.002% Tween-20; IS stands for internal standard peptide; p stands for phospho-peptide (product); Substrate stands for peptide.
Measurement: IC50 = 4675 nM (pChEMBL 5.329999923706055)
Descriptors: MW 453.51, ALogP 4.42, HBA 8, HBD 1, PSA 118.32, QED 0.42
Compound (SMILES): Cc1cc(=O)n(C)cc1S(=O)(=O)C[C@H]1C[C@@H](N(C)c2ncnc3[nH]ccc23)C1
InChIKey: XUBODOZNSMBJOD-OKILXGFUSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Caliper Enzyme Assay: Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK3 or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 0.088 nM (pChEMBL 10.0600004196167)
Descriptors: MW 401.49, ALogP 1.65, HBA 7, HBD 1, PSA 100.95, QED 0.7
Compound (SMILES): C/C(=N\OCCCc1ccccc1)c1ccc(C(=O)N2CC[C@](N)(COP(=O)(O)O)C2)cc1
InChIKey: PWSJXRMPENWESI-DMUURETOSA-N
Target: Sphingosine 1-phosphate receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99500
Assay: Receptor [35S] GTPgammaS Binding Assays: Compounds were loaded in a 384 Falcon v-bottom plate (0.5 μl/well in an 11 point, 3-fold dilution). Membranes prepared from S1P1/CHO cells or EDG3-Gal5-bla HEK293T cells (EDG3 equivalent S1P3) were added to the compound plate (40 l/well, final protein 3 μg/well) with MULTIDROP®. [35S]GTP (1250 Ci/mmol, Perkin Elmer) was diluted in assay buffer: 20 mM HEPES, pH7.5, 10 mM MgCl2, 150 mM NaCl, 1 mM EGTA (ethylene glycol tetraacetic acid), 1 mM DTT (Dithiothreitol), 10 μM GDP, 0.1% fatty acid free BSA, and 10 μg/ml Saponin to 0.4 nM. 40 μl of the [35S] GTP solution was added to the compound plate with a final concentration of 0.2 nM. The reaction was kept at room temperature for 45 min. At the end of incubation, all the mixtures in the compound plate were transferred to Millipore 384-well FB filter plates via the VELOCITY11 ® Vprep liquid handler. The filter plate was washed with water 4 times by using the manifold Embla plate washer and dried at 60° C. for 45 min. MicroScint 20 scintillation fluid (30 μl) was added to each well for counting on the Packard TOPCOUNT®. EC50 is defined as the agonist concentration that corresponds to 50% of the Ymax (maximal response) obtained for each individual compound tested.A smaller value for GTPγS S1P1 EC50 value indicated greater activity for the compound in the GTPγS S1P1 binding assay. Thus the compounds of the present invention may be used in treating, preventing, or curing various S1P1 receptor-related conditions. The compounds have potential use in treating, preventing, or curing autoimmune and inflammatory diseases such as multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, lupus, psoriasis, or minimizing or reducing rejection of transplanted organs.
Measurement: EC50 > 6250 nM (pChEMBL 5.203999996185303)
Descriptors: MW 475.48, ALogP 2.71, HBA 6, HBD 3, PSA 134.68, QED 0.21