lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1ccccc1NC(=O)Nc1ccc(C[C@@H]2NC(=O)C[C@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(C)C)NC2=O)cc1
InChIKey: FXYDXLJEJBXFBD-COJYKABBSA-N
Target: Integrin alpha1/beta2 complex (PROTEIN COMPLEX), Homo sapiens
UniProt: P56199
Assay: Antagonist activity at alpha1beta2 integrin receptor in human Jurkat E6.1 cells assessed as inhibition of ICAM-1-induced cell adhesion pre-incubated for 30 mins followed by 30 mins incubation in ICAM-1 coated wells by fluorescence plate reader assay
Measurement: IC50 = 1110 nM (pChEMBL 5.960000038146973)
Descriptors: MW 737.81, ALogP 1.27, HBA 8, HBD 9, PSA 261.23, QED 0.17
Compound (SMILES): COC(=O)c1cc(O)c2cc(NC(=O)NCCNC(=N)NC[C@H]3O[C@@H](n4cnc5c(N)ncnc54)[C@H](O)[C@@H]3O)ccc2c1
InChIKey: FOKMEBMJOQIKNH-ZDXOVATRSA-N
Target: PRMT5/MEP50 complex (PROTEIN COMPLEX), Homo sapiens
UniProt: O14744
Assay: Inhibition of N-terminal/C-terminal human recombinant full-length FLAG-tagged PRMT5 (2 to 637 residues)/C-terminal human His-tagged MEP50 (2 to 342 residues) using histone H2A as substrate and 3H-SAM as cosubstrate incubated for 60 mins by scintillation counter analysis
Measurement: IC50 = 9400 nM (pChEMBL 5.03000020980835)
Descriptors: MW 594.59, ALogP -0.39, HBA 13, HBD 9, PSA 254.88, QED 0.05
Compound (SMILES): COc1ccccc1Nc1cc(Nc2ccc(CC(=O)O[C@@H]3CCOC3)cn2)ncc1C(N)=O
InChIKey: JXLWZLUHIKGJID-MRXNPFEDSA-N
Target: JAK2/JAK1 (PROTEIN COMPLEX), Homo sapiens
UniProt: O60674
Assay: Inhibition of JAK1/2-mediated STAT1 phosphorylation in human M07E cells preincubated for 30 mins followed by IFN-alpha stimulation measured after 15 mins by flow cytometry
Measurement: IC50 = 48 nM (pChEMBL 7.320000171661377)
Descriptors: MW 463.49, ALogP 2.95, HBA 9, HBD 3, PSA 137.69, QED 0.41
Compound (SMILES): COc1ccccc1Nc1cc(Nc2ccc(CC(=O)O)cn2)ncc1C(N)=O
InChIKey: MYTVQTWPMPXBGM-UHFFFAOYSA-N
Target: JAK2/JAK1 (PROTEIN COMPLEX), Homo sapiens
UniProt: O60674
Assay: Inhibition of JAK1/2-mediated STAT1 phosphorylation in human M07E cells preincubated for 30 mins followed by IFN-alpha stimulation measured after 15 mins by flow cytometry
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 393.4, ALogP 2.7, HBA 7, HBD 4, PSA 139.46, QED 0.46
Compound (SMILES): Cc1ccccc1NC(=O)Nc1ccc(C[C@@H]2NC(=O)C[C@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(C)C)NC2=O)cc1
InChIKey: FXYDXLJEJBXFBD-COJYKABBSA-N
Target: Integrin alpha-5/beta-1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P08648
Assay: Agonist activity at alpha5beta1 integrin receptor in human K562 cells assessed as inhibition of fibronectin-induced cell adhesion pre-incubated for 30 mins followed by 1 hr incubation in fibronectin coated wells by fluorescence plate reader assay
Measurement: EC50 > 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 737.81, ALogP 1.27, HBA 8, HBD 9, PSA 261.23, QED 0.17
Compound (SMILES): COc1ccccc1Nc1cc(Nc2ccc(CC(=O)O[C@@H]3CCOC3)cn2)ncc1C(N)=O
InChIKey: JXLWZLUHIKGJID-MRXNPFEDSA-N
Target: JAK3/JAK1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P23458
Assay: Inhibition of JAK1/3-mediated STAT5 phosphorylation in human whole blood incubated for 30 mins followed by IL-2 addition measured after 15 mins by flow cytometry
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 463.49, ALogP 2.95, HBA 9, HBD 3, PSA 137.69, QED 0.41
Compound (SMILES): COc1ccccc1Nc1cc(Nc2ccc(CC(=O)O)cn2)ncc1C(N)=O
InChIKey: MYTVQTWPMPXBGM-UHFFFAOYSA-N
Target: JAK3/JAK1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P23458
Assay: Inhibition of JAK1/3-mediated STAT5 phosphorylation in human whole blood incubated for 30 mins followed by IL-2 addition measured after 15 mins by flow cytometry
Measurement: IC50 > 40000 nM (pChEMBL 4.3979997634887695)
Descriptors: MW 393.4, ALogP 2.7, HBA 7, HBD 4, PSA 139.46, QED 0.46
Compound (SMILES): Cc1ccccc1NC(=O)Nc1ccc(C[C@@H]2NC(=O)C[C@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(C)C)NC2=O)cc1
InChIKey: FXYDXLJEJBXFBD-COJYKABBSA-N
Target: Integrin alpha-4/beta-1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P13612
Assay: Binding affinity to alpha4beta1 integrin receptor (unknown origin) preincubated for 30 mins followed by 30 mins incubation in fibronectin coated wells by fluorescence based assay
Measurement: IC50 = 133 nM (pChEMBL 6.880000114440918)
Descriptors: MW 737.81, ALogP 1.27, HBA 8, HBD 9, PSA 261.23, QED 0.17
Compound (SMILES): CCc1cc2c(cc1Cl)nc1c(=O)[nH]c(=O)nc-1n2CCOCC(C)C
InChIKey: IOHJEWFKBRNJIO-UHFFFAOYSA-N
Target: HAT1/Rbap46 (PROTEIN COMPLEX), Homo sapiens
UniProt: O14929
Assay: Inhibition of C-terminal FLAG tagged full length human HAT1/Rbap46 transfected in HEK293F cells using histone H4 peptide as substrate and acetyl-CoA as cofactor preincubated for 10 mins followed by acetyl-CoA addition and measured after 1 hr by amplex red dye based fluorescence analysis
Measurement: IC50 = 19100 nM (pChEMBL 4.71999979019165)
Descriptors: MW 376.84, ALogP 2.47, HBA 6, HBD 1, PSA 89.87, QED 0.53
Compound (SMILES): Cc1ccccc1NC(=O)Nc1ccc(C[C@@H]2NC(=O)C[C@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(C)C)NC2=O)cc1
InChIKey: FXYDXLJEJBXFBD-COJYKABBSA-N
Target: Integrin alpha-4/beta-1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P13612
Assay: Binding affinity to alpha4beta1 integrin receptor (unknown origin) preincubated for 30 mins followed by 30 mins incubation in vascular cell adhesion molecule 1-induced cell adhesion coated wells by fluorescence based assay
Measurement: IC50 = 101 nM (pChEMBL 7)
Descriptors: MW 737.81, ALogP 1.27, HBA 8, HBD 9, PSA 261.23, QED 0.17
Compound (SMILES): Cc1ccccc1NC(=O)Nc1ccc(C[C@@H]2NC(=O)C[C@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(C)C)NC2=O)cc1
InChIKey: FXYDXLJEJBXFBD-COJYKABBSA-N
Target: Integrin alpha4/beta7 complex (PROTEIN COMPLEX), Homo sapiens
UniProt: P48169
Assay: Binding affinity to alpha4beta7 integrin receptor (unknown origin) preincubated for 30 mins followed by 30 mins incubation in mucosal vascular add-ressin cell adhesion molecule 1 coated wells by fluorescence based assay
Measurement: IC50 = 22.7 nM (pChEMBL 7.639999866485596)
Descriptors: MW 737.81, ALogP 1.27, HBA 8, HBD 9, PSA 261.23, QED 0.17
Compound (SMILES): Cc1ccccc1NC(=O)Nc1ccc(C[C@@H]2NC(=O)C[C@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(C)C)NC2=O)cc1
InChIKey: FXYDXLJEJBXFBD-COJYKABBSA-N
Target: Integrin alpha-M/beta-2 (PROTEIN COMPLEX), Homo sapiens
UniProt: P05107
Assay: Binding affinity to alphaMbeta2 integrin receptor (unknown origin) preincubated for 30 mins followed by 30 mins incubation in fibrinogen coated wells by fluorescence based assay
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 737.81, ALogP 1.27, HBA 8, HBD 9, PSA 261.23, QED 0.17
Compound (SMILES): Cc1ccccc1NC(=O)Nc1ccc(C[C@@H]2NC(=O)C[C@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(C)C)NC2=O)cc1
InChIKey: FXYDXLJEJBXFBD-COJYKABBSA-N
Target: Integrin alpha1/beta2 complex (PROTEIN COMPLEX), Homo sapiens
UniProt: P56199
Assay: Binding affinity to alpha1beta2 integrin receptor (unknown origin) preincubated for 30 mins followed by 30 mins incubation in ICAM-1 coated wells by fluorescence based assay
Measurement: IC50 = 897 nM (pChEMBL 6.050000190734863)
Descriptors: MW 737.81, ALogP 1.27, HBA 8, HBD 9, PSA 261.23, QED 0.17
Compound (SMILES): Cc1ccccc1NC(=O)Nc1ccc(C[C@@H]2NC(=O)C[C@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(C)C)NC2=O)cc1
InChIKey: FXYDXLJEJBXFBD-COJYKABBSA-N
Target: Integrin alpha-5/beta-1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P08648
Assay: Binding affinity to alpha5beta1 integrin receptor (unknown origin) preincubated for 30 mins followed by 30 mins incubation in fibronectin coated wells by fluorescence based assay
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 737.81, ALogP 1.27, HBA 8, HBD 9, PSA 261.23, QED 0.17
Compound (SMILES): CCS(=O)(=O)c1ccc(Cl)cc1Nn1cnc2cc(CN3CCN(C(C)C)CC3)c(C(F)(F)F)cc2c1=O
InChIKey: SJJYGHRRBQIVJC-UHFFFAOYSA-N
Target: Epithelial discoidin domain-containing receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q08345
Assay: LanthaScreen Assay: DDR1 binding activity was measured using the LanthaScreen (Registered trademark) Eu Kinase Binding Assay (manufactured by Life Technologies Corporation). The test compound and the Alexa Fluor® 647-labeled Kinase Tracer 178 (manufactured by Life Technologies Corporation) were added to a mixture of DDR1 and the LanthaScreen (Registered trademark) Eu-anti-GST antibody. After one-hour reaction at room temperature, the fluorescence resonance energy transfer was measured. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate relative to the test compound-free control.
Measurement: IC50 = 56 nM (pChEMBL 7.25)
Descriptors: MW 572.05, ALogP 4.26, HBA 8, HBD 1, PSA 87.54, QED 0.45
Compound (SMILES): CS(=O)(=O)c1ccc(-c2cc3c(N4CC(C(=O)NCc5ccc(Cl)cc5F)C4)ncnn3c2)cc1
InChIKey: WVWGFGQCDOAOHV-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Kit Wild Type Assay: In each well of a 384-well plate, 0.2 ng/ul final (2 nM) of wild type Kit (Carna Bioscience 08-156) was incubated in a total of 12.5 ul of buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 10 mM MgCl2, 1 mM DTT) with 1 uM Srctide (5-FAM-GEEPLYWSFPAKKK-NH2) and 400 uM ATP at 25 C for 90 minutes in the presence or absence of a dosed concentration series of compound (1% DMSO final concentration). The reaction was stopped by the addition of 70 ul of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 35 mM EDTA and 0.2% of Coating Reagent 3 (Caliper Lifesciences)). The plate was then read on a Caliper EZReader 2 (protocol settings: −1.9 psi, upstream voltage −700, downstream voltage −3000, post sample sip 35s).
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 513.98, ALogP 3.34, HBA 7, HBD 1, PSA 96.67, QED 0.42
Compound (SMILES): CC(C)n1nccc1-c1cc(-c2ccc(F)c(N3CCS(=O)(=O)CC3)c2)n2ncnc(N)c12
InChIKey: NVJYIQYVBCFFQJ-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: ADP-Glo Format PI3K Assay: The ADP-Glo format PI3K assays were performed in Proxiplate 384-well plates (Perkin Elmer #6008280). The final assay volume was 2 μl prepared from 1 μl additions of enzyme/PIP2:PS lipid (Invitrogen #PV5100) mixture and 1 μl ATP (provided in kit, Promega #V9101) and test compounds in assay buffer (50 mM HEPES pH 7.5, 3 mM MgCl2, 100 mM NaCl, 0.5 mM EGTA, 2 mM DTT, 0.03% CHAPS). The reaction was initiated by the combination of enzyme/lipid, ATP, and test compounds. The reaction mixture was incubated at room temperature for 30 minutes (PI3K Alpha, Beta, Gamma) or 3 hours for PI3K Delta. ADP-Glo (2 μl), followed by Kinase Detection reagent (4 μl), were added to reactions following the initial incubation and allowed to incubate 40 minutes at room temperature. The reaction mixture was analyzed on the TOPCOUNT (Perkin Elmer). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of enzyme in the assays are PI3K Alpha [0.5 nM], PI3K Beta [2 nM], PI3K Gamma [20 nM], PI3K Delta [0.5 nM]. ATP final concentrations are as follows: for Alpha [10 μM], for Beta [12.5 μM], for Gamma [6.5 μM], for Delta [100 μM]. Lipid final concentration was the same for all enzymes, [25 μM]. Dose response curves were generated to determine the concentration required to inhibit 50% of activity. Compounds were dissolved at 0.12 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. The IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 469.55, ALogP 2.8, HBA 9, HBD 1, PSA 111.41, QED 0.49
Compound (SMILES): COCCN1CC(c2ccccc2)[C@H](NC(=O)Nc2cnn3ccccc23)C1
InChIKey: JXXVKQOTWAPXMB-WHCXFUJUSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: TrkA Omnia Assay: Trk enzymatic selectivity was assessed using Omnia Kinase Assay reagents from Invitrogen Corp. Enzyme (TrkA from Invitrogen Corp.) and test compound (various concentrations) were incubated for 10 minutes at ambient temperature in a 384-well white polypropylene plate (Nunc catalog#267462). Omnia Tyr Peptide #4, as well as ATP, were then added to the plate. Final concentrations were as follows: 20 nM enzyme, 500 μM of ATP, 10 μM peptide substrate. The assay buffer consisted of 25 mM MOPS pH 7.5, 0.005% (v/v) Triton X-100 and 5 mM MgCl2. The production of phosphorylated peptide was monitored continuously for 70 minutes using a Molecular Devices FlexStation II384 microplate reader (excitation=360 nm; emission=485 nm). Initial rates were calculated from the progress curves. IC50 values were calculated from these rates using either a 4 or 5-parameter logistic curve fit.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 379.46, ALogP 2.57, HBA 5, HBD 2, PSA 70.9, QED 0.69
Compound (SMILES): COc1cc2ncnn2cc1-c1[nH]c2ccc(C3CCNCC3)cc2c1C(C)C
InChIKey: CJOVDQWVUBLRJG-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 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. 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 = 109 nM (pChEMBL 6.960000038146973)
Descriptors: MW 389.5, ALogP 4.48, HBA 5, HBD 2, PSA 67.24, QED 0.54
Compound (SMILES): C=CC(=O)Nc1cc(C)ccc1Nc1nc(Nc2nc(O)ncc2Cl)ncc1C(F)(F)F
InChIKey: LTSWGDYMLSQXLG-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P27361
Assay: Omnia Assay: The protocol below describes continuous-read kinase assays to measure potency of compounds against activated ERK1 enzyme. The mechanics of the assay platform are best described by the vendor (Invitrogen, Carlsbad, Calif.) on their website at the following URL: invitrogen.com/site/us/en/home.html.Briefly, a 1.25× stock of ERK1 enzyme (14-439-K) from Millipore (Billerica, Mass.), 5×ATP (AS001A) and ST17-Sox conjugated peptide substrate (KNZ1171C) were prepared in 1× kinase reaction buffer consisting of 20 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 5 mM β-glycerophosphate, 5% glycerol (10× stock, KB002A) and 0.2 mM DTT. 10 μL of ATP/ST17-sox peptide substrate mix was combined with 0.5 μL volume of 100% DMSO and serially diluted compounds were prepared in 100% DMSO in a Corning (#3574) 384-well, white, non-binding surface microtiter plate (Corning, N.Y.). Kinase reactions were started with the addition of 40 μL of ERK1 solution and monitored every 71 seconds for 30-240 minutes at λex360/λem485 in a Synergy plate reader from BioTek (Winooski, Vt.). At the conclusion of each assay, progress curves from each well were examined for linear reaction kinetics and fit statistics (R2, 95% confidence interval, absolute sum of squares). Initial velocity (0 minutes to 30+ minutes) from each reaction was determined from the slope of a plot of relative fluorescence units vs time (minutes or seconds) and then plotted against inhibitor concentration to estimate AppIC50 from log [Inhibitor] vs Response, Variable Slope model in GraphPad Prism from GraphPad Software (San Diego, Calif.).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 465.82, ALogP 4.56, HBA 8, HBD 4, PSA 124.95, QED 0.39
Compound (SMILES): Cc1cc(OCC2CC2)nc(-c2cc(F)c(N(C)CCCC(=O)O)c(F)c2)n1
InChIKey: PKSFUKUUIWRFTD-UHFFFAOYSA-N
Target: Free fatty acid receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5NUL3
Assay: Measurement of Activity of GPR120 Agonist (Cell-Based Assay): CHO-K1 cells expressing Ga16 and hGPR120 were dispensed into each well of a 96-well plate (3×104 cells/100 μl/well) and then incubated in 5% CO2, 37° C. incubator for 18 hours. Each well was treated with 100 μl of Calcium 5 dye (Molecular Devices) solution including 2% DMSO and then incubated in 5% CO2, 37° C. incubator for 1 hour. Serially diluted GPR120 agonists were prepared to a final concentration of 0.5% DMSO in a 96-well plate. Each well was treated with 50 μl of the agonist compounds using Plexstation II, and then fluorescence was measured at Ex 485 nm and Em 525 nm.Fluorescence increased by the serially diluted GPR120 agonists is calculated as a relative percent (%) value based on the fluorescence represented by the treatment of 1% DMSO only. EC50 refers to the concentration of agonist which shows 50% of maximum fluorescence increased by the treatment of agonist. The calculation of measurement was carried out by using statistical software (Prizm).
Measurement: EC50 < 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 391.42, ALogP 3.82, HBA 5, HBD 1, PSA 75.55, QED 0.7
Compound (SMILES): Cc1nn2c3c(nc2s1)CCN(C(=O)COc1ccc(NS(C)(=O)=O)nc1Cl)C3c1sc(C(C)C)nc1C
InChIKey: QWDPOSVZQBWYDO-UHFFFAOYSA-N
Target: Tryptophan 5-hydroxylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P17752
Assay: Biochemical In Vitro Assay: To generate the enzyme, full length human TPH1 is cloned into the plasmid pET20b(+) (Novagen) and expressed in E. coli. The bacterial cells are ruptured by sonication on ice and the lysate is cleared by centrifugation. The resulting protein in the pellet is re-extracted and TPH1 is purified from the obtained lysate by affinity chromatography using a pterin cosubstrate analog immobilized to the resin of the column. The protein is further purified by size exclusion chromatography to remove protein aggregates. The activity of TPH1 is determined by using a fluorescence assay. The enzyme activity assay is carried out at 15° C. with atmosphere oxygen for the duration of 60 minutes in a volume of 64 μl. The reaction is carried out in a 0.1 M Tris-HCl buffer, adjusted to pH 7.6, containing 1 mM DTT, 0.2 mg/mL catalase, 100 μM (±)-6-methyl-5,6,7,8-tetrahydropterine dihydrochloride, 40 μM L-tryptophan, and 40-80 nM of TPH1. The reaction is started by bringing together L-tryptophan with all the other reaction substituents and stopped by quenching with perchloric acid (HClO4). The amount of 5-hydroxy-L-tryptophan produced during the enzymatic reaction is determined by fluorescence readout. Fluorescence, as determined at 540 nm when excited at 300 nm wavelength, increases proportionally to the 5-hydroxy-L-tryptophan formed. Compounds are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into the assay plate. Fluorescence is measured for each well and the fluorescence at 540 nm wavelength is compared to the fluorescence of the vehicle in place of compound. Inhibitory activities of example compounds with respect to the TPH1 protein are determined by calculating the IC50 value (the concentration of compound needed to inhibit 50% of the enzyme activity).
Measurement: IC50 = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 596.16, ALogP 3.96, HBA 11, HBD 1, PSA 131.68, QED 0.32
Compound (SMILES): Nc1ncc(-c2ccc(C(=O)N3CCC(N4CCCC4)CC3)cc2)nc1C(=O)Nc1ccccc1
InChIKey: FWLGVYOSJMAVJJ-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Inhibition Assay: Compounds were screened for their ability to inhibit ATR kinase using a radioactive-phosphate incorporation assay. Assays were carried out in a mixture of 50 mM Tris/HCl (pH 7.5), 10 mM MgCl2 and 1 mM DTT. Final substrate concentrations were 10 M [γ-33P]ATP (3mCi 33P ATP/mmol ATP, Perkin Elmer) and 800 μM target peptide (ASELPASQPQPFSAKKK).Assays were carried out at 25° C. in the presence of 5 nM full-length ATR. An assay stock buffer solution was prepared containing all of the reagents listed above, with the exception of ATP and the test compound of interest. 13.5 μL of the stock solution was placed in a 96 well plate followed by addition of 2 μL of DMSO stock containing serial dilutions of the test compound (typically starting from a final concentration of 15 μM with 3-fold serial dilutions) in duplicate (final DMSO concentration 7%). The plate was pre-incubated for 10 minutes at 25° C. and the reaction initiated by addition of 15 μL [γ-33P]ATP (final concentration 10 μM).The reaction was stopped after 24 hours by the addition of 30 μL 0.1M phosphoric acid containing 2 mM ATP. A multiscreen phosphocellulose filter 96-well plate (Millipore, Cat no. MAPHN0B50) was pretreated with 100 μL 0.2M phosphoric acid prior to the addition of 45 μL of the stopped assay mixture. The plate was washed with 5×200 μL 0.2M phosphoric acid. After drying, 100 μL Optiphase SuperMix liquid scintillation cocktail (Perkin Elmer) was added to the well prior to scintillation counting (1450 Microbeta Liquid Scintillation Counter, Wallac).
Measurement: Ki = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 470.58, ALogP 3.68, HBA 6, HBD 2, PSA 104.45, QED 0.59
Compound (SMILES): Cc1cc(C)nc([C@@H]2CC[C@H]2c2nc3c(c(C#N)nn3[C@@H](C)c3ccc(C(F)(F)F)nc3)c(=O)[nH]2)n1
InChIKey: JERSLDYYZIQFKX-NUEKZKHPSA-N
Target: High affinity cGMP-specific 3',5'-cyclic phosphodiesterase 9A (SINGLE PROTEIN), Homo sapiens
UniProt: O76083
Assay: Enzyme Assay: Assays for PDE 1 through 11 were performed in parallel at room temperature in 384-well microtiter plates with an incubation volume of 20.2 μL. Solutions of test compounds were prepared in DMSO and serially diluted with DMSO to yield 30 μL of each of ten solutions differing by 3-fold in concentration, at 32 serial dilutions per plate. 100% inhibition was determined by adding buffer in place of the enzyme and 0% inhibition is determined by using DMSO (1% final concentrations). A Labcyte POD 810 (Labcyte, Sunnyvale, Calif.) was used to dispense 200 nL from each well of the titration plate to make eleven copies of the assay plate for each titration, one copy for each PDE enzyme. A solution of each enzyme (dilution from aliquots, sufficient to produce 20% substrate conversion) and a separate solution of FAM-labeled cAMP or FAM-labeled cGMP from Molecular Devices (Sunnyvale, Calif., product # R7506 or cGMP#R7508), at a final concentration of 50 nM were made in the assay buffer (10 mM Tris HCl, pH 7.2, 10 mM MgCl2, 0.05% NaN3 0.01% Tween-20, and 1 mM DTT). Note that the substrate for PDE2 is 50 nM FAM cAMP containing 1000 nM of cGMP. The enzyme and the substrate were then added to the assay plates in two consecutive additions of 10 μL and then shaken to mix. The reaction was allowed to proceed at room temperature for 60 minutes. A binding solution was then made from the kit components, comprised of 80% Solution A, 20% Solution B and binding reagent at a volume of 1/600 the total binding solution. The enzymatic reaction was stopped by addition of 60 μL of the binding solution to each well of the assay plate. The plates were sealed and shaken for 10 seconds
Measurement: Ki = 23.3 nM (pChEMBL 7.630000114440918)
Descriptors: MW 494.48, ALogP 4.08, HBA 8, HBD 1, PSA 126.03, QED 0.45
Compound (SMILES): O=C(N[C@@H]1CCCNC1)c1sc2nccc3c2c1NC(=O)N3c1c(F)cc(Oc2ccccc2)cc1F
InChIKey: GBKUMVBAWSKPAU-CQSZACIVSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Lanthascreen Binding Assay: A BTK kinase lanthascreen binding assay monitors compound binding to unphosphorylated-BTK kinase domain (UP-BTK), by competing with a fluorescent labeled tracer. UP-BTK, consisting of the kinase domain of non-phosphorylated BTK protein (389-659aa), was produced in a Baculovirus/insect cell expression system. Into a 384-well plate, 2 ng of GST-tagged human BTK (389-659aa) was incubated with compound, 50 nM of Tracer 236 and 2 nM anti-GST antibody for 60 minutes using an optimized Lanthascreen assay. After 60 minutes, plates were read at 340 nM and 615/665 nM in an Infinite F500 (Tecan).
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 521.55, ALogP 5.53, HBA 6, HBD 3, PSA 95.59, QED 0.32
Compound (SMILES): CN1C(=O)COc2cc(-c3c(-c4ccc(C#N)cc4)nc(N4CCC(N(C)C)CC4)c4cncn34)cnc21
InChIKey: FKIBBIRJDQPQLK-UHFFFAOYSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: Demethylase Biochemical Assay: LANCE LSD1/KDM1A demethylase assay¿10 uL of 1 nM LSD-1 enzyme (ENZO BML-SE544-0050) in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 25 mM NaCl, 5 mM DTT) were preincubated for 1 hour at 25° C. with 0.8 uL compound/DMSO dotted in black 384 well polystyrene plates. Reactions were started by addition of 10 uL of assay buffer containing 0.4 uM Biotin-labeled Histone H3 peptide substrate: ART-K(Me1)-QTARKSTGGKAPRKQLA-GGK(Biotin) SEQ ID NO:1 (AnaSpec 64355) and incubated for 1 hour at 25° C. Reactions were stopped by addition of 10 uL 1x LANCE Detection Buffer (PerkinElmer CR97-100) supplemented with 1.5 nM Eu-anti-unmodified H3K4 Antibody (PerkinElmer TRF0404), and 225 nM LANCE Ultra Streptavidin (PerkinElmer TRF102) along with 0.9 mM Tranylcypromine-HCl (Millipore 616431). After stopping the reactions plates were incubated for 30 minutes and read on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 508.59, ALogP 3.22, HBA 9, HBD 0, PSA 102.89, QED 0.41
Compound (SMILES): CCn1c(-c2cnc(C)nc2)nc2c(O[C@H]3CCN(C(=O)C4CC(C)C4)C3)ncnc21
InChIKey: PNGBSTRVQGLGNA-PGEKIEPBSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Biochemical Assay: The PI3-Kinase biochemical assay was optimized using the HTRF kit provided by Upstate (Millipore). The assay kit contains six reagents: 1) 4× Reaction Buffer; 2) native PIP2 (substrate); 3) Stop (EDTA); 4) Detection Mix A (Streptavidin-APC); 5) Detection Mix B (Eu-labeled Anti-GST plus GST-tagged PH-domain); 6) Detection Mix C. In addition, the following items were obtained or purchased; PI3Kinase (alpha 14-602, beta 14-603, gamma 14-558 and delta 14-604 from Upstate; Millipore), dithiothreitol (Sigma, D-5545), Adenosine-5′ triphosphate (InVitrogen, Cat#AS001A), native PIP3 (PI(3,4,5)P3, diC8, H+, CELLSIGNALS, INC. Cat #907) DMSO (Sigma, 472301).PI3Kinase Reaction Buffer is prepared by dilution the stock 1:4 with de-ionized water. DTT, PIP2 and Biotin-PIP3 were added to 1536 assay plate at a final concentration of 5 mM, 5 mM and 25 nM on the day of use. Enzyme addition and compound pre-incubation are initiated by the addition of 1.25 ul of PI3K (at twice its final concentration) in the 1× reaction buffer to all wells using a BioRaptor. Plates are incubated at room temperature for 15 minutes. Reactions are initiated by addition of 1.25 ul of 2× substrate solution (PIP2 and ATP in 1× reaction buffer) using BioRaptor. Plates are incubated in humidified chamber at room temperature for one hour. Reactions are quenched by addition of 0.625 uL of stop solution to all wells using the BioRaptor. The quenched reactions are then processed to detect product formation by adding 0.625 uL of Detection Solution to all wells using the BioRaptor (Detection mix C, Detection Mix A, and Detection Mix B combined together in an 18:1:1 ratio prepared 2 hours prior to use). Following a one hour incubation in the dark, the HTRF signal is measured on the Envision plate reader set for 330 nm excitation and dual emission detection at 620 nM (Eu) and 665 nM (APC).
Measurement: IC50 = 1.3 nM (pChEMBL 8.890000343322754)
Descriptors: MW 421.51, ALogP 2.64, HBA 8, HBD 0, PSA 98.92, QED 0.62
Compound (SMILES): O=C(Nc1cc(CN2CCCC2)ccc1Cl)c1n[nH]c2ccc(-c3cccnc3)cc12
InChIKey: CNTNWKGZRSIPET-UHFFFAOYSA-N
Target: Proto-oncogene Wnt-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P04628
Assay: Wnt activity assay: Reporter cell lines were generated by stably transducing cells of cancer cell lines (e.g., colon cancer) with a lentiviral construct that include a Wnt-responsive promoter driving expression of the firefly luciferase gene.Lentiviral constructs were made in which the SP5 promoter, a promoter having eight TCF/LEF binding sites derived from the SP5 promoter, was linked upstream of the firefly luciferase gene. The lentiviral constructs included a hygromycin resistance gene as a selectable marker. The SP5 promoter construct was used to transduce SW480 cells, a colon cancer cell line having a mutated APC gene that generates a truncated APC protein, leading to de-regulated accumulation of β-catenin. A control cell line was generated using another lentiviral construct containing the luciferase gene under the control of the SV40 promoter which does not require β-catenin for activation.Cultured SW480 cells bearing a reporter construct were distributed at approximately 10,000 cells per well into 384 well multiwell plates. Compounds were then added to the wells in half-log dilutions using a three micromolar top concentration. A series of control wells for each cell type received only buffer and compound solvent. Twenty-four hours after the addition of compound, reporter activity for luciferases was assayed, for example, by addition of the BrightGlo luminescence reagent (Promega) and the Victor3 plate reader (Perkin Elmer). Readings were normalized to DMSO only treated cells, and normalized activities were then used for the IC50 calculations.
Measurement: IC50 = 5100 nM (pChEMBL 5.289999961853027)
Descriptors: MW 431.93, ALogP 5.13, HBA 4, HBD 2, PSA 73.91, QED 0.46
Compound (SMILES): COc1ncc(-c2nc(N3CCOCC3)nc3c(CN4CCn5ccnc5C4)c(C)sc23)cc1NS(C)(=O)=O
InChIKey: POZFIHGZHIHLMO-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Enzyme Activity Inhibition Assay: 1. Buffer preparation: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS.2. Compound was formulated in 100% DMSO in a concentration gradient, and added to a 384-well plate to a final DMSO concentration of 1%.3. PI3Kα, PI3Kδ, PI3Kβ and PI3Kγ enzymes were diluted to an optimum concentration with the following buffer: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS, 2 mM DTT, and transferred to a 384-well plate and incubated with the compound for a certain time.4. Substrate was diluted to an optimum concentration with the following buffer: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS, 2 mM DTT, 50 μM PIP2, 25 μM ATP; then added to a 384-well plate to initiate the reaction, and for PI3Kα, PI3Kβ and PI3Kγ at room temperature reacted for 1 hr, for PI3Kδ at room temperature reacted for 2 hrs. For PI3Kβ and PI3Kγ, it was still necessary to further add 10 μL ADP-Glo Detection Reagent, and then equilibrated at room temperature for 30 minutes.
Measurement: IC50 = 3.4 nM (pChEMBL 8.470000267028809)
Descriptors: MW 570.7, ALogP 2.49, HBA 12, HBD 1, PSA 127.6, QED 0.35
Compound (SMILES): CC(F)(F)c1cc(-n2cc(COc3nccc(CO)n3)nn2)ccc1Cl
InChIKey: ZTLFVMSXLOLVQF-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 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 381.77, ALogP 2.89, HBA 7, HBD 1, PSA 85.95, QED 0.71
Compound (SMILES): COc1cccc(OC)c1-n1c(NS(=O)(=O)[C@@H]2CO[C@@H](O)[C@H](O)C2)nnc1-c1ccc(C)o1
InChIKey: MVPMYGOPOBZUPO-XEKQOVJPSA-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 = 180 nM (pChEMBL 6.75)
Descriptors: MW 480.5, ALogP 1.06, HBA 11, HBD 3, PSA 158.17, QED 0.45
Compound (SMILES): CC(C)S(=O)(=O)c1ccc(-c2cnc(N)c(-c3nnc(NC(=O)[C@H]4N[C@H]5CCC4C5)o3)n2)cc1
InChIKey: SUODGKPOQXPOKM-HUUYTVAOSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Inhibition Assay: Compounds were screened for their ability to inhibit ATR kinase using a radioactive-phosphate incorporation assay. Assays were carried out in a mixture of 50 mM Tris/HCl (pH 7.5), 10 mM MgCl2 and 1 mM DTT. Final substrate concentrations were 10 μM [γ-33P]ATP (3 mCi 33P ATP/mmol ATP, Perkin Elmer) and 800 μM target peptide (ASELPASQPQPFSAKKK) (SEQ ID NO:1)).Assays were carried out at 25° C. in the presence of 5 nM full-length ATR. An assay stock buffer solution was prepared containing all of the reagents listed above, with the exception of ATP and the test compound of interest. 13.5 μL of the stock solution was placed in a 96 well plate followed by addition of 2 μL of DMSO stock containing serial dilutions of the test compound (typically starting from a final concentration of 15 μM with 3-fold serial dilutions) in duplicate (final DMSO concentration 7%). The plate was pre-incubated for 10 minutes at 25° C. and the reaction initiated by addition of 15 μL [γ-33P]ATP (final concentration 10 μM).
Measurement: Ki = 2550 nM (pChEMBL 5.590000152587891)
Descriptors: MW 483.55, ALogP 2.04, HBA 10, HBD 3, PSA 165.99, QED 0.47
Compound (SMILES): CC1(C)OCCN(C2CCN(c3nnc(N)[nH]3)CC2)[C@H]1Cc1ccc(Cl)cc1
InChIKey: HXKIZRXXUTUDQE-KRWDZBQOSA-N
Target: Acidic mammalian chitinase (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BZP6
Assay: Human AMCase Activity Assay: An enzymatic assay with recombinant human AMCase was used in order to establish inhibitory activity of the compounds (Boot et al., 2001, JBC: 276). The assay was run in the 96-well plate format, each reaction in the total volume of 100 μL. 4-Methylumbelliferyl B-D-N,N′-diacetylchitobioside hydrate was used as a substrate for the enzyme. Upon hydrolysis by AMCase, the substrate releases 4-methylumbelliferyl (4MU) that, when ionized in basic pH, emits fluorescence at 460 nm.Briefly, 40 μL of a substrate was added to each well, followed by 10 μL of compound dilution and 50 μL of hAMCase recombinant enzyme solution. The reaction was carried out in citrate buffer, pH 5.2, in the dark, at 37° C. for 60 minutes with shaking. After that time the reaction was stopped by adding 195 μL of Stop Buffer (pH 10.5) to each well. The fluorescence of the reaction product was measured in Perkin Elmer Envision fluorescent plate reader at an excitation wavelength of 355 nm. The IC50 values were calculated using GraphPad Prism.
Measurement: IC50 = 55000 nM (pChEMBL 4.260000228881836)
Descriptors: MW 404.95, ALogP 2.73, HBA 6, HBD 2, PSA 83.3, QED 0.81
Compound (SMILES): CCn1nc(C(=O)NC[C@]2(O)CC[C@@H](S(C)(=O)=O)CC2)c(Cl)c1-c1ccc([C@H](O)C(C)(C)C(F)(F)F)cc1OC(F)F
InChIKey: KVSKGBKUSMIDKV-HHCYOLOASA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor Assay: ThermoFluor® experiments were carried out using instruments owned by Janssen Research and Development, L.L.C. through its acquisition of 3-Dimensional Pharmaceuticals, Inc. 1,8-ANS (Invitrogen) was used as a fluorescent dye. Protein and compound solutions are dispensed into black 384-well polypropylene PCR microplates (Abgene) and overlayed with silicone oil (1 μL, Fluka, type DC 200) to prevent evaporation. Bar-coded assay plates are robotically loaded onto a thermostatically controlled PCR-type thermal block and then heated at a typical ramp-rate of 1° C./min for all experiments. Fluorescence was measured by continuous illumination with UV light (Hamamatsu LC6) supplied via fiber optic and filtered through a band-pass filter (380-400 nm; >6 OD cutoff). Fluorescence emission of the entire 384-well plate was detected by measuring light intensity using a CCD camera (Sensys, Roper Scientific) filtered to detect 500±25 nm, resulting in simultaneous and independent readings of all 384 wells. Images were collected at each temperature, and the sum of the pixel intensity in a given area of the assay plate was recorded versus temperature.
Measurement: Kd = 5.3 nM (pChEMBL 8.279999732971191)
Descriptors: MW 646.08, ALogP 4.89, HBA 8, HBD 3, PSA 130.75, QED 0.31
Compound (SMILES): Cc1ncccc1[C@H](Nc1cc(Cl)c2ncc(C#N)c(N[C@H](C)c3ccccc3)c2c1)C1=CN(C(C)C)NN1
InChIKey: JZXWUVIUXPGONS-QTEAWJPNSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: High Throughput Biochemical Assay: EGFR activity was measured using KinEASE (Cisbio), a time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay. In this assay, EGFR-catalyzes the phosphorylation of a universal Tyrosine kinase peptide substrate labeled with XL665. Europium conjugated phosphor-tyrosine specific antibody binds the resulting phosphorylated peptide. Formation of phosphorylated peptide is quantified by TR-FRET with Europium as the donor and XL665 the acceptor. The assay was performed in two main steps. The first step is the kinase reaction step and the second step is the detection step with TR-FRET reagents. In brief, test compounds 1:3 serially diluted in DMSO were delivered into Corning white, low volume, non-binding 384 well plates using the Echo 550 acoustic liquid dispenser (Labcyte®). EGFR enzyme (Human EGFR, cytoplasmic domain [669-1210] from Carna Biosciences Cat. No. 08-115) and substrates TK substrate-biotin (included in Cisbio HTRF KinEASE-TK kit Cat. No. 62TK0PEJ) were dispensed into assay plates using a Multi-Flo (Bio-Tek Instruments). The standard 10 μL reaction mixture contained 6 μM ATP (1×Km) or 12 μM ATP (2×Km), 1 μM biotinylated peptide, 0.3 nM EGFR (for 1×Km ATP) or 0.1 nM EGFR (for 2×Km ATP) in reaction buffer (10 mM MOPs, pH 7.0, 1.5% Glycerol, 0.5 mg/ml BSA, 10 mM Mg-Acetate, 1 mM DTT, 0.025% NP-40). After 60 min of incubation at room temperature, 10 μL of Stop and Detect Solution (1:400 Europium Cryptate labeled anti-phosphorylated peptide antibody solution and 125 nM strepavidin-XL665 Tracer in a 50 mM Hepes pH 7.0 detection buffer containing sufficient EDTA) was added. The plate was then further incubated for over 60 minutes at room temperature and read using an Envision 2103 Multilabeled reader (PerkinElmer) with excitation/emission/FRET emission at 340 nm/615 nm/665 nm, respectively.
Measurement: IC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 539.09, ALogP 6.36, HBA 8, HBD 4, PSA 100.93, QED 0.21
Compound (SMILES): N#Cc1ccc(-c2cc(CNC3COC3)cc3ncnn23)cc1
InChIKey: RPHRBKTVHVYIHZ-UHFFFAOYSA-N
Target: Prolyl hydroxylase EGLN2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96KS0
Assay: PHD1 Enzyme Assay: The IC50 values for the PHD1 enzyme (residues 1-407) were determined by mixing increasing amounts of a compound of the invention with a fixed amount of the enzyme (20 nM final concentration) and peptide substrate (Asp-Leu-Asp-Leu-Glu-Ala-Leu-Ala-Pro-Tyr-Ile-Pro-Ala-Asp-Asp-Asp-Phe-Gln-Leu, 1 μM final concentration) and 2-oxoglutarate (0.5 M final concentration) in an assay buffer comprising 30 mM 2-(N-morpholino)ethanesulfonic acid pH 6.0, 2 mM sodium ascorbate, 100 μM dithiothreitol, 2 mg/ml bovine serum albumin, 60 μg/ml catalase enzyme and 1 μM iron (II) sulphate (FeSO4). The reaction was conducted by pre-incubating the PHD1 enzyme in the presence of a compound of the invention for 60 minutes at room temperature. The activity of the free enzyme was measured by adding the peptide, the 2-oxoglutarate and sodium ascorbate (see above for final concentrations). The assay was quenched by the addition of 30% v/v trichloroacetic acid (final concentration 5%). The amount of product released was measured using a UPLC-MS (Agilent 1290 with an ABSciex 4000qTrap Mass Spectrometer). Data were analysed using the classical isotherm equation for the determination of IC50.
Measurement: IC50 = 3794 nM (pChEMBL 5.420000076293945)
Descriptors: MW 305.34, ALogP 1.76, HBA 6, HBD 1, PSA 75.24, QED 0.79
Compound (SMILES): Nc1ncnc2c1c(Cc1cccc(Cl)c1)nn2C1CC1
InChIKey: KAVMTMRPCDBIFF-UHFFFAOYSA-N
Target: Calmodulin-domain protein kinase 1 (SINGLE PROTEIN), Toxoplasma gondii
UniProt: Q9BJF5
Assay: Inhibition Assay: Certain of the compounds prepared as described above were assayed to determine their IC50 for inhibition of T. gondii CDPK1 (tgCDPK1). At least three independent replicates of the assay were conducted for each compound tested.
Measurement: IC50 = 17.2 nM (pChEMBL 7.760000228881836)
Descriptors: MW 299.77, ALogP 2.99, HBA 5, HBD 1, PSA 69.62, QED 0.81
Compound (SMILES): N#Cc1ccnc(N2CC[C@@H]3CCN(C(=O)c4ccccc4-n4ncnn4)C[C@@H]32)c1
InChIKey: VTNUMMZMVJCUET-LPHOPBHVSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: FLIPR Assay: The utility of the compounds in accordance with the present invention as orexin receptor OX1R and/or OX2R antagonists may be readily determined without undue experimentation by methodology well known in the art, including the FLIPR Ca2+ Flux Assay. In a typical experiment the OX1 and OX2 receptor antagonistic activity of the compounds of the present invention was determined in accordance with the following experimental method. For intracellular calcium measurements, Chinese hamster ovary (CHO) cells expressing the rat orexin-1 receptor or the human orexin-2 receptor, are grown in Iscove's modified DMEM containing 2 mM L-glutamine, 0.5 g/ml G418, 1% hypoxanthine-thymidine supplement, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat-inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into Becton-Dickinson black 384-well clear bottom sterile plates coated with poly-D-lysine. All reagents were from GIBCO-Invitrogen Corp. The seeded plates are incubated overnight at 37° C. and 5% CO2. Ala-6,12 human orexin-A as the agonist is prepared as a 1 mM stock solution in 1% bovine serum albumin (BSA) and diluted in assay buffer (HBSS containing 20 mM HEPES, 0.1% BSA and 2.5 mM probenecid, pH7.4) for use in the assay at a final concentration of 70 pM. Test compounds are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates, first in DMSO, then assay buffer. On the day of the assay, cells are washed 3 times with 100 μl assay buffer and then incubated for 60 min (37° C., 5% CO2) in 60 μl assay buffer containing 1 μM Fluo-4AM ester, 0.02% pluronic acid, and 1% BSA. The dye loading solution is then aspirated and cells are washed 3 times with 100 μl assay buffer. 30 μl of that same buffer is left in each well. Within the Fluorescent Imaging Plate Reader (FLIPR, Molecular Devices), test compounds are added to the plate in a volume of 25 μl, incubated for 5 min and finally 25 μl of agonist is added. Fluorescence is measured for each well at 1 second intervals for 5 minutes and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 70 pM Ala-6,12 orexin-A with buffer in place of antagonist. For each antagonist, IC50 value (the concentration of compound needed to inhibit 50% of the agonist response) is determined.
Measurement: IC50 = 110 nM (pChEMBL 6.960000038146973)
Descriptors: MW 400.45, ALogP 1.67, HBA 8, HBD 0, PSA 103.83, QED 0.66
Compound (SMILES): CC(C)(NC(=O)c1ccc2c(c1)OCc1cnccc1-2)c1ccccc1
InChIKey: IZQXGOOSNRXSNM-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50; i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 = 54.5 nM (pChEMBL 7.260000228881836)
Descriptors: MW 344.41, ALogP 4.31, HBA 3, HBD 1, PSA 51.22, QED 0.77