lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CNCCOc1ccc(/C(=C(/CCCO)c2ccccc2)c2ccc(O)cc2)cc1.Cl
InChIKey: IHDFRYVHRNBUGT-OQKDUQJOSA-N
Target: Steroid hormone receptor ERR2 (SINGLE PROTEIN), Homo sapiens
UniProt: O95718
Assay: Inhibition of fluorescien-conjugated coactivator PGC1a binding to ERRbeta (unknown origin) after 1 hr by TR-FRET assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 403.52, ALogP 4.72, HBA 4, HBD 3, PSA 61.72, QED 0.34
Compound (SMILES): C[C@H](Nc1nc(N)nc(N)c1Cl)c1nc2c(Cl)cc(F)cc2c(=O)n1-c1cnc(N)cn1
InChIKey: HHITXZQNEBTKLH-LURJTMIESA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The activities were measured using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. TR-FRET monitored the formation of 3,4,5-inositol triphosphate molecule that competed with fluorescently labeled PIP3 for binding to the GRP-1 pleckstrin homology domain protein. An increase in phosphatidylinositide 3-phosphate product resulted in a decrease in TR-FRET signal as the labeled fluorophore was displaced from the GRP-1 protein binding site. Class I PI3K isoforms were expressed and purified as heterodimeric recombinant proteins. All assay reagents and buffers for the TR-FRET assay were purchased from Millipore. PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2xKm ATP (75-500 uM), 2 uM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, and 1% (v/v) DMSO at the following concentrations for each isoform: PI3Kα, PI3Kβ, and PI3Kδ between 25 and 50 uM, and PI3Kγ at 2 nM. The compounds of Table 1 and Compound X ((S)-2,4-diamino-6-((1-(5-chloro-4-oxo-3-(pyridin-3-yl)-3,4-dihydroquinazolin-2-yl)ethyl)amino)pyrimidine-5-carbonitrile) and Compound Y ((S)-2,4-diamino-6-((1-(5-chloro-4-oxo-3-(pyridin-3-yl)-3,4-dihydroquinazolin-2-yl)ethyl)amino)pyrimidine-5-carbonitrile) were added to the assay solution and incubated for 30 minutes at 25° C. The reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 us delay and 500 us read window). The results were normalized based on positive (1 uM wortmanin) and negative (DMSO) controls, and the IC50 values for PI3K α, β, δ and γ were calculated from the fit of the dose-response curves to a four-parameter equation.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 477.29, ALogP 2.33, HBA 11, HBD 4, PSA 176.54, QED 0.34
Compound (SMILES): CCOc1cccc(-c2c[nH]c3ccc(-c4nnc(NC)s4)cc23)n1
InChIKey: YNQGEMKAJKCOIP-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Enzyme Assay: The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction.Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 uM. A reference compound was included on each assay plate in order to validate that plate; on
Measurement: IC50 = 7.131 nM (pChEMBL 8.149999618530273)
Descriptors: MW 351.44, ALogP 4.19, HBA 6, HBD 2, PSA 75.72, QED 0.56
Compound (SMILES): CN(C)Cc1ccc(CNC(=O)CCCCCN2CCN(c3ccccc3-c3ccccc3)CC2)cc1
InChIKey: UUWNDYITXWRHHK-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 6 (SINGLE PROTEIN), Homo sapiens
UniProt: P50406
Assay: Displacement of [3H]-LSD from human 5-HT6 receptor expressed in HEK293 cell membranes after 1 hr by microbeta counting analysis
Measurement: Ki = 1506 nM (pChEMBL 5.820000171661377)
Descriptors: MW 498.72, ALogP 5.41, HBA 4, HBD 1, PSA 38.82, QED 0.34
Compound (SMILES): COc1ccc(CNC(=O)COc2ccc3cc(C(O)(C(F)(F)F)C(F)(F)F)c(O)cc3c2)cc1OC
InChIKey: CFKVLXGECXVRLD-UHFFFAOYSA-N
Target: Receptor-type tyrosine-protein phosphatase C (SINGLE PROTEIN), Homo sapiens
UniProt: P08575
Assay: Inhibition of recombinant CD45 (unknown origin) using pNPP as substrate by spectrophotometric method
Measurement: IC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 533.42, ALogP 4.57, HBA 6, HBD 3, PSA 97.25, QED 0.37
Compound (SMILES): NS(=O)(=O)NCC1CCC(CNc2ncnc3c2nc(-c2ccccc2Cl)n3-c2ccc(Cl)cc2)CC1
InChIKey: FSGQETVZTNOSAH-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: Radioligand Displacement Assay (CP55940): Further characterization of select compounds was performed using radioligand displacement of [3H]1 and equilibrium dissociation constant (Ki) values were determined. Selectivity of these compounds at CB1 versus CB2 was also determined by obtaining Ki values at either receptor using displacement of [3H]CP55940 in membranes of CHO-K1 cells over-expressing either receptor. Data reported are average values from 3-6 measurements.
Measurement: Ki = 10719 nM (pChEMBL 4.96999979019165)
Descriptors: MW 560.51, ALogP 4.8, HBA 7, HBD 3, PSA 127.82, QED 0.28
Compound (SMILES): COc1nc2ccc(C(O)(c3ccc(C(F)(F)F)nc3)c3cncn3C)cc2c(Cl)c1Cc1ccc(-n2cccn2)cc1
InChIKey: LZOGZAYEBDXEJA-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor® Assay: 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. Reference wells contained RORγt without compounds, and the assay conditions were as follows: 0.065 mg/mL RORγt, 60 μM 1,8-ANS, 100 mM Hepes, pH 7.0, 10 mM NaCl, 2.5 mM GSH, 0.002% Tween-20. Project compounds were arranged in a pre-dosed mother plate (Greiner Bio-one) wherein compounds are serially diluted in 100% DMSO by 1:2 from a high concentration of 10 mM over 12 columns within a series (column 12 is a reference well containing DMSO, no compound). The compounds were robotically dispensed directly into assay plates (1×=46 nL) using a Hummingbird capillary liquid handling instrument (Digilab). Following compound dispense, protein and dye in buffer was added to achieve the final assay volume of 3 μL, followed by 1 μL of silicone oil.
Measurement: Kd = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 605.02, ALogP 6.1, HBA 8, HBD 1, PSA 90.88, QED 0.24
Compound (SMILES): COc1cccc(F)c1C(=O)N1CCC(N2CC(CC#N)(n3cc(-c4ncnc5[nH]ccc45)cn3)C2)CC1
InChIKey: USZNWFJQMFPPHA-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
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) with an N-terminal His tag 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 microL 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 hr 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 Fusion plate reader (Perkin Elmer, Boston, Mass.).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 514.57, ALogP 3.2, HBA 8, HBD 1, PSA 115.96, QED 0.42
Compound (SMILES): O=C(c1ccccc1-n1nccn1)N1CCOC[C@H]1Cc1cc(-n2nccn2)ccc1F
InChIKey: HVGUQKUDHLWJBC-GOSISDBHSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: CYP3A4 Inhibition Assay: The CYP3A4 inhibition assay was performed using human liver microsomes and testosterone 6'-hydroxylation as a P450 isoform-specific marker. In a total volume of 150 uL, 14C-testosterone at a final concentration of 40 uM in a 100 mM phosphate buffer (pH 7.4) was incubated in a 96-well plate with 0.3 mg/mL of human liver microsomes in an Eppendorf thermomixer at 37° C. and 400 rpm. A 1.0 uL-aliquot of the 150-fold concentrated compound stock solution, prepared in DMSO, was added to yield final inhibitor concentrations of 0, 0.1, 0.5, 1.0, 5.0, 10, 25, and 50 uM. The reaction was initiated by addition of 15 uL of the NADPH-regenerating system containing the glucose-6-phosphate dehydrogenase and terminated after 7 min with a 75 uL-aliquot of methanol. After centrifugation at 465 g and 4° C. for 20 min, a 50 uL-aliquot of the supernatant was submitted to HPLC according to the method described below.
Measurement: IC50 = 20000 nM (pChEMBL 4.699999809265137)
Descriptors: MW 433.45, ALogP 2.07, HBA 8, HBD 0, PSA 90.96, QED 0.48
Compound (SMILES): N=C(NC(=N)N1CCN(C(=N)NC(=N)Nc2ccc(Cl)cc2Cl)CC1)Nc1ccc(Cl)cc1Cl
InChIKey: QOJPDZLAORGAEU-UHFFFAOYSA-N
Target: Cholinesterase (SINGLE PROTEIN), Equus caballus
UniProt: P81908
Assay: Competitive inhibition of equine serum butyrylcholinesterase in presence of varying butyrylthiocholine iodide substrate level preincubated for 20 mins followed by substrate addition measured every 1 min for 30 mins
Measurement: Ki = 410 nM (pChEMBL 6.389999866485596)
Descriptors: MW 544.28, ALogP 4.36, HBA 4, HBD 8, PSA 150, QED 0.21
Compound (SMILES): FC(F)(F)c1ccc(CN2CCN(c3cc(N4CCCC4)nc(N4CCCC4)n3)CC2)cc1
InChIKey: UTNIELHSQXTLFN-UHFFFAOYSA-N
Target: Caspase-4 (SINGLE PROTEIN), Homo sapiens
UniProt: P49662
Assay: Inhibition of human recombinant N-terminal His-tagged DTT-activated caspase-4 expressed in Escherichia coli using Ac-LEVD-AMC as substrate preincubated for 30 mins followed by substrate addition by fluorescence assay
Measurement: IC50 = 1520 nM (pChEMBL 5.820000171661377)
Descriptors: MW 460.55, ALogP 4.02, HBA 6, HBD 0, PSA 38.74, QED 0.67
Compound (SMILES): CN(C)c1ccc(C(=O)Nc2ccc(-c3n[nH]c(=O)c4ccccc34)cc2)cc1
InChIKey: PDOWPLWTKJLFMU-UHFFFAOYSA-N
Target: Rho-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13464
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 < 100 nM (pChEMBL 7)
Descriptors: MW 384.44, ALogP 3.91, HBA 4, HBD 2, PSA 78.09, QED 0.56
Compound (SMILES): Cc1ccc(Cl)cc1COc1ccc(S(=O)(=O)N2C[C@H](O)CC(C)(C)[C@@H]2C(=O)NO)cc1
InChIKey: WYKNWJFOHUYOQU-XLIONFOSSA-N
Target: Disintegrin and metalloproteinase domain-containing protein 17 (SINGLE PROTEIN), Homo sapiens
UniProt: P78536
Assay: Inhibition of TACE in human mononuclear cells assessed as inhibition of LPS-induced TNF-alpha production measured after 4 hrs in presence of LPS by ELISA
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 482.99, ALogP 2.88, HBA 6, HBD 3, PSA 116.17, QED 0.43
Compound (SMILES): c1cc(N2CCCNCC2)nc(-n2ncc3cnc(-c4cn[nH]c4)cc32)c1
InChIKey: CYOQMLSODZDBGH-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 0.086 nM (pChEMBL 10.069999694824219)
Descriptors: MW 360.43, ALogP 2.01, HBA 7, HBD 2, PSA 87.55, QED 0.58
Compound (SMILES): COc1cc(OC)cc(N(CCCNCC(F)(F)F)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: IQAIXSLQDLQWKO-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 >= 10000 nM (pChEMBL 5)
Descriptors: MW 500.53, ALogP 4.73, HBA 8, HBD 1, PSA 77.33, QED 0.32
Compound (SMILES): CNS(=O)(=O)c1ccc(OC/C(=C/F)CN)cc1
InChIKey: RVCAAPOJOTYELI-RMKNXTFCSA-N
Target: Amine oxidase [copper-containing] 3 (SINGLE PROTEIN), Rattus norvegicus
UniProt: O08590
Assay: Recombinant SSAO/VAP-1 Inhibition Assay in Rat Fat: Abdominal fat from BALB/c mice, Wistar or Sprague Dawley rats, which are tissues enriched with SSAO/VAP-1-were surgically removed. For every gram of animal abdominal fat tissue, 1 mL of 0.1 M NaPO4 buffer (pH 7.4) was added. Tissues were homogenized using IKA Ultra-Turrax T 10 homogenizer for 3 min, homogenate was centrifuged for 15 min at 3000×g. The middle layer (clear supernatant) was removed without disturbing the top layer (high fat content) or the debris on the bottom of the tube. SSAO/VAP-1 activity was determined by checking the fluorescent signal. Km/Vmax values were determined and the fat homogenate was aliquoted and stored at −80 °C until assays were performed. Assay was performed in a similar fashion as for human SSAO/VAP-1 (Example 5) except, the substrate (benzylamine) concentrations used for mouse fat homogenate and rat fat homogenate were 80 μM and 30 μM respectively: Briefly, in a standard 96 well plate assay 50 μL of purified human SSAO/VAP-1 (0.25 μg/mL) in 0.1 M NaPO4 buffer (pH 7.4) was added into each well. Test compounds were dissolved in DMSO and tested in a Concentration Response Curve (CRC) with 4-9 data points, typically in the micromolar or nanomolar range after incubation with human SSAO/VAP-1 for 30 min at 37 °C. After 30 min incubation, 50 μL of the reaction mixture containing 600 μM benzylamine (Sigma Aldrich), 120 μM Amplex Red (Sigma Aldrich) and 1.5 U/mL horseradish peroxidase (Sigma Aldrich) prepared in 0.1 M NaPO4 buffer (pH 7.4) were added to the corresponding well. The fluorescence unit (RFU) was read every 2.5 min for 30 min at 37 °C. excitation 565 nm and emission 590 (Optima; BMG labtech). The slope of the kinetics for each well was calculated using MARS data analysis software (BMG labtech) and this value was used to deduce the IC50 value (Dotmatics).
Measurement: IC50 < 100000 nM (pChEMBL 4)
Descriptors: MW 274.32, ALogP 0.79, HBA 4, HBD 2, PSA 81.42, QED 0.8
Compound (SMILES): COc1cc(OC)cc(N(CCCN2CCCC2(C)C)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1.Cl
InChIKey: MXVLJTMBTSLDPM-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 1513.56 nM (pChEMBL 5.820000171661377)
Descriptors: MW 500.65, ALogP 5.45, HBA 8, HBD 0, PSA 68.54, QED 0.3
Compound (SMILES): NS(=O)(=O)c1ccc(SCc2ccccc2-c2ccccc2)s1
InChIKey: HUPWCJRQUJXEQL-UHFFFAOYSA-N
Target: Carbonic anhydrase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00915
Assay: Inhibition of human recombinant cytosolic carbonic anhydrase 1 preincubated for 10 mins prior to testing by stopped-flow CO2 hydration assay
Measurement: Ki = 3600 nM (pChEMBL 5.440000057220459)
Descriptors: MW 361.51, ALogP 4.35, HBA 4, HBD 1, PSA 60.16, QED 0.69
Compound (SMILES): CN1CCC(Oc2ccc3c(c2)CN(C(=O)Nc2ccc(-c4n[nH]c(=O)c5ccccc45)cc2)C3)CC1
InChIKey: SECVORPAKLVROH-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: ROCK2 Kinase Activity Assays: HTRF assay: This assay used the CisBio HTRF KinEASE kit (kit 62ST2PEZ) and the kinase reaction containing 0.2 uM biotinylated substrate peptide (S2, CisBio), 5 uM, 100 uM or 1 mM of ATP, inhibitors (0.1-10,000 nM in 2% DMSO) and enzymes as in the 33P-ATP assay below. [33P]-ATP assay: ROCK2 activity was initially determined using a radioactive FlashPlate-based assay. In a 96-well format, biotinylated peptide substrates (2 mM final; S6-short for ROCK2), γ-[33P]-ATP (5 uM, 2 mCi/mmol), compounds (0.1-10000 nM in 2% DMSO), and ROCK2 (0.2 nM; Upstate) catalytic domains.
Measurement: Ki = 32.1 nM (pChEMBL 7.489999771118164)
Descriptors: MW 495.58, ALogP 4.61, HBA 5, HBD 2, PSA 90.56, QED 0.43
Compound (SMILES): CCC(=O)Nc1cc2c(Nc3ccc4c(ccn4Cc4ccccn4)c3)ncnc2cc1OC
InChIKey: YLFINAUVWATNCS-UHFFFAOYSA-N
Target: Receptor tyrosine-protein kinase erbB-4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15303
Assay: Irreversible inhibition of GST-tagged ERBB4 (unknown origin) (Gly-259 to Gly-690 residues) expressed in baculovirus infected Sf9 insect cells assessed as reduction in Glu/Tyr copolymer phosphorylation after 6 mins by ELISA
Measurement: IC50 = 95 nM (pChEMBL 7.019999980926514)
Descriptors: MW 452.52, ALogP 5.13, HBA 7, HBD 2, PSA 93.96, QED 0.36
Compound (SMILES): CO[C@H]1C[C@H](C(=O)Nc2ccc(-n3ccccc3=O)cc2F)C[C@@H]1NC(=O)c1ccc(Cl)s1
InChIKey: WTMBDSYSYGKVRA-BVGQSLNGSA-N
Target: Coagulation factor X (SINGLE PROTEIN), Homo sapiens
UniProt: P00742
Assay: Biochemical assay using fluorogenic peptide substrate.Step 1: Compound transfer to plate (50 nl).Step 2: Dilute FXa in Assay Buffer.Step 3: Dilute substrate in Assay Buffer.Step 4: Add equal volumes to Assay plate (total assay volume 10 uL).Step 5: Centrifuge plate, 1 min, 1000 rpm.Step 6: Detect fluorescence signal: ex: 320 nm; Em. 405 nm (EnVision), every 10 min. 0 min pre-incubation of enzyme with compound and 45 min reaction time after substrate addition.
Measurement: IC50 = 2.13 nM (pChEMBL 8.670000076293945)
Descriptors: MW 489.96, ALogP 3.85, HBA 6, HBD 2, PSA 89.43, QED 0.55
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3)ncnc2cc1OCCN1CCOCC1
InChIKey: FAJVOBZBVGQKFD-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human N-terminal GST His6-tagged thrombin cleavage site-fused EGFR (H672 to A1210 residues) expressed in baculovirus using poly-EY as substrate after 210 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 449.34, ALogP 4.4, HBA 7, HBD 1, PSA 68.74, QED 0.57
Compound (SMILES): C[C@H](NC(=O)C(=O)NC12CC3CC(CC(C3)C1)C2)C(=O)N[C@@H](CC(=O)O)C(=O)COC(=O)c1c(Cl)cccc1Cl
InChIKey: SLTSJDMMDKNHMN-CQHHIRSDSA-N
Target: Caspase-6 (SINGLE PROTEIN), Homo sapiens
UniProt: P55212
Assay: Inhibition of recombinant MCH2 (unknown origin) pre-incubated for 60 mins with Acetyl-Tyr-Val-Ala-Asp-amino-4-methylcoumarin substrate followed by enzyme addition by fluorescence based assay
Measurement: Ki = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 596.46, ALogP 2.27, HBA 7, HBD 4, PSA 167.97, QED 0.24
Compound (SMILES): O=C(CCCCNC(=O)c1nc(-c2nc(-c3ccccc3)cs2)sc1C1CC1)NO
InChIKey: TWIIELMPEVJFGQ-UHFFFAOYSA-N
Target: Protein deacetylase HDAC6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBN7
Assay: Enzyme Assay: Enzyme activity of HDAC1,3 is determined using the substrate Ac-Lys-Tyr-Lys(Ac)-AMC while the enzyme activity of HDAC6 is assayed using the substrate Boc-lys(Ac)-AMC. The reaction is carried out in flat-bottom, 96-well, or 384-well microplates. After the substrate was deacetylated by HDACs, the product AMC which was obtained from hydrolysis by trypsin can generate fluorescence signal. Measurement is taken in using an multilabe plate reader a 355 nm excitation filter and a 460 nm emission filter. The initial rate of fluorescence should accurately reflect the rate of product formation and enzyme activity. Sample processing: The sample was dissolved in DMSO and kept at a low temperature. DMSO in the final system is limited under a low concentration which won't affect the enzyme activity.
Measurement: IC50 = 185 nM (pChEMBL 6.730000019073486)
Descriptors: MW 442.57, ALogP 4.22, HBA 7, HBD 3, PSA 104.21, QED 0.26
Compound (SMILES): CN(CC(F)(F)F)C[C@H]1CC[C@H](N2CN(CC#N)C(=O)c3cnc4[nH]ccc4c32)CC1
InChIKey: UQXBHUJREXEPGF-SHTZXODSSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Assay: The inhibitory activity of the compounds of the present invention against JAK was measured.The respective enzymes (JAK1, JAK2, JAK3 and Tyk2) were purchased from Carna Biosciences, Inc.As a substrate of the enzymes (hereinafter referred to as substrate), LANCE Ultra ULight-JAK-1 (Tyr1023) Peptide (manufactured by PerkinElmer Inc.) was used.As an antibody for detecting phosphorylation of the substrate, LANCE Ultra Europium-anti-phospho tyrosine antibody (PT66) (manufactured by PerkinElmer Inc.) was used.The other reagents were purchased from the following.Adenosine triphosphate (ATP): Sigma-Aldrich4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES): DOJINDO LABORATORIESGlycol ether diamine tetraacetic acid (EGTA): DOJINDO LABORATORIES Magnesium chloride (MgCl2): Wako Pure Chemical Industries, Ltd.Dithiothreitol (DTT): Wako Pure Chemical Industries, Ltd.Tween 20: Sigma-AldrichEthylenediaminetetraacetic acid (EDTA): DOJINDO LABORATORIESThe compounds of the present invention, the enzymes (JAK1, JAK2, JAK3 and Tyk2), the substrate and ATP were used for assay as diluted with assay buffer.Â  As the assay buffer, one having the following composition was used.Â  HEPES (pH7.5): 50 mM EGTA: 1 mM MgCl2: 10 mM DTT: 2 mM Tween 20: 0.01% (wt/wt). The dilute concentration and the addition amount on a well plate as described hereinafter were adjusted so that the following final concentrations were achieved on the well plate.
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 434.47, ALogP 3.36, HBA 5, HBD 1, PSA 79.26, QED 0.73
Compound (SMILES): Cn1cc(-c2cc3c(cn2)cnn3-c2cccc(N3CC(O)C3)n2)cn1
InChIKey: ANEQDQCTWAKZMN-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 1.78 nM (pChEMBL 8.75)
Descriptors: MW 347.38, ALogP 1.4, HBA 8, HBD 1, PSA 84.89, QED 0.6
Compound (SMILES): O=C(Nc1ccccc1)Nc1ccc(C#Cc2ccc3nccn3n2)cc1
InChIKey: LPGJBMXKZTXNOS-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: PDGFRbeta: Automated FLIPR (Fluorometric Imaging Plate Reader) technology was used to screen for inhibitors of VEGF induced increases in intracellular calcium levels in fluorescent dye loaded endothelial cells. HUVEC (human umbilical vein endothelial cells) (Clonetics) were seeded in 384-well fibronectin coated black-walled plates overnight at 37° C./5% CO2. Cells were loaded with calcium indicator Fluo-4 for 45 minutes at 37° C. Cells were washed 2 times (Elx405, Biotek Instruments) to remove extracellular dye. For screening, cells were pre-incubated with test agents for 30 minutes, at a single concentration (10 uM) or at concentrations ranging from 0.0001 to 10.0 uM followed by VEGF165 stimulation (10 ng/mL). Changes in fluorescence at 516 nm were measured simultaneously in all 384 wells using a cooled CCD camera. Data were generated by determining max-min fluorescence levels for unstimulated, stimulated, and drug treated samples.
Measurement: IC50 = 5428 nM (pChEMBL 5.260000228881836)
Descriptors: MW 353.38, ALogP 3.77, HBA 4, HBD 2, PSA 71.32, QED 0.54
Compound (SMILES): O=C(COc1cccc(NC2CCOCC2)c1)NCC(O)CN1CCc2ccccc2C1
InChIKey: OAMWDJZRBPSBOE-UHFFFAOYSA-N
Target: Protein arginine N-methyltransferase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: O14744
Assay: Biochemical Assay: Molecular Biology: Full-length human PRMT5 (NM_006109.3) transcript variant 1 clone was amplified from a fetal brain cDNA library, incorporating flanking 5′ sequence encoding a FLAG tag (MDYKDDDDK) (SEQ ID NO.:4) fused directly to Ala 2 of PRMT5. Full-length human MEP50 (NM_024102) clone was amplified from a human testis cDNA library incorporating a 5′ sequence encoding a 6-histidine tag (MHHHHHH) (SEQ ID NO.:5) fused directly to Arg 2 of MEP50. The amplified genes were sublconed into pENTR/D/TEV (Life Technologies) and subsequently transferred by Gateway attL×attR recombination to pDEST8 baculvirus expression vector (Life Technologies).Protein Expression. Recombinant baculovirus and Baculovirus-Infected Insect Cells (BIIC) were generated according to Bac-to-Bac kit instructions (Life Technologies) and Wasilko, 2006, respectively. Protein over-expression was accomplished by infecting exponentially growing Spodoptera frugiperda (SF9) cell culture at 1.2×106 cell/ml with a 5000 fold dilution of BIIC stock. Infections were carried out at 27° C. for 72 hours, harvested by centrifugation, and stored at −80° C. for purification.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 439.56, ALogP 2.19, HBA 6, HBD 3, PSA 83.06, QED 0.56
Compound (SMILES): O=C(/C=C/c1ccc(Cl)cc1)NC[C@H]1CCN(CC(c2ccccc2)c2ccccc2)C(=O)[C@@H](CCN2CCCCC2)N1
InChIKey: JOSSSQYONSYDQE-HBLINEBUSA-N
Target: Melanocortin receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P41968
Assay: Radioligand Binding Assay: Radioligand binding assays were carried out using commercial or in-house prepared hMC1R, hMC3R and hMC4R membranes and [125I] NDP-MSH, as per the hMC5R procedure in Example 102. In-house plasma membranes were prepared from transfected mammalian cells (prepared as in Example 109, using plasmid DNA containing the human MC1R, MC3R or MC4R gene or other gene of interest in a plasmid vector with a mammalian origin of replication): Adherent cells were washed with warm Hanks buffered saline solution (HBSS). 1 mL of cold HBSS was added to each flask and the cells were scraped off with a rubber policeman. The scraped cells were added to a 50 mL tube on ice. The plates were then rinsed twice with 5 mL cold HBSS and this was also added to the tube. The cells were centrifuged at 1000×g for 5 mins in a bench top centrifuge and the supernatant was decanted. The remaining cell pellet was resuspended in 0.25 M sucrose. The cell suspension was centrifuged again as previously and the pellet resuspended in 5 mL of 0.25 M sucrose containing protease inhibitors. The cells were homogenised by a 10 second pulse with an Ika disperser followed by 30 seconds on ice. The homogenisation and ice incubation was repeated three times. The mixture was then centrifuged at 1260×g for 5 mins. The supernatant was decanted into another centrifuge tube, to which a buffer containing 50 mM Tris, pH 7.4, 12.5 mM MgCl2, 5 mM EGTA and protease inhibitors was added to make the volume up to 30 mL. This was centrifuged at 30,000×g for 90 mins at 4° C. The resulting pellet was resuspended in 1 mL of the buffer above also containing 10% glycerol. Membranes were aliquoted into cryovials which were snap-frozen in a dry-ice/ethanol bath before being stored at −80° C. until required for use.
Measurement: IC50 = 1500 nM (pChEMBL 5.820000171661377)
Descriptors: MW 599.22, ALogP 5.74, HBA 4, HBD 2, PSA 64.68, QED 0.28
Compound (SMILES): COc1ncc(-c2ccc3nccc(-c4ccc(Cn5cncn5)cc4)c3c2)cc1NS(=O)(=O)c1ccc(F)cc1F
InChIKey: AQYTWWQAZAOONP-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42338
Assay: Inhibition of PI3Kbeta (unknown origin) using PIP2 as substrate after 1 hr in presence of ATP by ADP-glo based luminescence assay
Measurement: IC50 = 1.7 nM (pChEMBL 8.770000457763672)
Descriptors: MW 584.61, ALogP 5.69, HBA 8, HBD 1, PSA 111.89, QED 0.24
Compound (SMILES): Cc1c(-c2ncnc3[nH]c(-c4ccc(CN5CCN(C)CC5)cc4)cc23)cccc1N1CCOc2cc(C3CC3)ccc2C1=O
InChIKey: UNPRODJUZQZSMD-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Inhibition of recombinant human full length BTK preincubated for 10 mins followed by Tk-biotin peptide substrate addition after 90 mins by TR-FRET assay
Measurement: IC50 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 598.75, ALogP 6.26, HBA 6, HBD 1, PSA 77.59, QED 0.25
Compound (SMILES): CCCN1CCC(c2ccc(Nc3nccc(-c4c(-c5cccc(C(=O)Nc6c(F)cccc6F)c5)nc5ccccn45)n3)c(OCC)c2)CC1
InChIKey: OIEHKLZTMMAROD-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human full length EGFR expressed in baculovirus expression system assessed as decrease in transfer of gamma-phosphate from ATP onto tyrosine residue of biotinylated peptide substrate preincubated for 1 hr followed by substrate addition measured after 90 mins by HTRF method
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 687.79, ALogP 8.72, HBA 8, HBD 2, PSA 96.68, QED 0.14
Compound (SMILES): COc1cc(C(=O)N2CCOC[C@H]2Cc2cccc(-n3nccn3)c2)c(-n2nccn2)cc1F
InChIKey: ASCMSCIVNMZFPN-GOSISDBHSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: CYP3A4 Inhibition Assay: The CYP3A4 inhibition assay was performed using human liver microsomes and testosterone 6'-hydroxylation as a P450 isoform-specific marker. In a total volume of 150 uL, 14C-testosterone at a final concentration of 40 uM in a 100 mM phosphate buffer (pH 7.4) was incubated in a 96-well plate with 0.3 mg/mL of human liver microsomes in an Eppendorf thermomixer at 37° C. and 400 rpm. A 1.0 uL-aliquot of the 150-fold concentrated compound stock solution, prepared in DMSO, was added to yield final inhibitor concentrations of 0, 0.1, 0.5, 1.0, 5.0, 10, 25, and 50 uM. The reaction was initiated by addition of 15 uL of the NADPH-regenerating system containing the glucose-6-phosphate dehydrogenase and terminated after 7 min with a 75 uL-aliquot of methanol. After centrifugation at 465 g and 4° C. for 20 min, a 50 uL-aliquot of the supernatant was submitted to HPLC according to the method described below.
Measurement: IC50 = 22000 nM (pChEMBL 4.659999847412109)
Descriptors: MW 463.47, ALogP 2.08, HBA 9, HBD 0, PSA 100.19, QED 0.43
Compound (SMILES): O=C(O)CCCOc1ccc(-c2cc(OC(F)(F)F)ccc2Cl)cc1
InChIKey: FGXVJHYCASUBMZ-UHFFFAOYSA-N
Target: Free fatty acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14842
Assay: Agonist activity at human GPR40 measured after 60 mins by IP1-HTRF assay
Measurement: EC50 = 1042 nM (pChEMBL 5.980000019073486)
Descriptors: MW 374.74, ALogP 5.15, HBA 3, HBD 1, PSA 55.76, QED 0.68
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3)ncnc2cc1OCC1CN(C(=O)c2cccnc2Cl)CCO1
InChIKey: HJONXIJNTCIJTH-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human N-terminal GST His6-tagged thrombin cleavage site-fused EGFR (H672 to A1210 residues) expressed in baculovirus using poly-EY as substrate after 210 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 574.85, ALogP 5.66, HBA 8, HBD 1, PSA 98.7, QED 0.28
Compound (SMILES): C[C@@H](Nc1ncnc2[nH]c(-c3ccc(CNCc4ccc(C(=O)NO)cc4)cc3)cc12)c1ccccc1
InChIKey: ZMEJPUFHDFOMJY-LJQANCHMSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of N-terminal GST-His6-thrombin cleavage site-fused human VEGFR2 (Asp805 to Val1356 residues) expressed in baculovirus expression system using biotinylated gastrin precursor at Tyr87 as substrate preincubated for 5 mins followed by substrate addition measured after 30 mins by DELFIA method
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 492.58, ALogP 5.21, HBA 6, HBD 5, PSA 114.96, QED 0.14
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)OCC3CCCC3)CC2)C1
InChIKey: YVEHYEHIMAWATH-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: In vitro JAK Kinase Assay: The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag 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 microL 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 hr 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 Fusion plate reader (Perkin Elmer, Boston, Mass.)
Measurement: IC50 < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 488.6, ALogP 3.54, HBA 8, HBD 1, PSA 115.96, QED 0.56
Compound (SMILES): CN(C(=O)CC#N)C1CCCN(c2ncnc3[nH]ccc23)C1
InChIKey: JEYFUWJVRMLTRZ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Lck (SINGLE PROTEIN), Homo sapiens
UniProt: P06239
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human Lck using TK-substrate-biotin as substrate incubated for 60 mins in presence of ATP by fluorescence method
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 298.35, ALogP 1.3, HBA 5, HBD 1, PSA 88.91, QED 0.92
Compound (SMILES): CC(C)c1nn(C)c(C(C)C)c1NS(=O)(=O)c1ccc(-c2cccc(CN(C)C)c2)cc1
InChIKey: OOAAJIDBDSUGSI-UHFFFAOYSA-N
Target: Glycylpeptide N-tetradecanoyltransferase (SINGLE PROTEIN), Leishmania major
UniProt: Q4Q5S8
Assay: Enzyme Inhibition Assay: Measurements were performed using a modification of the scintillation proximity assay platform described previously by Georgopapadakou, N. H. et al. (22nd International Congress on Chemotherapy, 2001, Abstract P16.001). Compounds were solubilised in DMSO at a top concentration of 10 mM and serially diluted in half log steps to achieve a range of final assay concentrations of 100 uM to 1 nM. Compound at each concentration (100-fold final) was added to white 384 well plates in a volume of 0.5 ml. Human, A. fumigatus, T. brucei or L. major N-myristoyl transferase enzyme, dissolved to a working concentration of 10 nM in assay buffer (30 mM Tris/HCl pH 7.4, 0.5 mM EGTA, 0.5 mM EDTA, 1.25 mM DTT, 0.1% Triton X-100), was then added to columns 1 to 11 and 13 to 23 of the plates in a volume of 20 ml. To columns 12 and 24, 20 ml assay buffer was added to provide a no enzyme control. Following a 5 minute incubation at room temperature the substrates (GCGGSKVKPQPPQAK(Biotin)-Amide and myristoyl coenzyme A), dissolved in assay buffer, were added to all wells in a volume of 20 ml to start the reaction. The final concentrations of peptide and 3H-myristoyl coenzyme A were 0.5 mM and 125 nM respectively and the specific activity of the radiolabel was 8 Ci/mmol. Plates were then incubated at room temperature for up to 50 minutes (dependant upon the period of linearity for the different enzyme species) before SPA beads, suspended to 1 mg/ml in a stop solution (200 mM Phosphoric Acid/NaOH pH 4, 750 mM MgCl2), were added in a volume of 40 ml.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 454.64, ALogP 5.2, HBA 5, HBD 1, PSA 67.23, QED 0.51
Compound (SMILES): CC[C@H](Nc1ncnc(N)c1C#Cc1cnccn1)c1nc2cccc(C)c2c(=O)n1-c1cc(F)cc(F)c1
InChIKey: BRIIAMQHODWQFW-QFIPXVFZSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2xKm ATP (100-300 uM), 10 uM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 1% (v/v) DMSO at the following concentrations for each isoform: PI3K alpha, beta, delta, and gamma at 50 picomolar (pM) and PI3Kgamma at 2 nanomolar (nM). After an assay reaction time of 30 minutes at 25° C., reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 us delay and 500 us read window).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 524.54, ALogP 4.1, HBA 9, HBD 2, PSA 124.5, QED 0.33
Compound (SMILES): O=C1NN=C2CCCc3c2c2c1cccc2n3CCN1CCCCC1
InChIKey: KVSHPARHGQSONL-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: Inhibition of PARP-1 in human HeLa cells incubated for 18 hrs in presence of H2O2
Measurement: EC50 = 23 nM (pChEMBL 7.639999866485596)
Descriptors: MW 336.44, ALogP 2.91, HBA 4, HBD 1, PSA 49.63, QED 0.94
Compound (SMILES): CN(C)C/C=C/C(=O)Nc1cc2c(Nc3ccc(Cc4ccccn4)cc3)ncnc2cn1
InChIKey: JABKEJXCSLSSEV-FNORWQNLSA-N
Target: Receptor tyrosine-protein kinase erbB-4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15303
Assay: Irreversible inhibition of GST-tagged ERBB4 (unknown origin) (Gly-259 to Gly-690 residues) expressed in baculovirus infected Sf9 insect cells assessed as reduction in Glu/Tyr copolymer phosphorylation after 6 mins by ELISA
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 439.52, ALogP 3.81, HBA 7, HBD 2, PSA 95.93, QED 0.4
Compound (SMILES): CCc1ccccc1-n1c(Cn2nc(-c3cccc(O)c3)c3c(N)ncnc32)cc2cccc(C)c2c1=O
InChIKey: XBYYIIIYXKVBPU-UHFFFAOYSA-N
Target: PI3-kinase p110-alpha/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P42336
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110alpha/untagged recombinant full length human p85alpha expressed in baculovirus infected Sf21 cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 502.58, ALogP 5, HBA 8, HBD 2, PSA 111.85, QED 0.34
Compound (SMILES): CN[C@@H](C)C(=O)Nc1ccc(-c2c(-c3ccnc(OC)c3)nc3cc(C)ccn23)c(C#Cc2ccc3c(ccc(=O)n3C)c2)n1
InChIKey: JQQZFPQLXLLQNN-QFIPXVFZSA-N
Target: Cytochrome P450 2C19 (SINGLE PROTEIN), Homo sapiens
UniProt: P33261
Assay: Inhibition Assays: The final incubation volume contains TRIS buffer (0.1 M), MgCl2 (5 mM), a certain concentration of human liver microsomes dependent on the P450 isoenzyme measured (P450 2C9, P450 3A4: 0.1 mg/ml; P450 2D6: 0.2 mg/ml; P450 2C19: 0.5 mg/ml; P450 2C8: 0.05 mg/ml) and a certain concentration of the individual substrate for each isoenzyme (P450 2C9: Diclofenac 10 μM; P450 3A4: Midazolam 5 μM; P450 2D6: Dextromethorphan 5 μM; P450 2C19: S-Mephenytoin 70 μM; P450 2C8: Amodiaquine 1 μM).
Measurement: IC50 = 12000 nM (pChEMBL 4.920000076293945)
Descriptors: MW 597.68, ALogP 4.57, HBA 9, HBD 2, PSA 115.44, QED 0.27
Compound (SMILES): COc1cc(OC)cc(N(CC(C)C)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: LUDGMVDSCQQOMP-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of TEL-fused FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in presence of mouse IL3 after 72 hrs by Alamar blue assay
Measurement: IC50 = 141.25 nM (pChEMBL 6.849999904632568)
Descriptors: MW 417.51, ALogP 4.84, HBA 7, HBD 0, PSA 65.3, QED 0.43
Compound (SMILES): N[C@]1(C(=O)O)[C@@H]2[C@@H](C(=O)O)[C@@H]2[C@H](Sc2nc[nH]n2)[C@H]1OCc1ccc(Cl)c(Cl)c1
InChIKey: QHBLXVIRCXAIGL-WNAOHANSSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: Displacement of [3H]-LY459477 from human mGlu2 receptor expressed in hamster AV12 cell membranes co-expressing human EAAT1 after 90 mins by liquid scintillation counting method
Measurement: Ki = 7.43 nM (pChEMBL 8.130000114440918)
Descriptors: MW 459.31, ALogP 1.9, HBA 7, HBD 4, PSA 151.42, QED 0.49
Compound (SMILES): COC(=O)[C@@H](Cc1cc2ccnc(N)c2cc1OC(F)(F)F)N1CC[C@H](NS(=O)(=O)c2cnc(N(C)C)s2)C1=O
InChIKey: SZFZHTXOJIXHBU-JKSUJKDBSA-N
Target: Coagulation factor X (SINGLE PROTEIN), Homo sapiens
UniProt: P00742
Assay: Inhibition Assay: The compounds according to the invention are tested in a range of concentrations (10pM to 10 µM final) in assay buffer (50 mM TRIS, 100 mM NaCl, 0.1 % BSA, pH 7,5) with 0.1% maximal final DMSO concentration and deposited in a proportion of 25 µl per well, on 25 µL enzyme (human coagulation factor Xa: Enzyme Research Laboratories HFXa, final concentration 0.003Ul/ml). The reactive were mixed, centrifuged and incubated 10 minutes at 37°C in a 96 well microtiter plate. The enzyme reaction was started with 50 µL substrate (S-2765, Biogenic ref 821413 in a final concentration of 62.5µM final). The time course of the reaction was monitored at 405 nm in a microtiter plate reader (Tecan M200) for 20 minutes at 37°C. The percentage of inhibition of enzymatic activity (expressed as maximum rate of cleavage of the substrate) is calculated with respect to the enzymatic activity in the absence of inhibitor.
Measurement: IC50 = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 602.62, ALogP 1.9, HBA 11, HBD 2, PSA 157.05, QED 0.35