lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Nc1ncc(-c2ccccc2)nc1C(=O)Nc1cnccc1N1CCC(N)CC1
InChIKey: LLMXWBSEQIDLJG-UHFFFAOYSA-N
Target: Glycogen synthase kinase-3 beta (SINGLE PROTEIN), Homo sapiens
UniProt: P49841
Assay: GSKbeta Assay: Types of GSK-3 assay used to test the selectivity/off target potential compounds of the invention with respect to PKC Î±/Î¸ inhibition activity includes the following: Type 1: The GSK-3 specific peptide used in this assay was derived from the phosphorylation site of glycogen synthase and its sequence is: YRRAAVPPSPSLSRHSSPHQ(S)EDEEE (SEQ ID NO: 1). (S) is pre-phosphorylated as is glycogen synthase in vivo and the three consensus sites for GSK-3 specific phosphorylation are underlined. The buffer used to make up the glycogen synthase peptide and [γ-33P] ATP consisted of MOPS 25 mM, EDTA 0.2 mM, magnesium acetate 10 mM, Tween-20 0.01% and mercaptoethanol 7.5 mM at pH 7.00. The compounds were dissolved in dimethyl sulphoxide (DMSO) to a final concentration of 100 mM. Various concentrations were made up in DMSO and mixed with the substrate (GSK-3 peptide) solution (to a final concentration 20 uM) described in the above section along with rabbit or human GSK-3α and GSK-3β (final concentration 0.5 uM/mL enzyme). The reactions were initiated with the addition of [γ-33P] ATP (500 cpm/pmole) spiked into a mixture of ATP (final concentration of 10 uM). After 30 minutes at room temperature the reaction was terminated by the addition of 10 uL of H3PO4/O.OP/0 Tween-20 (2.5%). A volume (10 uL) of the mixture was spotted onto P-30 phosphocellulose paper (Wallac & Berthold, EG&G Instruments Ltd, Milton Keynes). The paper was washed four times in H3PO4 (0.5%), 2 minutes for each wash, air dried and the radioactive phosphate incorporated into the synthetic glycogen synthase peptide, which binds to the P-30 phosphocellulose paper, was counted in a Wallac microbeta scintillation counter Analysis of Data: Values for IC50 for each inhibitor were calculated by fitting a four-parameter logistic curve to the model: cpm=lower+(upper-lower)/(I+(concentration IC50)).
Measurement: IC50 = 0.2 nM (pChEMBL 9.699999809265137)
Descriptors: MW 389.46, ALogP 2.3, HBA 7, HBD 3, PSA 123.05, QED 0.63
Compound (SMILES): COc1nc2c(-c3c(C)ccc4ncccc34)cc(-c3c(C)noc3C)cc2[nH]1
InChIKey: ZHSYNUAULROSRV-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Amplified Luminescent Proximity Homogeneous Assay (ALPHA): Binding of the bromodomain BRD4-I to an acetylated histone H4 peptide was measured using a bead-based Amplified Luminescent Proximity Homogeneous Assay (ALPHA). The synthetic peptide containing amino acids 1-18 of histone H4 was acetylated at lysine 5, 8, 12, 16 and conjugated to biotin (SGRGACKGGACKGLGACKGGAACKRH-GSGSK-biotin[SEQ ID NO:1]) was purchased from Millipore. BRD4-I was expressed and purified from Escherichia coli as an N-terminal His6-tagged protein. Nickel-Chelate ALPHA acceptor beads (Perkin Elmer) were used to specifically bind BRD4-1 and ALPHA streptavidin donor beads (Perkin Elmer) were used because they specifically recognized the biotinylated H4 peptide. Binding of BRD4-1 to the peptide resulted in proximity of the donor and acceptor beads which leads to an increase in ALPHA signal whereas disruption of this protein-peptide interaction with a small molecule inhibitor resulted in a decrease in ALPHA signal. Assays were performed in 50 mM Hepes (pH 7.5), 150 mM NaCl, 0.1 mg/ml BSA, 0.01% (v/v) Brij, 0.5% (v/v) DMSO, 200 nM H4 peptide and 15 nM of BRD4-1 protein. After an assay reaction time of 60 minutes at 25° C., binding was measured with 20 ug/ml streptavidin donor beads and 20 ug/ml nickle-chelate acceptor beads. ALPHA signal was detected on an Envision plate reader (Ex: 320 nm; Em: 570 nm; Ex time: 180 ms). Data were normalized based on a positive (2 uM I-BET) and negative (DMSO) controls and IC50 values were calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations.
Measurement: IC50 = 29.9 nM (pChEMBL 7.519999980926514)
Descriptors: MW 384.44, ALogP 5.37, HBA 5, HBD 1, PSA 76.83, QED 0.45
Compound (SMILES): Cc1nn(C)c(C)c1S(=O)(=O)N1CCC(N(C)c2ccc(Cl)cc2)CC1
InChIKey: KBAAZVHDLADJSE-UHFFFAOYSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: Biological Assay: The ability of a compound to block the intracellular release of calcium mediated by PK1 in RBL2H3 cells expressing human PKR1 receptors is determined as a measure of the compound's antagonist activity in vitro.Approximately 10,000 cells per assay well are seeded in normal culture medium in a 384 well plate (Corning). Twenty-four hours after seeding, the cells are loaded with a calcium sensitive fluorescent dye by replacing the culture medium with assay buffer (1× Hanks buffered saline, 25 mM HEPES, 0.1% w/v fatty acid free BSA (bovine serum albumin), pH 7.4) containing 1 mM probenecid and 1× Calcium 5 Reagent (Molecular Devices). Cells are incubated at 37° C. for 1 hour to allow for dye uptake.To test for antagonist activity, test compounds at a final concentration range between 0.32 nM-10 μM (diluted in assay buffer) are added to the assay wells and allowed to incubate for 10 minutes prior to stimulation with PK1. After incubation with test compounds the assay plate is placed in a FLIPR Tetra (Molecular Devices) and PK1 (diluted in assay buffer) is added at the determined EC80 concentration (final). Ligand-dependent changes in intracellular calcium levels are determined by measuring changes in fluorescence of the dye at 525 nM following to excitation at 485 nM.
Measurement: IC50 = 900 nM (pChEMBL 6.050000190734863)
Descriptors: MW 396.94, ALogP 2.98, HBA 5, HBD 0, PSA 58.44, QED 0.8
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3)ncnc2cc1OCc1csc(-c2ccc(Cl)cc2)n1
InChIKey: KIHIMMDNUNDKTJ-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 7.300000190734863)
Descriptors: MW 543.86, ALogP 8.04, HBA 7, HBD 1, PSA 69.16, QED 0.22
Compound (SMILES): CC/C=C(\c1ccccc1)c1ccnc2[nH]c3ccc(-c4ccc(N5CCN(C)CC5)cc4)cc3c12
InChIKey: JVLJDNLPCBISRM-OAHLVZFQSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: ELISA-Based In Vitro Kinase Assay: GST-tagged recombinant wild-type or L1196M mutated ALK kinase (rALK) was expressed in Sf9 insect cells using the pBacPAK baculovirus vector system (Clontech) and purified using Glutathione Sepharose 4B affinity beads (GE Healthcare). Recombinant 3C protease was used to remove the GST tag. Purified ALK was used to screen inhibitors in the ELISA-based kinase assay, as follows: Nunc-Immuno 96-well plates were incubated overnight at 30° C. with coating solution containing 2 μg of a specific ALK peptide substrate (ARDIYRASFFRKGGCAMLPVK) in PBS. Wells were then washed with 200 μL of wash buffer (PBS-Tween 0.05%) and incubated with 4% BSA in PBS for at least 2 h at 30° C. The kinase reaction was performed in the presence of 50 mM Tris pH 7.5, 5 mM MnCl2, 5 mM MgCl2, 0.3 mM ATP and purified rALK in a total volume of 100 μL/well at 30° C. for 15 min. For inhibitor testing the reaction mix was preincubated with inhibitor or vehicle for 10 min at room temperature before transferring to the ELISA plate. After the reaction, the wells were washed 5 times with 200 μL of wash buffer. Phosphorylated peptide was detected using 100 μL/well of a mouse monoclonal anti-phosphotyrosine antibody (clone 4G10 UpstateBiotech Ltd) diluted 1:2000 in PBS+4% BSA. After 30 min incubation at room temperature the antibody was removed and wells were washed as described above. 100 μL of a secondary antibody (anti-mouse IgG, Horseradish Peroxidase linked whole antibody, Amersham Pharmacia Biotech) diluted 1:1000 in PBS+4% BSA was added to each well and the plate was incubated again for 30 min at room temperature before washing as above. The plate was developed using 100 μL/well TMB Substrate Solution (Pierce) and the reaction was stopped by adding an equal volume of 1M H2SO4. Finally, the absorbance was read at 450 nm using an ELISA plate reader (Bio-Rad).
Measurement: IC50 = 1500 nM (pChEMBL 5.820000171661377)
Descriptors: MW 472.64, ALogP 6.98, HBA 3, HBD 1, PSA 35.16, QED 0.3
Compound (SMILES): N#CC1=C(c2ccc(OC(F)(F)F)cc2)CC(c2ccc(OCCCC(F)(F)F)cc2)(C(F)(F)F)NC1=O
InChIKey: WQVUELUBRNMKQI-UHFFFAOYSA-N
Target: Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Mus musculus
UniProt: Q921V5
Assay: Inhibition of mouse recombinant MGAT2 expressed in Sf9 cell membrane using 2-oleoylglycerol and oleoyl-CoA as substrates incubated for 10 mins by LCMS assay
Measurement: IC50 = 38 nM (pChEMBL 7.420000076293945)
Descriptors: MW 552.39, ALogP 6.56, HBA 4, HBD 1, PSA 71.35, QED 0.31
Compound (SMILES): COC(=O)N1CC[C@](C)(N2CCc3c(-c4cnc(N)nc4)nc(N4CCOCC4)nc32)C1
InChIKey: KEPIQLTVJCPYMV-NRFANRHFSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Inhibition of recombinant human N-terminal Myr signal and C-terminal Flag-tagged PI3K p110alpha expressed in rat Rat1 cells
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 440.51, ALogP 0.95, HBA 10, HBD 1, PSA 122.83, QED 0.74
Compound (SMILES): CC[C@H](C)[C@H](NC(=O)OC)C(=O)N1CCC[C@H]1c1nc2ccc(-c3ccc4cc(-c5cnc([C@@H]6CCCN6C(=O)[C@@H](NC(=O)OC)C(C)C)[nH]5)ccc4c3)cc2[nH]1
InChIKey: YZCCEKSVVFTTBV-IVLWUQRZSA-N
Target: Nonstructural protein 5A (SINGLE PROTEIN), Hepatitis C virus
UniProt: Q5L478
Assay: Transient Replicon Assay: In a transient set-up, a Huh-7 lunet hepatoma cell line was transiently transfected with an autonomously replicating RNA encoding a bi-cistronic expression construct. This construct comprises a firefly luciferase reporter gene preceding the NS3-NS5B subgenomic region of HCV (genotype 1a H77 or 1b Con1). Translation of the HCV subgenomic region is mediated by an internal ribosome entry site of encephalomyocarditis virus. The construct is furthermore flanked by 5' and 3' untranslated regions of HCV (genotype 1a H77 or 1b Con 1, respectively), which allow for replication of the RNA. In addition to the wild-type constructs, site-directed mutations were introduced into the transient HCV genotype 1b replicon in the gene encoding for the non-structural protein 5A (NS5A). More precisely, amino acid residues 28, 30, 31 and 93 in NS5A were independently altered. Cells were plated in 384 well plates in the presence of test and control compounds, which were added in various concentrations. Following an incubation of two days, replication of the HCV subgenomic replicon RNA was measured by assaying luciferase activity (using standard luciferase assay substrates and reagents and a Perkin Elmer ViewLux™ ultraHTS microplate imager). HCV subgenomic replicon containing cells in the control cultures have high luciferase expression in the absence of any inhibitor. The inhibitory activity of the compound was monitored, enabling a dose-response curve for each test compound.
Measurement: EC50 < 0.013 nM (pChEMBL 10.88599967956543)
Descriptors: MW 776.94, ALogP 7.25, HBA 8, HBD 4, PSA 174.64, QED 0.11
Compound (SMILES): Cc1ccn(-c2cnc(-c3c[nH]c4ccc(-c5nnc(N)s5)cc34)cn2)n1
InChIKey: REMPPKCPFILUCR-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 = 836.27 nM (pChEMBL 6.079999923706055)
Descriptors: MW 374.43, ALogP 3.22, HBA 8, HBD 2, PSA 111.19, QED 0.5
Compound (SMILES): CN[C@H]1c2cc3c(ccn3S(=O)(=O)c3ccccc3)cc2[C@@H]1CCO
InChIKey: HPBCFIIQRFMWKG-HNAYVOBHSA-N
Target: 5-hydroxytryptamine receptor 6 (SINGLE PROTEIN), Homo sapiens
UniProt: P50406
Assay: Displacement of [3H]-LSD from human 5-HT6R expressed in CHO cell membranes by radioligand binding assay
Measurement: Ki = 158.49 nM (pChEMBL 6.800000190734863)
Descriptors: MW 356.45, ALogP 2.62, HBA 5, HBD 2, PSA 71.33, QED 0.74
Compound (SMILES): CC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(N)=O)C(C)C)C(C)C
InChIKey: WEOCQMMYNTZAEL-UFSVOBIMSA-N
Target: Tissue factor pathway inhibitor (SINGLE PROTEIN), Homo sapiens
UniProt: P10646
Assay: Inhibition of C-terminus 10x-His-taged human TFPI-1 (29 to 282 residues) assessed as suppression of TFPI-mediated inhibition of extrinsic tenase complex by measuring factor 10a activation using S-2222 substrate in presence of FVIIa, lipidated TF, phospholipid vesicles by micro-plate reader based assay
Measurement: EC50 = 12870 nM (pChEMBL 4.889999866485596)
Descriptors: MW 2484.98
Compound (SMILES): COC(=O)CCCCS(C)(=O)=NC(=O)c1cnc(N)c(C#Cc2cccc(NC(=O)c3ccc(Cl)c(C(F)(F)F)c3)c2)c1
InChIKey: AUWLXKBIQAOUOV-UHFFFAOYSA-N
Target: Platelet-derived growth factor receptor beta (SINGLE PROTEIN), Homo sapiens
UniProt: P09619
Assay: PDGFRbeta Kinase Assay: Biochemical PDGFRbeta kinase assays were performed in 96 well microtiter plates that were coated overnight with 75 ug of poly-Glu-Tyr (4:1) in 10 mM Phosphate Buffered Saline (PBS), pH 7.4. The coated plates were washed with 2 mls per well PBS+0.05% Tween-20 (PBS-T), blocked by incubation with PBS containing 1% BSA, then washed with 2 mls per wellPBS-T prior to starting the reaction. Reactions were carried out in 100 uL reaction volumes containing 36 uM ATP in kinase buffer (50 mM Hepes buffer pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2 and 0.2 mM Na3VO4). Test compounds were reconstituted in 100% DMSO and added to the reaction to give a final DMSO concentration of 5%. Reactions were initiated by the addition 20 ul per well of kinase buffer containing 200-300 ng purified cytoplasmic domain PDGFR-b protein (Millipore). Following a 60 minute incubation at 30° C., the reactions were washed 2 mls per well PBS-T. 100 ul of a monoclonal anti-phosphotyrosine antibody-peroxidase conjugate diluted 1:10,000 in PBS-T was added to the wells for 30 minutes. Following a 2 mls per well wash with PBS-Tween-20, 100 ul of 0-Phenylenediamine Dihydrochloride in phosphate-citrate buffer, containing urea hydrogen peroxide, was added to the wells for 7-10 minutes as a colorimetric substrate for the peroxidase. The reaction was terminated by the addition of 100 ul of 2.5N H2SO4 to each well and read using a microplate ELISA reader set at 492 nm.
Measurement: IC50 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 635.06, ALogP 5.57, HBA 7, HBD 2, PSA 140.81, QED 0.19
Compound (SMILES): O=[N+]([O-])c1ccc(C2Nc3c(cccc3C(F)(F)F)C3C=CCC32)cc1
InChIKey: VJDUKWHSBOBGIY-UHFFFAOYSA-N
Target: Prothrombin (SINGLE PROTEIN), Bos taurus
UniProt: P00735
Assay: High-Throughput Screening Assay: To identify novel inhibitors of hepsin, the Chembridge DIVERSet 10,000 compound high-diversity library was screened using an assay based on the cleavage of the chromogenic peptide. In addition, the NINDS II library of 1040 compounds (MicroSource Discovery) was screened to identify hepsin inhibitors among established drugs and known bioactive molecules. Screens were performed in a 96-well format at a final compound concentration of 2 uM. To minimize false positives, positions 1 and 12 of each row contained DMSO/buffer controls. As a measure of reproducibility, the Z' score for this assay was 0.78 (Zhang et al., 1999 J Biomol Screen 4:67).
Measurement: IC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 360.33, ALogP 5.44, HBA 3, HBD 1, PSA 55.17, QED 0.44
Compound (SMILES): O=C(CCCCNC(=O)c1csc(-c2cccs2)n1)NO
InChIKey: QFWNBURRPHCHGZ-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 = 608 nM (pChEMBL 6.21999979019165)
Descriptors: MW 325.42, ALogP 2.28, HBA 6, HBD 3, PSA 91.32, QED 0.41
Compound (SMILES): Cl.c1ccc(N(CC2CC2)c2ccc3ncc(-c4cn[nH]c4)nc3c2)cc1
InChIKey: XRTFMLXFXJOUEC-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 341.42, ALogP 4.57, HBA 4, HBD 1, PSA 57.7, QED 0.58
Compound (SMILES): O=C(Nc1csc(-c2nncn2C2CC2)n1)c1cc(-n2cnc(C3CC3)c2)c(N2CCN3C(=O)OCC3C2)cn1
InChIKey: AFELNRHIXXVTNY-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99683
Assay: Apoptosis Signal-Regulating Kinase Assay: The assay measures the phosphorylation level of a biotinylated peptide substrate by the ASK1 kinase using HTRF detection. This is a competitive, time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay, based on HTRF® KinEASE™-STK manual from Cisbio. Test compound, 1 μM STK3 peptide substrate, 4 nM of ASK1 kinase were incubated with 10 mM MOP buffer, pH. 7.0 containing 10 mM Mg-acetate, 0.025% NP-40, 1 mM DTT, 0.05% BSA and 1.5% glycerol for 30 minutes then 100 μM ATP was added to start the kinase reaction and incubated for 3 hr. Peptide antibody labeled with 1× Eu3+ Cryptate buffer containing 10 mM EDTA and 125 nM Streptavidin XL665 were added to stop the reaction and phosphorylated peptide substrate was detected using Envision 2103 Multilabeled reader from PerkinElmer. The fluorescence was measured at 615 nm (Cryptate) and 665 nm (XL665) and a ratio of 665 nm/615 nm was calculated for each well. The resulting TR-FRET level (a ratio of 665 nm/615 nm) was proportional to the phosphorylation level. Under these assay conditions, the degree of phosphorylation of peptide substrate was linear with time and concentration for the enzyme.
Measurement: IC50 = 2.7 nM (pChEMBL 8.569999694824219)
Descriptors: MW 558.63, ALogP 3.09, HBA 12, HBD 1, PSA 136.19, QED 0.36
Compound (SMILES): NC1CCC(NC2CC2c2ccc(F)c(F)c2)CC1
InChIKey: GADXVPHSTSJKLA-UHFFFAOYSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: Biological Assay: The compounds of the invention can be tested for their ability to inhibit LSD1. The ability of the compounds of the invention to inhibit LSD1 can be tested as follows. Human recombinant LSD1 protein was purchased from BPS Bioscience Inc (catalog reference number 50100: human recombinant LSD1, GenBank accession no. NM_015013, amino acids 158-end with N-terminal GST tag, MW: 103 kDa). In order to monitor LSD1 enzymatic activity and/or its inhibition rate by our inhibitor(s) of interest, di-methylated H3-K4 peptide (Anaspec) was chosen as a substrate. The demethylase activity was estimated, under aerobic conditions, by measuring the release of H2O2 produced during the catalytic process, using the Amplex Red hydrogen peroxide/peroxidase assay kit (Invitrogen).Briefly, a fixed amount of LSD1 was incubated on ice for 15 minutes, in the absence and/or in the presence of at least eight 3-fold serial dilutions of the respective inhibitor (e.g., from 0 to 75 uM, depending on the inhibitor strength). Tranylcypromine (Biomol International) was used as a control for inhibition. Within the experiment, each concentration of inhibitor was tested in duplicate. After leaving the enzyme interacting with the inhibitor, KM of di-methylated H3-K4 peptide was added to each reaction and the experiment was left for 30 minutes at 37° C. in the dark. The enzymatic reactions were set up in a 50 mM sodium phosphate, pH 7.4 buffer. At the end of the incubation, Amplex Red reagent and horseradish peroxidase (HPR) solution were added to the reaction according to the recommendations provided by the supplier (Invitrogen), and left to incubate for 5 extra minutes at room temperature in the dark. A 1 uM H2O2 solution was used as a control of the kit efficiency. The conversion of the Amplex Red reagent to resorufin due to the presence of H2O2 in the assay, was monitored by fluorescence (excitation at 540 nm, emission at 590 nm) using a microplate reader (Infinite 200, Tecan). Arbitrary units were used to measure level of H2O2 produced in the absence and/or in the presence of inhibitor. The maximum demethylase activity of LSD1 was obtained in the absence of inhibitor and corrected for background fluorescence in the absence of LSD1. The IC50 value of each inhibitor was calculated with GraphPad Prism Software.
Measurement: IC50 = 37 nM (pChEMBL 7.429999828338623)
Descriptors: MW 266.33, ALogP 2.68, HBA 2, HBD 2, PSA 38.05, QED 0.88
Compound (SMILES): Cc1ncoc1-c1nnc(SCCCN2CC[C@@]3(C[C@H]3c3ccc(F)cc3)C2)n1C
InChIKey: XRDPJIJIWYVCKW-PGRDOPGGSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Inhibition of human ERG transfected in HEK293 cells assessed as reduction in tail current by patch clamp assay
Measurement: IC50 = 602.56 nM (pChEMBL 6.21999979019165)
Descriptors: MW 427.55, ALogP 4.28, HBA 7, HBD 0, PSA 59.98, QED 0.41
Compound (SMILES): Cc1nnc2sc(C(=O)NCc3ccc(S(=O)(=O)C(F)F)cc3)c(N)c2c1C
InChIKey: KNZAIFIZJGFOCI-UHFFFAOYSA-N
Target: Muscarinic acetylcholine receptor M4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08173
Assay: Positive allosteric modulation of human M4 receptor expressed in CHO cells co-expressing Gqui5 by calcium mobilization assay
Measurement: EC50 = 251 nM (pChEMBL 6.599999904632568)
Descriptors: MW 426.47, ALogP 2.82, HBA 7, HBD 2, PSA 115.04, QED 0.65
Compound (SMILES): O=C(O)C12CCC(C(=O)N3CC[C@](c4ccc(C(F)(C(F)(F)F)C(F)(F)F)cc4)(S(=O)(=O)c4ccc(F)cc4)C3)(CC1)C2
InChIKey: XHTMLUDJBICVHB-STEQJIOHSA-N
Target: Nuclear receptor ROR-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P35398
Assay: Inverse agonist activity at human Gal4-fused RORalpha LBD (272 to 556 residues) expressed in human Jurkat cells measured after 18 hrs by steady-glo luciferase reporter gene assay
Measurement: EC50 > 40000 nM (pChEMBL 4.3979997634887695)
Descriptors: MW 639.56, ALogP 6.05, HBA 4, HBD 1, PSA 91.75, QED 0.31
Compound (SMILES): COCC(=O)N(CCN1[C@@H]2CC[C@H]1C[C@@H](c1cccc(C(N)=O)c1)C2)Cc1ccccc1.O=C(O)C(F)(F)F
InChIKey: ZTCRVOLCAAAEAJ-MGJLCPRGSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Displacement of [3H]-diprenorphine from human mu opioid receptor expressed in CHOK1 cell membranes
Measurement: Ki = 10 nM (pChEMBL 8)
Descriptors: MW 435.57, ALogP 3.17, HBA 4, HBD 1, PSA 75.87, QED 0.66
Compound (SMILES): COc1cc(OC)cc(N(CC2CC2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: XJOQXSSQHKPXSP-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 = 8317.64 nM (pChEMBL 5.079999923706055)
Descriptors: MW 415.5, ALogP 4.6, HBA 7, HBD 0, PSA 65.3, QED 0.44
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3)ncnc2cc1OCC1CN(CCCOC2CCCCO2)CCCO1
InChIKey: JODXPOKDEXJWRJ-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 591.54, ALogP 6.09, HBA 9, HBD 1, PSA 87.2, QED 0.27
Compound (SMILES): Cc1ccc(OC(c2ccc(Cl)c(Cl)c2)C2CNC2)cc1
InChIKey: XWYMOFXPTGWSON-UHFFFAOYSA-N
Target: Sodium-dependent dopamine transporter (SINGLE PROTEIN), Homo sapiens
UniProt: Q01959
Assay: Binding Assay: DA transporter binding assay for evaluating binding of the compound to the dopamine transporter was carried out using human recombinant dopamine transporter membrane (PerkinElmer Life and Analytical Sciences, USA) expressed in CHO-K1 cells and radioisotope [3H]WIN35,428 (PerkinElmer).That is, the test drug, 8 nM [3H]WIN35,428, dopamine transporter membrane (23 ug/well), and 50 mM Tris-HCl buffer (pH 7.4) containing 100 mM NaCl were added to obtain a reaction mixture with a final volume of 0.25 ml. After incubation for 120 minutes at 4° C., the mixture was quickly passed through a Filtermat A glass fiber filter pre-soaked with 0.5% (w/v) PEI (polyethyleneimine) using Inotech Harvester (Inotech) to terminate the reaction. After washing with cold washing buffer (50 mM Tris-HCl, pH 7.4, 0.9% NaCl) solution, the filter was covered with MeltiLex and sealed in a sample bag. After drying in an oven, radioactivity was counted using MicroBeta Plus (Wallac).
Measurement: IC50 = 168 nM (pChEMBL 6.78000020980835)
Descriptors: MW 322.24, ALogP 4.64, HBA 2, HBD 1, PSA 21.26, QED 0.89
Compound (SMILES): CCN(c1cc(Cl)cc(C(=O)NCc2c(C)cc(C)[nH]c2=O)c1C)[C@H]1CC[C@H](N2CCC2)CC1
InChIKey: QDFDIPYFARJPPY-HZCBDIJESA-N
Target: Histone-lysine N-methyltransferase EZH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15910
Assay: Inhibition Assay: Test compounds were serially diluted 3-fold in DMSO in a 10 point-curve and 1 uL was spotted into a 384-well microplate in duplicate using a Platemate Plus equipped with 384-channel head (Thermo Scientific). The final top concentration of test compounds in the assay was 10 uM. Positive control (100% inhibition standard) was 1 mM final concentration of SAH and negative control (0% inhibition standard) contained 1 uL of DMSO. Test compounds were then incubated for 30 minutes with 40 uL per well of wild-type EZH2 (final concentration was 4 nM), Y641F EZH2 (final concentration was 0.1 nM) and A677G and A687V EZH2 (for each, final concentration was 2nM) and peptide in 1x assay buffer (20 mM BICINE pH =7.6, 1 mM DTT, 0.002% TWEEN 20 (generic: polysorbate 20), 0.005% BSG). For the wild-type EZH2 and A677G EZH2 assays, biotinylated peptide H3:21-44 with unmethylated K27 was present at a final concentration of 200 nM, while in the A687V EZH2 assay, biotinylated peptide H3.
Measurement: IC50 = 5.8 nM (pChEMBL 8.239999771118164)
Descriptors: MW 485.07, ALogP 4.73, HBA 4, HBD 2, PSA 68.44, QED 0.6
Compound (SMILES): C[C@H](Nc1ncnc(N)c1C#Cc1ccccc1)c1nc2cccc(C#N)c2c(=O)n1-c1ccccc1
InChIKey: OBUNALJDPAWAHD-IBGZPJMESA-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 483.54, ALogP 4.2, HBA 8, HBD 2, PSA 122.51, QED 0.37
Compound (SMILES): CN(CC(N)=O)[C@H](CC1CCCCC1)C(=O)N[C@@H](Cc1ccccc1)C(=O)NCc1ccc(CN)cc1
InChIKey: VBCWKMGQHTXCLL-IZZNHLLZSA-N
Target: Kallikrein-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P06870
Assay: In Vitro Inhibition Assay: KLK1 inhibitory activity in vitro was determined using standard published methods (see e.g. Johansen et al., Int. J. Tiss. Reac. 1986, 8, 185; Shori et al., Biochem. Pharmacol., 1992, 43, 1209; Stürzebecher et al., Biol. Chem. Hoppe-Seyler, 1992, 373, 1025). Human KLK1 (Callbiochem) was incubated at 37° C. with the fluorogenic substrate H-DVal-Leu-Arg-AFC and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in optical absorbance at 410 nm and the IC50 value for the test compound was determined.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 507.68, ALogP 2.25, HBA 5, HBD 4, PSA 130.55, QED 0.33
Compound (SMILES): Cc1nc2cccc(-c3cc4c([nH]3)CCNC4=O)c2nc1NC(C)(C)CO
InChIKey: WEXPEOSYSQAIMZ-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Homogeneous Time Resolved Fluorescence Assay (HTRF): 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 384-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 22-point dosing curve having a high dose of 1 μM. A reference compound was included on each assay plate [Costar 3658] in order to validate that plate; on one plate of every assay run, two additional reference compounds were included. The Reaction Buffer consisted of 45 mM Hepes, pH 7.0, 15 mM NaCl, and 1 mM MgCl. The quench/detection buffer consisted of 50 mM Tris, 100 mM NaCl, 0.05% BSA, 0.1% Tween and 3 mM EDTA. Biotinylated BAD peptide (Biopeptide), 10 mM ATP (Sigma), Labeled p-BAD (S112) mAb (Cell Signalling and Perkin Elmer) [with 0.05% BSA and 2 mM DTT added] streptavidin:Allophycocyanin [Perkin Elmer]. Final concentrations either Pim-1 enzyme [5 pM], or Pim-2 enzyme [0.5 pM], DMSO [1%], BLC BAD (S112) [0.5 μM], ATP [1.5 μM], streptavidin:Allophycocyanin [0.002 mg/mL] and biotinylated-BAD (S112) mAb [100 pM]. Initial incubations were carried out at RT (22° C.) for 30 min for both Pim-1 and for Pim-2. Pim enzyme is added to compound in buffer, and plates are incubated of 30 min. Biotinylated BAD and ATP are added and plates are incubated for 1 h. A mixture of labeled p-BAD (S112) mAb and quench/detection buffer are added and incubated for 2 h. Fluorescence was measured by an HTRF® Envision microplate reader.
Measurement: IC50 = 2.5 nM (pChEMBL 8.600000381469727)
Descriptors: MW 365.44, ALogP 2.4, HBA 5, HBD 4, PSA 102.93, QED 0.57
Compound (SMILES): Cc1noc(C)c1-c1cc(C(O)(c2ccccc2)c2cccnc2)c2nc(C3CC3)[nH]c2c1
InChIKey: MJTFKNPNWQJOQD-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: HTRF Assay: Binding of the two tandem bromodomains, BRD4-1 and BRD4-2, to an acetylated histone H4 peptide was measured using a homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay. The synthetic peptide containing amino acids 1-18 of histone H4 was acetylated at lysine 5, 8, 12, 16 and conjugated to biotin (SGRGACKGGACKGLGACKGGAACKRH-GSGSK-biotin [SEQ ID NO:1]) was purchased from Millipore. BRD4-1 and BRD4-2 were expressed and purified from Escherichia coli as N-terminal His6-tagged proteins. An XL665 labeled anti-His antibody (Cisbio) was used to specifically bind BRD4 and a cryptate labeled streptavidin protein was used because it specifically recognized the biotinylated H4 peptide. Binding of BRD4 to the peptide resulted in an increase in FRET signal whereas disruption of this protein-peptide interaction with a small molecule inhibitor resulted in a decrease in FRET signal. Assays were performed in 50 mM Hepes (pH 7.5), 150 mM NaCl, 0.1 mg/ml BSA, 0.01% (v/v) Brij, 0.5% (v/v) DMSO and 200 nM H4 peptide at the following concentrations for each BRD4 isoform: 60 nM BRD4-1 and 120 nM BRD4-2. After an assay reaction time of 60 minutes at 25° C., binding was measured with 2 nM cryptate labeled streptavidin and 10 nM anti-His-XL665 antibody. TR-FRET signal was detected on an Envision plate reader (Ex: 320 nm; Em: 615/665 nm; 100 μs delay and 200 μs read window). Data were normalized based on a positive (2 μM I-BET) and negative (DMSO) controls and IC50 values were calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations.
Measurement: Ki = 10.4 nM (pChEMBL 7.980000019073486)
Descriptors: MW 436.52, ALogP 5.39, HBA 5, HBD 2, PSA 87.83, QED 0.39
Compound (SMILES): CCCCCCOc1c(O)cc(Br)c2c1CN1CCc3cc(OC)c(O)cc3[C@@H]1C2
InChIKey: VYGXIFKVFAKJNM-FQEVSTJZSA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Displacement of [3H]N-methylspiperone from human D3 receptor expressed in HEKT cell membranes after 90 mins by microbeta scintillation counting method
Measurement: Ki = 3171 nM (pChEMBL 5.5)
Descriptors: MW 476.41, ALogP 5.48, HBA 5, HBD 2, PSA 62.16, QED 0.52
Compound (SMILES): N=C(NC(=N)N1CCN(C(=N)NC(=N)Nc2ccccc2)CC1)Nc1ccccc1
InChIKey: CSZRZZFDERCVRH-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 = 950 nM (pChEMBL 6.019999980926514)
Descriptors: MW 406.5, ALogP 1.75, HBA 4, HBD 8, PSA 150, QED 0.28
Compound (SMILES): NC(=O)c1cccc([C@H]2C[C@H]3CC[C@@H](C2)N3CCN(CCc2ccc(O)cc2)C(=O)CO)c1.O=C(O)C(F)(F)F
InChIKey: LKDHMOSTLNPGJN-JCNHLJMTSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Displacement of [3H]-diprenorphine from human mu opioid receptor expressed in CHOK1 cell membranes
Measurement: Ki = 10 nM (pChEMBL 8)
Descriptors: MW 451.57, ALogP 2.27, HBA 5, HBD 3, PSA 107.1, QED 0.54
Compound (SMILES): CCCN(CCC)C(=O)C1=Cc2ccc(C(=O)Nc3cncc(OCC)c3)cc2N=C(N)C1
InChIKey: LTTLXAFKULCCIV-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: Pharmacological Assay: For TLR8 and TLR7 activity testing, HEK-Blue human TLR8 or TLR7 cells (Invivogen, San Diego, Calif., USA) are used, respectively. These cells are designed for studying the stimulation of human TLR8 or TLR7 by monitoring the activation of NF-κB. A SEAP (secreted embryonic alkaline phosphatase) reporter gene is placed under the control of the IFN-b minimal promoter fused to five NF-κB and AP-1-binding sites. Therefore the reporter expression is regulated by the NF-κB promoter upon stimulation of human TLR8 or TLR7 for 20 hours. The cell culture supernatant SEAP reporter activity was determined using Quanti Blue kit (Invivogen, San Diego, Ca, USA) at a wavelength of 640 nm, a detection medium that turns purple/blue in the presence of alkaline phosphatase.
Measurement: EC50 = 12000 nM (pChEMBL 4.920000076293945)
Descriptors: MW 449.56, ALogP 4.16, HBA 6, HBD 2, PSA 109.91, QED 0.6
Compound (SMILES): COc1ccccc1Oc1ccc(C(=O)N(Cc2ccc(C(=O)O)cc2)CC(F)F)cc1
InChIKey: YOVTYVMJDJNYKX-UHFFFAOYSA-N
Target: Lysophosphatidic acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92633
Assay: In Vitro LPA1 Receptor Assay: Engagement of the LPA1 receptor by its ligand, oleoyl-L-α-lysophosphatidic acid (LPA), leads to the release of intracellular stores of calcium into the cytoplasm mediated through a Gq-driven increase in inositol triphosphate (IP3) levels. Gq is a heterotrimeric G protein subunit that activates phospholipase C.The ability of test compounds to prevent LPA-driven intracellular release of stored calcium from RH7777 cells expressing human LPA1 receptors was assayed by stimulating the cells with LPA in the presence of various test compounds to measure the test compounds' antagonism, i.e. activity in vitro. Approximately 10,000 cells in normal culture medium (Dulbecco's Modified Eagle Media, 10% Foetal Calf Serum) were dispensed per assay well in a black clear bottom collagen coated 384 well plate (Beckton Dickinson). Cells were allowed to settle for thirty minutes at room temperature before being incubated overnight at 37° C., 5% CO2. Sixteen to twenty hours after dispensing, the cells were loaded with a calcium-sensitive fluorescent dye by replacing the culture medium with assay buffer (1× Hanks buffered saline, 25 mM HEPES, 0.1% w/v fatty acid free BSA (bovine serum albumin), pH7.4) containing 1.25 mM probenecid and 1× Calcium 5 Reagent (Molecular Devices). Cells were incubated at 37° C. for one hour to allow for dye uptake. To test for antagonist activity, test compounds at a final concentration range between 0.32 nM-10 μM (diluted in assay buffer) were added to the assay wells and allowed to incubate for twenty five minutes. After incubation with test compounds the assay plate was placed in a FLIPR Tetra system (Molecular Devices) and LPA (diluted in assay buffer) was added to give a final concentration equivalent to its determined EC80 against LPA1 receptor. Ligand-dependent changes in intracellular calcium levels were determined by measuring changes in fluorescence of the dye over 515-575 nM following excitation over 470-495 nM.
Measurement: IC50 = 43 nM (pChEMBL 7.369999885559082)
Descriptors: MW 441.43, ALogP 5.09, HBA 4, HBD 1, PSA 76.07, QED 0.5
Compound (SMILES): CCn1cc(-c2cc3c(cn2)cnn3-c2cccc(N3CCC[C@H](N)C3)n2)cn1
InChIKey: KSUJTDNWWVQOFC-KRWDZBQOSA-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 = 0.155 nM (pChEMBL 9.8100004196167)
Descriptors: MW 388.48, ALogP 2.63, HBA 8, HBD 1, PSA 90.68, QED 0.58
Compound (SMILES): COC(=O)CN[C@H](CC1CCCCC1)C(=O)N[C@@H](Cc1ccccc1)C(=O)NCc1ccc(CN)cc1
InChIKey: MLHZEVRUXMVMRU-FTJBHMTQSA-N
Target: Kallikrein-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P06870
Assay: In Vitro Inhibition Assay: KLK1 inhibitory activity in vitro was determined using standard published methods (see e.g. Johansen et al., Int. J. Tiss. Reac. 1986, 8, 185; Shori et al., Biochem. Pharmacol., 1992, 43, 1209; Stürzebecher et al., Biol. Chem. Hoppe-Seyler, 1992, 373, 1025). Human KLK1 (Callbiochem) was incubated at 37° C. with the fluorogenic substrate H-DVal-Leu-Arg-AFC and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in optical absorbance at 410 nm and the IC50 value for the test compound was determined.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 508.66, ALogP 2.59, HBA 6, HBD 4, PSA 122.55, QED 0.31
Compound (SMILES): CCCOc1ccc(CCc2nc3cc(-c4c(C)noc4C)ccc3n2CCN2CCOCC2)cc1
InChIKey: CGWBJJZOKGZCSJ-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: Displacement of biotinylated ligand from CBP (unknown origin) by TR-FRET assay
Measurement: IC50 = 190 nM (pChEMBL 6.71999979019165)
Descriptors: MW 488.63, ALogP 5.21, HBA 7, HBD 0, PSA 65.55, QED 0.31
Compound (SMILES): C[C@H](N)C/C=C\c1ccccc1
InChIKey: LLHJXESMOGGFAI-BSKOKIOFSA-N
Target: 5-hydroxytryptamine receptor 2A (SINGLE PROTEIN), Homo sapiens
UniProt: P28223
Assay: Agonist activity at human 5-HT2A receptor expressed in HEK293 cells assessed as induction of calcium mobilization incubated for 15 mins by calcium 5 dye-based fluorescence assay
Measurement: EC50 = 1600 nM (pChEMBL 5.800000190734863)
Descriptors: MW 161.25, ALogP 2.44, HBA 1, HBD 1, PSA 26.02, QED 0.72
Compound (SMILES): COc1ccncc1N1CCOc2cc(S(=O)(=O)Nc3nccs3)ccc21
InChIKey: IJASFADUIAWVTI-UHFFFAOYSA-N
Target: Sodium channel protein type 5 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q14524
Assay: In Vitro Assay: HEK 293 cells stably transfected with either Nav 1.7 or with Nav 1.5 were recorded in population patch-clamp mode with the IonWorks® Quattro automated electrophysiology system in accordance with the manufacturer's specifications (Molecular Devices, LLC, Sunnyvale, Calif.). Sodium channel currents were measured in response to a train of depolarizations that induced successively greater inactivation. Cells were held at −110 mV for three seconds (Nav 1.7) or half a second (Nav 1.5) from a holding voltage of −15 mV, then put through a series of 26 pulses of 150 msec duration to −20 mV at a frequency of 5 Hz. Cells were then left unclamped for a period of 3 to 8 minutes while a single concentration of test compound was added. Cells were then reclamped and put through the same voltage protocol. Current at the end of the 26th pulse to −20 mV was subtracted from the peak current evoked by the 26th pulse to −20 mV to correct for leak current.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 404.47, ALogP 2.88, HBA 8, HBD 1, PSA 93.65, QED 0.7
Compound (SMILES): CNC(C)C(=O)Nc1cc(-c2cccc3nc(C)c(C)n23)cc(NC(=O)Cc2ccccc2)n1
InChIKey: WYOZIYTYBAQHRF-UHFFFAOYSA-N
Target: Baculoviral IAP repeat-containing protein 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13490
Assay: DELFIA Assay: R3 domains of human XIAP (covering amino acids 241 to 356; XIAP BIR3) and cIAPl (covering amino acids 256 to 363; cIAPl BIR3) were expressed and purified from Ecoli as GST-fusion proteins. Peptide AVPIAQKSE-Lys(Biotin), representing the N-terminus of mature human SMAC (SMAC peptide), was used as interaction partner in the protein- peptide interaction assay.BIR3 domains (10 nM) were incubated with SMAC peptide (10 nM) in assay buffer (50 mM Tris, 120 mM NaCl, 0.1% BSA, 1 mM DTT, 0.05% TritonXlOO) for one hour at room temperature in the presence of inhibitiory compounds. The assay mixture was transferred to a strepatvidin coated plate and incubated for one hour at room temperature to allow binding of the biotinylated peptide and associated BIR3 domains to the plate. After several washing steps Eu labeled anti-GST antibody (e.g. Perkin Elmer DELFIA Eu-Nl- antiGST AD0250) was added to detect BIR3 domain-SMAC peptide interactions according to Perkin Elmer's instructions. Briefly, the antibody was added (dilution 1:5000 in Perkin Elmer DELFIA Assay Buffer 2013-01) and incubated for one hour. After 3 washing steps using Delfia Washing Buffer (Perkin Elmer DELFIA Wash 2013-05), Enhancement Solution (Perkin Elmer Enhancement Asolution 2013-02) was added and incubation continued for 10 minutes. Time resolved Europium fluoresecence was measured in a Wallac Victor using Standard assay settings.
Measurement: IC50 = 5346 nM (pChEMBL 5.269999980926514)
Descriptors: MW 456.55, ALogP 3.74, HBA 6, HBD 3, PSA 100.42, QED 0.39
Compound (SMILES): N#Cc1ccc(N2CCC(c3nnn4cnc5[nH]ccc5c34)CC2)cc1
InChIKey: MUUMCNOIQREEJX-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 343.39, ALogP 2.86, HBA 6, HBD 1, PSA 85.9, QED 0.6