lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(=O)N1CCC(Cn2nc(C)c3c(Cl)c(C(=O)Nc4ccc(C(F)(F)F)cc4)cnc32)CC1
InChIKey: GLRKZHQSHRKZNQ-UHFFFAOYSA-N
Target: Glucocorticoid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04150
Assay: Biological Evaluation: The assay was performed using H4 neuroglioma cells and using indirect immunofluorescence detection of the endogenous GR receptor. Fixative: 4% PFA. Permeabilization buffer: 0.3% Triton X-100 in PBS. Primary antibody: 1:200 in 5% BSA block. Secondary antibody: 1:800 in 5% BSA block. HCS Cell Mask: 1:6000 in PBS. DAPI: 100 ng/ml.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 493.92, ALogP 4.92, HBA 5, HBD 1, PSA 80.12, QED 0.56
Compound (SMILES): CN(C)[C@]1(c2ccccc2)CC[C@]2(CC1)CN(CCC(=O)Nc1ccccn1)C(=O)N2CC1(O)CCC1
InChIKey: UTRZUHSAZLJJLK-JSEFLNQRSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Receptor Binding Assay: MOP: The hMOP receptor binding assay was performed as homogeneous SPA-assay (scintillation proximity assay) using the assay buffer 50 mM TRIS-HCl (pH 7.4) supplemented with 0.052 mg/ml bovine serum albumin (Sigma-Aldrich Co. St. Louis. Mo.). The final assay volume (250 μl/well) included 1 nM of [N-allyl-2.3-3H]naloxone as ligand (PerkinElmer Life Sciences. Inc. Boston. Mass. USA). and either test compound in dilution series or 25 μM unlabelled naloxone for determination of unspecific binding. The test compound was diluted with 25% DMSO in H2O to yield a final 0.5% DMSO concentration. which also served as a respective vehicle control. The assay was started by adding wheat germ agglutinin coated SPA beads (GE Healthcare UK Ltd. Buckinghamshire. UK) which had been preloaded with hMOP receptor membranes (PerkinElmer Life Sciences. Inc. Boston. Mass. USA). After incubation for 90 minutes at RT and centrifugation for 20 minutes at 500 rpm the signal rate was measured by means of a 1450 Microbeta Trilux ß-counter (PerkinElmer Life Sciences/Wallac. Turku. Finland). Half-maximal inhibitory concentration (IC50) values reflecting 50% displacement of [3H]naloxone-specific receptor binding were calculated by nonlinear regression analysis and Ki values were calculated by using the Cheng-Prusoff equation.
Measurement: Ki = 160 nM (pChEMBL 6.800000190734863)
Descriptors: MW 505.66, ALogP 3.83, HBA 5, HBD 2, PSA 89.01, QED 0.57
Compound (SMILES): COc1cc(C2(NC(=O)[C@@H](CC(C)C)NC(=N)NC(=O)Cc3ccc(OC)c(OC)c3)CC2)ccc1F
InChIKey: NKNIKHHAQYAFOP-HXUWFJFHSA-N
Target: Cathepsin D (SINGLE PROTEIN), Homo sapiens
UniProt: P07339
Assay: In-Vitro Fluorescence Assay: In order to identify modulators of cathepsin D activity, a continuous enzymatic test was carried out with a synthetic peptide which carries a fluorescent group (MCA=(7-methoxycoumarin-4-yl)acetyl) which is quenched by energy transfer from a Dpn (2,4 dinitrophenyl) group on the same molecule, in Greiner 384-well nb microtitre plates. Cleavage of the peptidic substrate by cathepsin D causes an increase in the fluorescence intensity. In order to determine the efficacy of substances, the time-dependent increase in the fluorescence intensity in the presence of the substance was compared with the time-dependent increase in fluorescence in the absence of substances. The reference substance used was pepstatin A (Sigma-Aldrich). The substrate used was MCA-GKPILFFRLK(Dnp)d-RNH2 (Enzo Life Sciences, Lörrach). The enzyme employed was cathepsin D isolated from the human liver (Sigma-Aldrich) in a final concentration of 1.4 nM. The test was carried out in 100 mM sodium acetate buffer, 1.25% (v/v) of DMSO, 0.25% (w/v) of Chaps, pH 5.5. 2 μl of each substance solution with serially diluted substance concentration were added to in each case 4 μl of cathepsin D solution and incubated at room temperature for 10 min. The reaction was started by addition of 2 μl of substrate solution (final concentration 5 μM). After carrying out a starting-point fluorescence measurement (excitation wavelength 340 nm/emission wavelength 450 nm) using an Envision multilabel reader (Perkin Elmer), the reaction was incubated at room temperature for 60 min. The amount of peptide fragment cleaved off during the reaction time was subsequently measured by determination of the increase in the fluorescence intensity at 450 nm (excitation wavelength 340 nm).
Measurement: IC50 = 86 nM (pChEMBL 7.070000171661377)
Descriptors: MW 514.6, ALogP 3.25, HBA 6, HBD 4, PSA 121.77, QED 0.27
Compound (SMILES): COc1ccc(C)cc1CC[C@@H]1CCc2cc([C@H]3CC[C@](N)(COP(=O)(O)O)C3)ccc2C1
InChIKey: ZWPBTWBMHLDVCT-VFHOHQAYSA-N
Target: Sphingosine-1-phosphate lyase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O95470
Assay: S1P1 Binding Assay: Membranes were prepared from CHO cells expressing human S1P1. Cells pellets (1×109 cells/pellet) were suspended in buffer containing 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.5, 50 mM NaCl, 2 mM EDTA (Ethylenediaminetetraacetic acid) and Protease Inhibitor cocktail (Roche), and disrupted on ice using the Polytron homogenizer. The homogenate was centrifuged at 20,000 rpm (48,000 g) and the supernatant was discarded. The membrane pellets were resuspended in buffer containing 50 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM MgCl2, 2 mM EDTA and stored in aliquots at −80° C. after protein concentration determination. Membranes (2 μg/well) and 0.03 nM final concentration of 33P-S1P ligand (1 mCi/ml, Perkin elmer or American Radiolabeled Chemicals) diluted in assay buffer (50 mM HEPES, pH7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% fatty acid free BSA (bovine serum albumin), 1 mM NaF) were added to the compound plates (384 Falcon v-bottom plate (0.5 μl/well in a 11 point, 3-fold dilution). Binding was performed for 45 minutes at room temperature, terminated by collecting the membranes onto 384-well Millipore FB filter plates, and radioactivity was measured by TOPCOUNT®. The competition data of the test compounds over a range of concentrations was plotted as percentage inhibition of radioligand specific binding. The IC50 is defined as the concentration of competing ligand needed to reduce specific binding by 50%.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 473.55, ALogP 4.82, HBA 4, HBD 3, PSA 102.01, QED 0.48
Compound (SMILES): C[C@@H]1CN(CC2(CN[C@H]3CC3c3ccccc3)CC2)[C@@H](C)CN1C
InChIKey: VPGKXFVPHFPWDQ-XDKBUYFWSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: Enzyme Activity Assay: The purpose of this test is to test the in vitro inhibitory activities of the compounds against LSD1. The enzyme used in this experiment was human LSD1, and the standard substrate was histone H3(1-21)K4me2 peptide (10 μM). HCI-2509 (SP2509) was used as reference compound, and the fluorescence coupling enzyme assay was employed. The production of FAD-dependent H2O2 as a result of demethylase activity of LSD1 was measured by coupling with HRP and Amplex Red. Compounds as well as control compound HCl-2509 were tested in 10-dose IC50 mode with a 3-fold serial dilution in duplicate starting at 10 μm. All compounds were pre-incubated 30 mins with enzyme before added substrate to start reaction. Fluorescence measurement: Ex/Em=535/590 by EnVision.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 327.52, ALogP 2.94, HBA 3, HBD 1, PSA 18.51, QED 0.87
Compound (SMILES): Cc1ccn(-c2cc3ncccc3c(-c3ccn(C4(CC#N)CN(CC(F)(F)F)C4)n3)n2)n1
InChIKey: KQGPOCVUERTZHJ-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: JAK Caliper Enzyme Assay at 1mM ATP: Compounds were added to a 384-well plate. Reaction mixtures contained 10 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% BSA, 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20% to 30% phosphorylation. The assays were stopped with 15 μL of 180 mM HEPES, pH 7.4, 20 mM EDTA, and 0.2% Coating Reagent 3. The assay plates were placed on a Caliper Life Science LC3000 instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 767 nM (pChEMBL 6.119999885559082)
Descriptors: MW 452.44, ALogP 3.47, HBA 8, HBD 0, PSA 88.45, QED 0.46
Compound (SMILES): COc1ccc(F)cc1-c1ccnc2[nH]c(C3CCN(CC(=O)O)CC3)c(C#N)c12
InChIKey: VMAHADVDDPZDHO-UHFFFAOYSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P50750
Assay: LANCE ULight TR-FRET Kinase Assay: CDK9 enzyme activities were measured using LANCE ULight TR-FRET kinase assay reagents (PerkinElmer, Waltham, Mass.). Compounds were directly added in 100% DMSO to white low volume assay plates (Perkin Elmer Proxiplate 6008289) using a Labcyte Echo acoustic dispenser. Assay reagents in serine/threonine kinase assay buffer containing 20 mM HEPES, 10 mM MgCl2, 100 mM Na3VO4, and 0.0075% Triton X-100. were added for final reaction mixture concentrations of 1000 μM ATP, 100 nM U-light MBP peptide (Perkin Elmer TRF0109M) and reaction initiated with 4 nM CDK9/Cyclin T1 (Carna Biosciences 04-110). The kinase reaction was carried out for 30 minutes before addition of stopping buffer to a final of 20 mM EDTA and 0.5 nM of LANCE Ultra Europium anti-phospho-MBP antibody (PerkinElmer TRF0201M) in LANCE detection buffer (PerkinElmer CR97-100). The reaction was equilibrated for 1 hour and the signal read in the Perkin Elmer Envision in TR-FRET mode (excitation at 320 nm and emission at 615/665 nm).
Measurement: IC50 = 77 nM (pChEMBL 7.110000133514404)
Descriptors: MW 408.43, ALogP 3.51, HBA 5, HBD 2, PSA 102.24, QED 0.67
Compound (SMILES): C[C@]1(c2cc(/C=C(\F)c3ccc(C#N)cn3)ccc2F)N=C(N)S[C@@]2(C(=O)N3CC[C@@H](O)C3)C[C@H]21
InChIKey: IBWXJAVCODUFQB-GVZVELPGSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro Enzymatic BACE1 and BACE2 FRET (Fluorescence Resonance Energy Transfer) Assays: The cDNAs for both human recombinant BACE1 and 2 with C-terminal 6-His Tags were cloned into transient protein expression vectors, which were subsequently transfected into mammalian cell lines. These recombinant proteins were further purified using Ni-NTA affinity chromatography (Qiagen). The assay buffer used in these screens was 0.05M acetate, pH 4.5, 8% DMSO final, 100 uM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The Beta Secretase enzyme (0.02 nM for BACE1 and 0.64 nM for BACE2) is pre-incubated for one hour with inhibitors, typically in about 1 uL of DMSO according to a serial dilution, are added thereto. This assay is effectively started by the addition of FRET substrate (50 nM) and the combination is incubated for one hour. The FRET assay is terminated by addition of Tris buffer, which raises the pH to neutrality, and the fluorescence is determined. The FRET substrate is a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. Proteolytic cleavage of the FRET substrate releases quenching of fluorescence (excitation 488 nm and emission 590 nm).
Measurement: IC50 = 0.8 nM (pChEMBL 9.100000381469727)
Descriptors: MW 495.56, ALogP 3.19, HBA 7, HBD 2, PSA 115.6, QED 0.67
Compound (SMILES): COc1ccccc1S(=O)(=O)Nc1noc2cc(Cn3nccn3)cc(OC)c12
InChIKey: SLLZJLQWFIDXEV-UHFFFAOYSA-N
Target: Histone acetyltransferase KAT6A (SINGLE PROTEIN), Homo sapiens
UniProt: Q92794
Assay: Kat6A Assay 1: A. Compound preparation    1. Prepare 10 mM stock solutions in 100% DMSO from solid material    2. Serial dilute 10 mM, 1 mM or 0.1 mM compound stocks 3-fold in 100% DMSO for 11-point dose responseB. Reagent preparation    1. Prepare 1× assay buffer containing 10 mM Tris HCL pH 8.0, 2.5 mM NaCl, 0.5 mM EDTA, 0.005% BSG and 0.02% Tween-20    2. Dilute Histone peptide (CPC Scientific) and AcCoA (Sigma) together in assay buffer to 2×.    3. Dilute KAT enzyme in assay buffer to 2×.C. Enzyme reaction    1. Final reaction conditions for each KAT assay in a 20 ul assay reaction volume:  ii. KAT6A 15 nM, 1 uM AcCoA, 2 uM H3 1-21 peptide, 45-minute reaction 2. Add 0.5 ul of diluted compound to the assay plate (384-well V-bottom polypropylene plates) or 0.5 ul of DMSO for control wells.    3. Add 10 ul of 2× Histone peptide/2×AcCoA mix to the assay plate.    4. Add 10 ul of 2× enzyme to the assay plate.    5. Stop the reaction after the indicated time with the addition of 2 ul of 5% formic acid    6. Each reaction was analyzed using self-assembled monolayer desorption/ionization time-of-flight mass spectrometry (Mrksich, Milan (2008) Mass Spectrometry of Self-Assembled Monolayers: A New Tool for Molecular Surface Science ACS Nano 2008 2 (1), 7-18; SAMDI Tech, Inc. (Chicago, Ill.)).    7. Area under the curve (AUC) for both substrate and product peaks was determined for KAT5 at M.W. 2561 [Substrate+H]+ and 2603 [Product+H]+ with a +/−1 Da tolerance, respectively    8. Area under the curve (AUC) for both substrate and product peaks was determined for KAT6a, KAT6B, KAT7 and KAT8 at M.W. 2723 [Substrate+H]+ and 2765 [Product+H]+ with a +/−1 Da tolerance, respectively. 9. Percent conversion to product was calculated by: AUCProduct/(AUCsubstrate+AUCProduct).D. Data analysis    1. IC50 values were determined by fitting the % conversion at each inhibitor concentration to the 4-parameter IC50 equation using Pfizer proprietary curve fitting software.    2. Ki values were determined by fitting the % conversion at each inhibitor concentration to the Morrison equation for tightbinding competitive inhibitors using Pfizer proprietary curve fitting software.
Measurement: Ki = 2.9 nM (pChEMBL 8.539999961853027)
Descriptors: MW 415.43, ALogP 2.29, HBA 9, HBD 1, PSA 121.37, QED 0.49
Compound (SMILES): Nc1ncnn2c(-c3cccc(C4CC4C(=O)NC4CC4)c3)cc(-c3ccnn3C3CCOCC3)c12
InChIKey: JMFPWAGXGIPHDD-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: ADP-Glo Format PI3K Assay: The ADP-Glo format PI3K assays were performed in Proxiplate 384-well plates (Perkin Elmer #6008280). The final assay volume was 2 μl prepared from 1 μl additions of enzyme/PIP2:PS lipid (Invitrogen #PV5100) mixture and 1 μl ATP (provided in kit, Promega #V9101) and test compounds in assay buffer (50 mM HEPES pH 7.5, 3 mM MgCl2, 100 mM NaCl, 0.5 mM EGTA, 2 mM DTT, 0.03% CHAPS). The reaction was initiated by the combination of enzyme/lipid, ATP, and test compounds. The reaction mixture was incubated at room temperature for 30 minutes (PI3K Alpha, Beta, Gamma) or 3 hours for PI3K Delta. ADP-Glo (2 μl), followed by Kinase Detection reagent (4 μl), were added to reactions following the initial incubation and allowed to incubate 40 minutes at room temperature. The reaction mixture was analyzed on the TOPCOUNT (Perkin Elmer). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of enzyme in the assays are PI3K Alpha [0.5 nM], PI3K Beta [2 nM], PI3K Gamma [20 nM], PI3K Delta [0.5 nM]. ATP final concentrations are as follows: for Alpha [10 μM], for Beta [12.5 μM], for Gamma [6.5 μM], for Delta [100 μM]. Lipid final concentration was the same for all enzymes, [25 μM]. Dose response curves were generated to determine the concentration required to inhibit 50% of activity. Compounds were dissolved at 0.12 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. The IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 483.58, ALogP 3.58, HBA 8, HBD 2, PSA 112.36, QED 0.43
Compound (SMILES): Cn1cncc1COc1cc(F)cc(Oc2ccc(-c3nn(C4CCOCC4)c4ncnc(N)c34)cc2F)c1
InChIKey: PQXNPHOTXQJFMN-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Fluorescence Polarization-Based Kinase Assay: Fluorescence polarization-based kinase assays were performed in 384 well-plate format using histidine tagged recombinant human full-length Bruton Agammaglobulinemia Tyrosine Kinase (Btk) and a modified protocol of the KinEASE FP Fluorescein Green Assay supplied from Millipore. Kinase reaction were performed at room temperature for 60 minutes in presence of 250 μM substrate, 10 μM ATP and variable test article concentrations. The reaction was stopped with EDTA/kinase detection reagents and the polarization measured on a Tecan 500 instrument. From the dose-response curve obtained, the IC50 was calculated using Graph Pad Prisms® using a non linear fit curve.
Measurement: Ki < 100 nM (pChEMBL 7)
Descriptors: MW 533.54, ALogP 4.81, HBA 10, HBD 1, PSA 115.13, QED 0.32
Compound (SMILES): CN1CCN(Cc2ccc(NC(=O)c3n[nH]c4cc(F)c(-c5cnn(C)c5)cc34)cc2)CC1
InChIKey: WPWSODPGBNZXPW-UHFFFAOYSA-N
Target: Dual specificity tyrosine-phosphorylation-regulated kinase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: Q13627
Assay: Kinase Assay: DYRK1A: Each compound was dissolved in DMSO as a 10 mM stock and used to prepare compound source plates. Serial dilution (1:3, 11-point dose-response curves from 10 M to 0.00016 M) and compound transfer was performed using the ECHO 550 (Labcyte, Sunnyvale, Calif.) into 1536-well black-walled round bottom plates (Corning).The DYRK1A kinase assay was run using the Ser/Thr 18 peptide Z-lyte assay kit according to manufacturer's instructions (Life Technologiesa Division of Thermo-Fisher). This is a non-radioactive assay using fluorescence resonance energy transfer (FRET) between coumarin and fluorescein to detect kinase activity which is represented as a ratio of coumarin emission/fluorescein emission.Briefly, recombinant DYRK1A kinase, ATP and Ser/Thr peptide 18 were prepared in 1× Kinase buffer to final concentrations of 0.19 μg/mL, 30 μM, and 4 μM respectively. The mixture was allowed to incubate with the representative compounds for one hour at room temperature. All reactions were performed in duplicate. Unphosphorylated (0% Control) and phosphorylated (100% control) forms of Ser/Thr 18 served as control reactions. Additionally, an 11-point dose-response curve of Staurosporine (1 uM top) was run to serve as a positive compound control.After incubation, Development Reagent A was diluted in Development Buffer then added to the reaction and allowed to further incubate for one hour at room temperature. The plate was read at Ex 400 Em 455 to detect the coumarin signal and Ex 400 Em 520 to measure the signal (EnVision Multilabel Plate Reader, PerkinElmer).
Measurement: EC50 = 1 nM (pChEMBL 9)
Descriptors: MW 447.52, ALogP 3.1, HBA 6, HBD 2, PSA 82.08, QED 0.49
Compound (SMILES): Cc1cnc(-c2ncccn2)c(C(=O)N2C3CCC2C(Nc2ccc(C(F)(F)F)cn2)C3)c1
InChIKey: BSKLAEUQUDKPQQ-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P56718
Assay: Rat and Human Orexin 1 Receptor Radioligand Binding Assay: Human Embryonic Kidney 293 cells (HEK293) stably expressing rat orexin 1 receptor (Genebank accession number NM_001525) or Chinese ovary cells (CHO) stably expressing human orexin 1 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM (Hyclone, cat #SH30022), 10% FBS, 1× Pen/Strep, 1× sodium pyruvate, 10 mM HEPES, 600 μg/mL G418 and DMEM/F12 (Gibco, Cat #11039), 10% FBS, 1× Pen/Strep, 600 μg/mL G418 media, respectively on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1× with Calcium and Magnesium, Cat #SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2K×G, 5 min at 4° C.), the supernatant was aspirated and the pellets frozen and stored at −800 C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and homogenized for 45 sec prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H]-(1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone) (Moraveck Corporation, specific activity=35.3 Ci/mmol), diluted to a 10 nM concentration in PBS (4 nM final). Compounds were solubilized in 100% DMSO (Acros Organics, Cat. #61042-1000) and tested over a range of 7 concentrations (from 0.1 nM to 10 μM). The final concentration of DMSO in the reactions is equal to or less than 0.1%. Total and nonspecific binding was determined in the absence and presence of 10 μM almorexant. The total volume of each reaction is 200 μL (20 μL of diluted compounds, 80 μL of [3H]-(1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone) diluted in PBS and 100 μL of the cell suspension). Reactions were run for 60 min at room temperature and terminated by filtration through GF/C filter plates (PerkinElmer, Cat. #6005174) presoaked in 0.3% polyethylenimine using the cell harvester (PerkinElmer Filtermate). The plates were washed 3 times by aspirating 30 ml PBS through the plates. Plates were dried in 55° C. oven for 60 min, scintillation fluid was added, and the radioactivity was counted on a Topcount (Packard).
Measurement: Ki = 310 nM (pChEMBL 6.510000228881836)
Descriptors: MW 454.46, ALogP 4.12, HBA 6, HBD 1, PSA 83.9, QED 0.64
Compound (SMILES): CCC[C@H](N)c1cccc(-c2cc(COC)c3cnn(-c4cccc(CO)n4)c3c2)n1
InChIKey: DRVZUMURXBBBGB-FQEVSTJZSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Biochemical Assay: The biochemical assay is in a AlphaScreen format. The kinase reaction is based on the IRAK-4 phosphorylation of a biotin labeled peptide. The phosphopeptide is incubated with anti-phosphothreonine antibody as well as streptavidin- and protein A-coated beads. Binding of the protein-A coated beads to the antibody and the streptavidin beads to the peptide, leads to an energy transfer from one bead to the other, ultimately producing a luminescent/fluorescent signal.Generally, the kinase reaction is carried out at 1 nM IRAK4, 1.6 μM peptide, 1 mM ATP in reaction buffer 50 mM Hepes, 60 mM NaCl, 5 mM MgCl2, 0.25 mM MnCl2, 2 mM DTT, 0.01% BSA, 0.01% Tween-20) for 3.5 h at RT.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 417.51, ALogP 3.92, HBA 7, HBD 2, PSA 99.08, QED 0.45
Compound (SMILES): O=C1N(Cc2cc3cc(Cl)ccc3n2CCCN2CCS(=O)(=O)CC2)c2cnccc2C12CC2
InChIKey: PKMVHHVQVXQDBW-UHFFFAOYSA-N
Target: Protein M2-1 (SINGLE PROTEIN), Human respiratory syncytial virus A (strain A2)
UniProt: P04545
Assay: Viral Cytopathic Effect (CPE) Assay: To measure anti-RSV activity of compounds, 96-well plates are seeded with 6×103 cells per well in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). Cells are infected the next day with sufficient RSV Long strain (ATCC) to produce an approximately 80-90% cytopathic effect after 6 days, in the presence of serial half-log diluted compound in a total volume of 200 μA per well. The viability of cells is assessed after 6 days using Cell Counting kit-8 (Dojindo Molecular Technologies). The absorbance at 450 nm and referenced at 630 nm is measured to determine 50% effective concentration (EC50)The compounds of the present invention were tested for their anti-RSV activity, and the activation as described herein. The Examples were tested in the above assay and found to have EC50 of about 0.0001 μM to about 10 μM. Particular compound of formula (I) were found to have EC50 of about 0.0001 μM to about 1 μM. Further particular compound of formula (I) were found to have EC50 of about 0.0001 μM to about 0.1 μM.
Measurement: EC50 = 797 nM (pChEMBL 6.099999904632568)
Descriptors: MW 499.04, ALogP 3.39, HBA 6, HBD 0, PSA 75.51, QED 0.52
Compound (SMILES): CC(C)(O)Cn1c(CC2CCNCC2)nc2cc(Cl)c(Cl)cc21
InChIKey: BUEFUFWBOBQPDC-UHFFFAOYSA-N
Target: Voltage-dependent N-type calcium channel subunit alpha-1B (SINGLE PROTEIN), Homo sapiens
UniProt: Q00975
Assay: High-Throughput Cav2.2/Kir2.3 T-Type Fluorescent Assay: Cells were plated in 384-well, clear-bottom, black-walled, poly-D-lysine coated plates (Becton Dickinson, Franklin Lake, N.J.) 2 days prior to use in the FLIPR assay. 100 μL of cells (1.4×106 cell/mL) containing doxycyline (Sigma-Aldrich, 1.5 μg/mL; to induce channel expression) were added to each well using a Multidrop (Thermo Scientific, Waltham, Mass.) and were maintained in 5% CO2 incubator at 37° C. On the morning of the assay, cells were transferred to a 5% CO2 incubator at 29° C. Cells can then be washed with a wash buffer containing (in mM): 118 NaCl, 18.4 HEPES, 11.7 D-glucose, 2 CaCl2, 0.5 MgSO4, 4.7 KCl, 1.2 KH2PO4, pH adjusted to 7.2 with NaOH. 4.4 μM of the fluorescent indicator dye, Fluo-4 (Invitrogen), prepared in pluronic acid (Sigma-Aldrich), were loaded into the wells and incubated for 45 minutes at 29° C. in 5% CO2. Cells were then rinsed with either a 2 mM KCl closed-state buffer (in mM: 138.5 NaCl, 10 HEPES, 10 D-glucose, 1 CaCl2, and 2 KCl, with the pH adjusted to 7.4 with NaOH) when performing the closed-state assay or 12.5 mM KCl inactivated-state buffer (in mM: 128 NaCl, 10 HEPES, 10 D-glucose, 1 CaCl2, and 12.5 mM KCl, with the pH adjusted to 7.4 with NaOH) when performing the inactivated-state assay. Concentration-dependent response curves were generated from 5 mM stock solutions prepared in DMSO (Sigma-Aldrich) and diluted in either the 2 mM KCl buffer or 12.5 mM KCl buffer and incubated for 20 minutes at 29° C. in 5% CO2. Calcium entry was evoked with an addition of 130 mM KCl stimulation buffer (in mM: 10.5 NaCl, 10 HEPES, 10 D-glucose, 1 CaCl2, and 130 KCl, with the pH adjusted to 7.4 with NaOH) for both the closed-state or inactivated-state assay. A change in the Fluo-4 fluorescence signal was assessed using FLIPRTETRA™ instrument (Molecular Devices, Sunnyvale, Calif.) for 3 minutes following the elevation of extracellular KCl using an illumination wavelength of 470-495 nm with emissions recorded at 515-575 nm.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 356.3, ALogP 3.66, HBA 4, HBD 2, PSA 50.08, QED 0.88
Compound (SMILES): CC(=O)N1CCC(c2cc(-c3cnc(-n4ncc5cc(C#N)cnc54)cc3NC(C)C)on2)C1
InChIKey: LSEFDRAJFXMNQH-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IRAK4 Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 μL prepared from 15 μL additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij 35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction mixture was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 456.51, ALogP 3.5, HBA 9, HBD 1, PSA 125.76, QED 0.48
Compound (SMILES): COc1ccccc1N1CCN(CCC2CC3(CCCCC3)C(=O)O2)CC1
InChIKey: HDYLTZGIUMDPTD-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: P34969
Assay: Radiolabel Binding Assay: A stock concentration of 5 nM [3H]LSD (lysergic acid diethyl amide) is prepared in 50 mM Tris-HCl, 10 mM MgCl2, 1 mM EDTA, pH 7.4 (Assay Buffer). Aliquots (50 μl) of radioligand are dispensed into the wells of a 96-well plate containing 100 μl of Assay Buffer. Duplicate 50-μl aliquots of the compound of the disclosure test and chlorpromazine positive control reference compound serial dilutions are added.Membrane fractions of cells expressing recombinant 5HT7 receptors (50 μL) are dispensed into each well. The membranes are prepared from stably tranfected cell lines expressing 5HT7 receptors cultured on 10-cm plates by harvesting PBS-rinsed monolayers, resuspending and lysing in chilled, hypotonic 50 mM Tris-HCl, pH 7.4, centrifuging at 20,000×g, decanting the supernatant and storing at −80° C.; the membrane preparations are resuspended in 3 ml of chilled Assay Buffer and homogenized by several passages through a 26 gauge needle before using in the assay.
Measurement: IC50 = 72 nM (pChEMBL 7.139999866485596)
Descriptors: MW 372.51, ALogP 3.47, HBA 5, HBD 0, PSA 42.01, QED 0.74
Compound (SMILES): Cc1nc(C(=O)Nc2ccc3[nH]nc(-c4cnoc4)c3c2)c(C)c(C)c1C#N
InChIKey: MOCPNIMOZIMCOH-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LanthaScreen Kinase Activity Assay: The assay was carried out under the following protocol conditions: 1 mM compound in DMSO was serially diluted 1:3, 11 points in DMSO with a Biomek FX and 0.1 μL of the diluted compound was subsequently stamped into the assay plate (384-well format Lumitrac 200, Greiner, 781075) with an Echo Labcyte such that the final compound concentration in the assay was 10 μM to 169 μM. Subsequently, 5 μL of 2× kinase solution (2.9 nM final concentration) was added to the assay plate in assay buffer composed of 50 mM Tris pH 8.5 (Sigma, T6791), 5 mM MgCl2 (Fluka, 63020), 1 mM EGTA (Sigma, E3889), 0.01% BRIJ-35 (Sigma, P1254) and 2 mM DTT. The reaction was started by addition of 2×ATP/LRRKtide solution in assay buffer such that the final concentration was 400 nM LRRKtide and 25 μM ATP. After 60 min incubation at room temperature, the reaction was stopped by addition of 10 μL of 2× stop solution containing a final concentration of 2 nM anti-pERM antibody and 10 mM EDTA. After a 30 min incubation at RT, the TR-FRET signal was measured on a Wallac 2104 EnVision multilabel reader at an excitation wavelength of 340 nm and reading emission at 520 nm and 495 nm. The ratio of the 520 nm and 495 nm emission was used to analyze the data.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 372.39, ALogP 3.66, HBA 6, HBD 2, PSA 120.49, QED 0.57
Compound (SMILES): CCC(C)c1ccnc(-c2nc3nc(C(=O)O)nc(N[C@H](C)C4CCC4)c3n2Cc2ccc(C(F)(F)F)cc2)c1
InChIKey: BVPPXULSSUKQLD-ZYMOGRSISA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: FRET Assay: An HDM2 FRET assay was developed to assess the compounds' inhibitory activity towards binding of p53 protein. A truncated version of HDM2 with residues 17 to 125 (containing p53 binding surface, Science (1994) 265, 346-355), with N-terminal His and Thioredoxin tag was generated in pET32a expression vector and expressed in E. coli strain BL21(DE3)Rosetta. Protein was purified using Ni-affinity chromatography, followed by size exclusion chromatography using Superdex 75 26/60 column. To assess inhibition of p53 binding to HDM2, a FITC labeled 8-mer peptide (sequence: Ac-Phe-Arg-Dpr-Ac6c-(6-Br)Trp-Glu-Glu-Leu-NH2; Anal Biochem. 2004 Aug. 1; 331(1):138-46) with strong affinity towards p53 binding pocket of HDM2 was used. The HDM2 assay buffer contained 1× Phosphate Buffered Saline (Invitrogen, Cat#14190), 0.01% BSA (Jackson ImmunoResearch, Cat#001-000-162), 0.01% Tween-20. In the 1× assay buffer recombinant HDM2 protein, peptide and Lumi4-Tb Cryptate-conjugate mouse anti-6×His antibody (cisbio, Cat# Tb61HISTLB) were added and transferred to ProxiPlate PLUS (PerkinElmer, Cat#6008269), containing compounds so that final DMSO concentration is 0.1%. Final concentrations of reagents in the assay wells are 0.5 nM HDM2, 0.25 nM anti HIS (Tb label) antibody and 3 nM peptide. After two hour incubation at room temperature in a humidified chamber plates were read on EnVision plate reader with the following settings: excitation: UV, 340 nM, two emission filters: 520 nm and 495 nm respectively. Ratio of em520/em495 was used to calculate % inhibition and to obtain IC50 with 4-parameter logistic equation.
Measurement: IC50 = 3.647 nM (pChEMBL 8.4399995803833)
Descriptors: MW 552.6, ALogP 6.77, HBA 7, HBD 2, PSA 105.82, QED 0.24
Compound (SMILES): Cc1nc(Nc2cccc(-c3cccc(-c4nc5cc(CN6CC[C@@](C)(C(=O)O)C6)cc(C#N)c5o4)c3C)c2C)c2ncc(CN[C@@H](C)CO)cc2n1
InChIKey: ALZVBCBIJOBHLG-PRAKJKJKSA-N
Target: Tyrosine-protein phosphatase non-receptor type 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06124
Assay: Src Homology Region 2 Domain-Containing Phosphatase (SHP) Assay: U2OS/PD-L1 cells (DiscoveRx Corporation) were maintained in McCoy's 5A medium with addition of 10% FBS, 0.25 μg/ml Puromycin. After removing the culture media, the cell medium was replaced with assay medium (RPMI1640 medium with 1% FBS). The U2OS/PD-L1 cells were then added in 384-well black clear bottom assay plate (CELLCOAT® Tissue Culture Plates, Greiner Bio-One) at 5000 cells per well in 20 μL assay medium. Test compounds were prepared by serial dilution in DMSO and 125 nL compound were first transferred to the 384 REMP plate well (Thermofisher) by ECHO liquid handler (Labcyte) followed with addition of 27.5 μL assay medium. 5 μL/well compounds in the assay medium were transferred to the cell plate with 0.05% DMSO in the final assay at 0.5 μM. Jurkat-PD-1-SHP cells (DiscoveRx Corporation) were cultured in RPMI1640 medium supplemented with 10% FBS, 250 μg/ml Hygromycin B, 500 μg/ml G418. After the replacement of culture media with assay medium, 5,000 Jurkat-PD-1-SHP cells in 20 μL were dispensed into each well. The assay plate was incubated at 37° C., 5% CO2 for 2 hours before 2.5 μL PathHunter reagent 1 (DiscoveRx Corporation) were added to each well. The assay plate was shaken for 1 min at 350 rpm in the dark followed with addition of 10 μL PathHunter reagent 2 (DiscoveRx Corporation). Chemiluminescent signal was recorded with TopCount reader (Perkin Elmer) after incubation at room temperature for 1 hour. Wells with DMSO were served as the positive controls and wells containing no cells were used as negative controls. IC50 determination was performed by fitting the curve of percentage of control activity versus the log of the compound concentration using the GraphPad Prism 6.0 software.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 710.84, ALogP 6.81, HBA 11, HBD 4, PSA 173.32, QED 0.11
Compound (SMILES): CC(C)C(CNC(=O)c1ccc([N+](=O)[O-])s1)N1CCc2ccccc2C1
InChIKey: GYCJOMIBWJVPSS-UHFFFAOYSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Radioligand Binding Assay: For a primary screen of selected molecules, binding to Î¼OR was assessed by measuring competition against the radioligand 3H-diprenorphine (3H-DPN). Each compound was initially tested at 20 Î¼M and was incubated with 3H-DPN at a concentration equal to the Kd (0.4 nM) of the radioligand in Î¼OR containing Sf9 insect cell membranes. The reaction contained 40 fmol of Î¼OR and was incubated in a buffer of 20 mM HEPES pH 7.5, 100 mM sodium chloride, and 0.1% bovine serum albumin for 1 hour at 25Â° C. To separate free from bound radioligand, reactions were rapidly filtered over Whatman GF/B filters with the aid of a Brandel harvester and 3H-DPN counts were measured by liquid scintillation. Compounds with more than 25% of 3H-DPN radioactivity were further tested in full dose-response to determine the affinity (Ki) in HEK293 membranes. Subsequently, the 15 analogs were tested in full dose-response for affinity at the Î¼OR and the ÎºOR by the National Institutes of Mental Health Psychoactive Drug Screen Program (PDSP), as were the affinities of compounds 12, PZM21, and their stereoisomers at the Î¼OR, Î´OR, ÎºOR and nociception receptor.
Measurement: Ki = 820 nM (pChEMBL 6.090000152587891)
Descriptors: MW 373.48, ALogP 3.47, HBA 5, HBD 1, PSA 75.48, QED 0.62
Compound (SMILES): COCCN1C(=O)NC2(C1=O)c1cc(Br)ccc1CC21CCC(OC)CC1
InChIKey: MFJNLGWIAGPLAD-UHFFFAOYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE Assay: For each compound being tested, the BACE activity was monitored in a fluorescence quenching assay (FRET) using the ectodomain of BACE (aa 1-454) fused to a myc-his tag and secreted from HEK293/BACEect. cells into OptiMEM (Invitrogen) as enzyme source and a substrate peptide derived from the APP-Swedish mutation which possesses a Cy3-fluorophore at the N-terminus and a Cy5Q-quencher at the C-terminus (Cy3-SEVNLDAEFK-Cy5Q-NH2; Amersham). The substrate was dissolved at 1 mg/mL in DMSO.The assay was performed in the presence of 5 μl OptiMEM (supernatant collected over 24 hours and cleared from cellular debris by centrifugation) containing the ectodomain of BACE, 25 μl water containing the desired concentration of test compound and 1% DMSO, 1 μM substrate peptide, 20 mM NaOAc, pH 4.4 and 0.04% Triton-X100 in a total assay volume of 50 μl in a 384 well plate. In general, 25 μl of compound dilution were given to the plate followed by the addition of 10 μl of BACE containing OptiMEM diluted 1:2 in water with 0.2% Triton X-100. The reaction was started with the addition of 15 μl substrate in NaOAc buffer. The reaction was incubated at 30° C. in a fluorimeter and the cleavage of the substrate was recorded as kinetic for 60 min. at ex: 530 nm, em: 590 nm. Blank wells containing either no inhibitor or no enzyme were included on each plate.The intensity of fluorescence was regressed against time in order to derive velocities of reaction in all 384 wells. These velocities were used for calculating percent inhibition using an uninhibited control containing 1% DMSO and a fully inhibited control incubations performed in the absence of enzyme. IC50 values were calculated by fitting percent inhibition vs. inhibitor concentration using standard software like GraphPadPrism.
Measurement: IC50 = 48.9 nM (pChEMBL 7.309999942779541)
Descriptors: MW 437.33, ALogP 2.97, HBA 4, HBD 1, PSA 67.87, QED 0.74
Compound (SMILES): O=C(Nc1cccc(-c2nnc(-c3ccco3)o2)c1)c1cccc(Cl)c1
InChIKey: DDQOAVJOFRHUAJ-UHFFFAOYSA-N
Target: Lysocardiolipin acyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q6UWP7
Assay: In Vitro Acyltransferase Assays: ALCAT1 enzymatic activity was determined by measuring the conversion of monolysocardiolipin (MLCL) to cardiolipin or lysophosphatidylglycerol (LPG) to phosphatidylglycerol (PG) in an enzymatic reaction mixture that contained 50 mm Tris/HCl, pH 7.0, 100 μm lysophospholipids, 25 μm [14C]acyl-CoA (50 mCi/mmol, American Radiolabeled Chemicals, Inc), and membrane fraction (0.5-2.5 μg) in a total volume of 200 μl. The reaction was incubated at room temperature for 30 min. The lipids were extracted will chloroform, dried, and separated by thin layer chromatography (TLC) with chloroform:hexane:methanol:acetic acid (50:30:10:5, v/v) or chloroform:methanol:water (65:25:4, v/v). After separation, TLC plates were exposed to a Phosphorlmager screen to visualize the radiolabeled products with a Molecular Dynamics Typhoon Scanner (Sunnyvale, Calif.). In some experiments, NBD-CoA was used at 100 μM to replace the [14C]acyl-CoA in the enzymatic reaction. All quantitative data were expressed as mean±S.E. Statistical analyses for differences between two groups were carried out using a Student's t test.
Measurement: IC50 = 2000 nM (pChEMBL 5.699999809265137)
Descriptors: MW 365.78, ALogP 4.9, HBA 5, HBD 1, PSA 81.16, QED 0.56
Compound (SMILES): COCCN1C[C@@H](NC(=O)Nc2c(C)c(OCC(C)(C)O)nn2-c2ccccc2)[C@H](c2cc(F)cc(F)c2)C1
InChIKey: XFPRSHUYGGGGTL-BJKOFHAPSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: TrkA Omnia Assay: Trk enzymatic selectivity was assessed using Omnia Kinase Assay reagents from Invitrogen Corp. Enzyme (TrkA from Invitrogen Corp.) and test compound (various concentrations) were incubated for 10 minutes at ambient temperature in a 384-well white polypropylene plate (Nunc catalog#267462). Omnia Tyr Peptide #4, as well as ATP, were then added to the plate. Final concentrations were as follows: 20 nM enzyme, 500 μM of ATP, 10 μM peptide substrate. The assay buffer consisted of 25 mM MOPS pH 7.5, 0.005% (v/v) Triton X-100 and 5 mM MgCl2. The production of phosphorylated peptide was monitored continuously for 70 minutes using a Molecular Devices FlexStation II384 microplate reader (excitation=360 nm; emission=485 nm). Initial rates were calculated from the progress curves. IC50 values were calculated from these rates using either a 4 or 5-parameter logistic curve fit.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 543.62, ALogP 3.84, HBA 7, HBD 3, PSA 100.88, QED 0.36
Compound (SMILES): Cc1cc(N2CCNCC2)ccc1Nc1ncc2c(n1)-n1ccc(C(=O)Nc3c(C)cccc3C)c1CC2
InChIKey: AGPXGQNJJJOBLB-UHFFFAOYSA-N
Target: Dual specificity protein kinase TTK (SINGLE PROTEIN), Homo sapiens
UniProt: P33981
Assay: TTK Enzyme Assay: The inhibitory activity of compounds on biochemically purified full-length TTK (Life Technologies, Madison, Wis., U.S.A.) was determined in the IMAP® assay (Molecular Devices, Sunnyvale, Calif., U.S.A.). Compounds were dissolved in 100% dimethylsulfoxide (DMSO). At the day of the experiment, the compound stock was diluted in 3.16 fold steps in 100% DMSO, to obtain a 10-point dilution series, followed by further dilution in IMAP reaction buffer, which consists of 10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 0.01% Tween-20, 0.1% NaN3 and 1 mM freshly prepared dithiothreitol. Compound solution was mixed with an equal volume of full-length TTK enzyme in IMAP reaction buffer. After pre-incubation of 1 hour in the dark at room temperature, fluorescein-labeled MBP-derived substrate peptide (Molecular Devices) was added and ATP to start the reaction. Final enzyme concentration was 3.9 nM, final substrate concentration 50 nM, and final ATP concentration was 5 μM. The reaction was allowed to proceed for 2 hours at room temperature in the dark. The reaction was stopped by quenching with IMAP progressive binding solution according to the protocol of the manufacturer (Molecular Devices). Fluorescein polarization was measured on an Envision multimode reader (Perkin Elmer, Waltham, Mass., USA). Dose-response curves were fitted to a four-parameter logarithmic equation in XLfit 5 (ID Business Solutions, Ltd., Guildford, U.K.).
Measurement: IC50 = 0.7 nM (pChEMBL 9.149999618530273)
Descriptors: MW 507.64, ALogP 4.7, HBA 7, HBD 3, PSA 87.11, QED 0.36
Compound (SMILES): C=CC(=O)N(C)Cc1ccc(Oc2nc(Nc3ccc(OC)nc3)ncc2F)cc1
InChIKey: YLCGOORWBSZEPE-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Omnia Assay Protocol : Briefly, 10× stocks of EGFR-WT (PV3872) from Invitrogen and EGFR-T790M/L858R (40350) from BPS Bioscience, San Diego, Calif., 1.13×ATP (AS001A) and appropriate Tyr-Sox conjugated peptide substrates (KCZ1001) were prepared in 1× kinase reaction buffer consisting of 20 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 5 mM β-glycerophosphate, 5% glycerol (10× stock, KB002A) and 0.2 mM DTT (DS001A). 5 μL of each enzyme were pre-incubated in a Corning (#3574) 384-well, white, non-binding surface microtiter plate (Corning, N.Y.) for 30 min. at 27° C. with a 0.5 μL volume of 50% DMSO and serially diluted compounds prepared in 50% DMSO. Kinase reactions were started with the addition of 45 μL of the ATP/Tyr-Sox peptide substrate mix and monitored every 30-90 seconds for 60 minutes at λex360/λcm485 in a Synergy4 plate reader from BioTek (Winooski, Vt.).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 409.42, ALogP 3.7, HBA 7, HBD 1, PSA 89.47, QED 0.57
Compound (SMILES): COc1cc(-n2cc(C(=O)N(c3ccccc3)c3ccccc3)c3ccccc32)c(C(=O)N2Cc3ccccc3C[C@H]2CN2CCN(C)CC2)cc1O
InChIKey: XTDQACDZTHCWLA-DHUJRADRSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Fluorescence Polarisation Assay: Method A:The fluorescence polarisation tests were carried out on microplates (384 wells). The Bcl-2 protein, labelled (histag-Bcl-2 such that Bcl-2 corresponds to the UniProtKB® primary accession number: P10415), at a final concentration of 2.50×10−8 M, is mixed with a fluorescent peptide (Fluorescein-REIGAQLRRMADDLNAQY), at a final concentration of 1.50×10−8M in a buffer solution (NaPO4 20 mM, NaCl 50 mM, EDTA 1 mM, pH 7.4), in the presence or absence of increasing concentrations of test compounds. After incubation for 2 hours, the fluorescence polarisation is measured.Method B:The fluorescence polarisation tests were carried out on microplates (384 wells). The Bcl-2 protein, labelled (histag-Bcl-2 such that Bcl-2 corresponds to the UniProtKB® primary accession number: P10415), at a final concentration of 2.50×10−8 M, is mixed with a fluorescent peptide (Fluorescein-REIGAQLRRMADDLNAQY), at a final concentration of 1.00×10−8M in a buffer solution (Hepes 10 mM, NaCl 150 mM, Tween20 0.05%, pH 7.4), in the presence or absence of increasing concentrations of test compounds. After incubation for 2 hours, the fluorescence polarisation is measured.
Measurement: IC50 = 6.9 nM (pChEMBL 8.15999984741211)
Descriptors: MW 705.86, ALogP 7.14, HBA 7, HBD 1, PSA 81.49, QED 0.18
Compound (SMILES): C[C@@H]1C[C@H](N)C[C@H](c2ccncc2NC(=O)c2ccc(F)c3cc(N4CC[C@H]4C)cnc23)C1
InChIKey: PTMHYAMOGUYWLS-MLHJIOFPSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Enzyme Assay: Pim-1 and Pim-3 kinase assays20 μL reactions were run in white 384 well polystyrene plates dotted with 0.8 μL compound/DMSO in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM mgCl2, 0.01% BSA, 5 mM DTT), containing 0.05 μM Biotin-labeled BAD peptide substrate (AnaSpec 62269), 1 mM ATP, and 2.5 pM (Pim-1, Invitrogen PV3503) or 1.25 pM (Pim-3, Millipore 14-738) enzyme for 1 h at 25° C. Reactions were stopped by addition of 10 μL STOP Buffer (150 mM Tris, pH=7.5, 150 mM NaCl, 75 mM EDTA, 0.01% Tween-20, 0.3% BSA,) supplemented with Phospho-Bad (Ser112) Antibody (Cell Signaling 9291) diluted 666-fold, and Streptavidin donor beads (PerkinElmer 6760002) along with Protein-A acceptor beads (PerkinElmer 6760137) at 15 mL each. Supplementation of the STOP buffer with beads and stopping the reactions were done under reduced light. Prior to the stopping reactions STOP buffer with beads was preincubated for 1 h in the dark at room temperature. After stopping the reactions, plates were incubated for 1 h in the dark at room temperature before reading on a PHERAstar FS plate reader (BMG Labtech) under reduced light.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 447.56, ALogP 4.85, HBA 5, HBD 2, PSA 84.14, QED 0.6