lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCc1cccc(N2CCN(CCC(O)CCNC(=O)c3nc(C)c[nH]3)CC2)c1Cl
InChIKey: ULCOXAYPHBANNH-UHFFFAOYSA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Radioligand Binding Assay: Binding at dopamine D2-like receptors was determined using previously described methods. Membranes were prepared from HEK293 cells expressing human D2R, D3R or D4R, grown in a 50:50 mix of DMEM and Ham's F12 culture media, supplemented with 20 mM HEPES, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 1× antibiotic/antimycotic, 10% heat-inactivated fetal bovine serum, and 200 μg/mL hygromycin (Life Technologies, Grand Island, N.Y.) and kept in an incubator at 37° C. and 5% CO2. Upon reaching 80-90% confluence, cells were harvested using pre-mixed Earle's Balanced Salt Solution (EBSS) with 5 μM EDTA (Life Technologies) and centrifuged at 3000 rpm for 10 min at 21° C. The supernatant was removed and the pellet was resuspended in 10 mL hypotonic lysis buffer (5 mM MgCl2.6H2O, 5 mM Tris, pH 7.4 at 4° C.) and centrifuged at 20,000 rpm for 30 min at 4° C. The pellet was then resuspended in fresh EBSS buffer made from 8.7 g/L Earle's Balanced Salts without phenol red (US Biological, Salem, Mass.), 2.2 g/L sodium bicarbonate, pH to 7.4. A Bradford protein assay (Bio-Rad, Hercules, Calif.) was used to determine the protein concentration and membranes were diluted to 500 μg/mL and stored in a −80° C. freezer for later use.
Measurement: Ki = 11.2 nM (pChEMBL 7.949999809265137)
Descriptors: MW 433.98, ALogP 2.63, HBA 5, HBD 3, PSA 84.49, QED 0.57
Compound (SMILES): CC(C)(C)c1nc(N2CCC(F)(F)C2)c2nnn(Cc3ccccc3CCS(=O)(=O)F)c2n1
InChIKey: CPMRMMKZSDPTJL-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: Radioligand Binding Assay: he affinity of the compounds of the invention for cannabinoid receptors was determined using recommended amounts of membrane preparations (PerkinElmer) of human embryonic kidney (HEK) cells expressing the human CNR1 or CNR2 receptors in conjunction with 1.5 or 2.6 nM [3H]-CP-55,940 (Perkin Elmer) as radioligand, respectively. Binding was performed in binding buffer (50 mM Tris, 5 mM MgCl2, 2.5 mM EDTA, and 0.5% (wt/vol) fatty acid free BSA, pH 7.4 for CB1 receptor and 50 mM Tris, 5 mM MgCl2, 2.5 mM EGTA, and 0.1% (wt/vol) fatty acid free BSA, pH 7.4 for CB2 receptor) in a total volume of 0.2 mL for 1 h at 30° C. shaking. The reaction was terminated by rapid filtration through microfiltration plates coated with 0.5% polyethylenimine (UniFilter GF/B filter plate; Packard). Bound radioactivity was analyzed for Ki using nonlinear regression analysis (Activity Base, ID Business Solution, Limited), with the Kd values for [3H]CP55,940 determined from saturation experiments.
Measurement: Ki = 1357 nM (pChEMBL 5.869999885559082)
Descriptors: MW 482.53, ALogP 3.25, HBA 8, HBD 0, PSA 93.87, QED 0.5
Compound (SMILES): O=C(O)C[C@@H](NC(=O)c1cccc(COc2ccc(-c3ccn[nH]3)nc2)c1)c1ccccc1
InChIKey: XPJQNJSSGLOUEP-HSZRJFAPSA-N
Target: Cytochrome P450 4A11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q02928
Assay: Inhibition Test for Each Compound of the Present Invention Against 20-HETE Producing Enzymes (CYP4F2 and CYP4A11): In the CYP4F2 inhibition test, the reaction solution containing each compound [final concentration of 50 mM, KPO4 (pH 7.4), 2.5 μM luciferine derivative, and 1 mM NADPH] was added to an Escherichia coli membrane fraction (100 μg/mL protein) in which human CYP4F2 had been expressed. In the CYP4A11 inhibition test, the reaction solution containing each compound [final concentration of 100 mM, Tris-HCl (pH 7.5), 60 μM luciferine derivative, 1.3 mM NADP+, 3.3 mM Glucose 6-Phosphate, 3.3 mM MgCl2, and 0.4 U/mL Glucose 6-Phosphate dehydrogenase] was added to an Escherichia coli membrane fraction (100 μg/mL protein) in which human CYP4A11 had been expressed. Following this, the membrane fraction was left to stand at room temperature for 60 minutes to perform an enzymatic reaction. After the reaction, a luciferine detection reagent was added, and the luminescence value was measured using a plate reader. By using that value, the percent inhibition of 20-HETE producing enzyme (%) was calculated according to the equation described below, and the 50% inhibitory concentration (IC50 value) for each compound was calculated.
Measurement: IC50 = 130 nM (pChEMBL 6.889999866485596)
Descriptors: MW 442.48, ALogP 4, HBA 5, HBD 3, PSA 117.2, QED 0.36
Compound (SMILES): O=C(O)c1ccncc1NCc1ccc(Cl)c(Cl)c1
InChIKey: IRAZPDXDKSGWPW-UHFFFAOYSA-N
Target: Lysine-specific demethylase 5A (SINGLE PROTEIN), Homo sapiens
UniProt: P29375
Assay: Enzymatic Assay: The enzymatic assay of Jarid1A activity is based upon Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) detection. The ability of test compounds to inhibit the activity of Jarid1A was determined in 384-well plate format under the following reaction conditions: 1 nM Jarid1A, 300 nM H3K4me3-biotin labeled peptide (Anaspec cat #64357), 2 μM alpha-ketoglutaric acid in assay buffer of 50 mM HEPES, pH7.3, 0.005% Brij35, 0.5 mM TCEP, 0.2 mg/ml BSA, 50 μM sodium L-ascorbate, and 2 μM ammonium iron(II) sulfate. Reaction product was determined quantitatively by TR-FRET after the addition of detection reagent Phycolink Streptavidin-allophycocyanin (Prozyme) and Europium-anti-mono- or di-methylated histone H3 lysine 4 (H3K4me1-2) antibody (PerkinElmer) in the presence of 5 mM EDTA in LANCE detection buffer (PerkinElmer) at a final concentration of 25 nM and 1 nM, respectively.The assay reaction was initiated by the following: 2 μl of the mixture of 900 nM H3K4me3-biotin labeled peptide and 6 μM alpha-ketoglutaric acid with 2 μl of 11-point serial diluted inhibitor in 3% DMSO was added to each well of plate, followed by the addition of 2 μl of 3 nM Jarid1A to initiate the reaction. The reaction mixture was incubated at room temperature for 30 minutes, and terminated by the addition of 6 μl of 5 mM EDTA in LANCE detection buffer containing 50 nM Phycolink Streptavidin-allophycocyanin and 2 nM Europium-anti-H3K4me1-2 antibody. Plates were read by EnVisionMultilabel Reader in TR-FRET mode (excitation at 320 nm, emission at 615 nm and 665 nm) after 1 hour incubation at room temperature. A ratio was calculated (665/615) for each well and fitted to determine inhibition constant (IC50).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 297.14, ALogP 3.7, HBA 3, HBD 2, PSA 62.22, QED 0.9
Compound (SMILES): C[C@@H](NC(=O)c1cc(=O)[nH]c(-c2ccccc2)n1)c1ccc(OC(F)(F)F)cc1F
InChIKey: WJDZLVCXQVIGKH-LLVKDONJSA-N
Target: cGMP-dependent 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O00408
Assay: Fluorescence Polarization (FP) Assay: In a typical experiment the PDE2 inhibitory activity of the compounds of the present invention was determined in accordance with the following experimental method. Rhesus PDE2A3 was amplified from rhesus macaque brain cDNA (Biochain Institute, Hayward, Calif.) using primers based on human PDE2A sequence (accession NM_002599.3) where the forward primer containing a Kozak consensus was 5′-gccaccatggggcaggcatgtggc-3′ and the reverse primer was 5′-tcactcagcatcaaggctgca-3′. Amplification with Easy-A High-Fidelity PCR cloning enzyme (Stratagene, La Jolla, Calif.) was 95° C. for 2 minutes followed by thirty three cycles of 95° C. for 40 seconds, 52° C. for 30 seconds, and 72° C. for 2 minutes 48 seconds. Final extension was 72° C. for 7 minutes. The PCR product was TA cloned into pcDNA3.3-TOPO (Invitrogen, Carlsbad, Calif.) according to standard protocol. A consensus sequence was developed from multiple clones and then deposited into GenBank (EU812167). AD293 cells (Stratagene, La Jolla, Calif.) with 70-80% confluency were transiently transfected with rhesus PDE2A3/pcDNA3.3-TOPO using Lipofectamine 2000 according to manufacturer specifications (Invitrogen, Carlsbad, Calif.).
Measurement: Ki = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 421.35, ALogP 3.97, HBA 4, HBD 2, PSA 84.08, QED 0.61
Compound (SMILES): CN(CC(F)(F)F)C1CCCC(CS(=O)(=O)NNC(=O)c2cc(-c3cn(C)cn3)cc(Cl)c2F)C1
InChIKey: WKNFBVXQYYHKMB-UHFFFAOYSA-N
Target: Histone acetyltransferase KAT6A (SINGLE PROTEIN), Homo sapiens
UniProt: Q92794
Assay: KAT-6A Enzyme Assay: KAT-6A. Enzyme assay buffer was 50 mM Tris pH 8.0, 0.002% Tween20, 0.005% bovine skin gelatin, and 1 mM dithiothreitol (DTT). For determination of IC50values, compounds were serially diluted with 2% (v/v) DMSO in the final reaction, pre-incubating each dilution of each compound with 40 μL of assay buffer containing KAT-6A enzyme (12.5 nM final concentration). 10 μL of assay buffer containing 1 μM peptide substrate and 1 μM acetyl coenzyme A (final concentrations) was added. Reactions (50 μL total) were then carried out at 25° C. for 90 minutes. Reactions were terminated by the addition of 0.5% formic acid (final concentration), and a sample of each reaction was analyzed by SAMDI Tech, Inc. (Chicago, Ill.) using self-assembled monolayer desorption/ionization time-of-flight mass spectrometry (Mrksich, M. (2008) Mass spectrometry of self-assembled monolayers: a new tool for molecular surface science. ACS Nano 2, 7-18).
Measurement: IC50 = 30000 nM (pChEMBL 4.519999980926514)
Descriptors: MW 539.98, ALogP 3.5, HBA 6, HBD 2, PSA 96.33, QED 0.4
Compound (SMILES): c1cncc(-c2ccc3c(c2)C2(CC3)CC2NCC2CCNCC2)c1
InChIKey: HOWOJXPJAATPCI-UHFFFAOYSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: Biochemical Assay for LSD1 Activity (50 minutes): LSD1 demethylase reactions were carried out in 50 mM HEPES pH 7.4, 100 mM NaCl, 1 mM DTT, 0.01% Tween-20, and 0.1 mg/mL BSA. All enzymatic reactions were performed for either 50 minutes at room temperature in a 10-μL volume. Five microliters of 800 nM biotinylated H3K4me1 peptide solution was added to each well of a black 384 well Proxiplate containing 80 nL compound (final concentration of 0.8% DMSO). Reactions were initiated by the addition of a mixture containing 20 nM LSD1 and 20 nM FAD (5 μL). Enzyme activity was stopped by the addition of 5 μL of high salt buffer consisting of 50 mM HEPES pH 7.4, 1.5 M NaCl, 1 mM DTT, 0.01% Tween-20, and 0.1 mg/mL BSA. Capture of the product peptide by the anti-H3K4me0 antibody and Streptavidin APC was allowed to proceed for 60 mM at room temperature before measuring the TR-FRET signal. Europium-labeled antibody and Streptavidin APC were used at final concentrations of 0.003 nM and 100 nM, respectively (total assay volume of 20 μL). Plates were read on a Perkin Elmer EnVision. Percent inhibition was calculated using Max (no inhibitor) and Min (quenched with stop buffer) controls and inhibition curves plotted to determine IC50 values.
Measurement: IC50 = 53.8 nM (pChEMBL 7.269999980926514)
Descriptors: MW 333.48, ALogP 3.29, HBA 3, HBD 2, PSA 36.95, QED 0.9
Compound (SMILES): CC1(C)C(=N)N[C@](C)(c2cc(NC(=O)c3cnc(C(F)(F)F)cn3)ccc2F)CS1(=O)=O
InChIKey: YWTKWRVWZMDOMN-SFHVURJKSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vivo Inhibition of Beta-Secretase: Several animal models, including mouse, rat, dog, and monkey, may be used to screen for inhibition of beta-secretase activity in vivo following administration of a test compound sample. Animals used in this invention can be wild type, transgenic, or gene knockout animals. For example, the Tg2576 mouse model, prepared and conducted as described in Hsiao et al., 1996, Science 274, 99-102, and other non-transgenic or gene knockout animals are useful to analyze in vivo inhibition of Amyloid beta peptide (Abeta) production in the presence of inhibitory test compounds. Generally, 2 to 18 month old Tg2576 mice, gene knockout mice or non-transgenic animals are administered test compounds formulated in vehicles, such as cyclodextran, phosphate buffers, hydroxypropyl methylcellulose or other suitable vehicles. One to twenty-four hours following the administration of compound, animals are sacrificed, and brains as well as cerebrospinal fluid (CSF) and plasma are removed for analysis of A-beta levels and drug or test compound concentrations (Dovey et al., 2001, Journal of Neurochemistry, 76, 173-181) Beginning at time 0, animals are administered by oral gavage, or other means of delivery such as intravenous injection, an inhibitory test compound of up to 100 mg/kg in a standard, conventional formulation, such as 2% hydroxypropyl methylcellulose, 1% Tween80. A separate group of animals receive 2% hydroxypropyl methylcellulose, 1% Tween80 alone, containing no test compound, and serve as a vehicle-control group. At the end of the test period, animals are sacrificed and brain tissues, plasma or cerebrospinal fluid are collected. Brains are either homogenized in 10 volumes (w/v) of 0.2% diethylamine (DEA) in 50 mM NaCl (Best et al., 2005, Journal of Pharmacology and Experimental Therapeutics, 313, 902-908), or in 10 volumes of 0.5% TritonX-100 in Tris-buffered saline (pH at about 7.6). Homogenates are centrifuged at 355,000 g, 4° C. for 30 minutes. CSF or brain supernatants are then analyzed for the presence of A-beta peptide by specific sandwich ELISA assays based on ECL (Electrochemiluminescence) technology.
Measurement: IC50 = 7.4 nM (pChEMBL 8.130000114440918)
Descriptors: MW 473.45, ALogP 2.88, HBA 6, HBD 3, PSA 124.9, QED 0.59
Compound (SMILES): O=S(=O)(N1CCN(C2COC2)CC1)N(Cc1ccc(-c2nnc(C(F)(F)F)o2)cn1)c1ccccc1
InChIKey: RUCVFOIOCBMGFY-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: Enzyme Activity Inhibition Assay: HDAC enzyme activity was measured using a HDAC Fluorimetric Drug Discovery Kit (BML-AK511, 516, Enzo Life Science). For the HDAC1 enzyme activity test, human recombinant HDAC1 (BML-SE456) was used as an enzyme source, and Fluor de Lys-SIRT1 (BNL-KI177) was used as a substrate. A 5-fold dilution of the compound was seeded into a 96-well plate, and then 0.3 μg of the enzyme and 10 μM of the substrate were added to each well of the plate and allowed to react at 30 for 60 minutes. Then, Fluor de LysDeveloper II (BML-KI176) was added thereto and allowed to react for 30 minutes, after which the fluorescence value (Ex 360, Em 460) was measured using a multi-plate reader (Flexstation 3, Molecular Device). The HDAC6 enzyme was tested using human recombinant HDAC6 (382180) (Calbiochem) according to the same protocol as the HDAC1 enzyme activity test method.
Measurement: IC50 = 79 nM (pChEMBL 7.099999904632568)
Descriptors: MW 524.53, ALogP 2.42, HBA 8, HBD 0, PSA 104.9, QED 0.46
Compound (SMILES): CC(C)(C)c1nc(-c2cc(OCCN3CCOCC3)c(C3CC3)cn2)no1
InChIKey: NFJZDGARXUMYRP-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: Radioligand Binding Assay: The affinity of the compounds of the invention for cannabinoid CB1 receptors was determined using recommended amounts of membrane preparations (PerkinElmer) of human embryonic kidney (HEK) cells expressing the human CNR1 or CNR2 receptors in conjunction with 1.5 or 2.6 nM [3H]-CP-55,940 (Perkin Elmer) as radioligand, respectively. Binding was performed in binding buffer (50 mM Tris, 5 mM MgCl2, 2.5 mM EDTA, and 0.5% (wt/vol) fatty acid free BSA, pH 7.4 for CB1 receptor and 50 mM Tris, 5 mM MgCl2, 2.5 mM EGTA, and 0.1% (wt/vol) fatty acid free BSA, pH 7.4 for CB2 receptor) in a total volume of 0.2 ml for 1 h at 30° C. shaking. The reaction was terminated by rapid filtration through microfiltration plates coated with 0.5% polyethylenimine (UniFilter GF/B filter plate; Packard). Bound radioactivity was analyzed for Ki using nonlinear regression analysis (Activity Base, ID Business Solution, Limited), with the Kd values for [3H]CP55,940 determined from saturation experiments. The compounds of formula (I) show an excellent affinity for the CB2 receptor with affinities below 10 μM, more particularly of 1 nM to 3 μM and most particularly of 1 nM to 100 nM.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 372.47, ALogP 3.02, HBA 7, HBD 0, PSA 73.51, QED 0.77
Compound (SMILES): O=C(CSc1nonc1/C(=N/O)Nc1ccc(F)c(Br)c1)NCC(O)CN1CCOCC1
InChIKey: ZNIRTOCANGYFDY-UHFFFAOYSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: Enzyme Assay: HIS-tagged IDO1 protein was recombinantly expressed in Escherichia coli using ZYP5052 autoinduction media supplemented with 500 uM delta aminolevulinic acid for 48 h at 16° C. IDO1 protein was purified using Ni2+-affinity resin and size exclusion chromatography. Purified protein was then diluted in assay buffer (50 mM Tris pH 7.0, 1% glycerol, 20 uM methylene blue, 0.05% Tween-20, 20 mM sodium ascorbate, 100 units/mL catalase to obtain a final IDO1 concentration of 40 nM. IDO1 solution (30 uM) or buffer alone (30 uM) were dispensed to wells of the assay plate using a BioRAPTR liquid dispenser (Beckman Coulter). Assay plates containing compound and IDO1 enzyme were incubated at RT for 30 min. Afterwards, 10 uL of 400 uM tryptophan in assay buffer were added to each well of the assay plate using a BioRAPTR liquid dispenser. Plates were incubated at RT for 60 min and reactions were quenched by addition of 10 uL of 0.5 M methyl isonipecotate in dimethyl sulfoxide. Plates were sealed and incubated at 37° C. for 4 h or 50° C. for 2 h. The plates are allowed to cool and then centrifuged for 1 min at 1000 ug. The resulting fluorescence was measured in an Envision plate reader (Perkin Elmer) with a 400/25 nm excitation filter and an 510/20 nm emission filter.
Measurement: IC50 = 43.1 nM (pChEMBL 7.369999885559082)
Descriptors: MW 533.38, ALogP 1.12, HBA 10, HBD 4, PSA 145.34, QED 0.12
Compound (SMILES): N#Cc1ccc2[nH]cc(CCCN3CCN(c4ccc(-n5ccncc5=O)cc4)CC3)c2c1
InChIKey: RLANJTMXVDVGLS-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 1A (SINGLE PROTEIN), Homo sapiens
UniProt: P08908
Assay: Human 5-HT1A Receptor Binding Assay: Human HEK-293 cell homogenates (36 μg protein) were incubated at 22° C. for 60 minutes with 0.3 nM [3H]8-0H-DPAT (Perkin-Elmer) in the absence or presence of the test compound in a buffer solution containing 50 mM Tris-HCl (pH 7.4), 10 mM MgSO4, 0.5 mM EDTA and 2 μg/ml aprotinine.The non-specific binding value was determined by incubating the same mixture in the presence of 10 μM 8-OH-DPAT, which was used as the standard reference compound and tested in each experiment at several concentrations to obtain a competition curve.Following the incubation, the samples were filtered rapidly under vacuum through glass fiber filter (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters were dried and the retained radioactivity was measured in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard). The experimental results were expressed as a percent inhibition of the control radioligand specific binding.Data AnalysisBinding assay of [3H] 8-OH-DPAT (0.3 nM) with 5-HT1A receptor in human HEK-293 cell was tested by scintillation proximity assay of membrane. The test compounds were required to be tested at least three times in the case of the concentration thereof being greater than 6 log, and the obtained data were subjected to a nonlinear regression analysis via a curve of Hill equation, to obtain IC50 value, and then calculated by ChengPrusoff equation to obtain Ki value, and Ki was the inhibition constant.
Measurement: Ki = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 438.54, ALogP 3.34, HBA 6, HBD 1, PSA 80.95, QED 0.5
Compound (SMILES): CC(C)S(=O)(=O)c1ccc(Nc2nn([C@H]3CCCC[C@@H]3C#N)c3cc[nH]c(=O)c23)cc1
InChIKey: HHJVLPGKNFAVES-QAPCUYQASA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biochemical HTRF Assay Protocol: The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer #PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at room temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading. After normalization to untreated controls, the percent inhibition of the HTRF signal at each compound concentration was calculated.
Measurement: IC50 = 690 nM (pChEMBL 6.159999847412109)
Descriptors: MW 439.54, ALogP 3.91, HBA 7, HBD 2, PSA 120.64, QED 0.62
Compound (SMILES): CN1CCC(CN(CC(=O)NCc2cccc(Cl)c2F)C(=O)Cn2nc(C(N)=O)c3ccccc32)CC1
InChIKey: PCCZIDVXXHNOTB-UHFFFAOYSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Factor D Esterolytic Assay: An established esterolytic assay for the measurement of Factor D activity and inhibition of Factor D activity was used (Kam, C. M.; McRae, B. J.; Harper, J. W.; Niemann, M. A.; Volanakis, J. E.; Powers, J. C. Human complement proteins D, C2, and B Active site mapping with peptide thioester substrates. J Biol. Chem. 1987, 262, 3444-3451). For this assay Z-Lys-SBzl, 1.29 mM (Kim, S.; Narayana, S. V. L; Volanakis, J. E. Mutational analysis of the substrate binding site of human complement Factor D. Biochemistry. 1994, 33, 14393-14399) was used as the substrate for Factor D (104 mM). Hydrolysis of this compound by Factor D liberated a free sulfhydryl group which is then reacted with 5,5′-dithiobis(2nitrobenzoic acid) producing an intense yellow color (Habeeb, A. F. S. A. Reaction of protein sulfhydryl groups with Ellman's Reagent. Methods in Enzymol. 1976, 25, 457-464). The assays were performed in 96 well microtiter plates and rates of hydrolysis were monitored at 405 nm on a Biotek Synergy H1 plate reader. Hydrolysis rates were reported as change in mOD/min. The assay was conducted in 100 mM HEPES, 500 mM NaCl, pH 7.5 containing 10% DMSO in a final volume of 50 μL per well.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 529.02, ALogP 2.41, HBA 6, HBD 2, PSA 113.56, QED 0.44
Compound (SMILES): Cn1cc(-c2cc(S(C)(=O)=O)ccc2Oc2cccc(C(=O)N3CCN(CCO)CC3)c2)c2cc[nH]c2c1=O
InChIKey: HNXHZLXTYBSBGL-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: TR-FRET Assay (BD2: amino acids E352-E168): Compound dilution series were prepared in DMSO via a 3-fold serial dilution from 2.5 mM to 42 nM. Compounds were then diluted 6:100 in assay buffer (20 mM Sodium Phosphate, pH 6.0, 50 mM NaCl, 1 mM Ethylenediaminetetraacetic acid disodium salt dihydrate, 0.01% Triton X-100, 1 mM DL-Dithiothreitol) to yield 3× working solutions. Six microliters (μL) of the working solution was then transferred to white, low-volume assay plates (Costar #3673). A 1.5× assay mixture containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and the Alexa-647-conjugated probe molecule was also prepared. Twelve μL of this solution were added to the assay plate to reach a final volume of 18 μL. The final concentration of 1× assay buffer contains 2% DMSO, 50 μM-0.85 nM compound, 8 nM His-tagged bromodomain, 1 nM Europium-conjugated anti-His-tag antibody and 100 nM or 30 nM probe (for BDI or BDII, respectively). After a one-hour incubation at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 550.64, ALogP 2.48, HBA 8, HBD 2, PSA 124.94, QED 0.36
Compound (SMILES): Cc1c(Cl)c2c(Cl)c(C)c1-c1c(-c3ccc(F)cc3)sc3ncnc(c13)O[C@@H](C(=O)O)Cc1cc(ccc1OCc1ccnc([C@H]3CC[C@@](F)(COC[C@@H]4COCCO4)CC3)n1)OC[C@@H](CN1CCN(C)CC1)O2
InChIKey: IDSFBOVQTFYUNQ-WZDAGNNJSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07820
Assay: Exemplary MCL-1 Inhibitors Bind MCL-1 in the presence of 10% HS: The ability of the exemplary MCL-1 inhibitors of Examples 1 through 151 to bind MCL-1 was demonstrated using the Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay. Tb-anti-GST antibody was purchased from Invitrogen (Catalog No. PV4216). The ability of exemplary MCL-1 inhibitors Example 1 to Example 3 to compete with probe F-Bak for binding MCL-1 was demonstrated using a Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) binding assay.MethodFor the assay, an acoustic dispenser was used to prepare dilution series from 10 mM test compounds in 100% DMSO and directly transfer 160 nL into low volume 384-well assay plates. 8 μL of a protein/probe/antibody mix was then added to each well resulting in final concentrations listed below: Test compound: 11 three-fold dilutions beginning at 25 μMProtein: GST-MCL-1  1 nM Antibody Tb-anti-GST  1 nM Probe: F-Bak 100 nMThe samples were then mixed on a shaker for 1 minute and incubated for an additional 2 hours at room temperature. For each assay plate, a probe/antibody and protein/antibody/probe mixture were included as a negative and a positive control, respectively. Fluorescence was measured on the Envision (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak) and 495/510 nm (Tb-labeled anti-his antibody) emission filters. Dissociation constants (Ki) were determined using Wang's equation (Wang, 1995, FEBS Lett. 360:111-114). The TR-FRET assay can be performed in the presence of varying concentrations of human serum (HS) or fetal bovine serum (FBS). Compounds were tested in the presence of 10% HS.
Measurement: Ki = 0.24 nM (pChEMBL 9.619999885559082)
Descriptors: MW 1076.06
Compound (SMILES): O=C(N[C@H]1CCN(c2nccn3ccnc23)C1)c1cnc(N2CCC(O)CC2)cn1
InChIKey: ILDOVQXCAMOBDO-AWEZNQCLSA-N
Target: Atypical chemokine receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P25106
Assay: Buffer Binding Analysis: Target compounds can be tested to determine their ability to bind with CXCR7 sites on MCF-7 and/or MDA-MB-435S CXCR7 transfected cells. Efficiency-maxim/zed radioligand binding using filtration protocols as described in Dairaghi D J, et al., HHV8-encoded vMIP-I selectively engages chemokine receptor CCR5. Agonist and antagonist profiles of viral chemokines., J. Biol. Chem. 1999 Jul. 30; 274(31): 21569-74 and Gosling J. et al., Cutting edge: identification of a novel chemokine receptor that binds dendritic cell-and T cell-active chemokines including ELC. SLC, and TECK., J. Immunol. 2000 Mar. 15; 164(6):2851-6 was used.In these assays, MCF-7 and/or MDA-MB-435S cells are interrogated with the target compounds and the ability of these compounds to displace 125I radiolabeled SDF-1 assessed using the protocol described in Dairaghi and Gosling. The target compounds are added to the plate to the indicated concentration and were then incubated with cells followed by the addition of radiolabeled chemokine (125I SDF-1) for 3 hr at 4° C. in the following binding medium (25 mM HEPES, 140 mM NaCl, 1 mM CaCl2, 5 mM MgCl2 and 0.2% bovine serum albumin, adjusted to pH 7.1). All assays are then incubated for 3 hrs at 4° C. with gentle agitation. Following incubation in all binding assays, reactions are aspirated onto PEI-treated GF/B glass filters (Packard) using a cell harvester (Packard) and washed twice (25 mM HEPES, 500 mM NaCl, 1 mM CaCl2, 5 mM MgCl2, adjusted to pH 7.1). Scintillant (MicroScint 10, Packard) is added to the wells, and the filters counted in a Packard Topcount scintillation counter. Data are analyzed and plotted using GraphPad Prism (GraphPad Software).
Measurement: IC50 = 1500 nM (pChEMBL 5.820000171661377)
Descriptors: MW 408.47, ALogP 0.49, HBA 9, HBD 2, PSA 111.78, QED 0.64
Compound (SMILES): COc1ccc(C)c(-c2nc([C@@H]3CCCN3C(=O)c3ccc(Cl)cc3-n3nccn3)no2)c1
InChIKey: KOAIPQZPHNCZCQ-IBGZPJMESA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: In Vitro Assay: Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES. On the day of the assay, 50 uL of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/L, 5 mM probenecid (Sigma) and 3 uM of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at rt for 30 min before measurement.Within the Fluorescent Imaging Plate Reader (FLIPR Tetra, Molecular Devices), antagonists are added to the plate in a volume of 10 uL/well, incubated for 120 min and finally 10 uL/well of agonist is added. Fluorescence is measured for each well at 1 second intervals, and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 3 nM orexin-A with vehicle in place of antagonist. The IC50 value (the concentration of compound needed to inhibit 50% of the agonistic response) is determined and may be normalized using the obtained IC50 value of an on-plate reference compound. Optimized conditions were achieved by adjustment of pipetting speed and cell splitting regime. The calculated IC50 values may fluctuate depending on the daily cellular assay performance. Fluctuations of this kind are known to those skilled in the art. In the case where IC50 values have been determined several times for the same compound, the geometric mean has been given.
Measurement: IC50 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 464.91, ALogP 4.27, HBA 8, HBD 0, PSA 99.17, QED 0.43
Compound (SMILES): COc1cc(N2CCN(C(=O)[C@@H](C)C[C@@]3(C4CC4)NC(=O)NC3=O)C[C@@H]2C)cc(F)c1F
InChIKey: LTDJFOLMHATVKC-MZFXBISCSA-N
Target: A disintegrin and metalloproteinase with thrombospondin motifs 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UNA0
Assay: In Vitro Assay: TABLE IX: The basis for the assay is the cleavage of the substrate TBIS-1 (5 FAM-TEGEARGSVILLK (5TAMRA)K-NH2) (SEQ ID NO: 2) by human ADAMTS-5.For the dose response (10 point), 4 μL of a dilution series of compound (2 mM highest concentration, ⅕ dilution in DMSO further diluted 1 in 10 in water, corresponding to a final highest concentration of 20 μM), is transferred to 384 well Fluotrac 200 plate (Greiner, cat #781076) and incubated at room temperature for 30 min with a 26 μL buffer solution (50 mM Hepes pH 7.5, 100 mM NaCl, 5 mM CaCl2, 0.1% CHAPS) containing hADAMTS-5 (0.63 ng/μL) (it will be appreciated by the skilled person that the potency read out is independent of the enzyme concentration).The reaction is initiated by adding to the assay plate TBIS-1 (10 μL, 4.5 μM, Anaspec) in the same buffer.Finally, the fluorescence is read on the Envision (Perkin Elmer) after an incubation of 90 min at 37° C. (Excitation 485 nm, emission 530).
Measurement: IC50 = 37.5 nM (pChEMBL 7.429999828338623)
Descriptors: MW 450.49, ALogP 2.02, HBA 5, HBD 2, PSA 90.98, QED 0.65
Compound (SMILES): NS(=O)(=O)OC[C@H]1C[C@@H](Nc2ccnc3cc(-c4cnc5ccccc5c4)nn23)[C@H](O)[C@@H]1O
InChIKey: BOWWVKGCWVBOPJ-NVGWVTIJSA-N
Target: Ubiquitin-like modifier-activating enzyme 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P22314
Assay: Ubiquitin Activating Enzyme (UAE) HTRF Assay: The UAE enzymatic reaction totals 50 μL and contains 50 mmol HEPES (pH 7.5), 0.05% BSA, 2.5 mM MgCl2, 0.1 uM ATP, 8 nM GST-Ubc-2, 35 nM flag-ubiquitin, 1 nM recombinant human UAE or mouse UAE. Compounds for this hUAE IC50 assay are tested at 10 point 3-fold dilution. The top concentration for this assay is 1 μM. Each compound is ordered in duplicate on the same plate. The enzymatic reaction mixture is incubated for 90 minutes at room temperature (24 degrees C.) in a 384 well plate prior to termination with a stop solution (0.1 M HEPES/0.05% Tween20, 20 mmol EDTA, 410 mM KF, 0.5 nM Eu Cryptate anti-FLAG M2-K antibody (Cis-bio International), 8 ug/mL Anti-GST XL-APC (Prozyme)). After incubation for 120 minutes, quantification of the FRET is performed on the Pherostar (BMG).
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 470.51, ALogP 0.69, HBA 10, HBD 4, PSA 164.96, QED 0.32
Compound (SMILES): CCCS(=O)(=O)N1CC(CC#N)(n2ccc(-c3nc(-c4cnn(C)c4)cc4ncccc34)n2)C1
InChIKey: IATGYVIDQUYKMS-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
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 = 5399 nM (pChEMBL 5.269999980926514)
Descriptors: MW 476.57, ALogP 2.56, HBA 9, HBD 0, PSA 122.59, QED 0.4
Compound (SMILES): CN1CCC(N2CC(CNC3CC3c3ccccc3)C2)CC1
InChIKey: HWABSZBZAQXJFK-UHFFFAOYSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: Biochemical Assay: LANCE LSD1/KDM1A demethylase assay 10 μL of 1 nM LSD-1 enzyme (ENZO BML-SE544-0050) in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 25 mM NaCl, 5 mM DTT) were preincubated for 1 hour at 25° C. with 0.8 μL compound/DMSO dotted in black 384 well polystyrene plates. Reactions were started by addition of 10 μL of assay buffer containing 0.4 μM Biotin-labeled Histone H3 peptide substrate: ART-K(Me1)-QTARKSTGGKAPRKQLA-GGK(Biotin) SEQ ID NO:1 (AnaSpec 64355) and incubated for 1 hour at 25° C. Reactions were stopped by addition of 10 μL 1×LANCE Detection Buffer (PerkinElmer CR97-100) supplemented with 1.5 nM Eu-anti-unmodified H3K4 Antibody (PerkinElmer TRF0404), and 225 nM LANCE Ultra Streptavidin (PerkinElmer TRF102) along with 0.9 mM Tranylcypromine-HCl (Millipore 616431). After stopping the reactions plates were incubated for 30 minutes and read on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 299.46, ALogP 2.16, HBA 3, HBD 1, PSA 18.51, QED 0.9
Compound (SMILES): Nc1cc(C(CCOCc2ccccc2)c2cnn(Cc3ccc4c(c3)CCNC4)c2)c2nn[nH]c2n1
InChIKey: INRMKJGJIVQJET-UHFFFAOYSA-N
Target: Myeloperoxidase (SINGLE PROTEIN), Homo sapiens
UniProt: P05164
Assay: MPO Chlorination Assay (APF Assay): MPO chlorination activity was measured in 100 mM KPi (pH 7.4) by utilizing the non-fluorescent reagent Aminophenyl fluorescein (APF, Invitrogen Cat. #A36003). APF is cleaved by (OCl) to yield the fluorescent compound fluorescein. Reactions were carried out in 50 μL total volume by adding a 25 μL mixture of 200 pM myeloperoxidase and 120 mM NaCl to 100 nL inhibitor in 100% DMSO in a 384 well, non-binding surface clear bottom plate (Corning #3655). Enzyme, inhibitor, and chloride were preincubated for ten minutes at rt.After the ten minute preincubation, 25 μL of an APF mixture containing 10 mM APF, 120 mM NaCl and 10 μM H2O2 was added to the plate using the internal dispensing system of a Hammatsu FDSS 6000. Kinetic determinations were carried out immediately on the FDSS 6000 (3 minute kinetic read, 1 read every second, ex: 485 nm, em: 535 nm). IC50 values for inhibitors were calculated by taking the slope of the linear portion of the kinetic measurement (20 seconds to 80-120 secs).IC50 values were calculated by determining the slope of the linear portion of the kinetic trace (180-540 secs), and using that calculated slope to determine % inhibition occurring at each concentration of inhibitor using the following equation:Y = A + B - A 1 + ( C / x ) Dwhere A=minimal Y value (activity level of inhibited sample), B=maximal Y value (activity level of uninhibited sample), C=Log IC50, D=Hill Slope, x=concentration of inhibitor.The exemplified Examples disclosed below were tested in the MPO peroxidation assay described above and found to have MPO inhibitory activity. A range of IC50 values of ≤10 μM (10000 nM) was observed.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 494.6, ALogP 3.56, HBA 8, HBD 3, PSA 119.56, QED 0.27
Compound (SMILES): COc1ccc(C(O)c2cc(-c3ncnc4cc(N5CCOCC5)ccc34)c(F)cc2Cl)nn1
InChIKey: MOWXJLUYGFNTAL-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Patch Clamp Assay: The patch-clamp measurement was carried out at room temperature in whole-cell configuration on human embryonic kidney cells (HEK293) which have been transfected in a stable manner with the hERG gene.The whole-cell configurations were carried out using an automated patch clamp device (Patchliner, Nanion Technologies, Munich). This is a glass chip-based system with which automated whole-cell measurements on up to 8 cells simultaneously are possible. The glass chip has a hole of defined size to which the cell is transferred into the Gigaseal by application of a reduced pressure and brought into the whole-cell configuration. Buffer, cell suspension and test substances were added to microchannels of the chip using a Teflon-coated pipette. The cells were clamped to a holding potential of -80 mV. For measurement of substance-promoted inhibition of the Kv11.1 channel, the following voltage protocol was applied at 10-second intervals: 51 ms/-80 mV, 500 ms/+40 mV, 500 ms/-40 mV, 200 ms/-80 mV. The leakage current is subtracted by means of the P4 method. The cells were resuspended in extracellular buffer (EC) and applied to the chip. After the cell had been collected, the seal was improved by addition of a seal enhancer buffer. As soon as the whole-cell configuration had been reached, the seal enhancer buffer was washed out and replaced by extracellular buffer. The measurement started in EC for 1.5 min. DMSO (vehicle control, 0.1% of DMSO) was then applied, and the control current was recorded for 3 min. The test substance was subsequently added twice in the same concentration, and the potassium current was measured for 3.5 min in each case.
Measurement: Ki = 12500 nM (pChEMBL 4.900000095367432)
Descriptors: MW 481.92, ALogP 3.81, HBA 8, HBD 1, PSA 93.49, QED 0.46
Compound (SMILES): O=C(O)C1CC12CCN(c1ccc(Nc3nc(N4CCCCCC4)nc4cn[nH]c(=O)c34)cc1)CC2
InChIKey: VWCMDXCFCAPJBO-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Inhibition of Enzymatic JAK2 Kinase Activity: Two μL of 10% DMSO in blank, substrate control and positive control wells and 2 μL of test compound in test wells was added. Thirteen μL of assay buffer in blank and substrate control wells and 13 μL of Enzyme buffer mix in positive and test wells was added. The reaction mixture was incubated for 30 minutes at RT on a plate shaker. Ultra Light-pGT substrate (5 μL) [poly Glu-Tyr (4:1) labeled with U Light TM dye, a tyrosine kinase substrate] and ATP mix was added to all wells. The reaction plate was incubated for 60 minutes at RT on a plate shaker. The reaction was stopped by adding 40 mM EDTA (10 μL) in buffer. Ten μL of antibody was added to all the wells. The plate was read in a Wallac 1420 Multilabel Counter Victor 3 instrument (Ex: 340 nm Em: 615 & 665 nm).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 489.58, ALogP 3.53, HBA 8, HBD 3, PSA 127.34, QED 0.49
Compound (SMILES): COc1cc(OC)cc(N2CCN(C(=O)CCC3(C)NC(=O)NC3=O)CC2)c1
InChIKey: GHECMAZLBWUDCR-UHFFFAOYSA-N
Target: A disintegrin and metalloproteinase with thrombospondin motifs 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UNA0
Assay: In Vitro Assay: TABLE VIII: The basis for the assay is the cleavage of the substrate TBIS-1 (5 FAM-TEGEARGSVILLK (5TAMRA)K-NH2) (SEQ ID NO: 2) by human ADAMTS-5.For the dose response (10 point), 4 μL of a dilution series of compound (2 mM highest concentration, ⅕ dilution in DMSO further diluted 1 in 10 in water, corresponding to a final highest concentration of 20 μM), is transferred to 384 well Fluotrac 200 plate (Greiner, cat #781076) and incubated at room temperature for 30 min with a 26 μL buffer solution (50 mM Hepes pH7.5, 100 mM NaCl, 5 mM CaCl2, 0.1% CHAPS 1) containing hADAMTS-5 (1 ng/μL, affinity purified, followed by overnight digestion of 6His tag by thrombin and dialysis) (it will be appreciated by the skilled person that the potency read out is independent of the enzyme concentration).The reaction is initiated by adding to the assay plate TBIS-1 (10 μL, 4.5 μM, Anaspec) in the same buffer.Finally, the fluorescence is read on the Envision (Perkin Elmer) after an incubation of 45 min at 37° C. (Excitation 485 nm, emission 530).
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 390.44, ALogP 0.73, HBA 6, HBD 2, PSA 100.21, QED 0.69
Compound (SMILES): O=C(Nc1nc2cccc(-c3ccc(CC(=O)N4CCC4)cc3)n2n1)C1CC1
InChIKey: BNNRLFSZEKDCEJ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK2 Inhibition Assay: Recombinant human JAK2 catalytic domain (amino acids 808-1132; catalog number PV4210) was purchased from Invitrogen. 0.025 mU of JAK2 was incubated with 2.5 μg polyGT substrate (Sigma catalog number P0275) in kinase reaction buffer (5 mM MOPS pH 7.5, 9 mM MgAc, 0.3 mM EDTA, 0.06% Brij and 0.6 mM DTT, 1 μM non-radioactive ATP, 0.25 μCi 33P-gamma-ATP (GE Healthcare, catalog number AH9968) final concentrations) with or without 5 μL containing test compound or vehicle (DMSO, 1% final concentration), in a total volume of 25 μL, in a polypropylene 96-well plate (Greiner, V-bottom). After 90 min at 30° C., reactions were stopped by adding of 25 μL/well of 150 mM phosphoric acid. All of the terminated kinase reaction was transferred to prewashed (75 mM phosphoric acid) 96 well filter plates (Perkin Elmer catalog number 6005177) using a cell harvester (Perkin Elmer). Plates were washed 6 times with 300 μL per well of a 75 mM phosphoric acid solution and the bottom of the plates was sealed. 40 μL/well of Microscint-20 was added, the top of the plates was sealed and readout was performed using the Topcount (Perkin Elmer). Kinase activity was calculated by subtracting counts per minute (cpm) obtained in the presence of a positive control inhibitor (10 μM staurosporine) from cpm obtained in the presence of vehicle. The ability of a test compound to inhibit this activity was determined as:Percentage inhibition=((cpm determined for sample with test compound present−cpm determined for sample with positive control inhibitor) divided by (cpm determined in the presence of vehicle−cpm determined for sample with positive control inhibitor))*100%.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 375.43, ALogP 2.52, HBA 5, HBD 1, PSA 79.6, QED 0.74
Compound (SMILES): CN1CCN(c2cc(F)c(-c3ccnc(N4CCOCC4)c3)cc2NC(=O)c2c[nH]c(=O)cc2C(F)(F)F)CC1
InChIKey: IIHUZGGGNFKXJX-UHFFFAOYSA-N
Target: WD repeat-containing protein 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P61964
Assay: Surface Plasmon Resonance (SPR) Assay: Exemplary compounds of the application were dissolved in 100% DMSO at 10 mM, assayed fresh, and then stored at −20° C. for repeat studies and other experiments. Full length WDR5 with an N-terminal His tag and C-terminal AviTag (Avidity Inc.) was expressed in E. coli with coexpression of BirA to biotin labelled protein in vivo. Purification of the protein was performed using Ni-NTA. The purified WDR5 protein has a molecular weight of 41976 Da.SPR studies were performed using a Biacore T200 instrument (GE Health Sciences Inc.). Biotinylated WDR5 protein (approximately 3000 RU) was stably captured to streptavidin coupled SA chips according to the manufacture's protocol (GE Health Sciences Inc.). The running buffer used was HBS-EP (20 mM Hepes pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P-20) plus 5% DMSO with a flow rate of 40 μl/min. For SPR analysis, 5 different concentrations of each exemplary compound of the application were sprayed into 96 or 384 well plates using an HP D300 digital dispenser. The concentration ranged from about 195 nM to about 12 nM in a two-fold series. Concentration ranges were adjusted higher or lower for weaker or more potent compounds respectively when necessary. For the KD determinations, single cycle kinetic analysis was performed with an on time of 60 seconds, and an off time of 300 or 600 seconds. Curve fitting and KD calculations were performed with the Biacore T200 Evaluation software (GE Health Sciences Inc).
Measurement: Kd = 4.79 nM (pChEMBL 8.319999694824219)
Descriptors: MW 560.55, ALogP 3.44, HBA 7, HBD 2, PSA 93.8, QED 0.46
Compound (SMILES): Cc1c(-c2cccc(S(=O)(=O)N(C)C)c2)c2nc(O)ccc2n1C/C(F)=C/CN
InChIKey: RUJYRUBFVZJLCY-DHDCSXOGSA-N
Target: Amine oxidase [copper-containing] 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16853
Assay: Inhibit Human Recombinant Assay: Human recombinant SSAO/VAP-1 amine oxidase activity was determined using the coupled colorimetric method as described for monoamine oxidase, copper-containing amine oxidases and related enzymes (Holt A. and Palcic M., A peroxidase-coupled continuous absorbance plate-reader assay for flavin monoamine oxidases, copper-containing amine oxidases and related enzymes. Nat Protoc 2006; 1: 2498-2505). Briefly, a cloned cDNA template corresponding to residues 34-763 of human SSAO/VAP-1, and incorporating a mouse Ig kappa (κ) signal sequence, N-terminal flag epitope tag and tobacco etch virus (TEV) cleavage site, was assembled in a mammalian expression vector (pLO-CMV) by Geneart AG. This vector containing human SSAO/VAP-1 residues was transfected into CHO-K1 glycosylation mutant cell line, Lec 8. A clone stably expressing human SSAO/VAP-1 was isolated and cultured in large scale. Active human SSAO/VAP-1 was purified and recovered using immunoaffinity chromatography. This was used as the source for SSAO/VAP-1 activity. A high-throughput colorimetric assay was developed using either 96 or 384 well format. Briefly, in a standard 96 well plate assay 50 μL of purified human SSAO/VAP-1 (0.25 μg/mL) in 0.1 M sodium phosphate buffer (pH 7.4) was added into each well. Test compounds were dissolved in DMSO and tested in a Concentration Response Curve (CRC) with 4-11 data points, typically in the micromolar or nanomolar range after incubation with human SSAO/VAP-1 for 30 min at 37° C. After 30 min incubation, 50 μL of the reaction mixture containing 600 μM benzylamine (Sigma Aldrich), 120 μM Amplex Red (Sigma Aldrich) and 1.5 U/mL horseradish peroxidase (Sigma Aldrich) prepared in 0.1 M sodium phosphate buffer (pH 7.4) were added to the corresponding well. The fluorescence unit (RFU) was read every 2.5 min for 30 min at 37° C. excitation 565 nm and emission 590 (Optima; BMG labtech).
Measurement: IC50 > 3000 nM (pChEMBL 5.5229997634887695)
Descriptors: MW 418.49, ALogP 2.78, HBA 6, HBD 2, PSA 101.45, QED 0.64