lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCOCc1c(-c2cc3cc(C)cc(OC)c3s2)c2c(N)ncnn2c1C(=O)N1CCS(=O)(=O)C1
InChIKey: WTMSECTVVFONAK-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: High ATP Kinase Assay: For the assay, 50 nl of each stock solution of the test compound in DMSO was pipetted into a black, low-volume 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany) 2 Î¼l of a solution of the above FGFR-1 fusion protein in aqueous assay buffer [8 mM MOPS pH 7.0, 10 mM magnesium acetate, 1.0 mM dithiothreitol, 0.05% (w/v) bovine serum albumin (BSA), 0.07% (v/v) Tween-20, 0.2 mM EDTA] was added, and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compound to the enzyme. Then, the kinase reaction was started by the addition of 3 ul of a solution of adenosine triphosphate (ATP, 3.3 mM; final concentration in the 5 ul assay volume=2 mM) and substrate (0.16 uM; final concentration in the 5 ul assay volume=0.1 uM) in assay buffer, and the resulting mixture was incubated for a reaction time of 15 min at 22° C. The concentration of FGFR-1 fusion protein was adjusted depending on the activity of the enzyme lot.
Measurement: IC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 515.62, ALogP 2.87, HBA 10, HBD 1, PSA 129.12, QED 0.42
Compound (SMILES): O=C(O)[C@H]1CC[C@H](C(=O)N2CC[C@](c3ccc(C(F)(C(F)(F)F)C(F)(F)F)cc3)(S(=O)(=O)c3ccc(-c4ccccc4)cc3)C2)CC1
InChIKey: NDFHVLDJJGLJQH-JYUFKMNQSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Scintillation Proximity Assay (SPA): The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 ug/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 uM to 0.28 nM for 10 min at room temperature in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% Glycerol (Sigma Cat#G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat #RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce [3H] 25-hydroxycholesterol specific binding by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 4621 nM (pChEMBL 5.329999923706055)
Descriptors: MW 685.66, ALogP 7.44, HBA 4, HBD 1, PSA 91.75, QED 0.26
Compound (SMILES): COC(=O)Nc1ccc(-c2[nH]c([C@@H]3C[C@H](C4CCN(S(C)(=O)=O)CC4)CN3C(=O)N3CCN(C(=N)N)CC3)nc2Cl)cc1
InChIKey: LEKOLCVJVAPJGW-FPOVZHCZSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: In Vitro Assay: Human Factor XIa (Haematologic Technologies Inc.) activity was measured at an enzyme concentration of 0.1 U/mL in 150 mM NaCl, 5 mM KCl, 1 mg/mL PEG6000, 50 mM HEPES-NaOH (pH7.4) with 300 μM S-2366 (pyroGlu-Pro-Arg-pNA, Chromogenix).
Measurement: IC50 = 5.4 nM (pChEMBL 8.270000457763672)
Descriptors: MW 636.18, ALogP 2.57, HBA 7, HBD 4, PSA 181.05, QED 0.29
Compound (SMILES): Cc1cc(Oc2ccnc(NC(=O)C3CC3)c2)ccc1NC(=O)Nc1cc(F)cc(C(F)(F)F)c1
InChIKey: IYSAFTLNAIBVBC-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition Assay: [Experimental method]: Use Lance@Ultra Ulight™-TK assay kit from PerKinElmer to evaluate the inhibitory activity of compounds at KDR kinase. [Instrument]: PerKinElmer's ENVISION plate reader. [Materials]: Optiplate-384 well plate (PerKinElmer), kinase buffer (50 mM Hepes pH7.5, 0.25 mM EGTA, 2 mM DTT, 0.01% Tween 20, 10 mM Mg2+, 0.5 mM Mn2+), KDR kinase (790-1356 AA, Crown Bioscience), KDR kinase substrate (PerKinElmer catalogue # TRF0127-M), Lance@Eu-W1024-anti-phosphotyrosine (PT66) (PerKinElmer, catalogue #AD0068), ATP (Invitrogen), DMSO (Sigma, catalogue #34869), purified water (Millipore, type: Milli-Q). [Study conditions]: Mix KDR (final concentration: 20 nM) and compound (final DMSO: 0.5%), pre-incubate for 20 min at 30° C.; then add ATP (final concentration: 90 μM) and the substrate (final concentration 50 μM). React for 2 hrs at 30° C. After the reaction, add antibody and reaction for 60 min at 30° C. Read the plate (615 nm, 665 nm), calculate the ratio of the value at 665 nm vs 615 nm and analyze the data.
Measurement: IC50 = 17.5 nM (pChEMBL 7.760000228881836)
Descriptors: MW 488.44, ALogP 6.33, HBA 4, HBD 3, PSA 92.35, QED 0.35
Compound (SMILES): CC(=O)N1CCN(C(=O)CN2C(=O)N(c3cccc(C(F)(F)F)c3)C3=C(C(=O)CC3)C2c2ccc(C#N)cc2)CC1
InChIKey: RZPMVHCHMCSONK-UHFFFAOYSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Neutrophil Elastase Assay: Materials: Human neutrophil elastase was purchased from Calbiochem (Cat. No.: 324681) and the elastase substrate MeOSuc-Ala-Ala-Pro-Val-AMC from Bachem (Cat. No.: I-1270). All other materials were of the highest grade commercially available. The following buffers were used: Compound buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5; Assay buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5, containing 0.01% BSA. Assay conditions: Test compounds were prediluted in DMSO and subsequently in compound buffer (5% DMSO final). 5 uL of these compound dilutions were mixed with 10 ul Neutrophil elastase (9 ng/ml in assay buffer) in a black 384 well OptiPlate (Perkin Elmer, Cat No.: 6007270) and incubated for 15 min at room temperature. Subsequently 10 uL substrate solution in assay buffer were added (250 uM final concentration) and the plates were incubated for 60 min at room temperature. After inactivation of the enzyme, fluorescence intensities were measured at 380 nm excitation and 460 nm emission wavelengths. Each plate contains wells with a high value control (DMSO+enzyme+substrate) and wells with a low value control (DMSO+inactivated enzyme+substrate). IC50 values were estimated using a sigmoidal concentration response curve with variable slope.
Measurement: IC50 = 2.9 nM (pChEMBL 8.539999961853027)
Descriptors: MW 565.55, ALogP 3.87, HBA 5, HBD 0, PSA 105.03, QED 0.56
Compound (SMILES): Cc1ccc(C(=O)N2CC(c3ccc(C#N)cc3)C2)cc1-c1[nH]c(C2(C)COC2)nc1C
InChIKey: YRSJYTYGSJQUDX-UHFFFAOYSA-N
Target: Fatty acid synthase (SINGLE PROTEIN), Homo sapiens
UniProt: P49327
Assay: Biochemical Assay: The FASN enzyme was isolated from SKBr3 cells. SKBr3 is a human breast cancer cell-line with high levels of FASN expression. It is estimated that FASN comprises about 25% of the cytosolic proteins in this cell line. SKBr3 cells were homogenized in a dounce homogenizer then centrifuged for 15 minutes at 4° C. to remove particulate matter. The supernatant was then analyzed for protein content, diluted to the appropriate concentration, and used to measure FASN activity. The presence of FASN was confirmed by western blot analysis. A similar method for isolation of FASN from SKBr3 cells is described in Teresa, P. et al. (Clin. Cancer Res. 2009; 15(24), 7608-7615). FASN activity of the SKBr3 cell extract was determined by measuring either NADPH oxidation or the amount of thiol-containing coenzyme A (CoA) released during the fatty acid synthase reaction. The dye CPM (7-diethylamino-3-(4'-maleimidyl-phenyl)-4-methylcoumarin) contains a thiol reactive group that increases its fluorescence emission on reaction with the sulfhydryl group of CoA.
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 426.52, ALogP 4.09, HBA 4, HBD 1, PSA 82.01, QED 0.68
Compound (SMILES): CC(=O)N1CCCC(OCc2cncc(-c3cnc4c(c3)CCN4C(N)=O)c2)C1
InChIKey: SPKYGHOUVSPBOJ-UHFFFAOYSA-N
Target: Cytochrome P450 11B2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P19099
Assay: Aldosterone Synthase Inhibition Assay: The aldosterone synthase inhibition assay employs cynomolgus adrenal gland mitochondria as the source of aldosterone synthase (CYP11B2). Mitochondria are prepared from frozen cynomolgus monkey adrenal glands according to Method A described in by J. D. McGarry et al. (Biochem. J., 1983, 214, 21-28), with a final resuspension in the AT buffer described in R. Yamaguchi et al. (Cell Death and Differentiation, 2007, 14, 616-624), frozen as aliquots in liquid nitrogen and stored at -80° C. until use. Assays are performed in 96-well format in a final volume of 60 uL/well, containing 100 mM potassium phosphate, pH 7.4, 1% (v/v) DMSO, and additionally, 2 uM of corticosterone and 6 mg of mitochondrial protein. Reactions are started by the addition of NADPH to 1 mM and allowed to proceed for 60-90 minutes at 37° C. Reactions are terminated by the addition of 60 uL of acetonitrile. One hundred microliters are then transferred to a glass filter plate and centrifuged at 570xg.
Measurement: IC50 = 48.5 nM (pChEMBL 7.309999942779541)
Descriptors: MW 395.46, ALogP 2.11, HBA 5, HBD 1, PSA 101.65, QED 0.85
Compound (SMILES): Cc1ccc([C@@H](NC(=O)Cc2ccc3oc([C@@H](O)c4c(C)noc4C)cc3c2)c2ccccc2)c(C)c1
InChIKey: DIURRJOJDQOMFC-IOWSJCHKSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Inverse agonist activity at RORgamma (unknown origin) expressed in human Jurkat cells assessed as inhibition of IL-17F production after 20 hrs by luciferase reporter gene assay
Measurement: IC50 = 63 nM (pChEMBL 7.199999809265137)
Descriptors: MW 494.59, ALogP 6.18, HBA 5, HBD 2, PSA 88.5, QED 0.28
Compound (SMILES): COc1ccc(C(=O)C2CCN(CC(=O)NCc3nc4c(c(=O)[nH]3)COCC4)CC2)cc1OC
InChIKey: SRVUVWKCPPRJQZ-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase tankyrase-1 (SINGLE PROTEIN), Homo sapiens
UniProt: O95271
Assay: Biochemical Assay: The autoparsylation activity of the TNKS 1/2 or PARP1/2 enzymes was measured by the liquid chromatography-mass spectrometry (LC/MS) detection of nicotinamide as readout. Compound activity in inhibiting the TNKS and PARP autoparsylation was evaluated by IC50 measurements. In the compound screening assays, the reaction is composed of 5 μL of compound in 8-point serial dilutions with concentrations ranging from 0.0086 to 18.75 μM, 20 nM of purified enzyme, and 250 μM of β-NAD+ in the 1× Assay Buffer. After 60 min incubation at room temperature, the reactions were quenched by the addition of 10 μL of 5× quenching solution (20% formic acid and 500 nM [d]-nicotinamide in water). For the background control wells, 10 μL of the 5× quenching solution per well was added prior to the addition of β-NAD+.
Measurement: IC50 = 42 nM (pChEMBL 7.380000114440918)
Descriptors: MW 470.53, ALogP 1.07, HBA 8, HBD 2, PSA 122.85, QED 0.55
Compound (SMILES): Cc1ccc(C2=C(c3nn[nH]n3)C(=O)NC(c3ccc(OCCCCCC(F)(F)F)cc3)(C(F)(F)F)C2)cc1
InChIKey: WDLHNOKAVCEXRE-UHFFFAOYSA-N
Target: Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q10469
Assay: MGAT LCMS Assay: The MGAT enzyme reactions were performed in CORNING Falcon 96-well Polypropylene plates, in a total volume of 60 uL of 50 mM Potassium Phosphate buffer pH 7.4, containing a final concentration of 100 uM 2-oleoylglycerol, 15 uM oleoyl-Coenzyme A and 0.0013 ug/uL Human or Mouse MGAT-2 or 0.0026 ug/uL Rat recombinant MGAT-2 membranes expressed in Sf9 cells. Assay plates were run through a fully automated robotics system and shaken for 5 seconds every minute for a total 10 minutes. The reactions were then quenched with 120 uL of ice cold methanol containing 1 ug/mL 1,2-distearoyl-rac-glycerol as the internal standard. Plates were shaken for 2 minutes and spun down to remove protein precipitation. After the spin, samples were transferred to LC/MS compatible PCR plates. For LC/MS analysis, a ThermoFisher Surveyor pump, utilizing a Waters Symmetry C8, 50x2.1 mm column, was used for the chromatography of enzyme products.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 553.51, ALogP 5.9, HBA 5, HBD 2, PSA 92.79, QED 0.25
Compound (SMILES): [C-]#[N+]c1ccc(N2C(=O)C3(CCCC3)N(c3ccc(C)cc3)C2=S)cc1C(F)(F)F
InChIKey: URHBUNBGEQPYFK-UHFFFAOYSA-N
Target: Androgen receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P10275
Assay: Reporter Assay: The compounds were subjected to tests using an artificial AR response reporter system in a hormone refractory prostate cancer cell line. In this system, the prostate cancer LNCaP cells were engineered to stably express about 5-fold higher level of AR than endogenous level. The exogenous AR has similar properties to endogenous AR in that both are stabilized by a synthetic androgen R1881. The AR-over expressed cells were also engineered to stably incorporate an AR response reporter and the reporter activity of these cells shows features of hormone refractory prostate cancer.
Measurement: IC50 = 147 nM (pChEMBL 6.829999923706055)
Descriptors: MW 429.47, ALogP 6.02, HBA 2, HBD 0, PSA 27.91, QED 0.43
Compound (SMILES): O=C(NC1CC=C(c2ccc(NC(=O)N3Cc4ccncc4C3)cc2)CC1)[C@@H]1CCCO1
InChIKey: VZXNRXCCBRFQHT-YANBTOMASA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 2:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1287128.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 12.8 nM (pChEMBL 7.889999866485596)
Descriptors: MW 432.52, ALogP 3.86, HBA 4, HBD 2, PSA 83.56, QED 0.77
Compound (SMILES): Cc1oc2c(c1CC(=O)/N=c1\scc(-c3ccc(OC(F)(F)F)c(Cl)c3)n1COP(=O)(O)O)c(=O)n(C)c(=O)n2C
InChIKey: MBFAPSKWPISCAC-OOAXWGSJSA-N
Target: Transient receptor potential cation channel subfamily A member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75762
Assay: Radiometric Calcium Assay: The inhibition of TRPA1 receptor activation was measured as inhibition of allylisothiocyanate (AITC) induced cellular uptake of radioactive calcium. Test compounds were dissolved in DMSO to prepare 10 mM stock solution and then diluted using plain medium with 0.1% BSA and 1.8 mM CaCl2 to get desired concentration. Final concentration of DMSO in the reaction was 0.5% (v/v). Human TRPA1 expressing CHO cells were grown in F-12 DMEM medium with 10% FBS, 1% penicillin-streptomycin solution, and 400 ug/ml of G-418. Cells were seeded 24 h prior to the assay in 96 well plates so as to get 50,000 cells per well on the day of experiment. Cells were treated with test compounds for 10 min followed by addition of AITC at a final concentration of 30 micromolar and 5 uCi/ml 45Ca+2 for 3 min at 25° C. Cells were washed and lysed using buffer containing 1% Triton X-100, 0.1% deoxycholate and 0.1% SDS. Radioactivity in the lysate was measured in Packard Top count.
Measurement: IC50 = 1180 nM (pChEMBL 5.929999828338623)
Descriptors: MW 638.9, ALogP 2.96, HBA 11, HBD 2, PSA 167.49, QED 0.29
Compound (SMILES): c1ccc2oc(-c3cnc4ccc(NCc5ccncc5)nn34)cc2c1
InChIKey: AJBOLFIHFBPBBR-UHFFFAOYSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BUB5
Assay: Kinase Assay: A recombinant fusion protein of Glutathione-S-Transferase (GST, N-terminally) and human full-length MKNK1 (amino acids 1-424 and T344D of accession number BAA 19885.1), expressed in insect cells using baculovirus expression system and purified via glutathione sepharose affinity chromatography, was purchased from Carna Biosciences (product no 02-145) and used as enzyme. As substrate for the kinase reaction the biotinylated peptide biotin-Ahx-IKKRKLTRRKSLKG (C-terminus in amide form) was used which can be purchased e.g. form the company Biosyntan (Berlin-Buch, Germany).For the assay 50 nL of a 100 fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 μL of a solution of MKNK1 in aqueous assay buffer [50 mM HEPES pH 7.5, 5 mM magnesium chloride, 1.0 mM dithiothreitol, 0.005% (v/v) Nonidet-P40 (Sigma)] was added and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compounds to the enzyme before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 μL of a solution of adenosine-tri-phosphate (ATP, 16.7 μM=>final conc. in the 5 μL assay volume is 10 μM) and substrate (0.1 μM=>final conc. in the 5 μL assay volume is 0.06 μM) in assay buffer and the resulting mixture was incubated for a reaction time of 45 min at 22° C. The concentration of MKNK1 was adjusted depending of the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical concentrations were in the range of 0.05 μg/ml. The reaction was stopped by the addition of 5 μL of a solution of TR-FRET detection reagents (5 nM streptavidine-XL665 [Cisbio Bioassays, Codolet, France] and 1 nM anti-ribosomal protein 56 (pSer236)-antibody from Invitrogen [#44921G] and 1 nM LANCE EU-W1024 labeled ProteinG [Perkin-Elmer, product no. AD0071]) in an aqueous EDTA-solution (100 mM EDTA, 0.1% (w/v) bovine serum albumin in 50 mM HEPES pH 7.5).
Measurement: IC50 = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 341.37, ALogP 4.15, HBA 6, HBD 1, PSA 68.25, QED 0.53
Compound (SMILES): C[C@@H]1CN(c2ccc(F)cc2)CCN1C(=O)c1ccc2c(=O)n(-c3ccc(F)cc3)c(CCCCC(=O)NS(=O)(=O)C3CC3)cc2c1
InChIKey: SFUMRSQYSSZVOI-HSZRJFAPSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Radioligand Binding Assay: Radioligand binding was performed using filtration or scintillation proximity assay (SPA) technology. SPA assays were done at room temperature in 10 mM HEPES pH 7.4, 1 mM EDTA containing 2 mM MnCl2 and 16 nM [3H]-PGD2 (PerkinElmer, Waltham, Mass.) (164 Ci mmol-1), in a final volume of 0.05 mL. Competing ligands were diluted in dimethylsulfoxide and added using very low volumes (500 nL). The reaction was initiated by the addition of a mixture of 3.52 ug of membrane protein prepared from a human embryonic kidney (HEK)-hCRTH2 cell line adhered to 140 ug of wheatgerm agglutinin SPA beads (PerkinElmer). Total and non-specific binding were determined in the absence and the presence of a CRTH2 antagonist, respectively. The reaction was routinely conducted for 60 minutes at room temperature followed by centrifugation for 5 minutes at 1000 RPM. The radioactivity was measured with a TopCountNXT (PerkinElmer). Filtration binding assays were done in a similar way with minor differences. The assay volume was 0.20 mL and competing ligands in dimethylsulfoxide were added in 2 uL. [3H]-PGD2 was used at 0.6 nM, and reactions were initiated by the addition of 10 ug of membrane protein. Reactions were terminated by filtration through GF/C filter plates (PerkinElmer) presoaked in 10 mM HEPES. After washing with buffer, plates were dried in a 50° C. oven for 1 h, scintillation cocktail was added and radioactivity was measured. Results determined using the SPA-based assay were similar to those from the filtration binding assay.
Measurement: Ki = 17.03 nM (pChEMBL 7.769999980926514)
Descriptors: MW 662.76, ALogP 4.94, HBA 7, HBD 1, PSA 108.79, QED 0.24
Compound (SMILES): C[C@@]1(c2nc(Nc3ncccc3C#N)ccc2F)CO[C@@](C)(C(F)(F)F)C(N)=N1
InChIKey: RNILDPYSUMNZGG-DLBZAZTESA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE-1 Inhibition Assay: Recombinant BACE-1 (extracellular domain, expressed in baculovirus and purified using standard methods) at 0.1 to 1 nM concentrations is incubated with the test compound at various concentrations for 1 hour at room temperature in 100 mM acetate buffer, pH 4.5, containing 0.1% CHAPS. Activity was measured using a final concentration of 3 uM of the fluorescence-quenched substrate Q-C(HSO3)-Ile-Asp-Leu-Ala-Val-Leu-Asp-HN-CH2-CH2-Mca, where Q=2-nitro-5-amino benzoic acid and Mca=7-methoxy-4-coumarinyl acetic acid. Catalytic turnover was monitored in a Spectramax Gemini fluorescence plate reader (Molecular Devices) in black 96-well microplates using excitation/emission wavelength of 325 nm and 400 nm, respectively. Fluorescence increase was followed for 15 min, in 1 minute's intervals.
Measurement: IC50 = 1500 nM (pChEMBL 5.820000171661377)
Descriptors: MW 408.36, ALogP 3.15, HBA 7, HBD 2, PSA 109.21, QED 0.76
Compound (SMILES): CN1C(=N)N[C@](C)(c2sc3c(-c4ccc(-c5cnco5)cc4F)nccc3c2Cl)CS1(=O)=O
InChIKey: PQCJKIZLGHAAMH-NRFANRHFSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: HTRF FRET Assay: Varying concentrations of inhibitors at 3× the final desired concentration in a volume of 10 μl are preincubated with purified human BACE1 catalytic domain (3 nM in 10 μl) for 30 minutes at 30° C. in reaction buffer containing 20 mM Na-Acetate pH 5.0, 10% glycerol, 0.1% Brij-35 and 7.5% DSMO. Reactions are initiated by addition of 10 μl of 600 nM QSY7-APPswe-Eu substrate (200 nM final) to give a final reaction volume of 30 in a 384 well Nunc HTRF plate. The reactions are incubated at 30° C. for 1.5 hours. The 620 nm fluorescence is then read on a Rubystar HTRF plate reader (BMG Labtechnologies) using a 50 millisecond delay followed by a 400 millisecond acquisition time window.
Measurement: Ki = 35.16 nM (pChEMBL 7.449999809265137)
Descriptors: MW 505.98, ALogP 4.43, HBA 7, HBD 2, PSA 112.18, QED 0.43
Compound (SMILES): COc1ncc(-c2ccc3ncnc(-c4cccc(C(=O)N5CCN(C(C)=O)CC5)c4)c3c2)cc1C#N
InChIKey: QYTXJLQBSYAMGR-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Inhibition of PI3Kdelta (unknown origin) assessed as reduction in ADP formation using phosphatidyl inositol as substrate after 30 to 60 mins by TR-FRET assay
Measurement: IC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 492.54, ALogP 3.54, HBA 7, HBD 0, PSA 112.31, QED 0.43
Compound (SMILES): CS(=O)(=O)N1CCC(c2ccc(CN(CC(F)(F)F)S(=O)(=O)Cc3ccccc3)c(F)c2)CC1
InChIKey: AWRRTBGEDHTOQO-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: In Vitro Assay: On day of the assay, 100 uL of 0.05% CHAPS (in deionized H2O) was added to all wells of the GFB Unifilter plate and allowed soak for 1 h. A wash buffer of 50 mM HEPES (pH 7.4), 150 mM NaCl, and 5 mM MgCl2 was prepared to wash the filter plate. To prepare an assay buffer, BSA was added to the wash buffer to reach 0.01% and DTT was added to reach 1 mM. For IC50 mode, 10 mM compound stocks were serially diluted in DMSO with DMSO to give 20x required final concentration in DMSO (15 uL compound+30 uL DMSO). The 20x compound stocks were diluted in DMSO with Assay Buffer 4-fold to reach 5x the final test concentration in 25% DMSO (10 uL compound+30 uL Assay Buffer). Solutions were mixed by aspiration several times with a pipette set on 50 uL volume. For the assay, 10 uL of 5x compound stock solutions in 25% DMSO were added to the assay plate in duplicate.
Measurement: IC50 = 65 nM (pChEMBL 7.190000057220459)
Descriptors: MW 522.59, ALogP 3.86, HBA 4, HBD 0, PSA 74.76, QED 0.49
Compound (SMILES): CC(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](C)C(=O)N[C@@H]1CSSC[C@@H](C(=O)O)NC(=O)[C@H](C)NC(=O)[C@H](Cc2ccccc2)NC(=O)[C@H](CCCNC(=N)N)NC1=O
InChIKey: WPHCAUSMODTBQF-PCYMNQSWSA-N
Target: C-X-C chemokine receptor type 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P61073
Assay: Inhibition of PE-conjugated-12G5 anti-CXCR4 antibody binding to CXCR4 in human CEM-CCRF cells preincubated for 30 mins followed by antibody addition by FACS Canto II cytofluorometric analysis
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 866.04, ALogP -3.31, HBA 12, HBD 14, PSA 364.8, QED 0.03
Compound (SMILES): C=CC(=O)N1CC2(CNc3c(C(N)=O)c(-c4ccc(Oc5ccccc5)cc4)nn3C2)C1
InChIKey: VIYKHGUZUDNUIC-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Time-Resolved Fluorescence Resonance Energy Transfer Assay: Compounds disclosed herein were tested for inhibition of Btk kinase activity in an assay based on time-resolved fluorescence resonance energy transfer methodology. Recombinant Btk was pre-incubated with the compounds disclosed herein at room temperature for 1 hour in an assay buffer containing 50 mM Tris pH7.4, 10 mM MgCl2, 2 mM MnCl2, 0.1 mM EDTA, 1 mM DT, 20 nM SEB, 0.1% BSA, 0.005% tween-20. The reactions were initiated by the addition of ATP (at the concentration of ATP Km) and peptide substrate (Biotin-AVLESEEELYSSARQ-NH2). After incubating at room temperature for 1 h, an equal volume of stop solution containing 50 mM HEPES pH7.0, 800 mM KF, 20 mM EDTA, 0.1% BSA, Eu cryptate-conjugated p-Tyr66 antibody and streptavidin-labeled XL665 was added to stop the reaction. Plates were further incubated at room temperature for 1 hour, and then the TR-FRET signals (ex337 nm, em 620 nm/665 nm) were read on BMG PHERAstar FS instrument. The residual enzyme activity in presence of increasing concentrations of compounds was calculated based on the ratio of fluorescence at 615 nm to that at 665 nm. The IC50 for each compound was derived from fitting the data to the four-parameter logistic equation by Graphpad Prism software.
Measurement: IC50 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 429.48, ALogP 2.88, HBA 6, HBD 2, PSA 102.48, QED 0.61
Compound (SMILES): O=C(CCCCCN1CCN(c2ccccc2-c2ccccc2)CC1)NCc1ccc(OCCN2CCOCC2)cc1
InChIKey: NKJBABKLZJYEME-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: P34969
Assay: Displacement of [3H]-5-CT from human 5-HT7b receptor expressed in HEK293 cell membranes after 1 hr by microbeta counting analysis
Measurement: Ki = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 570.78, ALogP 5.06, HBA 6, HBD 1, PSA 57.28, QED 0.27
Compound (SMILES): O=C1NCCc2[nH]c(-c3cccc4ccc(Nc5ccc(F)cc5F)nc34)cc21
InChIKey: FISXIELUYFZCMF-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Homogeneous Time Resolved Fluorescence Assay (HTRF): The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction. Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 μM. A reference compound was included on each assay plate in order to validate that plate; on one plate of every assay run, two additional reference compounds were included. The final buffer conditions were as follows: 60 mM Hepes, pH 7.0, 0.05% BSA, 2 mM DTT. Incubations were carried out at RT (22° C.) for 2 h for Pim-1, 1 hour and 30 min for Pim-3, and 45 min for Pim-2. The reaction was stopped by the addition of 3 mM EDTA, and fluorescence was measured by an HTRF® Rubystar microplate reader.
Measurement: IC50 = 2.8 nM (pChEMBL 8.550000190734863)
Descriptors: MW 390.39, ALogP 4.54, HBA 3, HBD 3, PSA 69.81, QED 0.48
Compound (SMILES): [C-]#[N+]c1ccc2c(c1)[C@@]1(N=C(C)C(N)=N1)[C@@]1(C2)C[C@@H](C)[C@H](OC)[C@@H](C)C1
InChIKey: XDEYYVVDSVTBMO-RSFMMIOLSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: Fluorescence Quench Assay: The assay was performed in the presence of OptiMEM (supernatant collected over 24 h and cleared from cellular debris by centrifugation) containing the ectodomain of BACE1, 25 μl water containing the desired 2-fold concentration of test compound and 2% 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 added to the plate followed by the addition of 10 μl of BACE1 containing OptiMEM diluted 1:10 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 rt (dark) in an Envision® multilabel reader (Perkin Elmer) and the cleavage of the substrate was recorded as kinetic for 60 min at ex: 485 nm, em: 538 nm. Blank wells containing no enzyme were included on each plate.
Measurement: IC50 = 3.5 nM (pChEMBL 8.460000038146973)
Descriptors: MW 350.47, ALogP 3.85, HBA 4, HBD 1, PSA 64.33, QED 0.78
Compound (SMILES): Nc1nc(-n2ncc3ccccc32)nc(NC2CCOCC2)c1Br
InChIKey: PXOKRGLRJOJXLP-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: [3H] cAMP SPA Assay: The analysis of the activity of the enzyme Phosphodiesterase [3H] cAMP SPA (Perkin Elmer #7090 TRKQ) kit was used for the study of Phosphodiesterase 10. The kit contains:Yttrium scintillation proximity assay (SPA) in beads: yttrium silicate in microspheres resuspended pellet in MiliQ water containing 18 mM zinc sulfate.    10×PDE assay buffer: 500 mM Tris/HCl; 83 mM MgCl2; 17 mM EGTA; pH 7.5.    Tracer [3H] cAMP: substrate for enzymatic reaction.The test is based on the preferential binding of linear nucleotides to the SPA beads in the presence of zinc sulfate, with respect to the binding of cyclic nucleotides, which is almost imperceptible. Therefore, in optimum conditions, the product of the enzymatic reaction binds directly to SPA, and the substrate of the enzyme does not.
Measurement: IC50 = 21.9 nM (pChEMBL 7.659999847412109)
Descriptors: MW 389.26, ALogP 2.75, HBA 7, HBD 2, PSA 90.88, QED 0.72
Compound (SMILES): CC1(C)CCN(c2cccc(Nc3cc(-c4ccccc4)nn4ccnc34)n2)C1
InChIKey: IJHAVTWLPGJNJI-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Inhibition Assay: Assay plates: 96-well MultiScreen 0.65 um filter plates (Millipore Cat. No.: MADVNOB10)Streptavidin coated beads: Streptavidin Sepharose, suspension 5.0 mL, in 50 mM EDTA/PBS diluted (1:100), (Amersham, Cat. No.: 17-5113-01)Compounds: 10 mM in 100% dimethylsulfoxide (DMSO), final conc.: compound 0.003-100 uM in 10% DMSOEnzyme: SYK RPA purified, truncated construct of Spleen Tyrosine Kinase aa 360-635, stock solution 1 mg/mL, MW: 31.2 KDa, final conc.:0.0005 uM. Peptide 1: biotinylated peptide is derived from a naturally occurring phosphor-acceptor consensus sequence (Biotin-EPEGDYEEVLE), special order from QCB, stock solution 20 mM, final conc.: 5.0 uM.ATP: Adenosine-5'-triphosphate 20 mM, (ROCHE Cat. No.: 93202720), final concentration: 20 uM Buffer: HEPES: 2-Hydroxyethyl piperazine-2-ethanesulfonic acid (Sigma, Cat. No.: H-3375)final concentration: 50 mM HEPES pH7.5BSA: Bovine Serum Albumin Fraction V, fatty acid free (Roche Diagnostics GmbH, Cat. No. 9100221).
Measurement: IC50 = 1047.83 nM (pChEMBL 5.980000019073486)
Descriptors: MW 384.49, ALogP 4.77, HBA 6, HBD 1, PSA 58.35, QED 0.55
Compound (SMILES): CSc1nc2c(c(Nc3ccc(C(C)(C)C)cc3)n1)CCN(c1ncccc1C(F)(F)F)CC2
InChIKey: KPFJDJSPOFTRKT-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: Functional Assay: HEK293 cells were transfected with human TRPV1 cloned in pcDNA3.1zeo(+) using the Effectene non-liposomal lipid based transfection kit (Qiagen) (hTRPV1/HEK293). hTRPV1/HEK293 cells were routinely grown as monolayers under selection in zeocin (200 μg/mL; Invitrogen) in Dulbecco's Modified Eagle Medium (DMEM, Gibco BRL) supplemented with 10% fetal bovine serum, and penicillin/streptomycin (50 units/mL) in 5% CO2 at 37° C. Cells were passaged frequently, every 3-5 days, to avoid overgrowth, depletion of essential medium components, or acidic medium exposure. Cells were passaged using a brief wash in 0.05% trypsin with 1 mM EDTA, followed by dissociation in divalent-free phosphate-buffered saline (Hyclone #SH30028.02). Dissociated cells were seeded onto poly-D-lysine coated black-walled 96-well plates (Biocoat; Becton Dickinson #354640) at about 40,000 cells per well and grown for approximately 1 day in culture medium to near confluency. The assay buffer was composed of 130 mM NaCl, 2 mM KCl, 2 mM MgCl2, 10 mM HEPES, 5 mM glucose, and either 2 mM or 20 μM CaCl2. On the day of the experiment, the culture medium was replaced with 2 mM calcium assay buffer using an automated plate washer (ELx405; Biotek, VT). The cells were incubated in 100 μL/well Fluo-3/AM (2 μM; TEFLabs #0116) with Pluronic F127 (100 μg/mL; Sigma #P2443) for 1 h at rt in the dark. After loading the cells, the dye solution was replaced with 50 μL/well of 20 μM calcium assay buffer using the ELx405 plate washer. Test compounds (50 μL/well) were added to the plate and incubated for 30 min. Intracellular Ca2+ levels were subsequently assayed using a Fluorometric Imaging Plate Reader (FLIPR™ instrument, Molecular Devices, CA) to simultaneously monitor Fluo-3 fluorescence in all wells (λexcitation=488 nm, λemission=540 nm) during challenge with agonist (capsaicin). The IC50 values were determined. Cells were challenged with 150 nM capsaicin and the fluorescence counts were captured following agonist addition at a sampling rate of 0.33 Hz. The contents of the wells were mixed 3 times (40 μL mix volume) immediately after the additions were made. Concentration dependence of block was determined by exposing each well of cells in duplicate rows of a 96 well plate to a serial dilution of test compound. The concentration series usually started at 10 Î¼M with a three-fold serial decrement in concentration. The magnitude of the capsaicin response was determined by measuring the change in fluo3 fluorescence before and 100 seconds after the addition of the agonist.
Measurement: IC50 = 2930 nM (pChEMBL 5.53000020980835)
Descriptors: MW 487.6, ALogP 6.26, HBA 6, HBD 1, PSA 53.94, QED 0.35
Compound (SMILES): CCOCc1cc2cnc(Nc3ccc(N4CCOCC4)cn3)nc2n(C2CCCC2)c1=O
InChIKey: AFNOMJNGXFOVCJ-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 4/cyclin D1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24385
Assay: Inhibition of Cdk4/Cyclin D (unknown origin) using retinoblastoma protein substrate and [gamma32P]ATP incubated for 15 mins by beta plate counter based assay
Measurement: IC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 450.54, ALogP 3.42, HBA 9, HBD 1, PSA 94.4, QED 0.59
Compound (SMILES): O=C(O)c1cccc(Nc2ccc(Cl)c(Cl)c2)c1
InChIKey: QCNWEXKXKUAGSV-UHFFFAOYSA-N
Target: Kynurenine 3-monooxygenase (SINGLE PROTEIN), Homo sapiens
UniProt: O15229
Assay: Inhibition of human KMO
Measurement: IC50 = 9500 nM (pChEMBL 5.019999980926514)
Descriptors: MW 282.13, ALogP 4.44, HBA 2, HBD 2, PSA 49.33, QED 0.87
Compound (SMILES): COc1cc(N2CC(C(F)(F)F)C2)c(F)cc1-n1c(=O)ccc2cc(S(=O)(=O)Nc3ccon3)ccc21
InChIKey: PRDKHLMNPASIBW-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Nav1.7 In Vitro PX Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to -10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes.
Measurement: IC50 = 495 nM (pChEMBL 6.300000190734863)
Descriptors: MW 538.48, ALogP 3.93, HBA 8, HBD 1, PSA 106.67, QED 0.37
Compound (SMILES): COC(=O)Nc1nc2cc(C3(O)c4ccccc4C(=O)N3Cc3ccc(C)cc3)ccc2[nH]1
InChIKey: ACJKPTOGQNRUEV-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition of B-Raf (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 442.48, ALogP 3.9, HBA 5, HBD 3, PSA 107.55, QED 0.44
Compound (SMILES): C=CCN(c1c(C)nn(C)c1C)S(=O)(=O)c1c(Cl)cc(Br)cc1Cl
InChIKey: GHTMBEKLBMVOOW-UHFFFAOYSA-N
Target: Glycylpeptide N-tetradecanoyltransferase (SINGLE PROTEIN), Trypanosoma brucei brucei (strain 927/4 GUTat10.1)
UniProt: Q388H8
Assay: Enzyme Inhibition Assay: Measurements were performed using a modification of the scintillation proximity assay platform described previously by Georgopapadakou, N. H. et al. (22nd International Congress on Chemotherapy, 2001, Abstract P16.001). Compounds were solubilised in DMSO at a top concentration of 10 mM and serially diluted in half log steps to achieve a range of final assay concentrations of 100 uM to 1 nM. Compound at each concentration (100-fold final) was added to white 384 well plates in a volume of 0.5 ml. Human, A. fumigatus, T. brucei or L. major N-myristoyl transferase enzyme, dissolved to a working concentration of 10 nM in assay buffer (30 mM Tris/HCl pH 7.4, 0.5 mM EGTA, 0.5 mM EDTA, 1.25 mM DTT, 0.1% Triton X-100), was then added to columns 1 to 11 and 13 to 23 of the plates in a volume of 20 ml. To columns 12 and 24, 20 ml assay buffer was added to provide a no enzyme control. Following a 5 minute incubation at room temperature the substrates (GCGGSKVKPQPPQAK(Biotin)-Amide and myristoyl coenzyme A), dissolved in assay buffer, were added to all wells in a volume of 20 ml to start the reaction. The final concentrations of peptide and 3H-myristoyl coenzyme A were 0.5 mM and 125 nM respectively and the specific activity of the radiolabel was 8 Ci/mmol. Plates were then incubated at room temperature for up to 50 minutes (dependant upon the period of linearity for the different enzyme species) before SPA beads, suspended to 1 mg/ml in a stop solution (200 mM Phosphoric Acid/NaOH pH 4, 750 mM MgCl2), were added in a volume of 40 ml.
Measurement: IC50 = 900 nM (pChEMBL 6.050000190734863)
Descriptors: MW 453.19, ALogP 4.49, HBA 4, HBD 0, PSA 55.2, QED 0.63
Compound (SMILES): N#Cc1ccc(N2C(=O)OC[C@H]2[C@@H](O)C(F)(F)F)cc1Cl
InChIKey: OCLUFVULSBZQOP-VHSXEESVSA-N
Target: Androgen receptor (SINGLE PROTEIN), Rattus norvegicus
UniProt: P15207
Assay: Biological Assay: In order to demonstrate the utility of the compounds of this invention, an androgen receptor binding assay was performed wherein many of the compounds of this invention are shown to demonstrate significant affinity for the androgen receptor. The assay was performed as specified by the manufacturer (Invitrogen, Madison, Wis.). Briefly, 1 ul of 10 mM compound was added to 500 ul of AR screening buffer in a 1.5 ml eppendorf tube to make a 2x10E-5M stock. 10-fold serial dilutions of the test compounds were prepared ranging in concentration from 10E-5M to 10E-12M. Each dilution was added in triplicate to a black 384-microtiter plate. The test compounds will be diluted 2-fold in the final reaction. 2x AR-Fluormone complex was prepared with 2 nM Flourmone AL Green and 30 nM AR. 25 ul of 2x complex was aliquoted to each reaction well, such that the final reaction volume was 50 ul per well.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 320.65, ALogP 2.46, HBA 4, HBD 1, PSA 73.56, QED 0.91
Compound (SMILES): COc1cccc(OC)c1C1CCCCC(=O)N1Cc1cccc(-c2ncccn2)c1
InChIKey: XKUGHTYMIFCAQZ-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: In Vitro Assay: Intracellular Calcium Measurements : In Vitro Assay: Intracellular Calcium Measurements:Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 g/ml G418, 100 U/ml penicillin, 100 g/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2. 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 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M 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.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 417.51, ALogP 4.8, HBA 5, HBD 0, PSA 64.55, QED 0.58
Compound (SMILES): COc1cc(C)cc(OC)c1-c1cc(Cl)cc(C(CC(=O)O)c2nnc(C)o2)c1
InChIKey: OYWDYVMHSSTKHM-UHFFFAOYSA-N
Target: Lysosomal protective protein (SINGLE PROTEIN), Homo sapiens
UniProt: P10619
Assay: Inhibition Assay: Recombinant human cathepsin A (residues 29-480, with a C-terminal 10-His tag; R&D Systems, #1049-SE) was proteolytically activated with recombinant human cathepsin L (R&D Systems, #952-CY). Briefly, cathepsin A was incubated at 10 ug/ml with cathepsin L at 1 ug/ml in activation buffer (25 mM 2-(morpholin-4-yl)-ethanesulfonic acid (MES), pH 6.0, containing 5 mM dithiothreitol (DTT)) for 15 min at 37° C. Cathepsin L activity was then stopped by the addition of the cysteine protease inhibitor E-64 (N-(trans-epoxysuccinyl)-L-leucine-4-guanidinobutylamide; Sigma-Aldrich, # E3132; dissolved in activation buffer/DMSO) to a final concentration of 10 uM.The activated cathepsin A was diluted in assay buffer (25 mM MES, pH 5.5, containing 5 mM DTT) and mixed with the test compound (dissolved in assay buffer containing (v/v) 3% DMSO) or, in the control experiments, with the vehicle in a multiple assay plate. After incubation for 15 min at room temperature.
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 416.86, ALogP 4.63, HBA 6, HBD 1, PSA 94.68, QED 0.6
Compound (SMILES): O=C(NC1CCc2cc(Cl)cc(Cl)c21)C1CCOc2ncsc21
InChIKey: OLPOMNJRPMMVAZ-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: YO-PRO Assay: Cells were grown to confluency in adherent culture at 37° C. in a humidified 5% CO2 incubator (split 1/5 every 3-4 days with DMEM, 10% FCS, 1% Penicillin/Streptomycin, 250 μg/ml Geneticin). Adherent cells were detached by incubation with Trypsine (1 ml per 165 cm2 dish) for 2 minutes, then washed off with 10 ml PBS (without Mg2+ and Ca2+), and resuspended in DMEM, 10% FCS, 1% Penicillin/Streptomycin, no Geneticin. 10,000 cells per well (48 hours before the assay) or 25,000 cells per well (Vi-cell XR (Beckman Coulter) (24 hours before the assay) in 50 μl full medium were seeded on 384-well black-wall, clear bottom plates, that were coated before with 10 μl per well Poly-L-Lysine, incubated for 30-60 minutes at 37° C. and washed once with PBS. Medium was removed from cells and 50 μl of assay buffer containing 0.5 μM YO-PRO-1 was added into the wells. Solutions of antagonist compounds were prepared by serial dilutions of a 10 mM DMSO solution of the antagonist into PBS using a BioMek (Beckman Coulter). Each concentration was performed in duplicate. For IC50 measurements 10 concentration points were measured (10 μM being the highest concentration followed by 9 serial dilution steps 1/3). The cells were incubated with the antagonists of the present invention together with ATP at a final concentration of 250 μM for 90 minutes. During this time period, four time points were taken. Each time point comprised the average of several measurements made within a few seconds. Fluorescence was measured in the FLIPR tetra (Molecular Devices) using the filters appropriate for YO-PRO-1 fluorescence (excitation 485/20, emission 530/25). The FLIPR tetra was equipped with Molecular Devices Screen Works system control software to define and run experimental protocols.
Measurement: IC50 = 1.8 nM (pChEMBL 8.739999771118164)
Descriptors: MW 369.27, ALogP 4.12, HBA 4, HBD 1, PSA 51.22, QED 0.87
Compound (SMILES): Cc1ccc2c(n1)OCC2NC(=O)Cc1cccc(C(F)(F)F)c1Cl
InChIKey: INLSXROHBSUFNR-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: In Vitro Assay: Cell line generation was performed in general according to established molecular cloning protocols. Specifically, RNA was extracted from human whole blood using the Qiagen RNeasy kit (Qiagen, CH) according to the manufacturer's instructions. Subsequently cDNA was made (Superscript II, Invitrogen AG, CH) and the human P2X7 gene (genbank ref. BC011913) was amplified with the following primers: ATCGCGGCCGCTCAGTAAGGACTCTTGAAGCCACT and CGCCGCTAGCACCACCATGCCGGCCTGCTGCAGCTGCA. The amplified sequence was subsequently ligated into a pcDNA3.1 (+) NotI, NheI digested plasmid. Human embryonic kidney (HEK) cells (ATCC CRL-1573, Manassas, Va., USA) were transfected with the pcDNA3.1 (+). hP2X7 plasmid using lipofectamine 2000 (Invitrogen AG, CH) according to the manufacturer's instructions. Following a 24 h exposure to DNA, cells were trypsinized and re-seeded at low density in the presence of 250 ug Geneticin. Geneticin resistant cells were then selected during two consecutive rounds of cloning by serial limiting dilution with visual inspection.
Measurement: IC50 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 370.76, ALogP 3.85, HBA 3, HBD 1, PSA 51.22, QED 0.89
Compound (SMILES): CCS(=O)(=O)N(CCN1CCOCC1)c1sc2ccccc2c1C(C)O
InChIKey: LFKMIVWZEUJCBT-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Patch Clamp Assay: For patch clamp experiments, HEK293 cells are stably transfected with canine TRPM8 and cultured in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin and 1 mg/ml G418. Cells are maintained at 37° C. and in 5% CO2.The extracellular solution contains (in mM): NaCl, 132; EGTA, 1; KCl, 5.4; MgCl2, 0.8; HEPES, 10; glucose, 10; pH=7.4. Recordings are performed using the conventional whole-cell patch clamp technique, 1-2 days after plating cells onto glass coverslips at densities appropriate for single cell recording. Currents are amplified by a patch clamp amplifier and filtered at 2 kHz (Axopatch 200B, Molecular Devices, Union City, Calif.). Menthol (100 μM) is applied to the cell at 0.5 ml/min via a gravity-fed perfusion system. Recordings involving menthol activation are performed at 22° C.
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 398.55, ALogP 2.44, HBA 6, HBD 1, PSA 70.08, QED 0.78