lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1nccn1Cc1ccc(Nc2nccc(-c3ccc(N4CC[C@H](F)C4)c(C#N)n3)n2)cn1
InChIKey: AEPKWENAKIFXHF-KRWDZBQOSA-N
Target: Inhibitor of nuclear factor kappa-B kinase subunit epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: Q14164
Assay: Time-Resolved Fluorescence-based Lanthascreen Assay: Kinase inhibition assays (10 μL) were performed at 20° C. in 384-well plate format. Compound IC50 values were determined at the apparent Km for ATP (20 μM) based on a radiometric assay (Invitrogen) using 8 or 10 point curves in duplicate. The final reaction conditions contained 400 nM fluorescein-IkBα substrate (DRHDSGLDSMKDE), 20 μM ATP, 2 nM or 8 nM IKKε or TBK1 kinase respectively, and 3% DMSO in kinase assay buffer consisting of 50 mM HEPES (pH 7.5), 10 mM MgCl, 1 mM EGTA, 0.01% Brij-35.Compound dilutions were prepared from 10 mM DMSO stocks by dilution into DMSO. Compound dilution series were further diluted in kinase assay buffer to give a 12% DMSO stock, the final concentration in the assay being 3% DMSO.The kinase phosphorylation assay was initiated by the addition of the kinase and the reaction was allowed to proceed for 1 h or 2.5 h for IKKε and TBK-1 kinase respectively.
Measurement: IC50 < 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 455.5, ALogP 3.65, HBA 9, HBD 1, PSA 108.44, QED 0.47
Compound (SMILES): CC(=O)Oc1cc2cccc(O)c(=O)c2c(O)c1O
InChIKey: WQKGPJPCPGXRTA-UHFFFAOYSA-N
Target: Toll-like receptor 1/2 (PROTEIN COMPLEX), Mus musculus
UniProt: Q9EPQ1
Assay: Antagonist activity at TLR1/2 in mouse RAW264.7 cells assessed as inhibition of Pam3CSK4-induced receptor activation by measuring NO production after 24 hrs
Measurement: IC50 = 4830 nM (pChEMBL 5.320000171661377)
Descriptors: MW 262.22, ALogP 1.24, HBA 6, HBD 3, PSA 104.06, QED 0.41
Compound (SMILES): O=C(c1cccc(Cc2n[nH]c(=O)c3c2CCCC3)c1)N1CCN(c2ccccc2Cl)CC1
InChIKey: GVWJZKMEBATZSU-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: PARP Enzyme Inhibition Assay: PARP1 assay was conducted in PARP assay buffer containing 50 mM Tris pH 8.0, 1 mM DTT, 4 mM MgCl2. PARP reactions contained 1.5 uM [3H]-NAD+ (1.6 uCi/mmol), 200 nM biotinylated histone H1, 200 nM sIDNA, and 1 nM PARP enzyme. Auto reactions utilizing SPA bead-based detection were carried out in 100 uL volumes in white 96 well plates. Reactions were initiated by adding 50 ul of 2xNAD+ substrate mixture to 50 uL of 2x enzyme mixture containing PARP and DNA. These reactions were terminated by the addition of 150 uL of 1.5 mM benzamide (~1000-fold over its IC50). 170 uL of the stopped reaction mixtures were transferred to streptavidin Flash Plates, incubated for 1 hour, and counted using a TopCount microplate scintillation counter.
Measurement: Ki = 10 nM (pChEMBL 8)
Descriptors: MW 462.98, ALogP 3.86, HBA 4, HBD 1, PSA 69.3, QED 0.64
Compound (SMILES): Cc1n[nH]c2cnc(C(=O)N3CCC[C@H]3c3ccc(Cl)cc3)cc12
InChIKey: FLEYJCJMMRLIBV-KRWDZBQOSA-N
Target: CDK8/Cyclin C (PROTEIN COMPLEX), Homo sapiens
UniProt: P24863
Assay: Inhibition of Alexa647 tracer binding to full length recombinant human His-tagged CDK8/cyclin C expressed in Baculovirus expression system preincubated for 20 mins followed by tracer addition and incubated in dark for 60 mins by FRET-based Lanthascreen assay
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 340.81, ALogP 3.9, HBA 3, HBD 1, PSA 61.88, QED 0.77
Compound (SMILES): Oc1ccc(/C=N/c2nc(-c3ccccc3)cs2)cc1
InChIKey: CWGDZTAFCAJDCC-LICLKQGHSA-N
Target: Urease (SINGLE PROTEIN), Canavalia ensiformis
UniProt: P07374
Assay: Inhibition Assay: Reaction mixtures consisting of 25 μL (1 unit/well) of Jack bean (Canavalia ensiformis) urease, 55 μL of buffer at pH 6.8, 100 mM of urea, and 5 μL of various concentrations of test compounds were incubated at 30° C. for 15 min in 96-well plates. Subsequently 45 μL phenol reagents (1% w/v phenol and 0.005% w/v sodium nitroprussside), and 70 μL of alkali reagent (0.5% w/v NaOH and 0.1% w/v NaOCl) were added to each well. Urease activity through indophenols method was confirmed by the production of ammonia, as described by Weatherburn [12]. After 50 min, the increasing absorbance at 630 nm was measured in a microplate reader SpectraMax M2 (Molecular Devices, CA, USA). All reactions were performed in triplicate in a final volume of 200 μL.
Measurement: IC50 = 20160 nM (pChEMBL 4.699999809265137)
Descriptors: MW 280.35, ALogP 4.27, HBA 4, HBD 1, PSA 45.48, QED 0.73
Compound (SMILES): CCOCc1c(-c2cc3cc(C)cc(OC)c3s2)c2c(N)ncnn2c1Cl
InChIKey: NIKUTJUSYMUTDM-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 = 14.5 nM (pChEMBL 7.840000152587891)
Descriptors: MW 402.91, ALogP 4.7, HBA 7, HBD 1, PSA 74.67, QED 0.52
Compound (SMILES): OC(c1cccnc1)(c1ccc2nc(Cl)c(Cc3cccc(Cl)c3)cc2c1)c1cncs1
InChIKey: ZEZHVKIBNFEYRX-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor® Assay: Bar-coded assay plates are robotically loaded onto a thermostatically controlled PCR-type thermal block and then heated at a typical ramp-rate of 1 °C./min for all experiments. Fluorescence was measured by continuous illumination with UV light (Hamamatsu LC6) supplied via fiber optic and filtered through a band-pass filter (380-400 nm; >6 OD cutoff). Fluorescence emission of the entire 384-well plate was detected by measuring light intensity using a CCD camera (Sensys, Roper Scientific) filtered to detect 500±25 nm, resulting in simultaneous and independent readings of all 384 wells. Images were collected at each temperature, and the sum of the pixel intensity in a given area of the assay plate was recorded versus temperature. Reference wells contained RORγt without compounds, and the assay conditions were as follows: 0.065 mg/mL RORγt, 60 μM 1,8-ANS, 100 mM Hepes, pH 7.0, 10 mM NaCl, 2.5 mM GSH, 0.002% Tween-20. Project compounds were arranged in a pre-dosed mother plate (Greiner Bio-one) wherein compounds are serially diluted in 100% DMSO by 1:2 from a high concentration of 10 mM over 12 columns within a series (column 12 is a reference well containing DMSO, no compound). The compounds were robotically dispensed directly into assay plates (1×=46 nL) using a Hummingbird capillary liquid handling instrument (Digilab). Following compound dispense, protein and dye in buffer was added to achieve the final assay volume of 3 μL, followed by 1 μL of silicone oil.
Measurement: Kd = 99 nM (pChEMBL 7)
Descriptors: MW 478.4, ALogP 6.27, HBA 5, HBD 1, PSA 58.9, QED 0.3
Compound (SMILES): Fc1cccc(-c2cc3nc4c(c(N5CCNCC5)n3n2)CCCC4)c1
InChIKey: AFYBBCQVYAHRRH-UHFFFAOYSA-N
Target: Beta-glucuronidase (SINGLE PROTEIN), Homo sapiens
UniProt: P08236
Assay: Activity Assay: PNPG Assay: Two ß-glucuronidase activity assays were employed to examine the potency of inhibitors both in vitro and in cell-based studies. An absorbance assay based on the conversion of p-nitrophenyl glucuronide (PNPG) to p-nitrophenol (PNP) (Szasz (1967) Clin Chem 13:752-759), which absorbs at 410 nm, was employed as the primary in vitro assay. A secondary assay involving the conversion of phenolphthalein glucuronide (PheG) to phenolphthalein (Phe), which absorbs at 540 nm, was also employed. Id. Both assays were validated in vitro and in living cells, and the wavelengths monitored did not overlap with absorbance characteristics of putative inhibitors.
Measurement: IC50 = 1204.6 nM (pChEMBL 5.920000076293945)
Descriptors: MW 351.43, ALogP 2.82, HBA 5, HBD 1, PSA 45.46, QED 0.77
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3)ncnc2cc1OCC1CN(Cc2ccccn2)CCO1
InChIKey: KOHLHBQFFZUSET-UHFFFAOYSA-N
Target: Ephrin type-B receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P54760
Assay: Inhibition of recombinant human N-terminal His6-tagged EphB4 kinase domain (E605 to E890 residues) expressed in baculovirus using poly-AEKY as substrate after 150 mins in presence of gamma33P-ATP by liquid scintillation spectrometry
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 526.42, ALogP 5.36, HBA 8, HBD 1, PSA 81.63, QED 0.33
Compound (SMILES): CCO[C@@H](CN1CCC2(CC1)CCN(C1=C(C)C(=O)OC1)C2=O)c1ccc2c(c1C)COC2=O
InChIKey: CWBAIUJUQLWRSX-QFIPXVFZSA-N
Target: ATP-sensitive inward rectifier potassium channel 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P48048
Assay: Thallium Flux Assay: The ROMK channel functional thallium flux assay is performed in 384 wells, using the FLIPR-Tetra instrument. HEK-hKir1.1 cells are seeded in Poly-D-Lysine microplates and kept in a 37° C.-10% CO2 incubator overnight. On the day of the experiment, the growth media is replaced with the FluxOR reagent loading buffer and incubated, protected from light, at ambient temperature (23-25° C.) for 90 min. The loading buffer is replaced with assay buffer+/-test compound followed by 30 min incubation at ambient temperature, where the Thallium/Potassium stimulant is added to the microplate.Step Protocol 1. Seed HEK-hKir1.1 cells (50 uA at 20,000 cells/well) in 384-well PDL coated Microplates 2. Allow cells to adhere overnight in humidified 37° C./10% CO2 incubator 3. Completely remove cell growth media from microplate and replace with 25 ul loading buffer 4. Incubate Microplate at room temperature, protected form light, for 90 min.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 468.55, ALogP 2.89, HBA 7, HBD 0, PSA 85.38, QED 0.59
Compound (SMILES): O=C(N[C@@H](Cc1ccc(F)cc1)C(=O)C(=O)N1CCC(O)CC1)c1cc2cc(Cl)ccc2[nH]1
InChIKey: VCKKIPCWUZKXJQ-FQEVSTJZSA-N
Target: Glycogen phosphorylase, liver form (SINGLE PROTEIN), Homo sapiens
UniProt: P06737
Assay: Inhibition of human recombinant liver glycogen phosphorylase A expressed in baculovirus infected Sf9 insect cells assessed as release of phosphate from glucose-1- phosphate in presence of 7.5 mM glucose by malachite green based assay
Measurement: IC50 = 200 nM (pChEMBL 6.699999809265137)
Descriptors: MW 471.92, ALogP 2.85, HBA 4, HBD 3, PSA 102.5, QED 0.48
Compound (SMILES): CCC(=O)Nc1nc2ccc(C3(O)c4ccccc4C(=O)N3c3ccc(F)cc3)cc2[nH]1
InChIKey: OROSVFNNWMXDQP-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 < 100 nM (pChEMBL 7)
Descriptors: MW 430.44, ALogP 3.9, HBA 4, HBD 3, PSA 98.32, QED 0.46
Compound (SMILES): COc1ccc(/C=C/C(=O)NCCC2CCN(Cc3ccccc3)CC2)c(OC)c1
InChIKey: AHWHBOQIIOPNCM-PKNBQFBNSA-N
Target: Cholinesterase (SINGLE PROTEIN), Homo sapiens
UniProt: P06276
Assay: Inhibition of human serum BuChE using butyrylthiocholine as substrate preincubated for 5 mins followed by substrate addition measured for 5 mins by Ellman's method
Measurement: IC50 = 4390 nM (pChEMBL 5.360000133514404)
Descriptors: MW 408.54, ALogP 4.14, HBA 4, HBD 1, PSA 50.8, QED 0.63
Compound (SMILES): c1ccc2c(N3CCC4(CCCC4)CC3)cncc2c1
InChIKey: RFERWVRPLUDUIY-UHFFFAOYSA-N
Target: Cytochrome P450 11B2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P19099
Assay: Inhibition of human CYP11B2-CLE9 expressed in chinese hamster V79 cells using 11-deoxycorticosterone as substrate preincubated for 1 hr followed by substrate addition measured for 3 hrs by HTRF assay
Measurement: IC50 = 106 nM (pChEMBL 6.96999979019165)
Descriptors: MW 266.39, ALogP 4.4, HBA 2, HBD 0, PSA 16.13, QED 0.76
Compound (SMILES): CC(C)(C)OC(=O)NCC#CCCNc1nc(-c2ccccc2)cn2c(-c3ccc(C(=O)NC4CC4)cc3)cnc12
InChIKey: JSCQAJABUJWLQU-UHFFFAOYSA-N
Target: Dual specificity protein kinase TTK (SINGLE PROTEIN), Homo sapiens
UniProt: P33981
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: 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 ul of a solution of Mps-1 in assay buffer [0.1 mM sodium-ortho-vanadate, 10 mM MgCl2, 2 mM DTT, 25 mM Hepes pH 7.7, 0.05% BSA, 0.001% Pluronic F-127] were added and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compounds to Mps-1 before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 ul of a solution of 16.7 adenosine-tri-phosphate (ATP, 16.7 uM=>final conc. in the 5 ul assay volume is 10 uM) and peptide substrate (1.67 uM=>final conc. in the 5 ul assay volume is 1 uM) in assay buffer and the resulting mixture was incubated for a reaction time of 60 min at 22° C. The concentration of Mps-1 in the assay was adjusted to the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical enzyme concentrations were in the range of about 1 nM (final conc. in the 5 ul assay volume).
Measurement: IC50 = 94.6 nM (pChEMBL 7.019999980926514)
Descriptors: MW 550.66, ALogP 5.29, HBA 7, HBD 3, PSA 109.65, QED 0.2
Compound (SMILES): O=C(c1ccc2cc(Oc3ccc(C(F)(F)F)cn3)ccc2n1)N1CCN2C[C@@H](F)C[C@@H]2C1
InChIKey: WVGGOYFEVTXPEO-DLBZAZTESA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FRET-Based Membrane Potential Assay: Two days prior to the experiment, frozen HEK293 cells stably expressing recombinant human Nav1.7 were quickly thawed and plated at 25,000 cells/well in growth medium [DMEM (Invitrogen #11965) with 10% HI FBS (Invitrogen #10082), 2 mM glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin (PSG, Sigma #G1146), and 500 ug/mL Geneticin (Invitrogen #10131)] in black-walled, clear-bottom 384-well poly-D-lysine-coated assay plates (Greiner Bio-One, Frickenhausen, Germany) and incubated in a humidified 5% CO2 incubator at 37° C. On the day of the assay, medium was removed by aspiration, and cells were washed with assay buffer [HBSS (Invitrogen, Carlsbad, Calif.) containing 20 mM HEPES (Invitrogen, Carlsbad, Calif.)]. After washing, 30 uL assay buffer containing the fluorescent voltage-sensor probe CC2-DMPE (Invitrogen, Carlsbad, Calif.) at 20 uM and 0.01% pluronic F-127 (Invitrogen, Carlsbad, Calif.) was added to the cells. Cells were incubated for 40 minutes at room temperature in the dark. Following the incubation, the cells were washed and 30 uL assay buffer containing 2.5 uM DiSBAC2(3) substrate (Invitrogen, Carlsbad, Calif.) and 0.5 mM VABSC-1 (Invitrogen, Carlsbad, Calif.) was added to the cells. The cells were incubated for 90 minutes at room temperature in the dark. Fluorescence readings were made using a FLIPR TETRA (Molecular Devices, Sunnyvale Calif.) equipped with voltage-sensor probe optics. At the start of each experiment the optimal (EC80) concentration of depolarizing agent (veratridine) was determined by testing a dilution curve of assay buffer containing veratridine (Sigma-Aldrich, St. Louis, Mo.) and 1 mg/mL scorpion venom (SVqq, from Leiurus quinquestriatus; Sigma-Aldrich, St. Louis, Mo.). Compounds were dissolved in dimethyl sulfoxide, and 8-point, 1:3 dilution concentration-response curves were prepared in duplicate in dimethyl sulfoxide, followed by preparation of 0.8 uL/well daughter plates of the dilutions. Test compounds in the daughter plate were diluted to (Ë¿3x) solutions in assay buffer immediately before assaying. Using the FLIPR TETRA, 20 uL of the (3x) compound solutions were first added to the cells, then 20 uL of depolarizing solution (3xEC80 veratridine+SVqq) were added 3 minutes later to activate the channel. Changes in fluorescence were measured at wavelengths of 440-480 nm and 565-625 nm over the course of the experimental run. Membrane depolarization was expressed as a ratio of the maximum F440-480 nm/F565-625 nm reading above average baseline F440-480 nm/F565-625 nm reading. IC50 values were calculated from curve fits of the ratio data using a four-parameter logistic Hill equation (Accelrys Assay Explorer 3.3 Client, Accelrys, San Diego, Calif.) with percent inhibition plotted against compound concentration.
Measurement: IC50 = 3360 nM (pChEMBL 5.46999979019165)
Descriptors: MW 460.43, ALogP 4.31, HBA 5, HBD 0, PSA 58.56, QED 0.55
Compound (SMILES): CNC(=O)N=S(C)(=O)Cc1cccc(Nc2ncc(F)c(-c3ccc(F)cc3OC)n2)c1
InChIKey: SOSTXYOXUQYPHG-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P50750
Assay: CDK9/CycT1 Kinase Assay: Recombinant full-length His-tagged human CDK9 and CycT1, expressed in insect cells and purified by Ni-NTA affinity chromatography, were purchased from Invitrogen (Cat. No PV4131). As substrate for the kinase reaction biotinylated peptide biotin-Ttds-YISPLKSPYKISEG (C-terminus in amid form) was used which can be purchased e.g. form the company JERINI Peptide Technologies (Berlin, 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 ul of a solution of CDK9/CycT1 in aqueous assay buffer [50 mM Tris/HCl pH 8.0, 10 mM MgCl2, 1.0 mM dithiothreitol, 0.1 mM sodium ortho-vanadate, 0.01% (v/v) Nonidet-P40 (Sigma)] were 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.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 461.49, ALogP 4.11, HBA 6, HBD 2, PSA 105.57, QED 0.57
Compound (SMILES): O=C(c1ccc2cc(Oc3ccc(C(F)(F)F)cn3)ccc2n1)N1CC(CO)CC(CO)C1
InChIKey: OQKKLRHGIGTVPK-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FRET-Based Membrane Potential Assay: Two days prior to the experiment, frozen HEK293 cells stably expressing recombinant human Nav1.7 were quickly thawed and plated at 25,000 cells/well in growth medium [DMEM (Invitrogen #11965) with 10% HI FBS (Invitrogen #10082), 2 mM glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin (PSG, Sigma #G1146), and 500 ug/mL Geneticin (Invitrogen #10131)] in black-walled, clear-bottom 384-well poly-D-lysine-coated assay plates (Greiner Bio-One, Frickenhausen, Germany) and incubated in a humidified 5% CO2 incubator at 37° C. On the day of the assay, medium was removed by aspiration, and cells were washed with assay buffer [HBSS (Invitrogen, Carlsbad, Calif.) containing 20 mM HEPES (Invitrogen, Carlsbad, Calif.)]. After washing, 30 uL assay buffer containing the fluorescent voltage-sensor probe CC2-DMPE (Invitrogen, Carlsbad, Calif.) at 20 uM and 0.01% pluronic F-127 (Invitrogen, Carlsbad, Calif.) was added to the cells. Cells were incubated for 40 minutes at room temperature in the dark. Following the incubation, the cells were washed and 30 uL assay buffer containing 2.5 uM DiSBAC2(3) substrate (Invitrogen, Carlsbad, Calif.) and 0.5 mM VABSC-1 (Invitrogen, Carlsbad, Calif.) was added to the cells. The cells were incubated for 90 minutes at room temperature in the dark. Fluorescence readings were made using a FLIPR TETRA (Molecular Devices, Sunnyvale Calif.) equipped with voltage-sensor probe optics. At the start of each experiment the optimal (EC80) concentration of depolarizing agent (veratridine) was determined by testing a dilution curve of assay buffer containing veratridine (Sigma-Aldrich, St. Louis, Mo.) and 1 mg/mL scorpion venom (SVqq, from Leiurus quinquestriatus; Sigma-Aldrich, St. Louis, Mo.). Compounds were dissolved in dimethyl sulfoxide, and 8-point, 1:3 dilution concentration-response curves were prepared in duplicate in dimethyl sulfoxide, followed by preparation of 0.8 uL/well daughter plates of the dilutions. Test compounds in the daughter plate were diluted to (Ë¿3x) solutions in assay buffer immediately before assaying. Using the FLIPR TETRA, 20 uL of the (3x) compound solutions were first added to the cells, then 20 uL of depolarizing solution (3xEC80 veratridine+SVqq) were added 3 minutes later to activate the channel. Changes in fluorescence were measured at wavelengths of 440-480 nm and 565-625 nm over the course of the experimental run. Membrane depolarization was expressed as a ratio of the maximum F440-480 nm/F565-625 nm reading above average baseline F440-480 nm/F565-625 nm reading. IC50 values were calculated from curve fits of the ratio data using a four-parameter logistic Hill equation (Accelrys Assay Explorer 3.3 Client, Accelrys, San Diego, Calif.) with percent inhibition plotted against compound concentration.
Measurement: IC50 = 14600 nM (pChEMBL 4.840000152587891)
Descriptors: MW 461.44, ALogP 3.5, HBA 6, HBD 2, PSA 95.78, QED 0.6
Compound (SMILES): CCOC(C)CNC(=O)c1ccc(NC(=O)N2Cc3ccccc3C2)cc1
InChIKey: GESIBHORGNKVPG-UHFFFAOYSA-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 = 29 nM (pChEMBL 7.539999961853027)
Descriptors: MW 367.45, ALogP 3.39, HBA 3, HBD 2, PSA 70.67, QED 0.82
Compound (SMILES): CC[C@H]1CC[C@H](Cn2c(N3CCO[C@H]4CCC[C@@H]43)nc3cc(-c4nnc(O)o4)nc(-c4cncc(Cl)c4)c32)CC1
InChIKey: QLEFQJWVYUDTEZ-MQQADFIWSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: FRET Assay: Methods: An HDM2 FRET assay was developed to assess the compounds' inhibitory activity towards binding of p53 protein. A truncated version of HDM2 with residues 17 to 125 (containing p53 binding surface, Science (1994) 265, 346-355), with N-terminal His and Thioredoxin tag was generated in pET32a expression vector and expressed in E. coli strain BL21(DE3)Rosetta. Protein was purified using Ni-affinity chromatography, followed by size exclusion chromatography using Superdex 75 26/60 column. To assess inhibition of p53 binding to HDM2, a FITC labeled 8-mer peptide (SEQ ID NO:1: Ac-Phe-Arg-Dpr-Ac6c-(6-Br)Trp-Glu-Glu-Leu-NH2; Anal Biochem. 2004 Aug. 1; 331(1):138-46) with strong affinity towards the p53 binding pocket of HDM2 was used. The HDM2 assay buffer contained 1x Phosphate Buffered Saline (Invitrogen, Cat#14190), 0.01% BSA (Jackson ImmunoResearch, Cat#001-000-162), 0.01% Tween-20. In the 1x assay buffer recombinant HDM2 protein, peptide and Lumi-4-Tb Cryptate-conjugate mouse.
Measurement: IC50 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 564.09, ALogP 5.88, HBA 10, HBD 1, PSA 115.22, QED 0.31
Compound (SMILES): O=C(O)Cc1cc2c(c(-c3ccc(F)c4c3CN(C(=O)OCc3ccccc3)CC4)c1)OCCO2
InChIKey: ABWSMUIKDSQQTJ-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Radioligand Displacement Assay: Binding assay was performed in a final assay volume of 250 ul. First, 25 ul of test compound, previously diluted in Binding-Buffer (Binding-Buffer: 50 mM Tris-Base, 100 mM NaCl, 1 mM EDTA, 0.1% BSA (protease free), 0.01% NaN3, 10 mM MnCl2, pH 7.0) was placed into each well. After addition of 75 ul Binding-Buffer, 50 ul of the radioligand 3H-PGD2 (at 2.5 nM (220.000 dpm/well) from ANAWA ART0662) was added to each well. Binding assay was started by addition of 100 ul CRTH2 membrane fragments, reaching a final concentration of 20 ug/well. For non-specific binding, PGD2 was added to the reaction mixture to 10 mM final concentration. This assay mix was incubated for 90 minutes at room temperature and then filtered through a GF/C filter 96-well plate which was pre-soaked for 3 hours in 0.5% polyethyleneimine (PEI). The filter-wells were washed three times with ice cold Binding-Buffer. Then, 40 ul of Microscint-40 (Packard) was added to each well and the retained radioactivity quantified in a Topcount (Packard).
Measurement: IC50 = 17.9 nM (pChEMBL 7.75)
Descriptors: MW 477.49, ALogP 4.59, HBA 5, HBD 1, PSA 85.3, QED 0.58
Compound (SMILES): CC[C@H](C)[C@H](NC(C)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@H](C(=O)N[C@@H](CC(=O)O)C(=O)NCC(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(N)=O)C(C)C)C(C)C
InChIKey: ILLOLVAXJJOVEF-IMBKOBLJSA-N
Target: Tissue factor pathway inhibitor (SINGLE PROTEIN), Homo sapiens
UniProt: P10646
Assay: Binding affinity to TFPI (unknown origin) incubated for 1.5 hrs using tracer peptide JBT0131 by screening ELISA
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 2381.77
Compound (SMILES): COC1CCN(C(=O)c2n[nH]c3cc(O)c(C(=O)N(C)c4ccc(N5CCOCC5)cc4)cc23)C1
InChIKey: FPSGBGPZYADLJN-UHFFFAOYSA-N
Target: Heat shock protein HSP 90-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P07900
Assay: Recombinant Hsp90 [His-Tev-huHsp90-alpha (9-236)] was immobilized on a Biacore CM5 chip at 25 degrees C and a flow rate of 30 uL/min using amine coupling at pH 4.50 according to Biacore standard protocol. Hsp90 was applied at a concentration of 20 ug/mL with 50uM 17-DMAG.Data sets were processed and analyzed using the software Biacore 4000 Evaluation. Solvent corrected and double-referenced association and dissociation phase data were fitted to a simple 1:1 interaction model with mass transport limitations
Measurement: Kd = 82.33 nM (pChEMBL 7.079999923706055)
Descriptors: MW 479.54, ALogP 2.24, HBA 7, HBD 2, PSA 111.23, QED 0.58
Compound (SMILES): Cn1cc(CNc2cc(OC[C@@H]3C[C@H]3c3nn(C)c4ccccc34)nn(C)c2=O)cn1
InChIKey: IKXWASVOIIOHNI-DOTOQJQBSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Fluorescence Polarization Assay: The fluorescence polarization assay for cyclic nucleotide phosphodiesterases was performed using an IMAP® FP kit supplied by Molecular Devices, Sunnyvale, Calif. (product #R8139). IMAP® technology has been applied previously to phosphodiesterase assays (Huang, W., et al., J. Biomol Screen, 2002, 7: 215).
Measurement: Ki = 0.197 nM (pChEMBL 9.710000038146973)
Descriptors: MW 419.49, ALogP 2.2, HBA 9, HBD 1, PSA 91.79, QED 0.49
Compound (SMILES): COc1ccc(CN(Cc2ccc(OC)c(OC)c2)S(=O)(=O)c2ccc(C(=O)O)cc2)cc1
InChIKey: XVRZXRNQAYUBII-UHFFFAOYSA-N
Target: Taste receptor type 2 member 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYV8
Assay: Fluorescence Polarization Assays: In another embodiment, Fluorescence Polarization ("FP") based assays may be used to detect and monitor ligand binding. Fluorescence polarization is a versatile laboratory technique for measuring equilibrium binding, nucleic acid hybridization, and enzymatic activity. Fluorescence polarization assays are homogeneous in that they do not require a separation step such as centrifugation, filtration, chromatography, precipitation, or electrophoresis. These assays are done in real time, directly in solution and do not require an immobilized phase. Polarization values can be measured repeatedly and after the addition of reagents since measuring the polarization is rapid and does not destroy the sample. Generally, this technique can be used to measure polarization values of fluorophores from low picomolar to micromolar levels.
Measurement: IC50 = 1470 nM (pChEMBL 5.829999923706055)
Descriptors: MW 471.53, ALogP 3.8, HBA 6, HBD 1, PSA 102.37, QED 0.48
Compound (SMILES): COc1ccc2ccn(CCCn3cc(NC(=O)c4ncoc4-c4cccc(C)c4)cn3)c2c1
InChIKey: DICIKTWZAQMHFE-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: In Vitro Assay: 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 μg/mL G418, 100 U/mL penicillin, 100 μg/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 μM 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 μl/well, incubated for 120 min and finally 10 μl/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.
Measurement: IC50 = 47 nM (pChEMBL 7.329999923706055)
Descriptors: MW 455.52, ALogP 5.15, HBA 7, HBD 1, PSA 87.11, QED 0.35
Compound (SMILES): O=C(/C=C/c1cccnc1)Nc1ccc(S(=O)(=O)c2ccc3ncccc3c2)cc1
InChIKey: IKAMYJCLMWXSAO-LFYBBSHMSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Biochemical Inhibition Assay: The NAMPT enzymatic reactions were carried out in Buffer A (50 mM Hepes pH 7.5, 50 mM NaCl, 5 mM MgCl2, and 1 mM THP) in 96-well V-bottom plates. The compound titrations were performed in a separate dilution plate by serially diluting the compounds in DMSO to make a 100x stock. Buffer A (89 uL) containing 33 nM of NAMPT protein was added to 1 uL of 100x compound plate containing controls (e.g. DMSO or blank). The compound and enzyme mix was incubated for 15 minutes at room temperature, then 10 uL of 10x substrate and co-factors in Buffer A were added to the test well to make a final concentration of 1 uM NAM, 100 uM 5-Phospho-D-ribose 1-diphosphate (PRPP), and 2.5 mM Adenosine 5'-triphosphate (ATP). The reaction was allowed to proceed for 30 minutes at room temperature, then was quenched with the addition of 11 uL of a solution of formic acid and L-Cystathionine to make a final concentration of 1% formic acid and 10 uM L-Cystathionine.
Measurement: IC50 = 5.4 nM (pChEMBL 8.270000457763672)
Descriptors: MW 415.47, ALogP 4.11, HBA 5, HBD 1, PSA 89.02, QED 0.5
Compound (SMILES): CN(C)[C@H]1c2cc3ccn(S(=O)(=O)c4ccccc4)c3cc2[C@H]1CCO
InChIKey: OCKYRBSJWIYEBM-OXQOHEQNSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Displacement of [3H]-methyl-spiperone from human dopamine D2L receptor expressed in HEK293 cell membranes after 1 hr by radioligand binding assay
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 370.47, ALogP 2.96, HBA 5, HBD 1, PSA 62.54, QED 0.75
Compound (SMILES): Cc1cc(NC(=O)c2ccc3cc4n(c3c2)[C@H](C)[C@H](C)NC4=O)no1
InChIKey: AMQAOXDPHJIPPA-WDEREUQCSA-N
Target: Ribosomal protein S6 kinase alpha-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15349
Assay: Kinase Glo Plus Assay: Human RSK2 protein, purchased from Invitrogen, is used to measure kinase activity utilizing Kinase Glo Plus (Promega) a homogeneous assay technology, which uses a luciferin-luciferase based ATP detection reagent to quantify residual ATP. The assay is performed using 0.75 nM His-RSK2, 0.75 .mu.M ATP and 1.0 uM S6 Kinase/RSK Substrate Peptide 1 (Upstate, catalog #12-124), in assay buffer consisting of 25 mM HEPES, pH 7.5, 10 mM MgCl2, 5 mM MnCl2, 50 mM KCl, 0.2% BSA, 0.01% CHAPS, 100 uM Na3VO4, 0.5 mM DTT, and 1% DMSO. Solutions of test compounds at various concentrations are prepared by 1:3 fold serial dilution of a 1 mM solution of compound in DMSO. The DMSO solutions are further diluted with assay buffer to a final concentration of DMSO of 5%. The assay is performed in a 384 well, white, non-binding plate (Corning, catalogue #3574). Solutions of test compounds (10 uL) are transferred to a dry assay plate, followed by addition of 20 uL kinase.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 338.37, ALogP 2.88, HBA 5, HBD 2, PSA 89.16, QED 0.75
Compound (SMILES): O=C1CC2(CCCC2)CN1c1ccc(C#Cc2ccccc2)cn1
InChIKey: UIRHDTFCRZTYRF-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P41594
Assay: Intracellular Ca2+ Mobilization Assay: A monoclonal HEK-293 cell line stably transfected with a cDNA encoding for the human mGlu5a receptor was generated; for the work with mGlu5 Positive Allosteric Modulators (PAMs), a cell line with low receptor expression levels and low constitutive receptor activity was selected to allow the differentiation of agonistic versus PAM activity. Cells were cultured according to standard protocols (Freshney, 2000) in Dulbecco's Modified Eagle Medium with high glucose supplemented with 1 mM glutamine, 10% (vol/vol) heat-inactivated bovine calf serum, Penicillin/Streptomycin, 50 μg/ml hygromycin and 15 μg/ml blasticidin (all cell culture reagents and antibiotics from Invitrogen, Basel, Switzerland). bout 24 hrs before an experiment, 5×104 cells/well were seeded in poly-D-lysine coated, black/clear-bottomed 96-well plates. The cells were loaded with 2.5 μM Fluo-4AM in loading buffer (1×HBSS, 20 mM HEPES) for 1 hr at 37 °C. and washed five times with loading buffer. The cells were transferred into a Functional Drug Screening System 7000 (Hamamatsu, Paris, France), and 11 half logarithmic serial dilutions of test compound at 37 °C. were added and the cells were incubated for 10-30 min. with on-line recording of fluorescence. Following this pre-incubation step, the agonist L-glutamate was added to the cells at a concentration corresponding to EC20 (typically around 80 μM) with on-line recording of fluorescence; in order to account for day-to-day variations in the responsiveness of cells, the EC20 of glutamate was determined immediately ahead of each experiment by recording of a full dose-response curve of glutamate. Responses were measured as peak increase in fluorescence minus basal (i.e. fluorescence without addition of L-glutamate), normalized to the maximal stimulatory effect obtained with saturating concentrations of L-glutamate.
Measurement: EC50 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 316.4, ALogP 3.78, HBA 2, HBD 0, PSA 33.2, QED 0.75
Compound (SMILES): C#CCN(CC#C)c1ccc(/C=C(\C#N)C(=O)O)c(OC)c1
InChIKey: AUELJRMSNKLDTD-GXDHUFHOSA-N
Target: Monocarboxylate transporter 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P53987
Assay: Inhibition Assay: The entire lactate uptake studies for the inhibition of MCT1 were carried out on RBE4 (Rat Brain Endothelial 4) cells. The expression of MCT1 on these cells was confirmed by Western Blotting. The cells were plated approximately 20-24 hours before the experiment, the number of cells being approximately 105 cells per well. The test compounds were dissolved in DMSO and diluted 1000 times using a solution of HEPES buffer (pH 7.43) which consists of 3 uM 14C-Lactate and 2 uM L-Lactate. The cells were washed twice with 500 uL of HEPES buffer and the cells were allowed to equilibrate for 15 minutes at 37° C. The HEPES buffer was removed and 250 uL of the test sample was added in triplicates. This was repeated for all the compounds, including the controls (CHC and DMSO). After 15 minutes, the compounds were removed from the well and 500 uL of ice-cold stop buffer (0.1 mM CHC solution in HEPES buffer) was added. The plate was kept on ice. Now, the HEPES buffer in one triplicate was removed and DMSO solution was added and immediately removed and ice-cold stop buffer was added. This was considered as "Zero". One triplicate was left blank, which was used for protein assay after lysing the cells. The cells were washed twice with ice-cold HEPES buffer and then 250 uL of 0.1M NaOH in 5% Triton-X solution was added and the plate is kept on a shaker for 40 minutes to lyse the cells. 150 uL of the lysed cells was added into 4 mL of the scintillation fluid in a scintillation vial and scintillation count was obtained in disintegrations per minute (dpm). The percent inhibition values were calculated by taking DMSO as minimum. Concentration study (usually 9-12 dilutions) was done to determine the IC50 of each compound.
Measurement: IC50 = 91 nM (pChEMBL 7.039999961853027)
Descriptors: MW 294.31, ALogP 1.76, HBA 4, HBD 1, PSA 73.56, QED 0.49
Compound (SMILES): Cc1c(Cl)cccc1NC(=O)c1cc(NC(=O)c2ccc(Cl)cc2Cl)cc2[nH]c(N(C)C)nc12
InChIKey: FUPIRZAOVRMXDC-UHFFFAOYSA-N
Target: Prostaglandin E synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O14684
Assay: Inhibition Assay: mPGES-1 microsome fractions were prepared from CHO-K1 cells transiently transfected with plasmid encoding the human mPGES-1cDNA. Microsomes were diluted with potassium phosphate buffer containing reduced glutathione (pH7.4), and DMSO containing test compound or DMSO alone was added (such that DMSO final concentration would be 1% in each) and incubated at 4° C. for 20 minutes. Then, the enzymatic reactions were initiated by the addition of PGH2 substrate (final concentration 1 μM) and incubated at 4° C. for 60 seconds. The reaction was terminated by the addition of a citrate solution (final citrate concentration 50 mM) containing ferric chloride (final concentration 1 mg/mL). PGE2 production in the enzyme reaction aliquot was measured using HTRF kit (Cisbio International, catalogue #62P2APEC).
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 516.82, ALogP 6.4, HBA 4, HBD 3, PSA 90.12, QED 0.29
Compound (SMILES): O=C(O)c1cccnc1Nc1ccc(F)cc1
InChIKey: CUEJKLHNOMZKTP-UHFFFAOYSA-N
Target: Dihydroorotate dehydrogenase (quinone), mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q02127
Assay: Inhibition of human recombinant DHODH
Measurement: IC50 = 2800 nM (pChEMBL 5.550000190734863)
Descriptors: MW 232.21, ALogP 2.66, HBA 3, HBD 2, PSA 62.22, QED 0.85
Compound (SMILES): COc1cc(-c2ccc(Cl)cc2)c(F)cc1-n1c(=O)ccc2cc(S(=O)(=O)Nc3ncccn3)ccc21
InChIKey: OMZWFOFADVNNIO-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 = 28 nM (pChEMBL 7.550000190734863)
Descriptors: MW 536.97, ALogP 5.05, HBA 7, HBD 1, PSA 103.18, QED 0.33
Compound (SMILES): CC1(C)CCC(CN2CCN(c3ccc(C(=O)NS(=O)(=O)c4ccc(NCCCNC5CC5)c([N+](=O)[O-])c4)c(Oc4cnc5[nH]ccc5c4)c3)CC2)=C(c2ccc(Cl)cc2)C1
InChIKey: JWICLIKRPNQLLC-UHFFFAOYSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Representative compounds were serially diluted in dimethyl sulfoxide (DMSO) starting at 50 uM (2x starting concentration; 10% DMSO) and 10 uL were transferred into a 384-well plate. Then 10 uL of a protein/probe/antibody mix was added to each well at final concentrations listed in TABLE 1. Protein:GST-Bcl-2, Probe: F-Bak Peptide Probe Acetyl-GQVGRQLAIIGDK(6-FAM)INR-amide(SEQ ID NO: 1), Protein(nM): 1, Probe (nM): 100, Antibody: Tb-anit-GST, Antibody (nm): 1.  The samples are then mixed on a shaker for 1 minute and incubated for an additional 3 hours at room temperature. For each assay, the probe/antibody and protein/probe/antibody were included on each assay plate as negative and positive controls, respectively. Fluorescence was measured on the ENVISION plate reader (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak peptide) and 495/510 nm (Tb-labeled anti-Histidine antibody) emission filters.
Measurement: Ki < 0.01 nM (pChEMBL 11)
Descriptors: MW 867.47, ALogP 8.38, HBA 11, HBD 4, PSA 174.83, QED 0.04
Compound (SMILES): NC(=O)c1nn(-c2ccccc2)c2c1CCc1ccc(NC(=O)c3ccccc3Cl)cc1-2
InChIKey: HGVZBVPWJMNPCP-UHFFFAOYSA-N
Target: Inhibitor of nuclear factor kappa-B kinase subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O14920
Assay: Inhibition of human recombinant C-terminal FLAG-epitope-tagged IKK2 expressed in baculovirus using biotinylated IkappaBalpha peptide as substrate assessed as decrease in [gamma-33P]-ATP incorporation in substrate after 30 mins by scintillation counting method
Measurement: IC50 <= 1000 nM (pChEMBL 6)
Descriptors: MW 442.91, ALogP 4.64, HBA 4, HBD 2, PSA 90.01, QED 0.48
Compound (SMILES): CC(C)(CO)NCCOc1ccc2c(c1)C(C)(C)c1[nH]c3cc(C#N)ccc3c1C2=O
InChIKey: QSYXOEHBHZYUKF-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Inhibition Assay: ALK-inhibiting activity was measured by following an activity of inhibiting phosphorylation by biotinylated peptide (EGPWLEEEEEAYGWMDF). For the detection of phosphorylation of the biotinylated peptide, time-resolved fluorescence measurement was performed using an anti-phosphorylated tyrosine antibody labeled with europium cryptate and streptavidin conjugated to XL665, i.e., an allophycocyanin derivative.
Measurement: IC50 = 3.595 nM (pChEMBL 8.4399995803833)
Descriptors: MW 417.51, ALogP 3.65, HBA 5, HBD 3, PSA 98.14, QED 0.53
Compound (SMILES): CCn1ccc(Nc2ncc3c(n2)-c2c(nn(C)c2Cc2cccc(-c4cnn(CCOC)c4)c2)CC3)n1
InChIKey: KZXBXGNMGTZACU-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: DELFIA Assay: The kinase activity is measured by DELFIA assay (dissociation-enhanced lanthanide fluorescence immunoassay, Perkin Elmer). The cytoplasmic kinase domain of human IGF-1R (amino acids 964-1370) is expressed as a fusion protein with a glutathione-S-transferase tag (IGF-1R-GST) in High Five.TM. Cells (Invitrogen). Enzyme activity is measured in the presence of substances and a control substance. Poly-glutamate-tyrosine peptide (pEY, Sigma Aldrich) and biotinylated pEY (bio-pEY) are used as reaction substrates.10 uL of substance in 25% DMSO are mixed with 30 uL of IGF-1R-GST solution (67 mM HEPES pH 7.4, 15 ug/mL pEY, 1.7 ug/mL bio-pEY, 13.3 mM MgCl2, 3.3 mM dithiothreitol, 0.0033% Brij 35, 2 ng IGF-1R-GST) in 96-well plates. The reactions are started with 10 uL of a 750 uM ATP solution. After 40 min at RT the reactions are stopped with 50 .mu.L of stop solution (250 mM EDTA, 20 mM HEPES pH 7.4). 90 uL from each reaction are transferred.
Measurement: IC50 = 0.1 nM (pChEMBL 10)
Descriptors: MW 509.62, ALogP 4.04, HBA 10, HBD 1, PSA 100.5, QED 0.32
Compound (SMILES): C=CC(=O)NC(C)(C)C1CCNc2c(C(N)=O)c(-c3ccc(Oc4ccccc4)cc3)nn21
InChIKey: YZFPHIKBWCHEDW-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 = 41 nM (pChEMBL 7.389999866485596)
Descriptors: MW 445.52, ALogP 3.88, HBA 6, HBD 3, PSA 111.27, QED 0.48
Compound (SMILES): O=C(C1CCS(=O)(=O)CC1)N1CCN(c2ccc(F)cn2)CC1c1ccc(F)c(F)c1
InChIKey: UOGDPSJQXZGRGE-UHFFFAOYSA-N
Target: Sodium- and chloride-dependent glycine transporter 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P48067
Assay: Cytostar-T Assay: In detail, SK-N-MC cells are seeded into 96-well Cytostar-T assay plates at a density of 200,000 cells/well and grown for 16-18 hours to confluence in growth medium as recommended by ATCC. Before starting the assay, cells are washed once with HBSS (Hank's buffered salt solution; Sigma, H8264) cont. 5 mM alanine (referred in here as HBSS/Ala) and afterwards the following reagents are added: 1.80 ul/well HBSS/A1a 2. 20 ul/well of HBSS/A1a containing 6x the concentration of compound in 6% DMSO 3. approx. 5-10 min incubation 4. 20 ul/well 3 uM glycine (3H-glycine (Perkin Elmer, NET004001MC, specific activity: 52 Ci/mmol; diluted 1:1 with unlabelled glycine) in HBSS/A1a. In the final assay, glycine concentration is 500 nM (250 nM derived from the 3H-glycine Perkin Elmer, 250 nM unlabelled glycine), DMSO concentration is 1%. The assay plate is immediately after addition of the 3H-glycine placed into a Micro-Beta Counter (Perkin Elmer) and the signal is recorded over 60 min.
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 453.49, ALogP 2.71, HBA 5, HBD 0, PSA 70.58, QED 0.71