lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1ccc(-n2nccn2)c(C(=O)N2CCC[C@@H](C)[C@H]2CNc2ncc(C#N)cc2F)c1
InChIKey: HMWCLKDXLYUSTH-IIBYNOLFSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Cell-based Functional Assay: Measurement of [Ca2+]i using a FLIPR: CHO-OX1 or CHO-OX2 cells are seeded into black-walled clear-base 384-well plates (Costar) at a density of 20,000 cells per well in F12-K medium supplemented with 10% FBS and selection antibiotic (500 ug/ml G418 or 15 ug/ml Blasticidin) and cultured overnight. The cells are incubated with equal volume of calcium4 loading buffer (Molecular Devices, Inc.) containing 2.5 mM probenecid at 37° C. for 30 min, followed by putative OX1 or OX2 receptor antagonists (dose-range 0.1 nM-10 μM) for another 30 min.
Measurement: IC50 = 83 nM (pChEMBL 7.079999923706055)
Descriptors: MW 433.49, ALogP 3.33, HBA 7, HBD 1, PSA 99.73, QED 0.66
Compound (SMILES): Cc1cnn(Cc2ccc(Cn3c(C)cc(C(=O)NCc4ccc(CN)cc4C)c3C)cc2)c1
InChIKey: PRNAVRPSAWUWHK-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Plasma kallikrein assay: Plasma kallikrein inhibitory activity in vitro was determined using standard published methods (see e.g. Johansen et al., Int. J. Tiss. Reac. 1986, 8, 185; Shori et al., Biochem. Pharmacol., 1992, 43, 1209; Stürzebecher et al., Biol. Chem. Hoppe-Seyler, 1992, 373, 1025). Human plasma kallikrein (Protogen) was incubated at 37° C. with the fluorogenic substrate H-DPro-Phe-Arg-AFC and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in optical absorbance at 410 nm and the IC50 value for the test compound was determined.
Measurement: IC50 = 28 nM (pChEMBL 7.550000190734863)
Descriptors: MW 455.61, ALogP 4.4, HBA 5, HBD 2, PSA 77.87, QED 0.41
Compound (SMILES): CCN(CC)Cc1ccc(-n2cnc(-c3nc(NC4CCN(S(C)(=O)=O)CC4)ncc3C(F)(F)F)c2)c(C(F)(F)F)c1
InChIKey: PLXOMHFCDSFJCV-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24864
Assay: HTRF Enzyme Activity Assay: CDK2/Cyclin E1 enzyme activity assays utilize full-length human CDK2 co-expressed as N-terminal GST-tagged protein with FLAG-Cyclin E1 in a baculovirus expression system (Carna Product Number 04-165). Assays were conducted in white 384-well polystyrene plates in a final reaction volume of 8 μL. CDK2/Cyclin E1 (0.25 nM) was incubated with the compounds of the Examples (40 nL serially diluted in DMSO) in the presence of ATP (50 μM or 1 mM) and 50 nM ULight-labeled eIF4E-binding protein 1 (THR37/46) peptide (PerkinElmer) in assay buffer (containing 50 mM HEPES pH 7.5, 1 mM EGTA, 10 mM MgCl2, 2 mM DTT, 0.05 mg/mL BSA, and 0.01% Tween 20) for 60 minutes at room temperature. The reactions were stopped by the addition of EDTA and Europium-labeled anti-phospho-4E-BP1 antibody (PerkinElmer), for a final concentration of 15 mM and 1.5 nM, respectively. HTRF signals were read after 1 hour at room temperature on a PHERAstar FS plate reader (BMG Labtech). Data was analyzed with IDBS XLFit and GraphPad Prism 5.0 software using a three or four parameter dose response curve to determine IC50 for each compound.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 619.64, ALogP 5.04, HBA 8, HBD 1, PSA 96.25, QED 0.34
Compound (SMILES): O=C1CS(=O)(=O)c2cccnc2N1Cc1cccc(C(F)(F)F)c1
InChIKey: XNKNGAGBNBQFPR-UHFFFAOYSA-N
Target: Intermediate conductance calcium-activated potassium channel protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O15554
Assay: KCa3.1 Assay: HEK293 cells over-expressing human KCa3.1 are used to measure the effects of compounds in inhibiting the KCa3.1 channel function. The assay is based on measuring the influx of Tl+ through KCa3.1 using a FLIPR dye assay (FLIPR Potassium Ion Channel Assay Kit #R8154 by Molecular Devices). The day before the assay HEK293/KCa3.1 cells are seeded on a 384 well plate. On the day of the assay, media is removed and replaced with Molecular Devices Potassium Ion Channel dye then incubated for 1 hour according to manufacturer's recommendations. The cells are then treated with compounds or DMSO control for 15 min, followed by addition of thallium (final concentration=1.5 mM) and ionomycin (final concentration=1 uM). The plate is read on HAMAMATSU 60000 measuring fluorescence signal for 50 seconds using (Em: 565-625 nm). Data analysis: HAMAMATSU 6000 data are exported using max-min value for frame 10 and 40 and corrected by spatial uniformity correction. Percent of control (POC) is calculated as 100×(max-minFlsample−max-minFllow)/(max-minFlhi−max-minFllow). Max-min Flsample is max-min value of each sample well containing test compound. Max-min Fllow is average max-min value of wells containing reference inhibitory compound.
Measurement: IC50 = 290 nM (pChEMBL 6.539999961853027)
Descriptors: MW 356.33, ALogP 2.42, HBA 4, HBD 0, PSA 67.34, QED 0.83
Compound (SMILES): Cc1cn(C(=O)c2c(-c3c(Cl)cc(Cl)cc3Cl)noc2C2(N(C)C)CC2)c2cccc(/C=C/C(=O)O)c12
InChIKey: ADKCWUPCLIWNNO-BQYQJAHWSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Inhibitory Activity Against IDH1R132H and IDH1R132C Enzymes: The IDH1R132H protein and the IDH1R132C protein were prepared as follows: the IDH1R132H or IDH1R132C gene was integrated into a pET24b(+) vector (Novagen) to prepare a construct for C-terminal fusion of 6× histidine tag. After transformation of Rosetta 2 (DE3) E. coli, the expression of the protein was induced with IPTG. The E. coli was collected and homogenized, followed by the affinity purification of the 6× histidine fusion protein using HisTrap HP columns (GE Healthcare Japan Corp.) and gel filtration purification using Superdex 200 columns (GE Healthcare Japan Corp.) to obtain the IDH1R132H or IDH1R132C protein of interest.IDH1R132H and IDH1R132C each convert 2-oxoglutarate and NADPH to D-2-hydroxyglutarate (2-HG) and NADP+. Therefore, the activity of the IDH1R132H and IDH1R132C enzymes can be measured by detecting NADPH levels.The enzyme inhibitory activity evaluation was carried out as follows: 40 μL each of reaction solutions containing different concentrations of each compound (100 mM Tris-HCl (pH 7.5), 150 mM NaCl, 20 mM MgCl2, 0.5 mg/mL bovine serum albumin, 1 mM reduced glutathione, 40 μM NADPH, 0.5 mM 2-oxoglutarate, 0.5% dimethyl sulfoxide, 50000 to 0.128 nM compound, and 12 nM IDH1R132H or 10 nM IDH1R132C as the enzyme) was added to each well of 384-well plates (Greiner Bio One International GmbH, #781096) and incubated at room temperature. While NADPH-derived fluorescence was occasionally monitored, the reaction was terminated by the addition of 5 μL of 0.5 M EDTA before consumption of NADPH. 5 μL of WST-8 reagent (Dojindo Laboratories, #CK04) was further added and mixed therewith. 15 minutes later, the absorbance at 450 nm was measured using a plate reader (PerkinElmer, Inc., EnVision). The observed absorbance value reflects the amount of residual NADPH. From the absorbance data, the enzyme inhibitory activity of each compound at each concentration was calculated and analyzed using medical statistical analysis software GraphPad Prism to calculate an IC50 value.
Measurement: IC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 558.85, ALogP 6.9, HBA 6, HBD 1, PSA 88.57, QED 0.26
Compound (SMILES): Fc1ccc(-n2cc(COc3ccc(Cl)cn3)nn2)cc1C(F)F
InChIKey: TXKOLHISXQEJFK-UHFFFAOYSA-N
Target: Glutamate NMDA receptor; GRIN1/GRIN2B (PROTEIN COMPLEX), Homo sapiens
UniProt: Q05586
Assay: NMDA receptor FLIPR assay: NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 149 mM NaCl, 4 mM KCl, 2 mM CaCl2, and 1.5 mM MgCl2, 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (149 mM NaCl (standard assay) or 150 mM (HTS assay), 4 mM KCl (standard assay) or 3 mM (HTS assay), 2 mM CaCl2, 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to EC80 (standard assay) or EC40 (HTS assay) is used to maximize the assay's signal window or the ability to detect NMDA receptor antagonists and negative allosteric modulators, respectively. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 56 nM (pChEMBL 7.25)
Descriptors: MW 354.72, ALogP 3.97, HBA 5, HBD 0, PSA 52.83, QED 0.7
Compound (SMILES): C#CC(C)(C)NC(=O)c1cc(NC(=O)Cc2ccc(Cl)cc2O)ccn1
InChIKey: YLJPRJILFJVBHR-UHFFFAOYSA-N
Target: Anoctamin-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5XXA6
Assay: Automated Whole-Cell Patch Clamp Assay to Detect TMEM16A Activity in Recombinant Cells: Compound activity was quantified by measuring the increase in current upon compound addition and expressing this as a percentage increase of baseline TMEM16A current level. Percentage increases in current were determined for each concentration and the data plotted as a function of concentration using either the Qpatch software or Graphpad Prism v6.05 providing the concentration which gave 50% of its maximal effect (EC50) and maximum efficacy (percentage of baseline increase). The method of calculating the results is illustrated in FIG. 1, which shows an example trace from the Qpatch TMEM16A assay. In FIG. 1, IBL equals baseline current, I[#1] equals the peak current during test compound concentration 1 incubation period and so on.
Measurement: EC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 371.82, ALogP 2.76, HBA 4, HBD 3, PSA 91.32, QED 0.7
Compound (SMILES): O=C(OCc1cc(Cl)cc(Cl)c1)N1CCC2(CC1)CN(S(=O)(=O)c1ccc3[nH]c(=O)oc3c1)C2
InChIKey: YBTQPVUNAIIYFN-UHFFFAOYSA-N
Target: Ataxin-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99700
Assay: Enzyme Inhibition Assay: ATX inhibition was measured by a fluorescence quenching assay using a specifically labeled substrate analogue (MR121 substrate). To obtain this MR121 substrate, BOC and TBS protected 6-amino-hexanoic acid (R)-3-({2-[3-(2-{2-[2-(2-amino-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propionylamino]-ethoxy}-hydroxy-phosphoryloxy)-2-hydroxy-propyl ester (Ferguson et al., Org Lett 2006, 8 (10), 2023) was labeled with MR121 fluorophore (CAS 185308-24-1,1-(3-carboxypropyl)-11-ethyl-1,2,3,4,8,9,10,11-octahydro-dipyrido[3,2-b:2′,3′-i]phenoxazin-13-ium) on the free amine of the ethanolamine side and then, after deprotection, subsequently with tryptophan on the side of the aminohexanoic acid.Assay working solutions were made as follows:Assay buffer (50 mM Tris-HCl, 140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 0.01%Triton-X-100, pH 8.0;ATX solution: ATX (human His-tagged) stock solution (1.08 mg/mL in 20 mM bicine, pH 8.5, 0.15 M NaCl, 10% glycerol, 0.3% CHAPS, 0.02% NaN3), diluted to 1.4-2.5×final concentration in assay buffer;MR121 substrate solution: MR121 substrate stock solution (800 M MR121 substrate in DMSO), diluted to 2-5×final concentration in assay buffer.Test compounds (10 mM stock in DMSO, 8 μL) were obtained in 384 well sample plates (Corning Costar #3655) and diluted with 8 μL DMSO. Row-wise serial dilutions were made by transferring 8 μL cpd solution to the next row up to row O. The compound and control solutions were mixed five times and 2 μL were transferred to 384 well assay plates (Corning Costar #3702). Then, 15 μL of 41.7 nM ATX solution was added (30 nM final concentration), mixed five times and then incubated for 15 minutes at 30° C. 10 μL of MR121 substrate solution was added (1 μM final concentration), mixed 30 times and then incubated for 15 minutes at 30° C. Fluorescence was then measured every 2 minutes for 1 hour (Perkin Elmer plate: vision multimode reader); light intensity: 2.5%; exp. time: 1.4 sec, Filter: Fluo_630/690 nm) and IC50 values were calculated from these readouts.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 526.4, ALogP 3.85, HBA 6, HBD 1, PSA 112.92, QED 0.55
Compound (SMILES): CCN1CCN(c2ccc(C(=O)Nc3n[nH]c4cnc(-c5c(F)cccc5OC)cc34)cc2)CC1
InChIKey: PUDXYRDTGTVPOC-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: HPK1 Kinase Binding Assay: A stock solution of 1 mM test compound was prepared in DMSO. The compound plate was prepared by 3-fold and 11-point serial dilutions. 0.1 μL of the compound in DMSO was transferred from the compound plate to the white 384 well polystyrene plates. The assay buffer contained 50 mM HEPES, pH 7.5, 0.01% Tween-20, 5 mM MgCl2, 0.01% BSA, and 5 mM DTT. 5 μl of 4 nM active HPK1 (SignalChem M23-11G) prepared in the buffer was added to the plate. The enzyme concentration given was based on the given stock concentration reported by the vender. 5 μl of 18 nM tracer 222 (ThermoFisher PV6121) and 4 nM LanthaScreen Eu-Anti GST antibody (ThermoFisher PV5595) were added. After one hour incubation at 25° C., the plates were read on a PHERAstar FS plate reader (BMG Labtech).
Measurement: Ki < 100 nM (pChEMBL 7)
Descriptors: MW 474.54, ALogP 4.17, HBA 6, HBD 2, PSA 86.38, QED 0.44
Compound (SMILES): Cc1cc2ncnn2cc1-c1[nH]c2ccc(C3CCN(CCS(N)(=O)=O)CC3)cc2c1C(C)C
InChIKey: ANTZCSUJIVJTNU-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 8.4 nM (pChEMBL 8.079999923706055)
Descriptors: MW 480.64, ALogP 3.78, HBA 6, HBD 2, PSA 109.38, QED 0.44
Compound (SMILES): CCOC(=O)N1Cc2cc(Cl)ccc2-n2c(C3CC4(C3)CN(c3nccc(C(F)(F)F)n3)C4)nnc2C1C
InChIKey: JLXZUBUMZUHYEV-UHFFFAOYSA-N
Target: Vasopressin V1a receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P37288
Assay: Vasopressin V1A Receptor Antagonist Assay: The purpose of this assay was to determine the inhibitory effect of synthesized compounds on the Vasopressin V1a receptor. The assay as performed in Chinese Hamster Ovary (CHO) cells expressing the human Arginine Vasopressin Receptor 1a (AVPR1a). Arginine Vasopressin (AVP) evokes an increase in intracellular calcium in CHO-AVPR1a cells which is measured in a fluorescence assay on the FLIPRTETRA using calcium sensitive dyes. Test compounds were assessed for their ability to affect the magnitude of the response to AVP, with antagonists showing a concentration-dependent reduction in the AVP-mediated fluorescence. Compounds were tested in duplicate in a 10-point, 1:3 dilution series starting at a nominal concentration of 3 μM in the assay.CHO-AVPR1a cells were maintained in routine culture in T175 Flasks at 37° C., 5% CO2. The growth medium consists of Ham's F12 media supplemented with 10% v/v fetal bovine serum, 1× non-essential amino acids, and 0.4 mg/ml Geneticin G418.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 547.97, ALogP 5.15, HBA 8, HBD 0, PSA 89.27, QED 0.45
Compound (SMILES): Cc1c(-c2cc3cc(NC(=O)O[C@H]4C[C@H]([C@@H](C)O)C4)ncc3c(N)c2F)cnc2c1NCCO2
InChIKey: UXHNAQGLGZVSBL-UMVBOHGHSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: TBD: TBD
Measurement: Ki = 0.017 nM (pChEMBL 10.770000457763672)
Descriptors: MW 467.5, ALogP 3.84, HBA 8, HBD 4, PSA 131.62, QED 0.43
Compound (SMILES): Oc1ccc(C(O)CN2C[C@@H]3C[C@@](O)(CC4CC4)C[C@@H]3C2)cc1
InChIKey: SPHVGMJUTPPTDD-ZMBPFFAYSA-N
Target: Glutamate receptor ionotropic, NMDA 2B (SINGLE PROTEIN), Homo sapiens
UniProt: Q13224
Assay: FDSS Assay: To each well of the plate, 10 μL test compound, control (MK801) or HHnoCa buffer was added to a final concentration of 10 μM with a final concentration of DMSO of 0.1%. Following 10 minutes pre-incubation in the dark, plates are loaded onto the Hamamatsu FDSS 6000. After collecting baseline fluorescence images, 3 μM glutamate, 3 glycine, and 1 mM Ca2+ in HHnoCa buffer is added to each well, and Ca2+ is recorded for 3 minutes. Data were processed by computing ratio of fluorescence at the end of data collection to baseline fluorescence to assess degree of Ca′ influx inhibition relative to that observed in MK801.
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 317.43, ALogP 2.3, HBA 4, HBD 3, PSA 63.93, QED 0.78
Compound (SMILES): Cc1cc(=O)c(C(=O)Nc2cc(Cl)c3ncsc3c2)nn1-c1ccccc1
InChIKey: LRBFOTVVOYSQMH-UHFFFAOYSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: ADP-Glo Kinase Assay: In order to measure RIPK1 activity the ADP-Glo kinase assay (Promega, Catalog #V7002) was used to measure the conversion of ATP to ADP. This enzymatic assay was performed in a 384-well white, Optiplate (Perkin Elmer, Catalog #6007299) with assay buffer consisting of 50 mM HEPES pH 7.5 (Gibco, Catalog #15630-080), 50 mM NaCl (Teknova, Catalog #S0252), 30 mM MgCl2 (Ambion, Catalog #AM9530G), 1 mM DTT (Santa Cruz Biotechnology, Catalog #sc-29089), 0.05% BSA (Sigma, Catalog #A3059-50G) and 0.02% CHAPS (Sigma, Catalog #C5070-5G). Stock solutions of the test compounds were prepared in 100% DMSO (Sigma, Catalog #D2650) and serially diluted 1:3 using 100% DMSO. Compounds were additionally diluted 1:40 in assay buffer, and 2 μL/well were transferred to the assay plate. 4 μL/well (final concentration of 5 nM) of RIPK1 protein (SignalChem, Catalog #R07-11G-05) diluted in assay buffer and added to the assay plate followed by a 10 minute preincutation at room temperature. 4 μL/well of ATP (Promega, Catalog #V7002) (final concentration of 50 μM) diluted in assay buffer were then added to the assay plate followed by a 6 hr reaction time. Final concentrations of RIPK1 and ATP refer to a 10 μL volume. Luminescence was measured using a BioTek Synergy NEO plate reader. IC50 values were calculated using a four-parameter logistic curve fit using Genedata Screener software.
Measurement: IC50 = 1178 nM (pChEMBL 5.929999828338623)
Descriptors: MW 396.86, ALogP 4.06, HBA 6, HBD 1, PSA 76.88, QED 0.57
Compound (SMILES): COc1ncc(-c2sc(NC(=O)CC3CCOCC3)nc2C)cc1S(=O)(=O)NC1CCCC(O)C1
InChIKey: IPCYUSHEARQTBO-UHFFFAOYSA-N
Target: Phosphatidylinositol 4-kinase alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P42356
Assay: Kinase Activity Assay: In some embodiments, the subject compounds inhibit a P14-kinase, as determined by a kinase activity assay, e.g., by an assay that determines the level of incorporation of radiolabeled phosphate from [γ-32P]-ATP into a substrate molecule after treatment with a subject compound, relative to a control, by measuring the beta-particle emission rate using a scintillation counter or phosphorimaging. In certain embodiments, the subject compounds have an IC50 value for PI4K-IIIβ of less than about 1 μM, less than about 0.2 μM, less than about 0.1 μM, less than about 10 nM, less than about 1 nM, or even less, such as described in Tables 2-3. In certain embodiments, the subject compounds have an IC50 value for PI4K-IIIα of less than about 50 μM, less than about 10 μM, less than about 1 μM, less than about 0.1 μM, less than about 10 nM, less than about 1 nM, or even less, such as described in Tables 2-3. In certain further embodiments, the subject compounds have an IC50 value for PI4K-IIIβ of 50 μM or less, [etc., etc.], 10 nM or less, 6 nM or less, or even less, such as described in Tables 2-3. In certain further embodiments, the subject compounds have an IC50 value for the PI3-kinase p110α-p85 complex of between about 8 and about 10 nM, between about 8 μM and about 10 μM, or even more.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 524.67, ALogP 2.86, HBA 9, HBD 3, PSA 139.74, QED 0.48
Compound (SMILES): CC(=O)N(C)C1CCN(S(=O)(=O)c2ccc(CNC(=O)c3ccc4nccn4c3)cc2)C1
InChIKey: LOIIZIOKHWHYQN-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Enzymatic Reaction 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 100× stock. Buffer A (89 μL) containing 33 nM of NAMPT protein was added to 1 μL of 100× compound plate containing controls (e.g. DMSO or blank). The compound and enzyme mixture was incubated for 15 min at rt, then 10 μL of 10× substrate and co-factors in Buffer A were added to the test well to make a final concentration of 1 μM NAM, 100 μM 5-Phospho-D-ribose 1-diphosphate (PRPP), and 2.5 mM Adenosine 5′-triphosphate (ATP). The reaction was allowed to proceed for 30 min at rt, then was quenched with the addition of 11 μL of a solution of formic acid and L-Cystathionine to make a final concentration of 1% formic acid and 10 μM L-Cystathionine. Background and signal strength was determined by addition (or non-addition) of a serial dilution of NMN to a pre-quenched enzyme and cofactor mix.
Measurement: IC50 = 20.3 nM (pChEMBL 7.690000057220459)
Descriptors: MW 455.54, ALogP 1.51, HBA 6, HBD 1, PSA 104.09, QED 0.61
Compound (SMILES): NS(=O)(=O)c1cccc(-c2ccc(CNC3CC(F)(F)C3)cc2)c1-c1nnn[nH]1
InChIKey: DZCHOFJQHMLORX-UHFFFAOYSA-N
Target: Metallo-beta-lactamase type 2 (SINGLE PROTEIN), Bacteria
UniProt: C7C422
Assay: Class B Enzyme Assay: The Class B enzyme activities were measured in the presence of the test inhibitor in a fluorescence assay against a commercially available substrate consisting of a cephalosporin core linking 7-hydroxycoumarin to fluorescein (CCF2-FA). The enzyme (NDM-1, IMP-1 or VIM-1) and the substrate were diluted in 100 mM KH2PO4 buffer (pH 7) containing 0.005% Tween-20 and 10 μM ZnSO4. In the assay, the final concentration of enzyme was 1 pM, 2 pM and 30 pM for NDM-1, IMP-1 and VIM-1, respectively, and the final concentration of CCF2-FA was 1.25 μM. The test inhibitor was dissolved in dimethylsulfoxide and diluted 1:50 in the assay, resulting in a final concentration range of 20 uM to 0.00063 uM. In a 384-well microplate, the test inhibitor was incubated with the metallo-β-lactamase enzyme and the substrate for 2 hours at 25° C. Fluorescence at 460 nm following excitation at 405 nm was measured. The IC50 value was determined from semi-logarithmic plots of enzyme inhibition versus inhibitor concentration, with a curve generated using a 4-parameter fit.
Measurement: IC50 = 4.54 nM (pChEMBL 8.34000015258789)
Descriptors: MW 420.45, ALogP 2.07, HBA 6, HBD 3, PSA 126.65, QED 0.56
Compound (SMILES): CN[C@@H](C)C(=O)N[C@H]1CCc2ccccc2N(Cc2c(OC)ccc3cc(CN4CCNCC4)ccc23)C1=O
InChIKey: ZHJANZNXKNLYSC-IDISGSTGSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay: The peptide AVPIAQKSEK-(ε-biotin)-OH (SEQ ID NO: 1) 1:2 TFA (Peptide A) was identified as a substrate for the TR-FRET assay by screening the 6× Histidine-tagged (SEQ ID NO: 2) BIR2 domain and BIR3 domain of XIAP against a set of 29 peptides synthesized based on sequences reported by Sweeny et al. (Biochemistry, 2006, 45, 14740 14748). The peptides were labeled with the fluorescent tags FITC or TAMRA and Kd values were determined by fluorescence polarization assay. The sequence AVPIAQKSEK (SEQ ID NO: 3) was identified as optimal for using in an assay. The peptide sequence was derivatized with biotin to provide AVPIAQKSEK-(ε-biotin)-OH (SEQ ID NO: 1) 1:2 TFA as the substrate for the TR-FRET assay.The XIAP protein sequence was obtained from the SWISS-PROT protein sequence database and the BIR2 and BIR3 domains were derived from that. The sequence of the BIR2 domain used for the TR-FRET assay is MRHHHHHHRDHFALDRPSETHADYLLRTGQVVDISDTIYPRNPAMYSEEARLKSFQNW PDYAHLTPRELASAGLYYTGIGDQVQCFACGGKLKNWEPGDRAWSEHRRHFPNCFFVL GRNLNIRSE (SEQ ID NO: 4).The sequence of the BIR3 domain used for the TR-FRET assay is MRHHHHHHRSDAVSSDRNFPNSTNLPRNPSMADYEARIFTFGTWIYSVNKEQLARAGF YALGEGDKVKCFHCGGGLTDWKPSEDPWEQHAKWYPGCKYLLEQKGQEYINNIHLTH SLEECLVRTT (SEQ ID NO: 5).Ten nanomolar of 6× Histidine-tagged (SEQ ID NO: 2) BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(ε-biotin)-OH (SEQ ID NO: 1) 1:2 TFA, in the presence of 50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/niL bovine serum albumin (BSA). Following a 45 min. incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations.
Measurement: IC50 = 5109 nM (pChEMBL 5.289999961853027)
Descriptors: MW 529.69, ALogP 2.83, HBA 6, HBD 3, PSA 85.94, QED 0.42
Compound (SMILES): CCn1nc(C(=O)NC[C@]2(N)CC[C@@H](S(C)(=O)=O)CC2)c(Cl)c1-c1ccc(CC(C)(C)C(F)(F)F)cc1OC(F)F
InChIKey: HZCILHSSPDEVKI-NDQYQSCMSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor Assay: ThermoFluor® experiments were carried out using instruments owned by Janssen Research and Development, L.L.C. through its acquisition of 3-Dimensional Pharmaceuticals, Inc. 1,8-ANS (Invitrogen) was used as a fluorescent dye. Protein and compound solutions are dispensed into black 384-well polypropylene PCR microplates (Abgene) and overlayed with silicone oil (1 μL, Fluka, type DC 200) to prevent evaporation. 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.
Measurement: Kd = 2.2 nM (pChEMBL 8.65999984741211)
Descriptors: MW 629.09, ALogP 5.37, HBA 7, HBD 2, PSA 116.31, QED 0.34
Compound (SMILES): Cn1ccc2c(C(=O)N[C@H]3CC[C@H](CCN4CCc5nc(OCC(C)(F)F)sc5CC4)CC3)cccc2c1=O
InChIKey: MMNIVLFMZPOKNE-MXVIHJGJSA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Binding Inhibition for Dopamine D3 Receptor: Buffer solution: 50 mM Tris-HCl (35409-45, Nacalai Tesque) (pH 7.4) containing 120 mM NaCl (31320-05, Nacalai Tesque), 1 mM MgCl.6HO (20909-55, Nacalai Tesque), 5 mM KCl (28514-75, Nacalai Tesque) and 2 mM CaCl) (067-31, NAKARAI CHEMICALS, LTD.) Radioligand: (final concentration) 2 nM [H]-Methylspiperone ([H���N-methyl-]-Methylspiperone, NET-856, 83.8 Ci/mmol, PerkinElmer)Non-specific ligand: (final concentration) 10 uM Butaclamol [(+)-Butaclamol Hydrochloride, D033, Sigma] SPA beads solution: SPA beads [WGA PVT SPA Scintillation Beads, RPNQ0001 (500 mg), RPNQ0060 (2 g), PerkinElmer] (0.2 mg/well) Incubation time and temperature: 120 min at 25° C.
Measurement: Ki = 0.19 nM (pChEMBL 9.720000267028809)
Descriptors: MW 558.7, ALogP 4.81, HBA 7, HBD 1, PSA 76.46, QED 0.43
Compound (SMILES): CC[C@H](C)Nc1cc(Nc2ccc(C(=O)N3CCC(N4CCOCC4)CC3)cc2OC)nc2[nH]cc(C(F)(F)F)c12
InChIKey: DGNJZUPLEZLZLO-SFHVURJKSA-N
Target: Dual specificity tyrosine-phosphorylation-regulated kinase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: Q13627
Assay: Enzyme Activity Assay: Table 5: Particularly, a substrate was added to a basic reaction buffer (20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO), to which cofactors necessary for the reaction were added. Then, DYRK1A kinase was added thereto, followed by mixing well. Each compound of Examples was added thereto by using acoustic technology (Echo550; nanoliter range). The mixture was left at room temperature for 20 minutes and then 33P-ATP (specific activity 10 mCi/ml) was added to initiate the reaction. After reacting at room temperature for 2 hours, spotting was performed on P81 exchange paper. Upon completion of the reaction, kinase activity was detected using a filter-binding method.
Measurement: IC50 > 100 nM (pChEMBL 7)
Descriptors: MW 574.65, ALogP 5.48, HBA 7, HBD 3, PSA 94.75, QED 0.33
Compound (SMILES): CC(NC(=O)c1ccccc1NC(=O)CN1C(=O)C(=O)N(Cc2ccccc2)C1=O)c1ccccc1
InChIKey: PWDOMNUZQXPHIM-UHFFFAOYSA-N
Target: Integrin alpha-4/beta-7 (PROTEIN COMPLEX), Homo sapiens
UniProt: P26010
Assay: ELISA assay : The studies began by screening for the optimal protein concentrations for the method. The studies were conducted in goat anti-mouse IgG coated black React-Bind 96 welled-plates (R&D Biosystems), referred to herein as the anti-mouse IgG plate. Twelve stock solutions were prepared containing a 1:1 stoichiometric mixture of gp120 and α4β7 integrin in PBS at pH 7.2 as given by 0 μM or control, 0.001 μM, 0.01 μM, 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM. 1 μM, 2.5 μM, 5 μM, 10 μM and 25 μM in protein (step 1, FIG. 1). A 200 μL aliquot of each stock solutions was then loaded across a 96 welled plate and treated either with 20 μL of PBS pH 7.2 (control) or 20 μL of a 100 μM stock solution of repandusinic acid (RA: compound whose anti-HIV activity is to be tested) in PBS pH 7.2 containing 1% DMSO. Three repetitions were run for both the control and positive or repandusinic acid treated experiments. The final concentration of repandusinic acid in each positive well was 10 μM. The plate was incubated for 4 h at 4° C. on a plate mixer at a speed that created a vortex in each well.
Measurement: IC50 = 0.64 nM (pChEMBL 9.1899995803833)
Descriptors: MW 484.51, ALogP 3.11, HBA 5, HBD 2, PSA 115.89, QED 0.38
Compound (SMILES): Cc1c(-c2[nH]c3ccc(C4CCN(C(=O)CN(C)C)CC4)cc3c2C(C)C)cn2ncnc2c1C
InChIKey: LSHVOUOIQDCYNF-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 0.7 nM (pChEMBL 9.149999618530273)
Descriptors: MW 472.64, ALogP 4.89, HBA 5, HBD 1, PSA 69.53, QED 0.45
Compound (SMILES): O=C(O)c1ccn(C(=O)N2C[C@H]3C[C@H](OCc4ccc(Cl)c(-c5nccs5)c4)C[C@H]3C2)n1
InChIKey: UQXZDOAAAOAECH-ZSHCYNCHSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: In Vitro Competitive Activity-Based Protein Profiling: Proteomes (mouse brain membrane fraction for mouse assays; human prefrontal cortex membrane fractions for human assays) (50 mL, 1.0 mg/mL total protein concentration) were preincubated with varying concentrations of inhibitors at 37° C. After 30 min, FP-Rh (1.0 mL, 50 mM in DMSO) was added and the mixture was incubated for another 30 min at 37° C. Reactions were quenched with SDS loading buffer (15 μL-4×) and run on SDS-PAGE. Following gel imaging, serine hydrolase activity was determined by measuring fluorescent intensity of gel bands corresponding to MAGL and FAAH using ImageJ 1.43u software. IC50 data from this assay is shown in Table 1. All compounds in Table 1 were more potent inhibitors of MAGL than FAAH.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 472.95, ALogP 4.25, HBA 7, HBD 1, PSA 97.55, QED 0.6
Compound (SMILES): CN1CCN(C(=O)Cn2cc(Nc3nc4c(N5C[C@@H]6C[C@H]5CN6C(=O)CCC(F)(F)F)cccn4n3)cn2)CC1
InChIKey: XXDCMHRRTBMSSF-OALUTQOASA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Enzyme Assays: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications. Ki values were then determined using the Morrison tight binding model (Morrison, J. F., Biochim. Biophys. Acta. 185:269-296 (1969); William, J. W. and Morrison, J. F., Meth. Enzymol., 63:437-467 (1979)) modified for ATP-competitive inhibition [Ki=Ki,app/(1+[ATP]/Km,app)].
Measurement: Ki = 19.8 nM (pChEMBL 7.699999809265137)
Descriptors: MW 560.59, ALogP 1.58, HBA 10, HBD 1, PSA 107.14, QED 0.46
Compound (SMILES): CC(C)(C)n1nc(-c2[nH]c3cc(C(=O)NC4CCOCC4)ccc3c2Cl)c2c(N)ncnc21
InChIKey: JONUNFKBZRKJSB-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: Enzyme Assay: Kinase activity was detected using CisBio HTRF kinEASE kit based on time-resolved fluorescence transfer (FRET). The assay was performed in 384-well white plates (Corning #3574) in a reaction volume of 10 μL containing 1× CisBio enzymatic buffer supplemented with a final concentration of 5 mM MgCl2, 1 mM DTT, 10 nM SEB and 0.01% Triton X100 for RET. The same buffer conditions were used for KDR with the addition of 2 mM MnCl2. RETV804M buffer used 1× CisBio enzymatic buffer supplemented with a final concentration of 2 mM MgCl2, 1 mM DTT, 20 nM SEB and 0.01% Triton X100.
Measurement: IC50 = 97 nM (pChEMBL 7.010000228881836)
Descriptors: MW 467.96, ALogP 3.87, HBA 7, HBD 3, PSA 123.74, QED 0.42
Compound (SMILES): CC1(C)CCCN(c2nc(N3CCOCC3)cc(=O)[nH]2)[C@@H](Cc2ccccc2)C1
InChIKey: BIARSFMNCDSVBD-IBGZPJMESA-N
Target: Serine-protein kinase ATM (SINGLE PROTEIN), Homo sapiens
UniProt: Q13315
Assay: Biological Assay: ATM: Recombinant, full length human FLAG-tagged ATM activity (Eurofins 14-933) was measured in an ELISA for p53 S15 phosphorylation, in a 384 well V-bottom polypropylene plate (Greiner Bio One 781280). Reactions contained 0.75 nM ATM, 25 nM full length myc tagged p53 (Eurofins 23-034), 10 μM UltraPure ATP (Promega V915B) and serial dilutions of inhibitors (0.1% DMSO final) in 25 mM HEPES, pH 7.5. 5 mM MgCl2, 2.5 mM MnCl2, 0.006% Brij-35, 0.5% glycerol, 0.1 mg/ml BSA, 0.5 mM DTT and were allowed to proceed for 30 min at 20° C. 70 mM EDTA final terminated the kinase reactions. An aliquot of the terminated reaction (25 μl) was transferred to a 384-well, high binding microplate (Greiner Bio One 781061), precoated with anti-myc capture antibody overnight (Millipore 05-724) diluted in 1:1000 in PBS, then blocked with Odyssey blocking buffer (LI-COR Biosciences 92740000) for 60 min at 20° C. The terminated reaction was allowed to capture for 2 h at 20° C. Phosphorylation was measured using an antibody specific to p53 S15 phosphorylation (1:5000 Abcam ab38497), anti-rabbit HRP (1:1000 Cell Signalling Technology, 7074) and TMB (ab171522). Antibodies were diluted in Odyssey blocking buffer and incubated for 60 min at 20° C. Three washes with PBS containing 0.05% (v/v) Tween 20 (PBS-T) were performed between each incubation. The TMB reaction was stopped with an equal volume of 0.2 M sulfuric acid and absorbance read at 450 nm on the Perkin Elmer Envision within 30 min of terminating the reaction. The percentage of inhibition was calculated for each concentration of compound using 0.1% DMSO control wells as 0% inhibition and 1 μM KU60019 as 100% inhibition.
Measurement: IC50 = 84.5 nM (pChEMBL 7.070000171661377)
Descriptors: MW 396.54, ALogP 3.23, HBA 5, HBD 1, PSA 61.46, QED 0.86
Compound (SMILES): COc1cc2c(cc1OC)[C@H]1CC(COC(=O)CC(C)(C)C(=O)O)[C@H](CC(C)C)CN1CC2
InChIKey: AIYVBFYJKHAPHD-MBYSJIBESA-N
Target: Synaptic vesicular amine transporter (SINGLE PROTEIN), Homo sapiens
UniProt: Q05940
Assay: Determining human Vmat2 Inhibitory Activity of a Compound: The human Ki's for the compounds listed in Table 11 were determined using a slightly modified procedure shown below (see data in column under the heading Ki nM). In a total volume of 0.15 mL in low-binding 96-well plates (Corning #3605), twelve concentrations of Compound 1-1 and R,R,R-DHTBZ were competed against 10 nM 3H-dihydrotetrabenezine (American Radiolabeled Chemicals, Kd 2.6 nM) on rat forebrain homogenate (100 μg membrane protein per well) or human platelet homogenate (15 μg membrane protein per well) in VMAT2 binding buffer (Dulbecco's phosphate buffered saline, 1 mM EDTA, pH 7.4). Following incubation at 25° C. for 90 minutes, bound radioligand was collected by rapid filtration onto GF/B glass fiber filters using a Unifilter-96 Harvester (PerkinElmer). Filter plates were pre-treated with 0.1% polyethylenimine and allowed to dry overnight, and following harvesting the filter plates were washed with 800 d VMAT2 binding buffer. Bound radioligand was quantified by scintillation counting using a Topcount NXT (PerkinElmer).
Measurement: Ki = 260 nM (pChEMBL 6.579999923706055)
Descriptors: MW 461.6, ALogP 4.33, HBA 6, HBD 1, PSA 85.3, QED 0.55
Compound (SMILES): CN(C)C(=O)[C@H]1CCCN1c1ccc(Nc2ncc(C(N)=O)c(NC3CC3)n2)cc1
InChIKey: CEWOXYZGVYNPJD-QGZVFWFLSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: In Vitro Assay: SYK tyrosine phosphorylation activity is measured using the LANCE Technology developed by Perkin Elmer Life and Analytical Sciences (Boston, Mass.). LANCE refers to homogeneous time resolved fluorometry applications using techniques such as time-resolved fluorescence resonance energy transfer assay (TR-FRET) (see generally for procedures in Perkin Elmer Application NoteHow to Optimize a Tyrosine Kinase Assay Using Time Resolved Fluorescence-Based LANCE Detection, wwww.perkinelmer.com/lifesciences). The assay principle involves detection of a phosphorylated substrate using energy transfer from a phosphospecific europium-labeled antibody to streptavidin-allophycocyanin as an acceptor.To test the ability of candidate molecules to inhibit SYK tyrosine phosphorylation activity, molecules are reconstituted in 30% DMSO and serially diluted 1:3 with the final dilution containing DMSO in the absence of the candidate molecule. The final DMSO concentration in the assay is 3%. Kinase assays are performed as a two part reaction. The first reaction is a kinase reaction and which comprises of a candidate molecule, full length active recombinant SYK enzyme (Millipore, CA) and biotin-labeled SYK-specific substrate biotin-DEEDYESP-OH. The second reaction involves termination of the kinase reaction and the simultaneous addition of the detection reagents-europium-labeled anti-phosphotyrosine reagent (Eu-W1024-PY100, Perkin Elmer, Boston, Mass.) and Streptavidin-Allophycocyanin detection reagent (SA-APC, Prozyme, CA). The kinase reaction is performed in a black U-bottom 96-well microtitre plate. The final reaction volume is 50 μL and contains a final concentration of 1 nM active SYK enzyme, 550 nM SYK-substrate, and 100 μM ATP diluted in a buffer containing 50 mM Tris pH 7.5, 5 mM MgCl2, and 1 mM DTT. The reaction is allowed to proceed for 1 hour at room temperature. The quench buffer contains 100 mM Tris pH 7.5, 300 mM NaCl2, 20 mM EDTA, 0.02% Brij35, and 0.5% BSA. The detection reagents are added to the reaction mixture at the following dilutions1:500 for Eu-W1024-PY100 and 1:250 for SA-APC. The kinase reaction is terminated by the addition of 50 μL quench buffer containing the detection reagents. The detection is allowed to proceed for 1 hr at room temperature.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 409.49, ALogP 1.95, HBA 7, HBD 3, PSA 116.48, QED 0.64
Compound (SMILES): Cc1c(F)cccc1[C@]1(C(=O)NO)Cc2nn(C)c(-c3ccc(F)cc3)c2C1
InChIKey: QOBAYSZNUZHZBS-OAQYLSRUSA-N
Target: Histone deacetylase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UKV0
Assay: HDAC9 Biochemicle Assay: The potency of the compounds is quantified by measuring the Histone Deacetylase 9 (HDAC9) enzymatic activity using the fluorogenic substrate, Boc-Lys(Tfa)-AMC. The substrate is deacetylated to Boc-Lys-AMC by HDAC9. Cleavage by trypsin results in the release of the fluorophore AMC from the deacetylated substrate. The fluorescence of the sample is directly related to the histone deacetylase activity in the sample.
Measurement: IC50 = 53 nM (pChEMBL 7.28000020980835)
Descriptors: MW 383.4, ALogP 3.22, HBA 4, HBD 2, PSA 67.15, QED 0.54
Compound (SMILES): CCC(=O)Nc1c[nH]/c(=N\c2ccc(Oc3ccnc(C(=O)OC)c3)cc2)n(Cc2ccc(Cl)cn2)c1=O
InChIKey: XPIIUVKXCXQJBF-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 cell transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 384-well microtiter plate at a concentration of 3000 cells/well and cultured in the medium (7.0% fetal bovine serum, 7.0% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 1.37 mM NaCl, 5.27 mM KCl, 0.9 mM MgCl2, 1.26 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.5% BSA, and 0.04% Pluronic F-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 5.27 mM KCl, 0.9 mM MgCl2, 1.26 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH7.5), and each well was added with 20 μL of this buffer. The plate was placed in High-Throughput Screening System FLIPR 384 (Molecular Device Co.). Measurement of fluorescence intensity by FLIPR 384 was started, and 20 μL of DMSO solutions containing different concentrations of the test compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 5.27 mM KCl, 0.9 mM MgCl2, 1.26 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 150 nM ATP solution (25 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 4 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement. The 50% inhibitory concentration (IC50) was calculated under the assumption that the specific maximum fluorescence intensity without test compound is 0% inhibition and that the specific maximum fluorescence intensity when the dilution buffer was added in place of ATP solution is 100% inhibition, to evaluate the inhibitory activity of the test compound.
Measurement: IC50 = 356 nM (pChEMBL 6.449999809265137)
Descriptors: MW 534.96, ALogP 3.83, HBA 9, HBD 2, PSA 140.56, QED 0.33