lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CN1CCC(N(C)C(=O)c2ccc(Nc3nc4c(N5CCC=C(c6ccccc6)CC5)cccn4n3)cc2)CC1
InChIKey: UPDDNQJFBWMCAD-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Enzyme Assay: 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.
Measurement: Ki = 7.53 nM (pChEMBL 8.119999885559082)
Descriptors: MW 535.7, ALogP 5.32, HBA 7, HBD 1, PSA 69.01, QED 0.36
Compound (SMILES): O=C(O)c1cccc(Cn2cc(-c3ccnc(-c4ncc(-c5ccccc5)[nH]4)c3)cn2)c1
InChIKey: SNAILYHFPHRTGA-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: PI3Kδ Scintillation Proximity Assay: The kinase reaction was conducted in polystyrene 384-well Greiner Bio-one white plate from Thermo Fisher Scientific in a final volume of 25 μL. Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 2%. The PI3Kδ assay was carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 μM PIP2, 2 μM ATP, 0.5 μCi [γ-33P] ATP, 3.4 nM PI3Kδ. Reactions were incubated for 120 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 163 mM potassium phosphate pH 7.8, 20% glycerol, 25 mM EDTA. The final concentration of SPA beads is 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1500 rpm for 10 min, the radioactivity of the product was determined by scintillation counting on Topcount (Perkin-Elmer). IC50 determination was performed by fitting the curve of percent of the solvent control activity versus the log of the inhibitor concentration using the GraphPad Prism 6.0 software.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 421.46, ALogP 4.75, HBA 5, HBD 2, PSA 96.69, QED 0.41
Compound (SMILES): N[C@@H](CCC1CC1)[C@](O)(Cc1cc(CCCC2CC2)ccn1)C(=O)O
InChIKey: AHRSBQQYNRZTPQ-AZUAARDMSA-N
Target: Leucyl-cystinyl aminopeptidase (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UIQ6
Assay: Inhibition Assay: The inventors have assumed that inhibition of nocturnal activity of placental leucine aminopeptidase (P-LAP), i.e. aminopeptidase that cleaves AVP, would maintain and/or increase an endogenous AVP level to enhance the antidiuretic effect, which would contribute to a decreased number of nocturnal voids, and have extensively studied compounds which inhibit P-LAP.
Measurement: IC50 = 45 nM (pChEMBL 7.349999904632568)
Descriptors: MW 346.47, ALogP 2.69, HBA 4, HBD 3, PSA 96.44, QED 0.57
Compound (SMILES): Cn1cc(-c2cc(-c3ccc(N4CCN(Cc5nccn5C)CC4)nc3)c3c(C#N)cnn3c2)cn1
InChIKey: DAYDTMNFSBMWPY-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of General Formula I were screened for their ability to inhibit wild type and V804M mutant RET kinase using CisBio's HTRF® KinEASE -TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Cat. No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Cat. No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Cat. No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 119 nM (pChEMBL 6.920000076293945)
Descriptors: MW 478.56, ALogP 2.72, HBA 10, HBD 0, PSA 96.1, QED 0.38
Compound (SMILES): Cc1cc(-c2cc3ncccc3c(-c3ccn(C4(CC#N)CN(CC(F)(F)F)C4)n3)n2)ccn1
InChIKey: FUOJTQLJUCRCAA-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Caliper Enzyme Assay at 1mM ATP: Compounds were added to a 384-well plate. Reaction mixtures contained 10 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% BSA, 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20% to 30% phosphorylation. The assays were stopped with 15 μL of 180 mM HEPES, pH 7.4, 20 mM EDTA, and 0.2% Coating Reagent 3. The assay plates were placed on a Caliper Life Science LC3000 instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 966 nM (pChEMBL 6.010000228881836)
Descriptors: MW 463.47, ALogP 4.35, HBA 7, HBD 0, PSA 83.52, QED 0.44
Compound (SMILES): O=C(N[C@H](C(=O)N1C[C@H](Cl)[C@H]2OCC(=O)[C@H]21)C1CCCCC1)c1ccc(-c2cnccn2)o1
InChIKey: QTCAQPIWOIDXIR-CCARFGRDSA-N
Target: Cathepsin K (SINGLE PROTEIN), Homo sapiens
UniProt: P43235
Assay: Enzyme Assay: Recombinant human cathepsins (CatS, CatK, CatL) were purchased from a Enzo Life Sciences. All assays were carried out in 96-well format using a buffer of 50 mM KH2PO4, 50 mM NaCl, 2 mM EDTA, 0.5 mM DTT and 1% Triton-X-100, pH 6.5 for Cathepsin S and a buffer of 50 mM NaOAc, 10 mM EDTA, 1 mM DTT and 0.01% Triton-X-100, pH 5.5 for CatK/L/B. For CatS, the enzyme (0.0007 mU/well) was incubated with fluorogeninc substrate (Z-VVR-AMC, 5 μM) at RT for 10 min. For CatK the enzyme (0.00175 mU/well) was incubated with fluorogeninc substrate (Z-FR-AMC, 40 μM) at RT for 10 min. For CatL, the enzyme (0.000874 mU/well) was incubated with fluorogeninc substrate (Z-VVR-AMC, 40 μM) at RT for 10 min. Flourogenic substrate turnover was detected using a microplate reader (Synergy H4, BioTek). Ki values were calculated using the Cheng Prusoff equation (Cheng & Prosoff 1973).
Measurement: Ki = 153 nM (pChEMBL 6.820000171661377)
Descriptors: MW 472.93, ALogP 2.2, HBA 7, HBD 1, PSA 114.63, QED 0.66
Compound (SMILES): CC1(C(=O)N2CCc3ccc(-c4nccc(Nc5ccc(-c6cn[nH]c6)cc5)n4)cc3C2)CC(F)(F)C1
InChIKey: NRQLONHNKNBGKZ-UHFFFAOYSA-N
Target: Rho-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13464
Assay: Biological Assay: The kinase IC50 was determined by a commercialized CISBIO kinase detection kit, HTRF KinEASE-STK S2 kit (62ST2PEC). ROCK2 (01-119) employed in the reaction was purchased from Carna Biosciences.Before the assay, the following working solutions as needed were formulated with corresponding reagents according to the instruction of the kinase detection kit: 1×kinase buffer, 5×STK-52 substrate working solution (1.5 μM) and 5×ATP working solution (1.5 μM), 5×ROCK2 kinase working solution, 4×Streptavidin-XL665 working solution, and 4×STK-Ab-Cryptate 2 detection solution. Then the assay was performed according to the following procedure.A solution of a compound at a concentration of 10000 nM was prepared with the 1×kinase buffer containing 2.5% DMSO. Gradient dilution of the solution of the compound was performed with the kinase buffer containing DMSO, so as to obtain solutions of a test compound at 9 different concentrations. In addition to wells of test compounds, a positive well (containing all the reagents except the compound) and a negative well (containing all the reagents except the test compound and kinase) were set. Except for the control wells (positive and negative wells), a solution of a test compound (4 μL) was added to each of the reaction wells, and a solution of 2.5% DMSO was added to the control wells. Then the substrate (2 μM, i.e., 2 μL. 5×STK-S2 substrate working solution) was added to each of the reaction wells. The 5×ROCK2 kinase working solution (2 μL, containing 1.4 ng ROCK2 kinase) was added to each of the reaction wells except for the negative well, the volume of which was made up with the 1×kinase buffer (2 μL). The 5×ATP working solution (2 μL) was added to each of the reaction wells, and the mixtures were incubated at room temperature for 2 hours. After the kinase reaction was complete, the 4×Streptavidin-XL665 working solution was added to each of the reaction wells, the solutions were mixed, followed by immediate addition of the 4×STK-Ab-Cryptate 2 detection solution (5 μL), and the mixtures were incubated at room temperature for 1 hour. The fluorescence signal was read on ENVISION (Perkinelmer) (excitation wavelength: 320 nm, and emission wavelength: 665 nm and 615 nm). The inhibitory rate in each well was calculated based on the fluorescence intensity value: ER (Emission Ratio)=(fluorescence intensity at 665 nm/fluorescence intensity at 615 nm); inhibitory rate=(ERpositive−ERtest compound)/(ERpositive−ERnegative)*100%. Curves were plotted and fitted to obtain the median inhibitory concentration (IC50) of each teat compound with the PRISM 5.0 software.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 500.55, ALogP 5.6, HBA 5, HBD 2, PSA 86.8, QED 0.37
Compound (SMILES): OC(c1ccc(F)c(-c2ncnc3cc(N4CCOCC4)ccc23)c1)c1nccc2ccoc12
InChIKey: XGIZNDWBXPNXFQ-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Patch Clamp Assay: The patch-clamp measurement was carried out at room temperature in whole-cell configuration on human embryonic kidney cells (HEK293) which have been transfected in a stable manner with the hERG gene.The whole-cell configurations were carried out using an automated patch clamp device (Patchliner, Nanion Technologies, Munich). This is a glass chip-based system with which automated whole-cell measurements on up to 8 cells simultaneously are possible. The glass chip has a hole of defined size to which the cell is transferred into the Gigaseal by application of a reduced pressure and brought into the whole-cell configuration. Buffer, cell suspension and test substances were added to microchannels of the chip using a Teflon-coated pipette. The cells were clamped to a holding potential of -80 mV. For measurement of substance-promoted inhibition of the Kv11.1 channel, the following voltage protocol was applied at 10-second intervals: 51 ms/-80 mV, 500 ms/+40 mV, 500 ms/-40 mV, 200 ms/-80 mV. The leakage current is subtracted by means of the P4 method. The cells were resuspended in extracellular buffer (EC) and applied to the chip. After the cell had been collected, the seal was improved by addition of a seal enhancer buffer. As soon as the whole-cell configuration had been reached, the seal enhancer buffer was washed out and replaced by extracellular buffer. The measurement started in EC for 1.5 min. DMSO (vehicle control, 0.1% of DMSO) was then applied, and the control current was recorded for 3 min. The test substance was subsequently added twice in the same concentration, and the potassium current was measured for 3.5 min in each case.
Measurement: Ki < 10000 nM (pChEMBL 5)
Descriptors: MW 456.48, ALogP 4.5, HBA 7, HBD 1, PSA 84.51, QED 0.43
Compound (SMILES): COC(=O)N1c2ccc3c(nc(CCn4ccccc4=O)n3CC(=O)NC3CCOCC3)c2CC[C@@H]1C
InChIKey: GHPRMWDIMPBOAL-SFHVURJKSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: HTRF Biochemical Assay for CBP and BRD4 Activity: The assay was performed in a final volume of 6 μL in assay buffer containing 50 mM Hepes (pH 7.5, (0.5M Hepes, pH 7.5 solution; Teknova H1575)), 0.5 mM GSH, 0.01% BGG (0.22 μM filtered, Sigma, G7516-25G), 0.005% BSA (0.22 μM filtered, EMD Millipore Corporation, 126575) and 0.01% Triton X-100 (Sigma, T9284-10L). Nanoliter quantities of 10-point, 3-fold serial dilution in DMSO were pre-dispensed into 1536 assay plates (Corning, #3724BC) for a final test concentration of 33 μM to 1.7 nM, top to lowest dose, respectively. 3 of 2× Protein and 3 μL of 2× Peptide Ligand were added to assay plates (pre-stamped with compound). Plates were incubated for varying times at room temperature prior to measuring the signal. TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) was measured on the PHERAstar (BMG, equipped with HTRF optic module [337/520/490]) or on the Envision (PerkinElmer, equipped with the TRF Laser unit, TRF dual mirror D400/D505 and emission filters M520 and M495). Data were reported as percent inhibition compared with control wells based on the following equation: % inh=1−((TR-FRET ratio−AveLow)/(AveHigh−AveLow)) where TR-FRET ratio=(Fluorescence at 520 nm/Fluorescence at 490 nm)*10000), AveLow=average TR-FRET ratio of no enzyme control (n=32), and AveHigh=average TR-FRET ratio of DMSO control (n=32). IC50 values were determined by curve fitting of the standard 4 parameter logistic fitting algorithm included in the Activity Base software package: IDBS XE Designer Model 205. Data is fitted using the Levenburg Marquardt algorithm. For all assay formats data were reported as percent inhibition compared with control wells based on the following equation: % inh=100*((FLU−AveLow)/(AveHigh−AveLow)) where FLU=measured Fluorescence, AveLow=average Fluorescence of no enzyme control (n=32), and AveHigh=average Fluorescence of DMSO control (n=32). IC50 values were determined by curve fitting of the standard 4 parameter logistic fitting algorithm included in the Activity Base software package: IDBS XE Designer Model 205. Data is fitted using the Levenburg Marquardt algorithm.
Measurement: IC50 = 50500 nM (pChEMBL 4.300000190734863)
Descriptors: MW 507.59, ALogP 2.64, HBA 8, HBD 1, PSA 107.69, QED 0.55
Compound (SMILES): COc1nnc(-c2cc(NC(=O)c3cnnc(C(C)(C)C#N)c3)ccc2C)cc1N1CCOCC1
InChIKey: VTHNGQWBKWIXPC-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: In Vitro Raf Activity: Raf and biotinylated Mek, kinase dead, were combined at 2× final concentrations in assay buffer (50 mM Tris, pH 7.5, 15 mM MgCl2, 0.01% BSA and 1 mM DTT) and dispensed 5 ml per well in assay plates (Greiner white 384 well assay plates #781207) containing 0.25 ml of 40× of a Raf kinase inhibitor test compound diluted in 100% DMSO. The plate was incubated for 60 minutes at room temperature. The Raf kinase activity reaction was started by the addition of 5 mL per well of 2×ATP diluted in assay buffer. After 3 hours (b-Raf(V600E)) or 1 hour (c-Raf). The reactions were stopped and the phosphorylated product was measured using a rabbit anti-p-MEK (Cell Signaling, #9121) antibody and the Alpha Screen IgG (ProteinA) detection Kit (PerkinElmer #6760617R), by the addition of 10 mL to the well of a mixture of the antibody (1:2000 dilution) and detection beads (1:2000 dilution of both beads) in Stop/bead buffer (25 mM EDTA, 50 mM Tris, pH 7.5, 0.01% Tween20). The additions were carried out under dark conditions to protect the detection beads from light. A lid was placed on top of the plate and incubated for 1 hour at room temperature, then the luminescence was read on a PerkinElmer Envision instrument. The concentration of each compound for 50% inhibition (IC50) was calculated by non-linear regression using XL Fit data analysis software.
Measurement: IC50 = 2.2 nM (pChEMBL 8.65999984741211)
Descriptors: MW 473.54, ALogP 3.14, HBA 9, HBD 1, PSA 126.15, QED 0.57
Compound (SMILES): Cc1c(Cl)cccc1-c1noc([C@@H]2CCCN2C(=O)c2cc(OC(F)(F)F)ccc2-n2nccn2)n1
InChIKey: DPNQFIAPEJNVKH-IBGZPJMESA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: In Vitro Assay: Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES. On the day of the assay, 50 uL of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/L, 5 mM probenecid (Sigma) and 3 uM of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at rt for 30 min before measurement.Within the Fluorescent Imaging Plate Reader (FLIPR Tetra, Molecular Devices), antagonists are added to the plate in a volume of 10 uL/well, incubated for 120 min and finally 10 uL/well of agonist is added. Fluorescence is measured for each well at 1 second intervals, and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 3 nM orexin-A with vehicle in place of antagonist. The IC50 value (the concentration of compound needed to inhibit 50% of the agonistic response) is determined and may be normalized using the obtained IC50 value of an on-plate reference compound. Optimized conditions were achieved by adjustment of pipetting speed and cell splitting regime. The calculated IC50 values may fluctuate depending on the daily cellular assay performance. Fluctuations of this kind are known to those skilled in the art. In the case where IC50 values have been determined several times for the same compound, the geometric mean has been given.
Measurement: IC50 = 614 nM (pChEMBL 6.210000038146973)
Descriptors: MW 518.88, ALogP 5.16, HBA 8, HBD 0, PSA 99.17, QED 0.36
Compound (SMILES): Nc1nc2cc(-c3ccc4ncc(N5CC(N6CCOCC6)C5)nc4c3)c(Cl)cc2o1
InChIKey: CLEAUKLYWGBLAL-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: PI 3-Kinase HTRF Assay: Briefly, the assay is a time resolved FRET assay that indirectly measures PIP3 product formed by the activity of a PI3-K. The kinase reaction is performed in a microtitre plate (e.g., a 384 well microtitre plate). The total reaction volume is approximately 20 ul per well. In the first step, each well receives 2 ul of test compound in 20% dimethylsulphoxide resulting in a 2% DMSO final concentration. Next, approximately 14.5 ul of a kinase/PIP2 mixture (diluted in 1x reaction buffer) is added per well for a final concentration of 0.25-0.3 ug/ml kinase and 10 uM PIP2. The plate is sealed and incubated for 15 minutes at room temperature. To start the reaction, 3.5 ul of ATP (diluted in 1x reaction buffer) is added per well for a final concentration of 10 uM ATP. The plate is sealed and incubated for 1 hour at room temperature. The reaction is stopped by adding 5 ul of Stop Solution per well and then 5 ul of Detection Mix is added per well. The plate is sealed, incubated for 1 hour at room temperature, and then read on an appropriate plate reader.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 436.9, ALogP 3.19, HBA 8, HBD 1, PSA 93.54, QED 0.52
Compound (SMILES): COc1cccc(-c2nc(Nc3ccncn3)c3cccn3n2)n1
InChIKey: VBAFOXIKWGSJME-UHFFFAOYSA-N
Target: TGF-beta receptor type-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P37173
Assay: Biological Assays: Assays are conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGFβR1 T204D or HIS-TGFβR2 WT, anti-HIS detection antibody, a labeled small molecule probe (Kd=<100 nM; koff=<0.001 s−1.) and test compounds in assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij35, 4 mM DTT, and 0.05 mg/ml BSA). The reaction is incubated for 1 hour at room temperature and the HTRF signal was measured on an Envision plate reader (Ex: 340 nm; Em: 520 nm/495 nm). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay are 1 nM HIS-TGFβR1 T204D or HIS-TGFβR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule probe (at Kd) and 0.5% DMSO. Dose response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 > 15000 nM (pChEMBL 4.823999881744385)
Descriptors: MW 319.33, ALogP 2.33, HBA 8, HBD 1, PSA 90.12, QED 0.62
Compound (SMILES): CN1C(=N)N[C@](C)(c2cc(NC(=O)c3ccc(F)cn3)ccc2F)[C@]2(CCN(S(=O)(=O)c3ccccc3)C2)S1(=O)=O
InChIKey: AGNMDKJKGFYLMR-CLJLJLNGSA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: Time-Resolved Endpoint proteolysis Assay: Inhibitor IC50's at purified human autoBACE-2 are determined in a time-resolved endpoint proteolysis assay that measures hydrolysis of the QSY7-EISEVNLDAEFC-Eu-amide FRET peptide substrate (BACE-HTRF assay). BACE-mediated hydrolysis of this peptide results in an increase in relative fluorescence (RFU) at 620 nm after excitation with 320 nm light. Inhibitor compounds, prepared at 3x the desired final concentration in 1xBACE assay buffer (20 mM sodium acetate pH 5.0, 10% glycerol, 0.1% Brij-35) supplemented with 7.5% DMSO are pre-incubated with an equal volume of autoBACE-2 enzyme diluted in 1xBACE assay buffer (final enzyme concentration 1 nM) in black 384-well NUNC plates for 30 minutes at 30° C. The assay is initiated by addition of an equal volume of the QSY7-EISEVNLDAEFC-Eu-amide substrate (200 nM final concentration, Km=8 uM for 4 uM for autoBACE-2) prepared in 1xBACE assay buffer supplemented with 7.5% DMSO and incubated for 90 minutes at 30° C. DMSO is present at 5% final concentration in the assay. Following laser excitation of sample wells at 320 nm, the fluorescence signal at 620 nm is collected for 400 ms following a 50 us delay on a RUBYstar HTRF plate reader (BMG Labtechnologies). Raw RFU data is normalized to maximum (1.0 nM BACE/DMSO) and minimum (no enzyme/DMSO) RFU values. IC50s are determined by nonlinear regression analysis (sigmoidal dose response, variable slope) of percent inhibition data with minimum and maximum values set to 0 and 100 percent respectively. Similar IC50s are obtained when using raw RFU data. The Ki values are calculated from the IC50 using the Cheng-Prusoff equation.
Measurement: Ki = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 604.66, ALogP 2.46, HBA 7, HBD 3, PSA 152.63, QED 0.4
Compound (SMILES): CCN1CCc2c(N)nn(C(=O)C3CCNc4ccccc43)c2C1
InChIKey: MLDUUCQJKVIFTD-UHFFFAOYSA-N
Target: Prothrombin (SINGLE PROTEIN), Homo sapiens
UniProt: P00734
Assay: Counterscreens for Selectivity: Thrombin. In a 96-well white opaque plate, 80 μl of assay buffer was added to each well. Test compounds were added, and serially diluted as above, to generate a concentration range of approximately 1-100 μM. Human alpha-thrombin (Enzyme Research Labs) was diluted in assay buffer to a final concentration of 12.5 nM. 80 μl of this enzyme solution was added to the assay wells. The enzyme/compound mixtures were incubated for 10 minutes at room temperature. Fluorogenic substrate (Boc-Val-Pro-Arg-7-amido-4-methylcoumarin; Sigma) was added to assay wells at a final concentration of 20 μM. The assay plate was spun for 1 minute at 1500 g and then read at 37° C. in a SpectraMax M2 plate reader (λex=380 nm and λem=460 nm). Activity was quantified as the rate of change in fluorescence, which corresponds to the rate of substrate cleavage. IC50 values were determined as the concentration of inhibitor that produced 50% of the rate of change of control wells without any inhibitor. For compounds with activity <1 μM, the assay was repeated with a lower concentration range, typically from 10-1000 nM.
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 325.42, ALogP 2.08, HBA 6, HBD 2, PSA 76.18, QED 0.88
Compound (SMILES): Cc1c(-c2[nH]c3ccc(C4CCN(C(=O)OC5CCN(C(C)C)C5)CC4)cc3c2C(C)C)cn2ncnc2c1C
InChIKey: XMTABVAQKKZWLF-UHFFFAOYSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
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 = 6.9 nM (pChEMBL 8.15999984741211)
Descriptors: MW 542.73, ALogP 6.42, HBA 6, HBD 1, PSA 78.76, QED 0.31
Compound (SMILES): N#CC[C@H]1CC[C@H](n2c(CC(=O)NC34CC5CC(CC(O)(C5)C3)C4)nc3cnc4[nH]ccc4c32)CC1
InChIKey: MPUPFFCJTANSDV-XTSASLLGSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Enzymatic Inhibition Assay: 11-point dosing series were made for each compound by serially diluting 1:3 or 1:4 in DMSO, with point 12 being a DMSO control. From the serial dilution plates, sample was transferred to a 384 wells assay plate (#781280, Greiner, Monroe, N.C.) using Labcyte Echo (Sunnyvale, Calif.), or Biosero ATS (San Diego, Calif.). The compounds were tested in duplicate. Column 12 was used for positive controls, and column 24 contained negative controls with no enzyme added. A compound from our internal collection, with inhibitory activity for JAK isoforms, was used as a reference compound. The final concentration of DMSO was ≤0.25% in a 20 μL reaction. Assay conditions for each of the proteins are summarized in Table 3. The enzyme reaction was initiated by the addition of 10 μL of 2× enzyme and ATP mixture to 10 μL of 2× substrate solution prepared in reaction buffer (50 mM MOPS pH 7.5, 10 mM MgCl2, 1 mM EDTA, 2 mM DTT, 0.002% Tween-20). The Tyk2 enzyme was pre-incubated with 2 mM ATP for 30 min prior to the reaction initiation. Immediately after the addition of the enzyme to the reaction mixture, the plate was centrifuged at 1000 rpm for 1 minute and incubated at 25° C. for 45 minutes for JAK 3 and 90 minutes for JAK1, JAK2 and Tyk2. The reaction was quenched by the addition of 20 μL of 0.5% TFA containing 0.15 μM of internal standard peptide using Multidrop Combi reagent dispenser (Thermo Scientific, Waltham, Mass.). Several wells in column 24 were typically used for the product standard curve. After the quench, the assay plate was centrifuged at 3000 rpm for 3 minutes and sealed with pierceable aluminum foil (Cat #06644-001, Agilent) using a PlateLoc (Agilent Technologies, Santa Clara, Calif.). The plates then were transferred on to the RapidFire for the MS analysis. Compound inhibition was assessed by a decrease of the phosphorylated product levels in sample wells compared to the non-inhibited enzyme reaction. The assay conditions for the above assays are shown in Table 3 and the results of Ex. 1-209 as tested in these assays are shown in Table 4.TABLE 3Assay conditions for JAK family enzyme assays* [p], [ATP], [Substrate], [IS],Enzyme nM μM μM nMJAK1-JH1JH2 8.0 12.5 200 100JAK2-JH1JH2 7.0 or 3.6 30 40 100JAK3-JH1JH2 2.0 150 40 100Tyk2-JH1JH2   25 or 14.7 50 200 100*Reaction buffer: 50 mM MOPS, pH 7.5 10 mM MgCl2, 1 mM EDTA, 2 mM DTT, 0.002% Tween-20; IS stands for internal standard peptide; p stands for phospho-peptide (product); Substrate stands for peptide.
Measurement: IC50 = 1.7 nM (pChEMBL 8.770000457763672)
Descriptors: MW 486.62, ALogP 4.3, HBA 6, HBD 3, PSA 119.62, QED 0.5
Compound (SMILES): O=C(O)C12CCC(C(=O)N3CCC4(S(=O)(=O)c5cccc(Cl)c5)c5ccc(C(F)(C(F)(F)F)C(F)(F)F)cc5CCC34)(CC1)CC2
InChIKey: YAPKXGXXZUYCIH-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Binding Assay : The binding of potential ligands to RORγ is measured by competition with [3H]25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA). The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 μg/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 μM to 0.28 nM, for 10 min at rt in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% glycerol (Sigma Cat # G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat # RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined.
Measurement: IC50 = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 696.08, ALogP 7.27, HBA 4, HBD 1, PSA 91.75, QED 0.33
Compound (SMILES): Oc1ccc2c3c(cnc2c1)-c1cccc(F)c1OC3c1ccc(OCCN2CC(CF)C2)cc1
InChIKey: UQBGEQPSMYFQHH-UHFFFAOYSA-N
Target: Estrogen receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P03372
Assay: Competition Binding Assay: Run the competition binding assay in a buffer containing 50 mM HEPES, pH 7.5, 1.5 mM EDTA, 150 mM NaCl, 10% glycerol, 1 mg/mL ovalbumin, and 5 mM DTT, using 0.025 μCi per well 3H-estradiol (118 Ci/mmol, 1 mCi/mL), 7.2 ng/well ERα (wild type), or 7.2 ng/well ERα (Y537S mutant) or 7.7 ng/well ERβ receptor. Add the test compound at 10 different concentrations ranging from 10,000 nM to 0.5 nM, and determine nonspecific binding in the presence of 1 μM of 1713 estradiol. Incubate the binding reaction (140 μL) for 4 hours at room temperature, and then add cold dextran-charcoal buffer (70 μL) (containing per 50 mL of assay buffer, 0.75 g of charcoal and 0.25 g of dextran) to each reaction. Mix the plates for 8 minutes on an orbital shaker at 4° C. and then centrifuge at 3000 rpm at 4° C. for 10 minutes. Transfer an aliquot (120 μL) of the mixture to another 96-well, white flat bottom plate (Costar) and add Perkin Elmer Optiphase Supermix scintillation fluid (175 μL) to each well. Seal the plates and shake vigorously on an orbital shaker. After an incubation of 2.5 hours, read the plates in a Wallac Microbeta counter.
Measurement: Ki = 15.8 nM (pChEMBL 7.800000190734863)
Descriptors: MW 474.51, ALogP 5.51, HBA 5, HBD 1, PSA 54.82, QED 0.4
Compound (SMILES): CN1CC(CS(=O)(=O)NNC(=O)c2cc(-c3cn(C)cn3)cc(Cl)c2F)CC1=O
InChIKey: OWTIBWGUWGMHFM-UHFFFAOYSA-N
Target: Histone acetyltransferase KAT6A (SINGLE PROTEIN), Homo sapiens
UniProt: Q92794
Assay: KAT-6A Enzyme Assay: KAT-6A. Enzyme assay buffer was 50 mM Tris pH 8.0, 0.002% Tween20, 0.005% bovine skin gelatin, and 1 mM dithiothreitol (DTT). For determination of IC50values, compounds were serially diluted with 2% (v/v) DMSO in the final reaction, pre-incubating each dilution of each compound with 40 μL of assay buffer containing KAT-6A enzyme (12.5 nM final concentration). 10 μL of assay buffer containing 1 μM peptide substrate and 1 μM acetyl coenzyme A (final concentrations) was added. Reactions (50 μL total) were then carried out at 25° C. for 90 minutes. Reactions were terminated by the addition of 0.5% formic acid (final concentration), and a sample of each reaction was analyzed by SAMDI Tech, Inc. (Chicago, Ill.) using self-assembled monolayer desorption/ionization time-of-flight mass spectrometry (Mrksich, M. (2008) Mass spectrometry of self-assembled monolayers: a new tool for molecular surface science. ACS Nano 2, 7-18).
Measurement: IC50 = 30000 nM (pChEMBL 4.519999980926514)
Descriptors: MW 443.89, ALogP 0.92, HBA 6, HBD 2, PSA 113.4, QED 0.64
Compound (SMILES): CC(=O)N[C@@H]1CCC[C@H](C(=O)Nc2cc(-c3cc(F)c4nn(C)c(C5CC5)c4c3)c(Cl)cn2)C1
InChIKey: IUMDSEQXNBJVCE-DOTOQJQBSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P50750
Assay: CDK4/CyclinD1 Enzymatic Activity Assay: The inhibitory activity of compounds was evaluated in vitro using TR-FRET assay with white 384-well low volume microplate (Greiner Bio-One). CDK4/Cyclin D1 catalyzed phosphorylation of peptide in the presence and absence of compounds was measured and used in IC50 determination. Recombinant protein complex CDK4/Cyclin D1, expressed from insect cell, was purchased from ProQinase. Testing compounds were dissolved in DMSO at 1 mM and tested in 9-dose IC50 mode. The reaction mixture was prepared by mixing CDK4/CyclinD1 (1 nM final), ULight-4E-BP1 (100 nM final, Perkinelmer, TRF0128-D), and ATP (2 mM final) in assay buffer (20 mM of HEPES pH 7.4, 1 mM of EGTA, 0.05% BSA, 0.005% Tween 20, and 1 mM TCEP). The compound of interest in DMSO was added to each well in 3-fold serial dilution by dispenser (TECAN D300E) to make a 9.9 μL of reaction mixture. After 20 minutes preincubation at room temperature, 0.1 μL MgCl2(10 mM final) was added to initiate the reaction. Following a 45 minutes incubation at 37° C., the reaction was stopped by addition of 2 μL of quenching buffer consisting of Lance detection buffer (Perkinelmer CR97-100C), LANCE Ultra Europium-anti-P-4E-BP1 (Perkinelmer, TRF0216-D), EDTA, and incubate at room temperature for additional 60 minutes in dark. The reaction signal was measured by Envision multimode plate reader (PerkinElmer, 2102-0010).
Measurement: IC50 = 4.12 nM (pChEMBL 8.380000114440918)
Descriptors: MW 483.98, ALogP 4.94, HBA 5, HBD 2, PSA 88.91, QED 0.54
Compound (SMILES): CC(C)Cc1ccc(C(C)c2nc(-c3ccncc3)no2)cc1
InChIKey: OKZXDENUVHWQMO-UHFFFAOYSA-N
Target: Sphingosine 1-phosphate receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99500
Assay: S1P1 assay: The method involves contacting the receptor with a suitable concentration of an inventive compound to bring about activation of the receptor. The contacting can take place in vitro, for example in carrying out an assay to determine the S1P receptor activation activity of an inventive compound undergoing experimentation related to a submission for regulatory approval.
Measurement: EC50 = 448 nM (pChEMBL 6.349999904632568)
Descriptors: MW 307.4, ALogP 4.48, HBA 4, HBD 0, PSA 51.81, QED 0.7
Compound (SMILES): NS(=O)(=O)OC[C@H]1C[C@@H](Nc2ccnc3cc(-c4ccnc(C(F)(F)F)c4)nn23)[C@H](O)[C@@H]1O
InChIKey: GYMPFXKRZWRPJD-HOLCONJYSA-N
Target: Ubiquitin-like modifier-activating enzyme 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P22314
Assay: Ubiquitin Activating Enzyme (UAE) HTRF Assay: The UAE enzymatic reaction totals 50 μL and contains 50 mmol HEPES (pH 7.5), 0.05% BSA, 2.5 mM MgCl2, 0.1 uM ATP, 8 nM GST-Ubc-2, 35 nM flag-ubiquitin, 1 nM recombinant human UAE or mouse UAE. Compounds for this hUAE IC50 assay are tested at 10 point 3-fold dilution. The top concentration for this assay is 1 μM. Each compound is ordered in duplicate on the same plate. The enzymatic reaction mixture is incubated for 90 minutes at room temperature (24 degrees C.) in a 384 well plate prior to termination with a stop solution (0.1 M HEPES/0.05% Tween20, 20 mmol EDTA, 410 mM KF, 0.5 nM Eu Cryptate anti-FLAG M2-K antibody (Cis-bio International), 8 ug/mL Anti-GST XL-APC (Prozyme)). After incubation for 120 minutes, quantification of the FRET is performed on the Pherostar (BMG).
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 488.45, ALogP 0.55, HBA 10, HBD 4, PSA 164.96, QED 0.39
Compound (SMILES): [C-]#[N+]C1(C)CCCC1N1CCC(c2ccc3[nH]c(-c4cc(C)c5ncnn5c4)c(C(C)C)c3c2)CC1
InChIKey: CEWIYRMTDZCNCO-UHFFFAOYSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
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 = 16.4 nM (pChEMBL 7.789999961853027)
Descriptors: MW 480.66, ALogP 6.72, HBA 4, HBD 1, PSA 53.58, QED 0.33
Compound (SMILES): CC(C)n1cc(CN2CCCC23CCN(C(=O)OC(C(F)(F)F)C(F)(F)F)CC3)c(-c2ccccc2F)n1
InChIKey: UZJMKDNAZICZCF-UHFFFAOYSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Mus musculus
UniProt: O35678
Assay: Preparation of Mouse Brain Proteomes from Inhibitor Treated Mice: Inhibitors were administered to wild-type C57B1/6J by oral gavage in a vehicle of polyethylene glycol. Each animal was sacrificed 4 h following administration and brain proteomes were prepared and analyzed according to previously established methods (See Niphakis, M. J., et al. (2011) ACS Chem. Neurosci. and Long, J. Z., et al. Nat. Chem. Biol. 5:37-44).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 550.52, ALogP 6.33, HBA 5, HBD 0, PSA 50.6, QED 0.41
Compound (SMILES): C[C@@H](c1ccc(C(F)(F)F)nc1)n1nc(C#N)c2c(=O)[nH]c(C3CCC3c3ncc(Br)cn3)nc21
InChIKey: HPFLLZGQKSUDNM-FNBIGBAISA-N
Target: High affinity cGMP-specific 3',5'-cyclic phosphodiesterase 9A (SINGLE PROTEIN), Homo sapiens
UniProt: O76083
Assay: Enzyme Assay: Assays for PDE 1 through 11 were performed in parallel at room temperature in 384-well microtiter plates with an incubation volume of 20.2 μL. Solutions of test compounds were prepared in DMSO and serially diluted with DMSO to yield 30 μL of each of ten solutions differing by 3-fold in concentration, at 32 serial dilutions per plate. 100% inhibition was determined by adding buffer in place of the enzyme and 0% inhibition is determined by using DMSO (1% final concentrations). A Labcyte POD 810 (Labcyte, Sunnyvale, Calif.) was used to dispense 200 nL from each well of the titration plate to make eleven copies of the assay plate for each titration, one copy for each PDE enzyme. A solution of each enzyme (dilution from aliquots, sufficient to produce 20% substrate conversion) and a separate solution of FAM-labeled cAMP or FAM-labeled cGMP from Molecular Devices (Sunnyvale, Calif., product # R7506 or cGMP#R7508), at a final concentration of 50 nM were made in the assay buffer (10 mM Tris HCl, pH 7.2, 10 mM MgCl2, 0.05% NaN3 0.01% Tween-20, and 1 mM DTT). Note that the substrate for PDE2 is 50 nM FAM cAMP containing 1000 nM of cGMP. The enzyme and the substrate were then added to the assay plates in two consecutive additions of 10 μL and then shaken to mix. The reaction was allowed to proceed at room temperature for 60 minutes. A binding solution was then made from the kit components, comprised of 80% Solution A, 20% Solution B and binding reagent at a volume of 1/600 the total binding solution. The enzymatic reaction was stopped by addition of 60 μL of the binding solution to each well of the assay plate. The plates were sealed and shaken for 10 seconds
Measurement: Ki = 0.12 nM (pChEMBL 9.920000076293945)
Descriptors: MW 545.32, ALogP 4.23, HBA 8, HBD 1, PSA 126.03, QED 0.41
Compound (SMILES): Cc1ncsc1C(CNC(=O)c1c(F)cccc1Cl)N1CCOC(c2ccc(F)cc2F)C1
InChIKey: QRIKVTXNYRWNNF-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: YO-PRO-1 Uptake Assay: YO-PRO®-1 is a fluorescent DNA-binding dye with a MW of 374 Da (Molecular Probes, cat. Y3603). This method is based upon the presumed ability of YO-PRO-1 to enter through the dilated or "large pore form" of P2X7 receptor and to bind to intracellular DNA whereupon it increases many fold its fluorescence intensity. The dye has an absorption spectrum compatible with excitation at 488 nm by argon laser sources and its emission wavelength is in the range of 515-575 nm. The aim of this assay was to validate the interaction of antagonists with P2X7 receptor using an alternative readout to Ca2+-sensitive fluorescent dyes.HEK-293 cells stably transfected with P2X7 receptor were seeded overnight in growth medium at 20,000 cells/well in 384-well plate. 24 hours later, the medium was removed, the cells were washed with Low Divalent Cation Assay Buffer (0.3 mM Ca2+ and 0 mM Mg2+) and then pre-loaded with 20 μL/w of 5 μM YO-PRO-1 dye. FLIPRTETRA fluorescence measurement immediately started. Then, 10 μL/w of test compounds and reference antagonist A438079 at 3×-concentration were injected with the FLIPRTETRA and the kinetic response over a period of five minutes was monitored. A second injection of 10 μL/w of 3× Bz-ATP EC80 (30 μM) was performed with the FLIPRTETRA and the signal of the emitted fluorescence was recorded for additional 60 minutes. All the experiment was carried out with a Low Divalent Cation Assay Buffer (0.3 mM Ca2+ and 0 mM Mg2+).The effect of the test compounds was measured as percent inhibition vs the reference antagonist and IC50 values were calculated accordingly.
Measurement: IC50 = 53 nM (pChEMBL 7.28000020980835)
Descriptors: MW 495.95, ALogP 5.07, HBA 5, HBD 1, PSA 54.46, QED 0.52
Compound (SMILES): C=C1C[C@@H](c2nc3cc(Cl)c(C)cc3[nH]2)N(C(=O)c2nc(C)sc2-c2ccc(C)c(C)c2)C1
InChIKey: VPGMOJNQLSGSJB-QFIPXVFZSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Inhibition 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 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 477.03, ALogP 6.72, HBA 4, HBD 1, PSA 61.88, QED 0.33
Compound (SMILES): Cn1c(SCCCN2C[C@H]3CCN(c4ccc(F)cc4)[C@H]3C2)nnc1-c1ccc(C(N)=O)nc1
InChIKey: BHNXJQYCDCKDKH-UTKZUKDTSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Functional Calcium Assay: Functional Calcium Assay at hD2 recombinant receptor. CHO cells stably expressing human dopamine receptor type 2, long variant (hD2L), coupled to Gα16 protein (CHO-Gα16-hD2L) were seeded into black walled clear-base 384-well plates at a density of 8,000 cells per well and grown overnight at 37° C. After washing with the assay buffer (20 mM HEPES, 145 mM NaCl, 5 mM KCl, 5.5 mM glucose, 1 mM MgCl2 and 2 mM CaCl2, pH 7.4) containing 2.5 mM Probenecid, cells were incubated with the cytoplasmic Ca2+ probe Fluo-4 AM at 1 μM (final concentration), 37° C. for 60 min. Plates were washed three times as above and placed into a Fluorometric Imaging Plate Reader (FLIPR Tetra, Molecular Devices) to monitor cell fluorescence (ex=470-495 nm, em=515-575 nm) before and after the addition of different concentrations of test compounds. Compounds of invention were dissolved in DMSO and 200-fold diluted with assay buffer plus 0.01% Pluronic F-127. Cells were exposed first to test compounds for 10 min, then to a submaximal concentration of the hD2 receptor agonist dopamine (EC80, 50-140 nM). The fluorescence before compound addition (baseline) and before and after addition of agonist challenge was monitored. The peak of Ca2+ stimulation (baseline subtracted) was plotted versus the concentration of test compound and the curve fitted using a four-parameter logistic equation (XLfit) to assess the agonist/antagonist potency and maximal response.
Measurement: Ki = 191 nM (pChEMBL 6.71999979019165)
Descriptors: MW 481.6, ALogP 2.81, HBA 8, HBD 1, PSA 93.17, QED 0.39