lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COCCN(CCCCc1ccc2c(n1)NCCC2)CC[C@H](Nc1ncnc2cc(Cl)ccc12)C(=O)O
InChIKey: FRLFLFWXQJDPEU-QHCPKHFHSA-N
Target: Integrin alpha-V/beta-6 (PROTEIN COMPLEX), Homo sapiens
UniProt: P06756
Assay: Solid Phase Integrin alphav/beta6 Binding Assay (B-1): Microplates were coated with recombinant human integrin αvβ6 (2 μg/mL) in PBS (100 μL/well 25° C., overnight). The coating solution was removed, washed with wash buffer (0.05% Tween 20; 0.5 mM MnCl2; in 1×TBS). The plate was blocked with 200 μL/well of Block Buffer (1% BSA; 5% sucrose; 0.5 mM MnCl2; in 1×TBS) at 37° C. for 2 h. Dilutions of testing compounds and recombinant TGFβ1 LAP (0.67 μg/mL) in binding buffer (0.05% BSA; 2.5% sucrose; 0.5 mM MnCl2; in 1×TBS) were added. The plate was incubated for 2 hours at 25° C., washed, and incubated for 1 hour with Biotin-Anti-hLAP. Bound antibody was detected by peroxidase-conjugated streptavidin. The IC50 values for testing compounds were calculated by four-parameter logistic regression.
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 527.07, ALogP 4.26, HBA 8, HBD 3, PSA 112.5, QED 0.27
Compound (SMILES): CCC(=O)NCCCCc1ccc(Cc2cc([C@@H]3O[C@H](SC)[C@@H](O)[C@H](O)[C@H]3O)ccc2C)cc1
InChIKey: DAKIYXKKXPNGFC-IONQDRQYSA-N
Target: Sodium/glucose cotransporter 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P31639
Assay: In Vitro Human SGLT2 Inhibition Assay: Human sodium/glucose co-transporter type 2 (SGLT2; accession number P31639; GI:400337) was cloned into pIRESpuro2 vector for mammalian expression (construct: HA-SGLT2-pIRESpuro2). HEK293 cells were transfected with the human HA-SGLT2-pIRESpuro2 vector and the bulk stable cell line was selected in presence of 0.5 μg/mL of puromycin. Human HA-SGLT2 cells were maintained in DMEM media containing 10% FBS, 1% GPS and 0.5 μg/mL of puromycin. The HEK293 cells expressing the human HA-SGLT2 were seeded in 384 well plates (30,000 cells/well) in DMEM media containing 10% FBS, 1% GPS and 0.5 μg/mL of puromycin, then incubated overnight at 37 C, 5% CO2. Cells were then washed with uptake buffer (140 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 5 mM Tris, 1 mg/mL bovine serum albumin (BSA), pH 7.3). Twenty microliters of uptake buffer with or without testing compounds were added to the cells. Then, 20 microliters of uptake buffer containing 14C-AMG (100 nCi) were added to the cells. The cell plates were incubated at 37° C., 5% CO2 for 1-2 hours. After washing the cells with uptake buffer, scintillation fluid was added (40 microliters/well) and 14C-AMG uptake was measured by counting radioactivity using a scintillation coulter (TopCoulter NXT; Packard Instruments).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 487.66, ALogP 3.28, HBA 6, HBD 4, PSA 99.02, QED 0.38
Compound (SMILES): C[C@@H](Nc1ncnc2c1cc(-c1ccnnc1)c(=O)n2C)c1cccc(C(F)F)c1F
InChIKey: GUBNZZJSMBVSEQ-LLVKDONJSA-N
Target: Son of sevenless homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07889
Assay: Surface Plasmon Resonance (SPR) Assay: Using a GE Biacore 8K SPR instrument, avi-tagged SOS1 catalytic domain protein was immobilized to a level of approximately 6000 response units (RU) on a streptavidin-coated SPR sensor chip in assay buffer containing 0.01 M HEPES, 0.15 M NaCl and 0.05% v/v Surfactant P20. In assay buffer containing 2% DMSO, concentration series of test compounds were generated spanning 5 μM to 4.9 nM over ten 2-fold dilutions. For each test compound, a separate 0 μM sample was generated for use during subsequent double reference subtraction. Serially for each test compound, individual dilution samples were flowed over the immobilized SOS1 protein at a flow rate of 50 μL/minute to monitor the association with SOS1. Dissociation of bound test compound from the SOS1 protein was immediately monitored by flowing assay buffer over the sensor surface and monitoring the decrease in binding signal back to the baseline level seen in the absence of compound. This was repeated for all compound dilutions in each series.
Measurement: IC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 426.4, ALogP 4.04, HBA 7, HBD 1, PSA 85.59, QED 0.52
Compound (SMILES): CN1C(=N)N[C@](C)(c2cc(NC(=O)c3ccc(F)cn3)ccc2F)[C@]2(CCN(S(=O)(=O)CC(F)(F)F)C2)S1(=O)=O
InChIKey: WGUDWIAWYZNTON-NHCUHLMSSA-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 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 610.59, ALogP 1.96, HBA 7, HBD 3, PSA 152.63, QED 0.44
Compound (SMILES): CCOC1CCN(Cc2c(OC)cc(C)c3[nH]ccc23)C(c2ccc(C(=O)O)nc2)C1
InChIKey: SHGLTVARVYZURW-UHFFFAOYSA-N
Target: Complement factor B (SINGLE PROTEIN), Homo sapiens
UniProt: P00751
Assay: Human Complement Factor B TR-FRET Assay: CVF-Bb complex prepared from purified cobra venom factor (1 uM), recombinant human complement factor B (expressed in drosophila cells and purified using standard methods) and human complement factor D (expressed in E. Coli, refolded and purified using standard methods). CVF-Bb complex at 3 nM concentration was incubated with test compound at various concentrations for 1 hour at room temperature in PBS pH 7.4 containing 10 mM MgCl2 and 0.05% (w/v) CHAPS. Human complement C3 substrate purified from plasma was added to a final concentration of 1 uM. After 1 hour incubation at room temperature, the enzyme reaction was stopped by addition of a cocktail of concentrated pan-protease inhibitors. The product of the reaction, C3a, was quantified by means of an enzyme-linked-immunosorbent assay. IC50 values were calculated from percentage of inhibition of CVF-Bb activity as a function of test compound concentration.
Measurement: IC50 = 1.8 nM (pChEMBL 8.739999771118164)
Descriptors: MW 423.51, ALogP 4.32, HBA 5, HBD 2, PSA 87.68, QED 0.59
Compound (SMILES): C[C@@H]1CC[C@@H](Oc2nccn3ccnc23)CN1C(=O)c1ccccc1-n1nccn1
InChIKey: OQONJHBJRAWTOY-HZPDHXFCSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: FLIPR Assay: In a typical experiment the OX1 and OX2 receptor antagonistic activity of the compounds of the present invention was determined in accordance with the following experimental method. For intracellular calcium measurements, Chinese hamster ovary (CHO) cells expressing the rat orexin-1 receptor or the human orexin-2 receptor, are grown in Iscove's modified DMEM containing 2 mM L-glutamine, 0.5 g/ml G418, 1% hypoxanthine-thymidine supplement, 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 Becton-Dickinson black 384-well clear bottom sterile plates coated with poly-D-lysine. All reagents are from GIBCO-Invitrogen Corp. The seeded plates are incubated overnight at 37° C. and 5% CO2. Ala-6,12 human orexin-A as the agonist is prepared as a 1 mM stock solution in 1% bovine serum albumin (BSA) and diluted in assay buffer (HBSS containing 20 mM HEPES, 0.1% BSA and 2.5 mM probenecid, pH7.4) for use in the assay at a final concentration of 70 pM. Test compounds are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates, first in DMSO, then assay buffer. On the day of the assay, cells are washed 3 times with 100 μl assay buffer and then incubated for 60 min (37° C., 5% CO2) in 60 μl assay buffer containing 1 μl M Fluo-4AM ester, 0.02% pluronic acid, and 1% BSA.
Measurement: IC50 = 2374 nM (pChEMBL 5.619999885559082)
Descriptors: MW 403.45, ALogP 2.38, HBA 8, HBD 0, PSA 90.44, QED 0.52
Compound (SMILES): C=CC(=O)N1CC(Oc2cc3c(Nc4ccc(Cl)cc4Cl)ncnc3cc2OC)C1
InChIKey: VURUUKGYNYHYRK-UHFFFAOYSA-N
Target: Receptor tyrosine-protein kinase erbB-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P04626
Assay: Cell Viability Assay and IC50 Estimation: Cell viability was determined using the Cell Titer Glo assay (Promega) as previously described. Briefly, 2000-3000 cells per well were plated in 384-well plates (Greiner Bio-One) in technical triplicate. Cells were treated with seven different concentrations of tyrosine kinase inhibitors or vehicle alone at a final volume of 40 μL per well. After 3 days hours, 11 μL of Cell Titer Glo was added to each well. Plates were shaken for 15 minutes, and bioluminescence was determined using a FLUOstar OPTIMA multi-mode micro-plate reader (BMG LABTECH). Bioluminescence values were normalized to DMSO treated cells, and normalized values were plotted in GraphPad Prism using non-linear regression fit to normalized data with a variable slope. IC50 values were calculated by GraphPad Prism at 50% inhibition.
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 445.31, ALogP 4.46, HBA 6, HBD 1, PSA 76.58, QED 0.56
Compound (SMILES): COC(=O)N1c2ccc3c(nc(CN4Cc5ccccc5CC4=O)n3[C@@H]3CCC[C@@H](C(=O)O)C3)c2CC[C@@H]1C
InChIKey: MWVOGVAMNZFYGO-CZTZKLFOSA-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 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 530.63, ALogP 4.84, HBA 6, HBD 1, PSA 104.97, QED 0.52
Compound (SMILES): C[C@@H]1CN(C(=O)[C@@](C)(O)C(F)(F)F)Cc2cnn(-c3ncc(F)cc3F)c21
InChIKey: KHHGSBWETSSXEC-ANRSDYALSA-N
Target: [Pyruvate dehydrogenase (acetyl-transferring)] kinase isozyme 2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q15119
Assay: Isothermal Titration Calorimetry: ITC measurements were performed with a VP-ITC micro calorimeter. In general titrations were performed by titrating the protein (50 μM) to the test compound (5 μM) in 12 μl injections. All binding experiments were carried out at 30° C. In general the test compounds were diluted form DMSO stock solutions into the measurement buffer with a maximum final concentration of 1% DMSO. The measurement buffer was 20 mM HEPES, 135 mM KCl, 1 mM TCEP, 2 mM MgCl2, 15 mM NaH2PO4, pH 7.5. The human PDHK2 (12-407) was produced in E. coli as his-tagged protein and purified by affinity chromatography. The tag was removed by side specific proteolysis. Before titration the protein buffer was changed to the measurement buffer containing the same DMSO concentration as the test compound dilution.
Measurement: IC50 < 300 nM (pChEMBL 6.5229997634887695)
Descriptors: MW 390.31, ALogP 2.3, HBA 5, HBD 1, PSA 71.25, QED 0.8
Compound (SMILES): CCOc1ncccc1-c1noc([C@@H]2CCCN2C(=O)c2cc(Cl)c(C)cc2-n2nccn2)n1
InChIKey: USGYMEPRBRHADY-SFHVURJKSA-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 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 479.93, ALogP 4.05, HBA 9, HBD 0, PSA 112.06, QED 0.41
Compound (SMILES): COCCOc1nc(Nc2ccc(CN3CCC(C)CC3)cc2)nc2[nH]cc(-c3ccc(OCc4ccccn4)cc3F)c12
InChIKey: YMNIYJFEUMDGFK-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: Mer Kinase Assay: Certain compounds of formula (I) were tested for their potency in inhibiting Mer kinase. Inhibition of Mer kinase activity by a test compound was calculated by AlphaScreen (PerkinElmer 6760620M). Standard assay conditions included 2 ng of recombinant Mer kinase (SignalChem) with 5 ng Poly-(GT)-Biotin (Cisbio) in the assay buffer, i.e., 10 μM ATP (Sigma), 8 mM MOPs, pH7.0 (Sigma M-1254), 0.2 mM EDTA (Sigma E-6758), 0.5% glycerol (J. T Baker 2136-1), 0.1 mg/ml BSA (Bio-Rad 500-0007), 0.01% 2-mercaptoethanol (BDH 441433A), 0.001% Brij35 (Sigma P1254), 10 mM MgCl2, and 10 mM MnCl2 in a final volume of 25 μL. Reactions were incubated at 30° C. for 30 minutes and stopped by adding 5 μL of 50 mM EDTA. Product analysis was performed using the AlphaScreen kit and counted with Enspire Alpha (PerkinElmer). Screen values of inhibitors were determined after carrying out assays with each compound at serially diluted concentrations in duplication. Results were analyzed using linear regression software (GraphPad Prism 4; GraphPad Software Inc.).
Measurement: IC50 < 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 596.71, ALogP 6.74, HBA 8, HBD 2, PSA 97.42, QED 0.15
Compound (SMILES): O=C(NC1CCC(F)(F)CC1)c1csc([C@H]2C[C@H]2NCC2CCOCC2)c1
InChIKey: KLTJYHBSHFQGGL-DLBZAZTESA-N
Target: Amine oxidase [flavin-containing] B (SINGLE PROTEIN), Homo sapiens
UniProt: P27338
Assay: Inhibitory Activity Assay: The MAO-B inhibitory activity evaluation described below followed the protocol of MAO-Glo (registered trademark) Assay of Promega KK. A test compound dissolved in DMSO was added to a reaction solution (100 mM HEPES (pH 7.5), 5% glycerol, 10% DMSO) containing MAO-B enzyme (Sigma-Aldrich Co. LLC.), and the mixture was reacted at room temperature for 15 min. MAO substrate (Promega KK) was added to start the reaction. After reaction at room temperature for 60 min, Luciferine detection reagent (Promega KK) (50 uL) was added to terminate the reaction. After reaction at room temperature for 20 min with stirring, the luminescence was measured by Envision (PerkinElmer). The MAO-B inhibitory rate (%) of the test compound was calculated by the following formula.inhibitory rate (%)=(1-(test compound count-blank)/(control-blank))x100The count of the MAO-B enzyme reaction mixture under compound non-addition conditions is indicated as control, and the count under compound non-addition and MAO-B enzyme non-addition conditions is indicated as blank. A concentration necessary for achieving 50% inhibitory rate was taken as IC50 value.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 398.52, ALogP 3.93, HBA 4, HBD 2, PSA 50.36, QED 0.76
Compound (SMILES): O=C(N[C@H]1CCC[C@@H]1Nc1nc2ccc(F)cc2s1)c1ncccc1-n1ccnn1
InChIKey: ZGCIMHAIPBFOLG-KBPBESRZSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: FLIPR Assay: Orexin antagonist activity was determined by measuring changes in intracellular calcium levels using a Ca2+ sensitive fluorescent dye. The changes in fluorescent signal were monitored by Fluorescent Imaging Plate Reader (FLIPR) technology available from Molecular Devices, LLC, U.S.A. Orexin mediated increases in intracellular Ca2+ concentration were readily detected upon activation with orexin-A. Twenty-four hours prior to the assay, RBL-2H3 cells stably expressing either human orexin receptor 1 or human orexin receptor 2 were seeded in cell culture medium in black, clear-bottom 384-well plates (commercially available from Corning Inc., U.S.A.) and grown overnight at 37° C., 5% CO2. On the day of the assay, cell culture media was removed and cells were loaded with Calcium 5 Dye (commercially sold by Molecular Devices, LLC, U.S.A.) for 1 hour at 37° C., 5% CO2. Test compounds (at 10 point half log concentration response curves from 10 uM) were added to cells for 15 minutes prior to the addition of orexin-A to all wells, to achieve a final concentration that produces approximately an 80% maximal response.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 423.48, ALogP 3.17, HBA 8, HBD 2, PSA 97.62, QED 0.51
Compound (SMILES): CC(C)(OCc1cc(-c2cccc(OC3CCC3)c2)n(-c2ccccc2Br)n1)C(=O)O
InChIKey: FXJGCZOUNBTECP-UHFFFAOYSA-N
Target: Monocarboxylate transporter 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P53985
Assay: Biological Activity Assay 4: MCT1 activity may be measured using BT-20 breast cancer cells that express high native levels of MCT1, but do not express MCT4 and are known to those with skill in the art. Preparation of BCECF loaded cells are as described for Assay 1. Lactate transport assay is as described for Assay 1, except 10 mM L-lactate (rather than 50 mM) is added.
Measurement: IC50 = 12000 nM (pChEMBL 4.920000076293945)
Descriptors: MW 485.38, ALogP 5.61, HBA 5, HBD 1, PSA 73.58, QED 0.45
Compound (SMILES): CCOc1cc(-c2ccc(N3CCC(C)(NC(=O)C4(N)CC4)CC3)nc2)c2c(C#N)cnn2c1
InChIKey: XUDZHYCPTLTWLD-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of Formula I were screened for their ability to inhibit wildtype 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; Catalog No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Catalog No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog 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 = 441 nM (pChEMBL 6.360000133514404)
Descriptors: MW 459.55, ALogP 2.63, HBA 8, HBD 2, PSA 121.57, QED 0.58
Compound (SMILES): COCc1nn(Cc2ccc3c(c2)CCN(C)C3)cc1C(=O)NCc1c(-n2ccc(C#N)n2)ccc(OC)c1F
InChIKey: NFYHGGHMDNEBTH-UHFFFAOYSA-N
Target: Kallikrein-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P06870
Assay: Determination of the IC50 for KLK1: KLK1 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; Sturzebecher et al., Biol. Chem. Hoppe-Seyler, 1992, 373, 1025). Human KLK1 (Callbiochem) was incubated at 25° C. with the fluorogenic substrate H-DVal-Leu-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 > 40000 nM (pChEMBL 4.3979997634887695)
Descriptors: MW 543.6, ALogP 3.2, HBA 9, HBD 1, PSA 110.23, QED 0.35
Compound (SMILES): CN[C@@H](C)C(=O)N[C@@H]1C(=O)N2C(C(=O)NC(c3ccccc3)c3ccccc3)CC[C@@H]2O[C@@H]1C
InChIKey: JTUKOFSAWIHEHS-HNYVQNJRSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: Biological Assays: 5000 PPC-1 cells were plated and grown overnight. Compounds were plated and 4 hrs later, TRAIL was added to half of the plate while RPMI was added to the other half of the plate as a control. Plates were return to the incubator for 24 hrs. Plates were removed from the incubator and placed on the bench for 30 min and then 25 uL of Cell Titer Glo were added per well. Plates were placed on a rocker and then read on a luminometer. 5000 MDA-MB-231 cells were plated per well. Compound was added and 4 hrs later, TRAIL was added at 5 ng/mL; RPMI was added for a minus TRAIL control. Plates were incubated an additional 24 hrs, removed to the bench for 30 min. and then 25 uL of cell titer glo was added per well. Plates were placed on a rocker and read on a luminometer. Data were fit using PRISM.
Measurement: Ki = 37500 nM (pChEMBL 4.429999828338623)
Descriptors: MW 464.57, ALogP 1.72, HBA 5, HBD 3, PSA 99.77, QED 0.58
Compound (SMILES): COc1cc2c(Nc3cc(Cl)c(Br)cc3F)ncnc2cc1OC[C@H]1CN2CCC[C@@H]2CO1
InChIKey: NGRFKPHUUUCTAT-ZIAGYGMSSA-N
Target: Ephrin type-A receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29317
Assay: AlphaScreen Assay: In AlphaScreen, when the donor and acceptor beads are close in proximity, a series of photochemical events will give rise to a fluorescent signal upon light activation. Here we use biotinylated poly-(Glu, Tyr) 4:1 as the kinase substrate, streptavidin-coated donor beads and anti-phosphortyrosine antibody PY100-coated acceptor beads. Upon phosphorylation, the peptide substrate can bind to both donor and acceptor beads, thus gives rise to fluorescence. Compounds, ATP, biotinylated poly-(Glu, Tyr) and kinases are mixed in a volume of 20 μL for 1 hr at ambient temperature using Greiner 384-well white clear bottom medium binding plates. Then 10 uL solution containing 15-30 mg/mL AlphaScreen beads, 75 mM Hepes, pH 7.4, 300 mM NaCl, 120 mM EDTA, 0.3% BSA and 0.03% Tween-20 is added to each well. After 2-16 hr incubation of the beads, plates are read in a Perkin Elmer AlphaQuest reader (available from PerkinElmer Life and Analytical Sciences, Inc., of Boston Mass.).
Measurement: IC50 = 10500 nM (pChEMBL 4.980000019073486)
Descriptors: MW 537.82, ALogP 5.18, HBA 7, HBD 1, PSA 68.74, QED 0.43
Compound (SMILES): Cc1ccc(-n2nccn2)c(C(=O)N2CCC[C@H]2c2ncc(-c3ccccc3C(F)(F)F)[nH]2)c1
InChIKey: GNFQVQGBHPTENM-NRFANRHFSA-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 = 117 nM (pChEMBL 6.929999828338623)
Descriptors: MW 466.47, ALogP 4.96, HBA 5, HBD 1, PSA 79.7, QED 0.46
Compound (SMILES): Cc1nc(C(=O)N2C(COc3ccc(F)cc3)CC3CC2C3)c(-c2ccccc2)s1
InChIKey: LNYCTJGRLILBQE-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Scintillation Proximity Assay (SPA) Binding Assay: CHO cells stably transfected with human Orexin type 1 receptors (CHO-hOX1) or HEK-293 cells transiently transfected with human Orexin type 2 receptors (HEK-hOX2) were collected after 16 h induction with 5 mM sodium butyrate. The cell pellets were re-suspended, homogenized in 15 mM Tris/HCl pH=7.5, 1 mM EGTA, 0.3 mM EDTA, 2 mM MgCl2, protease inhibitors and centrifuged at 40,000 g (20 min, 4° C.). After re-suspension, homogenization and centrifugation as above, the final pellets were re-suspended in 75 mM Tris/HCl pH=7.5, 1 mM EGTA, 0.3 mM EDTA, 12.5 mM MgCl2, 250 mM Sucrose, protease inhibitors, divided into aliquots and frozen down at −80° C.Compounds of invention were serially diluted in neat DMSO at 100-fold concentrations (1% DMSO final in the assay) and 2 μl/well were plated into 96-well Isoplates (Perkin Elmer).
Measurement: Ki = 107 nM (pChEMBL 6.96999979019165)
Descriptors: MW 422.52, ALogP 5.33, HBA 4, HBD 0, PSA 42.43, QED 0.56
Compound (SMILES): O=C(OCc1cnc(Cl)c(Cl)c1)N1CCC2(CC1)CN(C(=O)c1ccc3[nH]nnc3c1)C2
InChIKey: JUVRTXYUUUHSQR-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 = 622 nM (pChEMBL 6.210000038146973)
Descriptors: MW 475.34, ALogP 3.53, HBA 6, HBD 1, PSA 104.31, QED 0.58
Compound (SMILES): C[C@@H]1CC(F)(F)CN(C(=O)c2nn(C)nc2-c2ccc(Cl)cn2)[C@@H]1CNc1ncc(C(F)(F)F)cn1
InChIKey: SIBUGFDXKIZXFH-IAQYHMDHSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Orexin Receptor Cell-Based Functional Assay: Measurement of [Ca2+]i using a FLIPR: CHOOX1 or CHOOX2 cells were seeded into black-walled clear-base 384-well plates (Corning, catalog #3712) at a density of 20,000 cells per well in F12-K medium supplemented with 10% FBS and then incubated in a 5% CO2, 37° C. incubator overnight to reach 90% confluency. The cells were incubated with equal volume of calcium 6 loading buffer (Molecular Devices, Inc.) containing 2.5 mM probenecid at 37° C. for 2 h, followed by test compounds (dose-range 0.1 nM-10 μM) for another 30 min. The plates were then placed into a FLIPR (Molecular Devices, Inc.) to monitor fluorescence (λ excitation 488 nm, λ emission 540 nm) before and after the addition of EC90 of [OX].
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 530.89, ALogP 3.94, HBA 8, HBD 1, PSA 101.72, QED 0.5
Compound (SMILES): N#CC[C@H]1CC[C@H](n2c(CC(=O)N3CCC(O)CC3)nc3cnc4[nH]ccc4c32)CC1
InChIKey: AATRYKZHNGYMOX-WKILWMFISA-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 = 9.2 nM (pChEMBL 8.039999961853027)
Descriptors: MW 420.52, ALogP 3.08, HBA 6, HBD 2, PSA 110.83, QED 0.67
Compound (SMILES): CC(C)(O)c1cccc(-c2ncn(CC(F)F)c2-c2ccc3ncc(C#N)n3n2)c1
InChIKey: LCAOAERTWRJUNY-UHFFFAOYSA-N
Target: TGF-beta receptor type-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P37173
Assay: Biological Assay: Assays for the compounds reported below were conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGF-βR1 T204D or HIS-TGF-βR2 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-TGF-βR1 T204D or HIS-TGF-βR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule prode (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 408.41, ALogP 3.62, HBA 7, HBD 1, PSA 92.03, QED 0.54
Compound (SMILES): Cc1cccc(NS(=O)(=O)c2ccc(C)c(-c3cnc(N)c(-c4nc(C)no4)c3)c2)n1
InChIKey: PDLCAVUHYFSFFJ-UHFFFAOYSA-N
Target: Phosphatidylinositol 3-kinase catalytic subunit type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NEB9
Assay: Kinase Glo Luminescent Kinase Assay (Kglo) for PI 3-Kinase Alpha (A), PI 3-Kinase Beta (B), Vps34 (C), PI 4-Kinase Beta (D): The luminescence-based ATP detection reagent KinaseGlo was obtained from Promega, (Cat. No. V6714, Lot No. 236161) through Catalys, Wallisellen, Switzerland. L-alpha-phosphatidylinositol (PI, liver, bovine) was obtained from Avanti Polar Lipid (Cat. No. 840042C, Lot#LPI-274), phosphatidylinositol-4,5-bisphosphate (PIP(4,5)2) was obtained from Avanti Polar Lipid (Cat. No. 840046X). L-α-phosphatidylserine (PS) was obtained from Avanti Polar Lipid (Cat. No. 840032C), n-octylglucoside from Avanti Polar Lipid (Cat. No. 10634425001). Luminescence is a well established readout to determine ATP concentrations and can thus be used to follow the activity of many kinases regardless of their substrate. The Kinase Glo Luminescent Kinase Assay (Promega, Madison, Wis., USA) is a homogeneous HTS method of measuring kinase activity by quantifying the amount of ATP remaining in solution following a kinase reaction.50 nL of compound dilutions were dispensed onto black 384-well low volume Non Binding Styrene (NBS) plates (Costar Cat. No. NBS#3676). L-α-phosphatidylinositol (PI), provided as 10 mg/ml solution in methanol, was transferred into a glass tube and dried under a nitrogen beam. It was then resuspended in 3% OctylGlucoside (1-O-n-octyl-beta-D-glucopyranoside) by vortexing and stored at 4° C. 5 μl of a mix of PI/OctylGlucoside with the PI 3-kinase alpha and PI 3-kinase beta subtypes, or Vps34 or PI 4-kinase beta were added. Kinase reactions were started by the addition of 5 μl of an ATP-mix containing in a final volume 10 μl 10 mM TRIS-HCl pH 7.5, 3 mM MgCl2, 50 mM NaCl, 0.05% CHAPS, 1 mM DTT and 1 μM ATP at room temperature. Reactions were stopped with 10 μl of KinaseGlo and plates were read 10 mins later in a Synergy2 reader using an integration time of 0.1 seconds per well. 2.5 μM of NVP-BGT226 (1-(3-(trifluoromethyl)-4-(piperazin-1-yl)phenyl)-8-(6-methoxypyridin-3-yl)-3-methyl-1H-imidazo[4,5-c]quinolin-2(3H)-one) was added to the assay plates to generate the 100% inhibition of the kinase reaction, and the 0% inhibition was given by the solvent vehicle (90% DMSO in water). (1-(3-(trifluoromethyl)-4-(piperazin-1-yl)phenyl)-8-(6-methoxypyridin-3-yl)-3-methyl-1H-imidazo[4,5-c]quinolin-2(3H)-one) was used as a reference compound and included in all assay plates in the form of 16 dilution points in duplicate.IC50 values of the percentage inhibition of each compound at 8 concentrations (10, 3.0, 1.0, 0.3, 0.1, 0.030, 0.010 and 0.003 μM) n=2 were derived by fitting a sigmoidal dose-response curve to a plot of assay readout over inhibitor concentration as described. All fits were performed with the program XLfit4 (ID Business Solutions, Guildford, UK).
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 436.5, ALogP 3.5, HBA 8, HBD 2, PSA 136.89, QED 0.48
Compound (SMILES): CCn1cc(Nc2ncc(C(N)=O)c(NCc3c(F)ccc(F)c3Cl)n2)cn1
InChIKey: BEJOSQBBGMLEBM-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Kinase Activity Assay: JAK2 (the final concentration was 0.25 nM); ATP (the final concentration was 5 μM); ULight-labeled JAK-1 (Tyr1023) Peptide (the final concentration was 50 nM); the enzymatic reaction time was 1 hour. The maximum final concentration of compounds was 2.5 μM. After a 3-fold gradient dilution, 11 concentrations were obtained, with the minimum final concentration of 0.042 nM. The final DMSO concentration was 1%
Measurement: IC50 = 3.12 nM (pChEMBL 8.510000228881836)
Descriptors: MW 407.81, ALogP 3.08, HBA 7, HBD 3, PSA 110.75, QED 0.52
Compound (SMILES): COc1cc(-c2[nH]c3ccc(C4CCC(NC(C)C)CC4)nc3c2C(C)C)cn2ncnc12
InChIKey: KWMAAQNRLXTVKG-UHFFFAOYSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
Assay: TLR7/8/9 Inhibition Reporter Assays: 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 = 1.7 nM (pChEMBL 8.770000457763672)
Descriptors: MW 446.6, ALogP 5.43, HBA 6, HBD 2, PSA 80.13, QED 0.41
Compound (SMILES): CC(C)(C)c1cc(N2CCC(F)(F)C2)c2nnn(Cc3ccccc3SSc3ccccn3)c2n1
InChIKey: XXQYHMLSXSXKMW-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: Radioligand Binding Assay: he affinity of the compounds of the invention for cannabinoid receptors was determined using recommended amounts of membrane preparations (PerkinElmer) of human embryonic kidney (HEK) cells expressing the human CNR1 or CNR2 receptors in conjunction with 1.5 or 2.6 nM [3H]-CP-55,940 (Perkin Elmer) as radioligand, respectively. Binding was performed in binding buffer (50 mM Tris, 5 mM MgCl2, 2.5 mM EDTA, and 0.5% (wt/vol) fatty acid free BSA, pH 7.4 for CB1 receptor and 50 mM Tris, 5 mM MgCl2, 2.5 mM EGTA, and 0.1% (wt/vol) fatty acid free BSA, pH 7.4 for CB2 receptor) in a total volume of 0.2 mL for 1 h at 30° C. shaking. The reaction was terminated by rapid filtration through microfiltration plates coated with 0.5% polyethylenimine (UniFilter GF/B filter plate; Packard). Bound radioactivity was analyzed for Ki using nonlinear regression analysis (Activity Base, ID Business Solution, Limited), with the Kd values for [3H]CP55,940 determined from saturation experiments.
Measurement: Ki = 2577 nM (pChEMBL 5.590000152587891)
Descriptors: MW 512.66, ALogP 6.21, HBA 8, HBD 0, PSA 59.73, QED 0.28
Compound (SMILES): O=C(O)c1ccnc2c(Cl)c[nH]c12
InChIKey: UAFNSWUBMGTOQA-UHFFFAOYSA-N
Target: Lysine-specific demethylase 5B (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UGL1
Assay: Enzymatic Assay: The ability of test compounds to inhibit the activity of Jarid1B was determined in 384-well plate format under the following reaction conditions: 0.8 nM Jarid1B, 300 nM H3K4me3-biotin labeled peptide (Anaspec cat #64357), 2 μM alpha-ketoglutaric acid in assay buffer of 50 mM HEPES, pH7.3, 0.005% Brij35, 0.5 mM TCEP, 0.2 mg/ml BSA, 50 μM sodium L-ascorbate, and 2 μM ammonium iron(II) sulfate. Reaction product was determined quantitatively by TR-FRET after the addition of detection reagent Phycolink Streptavidin-allophycocyanin (Prozyme) and Europium-anti-mono- or di-methylated histone H3 lysine 4 (H3K4me1-2) antibody (PerkinElmer) in the presence of 5 mM EDTA in LANCE detection buffer (PerkinElmer) at a final concentration of 25 nM and 1 nM, respectively.The assay reaction was initiated by the following: 2 μl of the mixture of 900 nM H3K4me3-biotin labeled peptide and 6 μM alpha-ketoglutaric acid with 2 μl of 11-point serial diluted inhibitor in 3% DMSO was added to each well of the plate, followed by the addition of 2 μl of 2.4 nM Jarid1B to initiate the reaction. The reaction mixture was incubated at room temperature for 30 minutes, and terminated by the addition of 6 μl of 5 mM EDTA in LANCE detection buffer containing 50 nM Phycolink Streptavidin-allophycocyanin and 2 nM Europium-anti-H3K4me1-2 antibody.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 196.59, ALogP 1.91, HBA 2, HBD 2, PSA 65.98, QED 0.73
Compound (SMILES): O=C(OC1CN(c2nc3c(c(NC4CCOCC4)n2)[S@@+]([O-])CC3)C1)c1ccsc1
InChIKey: GKZRITVEZDWNGB-LJAQVGFWSA-N
Target: Tumor necrosis factor (SINGLE PROTEIN), Homo sapiens
UniProt: P01375
Assay: TNF-alpha RELEASE: Human peripheral blood mononuclear cells (PBMC) were isolated from fresh buffy coats by density centrifugation using lymphoprep tubes (Medinor). Frozen PBMC's were washed in serum free assay buffer (RPMI1640 with 25 mM HEPES, 1% pen/strep, 200 mM L glutamine, 0.5% human serum albumin) and living cells counted. Lipopolysaccharide (1 microg/ml; SIGMA) was added to the cells which were then transferred to 384 well tissue culture plates (5×105 c/ml) containing titrated test compounds. The cells were incubated for 18 hours at 37° C. in serum free assay buffer and the level of TNFalpha in the supernatant was quantitated by AlphaLISA (PerkinElmer) by measuring fluorescence intensity at 615 nm.Results were expressed as IC50 values calculated from inhibition curves using as controls the secretion in LPS stimulated wells and the secretion in cells incubated with 10 microM of a PDE4 inhibitor reference compound.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 434.54, ALogP 1.84, HBA 9, HBD 1, PSA 99.64, QED 0.56