lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): NC1=N[C@](CF)(c2cc(C#Cc3ccc(OC(F)F)cn3)ccc2F)C[C@@H](C(F)(F)F)O1
InChIKey: SXSDFPIELXYPDS-QFBILLFUSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro Enzymatic BACE FRET Assay: The assay buffer used in this screen is 0.05 M acetate, pH 4.2, 10% DMSO final, 100 uM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The Beta Secretase enzyme (0.2 nM) is pre-incubated for one hour with inhibitors, typically in about 1 uL of DMSO according to a serial dilution, are added thereto. This assay is effectively started by the addition of FRET substrate (50 nM) and the combination is incubated for one hour. The FRET assay is terminated with by addition of Tris buffer, which raises the pH to neutrality, and the fluorescence is determined. The FRET substrate is a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. Proteolytic cleavage of the FRET substrate releases quenching of fluorescence (excitation 488 nm and emission 425 nm).
Measurement: IC50 = 2960 nM (pChEMBL 5.53000020980835)
Descriptors: MW 461.34, ALogP 4.05, HBA 5, HBD 1, PSA 69.73, QED 0.55
Compound (SMILES): Cc1ccc(-c2noc(C3(O)CCC3)n2)cc1NC(=O)c1cnc2ccccn12
InChIKey: IMVBDLPHXQZTOB-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Cell Proliferation Assay: The compounds was tested for inhibition of SCF dependent proliferation using human Mo7e cells which endogenously express c-kit in a 384 well format. Three-fold serially diluted test compounds (Cmax=10 mM) were evaluated for their antiproliferative activity of Mo7e cells stimulated with human recombinant SCF. After 48 hours of incubation at 37° C., cell viability was measured by adding 25 uL of CellTiter Glo (Promega) to the cells and the luminescence was measured by a CLIPR CCD camera (Molecular Devices).
Measurement: IC50 = 144 nM (pChEMBL 6.840000152587891)
Descriptors: MW 389.42, ALogP 3.32, HBA 7, HBD 2, PSA 105.55, QED 0.55
Compound (SMILES): CS(=O)(=O)N1CCN(Cc2csc3c(-c4cccc5[nH]ncc45)nc(N4CCOCC4)nc23)CC1
InChIKey: KBDVZEHPFGRYTC-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Enzymatic Inhibitory Activity Assay:   1. Buffer preparation: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS.  2. Compound was formulated in 100% DMSO in a concentration gradient, deposited to a 384-well plate to make final DMSO concentration of 1%.  3. PI3Kα and PI3Kδ enzymes were diluted to be the optimum concentration with the following buffer: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS, 2 mM DTT. Transferred to a 384-well plate and incubated with the compound for a certain time.  4. Substrate was diluted to an optimum concentration with following buffer: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS, 2 mM DTT, 50 μM PIP2, Km ATP. The reaction was performed in a 384-well plate for 1 h at room temperature for PI3Kα and 2 hrs at room temperature for PI3Kδ.  5. Read the conversion rate using Caliper Reader, and calculate the inhibition rate as the average of two tests.
Measurement: IC50 = 57 nM (pChEMBL 7.239999771118164)
Descriptors: MW 513.65, ALogP 2.15, HBA 9, HBD 1, PSA 107.55, QED 0.43
Compound (SMILES): CC(=O)N1C2CCC1c1c(-c3cccc(C#N)c3)nn(Cc3ccc(F)cc3F)c1C2
InChIKey: IPAJIOJPIIOEBO-UHFFFAOYSA-N
Target: Potassium channel subfamily K member 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O14649
Assay: Biological Assay: Human TASK-1 channels were expressed in Xenopus oocytes. For this purpose, oocytes were isolated from Xenopus laevis and defoliculated. Subsequently, TASK-1-encoding RNA synthesized in vitro was injected into oocytes. After two days of TASK-1 protein expression, TASK-1 currents were measured by two-microelectrode voltage clamp. Data were acquired and analyzed using a TEC-10cx amplifier (NPI Electronic, Tamm, Germany) connected to an ITC-16 interface (Instrutech Corp., Long Island, USA) and Pulse software (HEKA Elektronik, Lambrecht, Germany). Oocytes were clamped to −90 mV and TASK-1 mediated currents were measured during 500 ms voltage pulses to 40 mV. Oocytes were continuously superfused with ND96 buffer containing: NaCl 96 mM, KCl 2 mM, CaCl2 1.8 mM, MgCl2 1 mM, HEPES 5 mM (pH adjusted to 7.4 with NaOH). All experiments were performed at room temperature.
Measurement: IC50 = 91 nM (pChEMBL 7.039999961853027)
Descriptors: MW 418.45, ALogP 4.36, HBA 4, HBD 0, PSA 61.92, QED 0.64
Compound (SMILES): CC(C)(C)NC(=O)c1cccc(Nc2ncnc(N3CC(Oc4ccc(F)cc4)C3)n2)c1
InChIKey: SYYVXAJIVBWQMV-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Cell-Based Assay: Compounds were tested on human Nav1.7 (hNav1.7) expressed stably in 293 cells, which have no endogenous sodium channels. Two forms of whole-cell patch-clamp assays were used. In both, peak current through Nav1.7 was evoked by a depolarizing voltage pulse, and currents were monitored as a function of time in response to exposure to several different concentrations of drug. Automated electrophysiology uses the PatchXpress 7000A system. Up to sixteen cells at a time were voltage-clamped in parallel, and the steady-state voltage dependence of inactivation was determined experimentally for each cell. For each cell, fractional inactivation was set at 20%, and peak current through Nav1.7 as a function of time was measured in response to increasing concentrations of test compound. Manual electrophysiology refers to conventional whole-cell patch-clamping (Hamill et al., 1981). After formation of the whole-cell clamp, cells were lifted off the bottom of the dish with the patch pipette and positioned directly in front of an array of glass microperfusion tubes each with internal diameter about 250 microns, with individual compound concentrations flowing from each tube. Current as a function of time in response to increasing drug concentrations was measured with Nav1.7 fully in the resting state (holding voltage set to −140 mV) and on the same cells with the holding voltage set to produce about 20% inactivation. Compounds were not applied for a prespecified time, but for as long as required to achieve a stable level of block.
Measurement: IC50 = 8390 nM (pChEMBL 5.079999923706055)
Descriptors: MW 436.49, ALogP 3.55, HBA 7, HBD 2, PSA 92.27, QED 0.61
Compound (SMILES): CN1C(=N)N[C@](C)(c2sc3c(-c4ccc(-c5ncco5)cc4)nccc3c2Cl)CS1(=O)=O
InChIKey: XXBWDOSFNJOFLD-NRFANRHFSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: HTRF FRET Assay: Varying concentrations of inhibitors at 3× the final desired concentration in a volume of 10 μl are preincubated with purified human BACE1 catalytic domain (3 nM in 10 μl) for 30 minutes at 30° C. in reaction buffer containing 20 mM Na-Acetate pH 5.0, 10% glycerol, 0.1% Brij-35 and 7.5% DSMO. Reactions are initiated by addition of 10 μl of 600 nM QSY7-APPswe-Eu substrate (200 nM final) to give a final reaction volume of 30 in a 384 well Nunc HTRF plate. The reactions are incubated at 30° C. for 1.5 hours. The 620 nm fluorescence is then read on a Rubystar HTRF plate reader (BMG Labtechnologies) using a 50 millisecond delay followed by a 400 millisecond acquisition time window.
Measurement: Ki = 115.7 nM (pChEMBL 6.940000057220459)
Descriptors: MW 487.99, ALogP 4.29, HBA 7, HBD 2, PSA 112.18, QED 0.45
Compound (SMILES): Cc1[nH]c(/C=C2\C(=O)Nc3ccc(F)cc32)c(C)c1C(=O)NCCCC(=O)NO
InChIKey: DJYRFGFGDIBQBL-ZROIWOOFSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of N-terminal GST-His6-thrombin cleavage site-fused human VEGFR2 (Asp805 to Val1356 residues) expressed in baculovirus expression system using biotinylated gastrin precursor at Tyr87 as substrate preincubated for 5 mins followed by substrate addition measured after 30 mins by DELFIA method
Measurement: IC50 <= 100 nM (pChEMBL 7)
Descriptors: MW 400.41, ALogP 2.28, HBA 4, HBD 5, PSA 123.32, QED 0.22
Compound (SMILES): O=C1NCC2(CCCC2)n2c1cc1cnc(Nc3ccc(N4CCC(N5CCCCC5)CC4)cn3)nc12
InChIKey: UEVWXXZKXKDLKI-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P24941
Assay: Caliper Assay: Selected compounds disclosed herein were tested in CDK4/cyclinD1, CDK6/CycD3 CDK2/CycA and CDK2/cyclinE kinase assays by Nanosyn (Santa Clara, Calif.) to determine their inhibitory effect on these CDKs. The assays were performed using microfluidic kinase detection technology (Caliper Assay Platform). The compounds were tested in 12-point dose-response format in singlicate at Km for ATP. Phosphoacceptor substrate peptide concentration used was 1 μM for all assays and Staurosporine was used as the reference compound for all assays. Specifics of each assay are as described below:CDK2/CyclinA: Enzyme concentration: 0.2 nM; ATP concentration: 50 μM; Incubation time: 3 hr.CDK2/CyclinE: Enzyme concentration: 0.28 nM; ATP concentration: 100 μM; Incubation time: 1 hr.CDK4/CyclinD1: Enzyme concentration: 1 nM; ATP concentration: 200 μM; Incubation time: 10 hr.CDK6/CyclinD3: Enzyme concentration: 1 nM; ATP concentration: 300 μM; Incubation time: 3 hr.
Measurement: IC50 = 2840 nM (pChEMBL 5.550000190734863)
Descriptors: MW 500.65, ALogP 4.04, HBA 8, HBD 2, PSA 91.21, QED 0.56
Compound (SMILES): CC1(C)CCC(CN2CCN(c3ccc(C(=O)NS(=O)(=O)c4ccc(NCC5CCOCC5)c([N+](=O)[O-])c4)c(Oc4ccccc4)c3)CC2)=C(c2ccc(Cl)cc2)C1
InChIKey: FDRWZCDJGQBDLH-UHFFFAOYSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Mus musculus
UniProt: P10417
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The inhibition constant (Ki) is the dissociation constant of an enzyme-inhibitor complex or a protein/small molecule complex, wherein the small molecule is inhibiting binding of one protein to another protein or peptide. Where the Ki for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-XL) is greater than the limits of detection of the assay used. Where the binding selectivity ratio for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the selectivity of a particular compound for Bcl-2 over Bcl-XL is at least as great as the number indicated. Where the Ki for a compound is represented as < (less than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-2) is lower than the limit of detection of the assay used. Inhibition constants were determined using Wang's equation (Wang Z-X).
Measurement: Ki = 0.049 nM (pChEMBL 10.3100004196167)
Descriptors: MW 828.43, ALogP 8.78, HBA 10, HBD 2, PSA 143.35, QED 0.1
Compound (SMILES): Cc1cncc(-c2cc3c(cn2)cnn3-c2ccc(C(F)F)c(N3CCC[C@H](N)C3)n2)n1
InChIKey: AFPFWSQRRIQPHP-HNNXBMFYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 0.036 nM (pChEMBL 10.4399995803833)
Descriptors: MW 436.47, ALogP 3.45, HBA 8, HBD 1, PSA 98.64, QED 0.52
Compound (SMILES): CC[C@@H](NC(=O)c1cc(C(=O)N2CCC[C@@H]2C)n2c1COCC2)c1cnc(OC)c(Cl)c1
InChIKey: UBUZPRWNQWEPLH-KBXCAEBGSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: hERG Inhibition Assay: The effect of compounds of the formula I on the cloned human cardiac hERG channel was evaluated in an in vitro model using a whole-cell patch-clamping technique. CHO (Chinese hamster ovary) cells stably expressing hERG, the potassium channel underlying IKr currents in human hearts, were grown in HAM's F-12 media supplemented with 10% fetal bovine serum, 1x penicillin/streptomycin and 500 ug/ml G418 (Invitrogen, Carlsbad, Calif., USA) in an atmosphere of 95% air and 5% carbon dioxide. Cells used for patch-clamping were seeded on glass or plastic coverslips 12 to 36 hours before use. hERG channel currents were recorded at room temperature using the whole-cell configuration of the patch clamp technique with an Axopatch 200B amplifier (Axon Instruments, Foster City, Calif., USA). Briefly, electrodes (3 to 6 MΩ resistance) were fashioned from TW150F glass capillary tubes (World Precision Instruments, Sarasota, Fla., USA) and filled with pipette solution (containing 120 mM potassium aspartate, 20 mM potassium chloride, 4 mM adenosine triphosphate disodium salt, 5 mM HEPES, 1 mM magnesium chloride; pH adjusted to 7.2 with potassium hydroxide). hERG currents were initiated by a positive voltage pulse (20 mV) followed by a negative pulse (-40 mV) and were recorded for off-line analyses. Once hERG current from a cell perfused with external solution (containing 130 mM sodium chloride, 5 mM potassium chloride, 2.8 mM sodium acetate, 1 mM magnesium chloride, 10 mM HEPES, 10 mM glucose, 1 mM calcium chloride; pH adjusted to 7.4 with sodium hydroxide) without the test compound, i.e. control solution, was stabilized, the cell was perfused with external solution containing the test compound at specific concentrations.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 460.96, ALogP 3.58, HBA 6, HBD 1, PSA 85.69, QED 0.71
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)c3ccc(C#N)cc3)CC2)C1
InChIKey: VQMJZRQFPBZMRK-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Inhibition of human N-terminal His-tagged JAK1 catalytic domain (837 to 1142 residues) expressed in baculovirus infected insect cells assessed as decrease in phosphorylation of biotinylated peptide substrate after 1 hr in presence of 1 mM ATP by HTRF method
Measurement: IC50 > 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 491.56, ALogP 2.92, HBA 8, HBD 1, PSA 130.52, QED 0.45
Compound (SMILES): O=C1NC(=O)/C(=C/c2cnn3c(NC4CC4)cc(N[C@H]4CC[C@H](NS(=O)(=O)C5CC5)CC4)nc23)N1
InChIKey: CFZXQTURHIVYLX-RKSCUSSWSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 75 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and the resid
Measurement: IC50 > 2500 nM (pChEMBL 5.6020002365112305)
Descriptors: MW 500.59, ALogP 1.29, HBA 9, HBD 5, PSA 158.62, QED 0.27
Compound (SMILES): COc1ccc(NC(=N)NC(=N)N2CCN(C(=N)NC(=N)Nc3ccc(OC)cc3)CC2)cc1
InChIKey: XGPJMZJKYNWFKH-UHFFFAOYSA-N
Target: Acetylcholinesterase (SINGLE PROTEIN), Electrophorus electricus
UniProt: O42275
Assay: Competitive inhibition of Electrophorus electricus acetylcholinesterase in presence of varying acetylthiocholine iodide substrate level preincubated for 20 mins followed by substrate addition measured every 1 min for 30 mins
Measurement: Ki = 25550 nM (pChEMBL 4.590000152587891)
Descriptors: MW 466.55, ALogP 1.76, HBA 6, HBD 8, PSA 168.46, QED 0.25
Compound (SMILES): O=C(Nc1ccc(C(F)(F)F)cn1)N1CCC(F)(c2ncc(-c3nc(CO)no3)cc2Cl)CC1
InChIKey: FCCGZAOSQOCGRD-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: Capsaicin-Based Assay: Two days prior to performing this assay, cells are seeded in poly-D-lysine-coated 96-well clear-bottom black plates (50,000 cells/well) in growth media containing 5 uM PonA (commercially available from Invitrogen) to induce expression of TRPV1. On the day of the assay, the plates are washed with 0.2 mL 1x Hank's Balanced Salt Solution (commercially available from Life Technologies) containing 1 mM CaCl2 and 20 mM HEPES, pH 7.4, and cells are loaded using 0.1 mL of wash buffer containing Fluo-4 (3 uM final). After one hour, the cells are washed twice with 0.2 mL of wash buffer and resuspended in 0.1 mL of wash buffer. The plates are transferred to a FLIPR for assay. 50 uL of test compound diluted with assay buffer (1x Hank's Balanced Salt Solution containing 1 mM CaCl2 and 20 mM HEPES, pH 7.4) are added to the cell plates and incubated for 2 min. The final concentration of the compound is adjusted to range from about 50 picoM to about 3 uM.
Measurement: IC50 = 16.6 nM (pChEMBL 7.78000020980835)
Descriptors: MW 500.84, ALogP 4.18, HBA 7, HBD 2, PSA 117.27, QED 0.52
Compound (SMILES): O=C(/C=C/c1cccnc1)Nc1ccc(S(=O)(=O)c2ccc(-n3cccn3)cc2)cc1
InChIKey: JMTXUTAREOGONG-YIXHJXPBSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Biochemical Inhibition Assay: The NAMPT enzymatic reactions were carried out in Buffer A (50 mM Hepes pH 7.5, 50 mM NaCl, 5 mM MgCl2, and 1 mM THP) in 96-well V-bottom plates. The compound titrations were performed in a separate dilution plate by serially diluting the compounds in DMSO to make a 100x stock. Buffer A (89 uL) containing 33 nM of NAMPT protein was added to 1 uL of 100x compound plate containing controls (e.g. DMSO or blank). The compound and enzyme mix was incubated for 15 minutes at room temperature, then 10 uL of 10x substrate and co-factors in Buffer A were added to the test well to make a final concentration of 1 uM NAM, 100 uM 5-Phospho-D-ribose 1-diphosphate (PRPP), and 2.5 mM Adenosine 5'-triphosphate (ATP). The reaction was allowed to proceed for 30 minutes at room temperature, then was quenched with the addition of 11 uL of a solution of formic acid and L-Cystathionine to make a final concentration of 1% formic acid and 10 uM L-Cystathionine.
Measurement: IC50 = 17.4 nM (pChEMBL 7.760000228881836)
Descriptors: MW 430.49, ALogP 3.75, HBA 6, HBD 1, PSA 93.95, QED 0.47
Compound (SMILES): CC(c1nn2c(C3CCOCC3)ncc2c(=O)[nH]1)N1CC(c2ccc(N3CCCC3)cc2)C1
InChIKey: FSNXFDJNCMPDLS-UHFFFAOYSA-N
Target: High affinity cGMP-specific 3',5'-cyclic phosphodiesterase 9A (SINGLE PROTEIN), Homo sapiens
UniProt: O76083
Assay: Inhibition Assay: A PDE9 assay may for example, be performed as follows: The assay is performed in 60 uL samples containing a fixed amount of the relevant PDE enzyme (sufficient to convert 20-25% of the cyclic nucleotide substrate), a buffer (50 mM HEPES 7.6; 10 mM MgCl2; 0.02% Tween20), 0.1 mg/ml BSA, 225 pCi of 3H-labelled cyclic nucleotide substrate, tritium labeled cAMP to a final concentration of 5 nM and varying amounts of inhibitors. Reactions are initiated by addition of the cyclic nucleotide substrate, and reactions are allowed to proceed for one hr at room temperature before being terminated through mixing with 15 uL 8 mg/mL yttrium silicate SPA beads (Amersham). The beads are allowed to settle for one hr in the dark before the plates are counted in a Wallac 1450 Microbeta counter.
Measurement: IC50 = 110 nM (pChEMBL 6.960000038146973)
Descriptors: MW 448.57, ALogP 3.07, HBA 7, HBD 1, PSA 78.76, QED 0.65
Compound (SMILES): Nc1ncc([C@H]2CNC(=O)C2)cc1-c1ccc(C(=O)N[C@H](CO)c2ccccc2)c(F)c1
InChIKey: DQDBBPDVJUXCCY-DYESRHJHSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Kinase Assay (New): Compound potency against activated ERK2 is determined using a kinase assay that measures ERK2-catalyzed phosphorylation of biotinylated ERKtide peptide substrate ([Biotin]-AHA-K-R-E-L-V-E-P-L-T-P-S-G-E-A-P-N-Q-A-L-L-R-[NH2], the peptide sequence derived from EGF receptor: SEQ ID NO:1). The assay is carried out in 50 mM HEPES [pH 7.5], 5 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 0.05% BSA using 0.25 nM ERK2, 200 nM ERKtide peptide and 35 uM ATP (all concentrations are final in the reaction) in a total volume of 10.25 uL. A 16-point, half-log dilution series of compounds at 41x final concentration is used for generating IC50 curves. Compound dilution series are prepared in 100% DMSO. ERK2 is preincubated with compounds for 30 minutes at ambient temperature. Reaction is initiated by addition of a substrate cocktail of ERKtide peptide and ATP and is allowed to proceed for 2-3 hours at ambient temperature. Reaction is terminated by addition of 10 uL of a 2x stop buffer consisting of 100 mM Tris-Cl [pH 7.5], 25 mM EDTA, 0.01% Tween 20, 10 ug/mL of AlphaScreen Protein A Acceptor Beads, 10 ug/mL of Streptavidin Donor Beads (PerkinElmer, Waltham, Mass.), and 1.4 ug/mL phospho-EGF Receptor (Thr669) antibody (Cat #3056, Cell Signaling Technology, Danvers, Mass.). Terminated reactions are read, after overnight incubation in the dark, on an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, Mass.), with excitation and emission wavelengths set to 680 nm and 570 nm, respectively. IC50 values are determined using a four-parameter fit.
Measurement: IC50 = 72.3 nM (pChEMBL 7.139999866485596)
Descriptors: MW 434.47, ALogP 2.54, HBA 5, HBD 4, PSA 117.34, QED 0.48
Compound (SMILES): N#Cc1ccc(-c2ccc(C[C@H](NC(=O)[C@@H]3Cc4cc5c(cc4CN3S(=O)(=O)c3ccc(N4CCOCC4)nc3)OC(c3ccc(OCc4ccc(Cl)c(Cl)c4)cc3)CO5)C(=O)O)cc2)cc1
InChIKey: IOFIDDARNLKUOU-HXCLOYEWSA-N
Target: Glucagon-like peptide 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P43220
Assay: cAMP Functional Assay: The efficacy of GLP-1 receptor agonists was studied in a cAMP functional assay using HEK-293 Cells expressing the cloned human GLP-1 receptor. GLP-1-expressing cells (10,000-20,000 cells per 0.1 mL) were plated in 96-well plates in Dulbecco's modified eagles media (DMEM) containing 10% fetal bovine serum, 2 mg/mL G418 and penicillin-streptomycin. Following overnight incubation of cells, media was removed, and compounds (at concentrations ranging from 0.0001 to 100 μM) were added to monolayer cells in Iscove's modified dulbecco's medium (IMDM), 100 μM RO 20-1724 PDE inhibitor, 0.1% BSA, 2% DMSO in a final volume of 100 μL, and incubated for 30 min at 37° C. 95%02, 5% CO2 in a humidified incubator. Alternatively, cells were harvested and combined with compounds in suspension using the buffer and protocol described above. cAMP was quantified using a homogenous time-resolved fluorescence detection system (cAMP dynamic, CIS bio International).
Measurement: EC50 = 48 nM (pChEMBL 7.320000171661377)
Descriptors: MW 960.89, ALogP 7.78, HBA 11, HBD 2, PSA 180.62, QED 0.11
Compound (SMILES): CN[C@@H](C)C(=O)Nc1ccc2c(n1)CN(C)C(=O)N2c1ccc(Cl)cc1
InChIKey: SOXBWWXKFGIPHB-NSHDSACASA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: In Vitro Competitive Displacement Binding Assay: Modified SMAC peptides and compounds were tested for their ability to displace the fluorescent tracer from either XIAP, cIAP-1 or cIAP-2. BIR3 domains of cIAP-1, cIAP-2 and XIAP were incubated with test compounds or SMAC based peptides and their respective peptide probes (Peptide Protein Research) in assay buffer (50 mM Hepes pH 7.5, 0.025% Tween-20, 0.01% BSA, and 1 mM DTT). Positive controls consisted of BIR3 proteins and tracer (no inhibition) and negative controls contained tracer only (100% inhibition). The samples were incubated at room temperature for 1 hr (XIAP and cIAP-2) or 3 hrs (cIAP-1) prior to being read in the BMG Pherastar in Fluorescence Polarization mode (FP 485 nm, 520 nm, 520 nm).
Measurement: IC50 = 505 nM (pChEMBL 6.300000190734863)
Descriptors: MW 373.84, ALogP 2.98, HBA 4, HBD 2, PSA 77.57, QED 0.86
Compound (SMILES): CC1(C)C[C@H](Nc2ncnc(N[C@@H]3C[C@@H](COS(N)(=O)=O)[C@@H](O)C3)n2)c2ccccc21
InChIKey: YKOZUSCXGQGYPB-RMHZUWNSSA-N
Target: NEDD8 activating enzyme (PROTEIN COMPLEX), Homo sapiens
UniProt: Q13564
Assay: Inhibition of recombinant human NAEalpha/N-terminal His-tagged NAEbeta expressed in baculovirus infected insect Sf9 cells using N-terminal FLAG tagged Nedd8 as substrate incubated for 105 mins in presence of N-terminal GST-tagged Ubc12 by FRET assay
Measurement: IC50 <= 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 448.55, ALogP 1.48, HBA 9, HBD 4, PSA 152.35, QED 0.49
Compound (SMILES): CC(C)Oc1ccc(-c2nc(-c3cccc4c3CC[C@H]4NC(=O)N3CCC(O)CC3)no2)cc1C#N
InChIKey: KIALPTLWECVGBJ-HSZRJFAPSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: Agonist activity at human S1P1 expressed in CRE-bla CHOK1 cells assessed as inhibition of forskolin-induced cAMP formation preincubated for 4 hrs followed by FRET-based beta-lactamase fluorescent substrate addition measured after 2 hrs
Measurement: EC50 = 0.5 nM (pChEMBL 9.300000190734863)
Descriptors: MW 487.56, ALogP 4.22, HBA 7, HBD 2, PSA 124.51, QED 0.55
Compound (SMILES): CC(=O)N1CCN(C(=O)N2CC[C@](c3ccc(C(OCc4ccc(F)cc4C#N)(C(F)(F)F)C(F)(F)F)cc3)(S(=O)(=O)c3ccc(F)cc3)C2)CC1
InChIKey: LZEQNVAHHXFCCI-HKBQPEDESA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Scintillation Proximity Assay (SPA): The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 ug/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 uM to 0.28 nM for 10 min at room temperature in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% Glycerol (Sigma Cat#G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat #RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce [3H] 25-hydroxycholesterol specific binding by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 106 nM (pChEMBL 6.96999979019165)
Descriptors: MW 758.69, ALogP 6.03, HBA 6, HBD 0, PSA 111.02, QED 0.22
Compound (SMILES): O=C(CC1([C@H]2COC[C@@H](C3CC3)N2S(=O)(=O)c2ccc(Cl)cc2)CC1)N1CC2CCC(C1)N2CCO
InChIKey: JIYFANCCHHINCA-BRLSKQCNSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: Inhibition of recombinant human CYP3A4 expressed in baculovirus infected insect cells using fluorescent substrate
Measurement: IC50 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 538.11, ALogP 2.35, HBA 6, HBD 1, PSA 90.39, QED 0.55
Compound (SMILES): CC(C)C[C@H](NC(=O)c1cc(-c2cccc(Cl)c2)c(Cl)cn1)C(N)=O
InChIKey: FGTMCYGSIWZUJC-HNNXBMFYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1×HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min. Compounds were added to a final assay volume of 100 μl and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 μl detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.
Measurement: EC50 = 466.3 nM (pChEMBL 6.329999923706055)
Descriptors: MW 380.28, ALogP 3.69, HBA 3, HBD 2, PSA 85.08, QED 0.8
Compound (SMILES): C[C@]1(N2Cc3cn(S(C)(=O)=O)nc3C2)CO[C@H](c2cc(F)ccc2F)[C@@H](N)C1
InChIKey: YEPZZFPPSSQKOU-CGTJXYLNSA-N
Target: Dipeptidyl peptidase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P27487
Assay: Fluorometric Assay: The utility of the compounds in accordance with the present invention as inhibitors of dipeptidyl peptidase-IV enzyme activity may be demonstrated by methodology known in the art. Inhibition constants are determined as follows. A continuous fluorometric assay is employed with the substrate Gly-Pro-AMC, which is cleaved by DPP-4 to release the fluorescent AMC leaving group. The kinetic parameters that describe this reaction are as follows: Km=50 uM; kcat=75 s-1; kcat/Km=1.5x106 M-1s-1. A typical reaction contains approximately 50 pM enzyme, 50 uM Gly-Pro-AMC, and buffer (100 mM HEPES, pH 7.5, 0.1 mg/ml BSA) in a total reaction volume of 100 uL. Liberation of AMC is monitored continuously in a 96-well plate fluorometer using an excitation wavelength of 360 nm and an emission wavelength of 460 nm. Under these conditions, approximately 0.8 uM AMC is produced in 30 minutes at 25 degrees C.
Measurement: IC50 = 37 nM (pChEMBL 7.429999828338623)
Descriptors: MW 412.46, ALogP 1.53, HBA 7, HBD 1, PSA 90.45, QED 0.82
Compound (SMILES): C[C@@H](Oc1nnc(-c2cc[nH]c(=O)c2)n1C)c1cc(-c2cccc(Cl)c2)on1
InChIKey: YGGDHMSWIMDIPD-LLVKDONJSA-N
Target: Metabotropic glutamate receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P41594
Assay: Antagonist activity against human mGluR5d expressed in cells by fluo-3 dye based FLIPR assay
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 397.82, ALogP 3.62, HBA 7, HBD 1, PSA 98.83, QED 0.55
Compound (SMILES): CCOc1cc(=O)n(C)cc1-c1cc(NC(=O)Cc2ccc(Oc3ccccc3)cc2)ccc1Oc1ccc(F)cc1F
InChIKey: ITZDGUNXXRKGPS-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Assay method B: Compound dilution series were prepared in DMSO via an approximately 3-fold serial dilution from one of the following: 0.47 mM to 7.8 nM. Compound dilutions were added directly into white, low-volume assay plates (Perkin Elmer Proxiplate 384 Plus# 6008280) using a Labcyte Echo in conjunction with Labcyte Access and Thermo Multidrop CombinL robotics. Compounds were then suspended in eight microliters (μL) of assay buffer (20 mM Sodium Phosphate, pH 6.0, 50 mM NaCl, 1 mM Ethylenediaminetetraacetic acid disodium salt dihydrate, 0.01% Triton X-100, 1 mM DL-Dithiothreitol) containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and Alexa-647-conjugated probe. The final concentration of 1X assay mixture for assay methods A, B and C contains 2% DMSO, 8 nM His-tagged bromodomain, 1 nM Europium-conjugated anti-His-tag antibody and 100 nM or 30 nM probe (for BDI or BDII, respectively) and compound concentration in the range of: 49.02 μM-15.63 nM for method A, 9.19 μM 150 pM for method B, and 0.92 μM 15 pM for method C. After a one-hour equilibration at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 1690 nM (pChEMBL 5.769999980926514)
Descriptors: MW 582.6, ALogP 7.5, HBA 6, HBD 1, PSA 78.79, QED 0.18
Compound (SMILES): COc1cc(C(=O)NCC2CCN(C)CC2)ccc1Nc1ncc2c(n1)-c1c(nn(C)c1-c1ccc(-c3cnn(C)c3)cc1)CC2
InChIKey: OSGQGIINHNRMCL-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: DELFIA Assay: The kinase activity is measured by DELFIA assay (dissociation-enhanced lanthanide fluorescence immunoassay, Perkin Elmer). The cytoplasmic kinase domain of human IGF-1R (amino acids 964-1370) is expressed as a fusion protein with a glutathione-S-transferase tag (IGF-1R-GST) in High Five.TM. Cells (Invitrogen). Enzyme activity is measured in the presence of substances and a control substance. Poly-glutamate-tyrosine peptide (pEY, Sigma Aldrich) and biotinylated pEY (bio-pEY) are used as reaction substrates.10 uL of substance in 25% DMSO are mixed with 30 uL of IGF-1R-GST solution (67 mM HEPES pH 7.4, 15 ug/mL pEY, 1.7 ug/mL bio-pEY, 13.3 mM MgCl2, 3.3 mM dithiothreitol, 0.0033% Brij 35, 2 ng IGF-1R-GST) in 96-well plates. The reactions are started with 10 uL of a 750 uM ATP solution. After 40 min at RT the reactions are stopped with 50 .mu.L of stop solution (250 mM EDTA, 20 mM HEPES pH 7.4). 90 uL from each reaction are transferred.
Measurement: IC50 = 0.16 nM (pChEMBL 9.800000190734863)
Descriptors: MW 617.76, ALogP 4.87, HBA 10, HBD 2, PSA 115.02, QED 0.25
Compound (SMILES): COc1ccc(Cl)cc1-c1cccc(C(CC(=O)O)c2nc(C)c(C)s2)c1
InChIKey: PQQQPMAQRRRTBF-UHFFFAOYSA-N
Target: Lysosomal protective protein (SINGLE PROTEIN), Homo sapiens
UniProt: P10619
Assay: Inhibition Assay: Recombinant human cathepsin A (residues 29-480, with a C-terminal 10-His tag; R&D Systems, #1049-SE) was proteolytically activated with recombinant human cathepsin L (R&D Systems, #952-CY). Briefly, cathepsin A was incubated at 10 ug/ml with cathepsin L at 1 ug/ml in activation buffer (25 mM 2-(morpholin-4-yl)-ethanesulfonic acid (MES), pH 6.0, containing 5 mM dithiothreitol (DTT)) for 15 min at 37° C. Cathepsin L activity was then stopped by the addition of the cysteine protease inhibitor E-64 (N-(trans-epoxysuccinyl)-L-leucine-4-guanidinobutylamide; Sigma-Aldrich, # E3132; dissolved in activation buffer/DMSO) to a final concentration of 10 uM.The activated cathepsin A was diluted in assay buffer (25 mM MES, pH 5.5, containing 5 mM DTT) and mixed with the test compound (dissolved in assay buffer containing (v/v) 3% DMSO) or, in the control experiments, with the vehicle in a multiple assay plate. After incubation for 15 min at room temperature.
Measurement: IC50 = 657 nM (pChEMBL 6.179999828338623)
Descriptors: MW 401.92, ALogP 5.7, HBA 4, HBD 1, PSA 59.42, QED 0.58
Compound (SMILES): CC(C)(O)CN1CC=C(c2cc3c(-c4cc(F)cc(N5CCOc6cc(C7CC7)ccc6C5=O)c4CO)ccnc3[nH]2)CC1
InChIKey: UEMXSGWWLNFGTQ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Recombinant Human BTK Inhibition Assay: The inhibition assays were performed using the generic tyrosine kinase assay kit from C is Bio (HTRF KinEASE-TK #62TK0PEB). The assay is based on the principle of fluorescence resonance energy transfer (FRET) between the Europium Cryptate labeled anti-phosphotyrosine antibody and streptavidin labeled fluorophore, XL-665. In an uninhibited kinase reaction the biotinylated peptide substrate (Tk-Biotin) gets phosphorylated at an intermediate tyrosine site which is captured by anti-phosphotyrosine antibody which eventually acts as a FRET donor. The streptavidin labeled XL-665 binds strongly to the Tk-Biotin substrate and act as a FRET acceptor. The resultant FRET signal is measured at 665 nm. A ratio of this specific fluorescence (665 nm) to the background fluorescence (620 nm), conferred by the free antibody, is directly proportional to the kinase activity. Inhibition of the kinase activity by a pharmacological intervention such as a Btk inhibitor of the present disclosure will result into decreased FRET signal.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 596.7, ALogP 5.64, HBA 6, HBD 3, PSA 101.92, QED 0.26
Compound (SMILES): C=CCCC(=O)Nc1ccc(NC(=O)N2Cc3ccccc3C2)cc1
InChIKey: UZDMGBIZEWMOCY-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 1:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1251667.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 25.4 nM (pChEMBL 7.590000152587891)
Descriptors: MW 335.41, ALogP 4.14, HBA 2, HBD 2, PSA 61.44, QED 0.81
Compound (SMILES): Cc1cc(-c2ccc(F)c(F)c2)cc(CNc2c(F)ccc(OCC(=O)O)c2F)c1C
InChIKey: UEKXHDFGIPGKNO-UHFFFAOYSA-N
Target: Prostaglandin E2 receptor EP2 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P43116
Assay: Cellular Functional Assay: Human CHO cells expressing the recombinant human prostanoid EP2 receptor were suspended in assay medium (HBSS buffer (Invitrogen) containing 20 mM HEPES (pH7.4) and 500 μM isobutyl-methylxanthine IBMX) and plated out to yield approximately 1×104 cells/well in a 96-well. Following this, cells were incubated with agonists for 30 min in the presence of the test compounds. For stimulated control measurements, separate assay wells contain 10 μM PGE2 (control specific agonist). All incubations were carried out at 37° C. in a 5% CO2 atmosphere. Following incubation, the amount of cAMP in each well was determined by HTRF method. The cells are lysed and the fluorescence acceptor (D2-labeled cAMP) and fluorescence donor (anti-cAMP antibody labeled with europium cryptate) are added.
Measurement: EC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 433.4, ALogP 5.6, HBA 3, HBD 2, PSA 58.56, QED 0.48
Compound (SMILES): Clc1ccc(CCN[C@H]2CC[C@H](c3nnn4cnc5[nH]ccc5c34)CC2)cc1
InChIKey: ZPAJIUBPKZGWEN-JCNLHEQBSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 2.5 nM (pChEMBL 8.600000381469727)
Descriptors: MW 394.91, ALogP 4.12, HBA 5, HBD 2, PSA 70.9, QED 0.53
Compound (SMILES): CC[C@@H](CN(C)S(=O)(=O)C1CC1)N1C(=O)[C@@](CC)(CC(=O)O)C[C@H](c2cccc(Cl)c2)[C@H]1c1ccc(Cl)cc1
InChIKey: LYZBKNYTGQNBNY-OCAOGGMISA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: Homogenous Time-Resolved Fluorescence Assay (HTRF2 Assay): The standard assay conditions for the in vitro HTRF assay consisted of a 50 ul total reaction volume in black 384-well Costar polypropylene plates in 1×PBS buffer pH 7.4, 1 mM DTT, 0.1% BSA, 2.5 nM GST-hMDM2 (aa 1-188), 5 nM biotinylated-p53 (aa 1-83), 1.8 nM SA-XLent (Cisbio; Bedford, Mass.), 0.6 nM anti-GST cryptate monoclonal antibody (Cisbio; Bedford, Mass.) and 200 mM KF. Amino acid residues 1-188 of human MDM2 were expressed as an amino-terminal glutathione-S-transferase (GST) fusion protein (GST-hMDM2) in Escherichia coli. Residues 1-83 of human p53 were expressed as an amino-terminal AviTag™-TrxA-6×His fusion protein (biotinylated p53) in E. coli. Each protein was purified from cell paste by affinity chromatography.Specifically, 10 uL of GST-hMDM2 was incubated with 10 ul of diluted compound (various concentrations, serially diluted) in 10% DMSO for 20 minutes at room temperature. 20 uL of biotinylated-p53 was added to the GST-hMDM2+compound mixture, and then incubated at room temperature for 60 min. 10 uL of detection buffer consisting of SA-XLent, anti-GST cryptate antibody and KF was added to GST-hMDM2, biotinylated-p53 and compound reaction and left at room temperature to reach equilibrium for >4 hrs. The final concentration of DMSO in the reaction was 2%. Time-resolved fluorescence readings were measured on a microplate multilabel reader. Percentage of inhibition was calculated relative to nutlin-3. All assay conditions remained the same as described above, with the exception of the following changes in reagent concentrations: 0.2 nM GST-hMDM2 (1-188), 0.5 nM biotinylated-p53 (1-83), 0.18 nM SA-XLent, and 100 mM KF.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 595.59, ALogP 6.12, HBA 4, HBD 1, PSA 94.99, QED 0.34
Compound (SMILES): O=C1CN(c2ccccc2F)CCN1CC(O)CN1CCc2ccccc2C1
InChIKey: DDJHZGLEVGJHEY-UHFFFAOYSA-N
Target: Protein arginine N-methyltransferase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: O14744
Assay: Inhibition of PRMT5 in human Z138 cells assessed as decrease in di-methyl arginine levels after 96 hrs by Western blot method
Measurement: EC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 383.47, ALogP 1.89, HBA 4, HBD 1, PSA 47.02, QED 0.86
Compound (SMILES): CNC(=O)c1ccnc2c(NS(=O)(=O)c3ccccc3)cccc12
InChIKey: ZPOYMOMUWHOHPK-UHFFFAOYSA-N
Target: Ferroportin (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NP59
Assay: Antagonist activity at C-terminal halo-tag fused human ferroportin expressed in MDCK cells assessed as inhibition of hepcidin-induced ferroportin internalization after overnight incubation by Draq5 staining-based fluorescence assay
Measurement: IC50 = 6240 nM (pChEMBL 5.210000038146973)
Descriptors: MW 341.39, ALogP 2.4, HBA 4, HBD 2, PSA 88.16, QED 0.76
Compound (SMILES): CCC(c1nc2c(cnn2C(C)C)c(=O)[nH]1)N1CC(Oc2ccccc2)C1
InChIKey: LQGBRKQMOXFYNN-UHFFFAOYSA-N
Target: High affinity cGMP-specific 3',5'-cyclic phosphodiesterase 9A (SINGLE PROTEIN), Homo sapiens
UniProt: O76083
Assay: Inhibition of human recombinant PDE9 expressed in insect Sf9 cells using [3H]-cGMP as substrate after 30 mins by scintillation proximity assay
Measurement: IC50 = 256 nM (pChEMBL 6.590000152587891)
Descriptors: MW 367.45, ALogP 2.91, HBA 6, HBD 1, PSA 76.04, QED 0.72
Compound (SMILES): COc1cc(-c2cccc(OC(F)(F)F)c2)c(F)cc1-n1c2c(ccc1=O)CN(S(=O)(=O)Nc1cccnn1)CC2
InChIKey: MBNQFIOGZKONLA-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Inhibition of human Nav1.7 expressed in HEK293 cells at holding potential yielding 20 to 50% inactivation measured after 3 mins by patchXpress electrophysiology assay
Measurement: IC50 = 2.3 nM (pChEMBL 8.640000343322754)
Descriptors: MW 591.54, ALogP 4.06, HBA 8, HBD 1, PSA 115.65, QED 0.32
Compound (SMILES): CNC(=O)C(OC(=O)N1CCN(C(c2ccc(Cl)cc2)c2ccc(Cl)cc2)CC1)C(F)(F)F
InChIKey: GFAFOZLWFGIFTF-UHFFFAOYSA-N
Target: Fatty-acid amide hydrolase 1 (SINGLE PROTEIN), Mus musculus
UniProt: O08914
Assay: In Vitro Competitive Activity-Based Protein Assay: Proteomes (mouse brain membrane fraction or cell lysates) (50 uL, 1.0 mg/mL total protein concentration) were preincubated with varying concentrations of inhibitors at 37 C. After 30 min, FP-Rh (1.0 uL, 50 uM in DMSO) was added and the mixture was incubated for another 30 min at 37 C. Reactions were quenched with SDS loading buffer (50 uL-4x) and run on SDS-PAGE. Following gel imaging, serine hydrolase activity was determined by measuring fluorescent intensity of gel bands corresponding to MAGL, ABHD6 and FAAH using ImageJ 1.43u software.Preparation of Mouse Brain Proteomes from Inhibitor Treated Mice. Inhibitors were administered to wild-type C57Bl/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 > 10000 nM (pChEMBL 5)
Descriptors: MW 504.34, ALogP 4.51, HBA 4, HBD 1, PSA 61.88, QED 0.65