lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cn1nccc1-c1cc(-c2cnn(C3(CC#N)CN(S(C)(=O)=O)C3)n2)c2cccnc2c1
InChIKey: QPNXDJOQDUCCFK-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK Caliper Enzyme Assay at 1mM ATP: Compounds were added to a 384-well plate. Reaction mixtures contained 10 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% BSA, 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20% to 30% phosphorylation. The assays were stopped with 15 μL of 180 mM HEPES, pH 7.4, 20 mM EDTA, and 0.2% Coating Reagent 3. The assay plates were placed on a Caliper Life Science LC3000 instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 448.51, ALogP 1.78, HBA 9, HBD 0, PSA 122.59, QED 0.46
Compound (SMILES): CCCCNNC(=O)c1ccc(OCC)c(OCC)c1
InChIKey: WSFDLHHIYJEKQK-UHFFFAOYSA-N
Target: Histone deacetylase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O15379
Assay: HDAC Inhibition: These SAR data indicate that a tripartite structure of this scaffold with a central C(O)NHNH unit flanked by a phenyl group and a short aliphatic chain increases HDAC inhibition. As for the phenyl group, the presence of a relatively bulky substituent at the para position relative to the carbonyl group also affords potent HDAC inhibitors.
Measurement: IC50 = 800 nM (pChEMBL 6.099999904632568)
Descriptors: MW 280.37, ALogP 2.52, HBA 4, HBD 2, PSA 59.59, QED 0.54
Compound (SMILES): O=C1NCc2nc(-c3c(F)cccc3F)cc(Nc3ccc(C(=O)N4C[C@H]5COC[C@H]5C4)cc3)c21
InChIKey: ODBNOQBNPBRPNZ-IYBDPMFKSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Caliper Assay: Tyk2 & JAK2: The caliper machine employs an off chip mobility shift assay to detect phosphorylated peptide substrates from kinase assays, using microfluidics technology. The assays are carried out at ATP concentration equivalent to the ATP Km, and at 1 mM ATP. Compounds are serially diluted in DMSO then further diluted in assay buffer (25 mM HEPES, pH 7.5, 0.01% Brij-35, 0.01% Triton, 0.5 mM EGTA). 5 ul of diluted compound was added into wells first, then 10 ul of enzyme mix was added into wells, followed by 10 uL of substrate mix (peptide and ATP in 10 mM MgCl2) to start reaction. Reaction was incubated at 28° C. for 25 min and then added 25 ul stop buffer (100 mM HEPES, 0.015% Brij-35, 50 mM EDTA), followed by reading with Caliper. JAK2 at 1 nM final concentration and TYK2 at 9.75 nM are from Carna, and substrates used are ATP at 20 and 16 uM, respectively. JAK2 assay uses peptide 22 and TYK2 uses peptide 30 (Caliper), each at 3 uM.
Measurement: Ki = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 476.48, ALogP 3.73, HBA 5, HBD 2, PSA 83.56, QED 0.6
Compound (SMILES): Cn1cc(-c2cnc(N)c(C(=O)N[C@H]3CCC[C@@H]3OCc3ccc(-c4ccc(CN)cc4)cc3)c2)cn1
InChIKey: KBUDNCDDBDPFJE-SVBPBHIXSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: Inhibition of N-terminal His-tagged recombinant human Axl (473 to end residues) expressed in baculovirus-infected Sf9 cells using biotin-labeled TK and ATP as substrate preincubated with enzyme for 30 mins followed by substrate addition and measured after 1 hr by HTRF assay
Measurement: IC50 = 1740 nM (pChEMBL 5.760000228881836)
Descriptors: MW 496.62, ALogP 4.06, HBA 7, HBD 3, PSA 121.08, QED 0.34
Compound (SMILES): C[C@@H]1CN(c2ccc(-c3n[nH]c4cnc(N5CC6CCC5CN6C(=O)N5CCCC5)nc34)cn2)CCN1
InChIKey: PUFNGSLFSIDSGP-QHGAOEGBSA-N
Target: Activin receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q04771
Assay: HTRF Assay (1 mM ATP): ALK2 (aa 147-end) was obtained from BPS biosciences. The enzymatic assays were conducted in white 384-well polystyrene plates in a final volume of 8 μL. The inhibitors were serially diluted in DMSO and added to the plate wells prior to addition of the other reaction components. The assays were carried out at 25° C. in the assay buffer (50 mM HEPES, pH 7.1, 10% Glycerol, 0.01% Brij50, 10 mM MgCl2, 1 mM EGTA, 5 mM DTT, and 0.01% BSA), containing 50 nM LANCE Ultra ULight-DNA Topoisomerase 2-alpha peptide (Perkin Elmer TRF0130), and 1 mM ATP. The final concentration of DMSO in the assay was 1% and the enzyme concentration was 2.5 nM for ALK2. The reactions were allowed to proceed for 2-4 hr after which, the reaction was quenched by addition of EDTA at a final concentration of 20 mM along with 1.5 nM LANCE Ultra Europium-anti-phospho-DNA Topoisomerase 2-alpha (Thr1342) antibody (Perkin Elmer TRF0218). The reaction was read on a PHERAstar FS plate reader (BMG Labtech). IC50 determination was performed by fitting percent control activity versus the log of the inhibitor concentration using the IDBS XLFit and GraphPad Prism 5.0 software.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 502.63, ALogP 2.08, HBA 8, HBD 2, PSA 109.41, QED 0.56
Compound (SMILES): CNC(=O)C(=N)c1c(N)cc(-c2cc(F)c(O)cc2C2CC2)nc1NCc1ccccc1N(C)S(C)(=O)=O
InChIKey: IJHPBWFDHMKENG-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK Caliper Enzyme Assay at 1 mM ATP: Test article was solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. An 11-point half log dilution series was created in DMSO with a top concentration of 600 μM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 resulting in a top final assay compound concentration of 10 μM and a 2% DMSO concentration.Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK3 or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 52 nM (pChEMBL 7.28000020980835)
Descriptors: MW 540.62, ALogP 3.17, HBA 8, HBD 5, PSA 161.5, QED 0.26
Compound (SMILES): COc1cc(O)c2c(c1)N(c1ccc3ncc(-c4cnn(C)c4)cc3c1)CCN(C(C)C)C2
InChIKey: ZTTACMJUSMUVNU-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Enzymatic Assay: FGFR3: In a final reaction volume of 30 uL, FGFR3 (h) (40 ng/ml) is incubated with 50 mM HEPES pH 7.5, 6 mM MnCl2, 1 mM DTT, 0.1 mM Na3VO4, 0.01% Triton-X-100, 500 nM Btn-Flt3 and 25 uM ATP in the presence of compound (1% DMSO final). After incubation for 60 minutes at room temperature the reaction is stopped with 2.27 nM EU-anti P-Tyr, 7 mM EDTA, 31.25 nM SA-XL-665 and 0.02% BSA which is present for 60 minutes at room temperature. Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) signal (ex340 nm. Em 620 nm, em 655 nm) is measured afterwards and results are expressed in RFU (Relative Fluorescence Units). In this assay, the inhibitory effect of different compound concentrations (range 10 uM to 0.1 nM) is determined and used to calculate an IC50 (M) and pIC50 (-log IC50) value.
Measurement: Kd = 1259 nM (pChEMBL 5.900000095367432)
Descriptors: MW 443.55, ALogP 4.71, HBA 7, HBD 1, PSA 66.65, QED 0.49
Compound (SMILES): CCc1c2c(nn1C)CN(C)CCCCn1c(C(=O)O)c(CCCOc3cccc4ccccc34)c3ccc(F)c-2c31
InChIKey: RYAPMXBEHQNOIT-UHFFFAOYSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Biological Assay 2: BCL-2 Assay: In the assay 11 different concentrations of each compound (0.1 nM, 0.33 nM, 1.1 nM, 3.8 nM, 13 nM, 44 nM, 0.15 μM, 0.51 μM, 1.7 μM, 5.9 μM and 20 μM) were typically measured as duplicates in the same microtiter plate. For that, 100-fold concentrated DMSO solutions were prepared by serial dilutions (1:3.4) of a 2 mM stock solution in a clear, 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany). From there, 50 nl were transferred in a dark test plate (Greiner Bio-One, Frickenhausen, Germany). The assay was initiated by addition of 2 μl of a 2,5-fold concentrated His-BCL-2 solution (usually for a 1 nM end concentration in 5 μl reaction volume) in aqueous assay buffer [50 mM Tris/HCl pH 7, 100 mM sodium chloride (NaCl), 50 mM potassium fluoride (KF), 0.005% Tween-20, 2 mM DTT, 0.1% bovine gamma globulin (BGG)] to the compounds in the assay plate. This was followed by a 10-minute incubation step at 22° C. for pre-equilibration of the putative complex between His-BCL-2 and the compounds. After that, 3 μl of a 1.67-fold concentrated solution (in assay buffer) consisting of Bad BH3-derived peptide (1 nM end concentration) and TR-FRET detection reagents [1.67 nM anti-His-Eu cryptate and 1.67 nM streptavidin-XL665 (both from Cisbio Bioassays, Codolet, France)], were added.
Measurement: IC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 568.69, ALogP 6.83, HBA 6, HBD 1, PSA 72.52, QED 0.22
Compound (SMILES): CCCCNc1ncc(-c2ccc(CNC3CC4CCC(C3)N4C)cn2)c(N[C@H]2CC[C@H](O)CC2)n1
InChIKey: UMJNEGIWFHRTLO-LDTFBWSASA-N
Target: Tyrosine-protein kinase receptor TYRO3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06418
Assay: Activity Assay: Briefly, activity assays were performed in a 384 well, polypropylene microplate in a final volume of 50 μL of 50 mM Hepes, Ph 7.4 containing 10 mM MgCl2, 1.0 mM DTT, 0.01% Triton X-100, 0.1% Bovine Serum Albumin (BSA), containing 1.0 μM fluorescent substrate and ATP at the Km for each enzyme. All reactions were terminated by addition of 20 μL of 70 mM EDTA. After a 180 min incubation, phosphorylated and unphosphorylated substrate peptides were separated in buffer supplemented with 1×CR-8 on a LabChip EZ Reader equipped with a 12-sipper chip. Data were analyzed using EZ Reader software. Assay conditions for MCE assays
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 493.7, ALogP 4.18, HBA 8, HBD 4, PSA 98.23, QED 0.37
Compound (SMILES): COC(=O)c1c(C#N)ccnc1O[C@@H]1CC[C@@H](C)N(C(=O)c2ccccc2-c2ncccn2)C1
InChIKey: WTEXMGOAFNNVRP-SJLPKXTDSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: FLIPR Ca2+ Flux 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 were 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 μM Fluo-4AM ester, 0.02% pluronic acid, and 1% BSA. The dye loading solution is then aspirated and cells are washed 3 times with 100 μl assay buffer. 30 μl of that same buffer is left in each well. Within the Fluorescent Imaging Plate Reader (FLIPR, Molecular Devices), test compounds are added to the plate in a volume of 25 μl, incubated for 5 min and finally 25 μl of agonist is added. Fluorescence is measured for each well at 1 second intervals for 5 minutes and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 70 pM Ala-6,12 orexin-A with buffer in place of antagonist.
Measurement: IC50 = 417 nM (pChEMBL 6.380000114440918)
Descriptors: MW 457.49, ALogP 3.27, HBA 8, HBD 0, PSA 118.3, QED 0.54
Compound (SMILES): C[C@H](Nc1nc(C(=O)N2CC(CC(=O)NC(C)(C)C)C2)c2sccc2n1)c1cncc(F)c1
InChIKey: CTNVMTHCGSYLTO-ZDUSSCGKSA-N
Target: Adenosine receptor A1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30542
Assay: Activity Assay: Compounds (Adenosine Receptor modulators) and activity assay.
Measurement: IC50 > 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 470.57, ALogP 3.78, HBA 7, HBD 2, PSA 100.11, QED 0.57
Compound (SMILES): CCc1cc(O)c(F)cc1-c1cc2[nH]nc(C(=O)NC)c2c(NCc2ccc(O)cc2N(C)S(C)(=O)=O)n1
InChIKey: LGAVUEXTUHVZEO-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK Caliper Enzyme Assay at 1 mM ATP: Test article was solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. An 11-point half log dilution series was created in DMSO with a top concentration of 600 μM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 resulting in a top final assay compound concentration of 10 μM and a 2% DMSO concentration.Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK3 or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 33.2 nM (pChEMBL 7.480000019073486)
Descriptors: MW 542.59, ALogP 3.11, HBA 8, HBD 5, PSA 160.54, QED 0.23
Compound (SMILES): O=C1Cc2cc(C(O)CN3CCN(c4cccc5sccc45)CC3)ccc2N1
InChIKey: HYDRTWYBNCKGDA-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 2A (SINGLE PROTEIN), Homo sapiens
UniProt: P28223
Assay: 5-HT2A Receptor Antagonism Activity Test: The 5-HT2A receptor antagonism activity test (The antagonism activity of test compounds on 5-HT2A receptor expressing human recombinant 5-HT2A receptor in CHO-K1 cells) was performed using FLIPR Calcium 5 Assay Kit (Product of Molecular Devices USA Inc.) according to the method documented in the kit instructions. The concentration of the test compounds was 0.1 nM-10000 nM and risperidone was used as a positive control. Test method is as follows: On the first day the seed cells are placed in T-175 flask containing 25 ml growth medium (F-12 nutrient mixture+10% FBS+1% penicillin/streptomycin+1.2% 50 mg/ml Geneticin) at a density of 14 million per bottle, cells were cultured under 37° C./5% CO2/humidified conditions for 24 hours; On the next day the seed cells were inoculated to the 384-well cell culture plate (each well containing 20,000 cells), the growth medium was replaced by 50 μL detecting medium (F-12 nutrient mixture+1.5% activated carbon-treated FBS), the cells were cultured under 37° C./5% CO2/humidified conditions for 16 hours; On the third day the medium was removed, each well of the cells plates were added with 24 μL freshly prepared loading dye solution (formulated according to the instructions), the plates were placed in an incubator and incubated under 37° C./5% CO2/humidified conditions for 120 minutes; transfer 6 μL test compound solution to the assay plate and gently shaking for one minute, incubated under 37° C./5% CO2/humidified conditions for 30 minutes; each well of the assay plate was added with 10 μL freshly prepared α-methyl-5-hydroxytryptamine solution (1.2 M, the final concentration of α-methyl-5-hydroxytryptamine is 300 nM), data were detected and analyzed by FLIPR (Product of US Molecular Devices Corporation). Inhibition rate of the compounds at different concentrations was calculated, IC50 value was calculated by the Excelfit software
Measurement: IC50 = 14.7 nM (pChEMBL 7.829999923706055)
Descriptors: MW 393.51, ALogP 3.25, HBA 5, HBD 2, PSA 55.81, QED 0.71
Compound (SMILES): Cc1ccccc1Oc1cc(Cl)ccc1O[C@H]1CCNC1
InChIKey: YVOYRBKOCCSWPI-AWEZNQCLSA-N
Target: 5-hydroxytryptamine receptor 2C (SINGLE PROTEIN), Homo sapiens
UniProt: P28335
Assay: Binding Assay: Receptor binding was performed using membrane fractions prepared from the HEK-293 cell line recombinantly expressing rat 5-HT7 receptors (NCBI accession NM_022938). Compound affinity for the rat 5-HT7 receptor subtype was evaluated by competitive radioligand binding assays using 5-carboxamido[3H]tryptamine ([3H]5-CT) (Amersham Biosciences, cat. 90000403) detection. HitHunter cAMP assays are in-vitro based competitive immunoassays. The assay was performed on the HEK-293 cell line stably transfected with r5-HT7 receptor. Cells were pre-incubated with test compounds for 10 minutes. For antagonist testing, the cells were then challenged with 100 nM 5-CT for 20 minutes. Cells were then lysed and cAMP measured according to manufacturers protocol (Amersham, cat. NET791250UC) or [3H]mesulergine (Amersham, cat. TRK1041). The assay was performed on membranes prepared from HEK-293 cells stably transfected with h5-HT6. Following centrifugation, membranes were resuspended and incubated for 60 min at room temperature with 1.7 nM [3H]LSD in the presence of increasing concentration of test compounds. Nonspecific binding was defined in the presence of 10 μM clozapine (Tocris, cat. TRK1068). Homogenized HEK-293 membranes expressing the human SERT were incubated in 50 mM Tris-HCl (pH 7.5), 120 mM NaCl, 5 mM KCl with [3H]-citalopram (3 nM) with or without test compounds. Nonspecific binding was determined in the presence of 10 μM fluoxetine. Radioactivity readouts and Ki values were performed as previously described for r5-HT7.
Measurement: Ki = 330 nM (pChEMBL 6.480000019073486)
Descriptors: MW 303.79, ALogP 4.18, HBA 3, HBD 1, PSA 30.49, QED 0.92
Compound (SMILES): CC1(C)C(N)=N[C@](C)(c2cc(/C=C(\F)c3cc4c(cn3)OCCO4)ccc2F)C(F)(F)S1(=O)=O
InChIKey: ISINGNQBVRWZCL-WHIQYKQFSA-N
Target: Cathepsin D (SINGLE PROTEIN), Homo sapiens
UniProt: P07339
Assay: In Vitro Enzymatic Cathepsin D (CatD) FRET Assay: Recombinant CatD was expressed in CHO cells. The assay buffer for CatD was 0.05 M citrate pH 3.5, 10% DMSO final, 5 mM CHAPS. The CatD enzyme (9 nM) was pre-incubated for one hour with inhibitors, typically in about 1 uL of DMSO according to a serial dilution, is added thereto. The assays was effectively started by the addition of different FRET substrates (20 nM for CatD) and the combination was incubated for one hour. The FRET assay was terminated with by addition of tris buffer, which raises the pH to neutrality, and the fluorescence was determined. The FRET substrate was a peptide with commercially available fluorophore and quencher, on opposite sides of the CatD cleavage site. The CatD substrate peptide sequence was based on sequence #1 of Table 1 from Gulnik et al., FEBS Lett. 413(2):379-384 (1997). Proteolytic cleavage of the FRET substrate released quenching of fluorescence (CatD excitation 500 nm and emission 580 nm).Alternatively, a CatD assay may also be run according to the procedure described in Yasuda et al., J. Biochem. 125(6): 1137-1143 (1999). In addition, the CatD and Cathepsin E assays are described in International Patent Application Publication No. WO2011069934.The in vitro CatD FRET assay data for each of the Examples is provided in Table 2, conducted by the first procedure described above. As shown by the high micromolar CatD data (very poorly active or inactive against CatD), the compounds disclosed herein possess the unexpected property of little to no ability to inhibit the activity of CatD. Thus, with this surprising selectivity profile, the compounds provided herein are believed to minimize, reduce or completely eliminate any risk of retinal atrophy and abnormal development of the eye and of the retinal pigmented epithelium as it relates to the normal function and activity of CatD.
Measurement: IC50 = 91300 nM (pChEMBL 4.039999961853027)
Descriptors: MW 499.49, ALogP 3.83, HBA 7, HBD 1, PSA 103.87, QED 0.64
Compound (SMILES): C[C@H](Nc1nc(C(=O)NC2CC[C@@H](O)C2)c2sccc2n1)c1ccccn1
InChIKey: YJKYOVAWMNRRSS-IAMFDIQRSA-N
Target: Adenosine receptor A3 (SINGLE PROTEIN), Homo sapiens
UniProt: P0DMS8
Assay: Activity Assay: Compounds (Adenosine Receptor modulators) and activity assay.
Measurement: IC50 > 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 383.48, ALogP 2.9, HBA 7, HBD 3, PSA 100.03, QED 0.63
Compound (SMILES): C[C@H](CCc1ccc(OC(=O)N(C)C)c(OC(F)(F)F)c1)N1Cc2cccc(F)c2C1
InChIKey: ZGNPRTXHIWGVPG-CQSZACIVSA-N
Target: Sigma non-opioid intracellular receptor 1 (SINGLE PROTEIN), Cavia porcellus
UniProt: Q60492
Assay: Competitive Radioligand Binding Assay: The affinity of candidate sigma-2 ligand compounds at sigma-1 and sigma-2 receptors was also determined by displacement of different known labeled sigma-2 or sigma-1 ligands. Filtration assays were conducted according the previously published procedure (Xu, et al., 2005). Test compounds were dissolved in N,N-Dimethylformamide (DMF), dimethyl sulfoxide (DMSO) or ethanol and then diluted in 50 mM Tris-HCl pH 7.4 buffer containing 150 mM NaCl and 100 mM EDTA. Membrane homogenates were made from guinea pig brain for sigma-1 binding assay and rat liver for sigma-2 binding assay. Membrane homogenates were diluted with 50 mM Tris-HCl buffer, pH 8.0 and incubated at 25° C. in a total volume of 150 uL in 96 well plates with the radioligand and test compounds with concentrations ranging from 0.1 nM to 10 uM. After incubation was completed, the reactions were terminated by the addition of 150 uL of ice-cold wash buffer (10 mM Tris HCl, 150 mM NaCl, pH 7.4) using a 96 channel transfer pipette (Fisher Scientific, Pittsburgh, Pa.) and the samples harvested and filtered rapidly through 96 well fiber glass filter plate (Millipore, Billerica, Mass.) that had been presoaked with 100 uL of 50 mM Tris-HCl buffer. Each filter was washed four times with 200 uL of ice-cold wash buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.4). A Wallac 1450 MicroBeta liquid scintillation counter (Perkin Elmer, Boston, Mass.) was used to quantitate the bound radioactivity. The sigma-1 receptor binding assays were conducted using guinea pig brain membrane homogenates (300 ug protein) and 5 nM [3H](+)-pentazocine (34.9 Ci/mmol, Perkin Elmer, Boston, Mass.), incubation time was 90 min at room temperature. Nonspecific binding was determined from samples that contained 10 μM of cold haloperidol. The sigma-2 receptor binding assays were conducted using rat liver membrane homogenates (300 ug protein) and 2 nM sigma-2 highly selective radioligand [3H]RHM-1 only (no other blockers) (America Radiolabeled Chemicals Inc. St. Louis, Mo.), 10 nM [3H]DTG (58.1 Ci/mmol, Perkin Elmer, Boston, Mass.) or 10 nM [3H]Haloperidol (America Radiolabeled Chemicals Inc., St. Louis, Mo.) in the presence of 1 uM (+)-pentazocine to block sigma-1 sites, incubation times were 6 minutes for [3H]RHM-1, 120 min for [3H]DTG and [3H]haloperidol at room temperature. Nonspecific binding was determined from samples that contained 10 uM of cold haloperidol.
Measurement: Ki = 520 nM (pChEMBL 6.28000020980835)
Descriptors: MW 440.44, ALogP 5.12, HBA 4, HBD 0, PSA 42.01, QED 0.58
Compound (SMILES): CC(C)n1nc(N2CC(CN[C@H]3C[C@H](Oc4ccc5c(c4)C(=O)N(C4CCC(=O)NC4=O)C5=O)C3)C2)c2cnc(Nc3ccnc(-c4cnn(S(=O)(=O)C5CC5)c4)n3)cc21
InChIKey: OSRDBKXMHYRQBG-QYYYJOFJSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: TR-FRET Assay for ERBB Kinases: All compounds and PROTACs were serially diluted in three-fold increments using 100% DMSO, followed by an intermediate 10-fold dilution using Buffer A (50 mM HEPES, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 1 mM DTT, and 0.1% Pluronic F-68). Two microliters of serially diluted compound or PROTAC were then transferred to black 384-well Proxiplates (PerkinElmer, #6008260) using an Integra Viaflo96. Next, 10 uL of protein kinase in Buffer A was added to each well of the assay plate and pre-incubated with compound for 10 minutes. Kinase reactions were then initiated by addition of 5 uL substrate mix containing 3 mM ATP and 30 uM fluorescein-labeled Poly-GluTyr (Thermo Fisher, #PV3610) in Buffer A and allowed to proceed for 10 minutes at room temperature. Reactions were quenched by addition of a 5 uL mixture containing 5 nM LanthaScreen® Tb-pY20 Antibody (Thermo Fisher, #PV3552) and 40 mM EDTA in Buffer A. Assay plates were then read using a Synergy2 (Biotek Instruments, Winooski, Vt.) via excitation thru a 340/20 nm bandpass filter and emission collected thru 490/10 nm (donor) and 520/25 nm (acceptor) bandpass filters. The final kinase concentrations used for each 15 uL reaction were as follows: 0.2 nM EGFR Exon20NPG (SignalChem, #E10-132GG), 0.1 nM wild type EGFR (BPS Bioscience, #40187), 0.3 nM EGFR L858R/T790M/C797S (BPS Bioscience, #40351), 0.1 nM EGFR L858R (BPS Bioscience, #40189), 0.4 nM L858R/T790M (BPS Bioscience, #40350), InM EGFR Dell9 (SignalChem, #E10-122JG), 10 nM EGFR Dell9 T790M (SignalChem, #E10-122KG), 0.3 nM Her2 (BPS Bioscience, #40230), 15 nM Her2 InsYVMA (SignalChem, #E27-13BG).
Measurement: IC50 = 2.3 nM (pChEMBL 8.640000343322754)
Descriptors: MW 834.92, ALogP 2.79, HBA 17, HBD 3, PSA 228.53, QED 0.14
Compound (SMILES): Cc1cnc(CN(C)C2CCC(c3ccc4[nH]c(-c5cn6ncnc6c(C)c5C)c(C(C)C)c4n3)CC2)cn1
InChIKey: VZZDIQXSJQZFEK-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
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 = 2.5 nM (pChEMBL 8.600000381469727)
Descriptors: MW 522.7, ALogP 6.27, HBA 7, HBD 1, PSA 87.89, QED 0.28
Compound (SMILES): C[C@H](Nc1ccc(S(=O)(=O)Nc2nccs2)cc1Cl)c1ccccc1F
InChIKey: XIUIGABZAWQPAQ-NSHDSACASA-N
Target: Sodium channel protein type 1 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P35498
Assay: Sodium Influx Assay (In Vitro Assay): This sodium influx assay employs the use of the cell permeable, sodium sensitive dye ANG2 to quantify sodium ion influx through sodium channels which are maintained in an open state by use of sodium channel modulators. This high throughput sodium influx assay allows for rapid profiling and characterization of sodium channel blockers.In general, Trex HEK293 cells were stably transfected with an inducible expression vector containing the full-length cDNA coding for the desired human sodium channel α-subunit and with an expression vector containing full length cDNA coding for the β1-subunit. Sodium channel expressing cell lines were induced with tetracycline (1 μg/mL) and plated on 384-well PDL-coated plates at a density of 25K-30K cells/well in culture media (DMEM, containing 10% FBS and 1% L-glutamine). After overnight incubation (37° C., 5% CO2), culture media was removed and cells were loaded with 5 uM ANG2 dye for 1-1.5 h in Buffer 1 (155 mM NMDG, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, adjusted with Tris to pH 7.4). Access dye was removed and cells were incubated with test compounds for 1 hr in buffer 1 containing sodium channel modulator(s) at room temperature. Hamamatsu FDSS μCell was used to perform a 1:1 addition of Na/K challenge buffer (140 mM NaCl, 20 mM HEPES, 1 mM CaCl2, 15 mM KCl, 1 mM MgCl2, 10 mM glucose, adjusted with Tris to pH 7.4) and simultaneously read plates at excitation wavelength of 530 nm and emission wavelength set at 558 nm. Percent inhibition of sodium ion influx was calculated for each test compound at each test concentration to determine the IC50 values.
Measurement: IC50 = 1542 nM (pChEMBL 5.809999942779541)
Descriptors: MW 411.91, ALogP 4.91, HBA 5, HBD 2, PSA 71.09, QED 0.6
Compound (SMILES): Cc1nc2c(F)cc(-c3nc(Nc4ccc5c(n4)CCN(CCN=C(N)N)C5)ncc3F)cc2n1C(C)C
InChIKey: FTFHCSWIOVPGGS-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P11802
Assay: LANCE Ultra ULight Assay: 1. Take 10 mM stock solution of the test compound, in 96-well compound plate, DMSO was used to dilute the compound to an initial concentration of 100x, then this concentration was used as the first concentration, 3-fold diluted to make 10 serial concentrations; 1 μL each serial dilution was then added to 19 μL 1x reaction buffer to prepare 5x compound for use; 2 μL 5x compound was transferred from 96-well plate to 384-well plate; compound-free control well was added with 2 μL the following liquid: 1x reaction buffer with the addition of 1 μL DMSO; 2 μL 250 mM EDTA was added to the Min control well.2.1x reaction buffer was used to formulate the kinase, substrate and ATP into a 2.5xenzyme/substrate mixture and 2.5xATP solution respectively. In the experiment, the final concentration of CDK4/CycD3 kinase is 0.76 ng/μL, the final concentration of ATP is 8 μM; the final concentration of CDK6/CycD3 kinase is 0.5 ng/μL, the final concentration of ATP is 50M; the final concentration of CDK2/CycA2 kinase is 0.86 ng/μL, the final concentration of ATP is 1 μM; the final concentration of CDK2/CycE1 kinase is 1.016 ng/μL, the final concentration of ATP is 20M; 2.5xenzyme/substrate mixture was added to a 384-well plate, incubated at room temperature for 5 minutes; then added with 2.5xATP solution, reacted at room temperature for 30 minutes.3. LANCE Detection Buffer was used, 1xto prepare 2xLANCE Ultra Europium-anti-phospho-eIF4E-binding protein 1 (Thr37/46) for use. After the enzymatic reaction was continued for 30 minutes, 10 mM EDTA was added to 384-well plate and the mixture was reacted at room temperature for 5 minutes. Then LANCE Ultra Europium-anti-phospho-eIF4E-binding protein 1 (Thr37/46) was added, reacted at room temperature for 1 hour.4. The 384-well plate was placed in HERAEUS Multifuge X1R centrifuge, centrifuged at 2000 rpm for 2 minutes; data were measured on EnVision, 337 nM wavelength laser was used as the excitation light, test at RFU665 nM and RFU615 nM, and RFU665 nM/RFU615 nM x10000 was used as the final data for analysis.
Measurement: IC50 = 0.99 nM (pChEMBL 9)
Descriptors: MW 520.59, ALogP 3.43, HBA 8, HBD 3, PSA 136.16, QED 0.25
Compound (SMILES): C=CC(=O)Nc1cnn(C[C@H]2CCCO2)c1-c1cc2c(cn1)CN(c1c(F)c(OC)cc(OC)c1F)C(=O)C21CC1
InChIKey: WTLHRQQAZRXBIR-QGZVFWFLSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Enzymatic Assay: The inhibitor potency of the exemplified compounds was measured in an enzyme assay that measures peptide phosphorylation using FRET measurements to detect product formation. Inhibitors were serially diluted in DMSO and a volume of 0.5 μL was transferred to the wells of a 384-well plate. For FGFR3, a 10 μL volume of FGFR3 enzyme (Millipore) diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 5 mM DTT, pH 7.5) was added to the plate and pre-incubated for a time between 5-10 minutes and up to 4 hours. Appropriate controls (enzyme blank and enzyme with no inhibitor) were included on the plate. The assay was initiated by the addition of a 10 μL solution containing biotinylated EQEDEPEGDYFEWLE peptide substrate (SEQ ID NO: 1) and ATP (final concentrations of 500 nM and 140 μM respectively) in assay buffer to the wells. The plate was incubated at 25° C. for 1 hr. The reactions were ended with the addition of 10 μL/well of quench solution (50 mM Tris, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.8; 30 mM EDTA with Perkin Elmer Lance Reagents at 3.75 nM Eu-antibody PY20 and 180 nM APC-Streptavidin). The plate was allowed to equilibrate for 1 hr before scanning the wells on a PheraStar plate reader (BMG Labtech).FGFR1, FGFR2, and FGFR4 are measured under equivalent conditions with the following changes in enzyme and ATP concentrations: FGFR1, 0.02 nM and 210 uM respectively, FGFR2, 0.01 nM and 100 uM, respectively, and FGFR4, 0.04 nM and 600 uM respectively. The enzymes were purchased from Millipore or Invitrogen.
Measurement: IC50 > 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 565.58, ALogP 4.12, HBA 8, HBD 1, PSA 107.81, QED 0.41
Compound (SMILES): CCC(CC)n1cc(-c2nc(-c3cnn(C4CN(C(=O)CN5CCCC5)C4)c3)cn3nccc23)cn1
InChIKey: PYPRSGPYVFWABX-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK3 Inhibition Assay: Compounds of Formula I were screened for their ability to inhibit JAK3 using the general enzyme inhibition assay method, in which the assay mixture contained 1 mM ATP, 10 μM Omnia® Y7 peptide (Catalog # IVGN KNZ3071C, Invitrogen Corporation, Carlsbad, Calif.) and 2 nM JAK3 in a total volume of 25 μL. JAK3 was purchased from Invitrogen Corporation, Carlsbad, Calif. (catalog # IVGN PV4080).
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 487.61, ALogP 3.3, HBA 9, HBD 0, PSA 89.38, QED 0.38
Compound (SMILES): O=c1c2cnc(C3CCOCC3)n2cc(Cc2ccccc2)n1Cc1cccc(F)c1
InChIKey: NTXQTICQWIKNOO-UHFFFAOYSA-N
Target: Dual specificity calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: P54750
Assay: PDE1 Inhibition Assay: PDE1A, PDE1B and PDE1C assays were performed as follows: the assays was performed in 60 μL samples containing a fixed amount of the PDE1 enzyme (sufficient to convert 20-25% of the cyclic nucleotide substrate), a buffer (50 mM HEPES pH 7.6; 10 mM MgCl2; 0.02% Tween20), 0.1 mg/ml BSA, 15 nM tritium labelled cAMP and varying amounts of inhibitors. Reactions were initiated by addition of the cyclic nucleotide substrate, and reactions were allowed to proceed for 1 h at room temperature before being terminated through mixing with 20 μL (0.2 mg) yttrium silicate SPA beads (PerkinElmer). The beads were allowed to settle for 1 h in the dark before the plates were counted in a Wallac 1450 Microbeta counter. The measured signals were converted to activity relative to an uninhibited control (100%) and IC50 values were calculated using XIFit (model 205, IDBS).
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 417.48, ALogP 4.17, HBA 5, HBD 0, PSA 48.53, QED 0.49
Compound (SMILES): COc1cc(F)cc2c1nc(N)n1nc(C3CC(C)CN(c4cn(C(C)(C)CO)nc4C)C3)nc21
InChIKey: LFWFKHQPUWTRMT-UHFFFAOYSA-N
Target: Adenosine receptor A2b (SINGLE PROTEIN), Homo sapiens
UniProt: P29275
Assay: Measurement of A2A and A2B Antagonism in cAMP Cell-Based Assay : The ability of compounds to antagonize human A2A and A2B adenosine receptors was determined using a kit to measure changes in intracellular cyclic AMP levels (LANCE cAMP 384 Kit, Perkin Elmer, Cat. No. AD0264). HEK293 cells recombinantly expressing either human A2A or A2B receptors, previously frozen in Recovery Medium (Life Technologies, Cat. No. 12648-010) were thawed and diluted into stimulation buffer (HBSS (Hyclone SH 30268.01), 5 mM HEPES (Gibco 15630-106), 200 nM rolipram (Tocris, Cat. No. 0905), and 1.5% (V/v) BSA stabilizer (kit component). The cell suspension was centrifuged at 200×g for 10 min and then resuspended in stimulation buffer, supplemented with a 1:10 000 dilution of Alexa Fluor 647 anti-cAMP antibody, to a density of 6.0×105 cells/mL. A Labcyte Echo 550 acoustic dispenser was used to transfer up to 25 nL of test compound dissolved in DMSO into the wells of a dry Optiplate-384 plate (Perkin Elmer, Cat. No. 6008289). All subsequent liquid additions were performed using a multichannel pipettor. Next, 5 μL of the cell suspension was added to the wells of the Optiplate-384 and incubated for 30 min. at 37° C. and 5% CO2 in a humidified environment. After this time 5 μL of either 300 nM or 600 nM adenosine (Sigma Cat. No. A9251) for A2A and A2B respectively was added and incubated for 30 minutes at 37° C. and 5% CO2 in a humidified environment. At this time detection mix was prepared by combining the LANCE Eu-W8044 labeled streptavidin and Biotin-cAMP in detection buffer according to the manufacturers protocol. 10 μL of the detection mix was added to each well of the Optiplate-384 which was covered with a plate seal and incubated under ambient conditions for 2 hours prior to reading the plate using an Envision (Perkin Elmer, Waltham, Mass.) multimode plate reader. Data was normalized by defining minimal effect as stimulation in the presence of 0.25% (v/v) DMSO and maximal effect as stimulation in the presence of 1 μM ZM241385 (Cayman, Cat. No. 1036). Curve fitting of the percent effect data versus the log of compound concentration used a 4-parameter concentration response curve fitting algorithm to calculate IC50 values.
Measurement: IC50 = 10.2 nM (pChEMBL 7.989999771118164)
Descriptors: MW 482.56, ALogP 2.87, HBA 10, HBD 2, PSA 119.62, QED 0.45
Compound (SMILES): CC(CC1(CCN)NC(=O)NC1=O)C(=O)N1CCN(c2cc(Cl)cc(Cl)c2)CC1
InChIKey: XKGHZMJCKNILID-UHFFFAOYSA-N
Target: Collagenase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P45452
Assay: In Vitro Assay: MMP-13:The basis for the assay is the cleavage of the substrate 390 MMP FRET Substrate I (Anaspec Cat # AS-27076) by human MMP13 (Chemicon, Cat #CC068).For the dose response (10 point), 4 μL of a dilution series of compound (20 μM highest concentration, 1/5 dilution in water), is transferred to 384 well Fluotrac 200 plate (Greiner, cat #781076) and incubated at room temperature for 30 min with a 26 μL buffer solution (50 mM Tris pH7.5, 150 mM NaCl, 10 mM CaCl2, 0.05% CHAPS, 5 μM ZnCl2) containing MMP13 (0.01 ng/IL) (it will be appreciated by the skilled person that the potency read out is independent of the enzyme concentration). Human MMP13 is preactivated by incubated the enzyme in the same buffer complemented with 1 mM freshly prepared p-Aminophenylmercuric acetate (AMPA) for 1 hour at 37° C.The reaction is initiated by adding to the assay plate 390 MMP FRET Substrate I (10 μL, 2.5 μM) in the same buffer.Finally, the fluorescence is read on the Envision (Perkin Elmer) after an incubation of 45 min at room temperature (Excitation 485 nm, Emission 530).
Measurement: IC50 > 14700 nM (pChEMBL 4.833000183105469)
Descriptors: MW 442.35, ALogP 1.6, HBA 5, HBD 3, PSA 107.77, QED 0.58
Compound (SMILES): Cn1cnc(-c2cc(Cl)c(F)c(C(=O)NNS(=O)(=O)C[C@@H]3CCC[C@@H](NC(=O)OC(C)(C)C)C3)c2)c1
InChIKey: WGFOUPFCONPGEL-GDBMZVCRSA-N
Target: Histone acetyltransferase KAT8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H7Z6
Assay: KAT-8 Enzyme Assay: Enzyme assay buffer was 50 mM Tris pH 8.0, 0.002% Tween20, 0.005% bovine skin gelatin, and 1 mM dithiothreitol (DTT). For determination of IC50values, compounds were serially diluted with 2% (v/v) DMSO in the final reaction, pre-incubating each dilution of each compound with 40 μL of assay buffer containing KAT-8 enzyme (12.5 nM final concentration). 10 μL of assay buffer containing 1 μM peptide substrate and 5 μM acetyl coenzyme A (final concentrations) was added. Reactions (50 μL total) were then carried out at 25° C. for 90 minutes. Reactions were terminated by the addition of 0.5% formic acid (final concentration), and a sample of each reaction was analyzed by SAMDI Tech, Inc. (Chicago, Ill.) using self-assembled monolayer desorption/ionization time-of-flight mass spectrometry (Mrksich, M. (2008) Mass spectrometry of self-assembled monolayers: a new tool for molecular surface science. ACS Nano 2, 7-18).
Measurement: IC50 = 30000 nM (pChEMBL 4.519999980926514)
Descriptors: MW 544.05, ALogP 3.53, HBA 7, HBD 3, PSA 131.42, QED 0.46
Compound (SMILES): Nc1nc2cc(-c3ccc4ncc(-c5cn[nH]c5)nc4c3)ccc2o1
InChIKey: ADBUPFTVWSNTHI-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Inhibition Assay of PI3K: An exemplary system is PI 3-Kinase (human) HTRF Assay from Upstate. The assay can be carried out according to the procedures suggested by the manufacturer. Briefly, the assay is a time resolved FRET assay that indirectly measures PIP3 product formed by the activity of a PI3-K. The kinase reaction is performed in a microtitre plate (e.g., a 384 well microtitre plate). The total reaction volume is approximately 20 ul per well. In the first step, each well receives 2 ul of test compound in 20% dimethylsulphoxide resulting in a 2% DMSO final concentration. Next, approximately 14.5 ul of a kinase/PIP2 mixture (diluted in 1× reaction buffer) is added per well for a final concentration of 0.25-0.3 ug/ml kinase and 10 μM PIP2. The plate is sealed and incubated for 15 minutes at room temperature. To start the reaction, 3.5 ul of ATP (diluted in 1× reaction buffer) is added per well for a final concentration of 10 μM ATP. The plate is sealed and incubated for 1 hour at room temperature. The reaction is stopped by adding 5 ul of Stop Solution per well and then 5 ul of Detection Mix is added per well. The plate is sealed, incubated for 1 hour at room temperature, and then read on an appropriate plate reader.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 328.33, ALogP 3.41, HBA 6, HBD 2, PSA 106.51, QED 0.51
Compound (SMILES): Cc1nc(C(=O)NC(C)(c2ccc(S(F)(F)(F)(F)F)cc2)C(C)C)cc(=O)[nH]1
InChIKey: GZEJHSDMMANXQL-UHFFFAOYSA-N
Target: GMP-stimulated 3',5'-cyclic nucleotide phosphodiesterase (SINGLE PROTEIN), Macaca mulatta
UniProt: B3VTJ2
Assay: Fluorescence Polarization (FP) Assay: In a typical experiment the PDE2 inhibitory activity of the compounds of the present invention was determined in accordance with the following experimental method. Rhesus PDE2A3 was amplified from rhesus macaque brain cDNA (Biochain Institute, Hayward, Calif.) using primers based on human PDE2A sequence (accession NM_002599.3) where the forward primer containing a Kozak consensus was 5′-gccaccatggggcaggcatgtggc-3′ and the reverse primer was 5′-tcactcagcatcaaggctgca-3′. Amplification with Easy-A High-Fidelity PCR cloning enzyme (Stratagene, La Jolla, Calif.) was 95° C. for 2 minutes followed by thirty three cycles of 95° C. for 40 seconds, 52° C. for 30 seconds, and 72° C. for 2 minutes 48 seconds. Final extension was 72° C. for 7 minutes. The PCR product was TA cloned into pcDNA3.3-TOPO (Invitrogen, Carlsbad, Calif.) according to standard protocol. A consensus sequence was developed from multiple clones and then deposited into GenBank (EU812167). AD293 cells (Stratagene, La Jolla, Calif.) with 70-80% confluency were transiently transfected with rhesus PDE2A3/pcDNA3.3-TOPO using Lipofectamine 2000 according to manufacturer specifications (Invitrogen, Carlsbad, Calif.).
Measurement: Ki = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 425.42, ALogP 5.04, HBA 3, HBD 2, PSA 74.85, QED 0.66
Compound (SMILES): COC(=O)c1ccc2c(c1)NC(=O)/C2=C(\Nc1ccc(C(=O)NOCCN2CCC(C#N)CC2)cc1)c1ccccc1
InChIKey: OFLUDTFEINFEBP-ZIADKAODSA-N
Target: Platelet-derived growth factor receptor beta (SINGLE PROTEIN), Homo sapiens
UniProt: P09619
Assay: PDGFRbeta Enzyme Inhibition: The inhibitory activities of compounds of the invention against PDGFRβ (PDGFRβ Kinase Enzyme System: Promega), were evaluated by mixing the PDGFRβ protein (6.25 ng/mL, 2 μL), substrate (Poly (4:1 Glu4, Tyr1), 100 ng/mL, 2 μL) with the test compound (2 μL at either 3 μM, 0.67 μM, 0.15 μM, 0.033 μM, 0.0073 μM, 0.0016 μM, 0.0036 μM or 0.00008 μM) for 90 min at 25° C. The kinase reaction was initiated by adding ATP (25 μM, 2 μL) and the mixture was incubated for 1 hr at 25° C. ADP-Glo reagent was added for 40 min (8 μL) then development reagent (16 μL) was added for 40 min prior to detection in a microplate reader (EnVision® Multilabel Reader, Perkin Elmer).
Measurement: Ki = 32.4 nM (pChEMBL 7.489999771118164)
Descriptors: MW 565.63, ALogP 4.3, HBA 8, HBD 3, PSA 132.79, QED 0.15
Compound (SMILES): CC1(C)Cc2cc(Nc3nn([C@]4(CC#N)CCCOC4)c4cc[nH]c(=O)c34)ccc2C1(O)C(F)(F)F
InChIKey: OTBGXODPXJHIIB-UXMRNZNESA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biochemical HTRF Assay : The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer #PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at room temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading.
Measurement: IC50 = 0.12 nM (pChEMBL 9.920000076293945)
Descriptors: MW 501.51, ALogP 4.22, HBA 7, HBD 3, PSA 115.96, QED 0.49