lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCc1cc(S(=O)(=O)C(F)(F)F)ccc1N(c1ccnc(N)n1)C(C)C
InChIKey: UQFGTZNEFMWCNU-UHFFFAOYSA-N
Target: Histamine H4 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H3N8
Assay: Binding Assay on Recombinant Human Histamine H4 Receptor: Receptor binding was assessed using isolated plasma membranes from SKNMC neuroblastoma cell lines stably expressing recombinant human H4 receptor. To isolate plasma membrane enriched in recombinant human H4 receptor, cells were collected from 100 culture dishes (150 mm) in PBS-EDTA. The cells were pellet by centrifugation (800 g) and resuspended in 5×TE buffer (50 mM Tris/5 mM EDTA). The cells were then homogenized and centrifuged at 1000 g for 10 min. The supernatant was collected and centrifuged for an additional 26000 g for 30 min. The supernatant was removed and the pellet was gently resuspended in 30 ml of 5×TE buffer. Protein concentration was then determined using Bradford protein assay (Bradford, M. M., Anal. Biochem., 1976, 72: 248-254). Isolated cell membranes were incubated with 2×KD (10 nM), [3H] histamine (Specific activity: 23 Ci/mmol), with or without test compounds for 45 min at 25° C. Nonspecific binding was defined with 100 μM cold histamine. Ki values were calculated based on an experimentally determined appropriate KD values according to Cheng and Prusoff (Biochem. Pharmacol. 1973, 22(23):3099-3108). Membranes were harvested by rapid filtration using the 96 well Brandel system or a cell harvester using a Whatman GF/C filter or filter plates treated with 0.5% polyethylenimine (PEI), and washed 4 times with ice-cold 50 mM Tris/5 mM EDTA buffer. Filters were then dried, mixed with scintillant and radioactive counts were determined.
Measurement: Ki = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 388.42, ALogP 3.46, HBA 6, HBD 1, PSA 89.18, QED 0.84
Compound (SMILES): O=C(Nc1ccc(Oc2cc(-c3ccncc3)cn3nccc23)c(F)c1)c1cccn(-c2ccc(F)cc2)c1=O
InChIKey: GFODQIAPQKSLPF-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: AXL Kinase Assay: The ability of compounds to inhibit the kinase activity of recombinant human baculovirus-expressed AXL was measured by homogeneous TRF (HTRF) using Cisbio's KinEASE assay system in white 384-well Optiplates. Assay buffer contained 1 mM DTT, 2 mM MnCl2, 2% DMSO, 50 nM supplement enzymatic buffer, and 1× enzymatic buffer. A 2× concentration of tyrosine kinase (TK) substrate-biotin/ATP mixture made in assay buffer was added to plates at 10 μL/well using the Multidrop Combi (Thermo Fisher Scientific, Waltham, Mass.). The final concentrations were 0.3 μM TK substrate-biotin, and 1.3 μM ATP. Compounds (100 nL), diluted in 100% DMSO on the Biomek FX, (Beckman Coulter, Inc., Brea, Calif.), were transferred to the assay plates using the Biomek FX pintool (2.5% final DMSO in assay). A 2× concentration (final=12 ng/mL) of GST-AXL (diluted in assay buffer) was added to plates at 10 uL/well using the Multidrop Combi. Plates were sealed, briefly shaken and incubated at 25° C. for 30 minutes. A 4× stock of Streptavidin-XL665 (final=18.8 nM) and a 1:100 diluted stock of TK antibody-cryptate were made in HTRF detection buffer and mixed together just prior to adding 20 μL/well on the Multidrop Combi. Plates were sealed, briefly shaken and incubated at 25° C. for 1 hour. The fluorescence of the resulting solution was measured using the PerkinElmer EnVision 2102 multi-label plate reader (PerkinElmer, Waltham, Mass.) with an excitation wavelength of 337 nm (laser) and emission wavelengths of 590 and 665 nm. Raw data was expressed as the ratio of 665/590×10,000.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 535.51, ALogP 5.87, HBA 7, HBD 1, PSA 90.52, QED 0.29
Compound (SMILES): O=P(O)(O)[C@H](Cc1ccccc1)S(=O)(=O)C[C@H]1O[C@@H](n2ncc3c(NC4CCCC4)nc(Cl)nc32)[C@H](O)[C@@H]1O
InChIKey: TXGGTRQBPNWULT-ZPXGPAEQSA-N
Target: 5'-nucleotidase (SINGLE PROTEIN), Homo sapiens
UniProt: P21589
Assay: Biochemical Assay: Assay Reaction ConditionsAssay Volume: 70 μlReaction Volume: 50 μlCD73: 0.3208 nMAMP: 15 μMAssay Buffer: 25 mM Tris-HCL, pH 7.4, 0.01% Brij-35, 0.01% BSA, 5 mM MgCl2Assay Procedure:Used 384 clear plate.Made dose titration of testing compounds in assay buffer, 10 points ½ log titrations in duplicates starting at 100 μM.Added 25 μl of CD73 to each well for a final concentration of 320 pM.Incubated at RT for 15 min.Added 25 μl of AMP to each well for a final concentration of 15 μM.Incubated at RT for 10 min.Added 10 μl of Malachite Green Reagent A, incubate at RT for 10 min.Added 10 of Malachite Green Reagent B, incubate at RT for 45 min.Read the Absorbance on Envision plate reader using excitation filter: Cy5 620 nM.The ability of the compounds disclosed herein to inhibit CD73 activity was quantified and the respective IC50 values were determined.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 602.01, ALogP 1.61, HBA 11, HBD 5, PSA 196.99, QED 0.18
Compound (SMILES): Cc1cc(-c2cnn(C)c2C(=O)Nc2cc3nc(-c4ccccc4)nn3cc2F)nc(C)n1
InChIKey: IHDXIKGBWVFMLB-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Phosphodiersterase Evaluation Assay: IMAP TR-FRET Phosphodiersterase Evaluation Assay Kit (Molecular Device) was used for the measurement. Ten μL of a diluted test compound having each concentration and 5 μL of the enzyme PDE10 (BPA Bioscience) that had been diluted to 2 ng/mL with 1×IMAP Reaction Buffer (prepared with 5×included with the kit, 10 mM Tris-HCl, pH=7.2, 10 mM MgCl2, 0.05% NaN3, and 0.1% BSA) containing 0.1% BSA were added to a 384-well plate (Corning). The obtained mixture was then pre-incubated at room temperature for 5 minutes. Thereafter, 5 μL of cAMP substrate solution which was included with the kit and diluted to 400 nM with 1×IMAP Reaction Buffer containing 0.1% BSA, was added to the reaction solution, and the obtained mixture was reacted at room temperature for 60 minutes. Thereafter, 60 μL of an IMAP TR-FRET Binding solution included with the kit was further added to the reaction solution, and the obtained mixture was then left for 3 hours or more. Subsequently, using ARVO SX (PerkinElmer), the fluorescent intensity of Terbium (Emission=490 nm) and TR-FRET (Emission=520 nm) in the reaction solution were measured at an excitation wavelength of 340 nm, so as to calculate the amount of the generated 5′-AMP. The count of a well to which a solvent had been added instead of the test compound was set at 0%, and the count of a well to which the enzyme PDE10 had not been added was set at 100%. Thus, the inhibitory activity of each test compound was calculated.
Measurement: IC50 < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 442.46, ALogP 3.6, HBA 8, HBD 1, PSA 102.89, QED 0.46
Compound (SMILES): FC(F)(F)c1cc(Oc2cccs2)nc(N2CCC3(CC2)CNCCO3)n1
InChIKey: BDPACDIUGGWICK-UHFFFAOYSA-N
Target: Sodium-dependent dopamine transporter (SINGLE PROTEIN), Homo sapiens
UniProt: Q01959
Assay: Scintillation Proximity Assay : The affinity of the compounds of the invention to the human DAT or NET or SERT transporters is assessed by using the [3H]WIN-35,428 or [3H]nisoxetine or [3H]citalopram binding assays in recombinant human DAT, NET and SERT membranes with the SPA technology. The final assay volume is 50 μL in 384 well plates.Briefly, 0.5 μL of test compound in neat DMSO or 0.5 μL of DMSO for total binding (TB) or 0.5 μL of indatraline 1 mM (10 μM final concentration) for non specific binding (NSB) are added to the assay plate. 50 μL of the SPA mixture is added to each well, containing: 30 μg/mL or 10 μg/mL or 25 μg/mL DAT, NET, SERT membranes, respectively; 5 nM [3H]WIN-35,428 or 5 nM [3H]nisoxetine or 1 nM [3H]citalopram, for DAT, NET, SERT assay, respectively; 2.5 mg/mL or 1 mg/mL or 4 mg/mL WGA-PVT SPA beads (PerkinElmer RPNQ0001, for DAT, NET, SERT assay, respectively. All components are added to Assay Buffer (20 mM HEPES pH 7.4, 145 mM NaCl, 5 mM KCl, 0.01% Pluronic F-127). 0.02% BSA was used for DAT binding only. Plates are sealed with Topseal A and centrifuged 1 min, 800 rpm. Plates are loaded into a 1450 Microbeta TriLux (Perkin-Elmer) plate reader and the radioactivity counted after at least 4 hrs or overnight incubation at room temperature. Curve fitting and IC50 estimations are performed using a four parameter model in XLfit (IDBS, Guilford, UK) for Microdoft Excel (Microsoft, Redmond, Wash.).
Measurement: IC50 = 45.7 nM (pChEMBL 7.340000152587891)
Descriptors: MW 400.43, ALogP 3.31, HBA 7, HBD 1, PSA 59.51, QED 0.85
Compound (SMILES): CCn1cc(S(=O)(=O)N2C[C@H](CNC(=O)C(C)(C)O)Oc3ccc(NC(=O)OC(C)(C)C(F)(F)F)cc32)c(OC(F)F)n1
InChIKey: CITCLBWGQPKYHW-AWEZNQCLSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Biochemical TR-FRET assay: The compounds of the invention inhibit RORgammaT activity. Activation of RORgammaT activity can be measured using, e.g., a biochemical TR-FRET assay. In such an assay, interaction of cofactor-derived peptides with human RORgammaT-Ligand Binding Domain (LBD) can be measured. The TR-FRET technique is a sensitive biochemical proximity assay that will give information concerning the interaction of a ligand with the LBD, in the presence of cofactor-derived peptides (Zhou et al., Methods 25:54-61, 2001).To identify novel antagonists of RORgammaT, an assay was developed which employs the interaction of RORgammaT with its co-activator peptide SRC1_2. This peptide mimics the recruitment of co-activators to RORgammaT through its interaction with the LXXLL (SEQ ID NO:1) (e.g., NR box) motifs (Xie et al., J. Immunol. 175: 3800-09, 2005; Kurebayashi et al., Biochem. Biophys. Res. Commun. 315: 919-27, 2004; Jin et al., Mol. Endocrinology 24:923-29, 2010). The RORγ-Ligand Binding Domain TR-FRET Assay was run according to the following protocol.HIS-tagged RORγ-LBD protein was recombinantly expressed in Escherichia coli. The RORγ-LBD protein was purified by Ni2+-affinity resin. Purified protein was then diluted in assay buffer (50 mM Tris pH 7.0, 50 mM KCl, 1 mM EDTA, 0.1 mM DTT, 100 μg/mL bovine serum albumin, delipidated) to obtain a RORγ-LBD final concentration of 3 nM. Europium tagged anti-HIS antibody was also added to this solution (1.25 nM). Separately, SF9 cells not expressing any recombinant protein were lysed (32,000 cells per μl in 25 mM Tris, 50 mM NaCl) and the previously frozen lysate was added to the diluted RORγ-LBD solution at a ratio of 0.75 μl SF9 lysate per 15 μl of diluted RORγ-LBD.
Measurement: IC50 = 5.84 nM (pChEMBL 8.229999542236328)
Descriptors: MW 643.59, ALogP 3.24, HBA 10, HBD 3, PSA 161.32, QED 0.33
Compound (SMILES): O=C(Nc1cc2c(cc1F)CN(c1cccc(-c3nncn3C3CC3)n1)C2=O)C1CC1
InChIKey: HHLBCYRTUQEKMV-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99683
Assay: ASK1 Kinase Assay: The ASK1 enzymatic assay was run following Promega ASK1 Kinase Enzyme System (Cat #V3881). The kit provides the protocol, enzymes and all reagents necessary to run an assay. Firstly, the compounds, enzyme, substrate and ATP were diluted in provided assay buffer. The final concentration of the enzyme was 50 nM, substrate (Myelin basic protein) 1 μg/ml and ATP 10 μM. The compound and the enzyme were pre-incubated in a 384 well white solid bottom plate (Greiner, Cat#784075) for 10 minutes. After incubation, the substrate and ATP were added and incubated for further 60 minutes. After 60 minutes, ADP-Glo™ was added and plate was incubated for another 40 minutes. After 40 minutes, Kinase Detection Reagent was added and the plate was incubated for 45 minutes. After 45 minutes, plate was read on Perkin Elmer EnVision using luminescence read (0.5 seconds/well).
Measurement: IC50 = 600 nM (pChEMBL 6.21999979019165)
Descriptors: MW 418.43, ALogP 3.32, HBA 6, HBD 1, PSA 93.01, QED 0.69
Compound (SMILES): C=CC(=O)N1CC(Nc2cncc(-c3cnc(Oc4cc(F)cc(F)c4)nc3)n2)C1
InChIKey: PZZLNYBCLHIHEG-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: BTK and EGFR in Vitro Assay: The BTK and EGFR biochemical assays utilize LANTHASCREEN® Eu Kinase Binding Assays from Thermo Fisher Scientific, measuring the binding of the kinase with a fluorinated tracer. The assay buffer consists of 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgCl2, 1 mM EGTA, and 1 mM DTT. The test compounds are diluted and added to the assay plate with a Labcyte ECHO® 555 liquid handler. The compounds are tested in 10 point dose response curves (100 μM-0.005 μM) with a maximum DMSO concentration of 1%. The assays are performed in 20 μL in low-volume 384-well white proxiplates. For the BTK assay, the concentration of full-length His-labeled BTK in the assay is 5 nM, the Eu-anti-His antibody is 2 nM, and the kinase tracer 236 is 60 nM. For the EGFR assay, the concentration of truncated (amino acids 668-1210) GST-labeled EGFR is 5 nM, the Eu-anti-GST antibody is 2 nM, and the kinase tracer 199 is 10 nM. The components of the assay (compound, enzyme/antibody, tracer) are assembled and incubated for 30 min prior to being read on a Perkin-Elmer EnVision with excitation at 340 nM, tracer emission at 665 nM, and antibody emission at 615 nM. The signal ratio is converted to percent inhibition using the following equation: % Inhibition=100−[(Test Compound−Median Minimum)/(Median Maximum−Median Minimum)×100].
Measurement: IC50 = 43.2 nM (pChEMBL 7.369999885559082)
Descriptors: MW 410.38, ALogP 2.81, HBA 7, HBD 1, PSA 93.13, QED 0.62
Compound (SMILES): Cc1ccc(CO)cc1NC1=NC(=O)NC2=NCC(c3cc(F)c(F)c(F)c3)N21
InChIKey: KYZIORCXNZGKMF-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Inhibition Assay: Evaluation of a human P2X7 receptor inhibitory activity Stably expressing cell line (1321N1 cell transfected with the human P2X7 receptor gene (GenBank accession number NM_002562.5 including T606C and G952A SNP)) was used. The cells were seeded in a 384-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (10% fetal bovine serum, 25 mM HEPES, 1% penicillin and streptomycin in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. After replacing with 20 μL of the HBSS buffer (20 mM HEPES, 55.6 mM D-glucose, 1× HBSS(−), pH7.4-7.5), 15 μL of 17.3 μM Yo-Pro solution in the HBSS buffer was added. The plate was placed in high-throughput cellular screening system FLIPR TETRA (Molecullar Devices, LLC.) and 15 μL of 130 μM BzATP solution in the HBSS buffer was added. Measurement of fluorescence intensity by FLIPR TETRA was started. After eight minutes, 15 μL of DMSO solutions containing different concentrations of the compound of the present invention as prepared by dilution with the HBSS buffer were dispensed to each well through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 20 minutes. The maximum fluorescence intensity without the compound of the present invention is calculated as 0% inhibition and the maximum fluorescence intensity when the reference compound was added is calculated as 100% inhibition.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 403.36, ALogP 2.81, HBA 5, HBD 3, PSA 89.32, QED 0.69
Compound (SMILES): NC(=O)c1cccc2[nH]c(N3CCN(c4cc(C(F)(F)F)ncn4)CC3)nc12
InChIKey: JOHQVCQUVDBUAX-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: HT Universal Chemiluminescent PARP Assay Kit: 1. 50 μl of 1×PARP buffer per well was added to infiltrate the histone, and the plate was incubated for 30 min at room temperature. Then the 1×PARP buffer in each well was aspirated, and the remaining liquid was tapped dry on paper towels.2. The diluted 5× solutions of Compounds (1) to (37) were added to respective wells (10 μl per well). The positive and negative control wells contained the 1×PARP buffer (containing 5% DMSO).3. The PARP enzyme was diluted in the 1×PARP buffer to give a concentration of 0.5 Unit per 15 μl, and then 15 μl of the enzyme solution was added to each well except that the negative control well was added exclusively with the 1×PARP buffer. The plate was incubated for 10 min at room temperature.4. 25 μl of the 1×PARP Cocktail was sequentially added to each well.5. The plate was incubated for 60 min at 27° C.6. After incubation, the reaction solution was aspirated from the wells, and the remaining liquid was tapped dry on paper towels. Then, the plate was washed 4 times with 0.1% Triton X-100 in PBS (200 μl per well per wash), and the remaining liquid was tapped dry on paper towels.7. Subsequently, the diluted 1× Strep-HRP solution was added to each well, and then the plate was incubated for 60 min at 27° C.8. After incubation, the reaction solution was aspirated from the wells, and the remaining liquid was tapped dry on paper towels. Then, the plate was washed 4 times with 0.1% Triton X-100 in PBS (200 μl per well per wash), and the remaining liquid was tapped dry on paper towels.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 391.36, ALogP 1.8, HBA 6, HBD 2, PSA 104.03, QED 0.71
Compound (SMILES): COc1ccc(-c2[nH]c(C3CCN(c4ncnc5[nH]c(-c6ccnc(OC)c6)cc45)CC3)nc2C(F)(F)F)cc1
InChIKey: KPCFDFASIXVVJW-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Biochemical Assay: Assays for biochemical cell-based activity of c-kit kinase are known in the art, for example, as described in U.S. Pat. Nos. 7,498,342 and 7,846,941, the disclosures of which are hereby incorporated by reference as it relates to such assays. The c-kit (or kinase domain thereof) is an active kinase in AlphaScreen. IC50 values are determined with respect to inhibition of c-Kit kinase activity, where inhibition of phosphorylation of a peptide substrate is measured as a function of compound concentration. Compounds to be tested were dissolved in DMSO to a concentration of 30 mM. These were diluted 30 μl into 130 μl of DMSO (4 mM) and 1 μl was added to an assay plate. These were then serially diluted 1:3 (50 μl to 100 μl DMSO) for a total of 8 points. Plates were prepared such that each kinase reaction is 30 μl in 1× kinase buffer (50 mM HEPES, pH 7.2, 5 mM MgCl2, 5 mM MnCl2, 0.01% NP-40, 0.2% BSA), 5% DMSO and 10 μM ATP. Substrate was 100 nM biotin-(E4Y)3 (Open Source Biotech, Inc.). C-kit kinase was at 0.1 ng per sample. After incubation of the kinase reaction for 1 hour at room temperature, 5 μl of donor beads (Streptavidin coated beads (Perkin Elmer Life Science) final concentration 1 μg/ml) in stop buffer (50 mM EDTA in 1× kinase buffer) was added, the sample was mixed and incubated for 30 minutes at room temperature before adding 5 ml of acceptor beads (PY30 coated beads (Perkin Elmer Life Science) final concentration 1 μg/ml) in stop buffer. The samples were incubated for 60 minutes at room temperature and the signal per well was read on AlphaQuest reader. Phosphorylated substrate results in binding of the PY30 antibody and association of the donor and acceptor beads such that signal correlates with kinase activity.
Measurement: Ki = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 549.56, ALogP 5.83, HBA 7, HBD 2, PSA 104.84, QED 0.27
Compound (SMILES): COc1cc(C(=O)N2CC3CCC2[C@@H]3N)cc2nc(-c3cc4ccc(Nc5cc(C)ncc5C)cc4n3CC3CC3)n(C)c12
InChIKey: JZHOCJAOSJYLMW-BYICHJTDSA-N
Target: Protein-arginine deiminase type-4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM07
Assay: Rapid-Fire Mass Spectrum (RFMS) Assay: Compounds were solubilized in 100% DMSO to achieve a 10 mM compound concentration. Compound stock solutions were stored at RT. A series of dilutions were prepared in DMSO and mixed 8 times with 20 μL mixing volume. Final top concentration of compound in the assay is 50 μM. Final assay conditions were as follows:Reaction volume: 26 μlAssay buffer: 25 mM hepes, pH 7.5, 5 mM NaCl, 1 mM DTT, 0.2 mg/ml BSA, 0.01% CHAPS, 50 μM Calcium, and 5 μM TPENFinal concentrations: 5 nM hPAD4 enzyme, 250 μM BAEE, and 0.5% DMSOTotal incubation time: 30 mins compound and enzyme preincubation at 37° C., 90 min enzyme/substrate reaction, 30 min reaction with phenyl glyoxal at 37° C.Stop solution: 40 μl 5% TCA in ACN
Measurement: IC50 = 120 nM (pChEMBL 6.920000076293945)
Descriptors: MW 589.74, ALogP 5.93, HBA 8, HBD 2, PSA 103.23, QED 0.24
Compound (SMILES): Cc1ccc(-c2noc(C)c2COc2cnc(C(=O)N3CC4(C3)CS(=O)(=O)C4)cn2)cn1
InChIKey: SRGDOSISIBITQO-UHFFFAOYSA-N
Target: Gamma-aminobutyric acid receptor subunit alpha-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14867
Assay: Electrophysiology: Electrophysiological experiments were performed on days 3 to 5 after the micro-injection of mRNA. During the experiment the oocytes were constantly superfused by a solution containing (in mM) NaCl 90, KCl 1, HEPES 5, MgCl2 1, CaCl2 1 (pH 7.4). Oocytes were impaled by two glass microelectrodes (resistance: 0.4 MΩ) which were filled with a solution containing KCl 1M+K-acetate 1.5 M and voltage-clamped to −80 mV. The recordings were performed at room temperature using the Roboocyte two-electrode voltage clamp system (Multichannelsystem). After an initial equilibration period of 1.5 min GABA was added for 1.5 min at a concentration evoking approximately 20% of a maximal current response (EC20). After another rest interval of 2.5 min GABA was again added evoking a response of similar amplitude and shape. 0.5 min after the onset of this second GABA application the test compound, at a concentration corresponding to approximatively 30 fold its Ki, was added while GABA was still present. Current traces were recorded at a digitization rate of 10 Hz during and shortly before and after the GABA application.
Measurement: Ki = 33.8 nM (pChEMBL 7.46999979019165)
Descriptors: MW 455.5, ALogP 1.59, HBA 9, HBD 0, PSA 128.38, QED 0.56
Compound (SMILES): COc1cccc(OC)c1-n1c(NS(=O)(=O)[C@H](C)[C@@H](O)c2oc(C)nc2C)nnc1-c1ccc(C)o1
InChIKey: KMTHXVFQDGGOJX-JLTOFOAXSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: GTPγS Binding: The human APJ receptor was cloned by polymerase chain reaction and the gene encoding the receptor was subcloned in pFLAG-CMV-3 expression vector (Sigma, Saint Louis, Mo. USA) in-house at Amgen. A GTPγS binding assay was performed on membranes prepared from CHO cells stably expressing human APJ receptor. The optimum experimental conditions for the concentrations of GDP, MgCl2, and NaCl in the assay buffer were initially determined. The assay was performed in assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, and 0.1% (w/v) BSA with 200 mM NaCl, 3 μM GDP] and membranes expressing human APJ receptor/well along with WGA PS beads. The reaction was initiated by addition of 0.2 nM [35S]GTPγS in the absence or presence of various ligands and incubated at RT for 90 min. Nonspecific binding was determined in the presence of 100 μM GTPγS and was always less than 0.2% of total binding.
Measurement: EC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 517.56, ALogP 3.32, HBA 11, HBD 2, PSA 154.74, QED 0.34
Compound (SMILES): CCCN(C(=O)c1cccc(C(F)(F)F)c1)c1cnc(C)c(-c2cnc(OC3CCOCC3)c(N3CCOCC3)c2)c1
InChIKey: LINYELHXLRSDKZ-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Raf Activity Assay: Raf and biotinylated Mek, kinase dead, were combined at 2x final concentrations in assay buffer (50 mM Tris, pH 7.5, 15 mM MgCl2, 0.01% BSA and 1 mM DTT) and dispensed 5 ml per well in assay plates (Greiner white 384 well assay plates #781207) containing 0.25 ml of 40x of a Raf kinase inhibitor test compound diluted in 100% DMSO. The plate was incubated for 60 minutes at room temperature. The Raf kinase activity reaction was started by the addition of 5 mL per well of 2xATP diluted in assay buffer. After 3 hours (b-Raf(V600E)) or 1 hour (c-Raf). The reactions were stopped and the phosphorylated product was measured using a rabbit anti-p-MEK (Cell Signaling, #9121) antibody and the Alpha Screen IgG (ProteinA) detection Kit (PerkinElmer #6760617R), by the addition of 10 mL to the well of a mixture of the antibody (1:2000 dilution) and detection beads (1:2000 dilution of both beads) in Stop/bead buffer (25 mM EDTA, 50 mM Tris, pH 7.5, 0.01% Tween20). The additions were carried out under dark conditions to protect the detection beads from light. A lid was placed on top of the plate and incubated for 1 hour at room temperature, then the luminescence was read on a PerkinElmer Envision instrument. The concentration of each compound for 50% inhibition (IC50) was calculated by non-linear regression using XL Fit data analysis software.
Measurement: IC50 = 2400 nM (pChEMBL 5.619999885559082)
Descriptors: MW 584.64, ALogP 5.92, HBA 7, HBD 0, PSA 77.02, QED 0.33
Compound (SMILES): Cc1ccc(S(=O)(=O)NC(CN)C2CC2)cc1-c1cnc2c(N)ncnn12
InChIKey: FGXQQSGYBNIOJM-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Scintillation Proximity Assay: PI3K-γ:[γ-33P]ATP (10 mCi/mL) and Wheat Germ Agglutinin (WGA) YSi SPA Scintillation Beads was purchased from Perkin-Elmer (Waltham, Mass.). Lipid kinase substrate, D-myo-Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)D (+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho linked (PIP2), CAS 204858-53-7, was purchased from Echelon Biosciences (Salt Lake City, Utah). PI3Kγ (p110γ) Recombinant Human Protein was purchased from Life technology (Grand Island, N.Y.). ATP, MgCl2, DTT, EDTA, MOPS and CHAPS were purchased from Sigma-Aldrich (St. Louis, Mo.).The kinase reaction was conducted in polystyrene 384-well Greiner Bio-one white plate from Thermo Fisher Scientific in a final volume of 25 μL. Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 2%. The PI3Kγ assay was carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 μM PIP2, 2 μM ATP, 0.5 μCi [γ-33P] ATP, 13 nM PI3Kγ. Reactions were incubated for 120 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 163 mM potassium phosphate pH 7.8, 20% glycerol, 25 mM EDTA. The final concentration of SPA beads is 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1500 rpm for 10 min, the radioactivity of the product was determined by scintillation counting on Topcount (Perkin-Elmer).
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 387.47, ALogP 0.7, HBA 8, HBD 3, PSA 141.29, QED 0.56
Compound (SMILES): C[C@H](CS(N)(=O)=O)NC(=O)c1ccc2c(-c3ccccc3Cl)nn(-c3ccc(F)cc3)c2c1
InChIKey: IHLUMLSKESFFBQ-CQSZACIVSA-N
Target: Diacylglycerol O-acyltransferase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96PD7
Assay: Enzymatic Activity Assay: DGAT2 activity was determined by measuring the amount of enzymatic product triolein (1,2,3-Tri(cis-9-octadecenoyl)glycerol) using the membrane prep mentioned above. The assay was carried out in deep well 384 plates in a final volume of 40 μL at room temperature. The assay mixture contained the following: assay buffer (100 mM Tris.Cl, pH 7.0, 20 mM MgCl2, 5% ethanol), 25 μM of diolein, 10 μM of oleoyl-CoA and 10 ng/μL of DGAT2 membrane.
Measurement: IC50 = 184 nM (pChEMBL 6.739999771118164)
Descriptors: MW 486.96, ALogP 3.89, HBA 5, HBD 2, PSA 107.08, QED 0.43
Compound (SMILES): CC(C)(C)N1CCC(N2C=C([C@@H](Nc3cc(Cl)c4ncc(C#N)c(Nc5ccc(F)c(Cl)c5)c4c3)c3cccc4c3CNC4)NN2)CC1
InChIKey: OAUBWRLLHUAXLE-DHUJRADRSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: High Throughput Biochemical Assay: EGFR activity was measured using KinEASE (Cisbio), a time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay. In this assay, EGFR-catalyzes the phosphorylation of a universal Tyrosine kinase peptide substrate labeled with XL665. Europium conjugated phosphor-tyrosine specific antibody binds the resulting phosphorylated peptide. Formation of phosphorylated peptide is quantified by TR-FRET with Europium as the donor and XL665 the acceptor. The assay was performed in two main steps. The first step is the kinase reaction step and the second step is the detection step with TR-FRET reagents. In brief, test compounds 1:3 serially diluted in DMSO were delivered into Corning white, low volume, non-binding 384 well plates using the Echo 550 acoustic liquid dispenser (Labcyte®). EGFR enzyme (Human EGFR, cytoplasmic domain [669-1210] from Carna Biosciences Cat. No. 08-115) and substrates TK substrate-biotin (included in Cisbio HTRF KinEASE-TK kit Cat. No. 62TK0PEJ) were dispensed into assay plates using a Multi-Flo (Bio-Tek Instruments). The standard 10 μL reaction mixture contained 6 μM ATP (1×Km) or 12 μM ATP (2×Km), 1 μM biotinylated peptide, 0.3 nM EGFR (for 1×Km ATP) or 0.1 nM EGFR (for 2×Km ATP) in reaction buffer (10 mM MOPs, pH 7.0, 1.5% Glycerol, 0.5 mg/ml BSA, 10 mM Mg-Acetate, 1 mM DTT, 0.025% NP-40). After 60 min of incubation at room temperature, 10 μL of Stop and Detect Solution (1:400 Europium Cryptate labeled anti-phosphorylated peptide antibody solution and 125 nM strepavidin-XL665 Tracer in a 50 mM Hepes pH 7.0 detection buffer containing sufficient EDTA) was added. The plate was then further incubated for over 60 minutes at room temperature and read using an Envision 2103 Multilabeled reader (PerkinElmer) with excitation/emission/FRET emission at 340 nm/615 nm/665 nm, respectively.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 686.67, ALogP 7.48, HBA 9, HBD 5, PSA 103.31, QED 0.14
Compound (SMILES): Cc1ccc(-c2cc(CN3CCC(C(F)(F)F)CC3)c3c(N)ncnn23)cc1C(=O)N[C@@H]1CN(C(=O)[C@@](C)(O)C(F)(F)F)C[C@@H]1F
InChIKey: QQFKAQGDZBIVCN-FYQCMVGJSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: RIPK1 HTRF Binding Assay: A solution was prepared containing 0.2 nM Anti GST-Tb (Cisbio, 61GSTTLB), 90.6 nM probe and 1 nM His-GST-TVMV-hRIPK1(1-324) in FRET Buffer (20 mM HEPES, 10 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 0.05 mg/mL BSA). Using Formulatrix Tempest, the detection antibody/enzyme/probe solution (2 mL) was dispensed into wells of a 1536 plate (Black Low Binding Polystyrene 1536 Plate (Corning, 3724)) containing 10 nL of compounds of interest at appropriate concentration in DMSO. The plate was incubated at rt for 1 h. FRET was measured using the EnVision plate reader (Excitation: 340 nM, Emission: 520 nM/495 nM). Total signal (0% inhibition) was calculated from wells containing 10 nL DMSO only. Blank signal (100% inhibition) calculated from wells containing 10 nL of 15 nM staurosporine and internal controls.
Measurement: IC50 = 9000 nM (pChEMBL 5.050000190734863)
Descriptors: MW 659.61, ALogP 3.65, HBA 8, HBD 3, PSA 129.09, QED 0.34
Compound (SMILES): Cc1noc(C)c1S(=O)(=O)Nc1cc(-c2ccccc2)ccc1C(=O)O
InChIKey: HZHGSKOQJLHWFR-UHFFFAOYSA-N
Target: ATP-citrate synthase (SINGLE PROTEIN), Homo sapiens
UniProt: P53396
Assay: ADP-Glo Assay: Materials and Instruments Used in the ADP-Glo AssayVendor Cat No.Materials ACL Sino Biological 11769-H07BCoANa2 Sigma C3144Potassium Citrate Sigma 89306ATP Promega V915BADP-GloTM Kinase Assay Promega V9102kitHEPES Life Technologies 15630080MgCl2 Sigma M1028Brij 35 detergent Merck 203728DTT Sigma 646563DMSO MP 196055Optiplate-384, White 384- Perkin Elmer 6007290well384 dilution plate Corning 3657Topseal A Perkin Elmer E534196-well plate Nunc 249944InstrumentPlate reader Perkin Elmer Envision 2104Centrifuge Eppendorf 5810RCompounds of the present invention were evaluated in an ADP-GLO assay as follows:a) Dilute cpd 1:3 in succession in DMSO by hand for each cpds for 12 ptsb) Add 0.1 μL diluted cpd solution to assay plate, each dose with 2 replicatesc) Centrifuge 1000 RPM for 1 mind) Add 5 μL ACL working solution to 384-well assay plate, centrifuge 1000 RPM for 1 mine) Incubate at 25° C. for 15 minf) Add 5 μL substrate working solution to initiate reactiong) Final ACL reaction concentrations: 3 nM ACL, 15 μM ATP, 3 μM CoA, 300 μM Citrate, 0.01% Brij35, 4 mM DTT, 1% DMSO;h) Reference final conc: 30 uM starting Conc, 3× dilution, 11+0 points. Test cpd conc: 30/100 uM starting Conc, 3× dilution, 11+0 points.i) Incubate at 25° C. for 60 minj) Add 10 μL ADP Glo reagent, centrifuge 1000 RPM for 1 mink) Incubate at 25° C. for 40 minl) Add 20 μL kinase detection reagent, centrifuge 1000 RPM for 1 minm) Incubate at 25° C. for 40 minn) Read on Envision for US LUM as RLU
Measurement: IC50 = 10250 nM (pChEMBL 4.989999771118164)
Descriptors: MW 372.4, ALogP 3.46, HBA 5, HBD 2, PSA 109.5, QED 0.71
Compound (SMILES): C=CC(=O)N1C(c2nc(-c3ccc(C(C)(O)c4ccccc4)cc3)c3c(N)nccn23)CC2CC21
InChIKey: UPDBAQYLSKJEIX-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: In Vitro Assay for the Inhibition of BTK Kinase Activity: 1: Test Principle:Mobility-Shift Assay, which is microfluidic chip technology, applies the basic concept of capillary electrophoresis to microfluidic environments. The substrate used for the experiment is a polypeptide with a fluorescent label. Under the action of enzyme in the reaction system, the substrate is transformed into a product, and the charge the substrate carries also changes accordingly. The use of the charge difference between the substrate and the product involved in Mobility-Shift Assay achieves the separating of the two, and they are tested respectively. The test results are expressed by conversion rates.2: Test Method:(1) preparation of samples to be tested: diluted with 100% DMSO to 50 times the final concentration of the reaction, i.e. 25 μmol/L;(2) dilution: 25 μmol/L is the initial concentration, then diluted with 4 times the concentration and diluted with 10 concentration gradients;(3) 100% DMSO was added to the positive control well and the negative control well, respectively;(4) the prepared compounds with 10 concentrations were diluted 10-fold with 1 xkinase buffer, respectively; wherein, the kinase buffer contained hydroxyethyl piperazine ethanesulfonic acid at a concentration of 50 mmol/L and a pH of 7.5, 0.01% dodecyl polyethylene glycol ether, 10 mmol/L magnesium chloride, 2 mmol/L dithiothreitol;(5) preparation of 2.5×enzyme solution: the kinase was added to 1×kinase buffer to form 2.5×enzyme solution;(6) preparation of 2.5×substrate solution: FAM-labeled polypeptide and ATP were added to the 1 xkinase buffer to form 2.5×substrate solution; (7) addition of the enzyme solution to the 384-well plate: 5 μl of 5×compound dissolved in 10% DMSO contained in the 384-well reaction plate, then 10 μl of 2.5×enzyme solution was added, the obtained system was incubated for 10 minutes at room temperature;(8) addition of the substrate solution to the 384-well plate: 10 μl of 2.5×substrate solution was added to the 384-well reaction plate;(9) kinase reaction and termination: after incubation at 28° C. for 1 hour, 25 μl stop solution was added to terminate the reaction; wherein, the stop solution contained hydroxyethyl piperazine ethanesulfonic acid at a concentration of 100 mmol/L and a pH of 7.5, 0.015% dodecyl polyethylene glycol ether, 0.2% surface reagent No. 3, 20 mmol/L ethylenediaminetetraacetic acid; (10) Caliper data reading: conversion rate data was read from Caliper.
Measurement: IC50 = 178 nM (pChEMBL 6.75)
Descriptors: MW 465.56, ALogP 4.08, HBA 6, HBD 2, PSA 96.75, QED 0.43
Compound (SMILES): CCOc1ccc(S(=O)(=O)N2C[C@@H](C)[C@@H](n3nc(CC(=O)O)c4ccccc43)C2)c(/C=C/CO)c1
InChIKey: VCEOLQNXVATEDP-FGWOIVPQSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Receptor Binding Inhibition Assay: Cell membrane was prepared from CRTH2-expressing K562 cell. The cell membrane (20 μg/well) was mixed with reaction buffer (50 mM Tris/HCl, pH 7.4, 10 mM MgCl2). Then, [3H]-PGD2 was added to the mixture and incubated for 60 min at room temperature. The concentration of [3H]-PGD2 was determined as equal to Kd calculated from Scatchard plot. Then, the mixture was filtrated with glass paper immediately. The filter paper was washed several times and radio activity on the filter was measured. Specific binding was defined as difference between total and non-specific binding (radio activity with 10 μM PGD2). Ki values for each compound were calculated from displacement curve.
Measurement: Ki < 100 nM (pChEMBL 7)
Descriptors: MW 499.59, ALogP 2.95, HBA 7, HBD 2, PSA 121.96, QED 0.46
Compound (SMILES): NS(=O)(=O)c1cccc(C2CCCNC2)c1-c1nn[nH]n1
InChIKey: IZWIOEPYZPYJMJ-UHFFFAOYSA-N
Target: Metallo-beta-lactamase type 2 (SINGLE PROTEIN), Bacteria
UniProt: C7C422
Assay: Class B Enzyme Assay: The Class B enzyme activities were measured in the presence of the test inhibitor in a fluorescence assay against a commercially available substrate consisting of a cephalosporin core linking 7-hydroxycoumarin to fluorescein (CCF2-FA). The enzyme (NDM-1, IMP-1 or VIM-1) and the substrate were diluted in 100 mM KH2PO4 buffer (pH 7) containing 0.005% Tween-20 and 10 μM ZnSO4. In the assay, the final concentration of enzyme was 1 pM, 2 pM and 30 pM for NDM-1, IMP-1 and VIM-1, respectively, and the final concentration of CCF2-FA was 1.25 μM. The test inhibitor was dissolved in dimethylsulfoxide and diluted 1:50 in the assay, resulting in a final concentration range of 20 uM to 0.00063 uM. In a 384-well microplate, the test inhibitor was incubated with the metallo-β-lactamase enzyme and the substrate for 2 hours at 25° C. Fluorescence at 460 nm following excitation at 405 nm was measured. The IC50 value was determined from semi-logarithmic plots of enzyme inhibition versus inhibitor concentration, with a curve generated using a 4-parameter fit.
Measurement: IC50 = 552 nM (pChEMBL 6.260000228881836)
Descriptors: MW 308.37, ALogP -0.02, HBA 6, HBD 3, PSA 126.65, QED 0.73
Compound (SMILES): CCOc1ncccc1-c1cc(NCc2ccn(C)n2)c2c(cnn2C(C)C)n1
InChIKey: KZFYUJZMDBTCDC-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 hr 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 hr 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 = 66 nM (pChEMBL 7.179999828338623)
Descriptors: MW 391.48, ALogP 3.82, HBA 8, HBD 1, PSA 82.68, QED 0.52
Compound (SMILES): Cc1ccc(CC(CNC(=O)CC(F)(F)F)n2c(=N)n(Cc3ccc(C)cc3)c3ccccc32)cc1
InChIKey: JWCQUTLXPMTBHQ-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: IP Accumulation Assay: HEK293 cells stably expressing OX2R are transfected with Gq alpha construct (6.4 ug per 10 cm dish) using Lipofectamine 3000 (ThermoFisher). 24 hours after transfection, cells are plated on poly-lysine-coated 96-well plates at 30,000 cells per well in Inositol-free DMEM+5% FCS with 5 uCi per ml Myo-[2-3H] Inositol (Perkin Elmer). Plates are incubated at 37° C. O/N in 8% C02 tissue culture incubator. Agonists are diluted serially in HBSS containing 10 mM Lithium Chloride. After removal of culture media, agonist solutions are added to the cells (100 ul/well) and incubated at 37° C. 45 min in 8% C02 incubator. Agonist solution is removed and cells are lysed on ice with 10 mM Formic Acid (40 ul/well) for 30 minutes. SPA Poly Lysine YSI beads (Perkin Elmer, cat #RPNQ0010) are added to the wells and mixed on a shaker for 30 minutes before reading on a Microbeta scintillation counter. Positive control curves run with all screens: orexin B and Yanagisawa (J. Med. Chem. 2015) agonist.
Measurement: EC50 = 5000 nM (pChEMBL 5.300000190734863)
Descriptors: MW 494.56, ALogP 5.44, HBA 4, HBD 2, PSA 62.81, QED 0.34
Compound (SMILES): O[C@H]1CCCN[C@@H]1CCCn1cnc2c(Cl)c(C(F)(F)F)ccc21
InChIKey: FKCAKVWJKNMRHV-YPMHNXCESA-N
Target: Ribose-phosphate pyrophosphokinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P60891
Assay: PRS Enzyme Activity Inhibition Experiment: In order to confirm the biological activities of the compounds prepared in Examples, % inhibition or IC50 values of PRS enzyme (phosphoribosylpyrophosphate synthetase enzyme) activities were calculated.Specifically, the portion corresponding to PRS in cDNA of EPRS was subcloned, and the obtained high-purity PRS protein was purified and used in the experiment. The compounds (1 μM) prepared in Examples were added into the reaction buffer (20 mM KPO4 (pH 7.4), 6 mM MgAc, 5 mM ATP, 400 mg/mL tRNA, 0.5 mM DTT, 20 mCi[3H]proline (1 mCi/mL)) and allowed to react at 37° C. for 5 to 10 minutes. The reaction was terminated with 3M paper that was in advance dried by addition of 5% TCA. The radioactivity was measured using a liquid scintillation counter.% Inhibition and IC50 values of the respective compounds were calculated and analyzed using Microsoft Excel or Sigma Plot 8.0.
Measurement: IC50 > 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 361.8, ALogP 3.6, HBA 4, HBD 2, PSA 50.08, QED 0.87
Compound (SMILES): O=C(Nc1ccc(OC(F)(F)F)c(F)c1)Nc1ccc(OC(F)(F)F)c(F)c1
InChIKey: VADYRBIKCJDUIN-UHFFFAOYSA-N
Target: Bifunctional epoxide hydrolase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34913
Assay: FRET-displacement assay: FRET assays to determine Ki for the compounds of Table I were carried out as described previously (Lee et al. Analytical Biochemistry 434 (2013) 259-268). In order to prevent leaching of fluorescence impurities from the plastic tube and non-specific binding to sEH inhibitors, the inhibitor stock solution (10 mM, DMSO) was stored in glass vials. In addition, sEH was diluted to desired concentration (20 nM) with sodium phosphate buffer (PB) (100 mM sodium phosphate, pH 7.4, 0.01% gelatin) to avoid loss of protein from non-specific binding to the cuvette surface. All buffer used in this assay was filtered by sterilized filtration unit (Millipore Durapore PVDF Membrane, pore size: 0.22 um).Measurement in 96-Well PlatesAll the measurement for FRET-based displacement assay in 96-well plate format were done in TECAN Infinite M1000 Pro 96 well fluorescence plate reader.Pre-Treatment of 96-Well PlateIn order to prevent non-specific binding of sEH or inhibitor on the 96-well plate, the 96 well plates were pre-incubated with PB with 0.1% gelatin overnight at rt. The gelatin coats the plate and prevents non-specific binding of sEH and sEH inhibitors to the plate. The buffer was discarded and the plate was dried before use.Assay ProcedureThe sEH stock was diluted to the desired concentration (20 nM) by PB (100 mM sodium phosphate, 0.1% gelatin, pH 7.4). ACPU (one equivalent to sEH, 10 mM, Ethanol) was added to the sEH solution and was incubated for 2 h at rt. The sEH-ACPU mixture (20 nM, 100 mM sodium phosphate, 0.1% gelatin, pH 7.4, 150 uL) was added to each well.The baseline fluorescence (F0) (λexcitation at 280 nm, λemission at 450 nm) of the samples was measured after the z-position and gain were optimized automatically by the fluorometers. The z and gain value was noted and will be used for the later fluorescent measurement. Because DMSO has been known to quench fluorescence. 1% DMSO in PB was served as a control (FDMSO). The desired concentration of inhibitors which is the concentration that 100% of sEH was bound to inhibitor, was added at the first well and was further diluted by 2-fold across the rest of the wells. Based on our study, 12 datum points which correspond to 12 different concentrations of the inhibitor, provide significant data to calculate the accurate Ki for the inhibitors. The samples were incubated at 30° C. for 1.5 h. Then, the fluorescence (λexcitation at 280 nm, λemission at 450 nm) of the samples was measured using the z-position and gain values that previously obtained.
Measurement: Ki = 1.95 nM (pChEMBL 8.710000038146973)
Descriptors: MW 416.22, ALogP 5.41, HBA 3, HBD 2, PSA 59.59, QED 0.66
Compound (SMILES): CC(=O)N[C@@H](C)COc1cnc(Oc2ccc(C(=O)N3CCCCC3)c(F)c2F)cn1
InChIKey: BYSLYPCCXBGOFW-ZDUSSCGKSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Inhibitory Activity on Human ACC1 and the ACC2: Recombinant human ACC1 and recombinant human ACC2, which were prepared by the method mentioned above, were preincubated with assay buffer solution (50 mM HEPES-KOH (pH 7.4), 10 mM magnesium chloride, 6-10 mM potassium citrate, 4 mM reduced form of glutathione, 1.5 mg/ml bovine serum albumin) for one hour. Then, 0.2 μL of each this invention compound solution (in DMSO) were dispensed to 384-well microplate, 5 μL of the preincubated enzyme solution and 5 μL of substrate solution (50 mM HEPES-KOH (pH 7.4), 1 mM ATP, 0.8 mM acetyl CoA and 25-50 mM potassium bicarbonate) were added to microplate. After centrifugation and shaking, the reaction mixtures were incubated in a humidified box at room temperature for 1 to 3 hours. After the incubation, the enzyme reactions were stopped by the addition of EDTA. Then, after the samples were cocrystallized with CHCA (α-cyano-4-hydroxy cinnamic acid) matrices on MALDI target plate, by using the matrix assist laser deionization time-of-flight mass spectrometer (MALDI-TOF MS), samples were measured in reflector negative mode. Deprotonated ions of acetyl CoA (AcCoA) of substrate and malonyl CoA (MalCoA) of the reaction product were detected, then, the conversion rates of acetyl CoA to malonyl CoA was calculated by the intensity of [MalCoA-H]−/(Intensity of [MalCoA-H]−+Intensity of [AcCoA-H]−) using each signal strength. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate of the enzymatic reaction at each concentration of the compounds. In addition, potassium citrate concentrations in assay buffer solution, potassium hydrogen carbonate concentrations in substrate solution and incubation time were adjusted by each lot of enzyme.
Measurement: IC50 = 775 nM (pChEMBL 6.110000133514404)
Descriptors: MW 434.44, ALogP 3.08, HBA 6, HBD 1, PSA 93.65, QED 0.72
Compound (SMILES): CNC(=O)[C@H]1CC[C@H](NC(=O)c2cnc(-c3cnn4cc(Cl)cnc34)cc2NC(C)C)CC1
InChIKey: NCYISWIEQQSSSE-KOMQPUFPSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IRAK4 Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 μL prepared from 15 μL additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij 35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction mixture was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 469.98, ALogP 3.3, HBA 7, HBD 3, PSA 113.31, QED 0.51