lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1ccc(NC(=O)c2cnnc(C(C)(C)C#N)c2)cc1-c1cc(N2CCOCC2)c(=O)n(C)n1
InChIKey: JBVJRGVLOBEGQX-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 = 8.4 nM (pChEMBL 8.079999923706055)
Descriptors: MW 473.54, ALogP 2.44, HBA 9, HBD 1, PSA 126.03, QED 0.6
Compound (SMILES): COCCC(=O)N1CCC2(CC1)CC2c1nc(-c2ccc(C(=O)Nc3ccccn3)cc2)c2c(N)nccn12
InChIKey: MUIARQGPXMXYBV-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Btk Enzyme Activity Assay A: Btk enzyme activity is measured using the IMAP (immobilized metal ion affinity-based fluorescence polarization) assay as outlined below. Btk enzyme (His-Btk (Millipore catalog #14-552), is diluted to 0.4 U/mL in KR buffer (10 mM Tris-HCl, 10 mM MgCl2, 0.01% Tween-20, 0.05% NaN3, 1 mM DTT, 2 mM MnCl2, pH 7.2).Serial dilution log 10 from 2 mM to 63.2 nM of test compounds are made in 100% DMSO. The dilutions in DMSO are then diluted 50-fold in KR-buffer. Final compound concentration range in the assay from 10 μM to 0.316 nM. 5 μL/well of test compound in KR buffer (final DMSO concentration in the assay is 1%) is mixed with 5 μl/well of 0.4 U/mL Btk enzyme (final concentration in the assay is 0.1 U/mL). Test compounds and Btk enzyme are pre-incubated 60 minutes at room temperature, before adding 5 μL/well of 200 nM Fluorescin labeled substrate peptide (Blk/Lyntide substrate, e.g. #R7188/#R7233, Molecular Devices) in KR-buffer. Final peptide substrate concentration in assay is 50 nM. The kinase assay is started by adding 5 μL/well of 20 μM ATP in KR-buffer (final ATP concentration is 5 μM ATP, Km ATP in Btk IMAP assay). Following incubation for 2 h at room temperature the enzyme reaction is stopped by adding 40 μL/well IMAP Progressive Binding Solution (according to suppliers (Molecular Devices) protocol using 75% 1× buffer A and 25% 1x buffer B with 1:600 Progressive Binding Solution). After 60 min incubation at room temperature in the dark the FP signal is read. Fluorescence at 535 nm is measured using parallel and perpendicular filters to determine differences in rotation due to binding of the phosphorylated substrate peptide to the beads. Values are calculated as percentage of the difference in readout (ΔmPi) of the controls with and without ATP. EC50 values are determined by curve fitting of the experimental results using Activity Base.
Measurement: EC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 525.61, ALogP 3.76, HBA 8, HBD 2, PSA 127.74, QED 0.38
Compound (SMILES): COc1ccccc1C1CC(=O)C(=CN(C)C)C(=O)C1
InChIKey: UCHLDYRNDOHPIG-RAXLEYEMSA-N
Target: Microtubule-associated protein 1 light chain 3 beta (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZQ8
Assay: Molecular Level Experiments of Targeting LC3B : By constructing a prokaryotic expression system, the LC3B protein was expressed and purified, and a preliminary screening and verification platform was established using fluorescence polarization experiments to determine the activity of synthesized small compound libraries.The recombinant protein GST-LC3B (final concentration 180 nM, SEQ ID NO: 1) and N-terminal FITC-labeled peptide (SEQ ID NO: 2, final concentration 18 nM) were placed in the FP buffer (50 mM HEPES pH 7.5, 0.1 mg/mL BSA and 1 mM DTT), to which a compound serially diluted with the FP buffer was added. Then the resulting mixture was incubated at 25° C. in the dark. The fluorescence polarization value (PerkinElmer Envision, emission wavelength 480 nm; absorption wavelength 535 nm) was monitored, and the IC50 value was calculated using the GraphPad Prism 6.0 program.
Measurement: IC50 = 9000 nM (pChEMBL 5.050000190734863)
Descriptors: MW 273.33, ALogP 2.16, HBA 4, HBD 0, PSA 46.61, QED 0.63
Compound (SMILES): O=C([C@H]1C[C@@](c2ccc(C(OCc3c(F)cccc3F)(C(F)(F)F)C(F)(F)F)cc2)(S(=O)(=O)c2ccc(F)cc2)C1)N1CC(O)C1
InChIKey: HGWUXMSDKQISPM-NZFXNERUSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: SPA Binding Assay: The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromotography. 15 ug/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 μM to 0.28 nM for 10 min at room temperature in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% Glycerol (Sigma Cat# G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat # RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer).
Measurement: IC50 = 39 nM (pChEMBL 7.409999847412109)
Descriptors: MW 681.57, ALogP 5.92, HBA 5, HBD 1, PSA 83.91, QED 0.24
Compound (SMILES): COc1cc2ncc(C#N)c(N3CCCC(F)(CNS(N)(=O)=O)CC3)c2cc1OC
InChIKey: QVODRGHKOPSVHR-UHFFFAOYSA-N
Target: Ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P22413
Assay: ENPP1 Assay: Table 1: Assay Buffer: 1 mM CaCl2, 0.2 mM ZnCl2, 50 mM Tris, pH 9.0 Substrate: 1.5 mM Thymidine 5′-monophosphate disodium salt hydrate (Sigma: T4510)Assay Cone.: 150 μM Enzyme: 1 ng/μL Recombinant Human ENPP-1 Protein (purified in-house)Assay Cone.: 5 ng/well DMSO 96-well clear assay plates Protocol: A ten-point serial dilution of drugs was prepared in 10× in assay buffer with the final assay concentrations starting at 10 μM, 3 μM, 1 μM, 0.3 μM . . . 0 μM. A dilution of DMSO was included as a control. The assay plate was set up as follows with each well in duplicate: 75 μL assay buffer+10 μL ENPP1 inhibitor or DMSO Dilutions+10 μL Substrate+5 μL Enzyme (5 ng). Both the enzyme and substrate were added to opposite sides of the well to ensure that there was no interaction until all wells had both components. The plate was then centrifuged gently for 10 seconds, followed by an incubation at 37° C. for 45 minutes. The reaction was quantified by measuring absorbance at 405 nm using the Envision Plate Reader.
Measurement: IC50 = 10.2 nM (pChEMBL 7.989999771118164)
Descriptors: MW 437.5, ALogP 1.62, HBA 7, HBD 2, PSA 130.57, QED 0.7
Compound (SMILES): O=C(c1ccc(Nc2nc3c(N4CCC(O)(c5ccc(Cl)cc5)CC4)cccn3n2)cc1)N1CCN(c2ccccn2)CC1
InChIKey: UHPZUWPOFMDRRL-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 11.4 nM (pChEMBL 7.940000057220459)
Descriptors: MW 609.13, ALogP 4.97, HBA 9, HBD 2, PSA 102.13, QED 0.28
Compound (SMILES): CN1CCC([C@@H](Cc2ccccc2)S(=O)(=O)N2[C@@H]3CC[C@H]2C[C@@H](NC(=O)c2cc(C4CC4)on2)C3)CC1
InChIKey: APNXYNIZVXMGAE-JPYYHWIRSA-N
Target: Histone-lysine N-methyltransferase SMYD3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H7B4
Assay: SMYD3 Biochemical Assay: The assays were all performed in a buffer consisting of 25 mM Tris-Cl pH 8.0, 1 mM TCEP, 0.005% BSG, and 0.005% Tween 20, prepared on the day of use. Compounds in 100% DMSO (1 ul) were spotted into a 384-well white opaque OptiPlate using a Bravo automated liquid handling platform outfitted with a 384-channel head (Agilent Technologies). DMSO (1 ul) was added to Columns 11, 12, 23, 24, rows A-H for the maximum signal control and 1 ul of SAH, a known product and inhibitor of SMYD3, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control. A cocktail (40 ul) containing the SMYD3 enzyme was added by Multidrop Combi (Thermo-Fisher). The compounds were allowed to incubate with SMYD3 for 30 min at room temperature, then a cocktail (10 ul) containing SAM and MEKK2 was added to initiate the reaction (final volume=51 ul). The final concentrations of the components were as follows: SMYD3 was 0.4 nM, 3H-SAM was 8 nM, MEKK2 was 12 nM, SAH in the minimum signal control wells was 1 mM, and the DMSO concentration was 2%. The assays were stopped by the addition of non-radiolabeled SAM (10 ul) to a final concentration of 100 uM, which dilutes the 3H-SAM to a level where its incorporation into MEKK2 is no longer detectable. Radiolabeled MEKK2 was detected using a scintillation proximity assay (SPA). 10 uL of a 10 mg/mL solution of SPA beads in 0.5 M citric acid was added and the plates centrifuged at 600 rpm for 1 min to precipitate the radiolabeled MEKK2 onto the SPA beads. The plates were then read in a PerkinElmer TopCount plate reader to measure the quantity of 3H-labeled MEKK2 as disintegrations per minute (dpm) or alternatively, referred to as counts per minute (cpm).% Inhibition Calculation% ⁢ ⁢ inh = 100 - ( dpm cmpd - dpm min dpm max - dpm min ) × 100Where dpm=disintegrations per minute, cmpd=signal in assay well, and min and max are the respective minimum and maximum signal controls.Four-Parameter IC50 FitY = Bottom + ( Top - Bottom ) ( 1 + ( X IC 50 ) Hill ⁢ ⁢ CoefficientWhere top and bottom are the normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit. The Hill Coefficient normally allowed to float but may also be fixed at 1 in a 3-parameter fit. Y is the % inhibition and X is the compound concentration.
Measurement: IC50 = 13.9 nM (pChEMBL 7.860000133514404)
Descriptors: MW 526.7, ALogP 3.56, HBA 6, HBD 1, PSA 95.75, QED 0.57
Compound (SMILES): Cc1cc(-n2ncc3cc(NC(=O)NCC4CNC4)ncc32)ccn1
InChIKey: WRPFQFBWUMZKHK-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 μM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 μL) was dispensed, followed by the addition of 15 μL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 μL kinase buffer containing 2.45 μM ERK2 IMAP substrate peptides and 75 μM ATP. The final reaction in each well of 25 μL consists of 0.5 nM hERK2, 900 nM unlabeled peptide, 80 nM labeled-peptide, and 30 μM ATP. Phosphorylation reactions were allowed to proceed for 60 minutes and were immediately quenched by the addition of 60 μL IMAP detection beads (1:1000 dilutions) in IMAP binding buffer (Molecular Devices) with 24 mM NaCl. Plates were read on EnVision reader after 60 minutes binding equilibration using Fluorescence Polarization protocol (Perkin Elmer).
Measurement: IC50 = 102 nM (pChEMBL 6.989999771118164)
Descriptors: MW 337.39, ALogP 1.46, HBA 6, HBD 3, PSA 96.76, QED 0.67
Compound (SMILES): CC(C)c1c(-c2cc(OC(F)F)c3ncnn3c2)[nH]c2ccc(C3CCNCC3)cc12
InChIKey: YQSCECQBUSCWPH-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 0.7 nM (pChEMBL 9.149999618530273)
Descriptors: MW 425.48, ALogP 5.07, HBA 5, HBD 2, PSA 67.24, QED 0.47
Compound (SMILES): COc1cccc(CN2C(=O)N(c3ccc(-c4cn[nH]c4)cc3)CC23CCN(CC(O)CO)CC3)c1
InChIKey: NLFNRYNOZYNMOP-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH) (SEQ ID NO. 1). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound.
Measurement: IC50 < 3 nM (pChEMBL 8.52299976348877)
Descriptors: MW 491.59, ALogP 2.72, HBA 6, HBD 3, PSA 105.16, QED 0.45
Compound (SMILES): O=S(=O)(Nc1cnc2[nH]c(-c3ccccc3)cc2c1)c1cn[nH]c1
InChIKey: SAJBUOAKZXOLIR-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Exemplary c-Kit Mutant Biochemical Assay: The c-kit mutant D816V (or kinase domain thereof) is an active kinase in AlphaScreen. IC50 values are determined with respect to inhibition of c-Kit mutant D816V 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 20 mM. These were diluted 30 μl into 120 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 20 μl in 1× kinase buffer (25 mM HEPES, pH 7.2, 8 mM MgCl2, 2 mM MnCl2, 50 mM NaCl, 0.01% Brij, 1 mM DTT, 0.01% BSA), 5% DMSO and 10 μM ATP. Substrate was 30 nM biotin-(E4Y)10 (EMD Millipore, Cat#12-440). C-kit mutant D816V kinase was at 0.75 ng per sample. After incubation of the kinase reaction for 30 minutes at room temperature, 5 μl of donor beads (Streptavidin coated beads (Perkin Elmer Life Science) final concentration 7.5 μg/ml) in stop buffer (25 mM Hepes pH 7.5, 100 mM EDTA, 0.01% BSA) was added, the sample was mixed and incubated for 20 minutes at room temperature before adding 5 μl of acceptor beads (PY20 coated beads (Perkin Elmer Life Science) final concentration 7.5 ug/ml) in stop buffer. The samples were incubated for 60 minutes at room temperature and the signal per well was read on EnVision reader. Phosphorylated substrate results in binding of the PY20 antibody and association of the donor and acceptor beads such that signal correlates with kinase activity.
Measurement: Ki < 1000 nM (pChEMBL 6)
Descriptors: MW 339.38, ALogP 2.75, HBA 4, HBD 3, PSA 103.53, QED 0.53
Compound (SMILES): O=C(c1ccc2[nH]ccc2c1)N1CCN(c2nc3ccccc3c(=O)[nH]2)CC1
InChIKey: YZFJYXHOHWZAFZ-UHFFFAOYSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: MAGL Inhibitory Activity: Compounds of the present invention are MAGL inhibitors. Thus, in one aspect, the present invention provides the use of compounds of Formula (I) or a pharmaceutically acceptable salt thereof as described herein for inhibiting MAGL in a mammal.In a further aspect, the present invention provides compounds of Formula (I) or a pharmaceutically acceptable salt thereof as described herein for use in a method of inhibiting MAGL in a mammal.In a further aspect, the present invention provides the use of compounds of Formula (I) or a pharmaceutically acceptable salt thereof as described herein for the preparation of a medicament for inhibiting MAGL in a mammal.In a further aspect, the present invention provides a method for inhibiting MAGL in a mammal, which method comprises administering an effective amount of a compound or a pharmaceutically acceptable salt thereof of Formula (I) as described herein to the mammal.Compounds were profiled for MAGL inhibitory activity by measuring the enzymatic activity of MAGL by following the hydrolysis of 4-nitorphenylacetate resulting in 4-nitrophenol, which absorbs at 405-412 nm (G. G. Muccioli, G. Labar, D. M. Lambert, Chem. Bio. Chem. 2008, 9, 2704-2710).The assay was carried out in 384 well assay plates (black with clear bottom, non-binding surface treated, Corning Ref. 3655) in a total volume of 40 μL. Compound dilutions were made in 100% DMSO (VWR Chemicals 23500.297) in a polypropylene plate in 3-fold dilution steps to give a final concentration range in the assay from 25 μM to 1.7 nM. 1 μL compound dilutions (100% DMSO) were added to 19 μL MAGL (recombinant wild-type) in assay buffer (50 mM TRIS (GIBCO, 15567-027), 1 mM EDTA (Fluka, 03690-100 ml)). The plate was shaked for 1 min at 2000 rpm (Variomag Teleshake) and then incubated for 15 min at RT. To start the reaction, 20 μL 4-Nitrophenlyacetate (Sigma N-8130) in assay buffer with 6% EtOH was added. The final concentrations in the assay were 1 nM MAGL and 300 μM 4-Nitrophenylacetate. After shaking (1 min, 2000 rpm) and 5 min incubation at RT, the absorbance at 405 nm was measured for a fist time (Molecular Devices, SpectraMax Paradigm). A second measurement was then done after incubation for 80 min at RT. From the two measurements, the slope was calculated by subtracting the first from the second measurement.
Measurement: IC50 = 9500 nM (pChEMBL 5.019999980926514)
Descriptors: MW 373.42, ALogP 2.37, HBA 4, HBD 2, PSA 85.09, QED 0.56
Compound (SMILES): CN[C@@H](CN1CCC2(CC1)CCN(c1ccc(C#N)cn1)C2)c1ccc2c(c1C)COC2=O
InChIKey: CKGLDNYWQCGEEC-QHCPKHFHSA-N
Target: ATP-sensitive inward rectifier potassium channel 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P48048
Assay: Thallium Flux Assay: Assay Protocol: The ROMK channel functional thallium flux assay was performed in 384 wells, using the FLIPR-Tetra instrument. HEK-hKir1.1 cells were seeded in Poly-D-Lysine microplates and kept in a 37° C.-10% CO2 incubator overnight. On the day of the experiment, the growth media was replaced with the FluxOR reagent loading buffer and incubated, protected from light, at ambient temperature (23-25° C.) for 90 min. The loading buffer was replaced with assay buffer±test compound followed by 30 min incubation at ambient temperature, where the thallium/potassium stimulant was added to the microplate.
Measurement: IC50 = 61.3 nM (pChEMBL 7.210000038146973)
Descriptors: MW 445.57, ALogP 3.19, HBA 7, HBD 1, PSA 81.49, QED 0.71
Compound (SMILES): COCCS(=O)(=O)C(C(=O)NCC(=O)NC1CC1)c1nc2cc(C(F)(F)F)c(-c3cnc(C)nc3)cc2s1
InChIKey: IXDCDFYGTLGZFQ-UHFFFAOYSA-N
Target: Endothelial lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5X9
Assay: Biological Activity: Endothelial lipase (EL) and hepatic lipase (HL) activities were measured using a fluorescent substrate, A10070, (Invitrogen, CA) doped into an artificial vesicle containing DMPG (Avanti Polar Lipids) as the excipient. Vesicles were prepared by combining 571 μL of 29 mM DMPG in a 1:1 mixture of MeOH and CHCl3 with 2000 μL of 1 mM A10070 in a 1:1 mixture of MeOH and CHCl3. The mixture was dried under nitrogen in multiple vials then resuspended in 20 mL total volume of 50 mM HEPES pH 8.0 buffer containing 50 mM NaCl and 0.2 mM EDTA. The sample was allowed to sit at room temperature for 15 min and then was sonicated 3×4 mins on ice with a Branson Sonicator using duty cycle 1. This preparation provides vesicles with a mole fraction of 0.11 for the FRET substrate.The enzymatic assay was measured using 384-well white Optiplates. Each well contained 20 μL of assay buffer (50 mM HEPES pH 8.0, 50 mM NaCl and 1 mM CaCl2) and 0.25 μL of a DMSO solution containing a compound of interest. EL or HL (10 μL) was added and allowed to incubate with the compound for 30 min at 37° C. The source of EL was conditioned media obtained from HT-1080 cells that were transformed using RAGE technology (Athersys) to overexpress endogenous EL, and HL was partially purified from conditioned media obtained from COS cells overexpressing HL. The reaction was started by the addition of 10 μL of a 1:10 dilution of vesicles. The final total reaction volume was 20.25 μL. The reaction rates were measured on a Gemini plate reader with an excitation wavelength of 490 nm and an emission wavelength of 530 nm. Readings were taken over a period of 60 minutes, and the slope between 300 and 900 secs of the readout was used to calculate the rate of the reaction.
Measurement: IC50 = 56 nM (pChEMBL 7.25)
Descriptors: MW 571.6, ALogP 2.58, HBA 9, HBD 2, PSA 140.24, QED 0.38
Compound (SMILES): C[C@@H]1CC[C@@H](Oc2nccc3c2COC3=O)CN1C(=O)c1ccc(F)cc1CS(C)(=O)=O
InChIKey: RIWWYCCSJBBACE-CZUORRHYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Intracellular Calcium 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. For each antagonist, IC50 value (the concentration of compound needed to inhibit 50% of the agonist response) is determined.
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 462.5, ALogP 2.51, HBA 7, HBD 0, PSA 102.87, QED 0.63
Compound (SMILES): NCCS(=O)(=O)c1ccc(-c2cccc3[nH]c([C@H](N)CNC(=O)C(F)(F)F)nc23)c(-c2nn[nH]n2)c1S(N)(=O)=O
InChIKey: IYNKBLIJSWYECH-LLVKDONJSA-N
Target: Metallo-beta-lactamase type 2 (SINGLE PROTEIN), Bacteria
UniProt: C7C422
Assay: Enzyme Activity: 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; for a review, see: Meine, M.-R.; Llarrull, L. I.; Vila, A. J. Antibiotics, 2014, 3, 285-316) 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 μM to 0.00063 μM. 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 = 1.02 nM (pChEMBL 8.989999771118164)
Descriptors: MW 602.58, ALogP -0.53, HBA 11, HBD 6, PSA 258.58, QED 0.14
Compound (SMILES): CCNCc1ccc(-c2cc(Nc3cnc(C#N)cn3)n[nH]2)c(OC)c1
InChIKey: PEEHUAJISNCEQA-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase Chk1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14757
Assay: Kinase Inhibiting Activity Assay: Chk-1 Kinase Inhibiting ActivityThe compounds of the invention were tested for activity against Chk-1 kinase using the materials and protocols set out below.Reaction Buffer:Base Reaction buffer: 20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO*Required cofactors are added individually to each kinase reactionReaction Procedure:(i) Prepare indicated substrate in freshly prepared Base Reaction Buffer(ii) Deliver any required cofactors to the substrate solution above(iii) Deliver indicated kinase into the substrate solution and gently mix(iv) Deliver compounds in DMSO into the kinase reaction mixture(v) Deliver 33P-ATP (specific activity 0.01 μCi/μl final) into the reaction mixture to initiate the reaction.(vi) Incubate kinase reaction for 120 minutes at room temperature(vii) Reactions are spotted onto P81 ion exchange paper (Whatman #3698-915)(viii) Wash filters extensively in 0.1% phosphoric acid.(ix) Dry filters and measure counts in scintillation counterKinase Information:CHK-1 Genbank Accession # AF016582Recombinant full length construct, N-terminal GST tagged, purified from insect cells.No special measures were taken to activate this kinase.Final concentration in assay=0.5 nMSubstrate: CHKtidePeptide sequence: [KKKVSRSGLYRSPSMPENLNRPR]Final concentration in assay=20 μM
Measurement: IC50 = 0.058 nM (pChEMBL 10.239999771118164)
Descriptors: MW 349.4, ALogP 2.6, HBA 7, HBD 3, PSA 111.54, QED 0.6
Compound (SMILES): Cn1cccc1-c1c(F)c(Cl)c(-c2ccc3nc(NC(=O)[C@@H]4C[C@@H]4F)cn3c2)c2cn[nH]c12
InChIKey: BZJJSNGNGHTBCP-OCCSQVGLSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: ADP-Glo kinase assay: HPK1 kinase activity was measured by Promega's ADP-Glo kinase assay. In this assay, 5 ng of recombinant human HPK1 (signalchem) is incubated with 5 μL of compounds (0.5% DMSO), 5 μL of MBP (0.5 μg/μl) and 5 μL of ATP (25 μM) in buffer (40 mM Tris, 7.5; 20 mM MgCl2; 0.1 mg/ml BSA; 5 μM DTT). The assay was started by incubating the reaction mixture in a 96-well plate at 30′ C for 40 minutes. After the incubation, 25 uL ADP-Glo reagent was added and the reaction was incubated at room temperature for 40-min to stop the reaction and degrade residual ATP. The ADP product was then converted to ATP by adding 50 uL per well of detection reagent. Luminescence was detected after 30-min room temperature incubation with the Molecular device I3X plate reader. The IC50 values were calculated from a series of percent inhibition values determined at a range of inhibitor concentration using software routines as implemented in the GraphPad Prism 7 software and SigmaPlot13.0.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 466.88, ALogP 4.97, HBA 5, HBD 2, PSA 80.01, QED 0.39
Compound (SMILES): C[C@@H]1CN(c2ccc3c(c2)OC(F)(F)O3)CCN1C(=O)NCCC1CCN(Cc2ccccc2)CC1
InChIKey: FMRBQUOHMPZAGS-HXUWFJFHSA-N
Target: Muscarinic acetylcholine receptor M4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08173
Assay: Binding Assay: Binding affinity (Ki) of compounds was measured by inhibition of radioligand binding to membranes from CHO cells expressing human M1, M2, M3, M4 and M5 receptors. Membranes were prepared by nitrogen cavitation and differential centrifugation as previously described (Hoare et al., Mol. Pharmacol. 2003 March; 63(3): 751-65). The radioligand employed was tritiated N-methylscopolamine, used at a concentration of 1.5 nM. A dose-response of twelve concentrations of compound was used, ranging from 10 Î¼M to 32 Î¼M. The assay buffer was 50 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 1 mM ethylenediaminetetraacetic acid, pH-adjusted to pH 7.4.
Measurement: Ki < 10 nM (pChEMBL 8)
Descriptors: MW 500.59, ALogP 4.53, HBA 5, HBD 1, PSA 57.28, QED 0.63
Compound (SMILES): C[C@@H](Nc1nc(C(=O)O)nc2nc(N3CCCC3c3ccccc3)n(Cc3ccc(C(F)(F)F)cc3)c12)C1CCC1
InChIKey: GGVYFLFTMZSDDI-FKSKYRLFSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: FRET Assay: An HDM2 FRET assay was developed to assess the compounds' inhibitory activity towards binding of p53 protein. A truncated version of HDM2 with residues 17 to 125 (containing p53 binding surface, Science (1994) 265, 346-355), with N-terminal His and Thioredoxin tag was generated in pET32a expression vector and expressed in E. coli strain BL21(DE3)Rosetta. Protein was purified using Ni-affinity chromatography, followed by size exclusion chromatography using Superdex 75 26/60 column. To assess inhibition of p53 binding to HDM2, a FITC labeled 8-mer peptide (sequence: Ac-Phe-Arg-Dpr-Ac6c-(6-Br)Trp-Glu-Glu-Leu-NH2; Anal Biochem. 2004 Aug. 1; 331(1):138-46) with strong affinity towards p53 binding pocket of HDM2 was used. The HDM2 assay buffer contained 1× Phosphate Buffered Saline (Invitrogen, Cat#14190), 0.01% BSA (Jackson ImmunoResearch, Cat#001-000-162), 0.01% Tween-20. In the 1× assay buffer recombinant HDM2 protein, peptide and Lumi4-Tb Cryptate-conjugate mouse anti-6×His antibody (cisbio, Cat# Tb61HISTLB) were added and transferred to ProxiPlate PLUS (PerkinElmer, Cat#6008269), containing compounds so that final DMSO concentration is 0.1%. Final concentrations of reagents in the assay wells are 0.5 nM HDM2, 0.25 nM anti HIS (Tb label) antibody and 3 nM peptide. After two hour incubation at room temperature in a humidified chamber plates were read on EnVision plate reader with the following settings: excitation: UV, 340 nM, two emission filters: 520 nm and 495 nm respectively. Ratio of em520/em495 was used to calculate % inhibition and to obtain IC50 with 4-parameter logistic equation.
Measurement: IC50 = 31.88 nM (pChEMBL 7.5)
Descriptors: MW 564.61, ALogP 6.53, HBA 7, HBD 2, PSA 96.17, QED 0.25
Compound (SMILES): COc1cccc(N2CCN(c3c(C#N)ccc(COC(=O)NC(=N)N)c3F)CC2)c1
InChIKey: NTQXPSYTSSDDLR-UHFFFAOYSA-N
Target: Amine oxidase [copper-containing] 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16853
Assay: Biochemical Assay: The MAO-Glo Assay (commercial available from PROMEGA, #V1402) provides a sensitive method for the measurement of monoamine oxidase (MAO) activity (Valley, M. P. et al., 2006, Anal. Biochem. 359: 238-246) from a variety of tissues, biofluids or recombinant expressed or purified enzymes. As substrate a derivate of the beetle luciferin ((4S)-4,5-dihydro-2-(6-hydroxybenzothiazolyl)-4-thiazole-carboxylic acid) is used, which is oxidized at a primary amine moiety. After a spontaneous elimination and a catalyzed esterase reaction, the turnover of the luciferine by the luciferase is recorded as a signal of AOC3 activity.For the determination of AOC3 activity or compound inhibition potency, the compound inhibitors are dissolved in DMSO and adjusted to the respective assay concentration with reaction buffer (50 mM HEPES, 5 mM KCl, 2 mM CaCl2), 1.4 mM MgCl2, 120 mM NaCl, 0.001% (v/v) Tween 20, 100 μM TCEP, pH 7.4). An aliquot of 3 μL of the compound dilution is added to a 384 well plate (Optiplate, PS, flat bottom, white, PERKIN ELMER, #6007290) with a final DMSO concentration of 6.6%. Recombinant CHO cells, overexpressing the human (1500 cells/well), mouse (1000 cells/well) or rat (500 cells/well) AOC3 enzyme are diluted in reaction buffer and added in a volume of 15 μL to the wells. After incubation for 20 minutes at 37° C., 2 μL of MAO substrate (dissolved in DMSO at 16 mM, adjusted to assay concentration in reaction buffer to a final assay concentration of 20 μM) is added and further incubated for 60 minutes at 37° C. The turnover of the substrate is determined by the addition of 20 μL of the detection-mix which was generated by the addition of reconstitution buffer with esterase (PROMEGA, #V1402) to the luciferine detection reagent (PROMEGA, #V1402). After an incubation period of 20 minutes, the luminescent signal is measured with Envision 2104 Multilabel Reader (PERKIN ELMER).Alternative assays for the determination of the AOC3 enzymatic activity could be the extraction of 14C-labelled benzylamine reaction product or the Amplex Red Monoamine Oxidase reaction (Molecular Probes, Netherlands) as described in Gella et al. (Gella, A. et al., 2013, J. Neural Transm. 120: 1015-1018).
Measurement: IC50 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 426.45, ALogP 2.15, HBA 7, HBD 3, PSA 127.7, QED 0.49
Compound (SMILES): N#Cc1cc(F)cc(-c2cccc(-c3nc(C4CCCN4C#N)no3)c2)c1
InChIKey: NDFRWSFZHWXZPM-UHFFFAOYSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 30 (SINGLE PROTEIN), Homo sapiens
UniProt: Q70CQ3
Assay: Biochemical IC50 Assay: Dilution plates were prepared at 21 times the final concentration (2100 μM for a final concentration of 100 μM) in 50% DMSO in a 96-well polypropylene V-bottom plate (Greiner #651201). A typical 8-point dilution series would be 100, 30, 10, 3, 1, 0.3, 0.1, 0.03 μM final. Reactions were performed in duplicate in black 384 well plates (small volume, Greiner 784076) in a final reaction volume of 21 μl. Either 1 μl of 50% DMSO or diluted compound was added to the plate. USP30 was diluted in reaction buffer (40 mM Tris, pH 7.5, 0.005% Tween 20, 0.5 mg/ml BSA, 5 mM beta-mercaptoethanol) to the equivalent of 0.05 μl/well and 10 μl of diluted USP30 was added to the compound. Enzyme and compound were incubated for 30 min at room temp. Reactions were initiated by the addition of 50 nM of TAMRA labelled peptide linked to ubiquitin via an iso-peptide bond as fluorescence polarisation substrate. Reactions were read immediately after addition of substrate and following a 2 h incubation at room temperature. Readings were performed on a Pherastar Plus (BMG Labtech). λ Excitation 540 nm; λ Emission 590 nm.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 359.36, ALogP 4.03, HBA 6, HBD 0, PSA 89.74, QED 0.66
Compound (SMILES): CNC(=O)N1CCn2c(C3CC3)nc(-c3cccc4cc(-c5cnc(C)s5)ncc34)c2C1
InChIKey: GDTKVWDPFAIKAW-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: CBP AlphaScreen Assay: 5 nM GST-CBP(1081-1197) and 20 nM biotin-H4(1-21) Ac-K5/8/12/16 (AnaSpec. 64989) were incubated with varying concentrations of CBP inhibitors in 15 μL of buffer containing 50 mM HEPES 7.5, 100 nM NaCl, 1 mM TCEP, and 0.003% Tween-20. After 30 minutes incubation at room temperature, 15 μL of detection buffer (BPS Bio. 33006) containing 7 μg/mL of Glutathione AlphaLisa acceptor beads (Perkin Elmer AL109) and 14 μg/mL of Streptavidin donor beads (Perkin Elmer 676002) was then added to the previous mixture. The reaction was incubated for an additional 2 hours at room temperature, and the AlphaScreen signal was quantified using the Envision Multilabel plate reader. As negative control, GST-CBP(1081-1197) was incubated with the non-acetylated biotin-H4(1-21) peptide(AnaSpec. 62555) and in presence of 0.25% of final DMSO concentration.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 444.56, ALogP 4.56, HBA 6, HBD 1, PSA 75.94, QED 0.5
Compound (SMILES): C[C@H]1C(=O)CC[C@H]2[C@]1(C)CC[C@]13C[C@@]14CC[C@@]1(C)CCC(C)(C)C[C@]15C[C@]54CC[C@@]23C
InChIKey: HVUKEPJWWTWQAX-HLXBWPQESA-N
Target: Tyrosine-protein phosphatase non-receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P18031
Assay: Metabolic Measurements: Glucose in tail blood was measured using a glucometer (One-Touch Basic; Lifescan, CA). For glucose tolerance tests (GTTs), mice were fasted for 10 hours and then injected with 20% D-glucose (2 mg/g body weight) and the blood glucose was monitored immediately before and at 15, 30, 60 and 120 mins following the injection. For insulin tolerance tests (ITTs), 4-h fasted animals were given insulin (0.75 mU/g) and blood glucose was measured immediately before and at 30, 60 and 120 minutes postinjection. Serum insulin, cholesterol, triglycerides (Stanbio Labs, TX), BDNF (Abnova), IGF1 and IGFBPs (R&D Systems) were determined by enzyme-linked immunosorbent assay.
Measurement: Ki = 4000 nM (pChEMBL 5.400000095367432)
Descriptors: MW 422.7, ALogP 7.97, HBA 1, HBD 0, PSA 17.07, QED 0.39
Compound (SMILES): CC(C)(C)[S@@+]([O-])NC(CCC1CC1)(c1cccc(C#N)c1)c1ccc(F)c(NC(=O)[C@H]2CCCN2C(=O)OCc2ccccc2)c1
InChIKey: QQQBZZFKCABGOT-BJXIDKMJSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Activity Assay: The effect of compounds of the invention on human plasma kallikrein activity was determined using the chromogenic substrates. In these experiments, 2 nM kallikrein was incubated with 80 μM S2302 (H-D-Pro-Phe-Arg-p-nitroaniline) in the absence or presence of increasing concentrations of compounds of the invention in a final volume of 200 μL Tris-HCl buffer (200 mM NaCl; 2.5 mM CaCl2; 50 mM Tris-HCl, pH 7.8).After incubation at 30° C., the activity of kallikrein was measured as a change in absorbance at OD 405 nm using BioTek PowerWave X340 Microplate Reader.
Measurement: Ki > 100 nM (pChEMBL 7)
Descriptors: MW 644.81, ALogP 6.92, HBA 6, HBD 2, PSA 117.52, QED 0.22
Compound (SMILES): CNC1CC=C(c2cnc(N)c3c(-c4ccc(NS(=O)(=O)c5cc(F)ccc5F)c5ccccc45)nc(C(C)C)n23)CC1
InChIKey: KKJXZLDORGYBOD-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase/endoribonuclease IRE1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75460
Assay: Biochemical Assay: The kinase reactions were performed in 384 well white ProxiPlate-384 Plus plates (PERKIN Elmer 6008280) using 25 mM MOPS assay buffer with 1 mM dithiothreitol, 25 mM MgCl2, 12.5 mM i-glycerophosphate, 5 mM EGTA, and 50 ug/mL BSA. Test compounds were prepared on the day of assay and dispensed using D300 digital dispenser as a 10-point 1/2 log dilution series in duplicate, normalised to a final DMSO concentration of 3%. Test compounds were pre-incubated for 30 min at room temperature with 10 nM IRE1alpha kinase (E31-11G from Signal Chem) in 2.5 uL of assay buffer and the reaction started by addition of 2.5 uL of ATP in assay buffer, to give a final ATP concentration of 100 uM and 5 nM IRE1alpha kinase. After 4 hours incubation at room temperature the reactions were stopped and the kinase activity determined using the ADP-Glo reagent from Promega, according to the manufacturer's instructions. Luminescence was measured on a luminometer (EnVision, PerkinElmer) and IC50 values calculated by fitting a sigmoidal curve to percent inhibition of control versus Log 10 of compound concentration.
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 602.71, ALogP 6.49, HBA 7, HBD 3, PSA 114.41, QED 0.2
Compound (SMILES): C[C@@H](COc1ccc([C@H]2c3ccc(O)cc3CC[C@H]2c2ccccc2)cc1)N1CC[C@@H](CF)C1
InChIKey: XQBKILUAMDIMEC-VZYCLNPTSA-N
Target: Estrogen receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P03372
Assay: Biological Evaluation: The relative efficacies of Formula I compounds as inhibitors of an enzyme activity (or other biological activity) can be established by determining the concentrations at which each compound inhibits the activity to a predefined extent and then comparing the results. Typically, the preferred determination is the concentration that inhibits 50% of the activity in a biochemical assay, i.e., the 50% inhibitory concentration or IC50. Determination of IC50 values can be accomplished using conventional techniques known in the art. In general, an IC50 can be determined by measuring the activity of a given enzyme in the presence of a range of concentrations of the inhibitor under study. The experimentally obtained values of enzyme activity then are plotted against the inhibitor concentrations used. The concentration of the inhibitor that shows 50% enzyme activity (as compared to the activity in the absence of any inhibitor) is taken as the IC50 value. Analogously, other inhibitory concentrations can be defined through appropriate determinations of activity.
Measurement: EC50 = 0.037 nM (pChEMBL 10.430000305175781)
Descriptors: MW 459.61, ALogP 6.31, HBA 3, HBD 1, PSA 32.7, QED 0.45
Compound (SMILES): O=C(Cc1ccc(-c2ccnnc2)cc1)Nc1ccc(-c2ccccc2)cn1
InChIKey: TUVDODYIZQKXKU-UHFFFAOYSA-N
Target: Protein Wnt-3a (SINGLE PROTEIN), Homo sapiens
UniProt: P56704
Assay: Wnt-Luc Reporter Assay: Human embryonic kidney 293 cells (obtained from American Type Culture Collection, ATCC, Manassas, Va.) are cultured in DMEM medium (Gibco/Invitrogen, Carlsbad, Calif.) supplemented with 10% FBS (Gibco/Invitrogen, Carlsbad, Calif.), 50 unit/mL penicillin and 50 μg/mL of streptomycin (Gibco/Invitrogen, Carlsbad, Calif.) at 37° C. with 5% CO2 in air atmosphere. 293 cells in a 10 cm dish are co-transfected with 8 μg of STF-reporter plasmid containing a luciferase gene driven by Wnt-responsive elements and 2 μg of pcDNA3.1-Neo (Gibco/Invitrogen, Carlsbad, Calif.) with 30 μL of FuGENE6 (Roche Diagnostics, Indianapolis, Ind.) following the manufacturer's protocol. Stable cell lines (293 Wnt-Luc) were selected with 400 μg/mL of G418 (Gibco/Invitrogen, Carlsbad, Calif.). The 293 Wnt-Luc cells and L-cell Wnt3a cells (obtained from American Type Culture Collection, ATCC, Manassas, Va.) are trypsinized and co-cultured into a 384-well plate with DMEM medium supplemented with 2% FBS, and treated with different concentrations of a compound of the invention. After 24 hours, the firefly luciferase activities are assayed with the Bright-Glo Luciferase Assay System (Promega, Madison, Wis.). The IC50 is measured when the effect of the compound reduces the luminescence signal by 50%.
Measurement: IC50 = 0.28 nM (pChEMBL 9.550000190734863)
Descriptors: MW 366.42, ALogP 4.39, HBA 4, HBD 1, PSA 67.77, QED 0.57
Compound (SMILES): COc1ccccc1-c1cc(Nc2ccc(N3CCOCC3)c(CO)c2)ncn1
InChIKey: IJNQPWVZGIBNSB-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P50750
Assay: Fluorescent Polarization Assay: The activity of the compounds described in the present invention can be determined by measuring the phosphorylation of a fluorescently-labeled peptide by human CDK9/CyclinT kinase complex by fluorescent polarization using a commercially available IMAP Screening Express Assay Kit (Molecular devices).Test compounds were diluted in 100% DMSO to 5 mM stock concentration, then further dilutions were made in H2O or 100% DMSO to desirable concentrations. Each reaction consisted of 5 nM enzyme: CDK9/CyclinT (Proqinase cat#0371-0345-1), 400 nM TAMRA-Rbtide (synthetic 15-mer peptide derived from human retinoblastoma tumor suppressor protein labeled with TAMRA dye, Genecust Europe), 12 μM ATP (=Kmapp, Sigma-Aldrich) and kinase buffer: 20 mM MOPS pH 7 (Sigma-Aldrich), 1 mM DTT (Sigma-Aldrich), 10 mM MgCl2 (Sigma-Aldrich), 0.01% Tween 20 (Sigma-Aldrich).For each reaction, 4 or 6 μl containing TAMRA-Rbtide, ATP and kianse buffer were combined with 2 μl diluted compound in H2O or 0.028 μl compound in 100% DMSO. The kinase reaction was started by the addition of 2 μl diluted enzyme.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 392.46, ALogP 3.22, HBA 7, HBD 2, PSA 79.74, QED 0.67
Compound (SMILES): CS(=O)(=O)N1CC=C(c2cc3c(-c4ccc(F)c(F)c4F)ccnc3[nH]2)CC1
InChIKey: KUROHDVCHHZQRT-UHFFFAOYSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P50750
Assay: LANCE ULight TR-FRET Kinase Assay: CDK9 enzyme activities were measured using LANCE ULight TR-FRET kinase assay reagents (PerkinElmer, Waltham, Mass.). Compounds were directly added in 100% DMSO to white low volume assay plates (Perkin Elmer Proxiplate 6008289) using a Labcyte Echo acoustic dispenser. Assay reagents in serine/threonine kinase assay buffer containing 20 mM HEPES, 10 mM MgCl2, 100 mM Na3VO4, and 0.0075% Triton X-100. were added for final reaction mixture concentrations of 1000 μM ATP, 100 nM U-light MBP peptide (Perkin Elmer TRF0109M) and reaction initiated with 4 nM CDK9/Cyclin T1 (Carna Biosciences 04-110). The kinase reaction was carried out for 30 minutes before addition of stopping buffer to a final of 20 mM EDTA and 0.5 nM of LANCE Ultra Europium anti-phospho-MBP antibody (PerkinElmer TRF0201M) in LANCE detection buffer (PerkinElmer CR97-100). The reaction was equilibrated for 1 hour and the signal read in the Perkin Elmer Envision in TR-FRET mode (excitation at 320 nm and emission at 615/665 nm).
Measurement: IC50 = 130 nM (pChEMBL 6.889999866485596)
Descriptors: MW 407.42, ALogP 3.7, HBA 3, HBD 1, PSA 66.06, QED 0.67