lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): NC(=O)CN1C(=O)OC(c2ccc(F)cc2)(c2ccc(F)cc2)[C@@H]1c1ccccc1F
InChIKey: OAZDBSGPAMIQCO-NRFANRHFSA-N
Target: Intermediate conductance calcium-activated potassium channel protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O15554
Assay: Electrophysiology Assay: Compounds were tested using a PatchXpress® (Model 7000A, Molecular Devices, Union City Calif.) on CHO cells over-expressing h KCa3.1 at ChanTest Corporation, 14656 Neo Parkway, Cleveland, Ohio.The compounds I of the present invention exhibit IC50 values in the patch clamp assay of 10 nM to 10 μM, preferably 10 nM to 1 μM for patch clamp.
Measurement: IC50 = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 426.39, ALogP 4.03, HBA 3, HBD 1, PSA 72.63, QED 0.67
Compound (SMILES): Cc1c(-c2[nH]c3ccc(N4CCN(CCCS(C)(=O)=O)[C@H](C)C4)nc3c2C(C)C)cn2ncnc2c1C
InChIKey: WCODJMMIHBBLOC-GOSISDBHSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
Assay: TLR7/8/9 Inhibition Reporter Assays: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 0.46 nM (pChEMBL 9.34000015258789)
Descriptors: MW 523.71, ALogP 3.96, HBA 8, HBD 1, PSA 99.49, QED 0.39
Compound (SMILES): CN1CCN(c2ccc(-c3n[nH]c4ccc(-c5cccc(C(=O)NCc6ccncc6)c5F)nc34)cc2)CC1
InChIKey: ITZHXMQAXDZDMV-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: HPK1 Kinase Binding Assay: A stock solution of 1 mM test compound was prepared in DMSO. The compound plate was prepared by 3-fold and 11-point serial dilutions. 0.1 μL of the compound in DMSO was transferred from the compound plate to the white 384 well polystyrene plates. The assay buffer contained 50 mM HEPES, pH 7.5, 0.01% Tween-20, 5 mM MgCl2, 0.01% BSA, and 5 mM DTT. 5 μl of 4 nM active HPK1 (SignalChem M23-11G) prepared in the buffer was added to the plate. The enzyme concentration given was based on the given stock concentration reported by the vender. 5 μl of 18 nM tracer 222 (ThermoFisher PV6121) and 4 nM LanthaScreen Eu-Anti GST antibody (ThermoFisher PV5595) were added. After one hour incubation at 25° C., the plates were read on a PHERAstar FS plate reader (BMG Labtech). Ki values were determined.
Measurement: Ki < 100 nM (pChEMBL 7)
Descriptors: MW 521.6, ALogP 4.51, HBA 6, HBD 2, PSA 90.04, QED 0.34
Compound (SMILES): CC(C)c1c[nH]c2nccc(Oc3ccc(NC4=NCC(F)(CO)CO4)cc3F)c12
InChIKey: YFMUBHHQFKSMGH-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: High ATP Kinase Assay: For the assay 50 nl of a 100 fold concentrated solution of the test compound in DMSO was pipetted into either a black low volume 384 well microtiter plate or a black 1536 well microtiter plate (both Greiner Bio-One, Frickenhausen, Germany), 2 μL of a solution of TBK1 in aqueous assay buffer [50 mM HEPES pH 7.0, 10 mM MgCl2, 1.0 mM dithiothreitol, 0.05% (w/v) bovine serum albumine, 0.01% (v/v) Nonidet-P40 (Sigma), protease inhibitor mixture (Complete w/o EDTA from Roche, 1 tablet per 5 mL)] were added and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compounds to the enzyme before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 μL of a solution of adenosine-tri-phosphate (ATP, 1.67 mM=>final conc. in the 5 μL assay volume is 1 mM) and substrate (1.67 μM=>final conc. in the 5 μL assay volume is 1 μM) in assay buffer and the resulting mixture was incubated for a reaction time of 30 min at 22° C. The concentration of TBK1 was adjusted depending of the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical concentrations were in the range of 0.002-0.004 pg/mL. The reaction was stopped by the addition of 3 μL of a solution of TR-FRET detection reagents (0.33 μM streptavidine-XL665 [Cisbio Bioassays, Codolet, France], 2.5 nM anti-phosho-Serine antibody [Merck Millipore, STK antibody, cat. #35-002] and 1.25 nM LANCE EU-W1024 labeled anti-mouse IgG antibody [Perkin-Elmer, product no. AD0077]) in an aqueous EDTA-solution (167 mM EDTA, 0.13% (w/v) bovine serum albumin in 100 mM HEPES/NaOH pH 7.5).The resulting mixture was incubated 1 h at 22° C. to allow the formation of complex between the phosphorylated biotinylated peptide and the detection reagents.
Measurement: IC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 416.43, ALogP 4.12, HBA 6, HBD 3, PSA 91.76, QED 0.58
Compound (SMILES): O=c1[nH]ncc(N2Cc3cccc(OC4CCNC4)c3C2)c1Cl
InChIKey: CTIOPZOTSYMTNZ-UHFFFAOYSA-N
Target: Protein mono-ADP-ribosyltransferase TIPARP (SINGLE PROTEIN), Homo sapiens
UniProt: Q7Z3E1
Assay: Enzymatic Assay for Inhibition of PARP7: Displacement of Probe A, a biotinylated probe binding to the TIPARP active site, was measured using a time-resolved fluorescence energy transfer (TR-FRET) assay. 20 nL of a dose response curve of each test compound was spotted in black 384-well polystyrene proxiplates (Perkin Elmer) using a Mosquito (TTP Labtech). Reactions were performed in a 8 μL volume by adding 6 μL of TIPARP and Probe A in assay buffer (20 mM HEPES pH=8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002% Tween20), incubating with test compound at 25° C. for 30 min, then adding 2 μL of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin (Perkin Elmer). The final concentrations of TIPARP and Probe A were 6 nM and 2 nM, respectively. The final concentration of ULight-anti 6xHis and LANCE Eu-W1024 labeled streptavidin were 4 nM and 0.25 nM, respectively. Reactions were incubated at 25° C. for an additional 30 min, then read on an Envision platereader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 320 nm and emission of 615 nm and 665 nM with a 90 μs delay. The ratio of the 665/615 nm emission were calculated for each well to determine the amount of complex of TIPARP and Probe A in each well. Control wells containing a negative control of 0.25% DMSO vehicle or a positive control of 100 μM Example 190 were used to calculate the % inhibition as described below:% ⁢ ⁢ inhibition = 100 × TRF cmpd - TRF min TRF max - TRF minwhere TRFcmpd is the TR-FRET ratio from the compound treated well, TRFmin is the TR-FRET ratio from the Example 190-treated positive control well and TRFmax is the TR-FRET ratio from the DMSO-treated negative control well.The % inhibition values were plotted as a function of compound concentration and the following 4-parameter fit was applied to derive the IC50 the values:Y = Bottom + ( Top - Bottom ) ( 1 + ( X IC 50 ) Hill ⁢ ⁢ Coefficientwhere top and bottom are normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit. The Hill Coefficient is 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 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 332.79, ALogP 1.68, HBA 5, HBD 2, PSA 70.25, QED 0.89
Compound (SMILES): COc1cccc([C@H](C)NC(=O)N2CCN(c3ncnc4[nH]c(C)cc34)CC2)c1
InChIKey: CHOUVMUAILVFNK-HNNXBMFYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Cell-Based Assays of c-Kit Mutant Kinase Activity: The c-Kit mutant D816V inhibitors were assessed using an engineered BaF3-FL KIT D816V or BaF3-FL KIT V560G/D816V cell line. The BaF3-FL KIT D816V cell lines were created by introduction of KIT mutant (D816V) full length constructs that render the cells dependent on the introduced kinase for growth. Inhibitors of c-Kit mutant D816V kinase reduce or eliminate the mediated c-kit mutant D816V kinase activation, resulting in reduced cell proliferation of the BaF3-FL Kit mutant D816V cells. This inhibition is measured by the effect of compound concentration on cell growth to assess IC50 values. BaF3-FL KIT D816V cells were seeded at 1×104 cells per well of a 96 well cell culture plate in 50 μl of cell culture medium of RPMI Medium 1× (Invitrogen #11875-093) supplemented with 10% FBS (Invitrogen #10438), 1% Non Essential Amino Acids (Invitrogen #11140), 1% Penicillin Streptomycin (Invitrogen #15140), 1% L-Glutamine (Invitrogen #25030-081). Compounds were dissolved in DMSO at a concentration of 5 mM and were serially diluted 1:3 for a total of eight points and added to the cells to a final maximum concentration of 10 μM in 100 μl cell culture medium (final concentration 0.2% DMSO). Cells were also treated with Dasatinib as a positive control. The cells were incubated at 37° C., 5% CO2 for three days. ATPlite Buffer (Perkin Elmer #6016739) and substrate were equilibrated to room temperature, and enzyme/substrate Recombinant Firefly Luciferase/D-Luciferin was reconstituted. The cell plates were equilibrated to room temperature for 30 minutes, then lysed by addition of 25 uL per well of the ATPlite Reagent. The plate was mixed for 5 minutes on a plate shaker to lyse the cells. The plates were read on a Tecan Safire using Luminescence protocol modified to read 0.1s per well. The luminescence reading assesses the ATP content, which correlates directly with cell number such that the reading as a function of compound concentration is used to determine the IC50 value.
Measurement: Ki = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 394.48, ALogP 2.87, HBA 5, HBD 2, PSA 86.38, QED 0.71
Compound (SMILES): C[C@H](O)C(=O)N1CC[C@H](Oc2ccc(-c3ncnc(Nc4ccc(N5CCC[C@@H](O)C5)cc4)n3)cc2C#N)[C@H](F)C1
InChIKey: IFTBMEURFLPLJP-CYQFXYHLSA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: TBK1 and IKKE Biological Assays: Enzymatic activity of IKKε and TBK1 was measured using a homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay that monitors enzyme dependent phosphorylation of a biotinylated serine/threonine peptide substrate. An increase in the amount of phosphorylated peptide results in an increase in TR-FRET signal. TBK1 and IKKε were expressed and purified as full length recombinant proteins. Detection reagents for the assay were purchased from Cisbio. TBK1 and IKKε enzymes were assayed under initial rate conditions in the presence of 2×Km ATP (40-80 μM) and 1 μM peptide, hepes (pH 7), 0.1 mM orthovanadate, 0.02% NaN3, 0.01% BSA, 10 mM MgCl2, 0.01% (v/v) tritonX, 1 mM dithiothreitol, 0.5% (v/v) DMSO at the following concentrations for each enzyme: TBK1 at 2.5 nM and IKKε at 0.3 nM. After an assay reaction time of 240 minutes at 25° C., reactions were terminated with EDTA.Amount of phosphorylated peptide was determined by the addition of 125 nM streptavidin XL665 and europium cryptate labeled anti-phospho monoclonal antibody and the resulting TR-FRET signal was recorded on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 200 μs read window). Data was normalized based on a positive (1 μM Staurosporine) and negative (DMSO) controls and IC50 values calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations.
Measurement: IC50 = 4000 nM (pChEMBL 5.400000095367432)
Descriptors: MW 561.62, ALogP 2.81, HBA 10, HBD 3, PSA 147.73, QED 0.39
Compound (SMILES): CO[C@@H]1CC1NC(=O)c1cnn2c(N(C)C(=O)OC(C)(C)C)cc(Nc3cccn([C@H]4C[C@H](F)C4)c3=O)nc12
InChIKey: BLNXRIOVZFCINH-HGFASETLSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: TYK2 JH2 Domain Binding Assay: The binding reaction was assembled by combining 16 μl of DNA-tagged kinase extract, 3.8 μl liganded affinity beads, and 0.18 μl test compound (PBS/0.05% Tween 20/10 mM DTT/0.1% BSA/2 μg/ml sonicated salmon sperm DNA)]. Extracts were used directly in binding assays without any enzyme purification steps at a ≥10,000-fold overall stock dilution (final DNA-tagged enzyme concentration <0.1 nM). Extracts were loaded with DNA-tag and diluted into the binding reaction in a two step process. First extracts were diluted 1:100 in 1× binding buffer (PBS/0.05% Tween 20/10 mM DTT/0.1% BSA/2 μg/ml sonicated salmon sperm DNA) containing 10 nM DNA-tag. This dilution was allowed to equilibrate at room temperature for 15 minutes and then subsequently diluted 1:100 in 1× binding buffer. Test compounds were prepared as 111× stocks in 100% DMSO. Kds were determined using an 11-point 3-fold compound dilution series with three DMSO control points. All compounds for Kd measurements are distributed by acoustic transfer (non-contact dispensing) in 100% DMSO
Measurement: Kd < 0.2 nM (pChEMBL 9.699000358581543)
Descriptors: MW 541.58, ALogP 3.2, HBA 10, HBD 2, PSA 132.09, QED 0.47
Compound (SMILES): CCCN(C(=O)/C=C/c1ccc(OC)cc1)c1ccccc1
InChIKey: GOKYNSVWGIDZNN-SDNWHVSQSA-N
Target: Transient receptor potential cation channel subfamily M member 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q7Z2W7
Assay: Assay on TRPM8 Modulators: A test comparable with that previously described in the literature by Behrendt H. J. et al., Br. J. Pharmacol. 141, 2004, 737-745, is carried out. The agonisation or antagonisation of the receptor can be quantified by means of a Ca2+-sensitive dye (e.g. FURA, Fluo-4, etc.). Agonists on their own bring about an increase in the Ca2+-signal; antagonists in the presence of, for example, menthol bring about a reduction in the Ca2+-signal (in each case detected by means of the Fluo-4 dye, which due to the Ca2+ has other fluorescent properties).To begin with, in a manner known per se, in cell culture flasks a fresh culture of transformed HEK cells is prepared. The HEK293-TRPM8 test cells are removed using trypsin from the cell culture flasks and 40 000 cells/well are sown with 100 μl medium in 96-well plates (Greiner #655948 Poly-D-lysine coated). In order to induce the TRPM8 receptor the growth medium tetracycline is mixed in (DMEM/HG, 10% FCS tetracycline-free, 4 mM L-glutamine, 15 μg/ml blasticidin, 100 μg/ml hygromycin B, 1 μg/ml tetracycline). The next day the cells are charged with Fluo-4Am dye and the test is performed. The procedure is as follows:    Addition of 100 μl/well of dye solution Ca-4 Kit (RB 141, Molecular Devices) per 100 μl of medium (DMEM/HG, 10% FCS tetracycline-free, 4 mM L-glutamine, 15 μg/ml blasticidin, 100 μg/ml hygromycin B, 1 μg/ml tetracycline).    Incubation in the incubator for 30 minutes/37° C./5% CO2, 30 minutes/RT.    Preparation of the test substances (different concentrations in 200 μl HBSS buffer), and of positive controls (different concentrations of menthol or icilin or ionomycin in 200 μl HBSS buffer) and negative controls (just 200 μl of HBSS buffer).    Addition of the test substances in quantities of 50 μl/well and measurement of the change in fluorescence (e.g. in the FLIPR assay device, Molecular Devices or NovoStar, BMG) at 485 nm excitation, 520 nm emission, and evaluation of the effective strength of the various substances/concentrations and determination of the EC50 values.The test substances are used in triplicate in concentrations of between 0.1 and 200 μm in the assay. Normally the compounds are kept ready in DMSO solutions and diluted for the assay to a maximum DMSO concentration of 2%.
Measurement: EC50 = 1700 nM (pChEMBL 5.769999980926514)
Descriptors: MW 295.38, ALogP 4.15, HBA 2, HBD 0, PSA 29.54, QED 0.75
Compound (SMILES): Cc1cc(CC#N)cc(F)c1-c1ncc2[nH]nc(-c3cnc(N4CCN(C)CC4)nc3)c2n1
InChIKey: BQVWGPRXHJKTAB-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: Kinase Binding Assay: A stock solution of 1 mM test compound was prepared in DMSO. The compound plate was prepared by 3-fold and 11-point serial dilutions. 0.1 μL of the compound in DMSO was transferred from the compound plate to the white 384 well polystyrene plates. The assay buffer contained 50 mM HEPES, pH 7.5, 0.01% Tween-20, 5 mM MgCl2, 0.01% BSA, and 5 mM DTT. 5 μl of 4 nM active HPK1 (SignalChem M23-11G) prepared in the buffer was added to the plate. The enzyme concentration given was based on the given stock concentration reported by the vender. 5 μl of 18 nM tracer 222 (ThermoFisher PV6121) and 4 nM LanthaScreen Eu-Anti GST antibody (ThermoFisher PV5595) were added. After one hour incubation at 25° C., the plates were read on a PHERAstar FS plate reader (BMG Labtech).
Measurement: Ki < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 443.49, ALogP 2.74, HBA 8, HBD 1, PSA 110.51, QED 0.51
Compound (SMILES): Cc1c(F)cccc1C1(C(=O)NO)CCCC1
InChIKey: NKYNMYBZTUJQRI-UHFFFAOYSA-N
Target: Histone deacetylase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P56524
Assay: Inhibition Assay: The potency of Class IIa Histone Deacetylase (HDAC) inhibitors is quantified by measuring the Histone Deacetylase 4 (HDAC4) catalytic domain enzymatic activity using the fluorogenic substrate, Boc-Lys(Tfa)-AMC. The substrate is deacetylated to Boc-Lys-AMC by HDAC4. Cleavage by trypsin results in the release of the fluorophore AMC from the deacetylated substrate. The fluorescence of the sample is directly related to the histone deacetylase activity in the sample.Serially dilute HDAC inhibitor compounds. Serial dilutions of the HDAC inhibitors and control reference compound (1-(5-(3-((4-(1,3,4-oxadiazol-2-yl)phenoxy)methyl)-1,2,4-oxadiazol-5-yl)thiophen-2-yl)-2,2,2-trifluoroethanone) are made by first resuspending the lyophilized compound to a final concentration of 10 mM in 100% dimethyl sulfoxide (DMSO). Stocks of 6 μl aliquots of the 10 mM compound in DMSO are prepared and stored at −2° C.
Measurement: IC50 = 270 nM (pChEMBL 6.570000171661377)
Descriptors: MW 237.27, ALogP 2.45, HBA 2, HBD 2, PSA 49.33, QED 0.61
Compound (SMILES): O=S(=O)(C1CCCCC1)N1CC(Nc2ccc(F)c(F)c2)C(c2ccccc2)C1
InChIKey: CHCDKHQODFVYFC-UHFFFAOYSA-N
Target: Alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P26572
Assay: Radioligand Binding Assay: Radioligand binding assays using recombinant CHO cell membranes expressing human GlyT1: Radioligand binding to human GlyT1c transporter-expressing membranes was determined as described in Mezler et al., Molecular Pharmacology 74:1705-1715, 2008.
Measurement: Ki < 10000 nM (pChEMBL 5)
Descriptors: MW 420.53, ALogP 4.51, HBA 3, HBD 1, PSA 49.41, QED 0.77
Compound (SMILES): CCN(CCN1CCCC1)c1nc2nc(-c3noc(=O)[nH]3)nc(N[C@H](C)C3CCC3)c2n1Cc1ccc(C(F)(F)F)cc1
InChIKey: RDSVGBUZXRAKJJ-GOSISDBHSA-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 = 23.87 nM (pChEMBL 7.619999885559082)
Descriptors: MW 599.66, ALogP 4.76, HBA 10, HBD 2, PSA 121, QED 0.25
Compound (SMILES): Cc1noc(-c2sccc2NC(=O)Cc2cccc3ccccc23)n1
InChIKey: OICBMTSZGYFITJ-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 10 (SINGLE PROTEIN), Homo sapiens
UniProt: P53779
Assay: HTRF Assay: (1) Assay buffer #1 with 0.0025% Tween and 1 mM DTT was prepared using JNK buffer stock solution.(2) Assay buffer #2 with 0.025% BSA was prepared using the assay buffer #1.(3) Test compound solution preparation: 5× compound solution was prepared using assay buffer #1 with 5% DMSO. The compound solution (10 μl/well) was added to a 384-well plate (Corning, Cat No. 3654).(4) aJNK1, 2 or 3 preparation: aJNK stock (100 μg/ml) at −80° C. was thawed on ice and an aJNK (1.6 ng/ml) solution was prepared using assay buffer #2. The aJNK solution (20 μl/well) was added to the plate and the plate was shaken. The enzyme was incubated with the compound at RT for 10 min.(5) ATP/substrate solution preparation: ATP and substrate stocks were thawed on ice. 2.5×ATP/substrate (e.g., 25 μM or 2.5 mM ATP/50 nM EPIW-1) was prepared using assay buffer #1. The ATP/substrate (20 μl/well) was added to the plate and the plate was shaken. The plate was incubated at RT for 15 min. Note: In an exemplary assay, the final ATP concentration was about 1 mM.(6) EDTA preparation: 30 mM EDTA was prepared using 0.5M EDTA stock and assay buffer #1. The EDTA (10 μl/well) was added to the plate to quench the enzyme reaction and the plate was shaken well.(7) Detection reagent preparation: Eu-63 and SA-XL stocks were thawed on ice. 4× Eu-63/SA-XL (2 nM Eu-63/9.4 nM SA-XL) was prepared using JNK detection buffer. The Eu-63/SA-XL solution (20 μl/well) was added to the plate and the plate was shaken. The plate was incubated at RT for 1 hr before reading the plate on LJL using ratiometric method named HTRF.
Measurement: IC50 = 5050 nM (pChEMBL 5.300000190734863)
Descriptors: MW 349.42, ALogP 4.44, HBA 5, HBD 1, PSA 68.02, QED 0.59
Compound (SMILES): Cc1cccc(OCC(C)C)c1-n1nc2c(c1-c1ccc(NC(N)=O)cc1)CN(Cc1cc(F)ccc1Cl)CC2
InChIKey: IDKVXOUTEVPGDY-UHFFFAOYSA-N
Target: C5a anaphylatoxin chemotactic receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21730
Assay: chemotaxis assay: Optionally, compounds may be further assayed for their ability to inhibit chemotaxis in cells. Chemotaxis assays are performed using 5 μm pore polycarbonate, polyvinylpyrrolidone-coated filters in 96-well chemotaxis chambers (Neuroprobe; Gaithersburg, Md.) using chemotaxis buffer (Hank's balanced salt solution (HBSS) and 1% FBS). C5aR ligands (i.e., C5a, R&D Systems; Minneapolis, Minn.) are use to evaluate compound mediated inhibition of C5aR mediated migration. Other chemokines (i.e., SDF-1α; R&D Systems; Minneapolis, Minn.) are used as specificity controls. The lower chamber is loaded with 29 μl of chemokine (i.e., 0.03 nM C5a) and varying amounts of compound; the top chamber contains 100,000 U937 or neutrophil cells in 20 μl. The chambers are incubated 1.5 hours at 37° C., and the number of cells in the lower chamber quantified either by direct cell counts in five high powered fields per well or by the CyQuant assay (Molecular Probes), a fluorescent dye method that measures nucleic acid content and microscopic observation.
Measurement: IC50 < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 562.09, ALogP 6.72, HBA 5, HBD 2, PSA 85.41, QED 0.25
Compound (SMILES): N#Cc1cc2c(cc1Oc1ccc(C(=O)NCCc3ccccc3)cc1)OCCC2C(=O)O
InChIKey: ZOOMZPGPIDJKPJ-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Binding Inhibition Assay (1% HSA): The coding sequence of human DP2 was introduced into the human Leukemic cell line K562 by electroporation and stable clones expressing DP2 were obtained by limiting dilution followed by cell surface staining with a rat monoclonal antibody specific for human DP2. Membranes were prepared from one of these DP2 expressing clones and used to determine the ability of compounds of the present invention to inhibit binding of prostaglandin D2 (PGD2) to its receptor DP2 in the presence of one or more of the following serum protein concentrations, 1% HSA, by the following procedure. Membranes (1.25 μg/well for 1% HSA) were mixed with 3H-labeled PGD2 and various concentrations of test compounds in 150 μL of binding buffer (50 mM Tris-HCl, pH 7.4, 40 mM MgCl2, 0.1% bovine serum albumin, 0.1% NaN3) in 96-well U-bottom polypropylene plates. After incubation for 60 minutes at room temperature, the assay was transferred to a filtration plate (#MAFB; Millipore Corporation, Bedford, Mass.), and washed three times with binding buffer. Radioactivity was measured by a scintillation counter (TopCount; PerkinElmer Life Sciences, Boston, Mass.). Nonspecific binding was determined by incubations in the presence of 1 μM unlabeled PGD2 or 5 μM of a known DP2 antagonist. EC50 values for inhibition of binding were determined for each compound tested from the inflexion point of a standard 4-parameter logistical curve fitted to the values obtained. Compounds of the invention had EC50 values less than 5 micromolar in one or more of the binding assays.
Measurement: EC50 = 37.4 nM (pChEMBL 7.429999828338623)
Descriptors: MW 442.47, ALogP 4.27, HBA 5, HBD 2, PSA 108.65, QED 0.57
Compound (SMILES): C[C@H](NC(=O)[C@@H](CCc1ccccc1)NCc1ccc(N2CCOCC2)cc1)C(=O)NCc1ccc(C(=N)N)cc1
InChIKey: QDWHWLJJDFWNPU-MUAVYFROSA-N
Target: Mannan-binding lectin serine protease 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00187
Assay: Enzymatic Assay for MASP-2: The assay was run at room temperature in an assay buffer containing 20 mM Hepes, pH 7.4, 140 mM NaCl and 0.1% Tween 20. Assay parameters were adjusted such that the assay was linear with respect to time, enzyme and substrate concentrations. Under these optimized assays conditions, IC50 values were equivalent to Ki values, except in a few cases of tight binding inhibitors. Cases of tight binding or possible slow binding inhibitors were handled by the methods described in Copeland R. A. (2013) Evaluation of Enzyme Inhibitors in Drug Discovery. 2nd Ed., John Wiley and Sons, Inc., Chapters 5-7.
Measurement: Ki < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 556.71, ALogP 2.72, HBA 6, HBD 5, PSA 132.57, QED 0.16
Compound (SMILES): COC1CCN(c2nccc(Nc3cc4c(cn3)nc(COCCCN3CCN(CCCOc5ccc6c(c5)C(=O)N(C5CCC(=O)NC5=O)C6=O)CC3)n4C(C)C)n2)CC1
InChIKey: INRMIESAVNLNEO-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: TR-FRET Assay for ERBB Kinases: All compounds and PROTACs were serially diluted in three-fold increments using 100% DMSO, followed by an intermediate 10-fold dilution using Buffer A (50 mM HEPES, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 1 mM DTT, and 0.1% Pluronic F-68). Two microliters of serially diluted compound or PROTAC were then transferred to black 384-well Proxiplates (PerkinElmer, #6008260) using an Integra Viaflo96. Next, 10 uL of protein kinase in Buffer A was added to each well of the assay plate and pre-incubated with compound for 10 minutes. Kinase reactions were then initiated by addition of 5 uL substrate mix containing 3 mM ATP and 30 uM fluorescein-labeled Poly-GluTyr (Thermo Fisher, #PV3610) in Buffer A and allowed to proceed for 10 minutes at room temperature. Reactions were quenched by addition of a 5 uL mixture containing 5 nM LanthaScreen® Tb-pY20 Antibody (Thermo Fisher, #PV3552) and 40 mM EDTA in Buffer A. Assay plates were then read using a Synergy2 (Biotek Instruments, Winooski, Vt.) via excitation thru a 340/20 nm bandpass filter and emission collected thru 490/10 nm (donor) and 520/25 nm (acceptor) bandpass filters. The final kinase concentrations used for each 15 uL reaction were as follows: 0.2 nM EGFR Exon20NPG (SignalChem, #E10-132GG), 0.1 nM wild type EGFR (BPS Bioscience, #40187), 0.3 nM EGFR L858R/T790M/C797S (BPS Bioscience, #40351), 0.1 nM EGFR L858R (BPS Bioscience, #40189), 0.4 nM L858R/T790M (BPS Bioscience, #40350), InM EGFR Dell9 (SignalChem, #E10-122JG), 10 nM EGFR Dell9 T790M (SignalChem, #E10-122KG), 0.3 nM Her2 (BPS Bioscience, #40230), 15 nM Her2 InsYVMA (SignalChem, #E27-13BG).
Measurement: IC50 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 837.98, ALogP 3.56, HBA 16, HBD 2, PSA 189.48, QED 0.12
Compound (SMILES): COC(=O)N[C@H]1CCC[C@@H]1[C@](C#N)(c1ccccc1)C1CCN(CC2CN(c3ccc(S(=O)(=O)C(F)(F)F)cc3)C2)CC1
InChIKey: DCFDPTPAVDCHET-TWLDFKIOSA-N
Target: Menin (SINGLE PROTEIN), Homo sapiens
UniProt: O00255
Assay: Fluorescence Polarization (FP) Competitive Binding Assay: A fluorescence polarization (FP) competitive binding assay was used to determine the binding affinities of representative menin inhibitors. A FAM labeled fluorescent probe was designed and synthesized based on a MLL1 peptide (FAM-MM2). Equilibrium dissociation constant (Kd) value of FAM-MM2 to menin protein was determined from protein saturation experiments by monitoring the total fluorescence polarization of mixtures composed with the fluorescent probe at a fixed concentration and the protein with increasing concentrations up to full saturation. Serial dilutions of the protein were mixed with FAM-MM2 to a final volume of 200 μl in the assay buffer (PBS with 0.02% Bovine γ-Globulin and 4% DMSO. 0.01% Triton X-100 was added right before assays). Final FAM-MM2 concentration was 2 nM. Plates were incubated at room temperature for 30 minutes with gentle shaking to assure equilibrium. FP values in millipolarization units (mP) were measured using the Infinite M-1000 plate reader (Tecan U.S., Research Triangle Park, N.C.) in Microfluor 1 96-well, black, v-bottom plates (Thermo Scientific, Waltham, Mash.) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Kd value of FAM-MM2, which was calculated by fitting the sigmoidal dose-dependent FP increases as a function of protein concentrations using Graphpad Prism 6.0 software (Graphpad Software, San Diego, Calif.), is determined as 1.4 nM.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 618.72, ALogP 5.11, HBA 7, HBD 1, PSA 102.74, QED 0.44
Compound (SMILES): CC1(C)OCCN(c2ccc(Nc3cc(-c4c(F)cccc4F)nc4c3C(=O)NC4)nc2)C1=O
InChIKey: WCSSPRUGMSWNCQ-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Tyk2 & JAK2 Caliper Assay: The caliper machine employs an off chip mobility shift assay to detect phosphorylated peptide substrates from kinase assays, using microfluidics technology. The assays are carried out at ATP concentration equivalent to the ATP Km, and at 1 mM ATP. Compounds are serially diluted in DMSO then further diluted in assay buffer (25 mM HEPES, pH 7.5, 0.01% Brij-35, 0.01% Triton, 0.5 mM EGTA). 5 ul of diluted compound was added into wells first, then 10 ul of enzyme mix was added into wells, followed by 10 uL of substrate mix (peptide and ATP in 10 mM MgCl2) to start reaction. Reaction was incubated at 28° C. for 25 min and then added 25 ul stop buffer (100 mM HEPES, 0.015% Brij-35, 50 mM EDTA), followed by reading with Caliper. JAK2 at 1 nM final concentration and TYK2 at 9.75 nM are from Carna, and substrates used are ATP at 20 and 16 uM, respectively. JAK2 assay uses peptide 22 and TYK2 uses peptide 30 (Caliper), each at 3 uM.
Measurement: Ki < 10 nM (pChEMBL 8)
Descriptors: MW 465.46, ALogP 3.55, HBA 6, HBD 2, PSA 96.45, QED 0.61
Compound (SMILES): Cc1cc2c(-c3ccnc(CN)c3)cc(COc3ccccc3CC(=O)O)cc2o1
InChIKey: VORBWAPIZUYXOP-UHFFFAOYSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Factor D Esterolytic Assay: An established esterolytic assay for the measurement of Factor D activity and inhibition of Factor D activity was used (Kam, C. M.; McRae, B. J.; Harper, J. W.; Niemann, M. A.; Volanakis, J. E.; Powers, J. C. Human complement proteins D, C2, and B Active site mapping with peptide thioester substrates. J Biol. Chem. 1987, 262, 3444-3451). For this assay Z-Lys-SBzl, 1.29 mM (Kim, S.; Narayana, S. V. L; Volanakis, J. E. Mutational analysis of the substrate binding site of human complement Factor D. Biochemistry. 1994, 33, 14393-14399.) was used as the substrate for Factor D (104 mM). Hydrolysis of this compound by Factor D liberated a free sulfhydryl group which is then reacted with 5,5′-dithiobis(2nitrobenzoic acid) producing an intense yellow color (Habeeb, A. F. S. A. Reaction of protein sulfhydryl groups with Ellman's Reagent. Methods in Enzymol. 1976, 25, 457-464). The assays were performed in 96 well microtiter plates and rates of hydrolysis were monitored at 405 nm on a Biotek Synergy H1 plate reader. Hydrolysis rates were reported as change in mOD/min. The assay was conducted in 100 mM HEPES, 500 mM NaCl, pH 7.5 containing 10% DMSO in a final volume of 50 μL per well.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 402.45, ALogP 4.47, HBA 5, HBD 2, PSA 98.58, QED 0.47
Compound (SMILES): COc1ccc(Cn2c(=O)n3ncnc3c3c4c(sc32)N(C(=O)C2CC2)CCC4)cc1
InChIKey: YYOFDWNEAOKVRN-UHFFFAOYSA-N
Target: Dual specificity calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1B (SINGLE PROTEIN), Homo sapiens
UniProt: Q01064
Assay: IMAP TR-FRET assay: The IMAP TR-FRET PDE assay was optimized for concentration of enzyme, Calmodulin, cAMP or cGMP substrate, DMSO tolerance, and incubation time. Into each well of a solid white 1536 well plate (Corning) was dispensed 250 pg full-length recombinant NH-terminal GST tagged human PDE1b enzyme (BPS Bioscience Cat #60011, San Diego, Calif.) in 2.5 μL IMAP BSA reaction buffer (Molecular Devices, Sunnyvale, Calif.) containing 10 U/mL Calmodulin and 2.5 mM CaCl2 (Sigma Aldrich.) After a brief centrifugation, 30 nL compound was added by transfer from 1 mM stock in DMSO using a Kalypsys 1536 Pintool. Plates were incubated for 5 minutes at room temperature before dispensing 1.5 μL of 533 nM 5-carboxy fluorescein (FAM)-labeled cAMP (Molecular Devices, Sunnyvale, Calif.) for a final concentration of 200 nM. After a brief centrifugation, the plates were incubated for 30 minutes at room temperature. The assay was terminated by adding 5 μL IMAP binding reagent/Tb complex (Molecular Devices, Sunnyvale, Calif.) to each well. Plates were incubated 1 hour at room temperature and read on a Viewlux multimode plate reader (Perkin Elmer). The instrument was set to excite using the DUG11 filter and measure using 490/10 nm and 520/10 nm filters.
Measurement: EC50 < 100 nM (pChEMBL 7)
Descriptors: MW 435.51, ALogP 2.85, HBA 8, HBD 0, PSA 81.73, QED 0.49
Compound (SMILES): C[C@@H]1CN(S(C)(=O)=O)CC[C@@H]1Nc1ncc(C(F)(F)F)c(-c2cn(-c3ccc(CO)cc3Cl)cn2)n1
InChIKey: ONDDVOHVPKZOHC-DYVFJYSZSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24864
Assay: HTRF Enzyme Activity Assay: CDK2/Cyclin E1 enzyme activity assays utilize full-length human CDK2 co-expressed as N-terminal GST-tagged protein with FLAG-Cyclin E1 in a baculovirus expression system (Carna Product Number 04-165). Assays were conducted in white 384-well polystyrene plates in a final reaction volume of 8 μL. CDK2/Cyclin E1 (0.25 nM) was incubated with the compounds of the Examples (40 nL serially diluted in DMSO) in the presence of ATP (50 μM or 1 mM) and 50 nM ULight-labeled eIF4E-binding protein 1 (THR37/46) peptide (PerkinElmer) in assay buffer (containing 50 mM HEPES pH 7.5, 1 mM EGTA, 10 mM MgCl2, 2 mM DTT, 0.05 mg/mL BSA, and 0.01% Tween 20) for 60 minutes at room temperature. The reactions were stopped by the addition of EDTA and Europium-labeled anti-phospho-4E-BP1 antibody (PerkinElmer), for a final concentration of 15 mM and 1.5 nM, respectively. HTRF signals were read after 1 hour at room temperature on a PHERAstar FS plate reader (BMG Labtech). Data was analyzed with IDBS XLFit and GraphPad Prism 5.0 software using a three or four parameter dose response curve to determine IC50 for each compound.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 544.99, ALogP 3.58, HBA 8, HBD 2, PSA 113.24, QED 0.49
Compound (SMILES): CN(C)Cc1cncc(-c2cc3c(-c4nc5c(-c6ccsc6)ccnc5[nH]4)n[nH]c3cc2F)c1
InChIKey: GSDFEBIUWXNBOO-UHFFFAOYSA-N
Target: Proto-oncogene Wnt-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P04628
Assay: The screening assay for Wnt activity: Reporter cell lines can be generated by stably transducing cancer cell lines (e.g., colon cancer) or primary cells (e.g., IEC-6 intestinal cells) with a lentiviral construct that includes a Wnt-responsive promoter driving expression of the firefly luciferase gene.SW480 colon carcinoma cells were transduced with a lentiviral vector expressing luciferase with a human Sp5 promoter consisting of a sequence of eight TCF/LEF binding sites. SW480 cells stably expressing the Sp5-Luc reporter gene and a hygromycin resistance gene were selected by treatment with 150 μg/ml of hygromycin for 7 days. These stably transduced SW480 cells were expanded in cell culture and used for all further screening activities. For Sp5-Luc reporter gene assays, the cells were plated at 10,000 cells/well in 96-well plates with growth medium containing 10% fetal calf serum and incubated overnight at 37° C. and 5% CO2. Each compound was dissolved in DMSO as a 10 mM stock in standard j-vials and used to prepare compound source plates in dose-response format with 3-fold serial dilutions and a 10 mM top concentration. Compound transfer from serially diluted source plates to assay plates containing the cells was accomplished using a pintool (Multimek 96, Beckman equipped with V&P Scientific FP1S50H pins) based liquid handling protocol. This protocol used a slotted pin to transfer 50 nl of compound from a source plate well to an assay plate well containing 50 μl of cells in growth medium. The 1000-fold dilution resulted in a final DMSO concentration of 0.1% on the cells in each well. Control wells received 50 nl of DMSO treatment for normalization and calculating IC50 values. The treated cells were incubated at 37° C. and 5% CO2 for an additional forty-two hours. Following incubation, the growth medium was removed and 50 μl of BrightGlo luminescence reagent (Promega) was added to each well of the 96-well assay plates. The plates were placed on an orbital shaker for 5 min and then luminescence was quantified on the Victor3 (Perkin Elmer) plate reader. Readings were normalized to DMSO only treated cells, and normalized activities were utilized for IC50 calculations using the dose-response log (inhibitor) vs. responsevariable slope (four parameters) nonlinear regression feature available in GraphPad Prism 5.0 or 6.0.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 469.55, ALogP 5.49, HBA 6, HBD 2, PSA 86.38, QED 0.35
Compound (SMILES): Cc1nc2ccc(N(C)C(=O)Cn3nc(-c4cccc(Cl)c4)c4ccc(OC5CCCCC5)cc4c3=O)cc2o1
InChIKey: IYEFVRHTAWRFDE-UHFFFAOYSA-N
Target: Cystic fibrosis transmembrane conductance regulator (SINGLE PROTEIN), Homo sapiens
UniProt: P13569
Assay: CFTR-YFP Assay: This protocol is designed to selectively screen small molecule compounds for F508del CFTR corrector activities in the YFP (yellow fluorescent protein) flux assay. In this protocol, the cells are incubated with testing compounds for 24 hours, washed with PBS, stimulated with forskolin and a standard potentiator, and fluorescence in each plate well is measured kinetically read on a 384-well plate reader, such as the Hamamatsu FDSS-6000. YFP fluorescence intensity values are acquired at high speed before and after iodide buffer is injected to the assay cells. Iodide enters the cells via active CFTR channels in the plasma membrane, and quenches the YFP fluorescence. The rate of fluorescence quenching is proportionate to the total CFTR activity in the cell membrane. F508del CFTR correctors increase the number of CFTR molecules in the testing cell plasma membrane, and thereby accelerate YFP quenching.
Measurement: EC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 557.05, ALogP 6.54, HBA 7, HBD 0, PSA 90.46, QED 0.24
Compound (SMILES): C=CC(=O)Nc1cc(C)ccc1Nc1nc(Nc2cc(OC)nc(N3CCC3)c2)ncc1C(F)(F)F
InChIKey: LCUPSSOLCGNKKF-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P27361
Assay: Omnia Assay: The protocol below describes continuous-read kinase assays to measure potency of compounds against activated ERK1 enzyme. The mechanics of the assay platform are best described by the vendor (Invitrogen, Carlsbad, Calif.) on their website at the following URL: invitrogen.com/site/us/en/home.html.Briefly, a 1.25× stock of ERK1 enzyme (14-439-K) from Millipore (Billerica, Mass.), 5×ATP (AS001A) and ST17-Sox conjugated peptide substrate (KNZ1171C) were prepared in 1× kinase reaction buffer consisting of 20 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 5 mM β-glycerophosphate, 5% glycerol (10× stock, KB002A) and 0.2 mM DTT. 10 μL of ATP/ST17-sox peptide substrate mix was combined with 0.5 μL volume of 100% DMSO and serially diluted compounds were prepared in 100% DMSO in a Corning (#3574) 384-well, white, non-binding surface microtiter plate (Corning, N.Y.). Kinase reactions were started with the addition of 40 μL of ERK1 solution and monitored every 71 seconds for 30-240 minutes at λex360/λem485 in a Synergy plate reader from BioTek (Winooski, Vt.). At the conclusion of each assay, progress curves from each well were examined for linear reaction kinetics and fit statistics (R2, 95% confidence interval, absolute sum of squares). Initial velocity (0 minutes to 30+ minutes) from each reaction was determined from the slope of a plot of relative fluorescence units vs time (minutes or seconds) and then plotted against inhibitor concentration to estimate AppIC50 from log [Inhibitor] vs Response, Variable Slope model in GraphPad Prism from GraphPad Software (San Diego, Calif.).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 499.5, ALogP 5.03, HBA 8, HBD 3, PSA 104.3, QED 0.37
Compound (SMILES): C[C@]1(c2cc(/C=C(\F)c3ccc(C#N)cn3)cnc2F)N=C(N)S[C@@]2(C(=O)N3CCOCC3)C[C@H]21
InChIKey: SHKIDCBHVZAMLK-ZEHMKQDJSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro Enzymatic BACE1 and BACE2 FRET (Fluorescence Resonance Energy Transfer) Assays: The cDNAs for both human recombinant BACE1 and 2 with C-terminal 6-His Tags were cloned into transient protein expression vectors, which were subsequently transfected into mammalian cell lines. These recombinant proteins were further purified using Ni-NTA affinity chromatography (Qiagen). The assay buffer used in these screens was 0.05M acetate, pH 4.5, 8% DMSO final, 100 uM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The Beta Secretase enzyme (0.02 nM for BACE1 and 0.64 nM for BACE2) is pre-incubated for one hour with inhibitors, typically in about 1 uL of DMSO according to a serial dilution, are added thereto. This assay is effectively started by the addition of FRET substrate (50 nM) and the combination is incubated for one hour. The FRET assay is terminated by addition of Tris buffer, which raises the pH to neutrality, and the fluorescence is determined. The FRET substrate is a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. Proteolytic cleavage of the FRET substrate releases quenching of fluorescence (excitation 488 nm and emission 590 nm).
Measurement: IC50 = 16.5 nM (pChEMBL 7.78000020980835)
Descriptors: MW 496.54, ALogP 2.85, HBA 8, HBD 1, PSA 117.49, QED 0.65
Compound (SMILES): CN(Cc1ccc(Cl)c(C#N)c1)C(=O)Cn1cnc2ccc(N3CCCN(CC4CCOC4)CC3)cc2c1=O
InChIKey: RTRUZGGLJQBPME-UHFFFAOYSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Human ATX Enzyme Inhibition Assay: ATX inhibition was measured by a fluorescence quenching assay using a specifically labeled substrate analogue (MR121 substrate). To obtain this MR121 substrate, BOC and TBS protected 6-amino-hexanoic acid (R)-3-({2-[3-(2-{2-[2-(2-amino-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propionylamino]-ethoxy}-hydroxy-phosphoryloxy)-2-hydroxy-propyl ester (Ferguson et al., Org. Lett. 2006, 8, 2023) was labeled with MR121 fluorophore [CAS RN 185308-24-1], 1-(3-carboxypropyl)-11-ethyl-1,2,3,4,8,9,10,11-octahydro-dipyrido[3,2-b:2′,3′-i]phenoxazin-13-ium) on the free amine of the ethanolamine side and then, after deprotection, subsequently with tryptophan on the side of the aminohexanoic acid.Assay working solutions were made as follows:Assay buffer (50 mM Tris-HCl, 140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 0.01% Triton-X-100, pH 8.0;ATX solution: ATX (human His-tagged) stock solution (1.08 mg/mL in 20 mM bicine, pH 8.5, 0.15 M NaCl, 10% glycerol, 0.3% CHAPS, 0.02% NaN3), diluted to 1.4-2.5× final concentration in assay buffer;MR121 substrate solution: MR121 substrate stock solution (800 μM MR121 substrate in DMSO), diluted to 2-5× final concentration in assay buffer.Test compounds (10 mM stock in DMSO, 8 μL) were obtained in 384 well sample plates (Corning Costar #3655) and diluted with 8 μL DMSO. Row-wise serial dilutions were made by transferring 8 μL cpd solution to the next row up to row O. The compound and control solutions were mixed five times and 2 μL were transferred to 384 well assay plates (Corning Costar #3702). Then, 15 μL of 41.7 nM ATX solution was added (30 nM final concentration), mixed five times and then incubated for 15 minutes at 30° C. 10 μL of MR121 substrate solution was added (1 μM final concentration), mixed 30 times and then incubated for 15 minutes at 30° C. Fluorescence was then measured every 2 min for 1 h (Perkin Elmer plate: vision multimode reader); light intensity: 2.5%; exp. time: 1.4 s, Filter: Fluo_630/690 nm) and IC50 values were calculated from these readouts.
Measurement: IC50 = 111 nM (pChEMBL 6.960000038146973)
Descriptors: MW 549.08, ALogP 3.13, HBA 8, HBD 0, PSA 94.7, QED 0.45
Compound (SMILES): O=C(O)c1ccn(C(=O)N2CCN(Cc3cccc(Oc4ccc(C(F)(F)F)cn4)c3)CC2)n1
InChIKey: GYCMYXNHMIYPQR-UHFFFAOYSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: In Vitro Competitive Activity-Based Protein Profiling: Proteomes (mouse brain membrane fraction for mouse assays; human prefrontal cortex membrane fractions for human assays) (50 mL, 1.0 mg/mL total protein concentration) were preincubated with varying concentrations of inhibitors at 37° C. After 30 min, FP-Rh (1.0 mL, 50 mM in DMSO) was added and the mixture was incubated for another 30 min at 37° C. Reactions were quenched with SDS loading buffer (15 μL-4×) and run on SDS-PAGE. Following gel imaging, serine hydrolase activity was determined by measuring fluorescent intensity of gel bands corresponding to MAGL and FAAH using ImageJ 1.43u software. IC50 data from this assay is shown in Table 1. All compounds in Table 1 were more potent inhibitors of MAGL than FAAH.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 475.43, ALogP 3.57, HBA 7, HBD 1, PSA 100.79, QED 0.6