lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cn1cc(-c2cnc(Nc3cc(C(F)(F)F)cc(C(F)(F)F)c3)nc2Nc2ccc3c(c2)CNCC3)cn1
InChIKey: VBMCYUGHTBTLMB-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: Verification on Inhibitory Activities of TAM Receptor Inhibiting Compounds on Tyro 3, Axl, and Mer: For evaluation of inhibitory activities of the compounds according to the present invention on Tyro 3, Axl, and Mer, the following test was carried out.The specific test method followed the method provided by Cisbio. The compounds of the examples were prepared at various concentrations, followed by addition of Tyro 3, Axl, or Mer and substrate peptides, and then ATP was added to initiate a reaction. After 1 hour, the reaction was stopped by addition of a solution containing EDTA. Thereafter, the amount of phosphorylated peptides was measured. Here, the phosphorylated peptides were treated with an europium (Eu)-labeled antibody recognizing phosphorylated peptides, and then after 1 hour, excited by irradiation of the light of a wavelength of 320 or 340 nm using an Envision Reader. The amount of light emitted at 665 nm was measured to investigate the degrees of inhibition of TAM receptors. The TAM receptor inhibitory activities (IC50 values) of the respective compounds were analyzed using the GraphPad Prism program, and tabulated in Tables 1 to 3 below. LDC1267 (CAS no. 1361030-48-9, Calbiochem), which is a TAM receptor inhibitor compound, was used as a positive control.
Measurement: IC50 = 3600 nM (pChEMBL 5.440000057220459)
Descriptors: MW 533.48, ALogP 6.05, HBA 7, HBD 3, PSA 79.69, QED 0.27
Compound (SMILES): CS(=O)(=O)N1CC(C(=O)N(Cc2ccc(-c3noc(C(F)(F)F)n3)cn2)c2cccc(F)c2)C1
InChIKey: SARHXDDKSAAZIO-UHFFFAOYSA-N
Target: Histone deacetylase 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BY41
Assay: Enzymatic Assay: HDAC8 reagents: HDAC8 was purchased from Enzo Life Sciences [catalog #BML-SE145]. Assays were performed with buffer containing 20 mM HEPES, pH 8.0 [Boston BioProducts, catalog #BB-104, 1M stock], 100 mM NaCl [Sigma, catalog #S5150, 5M stock], 20 mM KCl [BioChemika, catalog #87526, 4M stock], 1 mM MgCl2 [Fluka, catalog #63020, 1M stock], 0.05% BSA (Fraction V) [Invitrogen, catalog #15260, 7.5% stock], and 0.1% n-Octyl-β-D-glucopyranoside (N-OG) [Anatrace, catalog #O311, 10% stock]. The HDAC8 enzyme was run at the final concentration of 1.333 nM. Fluor-de-Lys substrate [BioMol Research Laboratories, catalog #KI-178], used to evaluate enzyme activity, was added at the final concentration of 200 uM. To enable detection of the signal, Developer II [BioMol Research Laboratories, catalog #KI-176] was added at a 1:200 dilution to the stop solution, which also included 20 uM SAHA [Sigma, catalog #SML0061] to ensure complete termination of the reaction.
Measurement: IC50 = 1300 nM (pChEMBL 5.889999866485596)
Descriptors: MW 499.45, ALogP 2.71, HBA 7, HBD 0, PSA 109.5, QED 0.48
Compound (SMILES): CN(C)CC(NC(=O)N1Cc2nc(Nc3ccccc3)ncc2C1(C)C)c1ccccc1
InChIKey: QDIMOURDMVMOHZ-UHFFFAOYSA-N
Target: Ribosomal protein S6 kinase alpha-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15418
Assay: Inhibition Assay: Lists compounds that were tested for inhibition of RSK; the IC50 values are in micromolar units, and refer to inhibition of RSK1 and RSK2, respectively. Where multiple measurements were made, each value is reported.
Measurement: IC50 = 17710 nM (pChEMBL 4.75)
Descriptors: MW 430.56, ALogP 4.28, HBA 5, HBD 2, PSA 73.39, QED 0.61
Compound (SMILES): CNCCNc1cncc(-c2ccc3[nH]c(-c4ccnc(C)c4)c(C(C)C)c3c2)c1
InChIKey: GNMBLQXVQDFNOY-UHFFFAOYSA-N
Target: Toll-like receptor 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR96
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-κc Band 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 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-Bue 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 = 162 nM (pChEMBL 6.789999961853027)
Descriptors: MW 399.54, ALogP 5.36, HBA 4, HBD 3, PSA 65.63, QED 0.37
Compound (SMILES): C=CC(=O)Nc1cn(CCOC)nc1-c1cc2c(cn1)CN(c1c(F)c(OC)cc(OC)c1F)C(=O)C21CC1
InChIKey: LSIUJFBRLCBDGK-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Enzymatic Assay: The inhibitor potency of the exemplified compounds was measured in an enzyme assay that measures peptide phosphorylation using FRET measurements to detect product formation. Inhibitors were serially diluted in DMSO and a volume of 0.5 μL was transferred to the wells of a 384-well plate. For FGFR3, a 10 μL volume of FGFR3 enzyme (Millipore) diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 5 mM DTT, pH 7.5) was added to the plate and pre-incubated for a time between 5-10 minutes and up to 4 hours. Appropriate controls (enzyme blank and enzyme with no inhibitor) were included on the plate. The assay was initiated by the addition of a 10 μL solution containing biotinylated EQEDEPEGDYFEWLE peptide substrate (SEQ ID NO: 1) and ATP (final concentrations of 500 nM and 140 μM respectively) in assay buffer to the wells. The plate was incubated at 25° C. for 1 hr. The reactions were ended with the addition of 10 μL/well of quench solution (50 mM Tris, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.8; 30 mM EDTA with Perkin Elmer Lance Reagents at 3.75 nM Eu-antibody PY20 and 180 nM APC-Streptavidin). The plate was allowed to equilibrate for 1 hr before scanning the wells on a PheraStar plate reader (BMG Labtech).FGFR1, FGFR2, and FGFR4 are measured under equivalent conditions with the following changes in enzyme and ATP concentrations: FGFR1, 0.02 nM and 210 uM respectively, FGFR2, 0.01 nM and 100 uM, respectively, and FGFR4, 0.04 nM and 600 uM respectively. The enzymes were purchased from Millipore or Invitrogen.
Measurement: IC50 > 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 539.54, ALogP 3.59, HBA 8, HBD 1, PSA 107.81, QED 0.41
Compound (SMILES): O=C1NC(=O)C(CCC(=O)N2CCN(c3ccc(F)c(Cl)c3)CC2)(C2CC2)N1
InChIKey: YLVNKSYPWFWMTF-UHFFFAOYSA-N
Target: 72 kDa type IV collagenase (SINGLE PROTEIN), Homo sapiens
UniProt: P08253
Assay: In Vitro Assay: MMP2: Protocol 2: The basis for the assay is the cleavage of the substrate 390 MMP FRET substrate I (Anaspec, Catalog n#: AS-27076) by human MMP2 (R&D SYSTEMS INC., Cat #902-MP).For the dose response (10 point), 4 μL of a dilution series of compound (2 mM highest concentration, 1/5 dilution in DMSO further diluted 1 in 10 in water, corresponding to a final highest concentration of 20 μM), is transferred to 384 well Fluotrac 200 plate (Greiner, cat #781076) and incubated at room temperature for 30 min with a 26 μL buffer solution (45 mM Tris pH 7.5, 9 mM CaCl2, 135 mM NaCl, 0.045% Brij35) containing MMP2 (0.03 ng/L) (it will be appreciated by the skilled person that the potency read out is independent of the enzyme concentration).The reaction is initiated by adding to the assay plate 390 MMP FRET substrate I (10 μL, 2.5 μM, Anaspec) in the same buffer.Finally, the fluorescence is read on the Envision (Perkin Elmer) after an incubation of 30 min at room temperature (Excitation 485 nm, Emission 530).
Measurement: IC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 408.86, ALogP 1.9, HBA 4, HBD 2, PSA 81.75, QED 0.73
Compound (SMILES): C=CC(=O)Nc1ccccc1Nc1ncc2c(n1)N(C)C(=O)N(c1c(Cl)c(OC)cc(OC)c1Cl)C2
InChIKey: WILHHSWUSOIGGD-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: Omnia Assay Protocol for Potency Assessment Against FGFR 4 Enzyme: A 10× stock solution of FGFR4-WT (PR4380C or P3054), (from Invitrogen, Carlsbad, Calif.) corresponding to method a in Table 10, was prepared as described below. Alternatively, a 10× stock solution of FGFR4-WT (F01-11G), (SignalChem, Richmond, BC) corresponding to method b in Table 10, was prepared as described below. A solution of 1.4×ATP (AS001A) and 5× Tyr-Sox conjugated peptide substrate (KNZ3101) were prepared in 1× kinase reaction buffer consisting of 20 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 5 mM P3-glycerophosphate, 5% glycerol (10× stock, KB002A) and 0.2 mM DTT (DS001A). 5 μL of FGFR4 was pipetted into a Corning (#3574) 384-well, white, non-binding surface microtiter plate (Corning, N.Y.), containing a 0.5 μL volume of 100% DMSO. The serially diluted compounds were prepared on a Tecan EVO100. A second addition of 10 μl of Tyr-Sox FGFR4 substrate was added to each well and the kinase reactions were started with the addition of 35 μL of 1.4×ATP. The reactions were monitored every 71 seconds for 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 −60 minutes) from each reaction was determined from the slope of a plot of relative fluorescence units vs time (seconds) and then plotted against inhibitor concentration to estimate IC50 from log[Inhibitor] vs Response (Variable Slope model in GraphPad Prism from GraphPad Software (San Diego, Calif.)).
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 529.38, ALogP 5.25, HBA 7, HBD 2, PSA 108.92, QED 0.4
Compound (SMILES): O=C(NC1CCNCC1)c1cccc(-c2nccc(Nc3ccc4[nH]ncc4c3)n2)c1
InChIKey: NCDYWCMXEYRYBW-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: Z′-LYTE Kinase Assay: Purified active ROCK1 and ROCK2 were obtained from Invitrogen (catalog numbers ROCK1, PV3691 and ROCK2, PV3759). The kit components include a coumarin and fluorescein labeled peptide based on myosin light chain 2 (KKRPQRRYSNVF), a proprietary protease containing development reagent and a proprietary Stop buffer used to terminate the development reaction. The inhibitory activities of compounds are measured according to the manufactures protocol. Briefly, decreasing concentrations of test compounds or the known ROCK inhibitor Y-27963, are added, from 10 uM to 2.56×10−5 uM to reaction buffer containing 50 mM HEPES pH 7.5, 10 mM MgCl2, 5 mM EGTA, and 0.05% Brij-35 and of ROCK1 at 0.18 ug/mL or ROCK2 at 0.8 ug/mL in assay dilution buffer. This mixture is overlayed into a white 96-well half area plate and the reaction is initiated with the addition of 5 uM ATP for ROCK1 or 12 uM ATP for ROCK2. The assay proceeds at room temperature for 1 hour followed by the addition of development reagent, and further incubation for 1 hour at room temperature. STOP reagent is then added and the reaction and immediately the coumarin and fluorescein emission signals are read on a Tecan Infinite M1000 fluorescence plate reader (excitation: 400 nm; emission 445 and 520 nm, respectively).
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 413.49, ALogP 3.25, HBA 6, HBD 4, PSA 107.62, QED 0.4
Compound (SMILES): CNC(=O)c1ncc(Nc2nccc(N3C[C@H]4CC[C@@H](C3)N4CC3CC3(F)F)n2)cc1C
InChIKey: LRZYHCZKTOWDMP-ZXFUBFMLSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Caliper Enzyme Assay: Compounds were added to a 384-well plate. Reaction mixtures contained 10 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% BSA, 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20% to 30% phosphorylation. The assays were stopped with 15 μL of 180 mM HEPES, pH 7.4, 20 mM EDTA, and 0.2% Coating Reagent 3. The assay plates were placed on a Caliper Life Science LC3000 instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 418 nM (pChEMBL 6.380000114440918)
Descriptors: MW 443.5, ALogP 2.59, HBA 7, HBD 2, PSA 86.28, QED 0.71
Compound (SMILES): Cc1cc(-c2[nH]c3ccc(C(=O)N(C)Cc4cccc(N)c4)cc3c2C(C)C)cc(C)n1
InChIKey: YHQIPROWUIBXTC-UHFFFAOYSA-N
Target: Toll-like receptor 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR96
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 = 27053 nM (pChEMBL 4.570000171661377)
Descriptors: MW 426.56, ALogP 5.82, HBA 3, HBD 2, PSA 75.01, QED 0.39
Compound (SMILES): COc1ncc(-c2cc3c(n2C)C(c2ccc(Cl)cc2)N(c2cc(Cl)c(=O)n(C)c2)C3=O)c(OC)n1
InChIKey: IFFAPYISKKZOPO-UHFFFAOYSA-N
Target: Bromodomain-containing protein 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15059
Assay: TR-FRET In-Vitro Binding Assay: All assays were performed in 384-well microtiter plates. Each assay plate contained 8-point serial dilutions for 40 test compounds, plus 16 high- and 16 low controls. Liquid handling and incubation steps were done on an Innovadyne Nanodrop Express equipped with a robotic arm (Thermo CatX, Perkin Elmer/Caliper Twister II) and an incubator (Liconic STX40, Thermo Cytomat 2C450). The assay plates were prepared by addition of 50 nl per well of compound solution in 90% DMSO HummingBird nanodispenser (Zinsser Analytic). The assay was started by stepwise addition of 4.5 μL per well of bromo domain protein (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 45 nM His-Brd2(60-472) or 45 nM His-Brd3(20-477) or 45 nM His-Brd4(44-477) all proteins produced in-house) and 4.5 μL per well of peptide solution (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 60 nM acetyl-histone H4 (AcK 5, 8, 12, 16) (Biosyntan GmbH)). Reactions were incubated at 30° C. for 35 minutes. Subsequently 4.5 μL per well detection mix (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 3 nM Eu-labeled anti-His6 antibody, 21 nM streptavidin-allophycocyanin) were added. After 35 minutes incubation at 30° C., plates were measured in a Perkin Elmer EnVision multilabel reader. Concentrations causing 50% inhibition (1050 values) were determined from percent inhibition values at different compound concentrations by non-linear regression analysis.
Measurement: IC50 = 59 nM (pChEMBL 7.230000019073486)
Descriptors: MW 526.38, ALogP 4.25, HBA 8, HBD 0, PSA 91.48, QED 0.39
Compound (SMILES): Cn1ncc(NC(=O)[C@@H]2CCCC[C@H]2C(=O)c2ccc(-c3ccn[nH]3)cc2)c1S(C)(=O)=O
InChIKey: DMHSDGHSQKYUDC-IAGOWNOFSA-N
Target: Flap endonuclease 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P39748
Assay: Binding Assay: Compounds were tested in a competition binding assay using 3H-MK591 as tracer. (Preparation of MK-591 is described in Bioorg. Med. Chem. Lett. 1999, 9, 2391). A 100,000×g pellet from COS-7 cells stably transfected with a plasmid expressing human ALOX5AP was the source of FLAP. Membrane pellets were resuspended in buffer (100 mM Tris-HCl, 0.05% Tween-20, 140 mM NaCl, 2 mM EDTA, 0.5 mM DTT, 5% Glycerol, pH 7.5) to give a final protein concentration of 12 mg/mL (2 μg/well). To perform assays, 1.4 μL compounds were dispensed into 96-well plates in 3-fold dilution series in triplicate. 84 μL radioligand (25000 CPM, 2 nM final concentration in assay) was then added followed by 84 μL membrane suspension and incubation at rt for 60 min. Following filtration, filter plates were dried 12 h at RT (or 50° C. for 1 hour). 50 μL scintillant was then added, the filterplates were sealed and radioactivity was measured in a microbeta counter. Specific binding was defined as total binding minus non-specific binding. Total binding was defined as 3H-MK591 bound to membranes in the absence of competitor, non-specific binding was defined as 3H-MK591 in the presence of 0.1 mM MK-591.
Measurement: IC50 = 45 nM (pChEMBL 7.349999904632568)
Descriptors: MW 455.54, ALogP 2.84, HBA 7, HBD 2, PSA 126.81, QED 0.55
Compound (SMILES): Cc1ncc(-c2ccc3cnc(NC(=O)N4CCOCC4)cc3c2)n1C
InChIKey: ROCBPXRBDJBMDL-UHFFFAOYSA-N
Target: Glycogen synthase kinase-3 beta (SINGLE PROTEIN), Homo sapiens
UniProt: P49841
Assay: Fluorescence Resonance Energy Transfer (FRET) Assay: Briefly, recombinant GSK3β kinase, ATP and Ser/Thr peptide 09 are prepared in 1Ã Kinase buffer to final concentrations of 0.04 μg/mL, 46 μM, and 4 μM respectively. The mixture is allowed to incubate with the representative compounds for one hour at room temperature. All reactions were performed in duplicate. Unphosphorylated and phosphorylated forms of Ser/Thr 18 serve as control reactions.After incubation, diluted Development Buffer is added to the reaction and allowed to further incubate for one hour at room temperature. The plate is read at Ex 400 Em 455 to detect the coumarin signal and Ex 400 Em 520 to measure the signal (EnVision Multilabel Plate Reader, PerkinElmer).
Measurement: EC50 = 1246 nM (pChEMBL 5.900000095367432)
Descriptors: MW 351.41, ALogP 2.81, HBA 5, HBD 1, PSA 72.28, QED 0.77
Compound (SMILES): CNC(=O)N1CCC(CS(=O)(=O)NNC(=O)c2cc(-c3cn(C)cn3)cc(Cl)c2F)C1
InChIKey: AJBMMUNILOMGSS-UHFFFAOYSA-N
Target: Histone acetyltransferase KAT7 (SINGLE PROTEIN), Homo sapiens
UniProt: O95251
Assay: KAT-7 Enzyme Assay: Enzyme assay buffer was 50 mM Tris pH 8.0, 0.002% Tween20, 0.005% bovine skin gelatin, and 1 mM dithiothreitol (DTT). For determination of IC50values, compounds were serially diluted with 2% (v/v) DMSO in the final reaction, pre-incubating each dilution of each compound with 40 μL of assay buffer containing KAT-7 enzyme (6 nM final concentration). 10 μL of assay buffer containing 1 μM peptide substrate and 2 μM acetyl coenzyme A (final concentrations) was added. Reactions (50 μL total) were then carried out at 25° C. for 120 minutes. Reactions were terminated by the addition of 0.5% formic acid (final concentration), and a sample of each reaction was analyzed by SAMDI Tech, Inc. (Chicago, Ill.) using self-assembled monolayer desorption/ionization time-of-flight mass spectrometry (Mrksich, M. (2008) Mass spectrometry of self-assembled monolayers: a new tool for molecular surface science. ACS Nano 2, 7-18).
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 472.93, ALogP 1.11, HBA 6, HBD 3, PSA 125.43, QED 0.54
Compound (SMILES): Cc1cc(-c2[nH]c3ccc(C(C)(C)C(=O)NC[C@H]4CC[C@H](N)CC4)cc3c2C(C)C)ccn1
InChIKey: LINLYQDATVBZNX-XYWHTSSQSA-N
Target: Toll-like receptor 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR96
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 = 1804 nM (pChEMBL 5.739999771118164)
Descriptors: MW 446.64, ALogP 5.57, HBA 3, HBD 3, PSA 83.8, QED 0.46
Compound (SMILES): CCn1cc(CN2C(=O)N(c3c(F)c(OC)cc(OC)c3F)Cc3cnc(-c4cn(C)nc4C)cc32)cn1
InChIKey: HQOORGXQDKMTBO-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: FGFR Enzymatic Assay 1: FGFR1, FGFR2, and FGFR4 are measured under equivalent conditions with the following changes in enzyme and ATP concentrations: FGFR1, 0.02 nM and 210 uM respectively, FGFR2, 0.01 nM and 100 uM, respectively, and FGFR4, 0.04 nM and 600 uM respectively. The enzymes were purchased from Millipore or Invitrogen. GraphPad prism3 was used to analyze the data. The IC50 values were derived by fitting the data to the equation for a sigmoidal dose-response with a variable slope. Y=Bottom+(Top−Bottom)/(1+10^((Log IC50−X)*HillSlope)) where X is the logarithm of concentration and Y is the response. Compounds having an IC50 of 1 μM or less are considered active. The compounds of the invention were found to be selective inhibitors of FGFR3 and/or FGFR4 according to the FGFR Enzymatic Assays. Compounds of Formula (I′) and (I) and all the compounds as described herein have been tested and exhibit an IC50 of less than 1 μM. Table 1 provides IC50 data for compounds of the invention assayed in the FGFR Enzymatic Assay after dilution in assay buffer, added to the plate and pre-incubated for 4 hours.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 523.54, ALogP 4.45, HBA 8, HBD 0, PSA 90.54, QED 0.36
Compound (SMILES): [C-]#[N+]CN1C2CCC1CC(c1ccc3[nH]c(-c4cn5ncnc5c(C)c4C)c(C(C)C)c3c1)C2
InChIKey: SHSLULYZPWJOFX-UHFFFAOYSA-N
Target: Toll-like receptor 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR96
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 = 7530 nM (pChEMBL 5.119999885559082)
Descriptors: MW 452.61, ALogP 6.21, HBA 4, HBD 1, PSA 53.58, QED 0.37
Compound (SMILES): CCn1c(-c2cccnc2C(C)OC)c2c3cc(ccc31)-c1cccc(c1)C[C@H](NC(=O)[C@H](C(C)C)N(C)C(=O)[C@H]1CCN(C(=O)C#CC3CCO3)C1)C(=O)N1CCC[C@H](N1)C(=O)OCC(C)(C)C2
InChIKey: OSPHUDXDINZSCP-FUTRASTKSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Biochemical Assay: A variety of Ras proteins may be inhibited by compounds of the present invention (e.g., K-Ras, N-Ras, H-Ras, and mutants thereof at positions 12, 13 and 61, such as G12C, G12D, G12V, G12S, G13C, G13D, and Q61L, and others described herein). The purpose of this biochemical assay is to measure the ability of test compounds to covalently label nucleotide-loaded K-Ras isoforms. In assay buffer containing 12.5 mM HEPES pH 7.4, 75 mM NaCl, 1 mM MgCl2, 1 mM BME, 5 uM Cyclophilin A and 2 uM test compound, a 5 uM stock of GMP-PNP-loaded K-Ras (1-169) G12C is diluted 10-fold to yield a final concentration of 0.5 uM; with final sample volume being 100 uL. The sample is incubated at 25�� C. for a time period of up to 24 hours prior to quenching by the addition of 10 uL of 5% Formic Acid. Quenched samples are centrifuged at 15000 rpm for 15 minutes in a benchtop centrifuge before injecting a 10 uL aliquot onto a reverse phase C4 column and eluting into the mass spectrometer with an increasing acetonitrile gradient in the mobile phase. Analysis of raw data may be carried out using Waters MassLynx MS software, with % bound calculated from the deconvoluted protein peaks for labeled and unlabeled K-Ras.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 942.17, ALogP 5.87, HBA 11, HBD 2, PSA 164.64, QED 0.15
Compound (SMILES): Cc1cc(-c2[nH]c3ccc(C4CCN(C5CCCC5(C)C#N)CC4)cc3c2C(C)C)cn2ncnc12
InChIKey: LRGXVIPVHAWSGO-UHFFFAOYSA-N
Target: Toll-like receptor 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR96
Assay: Biological 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.
Measurement: IC50 = 22305 nM (pChEMBL 4.650000095367432)
Descriptors: MW 480.66, ALogP 6.57, HBA 5, HBD 1, PSA 73.01, QED 0.36
Compound (SMILES): FC(F)(F)c1cccc(-c2ccc([C@H]3C[C@@H]3NCC3CCNCC3)cc2)c1
InChIKey: GJXFBKDQIXYBIK-RTWAWAEBSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: Biological Assay: Briefly, a fixed amount of LSD1 was incubated on ice for 15 minutes, in the absence and/or in the presence of at least eight 3-fold serial dilutions of the respective test compound (e.g., from 0 to 75 μM, depending on the inhibitor strength). Tranylcypromine (Biomol International) was used as a control for inhibition. Within the experiment, each concentration of inhibitor was tested in duplicate. After leaving the enzyme interacting with the inhibitor, KM of di-methylated H3-K4 peptide was added to each reaction and the experiment was left for 30 minutes at 37° C. in the dark. The enzymatic reactions were set up in a 50 mM sodium phosphate, pH 7.4 buffer. At the end of the incubation, Amplex® Red reagent and horseradish peroxidase (HPR) solution were added to the reaction according to the recommendations provided by the supplier (Invitrogen), and left to incubate for 5 extra minutes at room temperature in the dark. A 1 μM H2O2 solution was used as a control of the kit efficiency. The conversion of the Ampiex® Red reagent to resorufin due to the presence of H2O2 in the assay, was monitored by fluorescence (excitation at 540 nm, emission at 590 nm) using a microplate reader (Infinite 200, Tecan). Arbitrary units were used to measure level of H2O2 produced in the absence and/or in the presence of inhibitor.
Measurement: IC50 = 84 nM (pChEMBL 7.079999923706055)
Descriptors: MW 374.45, ALogP 4.82, HBA 2, HBD 2, PSA 24.06, QED 0.78
Compound (SMILES): CNCC(c1ccc(Br)c(Cl)c1)[C@H](O)c1ccccc1
InChIKey: YYXLUZNBTYTCJI-FQNRMIAFSA-N
Target: Sodium-dependent serotonin transporter (SINGLE PROTEIN), Homo sapiens
UniProt: P31645
Assay: Uptake Assay Protocol: SERT: This protocol was designed to measure inhibition of uptake by the human serotonin transporter. The reagents were human SERT (HEK293F) cells, fluoxetine (Sigma), nomifensine, neurotransmitter transporter uptake assay kit (Molecular Devices), freestyle 293 expression medium (Invitrogen), 10× Hank's Balanced Salt Solution (HBSS; Invitrogen), 1 M HEPES (Mediatech), Biocoat poly-D-lysine 96-well, black, clear plates (Becton, Dickinson), and 500 μL polypropylene U-bottom 96-well plates (Fisher). The Assay Buffer (AB) was 1λ HBSS and 0.02 M HEPES.The HEK293F cells were transfected with the human serotonin transporter and frozen in 1 mL aliquots at about 1E+07 cells/mL. On the day of the experiment, the cells were removed from −80° C. or liquid nitrogen and thawed in a room temperature water bath. The cells were dilute to about 1-2E+06 with Freestyle medium. A 1 mL sample (1:2 dilution) was prepared, and the cells were counted. The cells were spun at 1100 rpm for 5 minutes, and the medium was aspirated off. The cells were resuspend in medium at 1.5E+06 cells/mL for about 60,000 cells per well. 40 μL of cells were dispensed per well in the Biocoat plates. The plates were spun at 1100 rpm for 1 minute to improve homogeneity of the cell layer and were incubated at 37° C. for a minimum of 3 hours.
Measurement: Ki = 6.3 nM (pChEMBL 8.199999809265137)
Descriptors: MW 354.68, ALogP 4.14, HBA 2, HBD 2, PSA 32.26, QED 0.85
Compound (SMILES): CCc1cc(CN2CCCC23CCN(C(=O)OC(C(F)(F)F)C(F)(F)F)CC3)ccc1F
InChIKey: VWUFTHCPWDGASQ-UHFFFAOYSA-N
Target: Monoacylglycerol lipase ABHD6 (SINGLE PROTEIN), Mus musculus
UniProt: Q8R2Y0
Assay: In Vitro Competitive Activity-Based Protein Profiling Assay: MAGL, ABHD6 and FAAH: Proteomes (mouse brain membrane fraction or cell lysates) (50 μL, 1.0 mg/mL total protein concentration) were preincubated with varying concentrations of inhibitors at 37° C. After 30 min, FP-Rh (1.0 μL, 50 μM in DMSO) was added and the mixture was incubated for another 30 min at 37° C. Reactions were quenched with SDS loading buffer (50 μL-4X) and run on SDS-PAGE. Following gel imaging, serine hydrolase activity was determined by measuring fluorescent intensity of gel bands corresponding to MAGL, ABHD6 and FAAH using ImageJ 1.43 u software.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 470.43, ALogP 5.45, HBA 3, HBD 0, PSA 32.78, QED 0.55
Compound (SMILES): C=CC(=O)N1CCCN(C(=O)c2cc(Sc3cnc(NC(=O)c4cccc(CN[C@H](C)C(C)(C)C)c4)s3)c(C)cc2OC)CC1
InChIKey: ONKCBNMZBWUKKB-HSZRJFAPSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: In Vitro Assay: The potency of inhibition is measured by binding affinity.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 649.88, ALogP 6.25, HBA 8, HBD 2, PSA 103.87, QED 0.25
Compound (SMILES): CC(C)CN1CCC(F)(C(=O)Nc2cc3cc(-c4nncn4C)ccc3cn2)CC1
InChIKey: ZKOWFDJIWGKOEU-UHFFFAOYSA-N
Target: Dual specificity tyrosine-phosphorylation-regulated kinase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: Q13627
Assay: The screening assay for DYRK1A kinase activity: Each compound was dissolved in DMSO as a 10 mM stock and used to prepare compound source plates. Serial dilution (1:3, 11-point dose-response curves from 10 μM to 0.00016 μM) and compound transfer was performed using the ECHO 550 (Labcyte, Sunnyvale, Calif.) into 1536-well black-walled round bottom plates (Corning).The DYRK1A kinase assay was run using the Ser/Thr 18 peptide Z-lyte assay kit according to manufacturer's instructions (Life Technologiesa Division of Thermo-Fisher). This is a non-radioactive assay using fluorescence resonance energy transfer (FRET) between coumarin and fluorescein to detect kinase activity which is represented as a ratio of coumarin emission/fluorescein emission.Briefly, recombinant DYRK1A kinase, ATP and Ser/Thr peptide 18 were prepared in 1× Kinase buffer to final concentrations of 0.19 μg/mL, 30 μM, and 4 μM respectively. The mixture was allowed to incubate with the representative compounds for one hour at room temperature. All reactions were performed in duplicate. Unphosphorylated (0% Control) and phosphorylated (100% control) forms of Ser/Thr 18 served as control reactions. Additionally, an 11-point dose-response curve of Staurosporine (1 uM top) was run to serve as a positive compound control.After incubation, Development Reagent A was diluted in Development Buffer then added to the reaction and allowed to further incubate for one hour at room temperature. The plate was read at Ex 400 Em 455 to detect the coumarin signal and Ex 400 Em 520 to measure the signal (EnVision Multilabel Plate Reader, PerkinElmer).The Emission ratio (Em) was calculated as a ratio of the coumarin (C) emission signal (at 445 nm)/Fluorescein (F) emission signal (at 520 nm). The percent phosphorylation was then calculated using the following formula: [1−((Em ratio×F100%)−C100%)/((C0%−C100%)+(Em ratio×(F100%−F0%)))]. Dose-response curves were generated and inhibitory concentration (IC50) values were calculated using non-linear regression curve fit in the Dotmatics' Studies Software (Bishops Stortford, UK).
Measurement: EC50 = 10.8 nM (pChEMBL 7.96999979019165)
Descriptors: MW 410.5, ALogP 3.43, HBA 6, HBD 1, PSA 75.94, QED 0.7
Compound (SMILES): C=CC(=O)Nc1cccc(Nc2nc(Nc3ccccc3)ncc2C#N)c1
InChIKey: DIOMEEGYXLOHPP-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ITK/TSK (SINGLE PROTEIN), Homo sapiens
UniProt: Q08881
Assay: Omnia Assay: Briefly, 10x stocks of EGFR-WT (PV3872) from Invitrogen and EGFR-T790M/L858R (40350) from BPS Bioscience, San Diego, Calif., 1.13×ATP (AS001A) and appropriate Tyr-Sox conjugated peptide substrates (KCZ1001) 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 (DS001A). 5 μL of each enzyme were pre-incubated in a Corning (#3574) 384-well, white, non-binding surface microtiter plate (Corning, N.Y.) for 30 min. at 27° C. with a 0.5 μL volume of 50% DMSO and serially diluted compounds prepared in 50% DMSO. Kinase reactions were started with the addition of 45 μL of the ATP/Tyr-Sox peptide substrate mix and monitored every 30-90 seconds for 60 minutes at λex360/λem485 in a Synergy4 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) and then plotted against inhibitor concentration to estimate IC50 from log[Inhibitor] vs Response, Variable Slope model in GraphPad Prism from GraphPad Software (San Diego, Calif.).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 356.39, ALogP 3.96, HBA 6, HBD 3, PSA 102.73, QED 0.58
Compound (SMILES): CC#CC(=O)N[C@@H](C)c1nc(-c2ccc(C(=O)Nc3ccccn3)c(OC)c2)c2c(N)nccn12
InChIKey: LHFAKDSSXLAPAE-HNNXBMFYSA-N
Target: Tyrosine-protein kinase Tec (SINGLE PROTEIN), Homo sapiens
UniProt: P42680
Assay: IMAP (Immobilized Metal Ion Affinity-Based Fluorescence Polarization) Assay: TEC enzyme (Millipore #14-801M), is diluted to 2 U/mL in KR buffer (10 mM Tris-HCl, 10 mM MgCl2, 0.01% Tween-20, 0.1% NaN3, 1 mM DTT, 2 mM MnCl2, pH 7.5).Serial dilutions 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 ranged from 10 μM to 0.316 nM.The assay is performed as follows: 5 μL/well of test compound in KR buffer (final DMSO concentration in the assay is 1%) is mixed with 5 μL/well of 2 U/mL TEC enzyme (final concentration in the assay is 0.5 U/mL (6.3 nM)). Test compounds and TEC enzyme are pre-incubated 60 minutes at room temperature, before adding 5 μL/well of 200 nM Fluorescin labeled substrate peptide (Blk/Lyntide substrate #R7188, Molecular Devices) in KR-buffer. Final peptide substrate concentration in assay is 50 nM. The kinase assay is started by adding 5 L/well of 20 μM ATP in KR-buffer (final ATP concentration is 5 μM ATP, Km ATP in TEC IMAP assay). Following incubation for 2 hours at room temperature the enzyme reaction is stopped by adding 40 μL/well IMAP Progressive Binding Solution (according to suppliers (Molecular Devices) protocol using 60% 1× buffer A and 40% 1× buffer B with 800× diluted beads (Progressive Binding System, Molecular Devices #R8124). 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.
Measurement: IC50 = 2.2 nM (pChEMBL 8.65999984741211)
Descriptors: MW 469.51, ALogP 2.83, HBA 8, HBD 3, PSA 136.53, QED 0.37
Compound (SMILES): CCn1ccc(-c2cc3c(ccc4nnc(C)n43)n2Cc2cc(Cl)cc(CN3CCC3)c2)n1
InChIKey: VUKCSMRZCNPQKK-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: AlphaScreen Assay: BRD4-BD1 and BRD4-BD2 assays were conducted in white 384-well polystyrene plate in a final volume of 20 μL for BD1 and 40 μL for BD2. Inhibitors were first serially diluted in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 1.25% (BD1) and 0.83% (BD2). The assays were carried out at room temperature for 75 min. in the assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.05% CHAPS, 0.01% BSA), containing 50 nM Biotin-labeled tetra-acetylated histone H4 peptide (H4Ac4), 3.8 nM (BRD4-BD1, BPS Bioscience #31040) or 20 nM (BRD4-BD2, BPS Bioscience #31041). The reaction followed by the addition of 20 μL of assay buffer supplemented with Streptavidin donor beads (PerkinElmer 6760002) and GSH Acceptor beads (PerkinElmer-AL109C) at 4 μg/mL under reduced light. After plate sealing, the plate was incubated in the dark at room temperature for 75 min. before reading on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 459.99, ALogP 4.78, HBA 7, HBD 0, PSA 56.18, QED 0.37
Compound (SMILES): C[C@H](CO)Nc1n[nH]c2nccc(Oc3ccc(NC(=O)c4cc(C5CC5)nn(-c5ccc(F)cc5)c4=O)cc3F)c12
InChIKey: OXQCVUDNHCLLCA-OAHLLOKOSA-N
Target: Tyrosine-protein kinase receptor TYRO3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06418
Assay: Enzyme Assay: The affinity of compound binding to wild type and mutant human MET kinases is measured using Invitrogen's LanthaScreen Eu Kinase Binding technology. Briefly, GST-tagged recombinant human MET kinase domain from Signal Chem (see Table 8 below for concentration in assay) is incubated with 50 nM Alexa-Fluor® Tracer 236 (Invitrogen Cat No. PR9078A) and 2 nM Europium-anti-GST (Invitrogen Cat. No. A151 6) along with test compound in a buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, 1 mM DTT, and 2% DMSO. Compounds are typically prepared in a three-fold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-minute incubation at 22° C., the reaction is measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) calculated using a ratiometric emission factor. 100 POC is determined using no test compounds and 0 POC is determined using a concentration of control compound that completely inhibits the enzyme
Measurement: IC50 = 9800 nM (pChEMBL 5.010000228881836)
Descriptors: MW 573.56, ALogP 4.5, HBA 9, HBD 4, PSA 147.05, QED 0.2
Compound (SMILES): CCOc1ncccc1-c1cc(NCc2cnn(C)c2)c2c(cnn2CC)n1
InChIKey: GDQVLYVPAWKIKI-UHFFFAOYSA-N
Target: Dual specificity calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1C (SINGLE PROTEIN), Homo sapiens
UniProt: Q14123
Assay: PDE1 Inhibition Assay: PDE1A, PDE1B and PDE1C assays were performed as follows: the assays was performed in 60 μL samples containing a fixed amount of the PDE1 enzyme (sufficient to convert 20-25% of the cyclic nucleotide substrate), a buffer (50 mM HEPES pH 7.6; 10 mM MgCl2; 0.02% Tween20), 0.1 mg/ml BSA, 15 nM tritium labelled cAMP and varying amounts of inhibitors. Reactions were initiated by addition of the cyclic nucleotide substrate, and reactions were allowed to proceed for 1 hr at room temperature before being terminated through mixing with 20 μL (0.2 mg) yttrium silicate SPA beads (PerkinElmer). The beads were allowed to settle for 1 hr in the dark before the plates were counted in a Wallac 1450 Microbeta counter. The measured signals were converted to activity relative to an uninhibited control (100%) and IC50 values were calculated using XIFit (model 205, IDBS).
Measurement: IC50 = 440 nM (pChEMBL 6.360000133514404)
Descriptors: MW 377.45, ALogP 3.26, HBA 8, HBD 1, PSA 82.68, QED 0.53
Compound (SMILES): O=C(NC1CCNCC1F)c1cn(Cc2ccnc(F)c2)c2c(F)ccc(F)c12
InChIKey: WLZWNYJYLDDHQB-UHFFFAOYSA-N
Target: Muscarinic acetylcholine receptor M1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11229
Assay: Determination of Allosteric Potency EC50 Values for Muscarinic M1 Receptor: A stable CHO cell line expressing recombinant human Muscarinic M1 receptor and pCRE-Luc reporter system was used for cell-based assay. The assay offers a non-radioactive based approach to determine binding of a compound to GPCRs. In this specific assay, the level of intracellular cyclic AMP which is modulated by activation or inhibition of the receptor is measured. The recombinant cells harbor luciferase reporter gene under the control of cAMP response element.The above cells were grown in 96 well clear bottom white plates in Hams F12 medium containing 10% fetal bovine serum (FBS). Prior to the addition of compounds or standard agonist, cells were serum starved overnight. Increasing concentrations of test compounds were added along with EC20 of acetylcholine in OptiMEM medium to the cells. The incubation was continued at 37° C. in CO2 incubator for 4 hours. Medium was removed and cells were washed with phosphate buffered saline. The cells were lysed and luciferase activity was measured in a Luminometer. Luminescence units were plotted against the compound concentrations using Graphpad software. EC50 values of the compounds were defined as the concentration required in stimulating the luciferase activity by 50% in presence of EC20 of acetylcholine.
Measurement: EC50 = 1390 nM (pChEMBL 5.860000133514404)
Descriptors: MW 406.38, ALogP 2.93, HBA 4, HBD 2, PSA 58.95, QED 0.52
Compound (SMILES): Nc1sc(-c2c(F)cccc2F)nc1C(=O)Nc1cnc2c(c1N1CC[C@H](O)[C@@H](N)C1)CCC2
InChIKey: QKMVVDJYGIYDRY-YOEHRIQHSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Enzyme Assay: Pim-2 kinase assay-20 μL reactions were run in white 384 well polystyrene plates dotted with 0.8 μL compound/DMSO in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM MgCl2, 0.01% BSA, 5 mM DTT), containing 0.05 μM Fluorescein-labeled CREBtide peptide substrate (Invitrogen PV3508), 1 mM ATP, and 1 nM enzyme (Invitrogen PV3649) for 2 h at 25° C. Reactions were stopped by addition of 10 μL TR-FRET Dilution Buffer (Invitrogen PV3574) with 30 mM EDTA and 1.5 nM LanthaScreen Tb-CREB pSer133 antibody (Invitrogen PV3566). After 30 min. incubation at room temperature, plates were read on a PHERAstar FS plate reader.
Measurement: IC50 > 2000 nM (pChEMBL 5.698999881744385)
Descriptors: MW 486.55, ALogP 2.7, HBA 8, HBD 4, PSA 130.39, QED 0.45
Compound (SMILES): CCCN(C)CCC(=O)N1CCC(c2ccc3[nH]c(-c4cn5ncnc5c(C)c4C)c(C(C)C)c3c2)CC1
InChIKey: YCLZJXUCUKRSIC-UHFFFAOYSA-N
Target: Toll-like receptor 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR96
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 = 1000 nM (pChEMBL 6)
Descriptors: MW 514.72, ALogP 6.06, HBA 5, HBD 1, PSA 69.53, QED 0.31