lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCNCC[C@H](Oc1cccc2cnccc12)c1cccs1
InChIKey: AJMKPXNBSXISRO-KRWDZBQOSA-N
Target: Voltage-dependent calcium channel subunit alpha-2/delta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P54289
Assay: Binding Assay: Human α2δ-1 enriched membranes (2.5 μg) were incubated with 15 nM of radiolabeled [3H]-Gabapentin in assay buffer containing Hepes-KOH 10 mM, pH 7.4.NSB (non specific binding) was measured by adding 10 μM pregabalin. The binding of the test compound was measured at five different concentrations. After 60 min incubation at 27° C., binding reaction was terminated by filtering through Multiscreen GF/C (Millipore) presoaked in 0.5% polyethyleneimine in Vacuum Manifold Station, followed by 3 washes with ice-cold filtration buffer containing 50 mM Tris-HCl, pH 7.4. Filter plates were dried at 60° C. for 1 h and 30 μl of scintillation cocktail were added to each well before radioactivity reading.Readings were performed in a Trilux 1450 Microbeta radioactive counter (Perkin Elmer).
Measurement: Ki > 3000 nM (pChEMBL 5.5229997634887695)
Descriptors: MW 312.44, ALogP 4.42, HBA 4, HBD 1, PSA 34.15, QED 0.66
Compound (SMILES): NS(=O)(=O)c1ccc(CCN[C@H](C(=O)N2CCc3ccccc32)c2ccccc2)cc1
InChIKey: ZNDOJMRQYRETAN-QHCPKHFHSA-N
Target: Histone acetyltransferase p300 (SINGLE PROTEIN), Homo sapiens
UniProt: Q09472
Assay: Biochemical Assay: The full length p300 SPA assay was run following the same protocol as p300 HAT SPA assay, but used 6 nM purified full length p300 (purchased from Active Motif) in place of the purified p300-HAT domain.Select compounds were also evaluated in a H3K18Ac MSD cellular assay that measures the ability of compounds to inhibit the the acetylation of chromatin at H3K18, a process catalyzed by p300 and CBP. In a typical experiment the p300 HAT inhibitory activity inside cells of the compounds described herein was determined in accordance with the following experimental method. 20 k HCT-116 cells per well are plated in 75 μL RPMI+10% FBS media the night before treatment. Compounds plated in DMSO at 4× final concentration are resuspended in 30 μL RPMI+10% FBS, then 25 μL is combined with corresponding wells containing cells. Treated cells are incubated for 2 hr at 37° C., then lysed in 500 μL final volume and frozen at −80° C. MSD plates (Meso Scale Discovery) are coated overnight at 4° C. with 60 μL 1:500 α-total histone antibody (Millipore MAB3422) in PBS. Plates are then blocked with 5% BSA in TBST shaking at RT for 1 hr, washed, and 30 μL lysate added to each well for 2 hr shaking at RT. Plates are washed and 25 μL 1:216 α-H3K18ac antibody (CST 9675) in PBS added, then incubated for 1 hr shaking at RT. Plates are washed again, then 25 μL 1:1000 Sulfo-Tag goat α-rabbit antibody (Meso Scale Discovery R32Ab-1) in PBS is added for 1 hr shaking at RT. Plates are washed once more, then 150 μL 1× Read Buffer (MSD #R92TD-3) is added to all wells and read on MSD SECTOR Imager 2400 using the conventional read setup.
Measurement: IC50 = 1930 nM (pChEMBL 5.710000038146973)
Descriptors: MW 435.55, ALogP 2.8, HBA 4, HBD 2, PSA 92.5, QED 0.6
Compound (SMILES): COc1cn(Cc2ccc(F)c(Cl)c2)c(Nc2ccc(C#N)cc2C)cc1=O
InChIKey: WUMMHZLMXCUWHD-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Evaluation of a Human P2X7 Receptor Inhibitory Activity: Stably expressing cell line (1321N1 cell transfected with the human P2X7 receptor gene (GenBank accession number NM_002502.5 including T606C and G952A SNP)) was used. The cells were seeded in a 384-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (10% fetal bovine serum, 25 mM HEPES, 1% penicillin and streptomycin in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. After replacing with 20 μL of the HBSS buffer (20 mM HEPES, 55.6 mM D-glucose, 1×HBSS(−), pH7.4-7.5). 16 μL of 17.3 μM Yo-Pro solution in the HBSS buffer was added. The plate was placed in high-throughput cellular screening system FLIPR TETRA (Molecullar Devices, LLC.) and 15 μL of 130 μM BzATP solution in the HBSS buffer was added. Measurement, of fluorescence intensity by FLIPR TETRA was started. After eight minutes, 15 μL of DMSO solutions containing different concentrations of the compound of the present invention as prepared by dilution with the HBSS buffer were dispensed to each well through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 20 minutes. The maximum fluorescence intensity without the compound of the present invention is calculated as 0% inhibition and the maximum fluorescence intensity when the reference compound was added is calculated as 100% inhibition. Changing values of fluorescence intensity by the compound of the present invention were calculated by difference between maximum and minimum fluorescence intensity for 20 minutes.
Measurement: IC50 = 200 nM (pChEMBL 6.699999809265137)
Descriptors: MW 397.84, ALogP 4.62, HBA 5, HBD 1, PSA 67.05, QED 0.68
Compound (SMILES): CCCCCCCCCCCCCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)NO
InChIKey: TUERMRRNMUNKFA-CPCREDONSA-N
Target: Matrix metalloproteinase-9 (SINGLE PROTEIN), Homo sapiens
UniProt: P14780
Assay: Inhibition Assay: MMP-9: Recombinant MMP-9 expressed was obtained from Merck-Millipore (catalog number PF140-5UG, used in examples 1 to 9 and comparative examples) and Enzo (catalog number-SE360-0010, used in examples 10 to 16). DQ gelatin from pig skin fluorescein conjugated (MMP-9 substrate) was obtained from Life Technologies (catalog number E12055).Preparation of the MMP-9 for the activity assay: MMP-9 was provided as a stock solution (0.1 mg/mL). The enzyme was diluted in 1× reaction buffer to a final concentration of 5 μg/mL.Preparation of the DQ gelatin (substrate) solution for the activity assay: the solid form of the substrate was dissolved in water in order to obtain a stock solution of 1 mg/mL.Procedure: The enzymatic assays were performed in 96-well microtiter plate, which allowed simultaneous monitoring of multiple reactions. For each reaction, 84.6 μL of 1× reaction buffer (pH 7.6), 3.4 μL of MMP-9 (final concentration 85 ng/mL) and 2 μL of the corresponding new compound were added to each well. A stock solution of new compound was prepared in DMSO (100 mM), and dilutions were prepared from this stock solution with DMSO. Finally, to the mixture contained in each well 10 μL of the substrate were added (final concentration 50 μg/mL). The reaction was incubated for 4 hours at room temperature in an orbital shaker (100 rpm).
Measurement: IC50 = 1200 nM (pChEMBL 5.920000076293945)
Descriptors: MW 639.88, ALogP 5.92, HBA 5, HBD 5, PSA 143.63, QED 0.07
Compound (SMILES): Cc1ccc(-c2ccccc2C(=O)N2CC[C@H](C)[C@H]2c2nc3cc(Cl)c(C)cc3[nH]2)cc1
InChIKey: FVSDPGVQXOJCRW-GKVSMKOHSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Inhibition Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 μg/ml G418, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2.Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 443.98, ALogP 6.72, HBA 2, HBD 1, PSA 48.99, QED 0.38
Compound (SMILES): CCOC(=O)c1cc2ccc(OCc3cccc(Cl)c3)cc2nc1C
InChIKey: ASKYBHHJBVSRQM-UHFFFAOYSA-N
Target: Amine oxidase [flavin-containing] B (SINGLE PROTEIN), Homo sapiens
UniProt: P27338
Assay: Enzymatic Assay: The MAO enzymatic assay was performed according to the fluorometric method described by Matsumoto and colleagues (Matsumoto et al., Clin. Biochem. 1985, 18, 126-129) with the following modifications. Human recombinant MAO-A and MAO-B expressed in insect cells were used. For both assays, test compound and/or vehicle were preincubated with purified enzyme in phosphate buffer pH 7.4 for 15 minutes at 37° C. The reaction was initiated by addition of 50 μm kynuramine. Following a 60 minute incubation period, the reaction was terminated by the addition of 6 N NaOH. The amount of 4-hydroxyquinoline that formed was determined by spectrofluorimetrically at 325 nm/465 nm.
Measurement: EC50 < 100 nM (pChEMBL 7)
Descriptors: MW 355.82, ALogP 4.95, HBA 4, HBD 0, PSA 48.42, QED 0.61
Compound (SMILES): COc1cc(C)c2cc(Cc3c(Cl)ccc(C(=O)N4CCC(CC(=O)O)CC4)c3Cl)n(C)c2c1
InChIKey: SUJMEXATQSIBEY-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: transient transactivation assay: The primary screen was performed by transient transactivation assays. These cell-based assays were carried out using Cos-7 cells transfected with a chimeric human RORγ-Gal4 receptor expression plasmid and a 5Gal4 pGL3 TK Luc reporter plasmid. Transfections were performed by a chemical agent (Jet PEI). Transfected cells were distributed in 384-wells plates and were allowed to recover for 24 h. The culture medium was then removed and fresh medium containing the compounds to be tested was added (final concentration ranging from 10−4 M to 3 10−10 M). After an overnight incubation, luciferase expression was measured by adding SteadyGlo according to the manufacturer's instructions (Promega). T0901317 at 10−5 M was used as reference. Results were expressed as fold induction compared to basal level or as percentage activity compared to references taken as 100% (basal) or 0% (T0901317). Dose-effect curves and IC50 were calculated using the software Assay Explorer (MDL).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 503.43, ALogP 5.72, HBA 4, HBD 1, PSA 71.77, QED 0.47
Compound (SMILES): Cc1cc(S(=O)(=O)Nc2cscn2)ncc1NCc1c(F)cccc1CN1CCC1
InChIKey: GDTQBMQODPYSNY-UHFFFAOYSA-N
Target: Sodium channel protein type 5 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q14524
Assay: Sodium Influx Assay (In Vitro Assay): This sodium influx assay employs the use of the cell permeable, sodium sensitive dye ANG2 to quantify sodium ion influx through sodium channels which are maintained in an open state by use of sodium channel modulators. This high throughput sodium influx assay allows for rapid profiling and characterization of sodium channel blockers.In general, Trex HEK293 cells were stably transfected with an inducible expression vector containing the full-length cDNA coding for the desired human sodium channel α-subunit and with an expression vector containing full length cDNA coding for the β1-subunit. Sodium channel expressing cell lines were induced with tetracycline (1 μg/mL) and plated on 384-well PDL-coated plates at a density of 25K-30K cells/well in culture media (DMEM, containing 10% FBS and 1% L-glutamine). After overnight incubation (37° C., 5% CO2), culture media was removed and cells were loaded with 5 uM ANG2 dye for 1-1.5 h in Buffer 1 (155 mM NMDG, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, adjusted with Tris to pH 7.4). Access dye was removed and cells were incubated with test compounds for 1 hr in buffer 1 containing sodium channel modulator(s) at room temperature. Hamamatsu FDSS μCell was used to perform a 1:1 addition of Na/K challenge buffer (140 mM NaCl, 20 mM HEPES, 1 mM CaCl2, 15 mM KCl, 1 mM MgCl2, 10 mM glucose, adjusted with Tris to pH 7.4) and simultaneously read plates at excitation wavelength of 530 nm and emission wavelength set at 558 nm. Percent inhibition of sodium ion influx was calculated for each test compound at each test concentration to determine the IC50 values.
Measurement: IC50 = 30000 nM (pChEMBL 4.519999980926514)
Descriptors: MW 447.56, ALogP 3.6, HBA 7, HBD 2, PSA 87.22, QED 0.55
Compound (SMILES): CC1(O)CCN(c2nc(C(C)(C)C)nc3c2ncn3Cc2n[nH]cc2C(F)(F)F)C1
InChIKey: VJSGOMMBKDRBSR-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1x HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min. Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 uM to 0.13 nM cAMP. EC50 values were determined using Activity Base analysis (ID Business Solution, Limited). The EC50 values for a wide range of cannabinoid agonists generated from this assay were in agreement with the values published in the scientific literature. The compounds of the invention are CB2 receptor agonists with EC50 below 1 uM and selectivity versus CB1 in the corresponding assay of at least 10 fold. Particular compound of the invention are CB2 receptor agonists with EC50 below 0.05 uM and selectivity versus CB1 in the corresponding assay of at least 500 fold.
Measurement: EC50 = 22.1 nM (pChEMBL 7.659999847412109)
Descriptors: MW 423.44, ALogP 2.88, HBA 7, HBD 2, PSA 95.75, QED 0.67
Compound (SMILES): N#Cc1ccc(-c2cc(C(=O)NCc3cccc(Cl)c3)cc3ncnn23)cc1
InChIKey: JJYJJVFWDYJUSP-UHFFFAOYSA-N
Target: Prolyl hydroxylase EGLN2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96KS0
Assay: PHD1 Enzyme Assay: The IC50 values for the PHD1 enzyme (residues 1-407) were determined by mixing increasing amounts of a compound of the invention with a fixed amount of the enzyme (20 nM final concentration) and peptide substrate (Asp-Leu-Asp-Leu-Glu-Ala-Leu-Ala-Pro-Tyr-Ile-Pro-Ala-Asp-Asp-Asp-Phe-Gln-Leu, 1 μM final concentration) and 2-oxoglutarate (0.5 M final concentration) in an assay buffer comprising 30 mM 2-(N-morpholino)ethanesulfonic acid pH 6.0, 2 mM sodium ascorbate, 100 μM dithiothreitol, 2 mg/ml bovine serum albumin, 60 μg/ml catalase enzyme and 1 μM iron (II) sulphate (FeSO4). The reaction was conducted by pre-incubating the PHD1 enzyme in the presence of a compound of the invention for 60 minutes at room temperature. The activity of the free enzyme was measured by adding the peptide, the 2-oxoglutarate and sodium ascorbate (see above for final concentrations). The assay was quenched by the addition of 30% v/v trichloroacetic acid (final concentration 5%). The amount of product released was measured using a UPLC-MS (Agilent 1290 with an ABSciex 4000qTrap Mass Spectrometer). Data were analysed using the classical isotherm equation for the determination of IC50.
Measurement: IC50 = 307 nM (pChEMBL 6.510000228881836)
Descriptors: MW 387.83, ALogP 3.85, HBA 5, HBD 1, PSA 83.08, QED 0.58
Compound (SMILES): CNS(=O)(=O)N1CCc2c(nn(C(=O)C3CCNc4ccccc43)c2N)C1
InChIKey: OJHYULDHFKPDSI-UHFFFAOYSA-N
Target: Coagulation factor XII (SINGLE PROTEIN), Homo sapiens
UniProt: P00748
Assay: Factor XIIa (FXIIa) Inhibitory Activity: In a 96-well clear bottom plate, 80 μl of assay buffer was added to each well. Assay buffer consists of 0.5× Hank's Balanced Salt Solution (Invitrogen), buffered with 25 mM HEPES pH 7.4 (Invitrogen) and 0.5× Tris-buffered saline with Tween-20 0.05% (Santa Cruz Biotechnology). Test compounds were first dissolved in DMSO (Sigma) and then 4 μl were added to test wells containing assay buffer. Serial dilutions using an automated multi-channel pipette were used to generate a concentration range of approximately 1-100 μM. Human FXIIa (Enzyme Research Labs) was diluted in assay buffer to a final concentration of 12.5 nM. 80 μl of this enzyme solution was added to the assay wells. The enzyme/compound mixtures were incubated for 10 minutes at room temperature. Chromogenic substrate (Pefachrome XIIa; Enzyme Research Labs) was added to assay wells at a final concentration of 400 μM. The assay plate was spun for 1 minute at 1500 g and then read at 37° C. in a SpectraMax M2 plate reader (λ=405 nm). Activity was quantified as the rate of change in absorbance, which corresponds to the rate of substrate cleavage. IC50 values were determined as the concentration of inhibitor that produced 50% of the rate of change of control wells without any inhibitor. For compounds with activity <1 μM, the assay was repeated with a lower concentration range, typically from 10-1000 nM.
Measurement: IC50 = 2000 nM (pChEMBL 5.699999809265137)
Descriptors: MW 390.47, ALogP 0.53, HBA 7, HBD 3, PSA 122.35, QED 0.7
Compound (SMILES): N#Cc1ccc(-c2cnc3ccc(-c4ccc(C(=O)N5CCCNCC5)cc4)cn23)cc1
InChIKey: OPYNTMLXODTTLZ-UHFFFAOYSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BUB5
Assay: In Vitro Kinase Assay: MNK1 and MNK2 inhibitor activity was determined using recombinant kinase domains expressed in E. coli. MNK1 and MNK2 were expressed as GST fusion proteins and the GST tag was removed using PreScission protease. After concentration to 10-15 mg/ml the proteins were flash frozen in liquid nitrogen and stored at −80° C. MNK1 and MNK2 were activated using recombinant ERK2 which was activated using a constitutively active mutant of MEK1, both ERK2 and MEK1 were expressed in E. coli as N-terminally his tagged proteins. Recombinant ERK2 was activated by incubating 11.3 μM of the kinase with 1 μM MEK1 and 100 μM ATP. This reaction mixture was then used immediately for the activation of the MNKs. The activation of the MNK1 was performed by incubating 5.0 μM of MNK1 with 0.3 μM of activated ERK2 and 500 μM ATP at 30° C. for 6 hours. The activation of MNK2 was performed by incubating 50 μM of MNK2 with 3.0 μM of activated ERK2 and 500 μM ATP at 30° C. for 2 hours. The activated MNKs were stored at −20° C. until required for assay.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 421.5, ALogP 3.98, HBA 5, HBD 1, PSA 73.43, QED 0.54
Compound (SMILES): NC1CCN(Cc2ccc([C@H]3COc4ccccc4O3)cc2)CC1
InChIKey: ZSUDRFSFONYWNQ-HXUWFJFHSA-N
Target: Leukotriene A-4 hydrolase (SINGLE PROTEIN), Homo sapiens
UniProt: P09960
Assay: Enzyme Assay: The compounds of the invention are assessed for the ability to interact with human LTA4 hydrolase in an enzymatic assay that measures the ability of the enzyme to cleave the peptide bond of arginyl-aminomethylcoumarin (Arg-AMC). LTA4H Enzyme (1 nM final), Arg-AMC substrate (50 μM final), and compound are combined in a reaction buffer (50 mM Tris-HCl (pH 7.5), 100 mM KCl, 0.5% bovine serum albumin) at room temperature for 1 h. The formation of product is assessed by measuring the fluorescence of aminomethylcoumarin product (excitation wavelength 380 nm/emission wavelength 460 nm).
Measurement: IC50 = 2.28 nM (pChEMBL 8.640000343322754)
Descriptors: MW 324.42, ALogP 3.12, HBA 4, HBD 1, PSA 47.72, QED 0.94
Compound (SMILES): CNCCN1CCC(c2ccc3[nH]c(-c4ccnc(OC)c4)c(CC4CC4)c3c2)CC1
InChIKey: RKQRETHYYKXIND-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: 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.
Measurement: IC50 = 94 nM (pChEMBL 7.03000020980835)
Descriptors: MW 418.59, ALogP 4.59, HBA 4, HBD 2, PSA 53.18, QED 0.56
Compound (SMILES): Nc1cccc(S(=O)(=O)N2C3CCC[C@@H]2C[C@@H](NC(=O)c2cc(C4CC4)on2)C3)n1
InChIKey: UVTJUGSRDQBSET-GRKKQISMSA-N
Target: Histone-lysine N-methyltransferase SMYD3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H7B4
Assay: SMYD3 Biochemical Assay: The assays were all performed in a buffer consisting of 25 mM Tris-Cl pH 8.0, 1 mM TCEP, 0.005% BSG, and 0.005% Tween 20, prepared on the day of use. Compounds in 100% DMSO (1 ul) were spotted into a 384-well white opaque OptiPlate using a Bravo automated liquid handling platform outfitted with a 384-channel head (Agilent Technologies). DMSO (1 ul) was added to Columns 11, 12, 23, 24, rows A-H for the maximum signal control and 1 ul of SAH, a known product and inhibitor of SMYD3, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control. A cocktail (40 ul) containing the SMYD3 enzyme was added by Multidrop Combi (Thermo-Fisher). The compounds were allowed to incubate with SMYD3 for 30 min at room temperature, then a cocktail (10 ul) containing SAM and MEKK2 was added to initiate the reaction (final volume=51 ul). The final concentrations of the components were as follows: SMYD3 was 0.4 nM, 3H-SAM was 8 nM, MEKK2 was 12 nM, SAH in the minimum signal control wells was 1 mM, and the DMSO concentration was 2%. The assays were stopped by the addition of non-radiolabeled SAM (10 ul) to a final concentration of 100 uM, which dilutes the 3H-SAM to a level where its incorporation into MEKK2 is no longer detectable. Radiolabeled MEKK2 was detected using a scintillation proximity assay (SPA). 10 uL of a 10 mg/mL solution of SPA beads in 0.5 M citric acid was added and the plates centrifuged at 600 rpm for 1 min to precipitate the radiolabeled MEKK2 onto the SPA beads. The plates were then read in a PerkinElmer TopCount plate reader to measure the quantity of 3H-labeled MEKK2 as disintegrations per minute (dpm) or alternatively, referred to as counts per minute (cpm).% Inhibition Calculation% ⁢ ⁢ inh = 100 - ( dpm cmpd - dpm min dpm max - dpm min ) × 100Where dpm=disintegrations per minute, cmpd=signal in assay well, and min and max are the respective minimum and maximum signal controls.Four-Parameter IC50 FitY = Bottom + ( Top - Bottom ) ( 1 + ( X IC 50 ) Hill ⁢ ⁢ CoefficientWhere top and bottom are the normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit. The Hill Coefficient normally allowed to float but may also be fixed at 1 in a 3-parameter fit. Y is the % inhibition and X is the compound concentration.
Measurement: IC50 = 498 nM (pChEMBL 6.300000190734863)
Descriptors: MW 431.52, ALogP 2.03, HBA 7, HBD 2, PSA 131.42, QED 0.74
Compound (SMILES): CP(C)(=O)c1ncccc1-c1ccc(N2C(=O)[C@H](NC(=O)Nc3ccc(C(F)(F)F)cc3F)C[C@H]2CO)c(F)c1F
InChIKey: MRMBFIJJGQCQQO-IFXJQAMLSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: FPR2 and FPR1 Cyclic Adenosine Monophosphate (cAMP) Assays: A mixture of forskolin (5 μM final for FPR2 or 10 μM final for FPR1) and IBMX (200 μM final) were added to 384-well Proxiplates (Perkin-Elmer) pre-dotted with test compounds in DMSO (1% final) at final concentrations in the range of 0.020 nM to 100 μM. Chinese Hamster Ovary cells (CHO) overexpressing human FPR1 or human FPR2 receptors were cultured in F-12 (Ham's) medium supplemented with 10% qualified FBS, 250 μg/ml zeocin and 300 μg/ml hygromycin (Life Technologies). Reactions were initiated by adding 2,000 human FPR2 cells per well or 4,000 human FPR1 cells per well in Dulbecco's PBS (with calcium and magnesium) (Life Technologies) supplemented with 0.1% BSA (Perkin-Elmer). The reaction mixtures were incubated for 30 min at room temperature. The level of intracellular cAMP was determined using the HTRF HiRange cAMP assay reagent kit (Cisbio) according to manufacturer's instruction. Solutions of cryptate conjugated anti-cAMP and d2 fluorophore-labelled cAMP were made in a supplied lysis buffer separately. Upon completion of the reaction, the cells were lysed with equal volume of the d2-cAMP solution and anti-cAMP solution. After a 1-h room temperature incubation, time-resolved fluorescence intensity was measured using the Envision (Perkin-Elmer) at 400 nm excitation and dual emission at 590 nm and 665 nm. A calibration curve was constructed with an external cAMP standard at concentrations ranging from 1 μM to 0.1 pM by plotting the fluorescent intensity ratio from 665 nm emission to the intensity from the 590 nm emission against cAMP concentrations. The potency and activity of a compound to inhibit cAMP production was then determined by fitting to a 4-parametric logistic equation from a plot of cAMP level versus compound concentrations.
Measurement: EC50 = 1.5 nM (pChEMBL 8.819999694824219)
Descriptors: MW 600.46, ALogP 4.72, HBA 5, HBD 3, PSA 111.63, QED 0.28
Compound (SMILES): Cc1cc(-c2[nH]c3ccc(C4CCN(C(=O)C5CCOCC5)CC4)cc3c2C(C)C)ccn1
InChIKey: XWCBXPYIIDFVMO-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: 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.
Measurement: IC50 = 45.6 nM (pChEMBL 7.340000152587891)
Descriptors: MW 445.61, ALogP 5.79, HBA 3, HBD 1, PSA 58.22, QED 0.55
Compound (SMILES): CCC[C@H](N)c1cccc(-c2cc(C3=CCN(C(C)=O)CC3)c3cnn(-c4cccc(CO)n4)c3c2)n1
InChIKey: JWFIRLJLFLMMEL-VWLOTQADSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Biochemical Assay: The biochemical assay is in a AlphaScreen format. The kinase reaction is based on the IRAK-4 phosphorylation of a biotin labeled peptide. The phosphopeptide is incubated with anti-phosphothreonine antibody as well as streptavidin- and protein A-coated beads. Binding of the protein-A coated beads to the antibody and the streptavidin beads to the peptide, leads to an energy transfer from one bead to the other, ultimately producing a luminescent/fluorescent signal.Generally, the kinase reaction is carried out at 1 nM IRAK4, 1.6 μM peptide, 1 mM ATP in reaction buffer 50 mM Hepes, 60 mM NaCl, 5 mM MgCl2, 0.25 mM MnCl2, 2 mM DTT, 0.01% BSA, 0.01% Tween-20) for 3.5 h at RT.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 496.62, ALogP 4.41, HBA 7, HBD 2, PSA 110.16, QED 0.39
Compound (SMILES): C[C@H]1CC[C@@H](c2nc(-c3ccc(C(=O)Nc4cc(OCC(F)(F)F)ccn4)cc3)c3c(N)nccn23)CN1C(=O)C1(C)COC1
InChIKey: HJVBQWNEEVHOKR-GHTZIAJQSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Btk Enzyme Activity Assay C: BTK enzymatic activity was determined with the LANCE (Lanthanide Chelate Excite) TR-FRET (Time-resolved fluorescence resonance energy transfer) assay. In this assay, the potency (IC50) of each compound was determined from an eleven point (1:3 serial dilution; final compound concentration range in assay from 1 μM to 0.017 nM) titration curve using the following outlined procedure. To each well of a black non-binding surface Corning 384-well microplate (Corning Catalog #3820), 5 nL of compound (2000 fold dilution in final assay volume of 10 μL) was dispensed, followed by the addition of 7.5 μL of 1× kinase buffer (50 mM Hepes 7.5, 10 mM MgCl2, 0.01% Brij-35, 1 mM EGTA, 0.05% BSA & 1 mM DTT) containing 5.09 pg/μL (66.67 pM) of BTK enzyme (recombinant protein from baculovirus-transfected Sf9 cells: full-length BTK, 6HIS-tag cleaved). Following a 60 minute compound & enzyme incubation, each reaction was initiated by the addition of 2.5 μL 1× kinase buffer containing 8 μM biotinylated A5 peptide (Biotin-EQEDEPEGDYFEWLE-NH2), and 100 μM ATP. The final reaction in each well of 10 μL consists of 50 pM hBTK, 2 μM biotin-A5-peptide, and 25 μM ATP. Phosphorylation reactions were allowed to proceed for 120 minutes. Reactions were immediately quenched by the addition of 20 uL of 1× quench buffer (15 mM EDTA, 25 mM Hepes 7.3, and 0.1% Triton X-100) containing detection reagents (0.626 nM of LANCE-Eu-W1024-anti-phospho Tyrosine antibody, PerkinElmer and 86.8 nM of Streptavidin-conjugated Dylight 650, Dyomics/ThermoFisher Scientific). After 60 minutes incubation with detection reagents, reaction plates were read on a PerkinElmer EnVision plate reader using standard TR-FRET protocol. Briefly, excitation of donor molecules (Eu-chelate:anti-phospho-antibody) with a laser light source at 337 nm produces energy that can be transferred to Dylight-650 acceptor molecules if this donor:acceptor pair is within close proximity. Fluorescence intensity at both 665 nm (acceptor) and 615 nm (donor) are measured and a TR-FRET ratio calculated for each well (acceptor intensity/donor intensity). IC50 values were determined by 4 parameter robust fit of TR-FRET ratio values vs. (Log10) compound concentrations.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 623.64, ALogP 4.7, HBA 9, HBD 2, PSA 136.97, QED 0.3
Compound (SMILES): O=C(NCCCc1ccncc1)c1ccc2nc(-c3ccc(F)cc3)c(-c3ccc(F)cc3)nc2c1
InChIKey: NUMSZVQBTCXKGU-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: NAMPT Enzyme Assay: To measure the inhibition of NAMPT activity hNAMPT protein stock and anti 6His-Tb (Cisbio; Cat. No. 61HISTLB) is diluted to 3× final concentration with assay buffer (50 mM Tris-HCl (pH7.5), 1 mM DTT, 100 mM NaCl, 10 mM MgCl2, 0.005% Tween 20). To this solution is added test compounds or vehicle control (DMSO) and BodiPY ligand (structure below). The plate is shaken for 1-2 min, sealed and incubated for 1 hour at rt in the dark. The TR-FRET signal is measured using BMG Pherastar (Lanthascreen protocol on BMG Pherastar). Excitation was carried out at 320 nm, and the ratio of emission of BodiPY (520 nm) to terbium (486 nm) was determined. Concentration response curves are generated by calculating the excitation increase in test compound-treated samples relative to DMSO-treated controls. For the assay method described above, test compound percent inhibition values at a single concentration are calculated relative to control (DMSO) treated samples. Compound concentration response curves are fitted to generate IC50 values from those curves. One skilled in the art will appreciate that these values generated either as percentage inhibition at a single concentration or IC50 values are subject to experimental variation.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 480.52, ALogP 6, HBA 4, HBD 1, PSA 67.77, QED 0.29
Compound (SMILES): Nc1ncnc2ccc(NCCc3ccc(F)cc3)nc12
InChIKey: MAUQWFRCYUDOSP-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O95819
Assay: MAP4K4 Inhibition Assay: The kinase activity of purified human MAP4K4 kinase domain was measured by monitoring the phosphorylation of a peptide substrate derived from moesin protein (Leu-Gly-Arg-Asp-Lys-Tyr-Lys-Thr-Leu-Arg-Gln-Ile-Arg-Gln) fluorescently labeled on the N-terminus with 5-carboxyfluorescein using the Caliper LabChip technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (IC50), compounds were serially diluted in DMSO and added to 10 uL kinase reactions containing 1 nM purified MAP4K4 enzyme, 1 uM peptide substrate, 10 uM ATP, 10 mM MgCl2, 1 mM EGTA, 50 mM Hepes pH 7.2, 1 mM DTT, 0.01% Triton X-100, and 2% DMSO. Reactions were incubated at room temperature in Perkin Elmer Proxiplates for 45 minutes and stopped by the addition of 10 uL of an EDTA-containing solution (50 mM Hepes pH 7.2, 40 mM EDTA, 0.02% Triton X-100). The fraction of phosphorylated peptide was determined as a fraction of total peptide substrate using the Caliper Lab Chip 3000 according to the manufacturer's instructions. IC50 values were determined using the four-parameter non-linear fit model.
Measurement: IC50 = 70.1 nM (pChEMBL 7.150000095367432)
Descriptors: MW 283.31, ALogP 2.4, HBA 5, HBD 2, PSA 76.72, QED 0.77
Compound (SMILES): COc1ncccc1-c1nc2c([nH]1)[C@@H](c1ccc(Cl)cc1)N(c1cc(C)c3nnc(C)n3c1)C2=O
InChIKey: QIJUHAIEZZEKSX-OAQYLSRUSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: 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 ul 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 4.5ul 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 ul 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 (IC50 values) were determined from percent inhibition values at different compound concentrations by non-linear regression analysis.
Measurement: IC50 = 40 nM (pChEMBL 7.400000095367432)
Descriptors: MW 485.94, ALogP 4.54, HBA 7, HBD 1, PSA 101.3, QED 0.4
Compound (SMILES): CC(C)[C@@H]1CN(c2ccc(CO)c(S(C)(=O)=O)c2)CCN1c1nc(C(F)(F)F)c(C(N)=O)s1
InChIKey: VRJNLDNMVIUGAM-AWEZNQCLSA-N
Target: Oxysterols receptor LXR-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q13133
Assay: LXR alpha/beta Radioligand Binding Assay: Compounds of the invention were assessed in a competition binding assay where different concentrations of compounds were incubated with the LXR ligand binding domain (LBD) in the presence of radiolabeled LXR ligand [3H]TO901317. The amount of the LXR-LBD that complexed with [3H]T0901317 was measured by scintillation proximity assay (SPA) employing non-specific binding of LXR-LBD to poly-lysine coated Yttrium silicate beads. Partially purified LXR α or β LBD protein (15-45 nM) was incubated at rt for 30 min with 15 nM [3H]TO901317 (25-40 Ci/mmol) and different concentrations of test compounds in 80 μL of phosphate buffered saline (PBS) buffer containing 2.5% DMSO, 1% glycerol, 2 mM EDTA, 2 mM CHAPS and 5 mM DTT in 96-well plates. Poly-lysine SPA beads (50 μg) were added to each well and the total volume was adjusted to 120 μL. The plates were shaken on an orbital shaker for 20 min and then allowed to settle for 10 more min at rt before a brief centrifugation at 2,000 rpm for 1 min. The SPA signal was measured on a MicroBeta® liquid scintillation counter (Perkin Elmer, Waltham, Mass.), and the results were used for calculating IC50 values based on the total binding (DMSO control) and non-specific binding (5 μM of unlabeled TO901317) controls. The Ki values were calculated according to equation 1, where [RL] is the final concentration of [3H]TO901317 in the assay, and the Kd values of 20 nM and 10 nM of TO901317 for LBDs of LXRα and LXRβ, respectively, were determined by direct titration of the radioligand with these proteins.
Measurement: Ki = 1663 nM (pChEMBL 5.78000020980835)
Descriptors: MW 506.57, ALogP 2.51, HBA 8, HBD 2, PSA 116.83, QED 0.62
Compound (SMILES): Cc1cc(Nc2cc(C)[nH]n2)nc(-c2cnc(C(=O)N[C@@H](C)c3ccc(-n4cc(F)cn4)nc3)c(F)c2)n1
InChIKey: AMHDRUGDFXEYSY-HNNXBMFYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Wild Type Assay at KM: In each well of a 384-well plate, 7.5 nM-10 nM of wild type RET (ProQinase 1090-0000-1) is incubated in a total of 12.5 μL of buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 10 mM MgCl2, 1 mM DTT) with 1 μM CSKtide (FITC-AHA-KKKKD DIYFFFG-NH2) and 25 μM ATP at 25° C. for 120 minutes in the presence or absence of a dosed concentration series of compound (1% DMSO final concentration). The reaction is stopped by the addition of 70 μL of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 35 mM EDTA and 0.2% of Coating Reagent 3 (Caliper Lifesciences)). The plate is then read on a Caliper EZReader 2 (protocol settings: −1.7 psi, upstream voltage −500, downstream voltage −3000, post sample sip 35s). Data is normalized to 0% and 100% inhibition controls and the IC50 calculated using a 4-parameter fit in the CORE LIMS.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 516.52, ALogP 3.97, HBA 9, HBD 3, PSA 139.19, QED 0.3
Compound (SMILES): CN[C@@H](C)C(=O)N[C@H]1CN(C(=O)c2ccc(C(C)=O)cc2)c2ccccc2N(Cc2c(OCC(F)(F)F)c[n+]([O-])c3ccccc23)C1=O
InChIKey: NAPUAFOAUICUGC-FNZWTVRRSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay for BIR2 and BIR3: Ten nanomolar of 6× Histidine-tagged BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(c-biotin)-OH 1:2 TFA, in the presence of 50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/mL bovine serum albumin (BSA). Following a 45 min. incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations. Percentage of inhibition at each concentration was determined to generate an IC50 value for each test compound.
Measurement: IC50 = 41 nM (pChEMBL 7.389999866485596)
Descriptors: MW 663.65, ALogP 3.9, HBA 7, HBD 2, PSA 134.99, QED 0.16
Compound (SMILES): O=C(Nc1cc(C(F)(F)F)ccc1N1CCN(Cc2cscn2)CC1)c1ccc(-c2ccncc2)o1
InChIKey: GXESAQKBZJNLLD-UHFFFAOYSA-N
Target: SRSF protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96SB4
Assay: Inhibition Assay: Screening of a series of molecules identified compounds (Compounds 5, 8, 16 to 18 and 43 of Table 3) that had improved potency against SRPK1 against a series of analogous compounds in which the R3 group of the respective compounds of the present invention (as defined by Formula I) was replaced with a pyridyl ring (FIGS. 1 and 2, in which the reference compounds had an IC50 of 6.00 nM and a ΔTm of 12.8° C. (FIG. 1); an IC50 of 38.8 nM (FIG. 2a ) and an IC50 of 3.8 nM (FIG. 2b )) as determined by kinase assay or differential scanning fluorimetry (temperature difference, ΔTm is inversely proportional to the log Kd, i.e. an increased temperature difference indicates a higher affinity and therefore a more potent inhibitor). These included furan, oxazole, pyrazole, thiazole, methyl-pyrazole, and oxadiazole moieties. Replacement of the pyridine ring of indolyl compound 38 in Table 3 with a furan ring resulted in a drop in potency (23.9 nM for compound 38 vs. 65 nM for the resulting furan analogue).
Measurement: IC50 = 13.7 nM (pChEMBL 7.860000133514404)
Descriptors: MW 513.55, ALogP 5.39, HBA 7, HBD 1, PSA 74.5, QED 0.37
Compound (SMILES): CN(C)C/C=C/C(=O)N1CCC[C@@H](c2cn(-c3ccc(Oc4ccccc4F)cc3)c3c(N)n[nH]c(=O)c23)C1
InChIKey: NMAYPMKISGWRKK-ZAOIQFLMSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Cell Proliferation Assays: MTS testing kit was purchased from Promega. The DMEM, Fetal bovine serum and Penicillin-Streptomycin were purchased from Gibco. Dimethyl sulfoxide (DMSO) was purchased from Sigma.To investigate whether a compound is able to inhibit the activity of BTK in cells, a mechanism-based assay using DOHH2 (DSMZ catalog#: ACC47) and Mino (ATCC® Number CRL-3000) cells was developed. In this assay, inhibition of BTK was detected by the inhibition of DOHH2 and Mino cell proliferations. DOHH2 or Mino cells were cultured in culture flasks to 40-80% confluence in RPMI-1640 plus 10% fetal bovine serum. C ells were collected and plated onto 96-well plates at desired cell density (DOHH2: 5000 cells/well; Mino: 10000 cells/well). Plates were incubated overnight at 37° C., with 5% CO2 to adhere. Compounds were added to the plates, and the final compound concentrations were 10000, 3333, 1111, 270, 124, 41, 14, 4.6 and 1.5 nM. Plates were placed at 37° C., with 5% CO2 for 120 h (DOHH2) or 72 h (Mino). 20 μl MTS/100 μl medium mixture solution were added to each well and the plates were incubated for exactly 2 hours. The reaction was stopped by adding 25 μl 10% SDS per well. Absorbance at 490 nm and 650 nm (reference wavelength) were measured and IC50 was calculated using GraphPad Prism 5.0.
Measurement: IC50 = 151 nM (pChEMBL 6.820000171661377)
Descriptors: MW 530.6, ALogP 4.05, HBA 7, HBD 2, PSA 109.48, QED 0.35
Compound (SMILES): COc1ccc(Cc2nc(C(=O)N[C@@H](c3ccc(SC(F)(F)F)c(F)c3)C3CC3)cc(=O)[nH]2)cc1F
InChIKey: ZKTZRJWWEYFDOJ-JOCHJYFZSA-N
Target: GMP-stimulated 3',5'-cyclic nucleotide phosphodiesterase (SINGLE PROTEIN), Macaca mulatta
UniProt: B3VTJ2
Assay: Fluorescence Polarization (FP) Assay: In a typical experiment the PDE2 inhibitory activity of the compounds of the present invention was determined in accordance with the following experimental method. Rhesus PDE2A3 was amplified from rhesus macaque brain cDNA (Biochain Institute, Hayward, Calif.) using primers based on human PDE2A sequence (accession NM_002599.3) where the forward primer containing a Kozak consensus was 5′-gccaccatggggcaggcatgtggc-3′ and the reverse primer was 5′-tcactcagcatcaaggctgca-3′. Amplification with Easy-A High-Fidelity PCR cloning enzyme (Stratagene, La Jolla, Calif.) was 95° C. for 2 minutes followed by thirty three cycles of 95° C. for 40 seconds, 52° C. for 30 seconds, and 72° C. for 2 minutes 48 seconds. Final extension was 72° C. for 7 minutes. The PCR product was TA cloned into pcDNA3.3-TOPO (Invitrogen, Carlsbad, Calif.) according to standard protocol. A consensus sequence was developed from multiple clones and then deposited into GenBank (EU812167). AD293 cells (Stratagene, La Jolla, Calif.) with 70-80% confluency were transiently transfected with rhesus PDE2A3/pcDNA3.3-TOPO using Lipofectamine 2000 according to manufacturer specifications (Invitrogen, Carlsbad, Calif.).
Measurement: Ki = 3.8 nM (pChEMBL 8.420000076293945)
Descriptors: MW 525.5, ALogP 5.14, HBA 5, HBD 2, PSA 84.08, QED 0.31
Compound (SMILES): Cc1cc(OCCCc2c(C(F)(F)F)n(CCC(=O)O)c3ccccc23)cc(C)c1Cl
InChIKey: RAIMGGIZLUQNEE-UHFFFAOYSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07820
Assay: Fluorescence Polarization Anisotropy Competition Assay: Compound affinity was measured using a fluorescence polarization anisotropy competition assay. Anisotropy measurements were carried out in 384-well, black, flat-bottom plates (Greiner Bio-one, Monroe, N.C., USA). The assay was run using a fluorescein isothiocyanate-labeled BH3 peptide derived from Bak (FITC-AHx-GQVGRQLAIIGDDINR-NH2) that was purchased from GenScript (Piscataway, N.J.) at >95% purity and used without further purification. 10 nM FITC-Bak peptide and 14 nM recombinant Mcl-1 (residues 172-327) were added to assay buffer (3 mM dithiothreitol, 50 mM NaCl, 20 mM Tris, pH 7.5). For selectivity assays, 40 nM Bcl-2 (residues 1-207A96T,G110R, Δ35-91, replaced with Bcl-xL35-50) or 4 nM Bcl-xL (residues 1-209, loop 45-86 deleted) were incubated with 10 nM FITC-Bak in assay buffer.Compounds are diluted in DMSO in a 10-point, 3-fold serial dilution scheme. 2.5 uL compound is added to 47.5 uL of assay buffer containing FITC-Bak and protein, for a final DMSO concentration of 5% and a top concentration of 20 uM. A FITC-Bak peptide alone (100% inhibition) and peptide plus protein (0% inhibition) control is included on each assay plate. The plate was mixed and incubated for 90 minutes at room temperature. Anisotropy is measured at excitation wavelength 480 nm and emission wavelength 535 nm using an EnVision Multi-label plate reader (PerkinElmer, Wellesley, Mass., USA). Fluorescence anisotropy is plotted against compound concentration to generate an IC50 (inhibitor concentration at which 50% of bound peptide is displaced) by fitting the data to a 4-parameter logistic model using XLFit software (Guildford, Surrey, UK). IC50 is converted to a binding dissociation constant (Ki value)
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 453.89, ALogP 6.42, HBA 3, HBD 1, PSA 51.46, QED 0.4