lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCOC(=O)N1CC=C(c2cccn3nc(Nc4ccc(C(=O)NCCN5CCNCC5)cc4)nc23)CC1
InChIKey: WCVNPYRRGPWNJI-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 1.47 nM (pChEMBL 8.829999923706055)
Descriptors: MW 518.62, ALogP 2.35, HBA 9, HBD 3, PSA 116.13, QED 0.42
Compound (SMILES): CP(C)(=O)c1ccccc1-c1ccc(N2CC[C@@H](NC(=O)Nc3ncc(C(F)(F)F)cc3F)C2=O)c(F)c1F
InChIKey: UNMNZXSRCIOIGJ-QGZVFWFLSA-N
Target: fMet-Leu-Phe receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P21462
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 > 10000 nM (pChEMBL 5)
Descriptors: MW 570.43, ALogP 5.36, HBA 4, HBD 2, PSA 91.4, QED 0.32
Compound (SMILES): Cc1ncsc1-c1ccc(CNC(=O)[C@@H]2C[C@@H](O)CN2C(=O)[C@H](C(C)C)N2Cc3ccccc3C2=O)c(OCCOCCOCC#Cc2cccc3c2C(=O)N([C@@H](C(=O)Nc2nccs2)c2cc(F)ccc2O)C3)c1
InChIKey: NJLCDOIUFBCJLJ-RYXNNJGZSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: TR-FRET Assay for ERBB Kinases: All compounds and PROTACs were serially diluted in three-fold increments using 100% DMSO, followed by an intermediate 10-fold dilution using Buffer A (50 mM HEPES, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 1 mM DTT, and 0.1% Pluronic F-68). Two microliters of serially diluted compound or PROTAC were then transferred to black 384-well Proxiplates (PerkinElmer, #6008260) using an Integra Viaflo96. Next, 10 uL of protein kinase in Buffer A was added to each well of the assay plate and pre-incubated with compound for 10 minutes. Kinase reactions were then initiated by addition of 5 uL substrate mix containing 3 mM ATP and 30 uM fluorescein-labeled Poly-GluTyr (Thermo Fisher, #PV3610) in Buffer A and allowed to proceed for 10 minutes at room temperature. Reactions were quenched by addition of a 5 uL mixture containing 5 nM LanthaScreen® Tb-pY20 Antibody (Thermo Fisher, #PV3552) and 40 mM EDTA in Buffer A. Assay plates were then read using a Synergy2 (Biotek Instruments, Winooski, Vt.) via excitation thru a 340/20 nm bandpass filter and emission collected thru 490/10 nm (donor) and 520/25 nm (acceptor) bandpass filters. The final kinase concentrations used for each 15 uL reaction were as follows: 0.2 nM EGFR Exon20NPG (SignalChem, #E10-132GG), 0.1 nM wild type EGFR (BPS Bioscience, #40187), 0.3 nM EGFR L858R/T790M/C797S (BPS Bioscience, #40351), 0.1 nM EGFR L858R (BPS Bioscience, #40189), 0.4 nM L858R/T790M (BPS Bioscience, #40350), InM EGFR Dell9 (SignalChem, #E10-122JG), 10 nM EGFR Dell9 T790M (SignalChem, #E10-122KG), 0.3 nM Her2 (BPS Bioscience, #40230), 15 nM Her2 InsYVMA (SignalChem, #E27-13BG).
Measurement: IC50 = 6100 nM (pChEMBL 5.210000038146973)
Descriptors: MW 1056.21
Compound (SMILES): CCOc1cccc2cc(C(=O)N[C@@H]3C4CCN(CC4)C34CC4)sc12
InChIKey: VFIFSUHWHJHYJR-GOSISDBHSA-N
Target: 5-hydroxytryptamine receptor 3A (SINGLE PROTEIN), Homo sapiens
UniProt: P46098
Assay: [3H]BRL 43694 Competition Binding (h-5HT3 Assay: [3H]BRL 43694 competition binding assay was performed under contract by Cerep Poitiers, France following the methods described in Hope, A. G et al., Characterization of a human 5-hydroxytryptamine3 receptor type A (h5-HT3R-AS) subunit stably expressed in HEK 293 cells, Brit. J. Pharmacol., (1996) 118: 1237-1245.In brief, Chinese Hamster Ovary (CHO) cells stably expressing human 5-HT3 serotonin receptors, grown to confluence in 175 cm2 flasks. Following aspiration of the culture medium, cells were harvested by mechanical agitation in ice cold PBS containing (in mM): (150 NaCl, 8 K2HPO4, 2 KH2PO4, pH 7.4, 37° C.), centrifuged at 4,000 g for 10 min and subsequently stored as a cell pellet at −80 C. When required, the pellet was thawed and resuspended in ice cold homogenization buffer (Tris 50 mM, EGTA 5.0 mM, phenylmethylsulphonylfluoride 0.1 mM, pH 7.6) and homogenized. The homogenate was centrifuged at 48,000 g for 10 minutes at 40° C. The resulting pellet was resuspended in ice cold binding buffer comprising (in mM): NaCl 140, KCl 2.8, CaCl2 1.0; MgCl2, 2.0; HEPES 10 (pH 7.4) and centrifuged as above. The pellet was resuspended in ice cold binding buffer and the protein concentration was determined by the method of Lowry et al., Protein measurement with the Folin phenol reagent, J. Biol. Chem., (1953) 193, 265-275). The membrane homogenate was adjusted to a protein concentration of approximately 600 mg/mL in binding buffer. Assay tubes were loaded with equal volumes of binding buffer containing [3H]BRL 43694 and test compound and 0.5 mL of membrane homogenate in a total reaction volume of 1 ml. Binding was initiated by the addition of the membrane homogenate and allowed to proceed for 120 min. at room temperature. Bound and free radioligand were separated by the addition of 3 ml of ice-cold binding buffer and immediate vacuum filtration through pre-soaked (0.1% (v/v) polyethyleneimine) Whatman GF/B filters. Filters were washed with a further 2×3 mL applications of binding buffer and counted for radioactivity using a scintillation counter.The results were expressed as a percent inhibition of control specific binding obtained in the presence of the test compounds where Inhibition (%)=100−[(measured specific binding/control specific binding)×100].
Measurement: Ki = 290 nM (pChEMBL 6.539999961853027)
Descriptors: MW 356.49, ALogP 3.66, HBA 4, HBD 1, PSA 41.57, QED 0.91
Compound (SMILES): Cc1cc(Nc2nccc(N3C[C@H]4CC[C@@H](C3)N4C(=O)[C@@H]3CC3(F)F)n2)cnc1C(N)=O
InChIKey: MUZOPNUVNZDWFF-ZNMIVQPWSA-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 = 170 nM (pChEMBL 6.769999980926514)
Descriptors: MW 443.46, ALogP 1.86, HBA 7, HBD 2, PSA 117.34, QED 0.72
Compound (SMILES): Cc1coc(NC(=O)c2n[nH]c3cc(F)c(-c4cnn(C5CCN(C)CC5)c4)cc23)n1
InChIKey: OFMOKGPYRJCAKE-UHFFFAOYSA-N
Target: Glycogen synthase kinase-3 beta (SINGLE PROTEIN), Homo sapiens
UniProt: P49841
Assay: Kinase Assay: GSK3β: Each compound is 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 M to 0.0003 M) and compound transfer was performed using the ECHO 550 (Labcyte, Sunnyvale, Calif.) into 1536-well black-walled round bottom plates (Corning).The GSK3β kinase assay is run using the Ser/Thr 09 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 ratio of coumarin emission/fluorescein emission.Briefly, recombinant GSK3β kinase, ATP and Ser/Thr peptide 09 are prepared in 1× 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 (0% Control) and phosphorylated (100% control) 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 = 193 nM (pChEMBL 6.710000038146973)
Descriptors: MW 423.45, ALogP 3.38, HBA 7, HBD 2, PSA 104.87, QED 0.52
Compound (SMILES): CC1(C(=O)N2CC[C@@]3(S(=O)(=O)c4ccc(F)cc4)c4ccc(C(F)(C(F)(F)F)C(F)(F)F)cc4CC[C@@H]23)CCS(=O)(=O)CC1
InChIKey: CNZOURNMHUJTHK-RCZVLFRGSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Binding Assay : The binding of potential ligands to RORγ is measured by competition with [3H]25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA). The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 μg/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 μM to 0.28 nM, for 10 min at rt in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% glycerol (Sigma Cat # G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat # RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined.
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 673.64, ALogP 5.55, HBA 5, HBD 0, PSA 88.59, QED 0.31
Compound (SMILES): Cc1ccnc2ncn(Cc3nc([C@H]4C[C@](F)(c5ccc(F)cc5)C4)no3)c(=O)c12
InChIKey: SPQAQEDLUSSZTH-IPVJXJPOSA-N
Target: Transient receptor potential cation channel subfamily A member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75762
Assay: FLIPR Assay (90 minutes): The IC50 (effective concentration) of compounds on the human TRPA1 channel was determined using a FLIPR Tetra instrument. CHO cells expressing TRPA1 were plated into 384-well plates, incubated overnight at 37° C., and loaded with BD calcium indicator dye for 1 hr at 37° C. followed by 90 minutes at room temperature. The assay buffer was Hank's Balanced Salt Solution (HBSS) containing 20 mM HEPES (pH readjusted to 7.4) along with 0.02% BSA.Following dye load and plate cool down, compounds were added to the cells using FLIPR Tetra. Plates were then incubated with compounds for 10 minutes or 90 minutes at room temperature prior to adding agonist. Following this incubation, about an EC50 concentration of cinnamaldehyde (75) was added to active the channels and block of cinnamaldehyde induced calcium influx was measured.The IC50 results were fit with a standard Hill function, keeping the Hill coefficient (n) fixed to 1.5. Fixing the Hill coefficient will generally reduce variability of the IC50 determination. The IC50 results were individually examined to make sure the MIN and MAX points were set correctly prior to validation of the results.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 409.4, ALogP 3.41, HBA 7, HBD 0, PSA 86.7, QED 0.51
Compound (SMILES): Cc1ccc(Cl)c(C2(Nc3ncc(-c4nnc(C(F)F)o4)cn3)CC2)c1
InChIKey: LDWNTSLIHVHEOV-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: Enzymatic Activity Assay: All enzymatic reactions were conducted in duplicate at room temperature for 17 hours in a 50 μl mixture containing HDAC assay buffer (50 mM Tris-HCl, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.05% Tween 20, 5 μg BSA), an HDAC substrate, an HDAC enzyme, and a test compound. Compound dilution was prepared ten-fold higher than the final concentration of the compounds with 10% DMSO in HDAC assay buffer and 5 μl of the dilution was added to a 50 μl reaction so that the final concentration of DMSO is 1% in all of reactions. After enzymatic reactions, 50 μl of HDAC Developer was added to each well and the plate was incubated at room temperature for an additional 20 minutes. Fluorescence intensity was measured at an excitation of 360 nm and an emission of 460 nm using a Tecan Infinite M1000 or Biotek Synergy microplate reader.The fluorescent intensity data were analyzed using the computer software, Graphpad Prism. In the absence of the compound, the fluorescent intensity (Ft) in each data set was defined as 100% activity. In the absence of HDAC, the fluorescent intensity (Fb) in each data set was defined as 0% activity. The percent activity in the presence of each compound was calculated according to the following equation: % activity=(F−Fb)/(Ft−Fb), where F=the fluorescent intensity in the presence of the compound.
Measurement: IC50 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 377.78, ALogP 4.53, HBA 6, HBD 1, PSA 76.73, QED 0.7
Compound (SMILES): CC1(CNc2ncnc3ccc(NC(c4ccc(F)cc4)c4cn(C5CC5)nn4)cc23)CCOCC1
InChIKey: CVDCGMIOVXHTCP-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: High Throughput EGFR Biochemical Assay: EGFR activity was measured using KinEASE (Cisbio), a time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay. In this assay, EGFR-catalyzes the phosphorylation of a universal Tyrosine kinase peptide substrate labeled with XL665. Europium conjugated phosphor-tyrosine specific antibody binds the resulting phosphorylated peptide. Formation of phosphorylated peptide is quantified by TR-FRET with Europium as the donor and XL665 the acceptor. The assay was performed in two main steps. The first step is the kinase reaction step and the second step is the detection step with TR-FRET reagents. In brief, test compounds 1:3 serially diluted in DMSO were delivered into Corning white, low volume, non-binding 384 well plates using the Echo 550 acoustic liquid dispenser (Labcyte®). EGFR enzyme (Human EGFR, cytoplasmic domain [669-1210] from Carna Biosciences Cat. No. 08-115) and substrates TK substrate-biotin (included in Cisbio HTRF KinEASE-TK kit Cat. No. 62TK0PEJ) were dispensed into assay plates using a Multi-Flo (Bio-Tek Instruments). The standard 10 μL reaction mixture contained 6 μM ATP (1×Km) or 12 μM ATP (2×Km), 1 μM biotinylated peptide, 0.3 nM EGFR (for 1×Km ATP) or 0.1 nM EGFR (for 2×Km ATP) in reaction buffer (10 mM MOPS, pH 7.0, 1.5% Glycerol, 0.5 mg/ml BSA, 10 mM Mg-Acetate, 1 mM DTT, 0.025% NP-40). After 60 min of incubation at room temperature, 10 μL of Stop and Detect Solution (1:400 Europium Cryptate labeled anti-phosphorylated peptide antibody solution and 125 nM strepavidin-XL665 Tracer in a 50 mM Hepes pH 7.0 detection buffer containing sufficient EDTA) was added. The plate was then further incubated for over 60 minutes at room temperature and read using an Envision 2103 Multilabeled reader (PerkinElmer) with excitation/emission/FRET emission at 340 nm/615 nm/665 nm, respectively. Fluorescence intensities at 615 nm and 665 nm emission wavelengths were expressed as a ratio (665 nm/615 nm). Percent inhibition was calculated as follows: % Inhibition=100x(RatioSample−Ratio0% Inhibition)/(Ratio100% Inhibition−Ratio0% Inhibition) where 0.05% DMSO (0% inhibition) was the negative control and 100 μM Staurosporine and Gefitinib (100% inhibition) was used as the positive control.
Measurement: EC50 = 296 nM (pChEMBL 6.53000020980835)
Descriptors: MW 487.58, ALogP 5.13, HBA 8, HBD 2, PSA 89.78, QED 0.36
Compound (SMILES): c1ccc2cc(Oc3cnn[nH]3)ccc2c1
InChIKey: AATVOAHHHGJFOB-UHFFFAOYSA-N
Target: 2-Hydroxyacid oxidase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UJM8
Assay: HepaRG-CAR Cell-Based Assay for Quantitation of Glycolate Oxidase Inhibition: A HepaRG human hepatic cell line was transfected for stable overexpression of the constitutive androstane receptor (i.e., HepaRG-CAR cells), as reported by van der Mark et al. (Drug Metab. Dispos., 2017, 45:56-67. Overexpression of CAR in these cells resulted in higher levels of glycolate oxidase (GOX) expression compared to the parental HepaRG cells. HepaRG-CAR cells were plated in a 12-wells plate and incubated for 4 weeks until fully differentiated.To measure cellular glycolate flux, the HepaRG-CAR cells were incubated in Williams medium supplemented with 10% fetal bovine serum (FBS), 5 μg/mL insulin, 50 μM hydrocortisone hemisuccinate, 2 mM glutamine, 5000 U/mL penicillin and 5 mg/mL streptomycin. Test compounds were added to the medium at 0, 0.3, 1, 3 or 10 μM and incubated for 30 minutes, after which 500 μM glycolate was also added. After incubation for 48 hours, 400 μL medium was taken from the culture plate and added to 60 μL 37% HCl.Internal standards (2,2-d2 glycolate, 1,2-13C2 oxalate and 13C2-glyoxylate) and hydroxylamine were added followed by another 30 minute incubation at 80° C. The acids were extracted using ethyl acetate with NaCl. The organic phase was dried under nitrogen and derivatized with N-tert-butyldimethylsilyl-N-methyl trifluoroacetamide (MTBSTFA) for 30 minutes at 80° C. The amounts of glycolate, glyoxylate and oxalate were determined by gas chromatography-mass spectrometry (GC-MS) analysis, using a 25 meter CP-Sil 5 CB low bleed column. A standard curve was used to calculate the concentrations of each acid in the culture medium.
Measurement: IC50 = 2000 nM (pChEMBL 5.699999809265137)
Descriptors: MW 211.22, ALogP 2.75, HBA 3, HBD 1, PSA 50.8, QED 0.71
Compound (SMILES): Nc1ccc(-c2ccnc(F)c2)c(-c2ccncc2)n1
InChIKey: FDLDAJSLNATHTH-UHFFFAOYSA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: Adenosine A2B Receptor Subtype Competition Radioligand Binding Assay: The binding assay for adenosine A2B receptor subtype was carried out on human recombinant source (HEK-293 cells) and [3H]DPCPX as radioligand, according to assay disclosed by Fredholm et al. (International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors, Pharmacol Rev. 2001December; 53(4):527-52).
Measurement: Ki > 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 266.28, ALogP 2.93, HBA 4, HBD 1, PSA 64.69, QED 0.72
Compound (SMILES): COc1cccc(-c2nc(C(=O)N3CCC[C@@]3(C)c3nc4cc(Cl)c(C(F)(F)F)cc4[nH]3)c(-c3ccc(F)cc3)s2)c1
InChIKey: KXZFGVLRGRWKAF-LJAQVGFWSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
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 = 671 nM (pChEMBL 6.170000076293945)
Descriptors: MW 615.05, ALogP 8.32, HBA 5, HBD 1, PSA 71.11, QED 0.2
Compound (SMILES): c1ccc(Cc2noc(-c3cc(OCC4CC4)c(C4CC4)cn3)n2)cc1
InChIKey: IGDPJCVOFYOBAV-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: Radioligand Binding Assay: The affinity of the compounds of the invention for cannabinoid CB1 receptors was determined using recommended amounts of membrane preparations (PerkinElmer) of human embryonic kidney (HEK) cells expressing the human CNR1 or CNR2 receptors in conjunction with 1.5 or 2.6 nM [3H]-CP-55,940 (Perkin Elmer) as radioligand, respectively. Binding was performed in binding buffer (50 mM Tris, 5 mM MgCl2, 2.5 mM EDTA, and 0.5% (wt/vol) fatty acid free BSA, pH 7.4 for CB1 receptor and 50 mM Tris, 5 mM MgCl2, 2.5 mM EGTA, and 0.1% (wt/vol) fatty acid free BSA, pH 7.4 for CB2 receptor) in a total volume of 0.2 ml for 1 h at 30° C. shaking. The reaction was terminated by rapid filtration through microfiltration plates coated with 0.5% polyethylenimine (UniFilter GF/B filter plate; Packard). Bound radioactivity was analyzed for Ki using nonlinear regression analysis (Activity Base, ID Business Solution, Limited), with the Kd values for [3H]CP55,940 determined from saturation experiments. The compounds of formula (I) show an excellent affinity for the CB2 receptor with affinities below 10 μM, more particularly of 1 nM to 3 μM and most particularly of 1 nM to 100 nM.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 347.42, ALogP 4.39, HBA 5, HBD 0, PSA 61.04, QED 0.63
Compound (SMILES): O=C(O)C12CCC(C(=O)N3CCC4(S(=O)(=O)c5ccc(Cl)c(Cl)c5)c5ccc(C(F)(C(F)(F)F)C(F)(F)F)cc5CCC34)(CC1)CC2
InChIKey: GWCNASWFBUUXFB-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Binding Assay : The binding of potential ligands to RORγ is measured by competition with [3H]25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA). The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 μg/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 μM to 0.28 nM, for 10 min at rt in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% glycerol (Sigma Cat # G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat # RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined.
Measurement: IC50 = 318 nM (pChEMBL 6.5)
Descriptors: MW 730.53, ALogP 7.92, HBA 4, HBD 1, PSA 91.75, QED 0.32
Compound (SMILES): Cc1cc(C(C)Nc2ccc(Cl)nc2C(=O)O)c2oc(N3Cc4ccc5c(c4C3)OCO5)cc(=O)c2c1
InChIKey: DZVOSSKCUIZNKE-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: PI3K-Alpha kinase (PIK3CA) activity, wild-type and H1047R mutant and determining IC50 values for inhibitors: Recombinant, catalytically active human full length PIK3KA Wild-type and H1047R mutant were purchased as 1:1 complex of N-terminal 6×his tagged p110<(catalytic) and untagged p85<(regulatory subunit) from EMD Millipore Sigma (cat. no. 14-602M and 14-792M, respectively). The enzyme stocks were diluted to 5× stocks in buffer (20 mM HEPES pH 7.4, 100 mM NaCl, 0.5 mM EGTA, 0.01% triton-x-100) just before use. PIP2diC8 (Avanti Polar Lipids Inc., cat. no. 850185) or phosphoinositol-4,5-bisphosphate with phosphoserine (PIP2:PS) membrane (Thermo Fisher Scientific, cat. no. PV5100) was used as lipid substrates. PIP2diC8 lyophilized powder and PIP2:PS (1:19) membrane stock (1 mM in PIP2) were separately dissolved in milliQ water to a concentration of 250 uM and stored in −20° C. 10 mM stocks of compounds were serially diluted (3×) in neat DMSO and stored in a dessicator at room temperature. 5× compound stocks in 25% DMSO were prepared fresh from neat DMSO stocks. Wild-type (WT) and H1047R mutant protein, along with buffer components (except ATP), were incubated with or without compound at 27° C. for 1h. After incubation, the reaction was initiated by the addition of 5 uL of 125 uM ATP. A typical assay mixture (25 uL) comprised 40 mM HEPES buffer, pH 7.4, 25 mM MgCl2, 0.01% v/v triton-X-100, 5% v/v DMSO, 20 mM NaCl, 1-5 nM wt or H1047R, 25 uM ATP, and 50 uM PTP2diC8 or PTP2 in membrane. The reaction was allowed to proceed until about 1000 conversion (2.5 uM ADP) after which time, 10 uL of reaction mixture was quenched with 25 uL of transcreener reagent (Transcreener ADP2 FI assay kit, BellBrook labs, Cat. No. 3013). The contents were incubated at rt for 1h and fluorescence was measured using a plate reader (Paradigm, Molecular Devices). The same assay was also run at pH 6.0 or 6.4 using MOPS buffer (Fisher BioReagents, CAS 1132-61-2). A calibration curve was generated under identical buffer conditions with varying ADP amounts. Using that, the observed fluorescence was converted to uM ADP.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 519.94, ALogP 5.27, HBA 8, HBD 2, PSA 114.13, QED 0.34
Compound (SMILES): C=CC(=O)Nc1cccc(-c2cc(-c3ccc(Oc4cc(C(F)(F)F)ccn4)cc3)c(N)c3cncnc23)c1
InChIKey: AQWNMPBZLFJHDG-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Inhibition assay for BTK: To determine IC50 of a compound against a kinase, a series of concentrations of the compound were tested for the inhibition. IC50 was calculated by plotting the concentration of compound vs the percentage of inhibition in treated wells using GraphPad Prism 5.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 527.51, ALogP 6.88, HBA 6, HBD 2, PSA 103.02, QED 0.18
Compound (SMILES): CC[C@H](NC(=O)[C@H](C)NC)C(=O)N1C[C@@H](F)C[C@H]1Cc1c(-c2nc3cc(F)ccc3n2C[C@@H]2C[C@H](F)CN2C(=O)[C@@H](NC(=O)[C@H](C)NC)C2CCCCC2)[nH]c2cc(F)ccc12
InChIKey: APTRBTUTSFUIJT-IAECAYPDSA-N
Target: Baculoviral IAP repeat-containing protein 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13489
Assay: Biological Assay 1: affinity of compounds to cIAP1-BIR3, cIAP2-BIR3, XIAP-BIR3: 1) Affinity of compounds listed in table to cIAP1-BIR3, Ciap2-BIR3, XIAP-BIR3 was tested referring to Nikolovska-Colesks, Z. et.Al. (Analytical Biochemistry,2004,232:261-273) to obtain IC50 value. Generally speaking, in 10-dose IC50 mode, IAP antagonists with different concentrations were diluted in series by 3-fold and fluorescence labelled ARPFAQ-K (5-FAM)-NH2 peptide was used as probe to test compounds binding to protein BIR3 domain.2) Test condition: cIAP1-BIR3: 20 nM; cIAP2-BIR3: 60 nM; XIAP-BIR3: 30 nM; 5 nM probe in 100 mM tripotassium phosphate, pH 7.5, 0.1 mg/mL BSA, 0.005% Triton X100 and 0.5% DMSO.3) Test procedure: compounds were added into protein with different concentrations by using ECHO (LabCyte), and then the mixture was preincubated for 15 min. The probe was added and the final concentration was 5 nM. After incubating for 60 min, FP was tested and mP was calculated.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 866.02, ALogP 5.06, HBA 8, HBD 5, PSA 156.49, QED 0.1
Compound (SMILES): O=C(NS(=O)(=O)c1ccc(NCC2CCOCC2)c([N+](=O)[O-])c1)c1ccc(C2=CCC(N3CCCC3c3cccc(Cl)c3C3CC3)CC2)cc1Oc1cnc2[nH]ccc2c1
InChIKey: QUFFEBFYDGSUQY-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07817
Assay: Bcl-2/Bcl-X Fluorescence Polarization Assay: Compounds disclosed herein were tested for blocking of Bcl-2/Bcl-xl protein with its ligand in an assay based on fluorescence polarization methodology. Recombinant human 2.7 nM Bcl-2/1.3 nM Bcl-xl protein was pre-incubated with a serial dilution of compounds disclosed herein (maximum concentration is 1 μM for Bcl-2 assay, and 10 μM or 1 μM for Bcl-xl assay, 3-fold serially diluted, 10 points) at room temperature for 0.5 hour in an assay buffer containing 20 ml potassium phosphate buffer, pH 7.5, 50 mM NaCl, 1 mM EDTA, 0.05% Tween-20, 0.01% BSA. Then the FITC labeled Bak peptide Ac-GQVGRQLAIIGDK (FITC)INR-amide (1 nM for Bcl-2, 0.82 nM for Bcl-xl) was added to plate and further incubated at room temperature for 0.5 h. The FP signals (485 nm-520 nm-520 nm) were read on BMG PHERAstar FS or BMG PHERAstar FSX instalment. The inhibition percentage of Bcl-2/Bcl-xl interaction with its ligand in presence of increasing concentrations of compounds was calculated based on the FP signals. The IC50 for each compound was derived from fitting the data to the four-parameter logistic equation by Graphpad Prism software.
Measurement: IC50 = 5761 nM (pChEMBL 5.239999771118164)
Descriptors: MW 851.43, ALogP 9.52, HBA 10, HBD 3, PSA 168.79, QED 0.08
Compound (SMILES): COc1cc(CO)cc(S(=O)(=O)N2CCc3cccc(CCc4ccc(C(=O)O)cc4)c32)c1
InChIKey: GNQFVWHBHKWCJW-UHFFFAOYSA-N
Target: Prostaglandin E2 receptor EP4 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P35408
Assay: FLIPR Assay: Cells were seeded at a density of 5×104 cells per well in Biocoat® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 mM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 ml in each well. Plates were re-equilibrated to 37° C. for a few minutes.Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR™, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration. The peak increase in fluorescence intensity was recorded for each well. On each plate, four wells each served as negative (HBSS-HEPES buffer) and positive controls (standard agonists: BW245C (hDP); PGE2 (hEP1; hEP2/Gqs5; hEP3A/Gqi5; hEP4/Gqs5); PGF2a (hFP); carbacyclin (hIP); U-46619 (hTP), depending on receptor). The peak fluorescence change in each drug-containing well was then expressed relative to the controls.
Measurement: Ki = 8234 nM (pChEMBL 5.079999923706055)
Descriptors: MW 467.54, ALogP 3.42, HBA 5, HBD 2, PSA 104.14, QED 0.53
Compound (SMILES): N#CC1(c2cnc3c(C(=O)Nc4cncc(F)c4N4CCN(C5COC5)CC4)c(N)nn3c2)CC1
InChIKey: CSQPCWDGHLQJSD-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Inhibition Assay: Table 5: Compounds were screened for their ability to inhibit ATR kinase using a radioactive-phosphate incorporation assay. Assays were carried out in a mixture of 50 mM Tris/HCl (pH 7.5), 10 mM MgCl2 and 1 mM DTT. Final substrate concentrations were 10 μM [y-33P]ATP (3 mCi 33P ATP/mmol ATP, Perkin Elmer) and 800 μM target peptide(ASELPASQPQPFSAKKK) (SEQ ID NO:1).Assays were carried out at 25° C. in the presence of 5 nM full-length ATR. An assay stock buffer solution was prepared containing all of the reagents listed above, with the exception of ATP and the test compound of interest. 13.5 μL of the stock solution was placed in a 96 well plate followed by addition of 2 μL of DMSO stock containing serial dilutions of the test compound (typically starting from a final concentration of 15 μM with 3-fold serial dilutions) in duplicate (final DMSO concentration 7%). The plate was pre-incubated for 10 minutes at 25° C. and the reaction initiated by addition of 15 μL [γ-33P]ATP (final concentration 10 μM).
Measurement: Ki = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 477.5, ALogP 1.17, HBA 10, HBD 2, PSA 137.7, QED 0.55
Compound (SMILES): CCN1CCN(Cc2cc3c4c(cnc3[nH]2)CN(c2c(F)c(OC)cc(OC)c2F)C(=O)C4(C)C)CC1
InChIKey: CQZQLQVEMYIPJL-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P21802
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 5-10 minutes. 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).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 513.59, ALogP 3.82, HBA 6, HBD 1, PSA 73.93, QED 0.54
Compound (SMILES): Cc1cc(Nc2cc3ncccc3c(N[C@@H]3C[C@H]4CCC[C@@H](C3)N4Cc3cccc(O)c3)n2)n[nH]1
InChIKey: WSFQMCDKLICTAR-MZADTFQBSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biochemical JAK Assay: A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 and Tyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH 7.5, 0.01% Brij-35, 10 mM MgCl2, and 1 mM EGTA). Recombinant GST-tagged JAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained from Life Technologies.Serially diluted compounds were pre-incubated with each of the four JAK enzymes and the substrate in white 384-well microplates (Corning) at ambient temperature for lh. ATP was subsequently added to initiate the kinase reactions in 10 μL total volume, with 1% DMSO. The final enzyme concentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and 0.25 nM respectively; the corresponding Km ATP concentrations used are 25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200 nM for all four assays. Kinase reactions were allowed to proceed for 1 hour at ambient temperature before a 10 μL preparation of EDTA (10 mM final concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (Life Technologies, 2 nM final concentration) in TR-FRET dilution buffer (Life Technologies) was added. The plates were allowed to incubate at ambient temperature for lh before being read on the EnVision reader (Perkin Elmer). Emission ratio signals (520 nm/495 nm) were recorded and utilized to calculate the percent inhibition values based on DMSO and background controls.For dose-response analysis, percent inhibition data were plotted vs. compound concentrations, and IC50 values were determined from a 4-parameter robust fit model with the Prism software (GraphPad Software). Results were expressed as pIC50 (negative logarithm of IC50) and subsequently converted to pKi (negative logarithm of dissociation constant, Ki) using the Cheng-Prusoff equation.Test compounds having a higher pKi value in each of the four JAK assays show greater inhibition of JAK activity. Compounds of the invention tested in this assay typically exhibited pKi values between about 7 and about 10.3
Measurement: Ki < 0.1 nM (pChEMBL 10)
Descriptors: MW 469.59, ALogP 5.11, HBA 7, HBD 4, PSA 101.99, QED 0.31
Compound (SMILES): C[C@H](NC(=O)c1c(N)nn2cccnc12)c1cc2cccc(Cl)c2c(=O)n1-c1cccc(S(C)(=O)=O)c1
InChIKey: JFOUTONBSIXQAW-AWEZNQCLSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Inhibition Assay: Class I PI3-Ks can be either purchased (p110α/p85α, p110β/p85α, p110δ/p85α from Upstate, and p110γ from Sigma) or expressed as previously described (Knight et al., 2004). IC50 values are measured using either a standard TLC assay for lipid kinase activity (described below) or a high-throughput membrane capture assay. Kinase reactions are performed by preparing a reaction mixture containing kinase, inhibitor (2% DMSO final concentration), buffer (25 mM HEPES, pH 7.4, 10 mM MgCl2), and freshly sonicated phosphatidylinositol (100 μg/ml). Reactions are initiated by the addition of ATP containing 10 μCi of γ-32P-ATP to a final concentration of 10 or 100 μM and allowed to proceed for 5 minutes at room temperature. For TLC analysis, reactions are then terminated by the addition of 105 μL 1N HCl followed by 160 μL CHCl3:MeOH (1:1). The biphasic mixture is vortexed, briefly centrifuged, and the organic phase is transferred to a new tube using a gel loading pipette tip precoated with CHCl3. This extract is spotted on TLC plates and developed for 3-4 hours in a 65:35 solution of n-propanol:1M acetic acid. The TLC plates are then dried, exposed to a phosphorimager screen (Storm, Amersham), and quantitated. For each compound, kinase activity is measured at 10-12 inhibitor concentrations representing two-fold dilutions from the highest concentration tested (typically, 200 μM). For compounds showing significant activity, IC50 determinations are repeated two to four times, and the reported value is the average of these independent measurements.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 537, ALogP 3.16, HBA 9, HBD 2, PSA 141.45, QED 0.35
Compound (SMILES): Cc1ccc(/C=C/CC(=O)Nc2ccc(C(=O)Nc3ccc(F)cc3N)cc2)cn1
InChIKey: FFYPWXUBGIMTJU-NSCUHMNNSA-N
Target: Histone deacetylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92769
Assay: HDACs 1, 2, and 3 Enzymatic Inhibitions: HDACs assay was performed according to standard Protocols (Fluorgenic HDACs 1, 2, and 3 assay kit, BPS Bioscience). All of the compounds, with Chidamide and Entinostat as positive control, at doses ranging from 20 μM to 1.28 nM, were mixed with kit buffer and incubated at 37° C. for 1 hour. After 1 h, assay developer was added to the samples and the absorbance was read at fluorogenic wavelength. The relative inhibition to HDACs 1, 2, and 3 activities in each sample was determined. Example 10 Enzyme Inhibition Kinetic of HDAC3 by GNTbm-02 was Determined HDAC3
Measurement: IC50 = 510 nM (pChEMBL 6.289999961853027)
Descriptors: MW 404.45, ALogP 4.41, HBA 4, HBD 3, PSA 97.11, QED 0.53
Compound (SMILES): COC(=O)c1cnc(N)c(-c2cc3cc(NC(=O)Nc4cc(C(F)(F)F)ccc4F)ccc3s2)c1
InChIKey: LSCATNNDFBVKMR-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Biochemical Assay: Biochemical KDR kinase assays were performed in 96 well microtiter plates that were coated overnight with 75 μg/well of poly-Glu-Tyr (4:1) in 10 mM Phosphate Buffered Saline (PBS), pH 7.4. The coated plates were washed with 2 mls per well PBS+0.05% Tween-20 (PBS-T), blocked by incubation with PBS containing 1% BSA, then washed with 2 mls per well PBS-T prior to starting the reaction. Reactions were carried out in 100 μL reaction volumes containing 2.7 μM ATP in kinase buffer (50 mM Hepes buffer pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2 and 0.2 mM Na3VO4). Test compounds were reconstituted in 100% DMSO and added to the reaction to give a final DMSO concentration of 5%. Reactions were initiated by the addition 20 ul per well of kinase buffer containing 200-300 ng purified cytoplasmic domain KDR protein (BPS Bioscience, San Diego, Calif.). Following a 15 minute incubation at 30° C., the reactions were washed 2 mls per well PBS-T. 100 μl of a monoclonal anti-phosphotyrosine antibody-peroxidase conjugate diluted 1:10,000 in PBS-T was added to the wells for 30 minutes. Following a 2 mls per well wash with PBS-Tween-20, 100 μl of O-Phenylenediamine Dihydrochloride in phosphate-citrate buffer, containing urea hydrogen peroxide, was added to the wells for 7-10 minutes as a colorimetric substrate for the peroxidase. The reaction was terminated by the addition of 100 μl of 2.5N H2SO4 to each well and read using a microplate ELISA reader set at 492 nm. IC50 values for compound inhibition were calculated directly from graphs of optical density (arbitrary units) versus compound concentration following subtraction of blank values.
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 504.47, ALogP 6.13, HBA 6, HBD 3, PSA 106.34, QED 0.23
Compound (SMILES): Cc1nn(C)c2c(-n3nc(COC(C)(C)C(=O)O)cc3-c3cccc(OCC(C)(C)C)c3)cccc12
InChIKey: LONCIMGLEVRXRB-UHFFFAOYSA-N
Target: Monocarboxylate transporter 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O15427
Assay: MCT4-Mediated Lactate Transport in MDA-MB-231 Breast Cancer Cells: MDA-MB-231 breast cancer cells may be used to measure MCT4 activity in cells with high native levels of MCT4 and low levels of MCT1 and are known to those with skill in the art. MCT4 activity may be assessed by monitoring the intracellular pH change that accompanies lactate/proton symport, using the pH-sensitive fluorescent dye 2′,7′-bis-(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), in a manner similar to that previously reported for MCT1 and MCT4. The following is an exemplary procedure for assaying MCT4 activity of the compounds of Formula (I).
Measurement: IC50 = 2.4 nM (pChEMBL 8.619999885559082)
Descriptors: MW 490.6, ALogP 5.54, HBA 7, HBD 1, PSA 91.4, QED 0.35
Compound (SMILES): COc1cccc(-n2ccc(N(C(=O)c3cccc(O)c3)C3CCN(C)CC3)n2)c1
InChIKey: KGYTWKJBVJGHDW-UHFFFAOYSA-N
Target: Voltage-dependent calcium channel subunit alpha-2/delta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P54289
Assay: Human alpha2delta-1 Subunit of Cav2.2 Calcium Channel 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. 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 hour 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 > 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 406.49, ALogP 3.33, HBA 6, HBD 1, PSA 70.83, QED 0.7
Compound (SMILES): CC(C)CNc1ccc(C2=NNC(=O)OC2)cc1C(F)(F)F
InChIKey: UCSCXOFPNHJIGF-UHFFFAOYSA-N
Target: cGMP-inhibited 3',5'-cyclic phosphodiesterase 3B (SINGLE PROTEIN), Homo sapiens
UniProt: Q13370
Assay: PDE3B Enzyme Inhibition: The commercially available 3H-cAMP Scintillation Proximity Assay (SPA, Perkin Elmer) system was used for enzyme inhibition studies. For the determination of the in vitro effect of example compounds on the PDE3B reactions 2 μl of the respective example compound solution in DMSO (serial dilutions) were placed in wells of microtiter plates (Isoplate-96/200W; Perkin Elmer). 50 μl of a dilution of PDE3B cell extract from Sf9 cells overexpressing human full length PDE3B (SB Drug Discovery, UK) in buffer A (50 mM Tris/HCl pH 7.5, 8.3 mM MgCl2, 1.7 mM EDTA, 0.2% BSA) was added. The dilution of the PDE3B cell extract was chosen such that the reaction kinetics was linear and less than 70% of the substrate was consumed (typical dilution 1:6000). The reaction was started by addition of 50 μl (0.025 μCi) of 1:2000 in buffer A w/o BSA diluted substrate [8-3H]adenosine 3′, 5′-cyclic phosphate (1 μCi/μl; Perkin Elmer). After incubation at room temperature for 60 min, the reaction was stopped by addition of 25 μl of a suspension containing 18 mg/ml yttrium scintillation proximity beads (Perkin Elmer) in water. The microtiter plates were sealed and measured in a Microbeta scintillation counter (PerkinElmer Wallac). IC50 values were determined from sigmoidal curves by plotting percentage PDE3B activity vs log compound concentration.
Measurement: IC50 = 56 nM (pChEMBL 7.25)
Descriptors: MW 315.3, ALogP 3.22, HBA 4, HBD 2, PSA 62.72, QED 0.9