lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C1CCc2cc(/C=C/C(=O)N3CC4CC(n5cncn5)CC4C3)cnc2N1
InChIKey: KGQZNUABAXNLBH-DAFODLJHSA-N
Target: Enoyl-[acyl-carrier-protein] reductase [NADPH] FabI (SINGLE PROTEIN), Staphylococcus aureus
UniProt: Q6GI75
Assay: Enzyme Inhibition Assay: FabI enzyme inhibition assays were carried out in half-area, 384-well microtitre plates. Compounds were evaluated in 40-μl assay mixtures containing 100 mM NaADA, pH 6.5 (ADA=N-[2-acetamido]-2iminodiacetic acid), 250 μM crotonoyl-CoA, 625 μM NADH and 50 μg/ml S. aureus ATCC 29213 FabI. Inhibitors were typically varied over the range of 50 to 0.39 μM. The reaction mixtures were incubated for 30 minutes at room temperature and the reaction was stopped by adding 200 mM Tris buffer (pH 9.0) to create a pH-shift. The consumption of NADH was monitored by measuring the change in absorbance at 340. By comparing sample readings to those of negative (absence of compound) and positive (absence of enzyme) controls, the percent inhibition of enzymatic activity of the compounds was determined. A best-fit curve is fitted by a minimum of squares method.
Measurement: IC50 = 8900 nM (pChEMBL 5.050000190734863)
Descriptors: MW 378.44, ALogP 1.68, HBA 6, HBD 1, PSA 93.01, QED 0.82
Compound (SMILES): CN1CCC(CNc2nn3c(-c4cccc(N(C)C)c4)nnc3c3c2N(C)CCO3)CC1
InChIKey: CXINRXVREHWMLQ-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM-1 Biochemical Assay: The biochemical assay to measure PIM-1 activity relies on the ADP Hunter assay kit (DiscoveRx Corp., Cat. #90-0077), that determines the amount of ADP as direct product of the kinase enzyme activity.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 436.56, ALogP 2.44, HBA 9, HBD 1, PSA 74.06, QED 0.65
Compound (SMILES): Cc1cc(OCCCc2c3n(c4c(-c5c(C)nn(C)c5C)c(Cl)ccc24)CCCN(CCc2ccccc2OC=O)C3=O)cc(C)c1Cl
InChIKey: KHQVTKRAWDSHNQ-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 either a fluorescein isothiocyanate-labeled BH3 peptide derived from Bak (FITC-AHx-GQVGRQLAIIGDDINR-NH2) or a fluorescein isothiocyanate-labeled BH3 peptide derived from Bim (FITC-AHx-EARIAQELRRIGDEFNETYTR-NH2) that were purchased from GenScript (Piscataway, N.J.) at >95% purity and used without further purification. 10 nM FITC-Bak peptide and 15 nM recombinant Mcl-1 (residues 172-327) were added to assay buffer (3 mM dithiothreitol, 50 mM NaCl, 20 mM Tris, pH 7.5). The Bim based assay was run with 1 nM FITC-Bim peptide and 1.5 nM recombinant Mcl-1 (residues 172-327) added to assay buffer (20 mM TRIS pH 7.5, 50 mM NaCl, 3 mM DTT, 0.01% CHAPS). For selectivity assays, 40 nM Bcl-2 (residues 1-207A96T,G110R, Î35-91, 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.
Measurement: Ki < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 687.67, ALogP 8.22, HBA 7, HBD 0, PSA 78.59, QED 0.1
Compound (SMILES): CC(=O)N[C@H]1CC[C@H](COc2nc(Nc3cn(C(C)(C)C#N)nc3C)ncc2C)CC1
InChIKey: KUMLVPOADRGTPG-IYARVYRRSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LRRK2 Enzymatic Assay: LRRKtide substrate (peptide sequence RLGRDKYKTLRQIRQ, derived from human ezrin [amino acids 561-573], moesin [amino acids 539-553] and radixin [amino acids 558-570], obtained from SignalChem, catalogue #L10-58, reconstituted in 20 mM Tris-HCl at pH 7.5 to a final concentration of 1 mg/mL, assay concentration 20 μM) and recombinant human LRRK2 (catalytic domain only [amino acids 970-2527], GST-tagged, expressed in insect cells, obtained from ThermoFisher Scientific, catalogue #PV4874, 0.35 mg/mL, assay concentration 30 nM) were mixed in assay buffer (20 mM Hepes pH 7.5, 10 mM MgCl, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM NaVO, 2 mM DTT, 1% DMSO). Compounds of interest (in DMSO, serial 3-fold dilution from 10 μM to 0.5 nM) or control (1% DMSO) were dispensed into the kinase reaction mixture by Acoustic technology (Echo550; nanoliter range). After incubation at room temperature for 20 minutes, the kinase reaction was initiated by addition of [P]-ATP (Specific activity 10 μCi/μl) and the mixture was incubated at room temperature for 2 hours. The reaction was then stopped by spotting the reaction mixture on strips of phosphocellulose P81 paper. Following washing, the radioactivity of the P81 paper was measured and kinase activity data were expressed as the percent remaining kinase activity in test samples compared to vehicle (dimethyl sulfoxide) reactions.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 425.54, ALogP 3.37, HBA 8, HBD 2, PSA 117.75, QED 0.7
Compound (SMILES): CN1CCn2ncc(Nc3nccc(-c4cc(C#N)c5c(c4)[C@@](C)(CO)CN5)n3)c2C1
InChIKey: ARVHLTBACDZWGS-JOCHJYFZSA-N
Target: Mitogen-activated protein kinase kinase kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99558
Assay: Inhibition of Auto-Phosphorylation of Recombinant Human NF-kappaB-Inducing Kinase (NIK1/AP3K14) Activity: NIK/MAP3K14 auto-phosphorylation activity was measured using the AlphaScreen® (αscreen) format (Perkin Elmer). All compounds tested were dissolved in dimethyl sulfoxide (DMSO) and further dilutions were made in assay buffer. Final DMSO concentration was 100 (v/v) in assays. Assay buffer was 50 mM Tris pH 7.5 containing 1 mM EGTA (ethylene glycol tetraacetic acid), 1 mM DTT (dithiothreitol), 0.1 mM Na3VO4, 5 mM MgCl2, 0.01% Tween® 20. Assays were carried out in 384 well Alphaplates (Perkin Elmer). Incubations consisted of compound, 25 microM Adenosine-5′-triphosphate (ATP), and 0.2 nM NIK/MAP3K14. Incubations were initiated by addition of GST-tagged NIK/MAP3K14 enzyme, carried out for 1 h at 25° C. and terminated by addition of stop buffer containing anti-phospho-KK Ser176/180 antibody. Protein A Acceptor and Glutathione-Donor beads were added before reading using an EnVision® Multilabel Plate Reader (Perkin Elmer). Signal obtained in the wells containing blank samples was subtracted from all other wells and IC50's were determined by fitting a sigmoidal curve to % inhibition of control versus Log10 compound concentration.
Measurement: IC50 = 3.5 nM (pChEMBL 8.460000038146973)
Descriptors: MW 416.49, ALogP 2.08, HBA 9, HBD 3, PSA 114.92, QED 0.59
Compound (SMILES): COc1cccc(-c2c(C)nc3c(N4CC[C@@H](NC(C)=O)C4)cc(Cl)nn23)c1
InChIKey: VFLTYSAHDILNBC-OAHLLOKOSA-N
Target: Sphingomyelin phosphodiesterase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NY59
Assay: Fluorescence Based Assay: The fluorescence based assay to monitor the activity of human nSMase2 in the presence or absence of potential inhibitors has been described recently. Figuera-Losada, et al. Lysate of cells expressing recombinant nSMase2 is used to catalyze the hydrolysis of sphingomyelin (SM) to ceramide and phosphorylcholine. Phosphorylcholine undergoes dephosphorylation in a reaction catalyzed by alkaline phosphatase (4 U/mL) to produce choline which in turn is oxidized by choline oxidase (0.1 U/mL) to betaine and hydrogen peroxide (H2O2). Hydrogen peroxide is made to react with Amplex red (50 μM) in the presence of horseradish peroxidase (HRP, 1 U/mL) to generate the fluorescent molecule resorufin. Generation of fluorescence is monitored by measuring relative florescence units (RFU) with excitation at 530 nm and emission at 590 nm. Extent of fluorescence is directly proportional to the extent of SM hydrolysis. Substrate stock solution is prepared in 2% Triton X-100 and sonicated for 1 min. Reactions are carried out for 1 h at 37° C. in 100 mM Tris-HCl pH 7.4, 10 mM MgCl2, 0.2% Triton X-100. This assay has been optimized in 384-well format (50 μL total volume per well) under conditions where nSMase2-catalyzed hydrolysis of SM is linear with respect to nSMase2 concentration, time of incubation and SM concentration (FIGS. 3A-3C). The assay has high reliability (Z′=0.8-0.9). It is used for compound screening, IC50 determinations and mode of inhibition studies
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 399.88, ALogP 3.08, HBA 6, HBD 1, PSA 71.76, QED 0.73
Compound (SMILES): O=C(NCc1ccc(S(=O)(=O)N2CCC3(CCNCC3)CC2)cc1)c1ccc2nccn2c1
InChIKey: BMCCRDAEKSMZJI-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Enzymatic Reaction Assay: The NAMPT enzymatic reactions were carried out in Buffer A (50 mM Hepes pH 7.5, 50 mM NaCl, 5 mM MgCl2, and 1 mM THP) in 96-well V-bottom plates. The compound titrations were performed in a separate dilution plate by serially diluting the compounds in DMSO to make a 100× stock. Buffer A (89 μL) containing 33 nM of NAMPT protein was added to 1 μL of 100× compound plate containing controls (e.g. DMSO or blank). The compound and enzyme mixture was incubated for 15 min at rt, then 10 μL of 10× substrate and co-factors in Buffer A were added to the test well to make a final concentration of 1 μM NAM, 100 μM 5-Phospho-D-ribose 1-diphosphate (PRPP), and 2.5 mM Adenosine 5′-triphosphate (ATP). The reaction was allowed to proceed for 30 min at rt, then was quenched with the addition of 11 μL of a solution of formic acid and L-Cystathionine to make a final concentration of 1% formic acid and 10 μM L-Cystathionine. Background and signal strength was determined by addition (or non-addition) of a serial dilution of NMN to a pre-quenched enzyme and cofactor mix.
Measurement: IC50 = 19.2 nM (pChEMBL 7.71999979019165)
Descriptors: MW 467.6, ALogP 2.42, HBA 6, HBD 2, PSA 95.81, QED 0.6
Compound (SMILES): Cc1cc(-c2ccc3nnc(C(F)(F)F)n3n2)ccc1C(F)(F)F
InChIKey: SOQUSGATDMXNHT-UHFFFAOYSA-N
Target: Cytochrome P450 1A2 (SINGLE PROTEIN), Homo sapiens
UniProt: P05177
Assay: CYP1A2 Inhibition Assay: CYP1A2 Inhibition in Human Liver Microsomes. Pooled human liver microsomes were incubated with individual CYP, CYP1A2, isozyme-specific marker substrate (Phenacetin) in the presence of test compound at various concentrations (0.05, 0.15, 0.5, 1.5, 5, 15, 50 uM). The specific marker metabolites are measured with LC/MS/MS. The remaining enzymatic activities and inhibitory potency IC50 are determined. Procedure: Microsomes are removed out of the −80° C. freezer to thaw on ice and 20 μL of the substrates solution added to the corresponding wells. Then 20 μL PB was added to blank wells and 2 μL of the test compounds and positive control working solution added to the corresponding wells. 2 μL of solvent was added to No Inhibitor wells and Blank wells. Then 158 μL of the HLM working solution was added to all wells of the incubation plate. The plate was pre-warmed for about 10 min using a 37° C. water bath. Then 20 μL of the NADPH cofactor solution was added to all incubation wells, mixed and incubated for 10 minutes at 37° C. water bath. The reaction is terminated by adding 400 μL cold stop solution (200 ng/mL Tolbutamide and 200 ng/mL Labetalol in ACN). The samples were centrifuged at 4000 rpm for 20 minutes to precipitate protein. Finally 200 μL of the supernatant was transferred to 100 μL HPLC water, shaken for 10 min and analyzed by LC/MS/MS.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 346.23, ALogP 4.14, HBA 4, HBD 0, PSA 43.08, QED 0.62
Compound (SMILES): Cc1cnc(CNc2ncc(C(=O)N3CC4(CCOCC4)C3)cn2)cn1
InChIKey: DDOUGYMDNSYHSI-UHFFFAOYSA-N
Target: Pantetheinase (SINGLE PROTEIN), Homo sapiens
UniProt: O95497
Assay: Enzyme Assay: The test inhibitors were solubilized in DMSO to a stock concentration of 30 mM. On the day of the assay, dose response plates were prepared by diluting the inhibitors in DMSO at compound concentration 200-fold the final in-assay concentration. Intermediate concentrations were prepared by diluting in DMSO in a four-fold series for a total of 11 data points.To prepare a working solution of human vanin-1, the enzyme was diluted to 33.3 pM in the assay buffer consisting of 50 mM Tris-HCl pH=8.0, 50 mM KCl, 0.005% Brij-35 and 1.6 mM cysteamine. To begin the assay 100 nL was transferred from the compound plate to the assay plate. Next, 15 μL of the vanin-1 working solution were transferred to the assay plate. The inhibitor and enzyme were incubated at room temperature for 30 minutes. The enzyme reaction was then initiated by the addition of 5 μL of 200 μM pantetheine 7-amino-4-trifluoromethylcoumarin prepared in assay buffer. The final concentrations in the assay were 25 pM human vanin-1 and 50 uM substrate. The final concentration of DMSO was 0.5%. The assay plates were incubated for 60 minutes and before they were read on a Perkin Elmer EnVision Model 2103 using a 405 nm excitation wavelength and a 510 nm emission wavelength for detection.
Measurement: IC50 = 26.3 nM (pChEMBL 7.579999923706055)
Descriptors: MW 354.41, ALogP 1.44, HBA 7, HBD 1, PSA 93.13, QED 0.89
Compound (SMILES): CCCS(=O)(=O)Nc1cc(F)c(F)c(Oc2ccc3ncn(C)c(=O)c3c2C)c1Cl
InChIKey: QVMJUHSHJTUQMH-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: BRAF V600E and V600K Enzyme Assay: A competitive displacement assay was configured for B-Raf that monitors the amount of a fluorescently-tagged tracer bound to B-Raf via TR-FRET from an anti-tag Eu-labeled antibody also bound to B-Raf. For full-length FLAG-tagged B-Raf(V600E), the assay mixtures consisted of 25 mM K+HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, 1 mM DTT, 2% DMSO (from compound), 50 nM Tracer 1710 (ThermoFisher, PR9176A), 0.5 nM Eu anti-FLAG (M2)-cryptate Ab (Cisbio, 61FG2KLB) and 5 nM full-length, N-terminally FLAG-tagged B-Raf(V600E) (Origene Technologies, TP700031. When B-Raf(V600K) was assayed, the following substitutions were made: 15 nM Tracer 178 (ThermoFisher, PV5593), 2 nM Eu-anti-GST Ab (ThermoFisher, PV5595) and 5 nM N-terminally GST-tagged B-Raf(V600K) (331-end, SignalChem, B08-12DG). Compounds were typically diluted in DMSO across an 11-point dosing range created using a 3-fold serial dilution protocol at a top dose of 10 μM. The assay was run in 384-well, polystyrene, low-volume, non-treated, white microtiter plates (Costar 4512) in a final volume of 12 μL. Low control wells included 1 μM of a potent B-Raf inhibitor as a control.
Measurement: IC50 = 1.55 nM (pChEMBL 8.8100004196167)
Descriptors: MW 457.89, ALogP 4.12, HBA 6, HBD 1, PSA 90.29, QED 0.56
Compound (SMILES): CCOc1cc(O[C@H]2CCc3c(-c4ccccc4F)cccc32)c(Cl)cc1CN[C@@H](CO)C(=O)O
InChIKey: XHJGRRNVCLUXIH-ZEQRLZLVSA-N
Target: Programmed cell death 1 ligand 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZQ7
Assay: Enzyme-Linked Immunosorbent AssayâELISA: Plates were coated with 1 Î¼g/mL of human PD-L1 (obtained from R&D) in PBS overnight at 4Â° C. The wells were then blocked with PBS containing 2% BSA in PBS (W/V) with 0.05% TWEEN-20 for 1 hour at 37Â° C. The plates were washed 3 times with PBS/0.05% TWEEN-20 and the samples were diluted to 1:5 in dilution medium in the ELISA plates. Human PD-1 and biotin 0.3 Î¼g/mL (ACRO Biosystems) were added and incubated for 1 hour at 37Â° C. then washed 3 times with PBS/0.05% TWEEN-20. A second block was added with PBS containing 2% BSA in PBS (W/V)/0.05% TWEEN-20 for 10 min at 37Â° C. and was washed 3 times with PBS/0.05% TWEEN-20. Streptavidin-HRP was added for 1 hour at 37Â° C. then washed 3 times with PBS/0.05% TWEEN-20. TMB substrate was added and reacted for 20 min at 37Â° C. A stop solution (2 N aqueous H2SO4) was added. The absorbance was read at 450 nm using a micro-plate spectrophotometer.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 499.97, ALogP 5.15, HBA 5, HBD 3, PSA 88.02, QED 0.36
Compound (SMILES): CN1CCN([C@@H]2CCCN(c3cnc(C(N)=O)c(Nc4ccc(C5(C)CCN(C(=O)C6CC6)CC5)cc4)n3)C2)C1=O
InChIKey: ZVTYOOQKQKVLKZ-HSZRJFAPSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Btk In Vitro Inhibitory Assay: Human Btk kinase (Genbank accession # NP_000052) was purified from insect cells as a full-length construct containing a N-terminal 6×-His tag. Btk kinase activity was determined using a radiometric filter binding assay. Measurements are performed in a low μL reaction volume 384-well assay plates. BTK enzyme (8 nM final in reaction), inhibitor (at requested doses), and 0.2 mg/mL peptide substrate (Poly-Glu-Tyr, 4:1 ratio) are incubated in a reaction buffer composed of 20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO for 15 min. followed by addition of 1 μM ATP to start the assay. Kinase reactions are carried out for 120 min. at room temperature. The reaction was stopped by spotting of reaction sample onto P81 cationic exchange paper (Whatman). Unbound phosphate was removed by extensive washing of filters in 0.75% Phosphoric acid. After subtraction of background derived from control reactions containing inactive enzyme (via addition of saturating EDTA), kinase activity data for each dose of compound tested was expressed as the percent of remaining kinase activity in test samples compared to vehicle (dimethyl sulfoxide) reactions.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 560.7, ALogP 2.95, HBA 7, HBD 2, PSA 128, QED 0.53
Compound (SMILES): COCCn1cc(N2C[C@@H](C)n3c(c(CCCOc4cc(C)c(Cl)c(C)c4)c4ccc(Cl)c(-c5c(C)nn(C)c5C)c43)C2=O)c2cc(-c3ncn(CC4CCOCC4)n3)ccc21
InChIKey: LQANJABHSZJWQV-SSEXGKCCSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07820
Assay: TR-FRET Binding Assay: Measurements were carried out in OptiPlate-384 White Opaque 384-well plates (Perkin Elmer. Shelton, Conn.). 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, 300 nM FITC-Bak peptide, 1 nM recombinant Maltose Binding Protein (MBP) tagged Mcl-1 (residues 172-327) and 1 nM terbium conjugated-MBP antibody were added to assay buffer (4.5 mM Monobasic potassium phosphate, 15.5 mM Dibasic potassium phosphate, 1 mM Sodium EDTA, 50 mM NaCl, 1 mM DTT, 0.05% Pluronic F-68, pH 7.5). Compounds are diluted in DMSO in a 16-point, semilog serial dilution scheme. For the BAK FITC TR-FRET assay 50 nL compound is added to 20 μL of assay buffer containing Bak-FITC. MBP tagged Mcl-1 protein and antibody, for a final DMSO concentration of 0.25% and a top concentration of 10 μM. 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 3 hours at room temperature. TR-FRET signal (Delta F) is measured on a Biotek Cytation 3 multimode plate reader equipped with a filter cube containing an Ex 340/30 Em 620/10 nm filter and an Ex 340/30 Em 520 filter.
Measurement: Ki = 9.6 nM (pChEMBL 8.020000457763672)
Descriptors: MW 877.92, ALogP 10.1, HBA 11, HBD 0, PSA 106.39, QED 0.11
Compound (SMILES): Cc1c(Cl)cc(C(C)n2nc(C)c3c(N)ncnc32)c2c1CN(C(=O)CC#N)CCO2
InChIKey: BLGZEENVRQBMTL-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: PI3K Enzyme Assay: Lipid kinase substrate, phosphoinositol-4,5-bisphosphate (PIP2), are purchased from Echelon Biosciences (Salt Lake City, Utah). PI3K isoforms α, β, δ and γ are purchased from Millipore (Bedford, Mass.). ATP, MgCl2, DTT, EDTA, MOPS and CHAPS are purchased from Sigma-Aldrich (St. Louis, Mo.).The kinase reaction are conducted in clear-bottom 96-well plate from Thermo Fisher Scientific in a final volume of 24 μL. Inhibitors are first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay is 0.5%. The PI3K assays are carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. The reaction mixture is prepared containing 50 M PIP2, kinase and varying concentration of inhibitors. Reactions are initiated by the addition of ATP containing 2.2 μCi [γ-33P]ATP to a final concentration of 1000 μM. The final concentration of PI3K isoforms α, β, δ and γ in the assay were 1.3, 9.4, 2.9 and 10.8 nM, respectively. Reactions are incubated for 180 minutes and terminated by the addition of 100 μL of 1 M potassium phosphate pH 8.0, 30 mM EDTA quench buffer. A 100 μL aliquot of the reaction solution are then transferred to 96-well Millipore MultiScreen IP 0.45 m PVDF filter plate (The filter plate is prewetted with 200 μL 100% ethanol, distilled water, and 1 M potassium phosphate pH 8.0, respectively). The filter plate is aspirated on a Millipore Manifold under vacuum and washed with 18×200 μL wash buffer containing 1 M potassium phosphate pH 8.0 and 1 mM ATP. After drying by aspiration and blotting, the plate is air dried in an incubator at 37° C. overnight. Packard TopCount adapter (Millipore) is then attached to the plate followed with addition of 120 μL Microscint 20 scintillation cocktail (Perkin Elmer) in each well. After the plate sealing, the radioactivity of the product is determined by scintillation counting on Topcount (Perkin-Elmer). IC50 determination is performed by fitting the curve of percent control activity versus the log of the inhibitor concentration using the GraphPad Prism 3.0 software. Compounds with an IC50 of less than 10 μM in the assay of Example A2 are considered to be active.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 439.91, ALogP 2.92, HBA 8, HBD 1, PSA 122.95, QED 0.66
Compound (SMILES): CN(C)S(=O)(=O)c1ccc(-c2cnc3ncc(C4(c5ccc6ncccc6c5)CC4)n3c2)cc1
InChIKey: IWECQNLEWMVIMJ-UHFFFAOYSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: In Vitro c-Met Kinase Enzyme Assay: Compounds were screened in vitro for their ability to inhibit c-Met kinase activity. The IC50 values of compounds for the inhibition of c-Met kinase were determined as described in the literature with some modifications (Wang, X. et al, Mol. Cancer Ther. 2003, 2(11):1085-1092; Calic, M. et al., Croatica Chemical ACTA. 2005, 78(3):367-374). Briefly, histidine-tagged c-Met catalytic domain fusion protein (Invitrogen, #PV3143) was used for the assay. IC50 measurements were based on the degree of phosphorylation of poly Glu-Tyr (Sigma-Aldrich, #P0275) that was coated (0.01 mg/per well) on 96-well microplates (R&D systems, #DY990). The reaction was carried out in a 50 μL solution containing 50 mM HEPES (pH 7.5), 10 mM MnCl2, 10 mM MgCl2, 0.5 mM DTT, 100 μM Na3VO4, 5 μM ATP (Cell Signaling Technology, #9804) and serial dilutions of individual compounds. The reaction lasted for 25 minutes at 30° C. After the reaction was completed, the contents of the plates was discarded. Plates were then washed with TBS-T (250 μL/well, 5×) and then blocked with TBS-T containing 1% BSA for 2 hours. The contents of the plates was discarded, and 100 μL (per well) of peroxidase-labeled anti-phospho-tyrosine antibody (Sigma, #A5964) diluted (1:60,000) in 1% BSA containing TBS-T were then added and incubated for 1 hour. Plates were washed with TBS-T (250 mL/well, 5×) and followed by the color reaction using 100 μL (1:1 mixture) of H2O2 and tetramethylbenzidine (R&D Systems, #DY999). The reaction was stopped in minutes with 100 μL of 2 N H2SO4. The optical density was measured immediately using a microplate reader at 450 nm with wavelength correction at 540 nm. IC50 values were calculated with the GraphPad Prism software. The linear range (i.e., the time period over which the rate remained equivalent to the initial rate) was determined for the kinase and IC50 determinations were performed within this range.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 469.57, ALogP 4.27, HBA 6, HBD 0, PSA 80.46, QED 0.38
Compound (SMILES): Cc1ncc(NC(=O)c2cc(CN(C)C)cc(C(F)(F)F)c2)cc1-c1cnc(N(C)C)c(N2CCOCC2)c1
InChIKey: BHRMRRQALOPXNK-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Raf Activity Assay: Raf and biotinylated Mek, kinase dead, were combined at 2x final concentrations in assay buffer (50 mM Tris, pH 7.5, 15 mM MgCl2, 0.01% BSA and 1 mM DTT) and dispensed 5 ml per well in assay plates (Greiner white 384 well assay plates #781207) containing 0.25 ml of 40x of a Raf kinase inhibitor test compound diluted in 100% DMSO. The plate was incubated for 60 minutes at room temperature. The Raf kinase activity reaction was started by the addition of 5 mL per well of 2xATP diluted in assay buffer. After 3 hours (b-Raf(V600E)) or 1 hour (c-Raf). The reactions were stopped and the phosphorylated product was measured using a rabbit anti-p-MEK (Cell Signaling, #9121) antibody and the Alpha Screen IgG (ProteinA) detection Kit (PerkinElmer #6760617R), by the addition of 10 mL to the well of a mixture of the antibody (1:2000 dilution) and detection beads (1:2000 dilution of both beads) in Stop/bead buffer (25 mM EDTA, 50 mM Tris, pH 7.5, 0.01% Tween20). The additions were carried out under dark conditions to protect the detection beads from light. A lid was placed on top of the plate and incubated for 1 hour at room temperature, then the luminescence was read on a PerkinElmer Envision instrument. The concentration of each compound for 50% inhibition (IC50) was calculated by non-linear regression using XL Fit data analysis software.
Measurement: IC50 = 2.4 nM (pChEMBL 8.619999885559082)
Descriptors: MW 542.61, ALogP 4.69, HBA 7, HBD 1, PSA 73.83, QED 0.46
Compound (SMILES): COc1cncc(-c2ccnc(-c3ncc(C4CC4)[nH]3)c2)c1
InChIKey: LHORCQKYHNRUSS-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: PI3K-γ Scintillation Proximity Assay: The kinase reaction was conducted in polystyrene 384-well Greiner Bio-one white plate from Thermo Fisher Scientific in a final volume of 25 μL. Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 2%. The PI3Kγ assay was carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 μM PIP2, 2 μM ATP, 0.5 μCi [γ-33P] ATP, 13 nM PI3Kγ. Reactions were incubated for 120 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 163 mM potassium phosphate pH 7.8, 20% glycerol, 25 mM EDTA. The final concentration of SPA beads is 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1500 rpm for 10 min, the radioactivity of the product was determined by scintillation counting on Topcount (Perkin-Elmer). IC50 determination was performed by fitting the curve of percent of the solvent control activity versus the log of the inhibitor concentration using the GraphPad Prism 6.0 software.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 292.34, ALogP 3.42, HBA 4, HBD 1, PSA 63.69, QED 0.8
Compound (SMILES): Nc1nc2c(ncn2[C@@H]2O[C@@H]3COP(=O)(O)O[C@H]4[C@@H](F)[C@H](n5cnc6c(N)ncnc65)O[C@@H]4CNS(=O)(=O)O[C@H]3[C@@H]2F)c(=O)[nH]1
InChIKey: AWJQWNOYVSWPGJ-JBLCYPDHSA-N
Target: Stimulator of interferon genes protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q86WV6
Assay: STING SPA Binding Assay: The assay was run in 1536-well plates in a total volume of 8 μL per well by adding 8 nM [3H]-2′3′-cGAMP and 40 nM biotin-STING protein in assay buffer [25 mM HEPES (Corning 25-060-C1) pH 7.5, 150 mM NaCl (Sigma S5150), 0.5 mg/mL BSA (Gibco 15260-037), 0.001% Tween-20 (Sigma P7949), molecular grade water (Corning 46-000-CM)]. Test compounds (80 nL) were added with an acoustic dispenser (EDC Biosystems) in 100% DMSO for a final assay concentration of 1% DMSO. Plates were centrifuged for 1 min and incubated for 60 min at room temperature. Finally, (2 μL) polystyrene streptavidin SPA beads (PerkinElmer RPNQ0306) were added and plates were sealed and centrifuged for 1 min at room temperature. Plates were dark adapted for 2 h and read on a ViewLux (Perkin Elmer) for 12 min per plate. A saturation binding curve for [3<H]-2′3′cGAMP showed a KD of 3.6±0.3 nM for binding to STING, comparable to reported values for the natural ligand (Zhang et al., Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING (Molecular Cell 2013 51(2):10.1016/j.molcel.2013.05.022.).
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 677.5, ALogP -1.67, HBA 18, HBD 5, PSA 288.83, QED 0.15
Compound (SMILES): CN(C)CCN(Cc1ccccc1C(F)(F)F)C(=O)CNc1cccc2c1CCCN(CC1CC1)C2
InChIKey: WQSSKXPCBAZMOM-UHFFFAOYSA-N
Target: Atypical chemokine receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P25106
Assay: In Vitro Assay: The assay is using the PathHunter CHO-K1 CXCR7 β-arrestin cell line from DiscoverX. The system is based on the Enzyme Fragment Complementation Technology. Two complementing fragments of the β-galactosidase enzyme are expressed within stably transfected cells. The larger portion of β-gal, termed EA for Enzyme Acceptor, is fused to the C-terminus of b-arrestin 2. The smaller fragment, termed ProLink¿ tag, is fused to CXCR7 at the C-terminus. Upon activation, b-arrestin is recruited which forces the interaction of ProLink and EA, allowing complementation of the two fragments of b-gal and the formation of a functional enzyme which is capable of hydrolysing the substrate and generating a chemiluminescent signal. CHO-K1 CXCR7 β-arrestin cells are detached from culture dishes with a cell dissociation buffer (Invitrogen, #13151-014) and collected in growing medium (F12 HAMS 90% (v/v)/FCS 10% (v/v), Penicillin/streptomycin 1% (v/v)). 5000 cells per well (in 20 μl) are seeded in a 384 well plate (white-walled, clear bottom; BD Falcon #353274). The plate is incubated at 37° C./5% CO2 for 24 hours. Medium is then replaced by 20 μl OPTIMEM (Invitrogen #31985) for 3 to 4 hours. Test compounds are dissolved at 10 mM in DMSO and serially diluted in DMSO to 200× of the final concentration for dose response curves. Compounds are then diluted 1:33.3 in HBSS1X. 5 μl/well of HBSS1X/20 mM HEPES/0.2% BSA are added to the assay plate followed by addition of 5 μl/well of diluted compounds. CXCL12 (Peprotech #300-28A) may be used as a reference agonist. The plate is incubated for 90 minutes at 37° C. 12 μl of detection reagent (Path Hunter Detection Kit, DiscoveRx, #93-0001) is transferred to the assay plate and to the plate is incubated for 1 hour at room temperature. Luminescent signal is read in a microplate reader (FLUOstar Optima, bmg). The calculated EC50 values may fluctuate depending on the daily cellular assay performance. Fluctuations of this kind are known to those skilled in the art. Average EC50 values from several measurements are given as geometric mean values.
Measurement: EC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 502.63, ALogP 4.87, HBA 4, HBD 1, PSA 38.82, QED 0.5
Compound (SMILES): NCc1ccc(CNC(=O)c2cnn(Cc3csc(-c4ccccc4)n3)c2C(F)(F)F)cc1
InChIKey: ZOGFWBZPASWNGT-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Plasma kallikrein assay: Plasma kallikrein inhibitory activity in vitro was determined using standard published methods (see e.g. Johansen et al., Int. J. Tiss. Reac. 1986, 8, 185; Shori et al., Biochem. Pharmacol., 1992, 43, 1209; Stürzebecher et al., Biol. Chem. Hoppe-Seyler, 1992, 373, 1025). Human plasma kallikrein (Protogen) was incubated at 37° C. with the fluorogenic substrate H-DPro-Phe-Arg-AFC and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in optical absorbance at 410 nm and the IC50 value for the test compound was determined.
Measurement: IC50 = 488 nM (pChEMBL 6.309999942779541)
Descriptors: MW 471.51, ALogP 4.46, HBA 6, HBD 2, PSA 85.83, QED 0.42
Compound (SMILES): CS(=O)(=O)c1cc(C2CCC2)ccc1Nc1cc(NC(=O)[C@H]2CC2(F)F)nc2[nH]c(C(F)F)nc12
InChIKey: KMJOJYFMDXYAQQ-GFCCVEGCSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Binding Assay: Binding constants for compounds of the present invention against the JH2 domain were determined by the following protocol for a KINOMEscan® assay (DiscoveRx). A fusion protein of a partial length construct of human TYK2 (JH2domain-pseudokinase) (amino acids G556 to D888 based on reference sequence NP_003322.3) and the DNA binding domain of NFkB was expressed in transiently transfected HEK293 cells. From these HEK 293 cells, extracts were prepared in M-PER extraction buffer (Pierce) in the presence of Protease Inhibitor Cocktail Complete (Roche) and Phosphatase Inhibitor Cocktail Set II (Merck) per manufacturers' instructions. The TYK2 (JH2domain-pseudokinase) fusion protein was labeled with a chimeric double-stranded DNA tag containing the NFkB binding site (5′-GGGAATTCCC-3′) fused to an amplicon for qPCR readout, which was added directly to the expression extract (the final concentration of DNA-tag in the binding reaction is 0.1 nM).
Measurement: Kd > 10 nM (pChEMBL 8)
Descriptors: MW 511.5, ALogP 4.9, HBA 6, HBD 3, PSA 116.84, QED 0.39
Compound (SMILES): COc1nc(OC)nc(N2CCc3cccc(N4CCN(C(=O)Cn5cnc6cccnc65)[C@H](C)C4)c3C2)n1
InChIKey: LNGUZUIAZCFSNG-GOSISDBHSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: FLIPR Assay: The bioactivity of compounds is tested in a fluorometric imaging plate reader (FLIPR: Molecular Devices) using engineered CHO-K1 cells expressing the human CXCR3A (GenBank: AY242128) coupled to a G protein (Galpha(16)). Cells are plated the day prior to bioassay in F12 medium supplemented with 10% FBS and G418 and hygromycin antibiotics to maintain recombinant selection. At the day of bioassay, cells are washed and dye loaded for one hour with Fluo-4-AM (Invitrogen) in Hanks Balanced Salt Solution (Invitrogen), buffered with 20 mM Hepes at pH 7.4 and sodium bicarbonate (0.038%), containing 5 mM probenecid. This buffer, but lacking the dye and containing probenecid at a concentration of 2.5 mM, is also used for washing steps (wash buffer); or lacking both dye and probenecid but supplemented with 0.1% BSA for compound dilution steps (dilution buffer). Cells are washed free of excess dye and 60 microliter of wash buffer is added. Stock solutions of test compounds are made up at a concentration of 10 mM in DMSO, and serially diluted in dilution buffer to concentrations required for inhibition dose response curves. After a 10 minute incubation period at 37° C., 10 microliters of each compound dilution are transferred from a compound plate to the plate containing the recombinant cells in the FLIPR instrument according to the manufacturer's instructions. Following basal readings, 10 microliter CXCL10 agonist at a concentration of 20 nM (from Peprotech) is added, again using the FLIPR instrument. Changes in fluorescence are monitored before and after addition of the test compounds. Emission peak values above base level after CXCL10 addition are exported after base line subtraction.
Measurement: IC50 = 3.39 nM (pChEMBL 8.470000267028809)
Descriptors: MW 529.61, ALogP 1.93, HBA 11, HBD 0, PSA 114.63, QED 0.37
Compound (SMILES): CCS(=O)(=O)c1ccc(Cl)cc1Cn1cnc2c(Cl)c(CN3CCC[C@@H](C(N)=O)C3)c(C(F)(F)F)cc2c1=O
InChIKey: HHSVLGSNZJCLQJ-CQSZACIVSA-N
Target: Epithelial discoidin domain-containing receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q08345
Assay: LanthaScreen Assay: DDR1 binding activity was measured using the LanthaScreen (Registered trademark) Eu Kinase Binding Assay (manufactured by Life Technologies Corporation). The test compound and the Alexa Fluor® 647-labeled Kinase Tracer 178 (manufactured by Life Technologies Corporation) were added to a mixture of DDR1 and the LanthaScreen (Registered trademark) Eu-anti-GST antibody. After one-hour reaction at room temperature, the fluorescence resonance energy transfer was measured. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate relative to the test compound-free control.
Measurement: IC50 = 110 nM (pChEMBL 6.960000038146973)
Descriptors: MW 605.47, ALogP 4.26, HBA 7, HBD 1, PSA 115.36, QED 0.43
Compound (SMILES): Cc1nc([C@@H]2O[C@H](CO)[C@H](O)[C@H](n3cc(-c4ccc5scnc5c4)nn3)[C@H]2O)n(-c2cc(Cl)ccc2C(F)(F)F)n1
InChIKey: IUNVHNRVBRIHFO-YDXQKAQTSA-N
Target: Galectin-3 (SINGLE PROTEIN), Homo sapiens
UniProt: P17931
Assay: Biological Assay: The Gal-3 assays were performed in 384 white Opti plates in three replicates at room temperature with gentle shaking at 250-300 rpmFrom the original stocks, 2.525× working stock concentrations of His-tagged recombinant human Gal-3 (hGal-3) and that of B-ASF were prepared. From the working stock, 20 μl of hGal-3 (15 nM) and 20 μl B-ASF (15 nM) were added to the plates. In Negative Control, only hGal-3 was added. A concentration range of 50× working stocks were prepared for the compounds in 100% DMSO. Aliquots of 1 uL of the compounds were added to the wells and pre-incubated with 20 μl hGal-3 per well for 30 minutes Then 20 μl B-ASF wereadded and incubated for another 1 hour. To detect the signal, 5 μL (final conc. of 1.0 nM) terbium labelled Anti-His antibody was added and incubated for 30 min followed by adding 5 μL (final conc. of 20 nM) Streptavidin d2 and incubation for another 1 hour. The assay signal was detected using HTRF screen protocol (Excitation wavelength=340 nm, emission wavelength=615 nm/665 nm) on Envision 2104 Multilabel Reader. Data analysed using Toolset and Curve Master. Results are reported in the experimental section (IC50 in μM).
Measurement: IC50 = 87 nM (pChEMBL 7.059999942779541)
Descriptors: MW 608, ALogP 3.51, HBA 12, HBD 3, PSA 144.23, QED 0.27
Compound (SMILES): OC(c1cn(C2CCC2)nn1)c1c(C2CC2)sc2cncn12
InChIKey: UFIWPNSJOFTBKF-UHFFFAOYSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: Enzymatic Assay: IDO1: Recombinant full-length human IDO1 with a N-terminal hexahistidine tag expressed in E. coli and purified to homogeneity is incubated at a final concentration of 2 nM in assay buffer consisting of 37.5 mM phosphate buffer at pH6.5 supplemented with 10 mM sodium L-ascorbate, 0.45 μM methylene blue, 50 U/ml catalase, 0.01% BSA, and 0.01% Tween 20 (protocol modified from Seegers et al, JBS 2014). Example compounds are serially diluted in DMSO, further diluted in phosphate buffer, and added to the enzyme at final concentrations ranging from 10 μM to 0.5 nM. The final DMSO concentration is 0.6%. Following a pre-incubation of 30 minutes at RT, the reaction is started by the addition of L-tryptophan at a final concentration of 5 μM in assay buffer. After 30 minutes of incubation at RT, 3 μL of the reaction mixture are transferred to a 384 deep well plate containing 25 μL of deionized water. 100 μl of 200 nM L-Tryptophan-(indole-d5) in cold 100% methanol are added followed by a 10 minutes centrifugation at 4,000 rpm at 4° C. An additional 75 μL of deionized water are then added and followed by a 10 minutes centrifugation at 4,000 rpm at 4° C. The product of the reaction N′-Formylkynurenine (NFK) is quantified by LCMS and normalized to the L-Tryptophan-(indole-d5) signal. Samples with 0.6% DMSO (0% effect) and a TDO/IDO inhibitor (100% effect) are used as control samples to set the parameters for the non-linear regression necessary for the determination of the half-maximal inhibitory concentration (IC50) for each compound. For each compound concentration the percentage of activity compared to 0% and 100% effect is calculated as average ±STDEV (each concentration measured in duplicate). IC50 values and curves are generated with XLfit software (IDBS) using Dose-Response One Site model 203 (four parameter logistic curve model). When compounds are measured multiple times, mean values are given.
Measurement: IC50 = 183 nM (pChEMBL 6.739999771118164)
Descriptors: MW 315.4, ALogP 2.67, HBA 7, HBD 1, PSA 68.24, QED 0.8
Compound (SMILES): Cc1cnc(NC(=O)[C@H]2CCC[C@@H](NC(=O)C3COC3)C2)cc1-c1cnn2c1CC(C)(C)C2
InChIKey: BNMSUGRKZJRLFE-FUHWJXTLSA-N
Target: Cyclin-dependent kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P50750
Assay: Kinase Assay (ATP Concentration at Km): Activity of CDK9 was determined in-vitro using a mobility shift assay on a Caliper LC3000 reader (Caliper/PerkinElmer), which measures fluorescence of a phosphorylated and unphosphorylated fluorescent peptide substrate and calculates a ratiometric value to determine percent turnover. Phosphorylation of the peptide in the presence and absence of the compound of interest was determined. Enzyme/substrate/adenosine triphosphate (ATP) mix (3 nM CDK9/CycT1, 6 μM ATP, 1.5 μM CDK9 peptide substrate (FITC-X-GSRTPMY-NH2 (X: epsilon aminocaproic acid)), 50 mM HEPES (pH7.2), 1 mM dithiothreitol, 0.01% tween 20, 50 μg/mL bovine serum albumin, (final assay concentration)) (5 μl) was preincubated with 2 μl of compound for 15 minutes at 25° C. Reactions were initiated with 5 μl of 24 mM MgCl2 (10 mM final assay concentration) in buffer (50 mM HEPES (pH7.2), 1 mM dithiothreitol, 0.01% tween 20, 50 μg/mL bovine serum albumin, (final assay concentration) and incubated at 25° C. for 90 minutes and reactions were stopped by addition of 5 μl of Stop mix consisting of 65 mM HEPES (pH7.2), 35.5 mM EDTA, 0.227% Coatin Reagent 3 (Caliper/PerkinElmer), and 0.003% Tween. Phosphorylated and unphosphorylated substrate was detected by a Caliper LC3000 reader (Caliper/PerkinElmer) in the presence of separation buffer consisting of 100 mM HEPES (pH7.2), 15.8 mM EDTA, 0.1% Coatin Reagent 3 (Caliper/PerkinElmer), 0.015% Brij-35, 5% DMSO, and 5.6 mM MgCl2.
Measurement: IC50 < 3 nM (pChEMBL 8.52299976348877)
Descriptors: MW 451.57, ALogP 3.1, HBA 6, HBD 2, PSA 98.14, QED 0.73
Compound (SMILES): C[C@@H](Nc1nc(Cl)nc2c1cnn2[C@@H]1O[C@H](COC(CO)(CO)P(=O)(O)O)[C@@H](O)[C@H]1O)c1ccc(F)cc1
InChIKey: MLNYWHCSJVCYST-XOQFURIUSA-N
Target: 5'-nucleotidase (SINGLE PROTEIN), Homo sapiens
UniProt: P21589
Assay: Biochemical Assay: Assay Reaction ConditionsAssay Volume: 70 μlReaction Volume: 50 μlCD73: 0.3208 nMAMP: 15 μMAssay Buffer: 25 mM Tris-HCL, pH 7.4, 0.01% Brij-35, 0.01% BSA, 5 mM MgCl2Assay Procedure:Used 384 clear plate.Made dose titration of testing compounds in assay buffer, 10 points ½ log titrations in duplicates starting at 100 μM.Added 25 μl of CD73 to each well for a final concentration of 320 pM.Incubated at RT for 15 min.Added 25 μl of AMP to each well for a final concentration of 15 μM.Incubated at RT for 10 min.Added 10 μl of Malachite Green Reagent A, incubate at RT for 10 min.Added 10 of Malachite Green Reagent B, incubate at RT for 45 min.Read the Absorbance on Envision plate reader using excitation filter: Cy5 620 nM.The ability of the compounds disclosed herein to inhibit CD73 activity was quantified and the respective IC50 values were determined.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 577.89, ALogP 0.29, HBA 12, HBD 7, PSA 212.54, QED 0.13
Compound (SMILES): Cc1cnc([C@@H](CC(=O)NC[C@H](Cc2ccc(C(N)=O)c(F)c2)N(C)C)C2(C(F)(F)F)CC2)s1
InChIKey: MONMSWHHJDZEOD-DOTOQJQBSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: MOR cAMP Agonist and Antagonist Assays: CHO Tag-lite human mOR stable cell line from Cisbio (NCBI accession number: NM_000914.3, Bedford, Mass.) were seeded and grown to approximately 80% confluence in Ham F-12 with 10% FBS, 50 U/ml penicillin, 50 μg/ml streptomycin, 2 mM Hepes, and 1 mg/ml geneticin (Invitrogen, Carlsbad, Calif.). Cells were then harvested using accutase (Corning. Coming, N.Y.), centrifuged at 1300 RPM for 5 min, and plated at 5000 cells per 5 μL per well in a 5× dilution of stimulation buffer consisting of the HTRF cAMP Gi kit, water and IBMX at 0.5 mM (Cisbio, Bedford, Mass.) in white HTRF low volume 384 well plates (Cisbio, Bedford, Mass.). Plates were then incubated at 37° C. in 5% CO2 for 10 minutes. For the agonist assay, forskolin was added to a final concentration of 4 μM. For the antagonist assay, forskolin (4 μM) and DAMGO at EC90 final concentration were added. Test compounds were dissolved in DMSO and water, and then serially diluted to working concentrations such that the concentration of DMSO was less than 0.1%. Diluted test compounds were added at 2.5 μL per well, and plates were incubated at 37° C., and 5% CO2 for 15 minutes, and then at room temperature for 15 minutes. Next 5 μl/well of cAMP Eu-cryptate and 5 μl/well of anti-cAMP-d2 (both diluted 1:20 in lysis buffer) were added, and plates were incubated at room temperature for 1 hour. Following incubation, plates were read in a Synergy Neo2 multi-mode reader (Biotek, Winooski, Vt.). Plate reader settings were set to time resolved fluorescence with excitation at 330 nm and emissions of 620 nm and 665 nm. Emission fluorescence was normalized (665/620 nm signal×1000). For the agonist assay, data was normalized using the maximal DAMGO response. Measurements were performed in triplicate and the dose response was fit using nonlinear regression.
Measurement: IC50 = 1650 nM (pChEMBL 5.78000020980835)
Descriptors: MW 500.56, ALogP 3.79, HBA 5, HBD 2, PSA 88.32, QED 0.49
Compound (SMILES): Cc1oc2c(c1C)c1ncnn1c(=O)n2Cc1ccc(Cl)cc1
InChIKey: PBOQMKNJXRTEHB-UHFFFAOYSA-N
Target: Dual specificity calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1B (SINGLE PROTEIN), Homo sapiens
UniProt: Q01064
Assay: IMAP TR-FRET assay: The IMAP TR-FRET PDE assay was optimized for concentration of enzyme, Calmodulin, cAMP or cGMP substrate, DMSO tolerance, and incubation time. Into each well of a solid white 1536 well plate (Corning) was dispensed 250 pg full-length recombinant NH-terminal GST tagged human PDE1b enzyme (BPS Bioscience Cat #60011, San Diego, Calif.) in 2.5 μL IMAP BSA reaction buffer (Molecular Devices, Sunnyvale, Calif.) containing 10 U/mL Calmodulin and 2.5 mM CaCl2 (Sigma Aldrich.) After a brief centrifugation, 30 nL compound was added by transfer from 1 mM stock in DMSO using a Kalypsys 1536 Pintool. Plates were incubated for 5 minutes at room temperature before dispensing 1.5 μL of 533 nM 5-carboxy fluorescein (FAM)-labeled cAMP (Molecular Devices, Sunnyvale, Calif.) for a final concentration of 200 nM. After a brief centrifugation, the plates were incubated for 30 minutes at room temperature. The assay was terminated by adding 5 μL IMAP binding reagent/Tb complex (Molecular Devices, Sunnyvale, Calif.) to each well. Plates were incubated 1 hour at room temperature and read on a Viewlux multimode plate reader (Perkin Elmer). The instrument was set to excite using the DUG11 filter and measure using 490/10 nm and 520/10 nm filters.
Measurement: EC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 328.76, ALogP 2.96, HBA 6, HBD 0, PSA 65.33, QED 0.57