lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCc1nc2ccc(C3=CCN(C(=O)Nc4cccs4)CC3)cn2c1N(C)c1nc(-c2ccc(F)cc2)cs1
InChIKey: YNXIBRAUKJKILJ-UHFFFAOYSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Biochemical Assay: TABLE VI: (UniProtKB/SwissProt Sequence ref Q13822) biochemical assay using the fluorogenic autotaxin substrate FS-3 as substrate. FS-3 is a doubly labeled analog of LPC wherein the fluorophore is quenched through intramolecular energy transfer. Without hENPP2, the emission of the probe is quenched. If the substrate is hydrolyzed by hENPP2, the emission of the probe is not quenched anymore resulting in a fluorescence increase. Inhibition of hENPP2 by compounds will result in a decrease of the signal.10 μL of a dilution series of compound, starting from 20 μM highest concentration, 1/5 dilution, is added to the wells. hENPP2 is used at a final concentration of 0.4 μg/mL or 0.64 μg/mL (it will be appreciated by the skilled person that the potency read out is independent of the enzyme concentration). The enzyme is diluted in 50 mM Tris-HCl pH 8.0, 250 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 0.1% fatty acid free BSA in a total volume of 20 μL. Enzyme mixture is added to compounds and the resulting mixture is incubated for 30 min at room temperature under shaking. The reaction is started by the addition of 20 μL of 0.75 μM FS-3 diluted in the same buffer as described above and the mixture is incubated at 30° C. for 30 min. Fluorescence is read on the Envision (Excitation 485 nm, emission 520 nM).
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 558.71, ALogP 7.31, HBA 7, HBD 1, PSA 65.77, QED 0.24
Compound (SMILES): CN1C(=O)Cc2cc(-c3cncc(-c4ccc(=O)n(C5CCCCO5)c4)c3)ccc21
InChIKey: BJEJSENZHSNTBH-UHFFFAOYSA-N
Target: Bone morphogenetic protein receptor type-1A (SINGLE PROTEIN), Homo sapiens
UniProt: P36894
Assay: Luminescence-Based ADPGlo Kinase Activity Assay: Example 53: The assays were performed in 384-well, low volume microtiter assay plates in a final reaction volume of 6 ul. Dose-response curves were generated by incubating 10 nM of each kinase in 50 mM Hepes pH 7.5, 0.02% Tween 20, 0.02% BSA, 1 mM DTT, 10 uM Na3VO4, 10 mM ß-Glycerolphosphate, 1 mM MgCl2, 12 mM MnCl2 and 15 uM ATP for 60 min at 32° C. in the presence or absence of compound diluted in DMS. The amount of generated ADP is a measure of kinase activity and is quantified using the ADP-Glo Kinase Assay (Promega) according to manufacturer's instructions. ADP is converted to ATP by adding 3 ul of ADP-Glo Reagent and incubation at 32° C. for 60 min. ATP is subsequently converted into a bioluminescent signal by adding 6 ul luciferase assay reagents (Kinase detection buffer+Kinase Detection Substrate (Promega)) and further incubation at 32° C. for 60 min. For the measurement of luminescence a PHERAstar Multilabel Reader was used at a measurement interval time of 0.1 second (optical module for luminescence measurements in the 230 nm to 750 nm wavelength range). The luminescent signal positively correlates with kinase activity.
Measurement: IC50 > 15100 nM (pChEMBL 4.821000099182129)
Descriptors: MW 401.47, ALogP 3.8, HBA 5, HBD 0, PSA 64.43, QED 0.67
Compound (SMILES): CCc1nc2ccc(C3=CCN(S(=O)(=O)C(F)(F)F)CC3)cn2c1N(C)c1nc(-c2ccc(Cl)cc2)cs1
InChIKey: MYQJTEAFLDPJFI-UHFFFAOYSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Biochemical Assay: TABLE VI: (UniProtKB/SwissProt Sequence ref Q13822) biochemical assay using the fluorogenic autotaxin substrate FS-3 as substrate. FS-3 is a doubly labeled analog of LPC wherein the fluorophore is quenched through intramolecular energy transfer. Without hENPP2, the emission of the probe is quenched. If the substrate is hydrolyzed by hENPP2, the emission of the probe is not quenched anymore resulting in a fluorescence increase. Inhibition of hENPP2 by compounds will result in a decrease of the signal.10 μL of a dilution series of compound, starting from 20 μM highest concentration, 1/5 dilution, is added to the wells. hENPP2 is used at a final concentration of 0.4 μg/mL or 0.64 μg/mL (it will be appreciated by the skilled person that the potency read out is independent of the enzyme concentration). The enzyme is diluted in 50 mM Tris-HCl pH 8.0, 250 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 0.1% fatty acid free BSA in a total volume of 20 μL. Enzyme mixture is added to compounds and the resulting mixture is incubated for 30 min at room temperature under shaking. The reaction is started by the addition of 20 μL of 0.75 μM FS-3 diluted in the same buffer as described above and the mixture is incubated at 30° C. for 30 min. Fluorescence is read on the Envision (Excitation 485 nm, emission 520 nM).
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 582.07, ALogP 6.38, HBA 7, HBD 0, PSA 70.81, QED 0.26
Compound (SMILES): CNC(=O)c1ccc(N2CCN(Cc3ccc4nc(CC(F)F)c(=O)[nH]c4c3)CC2)cn1
InChIKey: GEWVKKFPLYRKPW-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UGN5
Assay: Fluorescence Anisotropy Binding Assay: PARP2: Recombinant full length PARP2 protein was diluted to 6 nM with 50 mM Tris pH 8, 0.001% Triton X100, 10 mM MgCl2, 150 mM NaCl and incubated for four hours with an equivalent volume of 2 nM fluorescent probe diluted with 50 mM Tris pH 8, 0.001% Triton X100, 10 mM MgCl2, 150 mM NaCl. The final DMSO concentration of the probe was kept below 1% (v/v).
Measurement: IC50 = 4400 nM (pChEMBL 5.360000133514404)
Descriptors: MW 442.47, ALogP 1.81, HBA 6, HBD 2, PSA 94.22, QED 0.6
Compound (SMILES): COc1cc(OC)c(F)c(N2Cc3cnc(NCCN4CCN(C)CC4)cc3C3(CC3)C2=O)c1F
InChIKey: JLLPQMNQJBQSRY-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).FGFR1 and FGFR2 were measured under equivalent conditions with the following changes in enzyme and ATP concentrations: FGFR1, 0.02 nM and 210 μM, respectively and FGFR2, 0.01 nM and 100 μM, respectively. The enzymes were purchased from Millipore or Invitrogen.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 487.55, ALogP 2.61, HBA 7, HBD 1, PSA 70.17, QED 0.64
Compound (SMILES): CN[C@@H](C)C(=O)NC1C(=O)N(Cc2c(C)ccc3ccccc23)c2ccccc2N(c2ccccc2)C1=O
InChIKey: SDKJKQSILYPXOB-QDLLFRBVSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay for BIR2 and BIR3: Ten nanomolar of 6× Histidine-tagged BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(c-biotin)-OH 1:2 TFA, in the presence of 50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/mL bovine serum albumin (BSA). Following a 45 min. incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations. Percentage of inhibition at each concentration was determined to generate an IC50 value for each test compound.
Measurement: IC50 > 54800 nM (pChEMBL 4.261000156402588)
Descriptors: MW 506.61, ALogP 4.45, HBA 4, HBD 2, PSA 81.75, QED 0.38
Compound (SMILES): Cc1ccccc1OC1CN(c2ccc(-c3cc(OCC(C)(C)O)cn4ncc(C#N)c34)cn2)C1
InChIKey: BRTTXFJHYHTTSG-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of Formula I were screened for their ability to inhibit wildtype and V804M mutant RET kinase using CisBio's HTRF KinEASE-TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Catalog No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Catalog No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 3403 nM (pChEMBL 5.46999979019165)
Descriptors: MW 469.55, ALogP 3.99, HBA 8, HBD 1, PSA 95.91, QED 0.44
Compound (SMILES): COCCN1CCN(C(=O)c2ccc(Nc3nc4c(C5=CCN(C(=O)CCC(F)(F)F)CC5)cccn4n3)cc2)CC1
InChIKey: BRCBELFWRIXMOC-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 = 0.32 nM (pChEMBL 9.489999771118164)
Descriptors: MW 585.63, ALogP 3.84, HBA 8, HBD 1, PSA 95.31, QED 0.41
Compound (SMILES): CC1(C)CC(Nc2ncc3cc(CCO)c(=O)n(C4CCCC4)c3n2)CCN1S(C)(=O)=O
InChIKey: PJNDWVHDRGLRDW-UHFFFAOYSA-N
Target: CDK6/cyclin D1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24385
Assay: CDK6/Cyclin D1 Mobility Shift Assay: The purpose of the CDK6/Cyclin D1 assay is to evaluate the inhibition (% inhibition, Kiapp and Ki values) in the presence of small molecule inhibitors by using a fluorescence based microfluidic mobility shift assay. CDK6/Cyclin D1 catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide 5-FAM-Dyrktide (5-FAM-RRRFRPASPLRGPPK) (SEQ ID NO:2). The mobility shift assay electrophoretically separates the fluorescently labeled peptides (substrate and phosphorylated product) following the kinase reaction. Both substrate and product are measured and the ratio of these values is used to generate % conversion of substrate to product by the LabChip EZ Reader. Typical reaction solutions contained 2% DMSO (± inhibitor), 10 mM MgCl2, 1 mM DTT, 2 mM ATP, 0.005% Tween 20 (TW-20), 3 μM 5-FAM-Dyrktide, 3 nM (active sites) CDK6/Cyclin D1 in 40 mM HEPES buffer at pH 7.5.Inhibitor Ki determinations for non-phosphorylated CDK6/CyclinD1 (LJIC-2003A2/1865) were initiated with the addition of ATP (50 μL final reaction volume), following a twelve minute pre-incubation of enzyme and inhibitor at 22° C. in the reaction mix. The reaction was stopped after 35 minutes by the addition of 50 μL of 25 mM EDTA. Ki determinations were made from a plot of the fractional velocity as a function of inhibitor concentration fit to the Morrison equation with the enzyme concentration as a variable.
Measurement: Ki = 12.3 nM (pChEMBL 7.909999847412109)
Descriptors: MW 463.6, ALogP 2.06, HBA 8, HBD 2, PSA 117.42, QED 0.67
Compound (SMILES): COc1ccc(CNC(=O)C2(O)CCN(c3cc(C)cc(Nc4cc(C)[nH]n4)n3)CC2)cc1
InChIKey: OBJQUSGHNMOYLI-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET V804L Gatekeeper Mutant Assay at KM: In each well of a 384-well plate, 7.5 nM-10 nM of mutant RET (ProQinase 1096-0000-1) was incubated in a total of 12.5 μL of buffer (100 mM HEPES pH 7.5, 0.015% BriJ 35, 10 mM MgCl2, 1 mM DTT) with 1 μM CSKtide (FITC-AHA-KKKKDDIYFFFG-NH2) (SEQ ID NO: 5) and 10 μM ATP at 25° C. for 120 minutes in the presence or absence of a dosed concentration series of compound (1% DMSO final concentration). The reaction was stopped by the addition of 70 μL of Stop buffer (100 mM HEPES pH 7.5, 0.015% BriJ 35, 35 mM EDTA and 0.2% of Coating Reagent 3 (Caliper Lifesciences)). The plate was then read on a Caliper EZReader 2 (protocol settings: −1.7 psi, upstream voltage −500, downstream voltage −3000, post sample sip 35 s). Data was normalized to 0% and 100% inhibition controls and the IC50 calculated using a 4-parameter fit in the CORE LIMS.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 450.54, ALogP 2.82, HBA 7, HBD 4, PSA 115.4, QED 0.44
Compound (SMILES): O=C(NO)c1ccc(Cn2c(=O)n(CCc3ccc(N4CCOCC4)cc3)c(=O)c3ccccc32)cc1
InChIKey: WMWNLURBMXMZLD-UHFFFAOYSA-N
Target: Histone deacetylase 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BY41
Assay: Enzymatic Assay: The IC50 values for aforementioned compounds against HDACs were determined. HDAC 1 to 11 can be assayed by using acetylated AMC-labeled peptide substrate. The Substrate 1, a fluorogenic peptide from p53 residues 379-382 (RHKKAc) is used for all MAC 1 to 11 but HDAC8, which has a substrate II (RHKAcKAc), a fluorogenic diacyl peptide based on residues 179-382 of p53. Compounds were tested in 10-dose 1050 mode in duplicate with 3-fold serial dilution starting at 10 uM.
Measurement: IC50 = 979 nM (pChEMBL 6.010000228881836)
Descriptors: MW 500.56, ALogP 2.41, HBA 8, HBD 2, PSA 105.8, QED 0.3
Compound (SMILES): CC(NC(=O)c1cc(=O)[nH]c(-c2ccccc2)n1)c1ccc(C(C)(C)C(F)(F)F)cc1
InChIKey: VYMVRVDCRFSJJK-UHFFFAOYSA-N
Target: cGMP-dependent 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O00408
Assay: Fluorescence Polarization (FP) Assay: In a typical experiment the PDE2 inhibitory activity of the compounds of the present invention was determined in accordance with the following experimental method. Rhesus PDE2A3 was amplified from rhesus macaque brain cDNA (Biochain Institute, Hayward, Calif.) using primers based on human PDE2A sequence (accession NM_002599.3) where the forward primer containing a Kozak consensus was 5′-gccaccatggggcaggcatgtggc-3′ and the reverse primer was 5′-tcactcagcatcaaggctgca-3′. Amplification with Easy-A High-Fidelity PCR cloning enzyme (Stratagene, La Jolla, Calif.) was 95° C. for 2 minutes followed by thirty three cycles of 95° C. for 40 seconds, 52° C. for 30 seconds, and 72° C. for 2 minutes 48 seconds. Final extension was 72° C. for 7 minutes. The PCR product was TA cloned into pcDNA3.3-TOPO (Invitrogen, Carlsbad, Calif.) according to standard protocol. A consensus sequence was developed from multiple clones and then deposited into GenBank (EU812167). AD293 cells (Stratagene, La Jolla, Calif.) with 70-80% confluency were transiently transfected with rhesus PDE2A3/pcDNA3.3-TOPO using Lipofectamine 2000 according to manufacturer specifications (Invitrogen, Carlsbad, Calif.).
Measurement: Ki = 103 nM (pChEMBL 6.989999771118164)
Descriptors: MW 429.44, ALogP 4.77, HBA 3, HBD 2, PSA 74.85, QED 0.61
Compound (SMILES): CCc1cccc(-c2cc(C(=O)NC)cc3c2CCN3C(=O)[C@@H]2CCCN2C#N)c1
InChIKey: YBGZJESLGRCDEE-NRFANRHFSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 30 (SINGLE PROTEIN), Homo sapiens
UniProt: Q70CQ3
Assay: USP30 Biochemical IC50 Assay: Dilution plates were prepared at 21 times the final concentration (2100 μM for a final concentration of 100 μM) in 50% DMSO in a 96-well polypropylene V-bottom plate (Greiner #651201). A typical 8-point dilution series would be 100, 30, 10, 3, 1, 0.3, 0.1, 0.03 μM final. Reactions were performed in duplicate in black 384 well plates (small volume, Greiner 784076) in a final reaction volume of 21 μl. Either 1 μl of 50% DMSO or diluted compound was added to the plate. USP30 was diluted in reaction buffer (40 mM Tris, pH 7.5, 0.005% Tween 20, 0.5 mg/ml BSA, 5 mMbeta-mercaptoethanol) to the equivalent of 0.05 μl/well and 10 μl of diluted USP30 was added to the compound. Enzyme and compound were incubated for 30 min at room temp. Reactions were initiated by the addition of 50 nM of TAMRA labelled peptide linked to ubiquitin via an iso-peptide bond as fluorescence polarisation substrate. Reactions were read immediately after addition of substrate and following a 2 hr incubation at room temperature. Readings were performed on a Pherastar Plus (BMG Labtech). λ Excitation 540 nm; λ Emission 590 nm.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 402.5, ALogP 3.11, HBA 4, HBD 1, PSA 76.44, QED 0.8
Compound (SMILES): O=C(O)c1ccc(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)cc1F
InChIKey: XIACOCZEICGICV-ZEQKJWHPSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Mus musculus
UniProt: P51450
Assay: General RORgamma SPA 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. The IC50 value is defined as the concentration of test compound needed to reduce [3H] 25-hydroxycholesterol specific binding by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 489 nM (pChEMBL 6.309999942779541)
Descriptors: MW 665.53, ALogP 6.48, HBA 4, HBD 1, PSA 91.75, QED 0.25
Compound (SMILES): C[C@@H](Oc1nc(-c2ccc3cc(C(=O)N(C)C)n(C)c3c2)cc2nn(C)cc12)[C@H]1CNC(=O)C1
InChIKey: ZXORLPNGQSWZPN-RHSMWYFYSA-N
Target: Aurora kinase B (SINGLE PROTEIN), Homo sapiens
UniProt: Q96GD4
Assay: Aurora B Kinase Assay: Recombinant human Aurora B (amino acids 1-344, clone number DU1773, Molecular weight 40.2 kDa, University of Dundee) was expressed as a fusion protein with an N-terminal His tag, affinity-purified and deep-frozen at a concentration of approx. 0.25-0.5 mg/ml in storage buffer (50 mM Tris-HCl pH 8; 25 mM Na-β-glycerophosphat; 0.1 mM EGTA; 150 mM NaCl; 0.03% Brij-35; 1 mM DTT and 10% glycerol) at −80° C. until use.The activity of the Aurora B kinase protein was determined using the ADP Glo® Luminescence Kinase test (Promega; V9103X). In this homogeneous test the amount of ADP remaining after the kinase reaction is quantified by a luciferin-luciferase reaction using luminescence. The luminescence signal obtained correlates with the amount of ADP still present and thus correlates with the activity of the protein kinase.
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 460.54, ALogP 2.73, HBA 7, HBD 1, PSA 94.28, QED 0.49
Compound (SMILES): CS(=O)(=O)CC1COCC(CCN2CCNC(=O)c3oc4ccc(C(F)(F)F)cc4c32)O1
InChIKey: OWXORYLIIMUPDP-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Pim-1 Inhibitory Activity Assay: The calculation of the Pim-1 inhibitory activity of the compound was performed using the following solution according to the protocol attached to ADP-Glo Kinase Assay (cat. V9102, Promega). The purified enzyme of human Pim-1 described above was used. (i) Preparation of Solution. The kinase buffer solution (50 mmol/L HEPES (pH 7.5), 5 mmol/L MgCl2, 1 mmol/L DTT, 0.05% BSA) was prepared by dissolving HEPES (Jena Bioscience), MgCl2 (Sigma-Aldrich), DTT (Sigma-Aldrich) and BSA (Sigma-Aldrich) in purified water. The ATP solution (288 μmol/L) was prepared by dissolving 100 mmol/L ATP (Promega) in the kinase buffer solution. The enzyme/substrate solution (0.2 mmol/L Pim-1, 30 μmol/L Pim2tide) was prepared by dissolving Pim-1 (described above) and Pim2tide (custom synthetic product by GenScript USA, the same product as PIM2tide cat. 12-542 by Millipore) in the kinase buffer solution. The test compound solution (containing 12.5% DMSO) was prepared by dissolving the DMSO solution of the test compound in the kinase buffer solution. The vehicle solution (containing 12.5% DMSO) was prepared by dissolving DMSO in the kinase buffer solution. (ii) Method. To a 384-well assay plate (Corning, 4513), the test compound solution or vehicle solution (control) was added by 1 μL/well, the enzyme/substrate solution or kinase buffer solution (blank) was added by 2 μL/well, and the ATP solution was added by 2 μL/well, and they were mixed. After enzymatically reacting at room temperature for 45 minutes, ADP-Glo Reagent (Promega) was added by 5 μL/well, and they were mixed. After reacting at room temperature for 60 minutes, Kinase Detection Reagent (Promega) was added by 10 μL/well, and they were mixed. After reacting at room temperature for 30 minutes, the luminescence amount of each well was measured for 10 msec with a multi-label plate reader EnVision (PerkinElmer).
Measurement: Ki = 5.36 nM (pChEMBL 8.270000457763672)
Descriptors: MW 476.47, ALogP 2.22, HBA 7, HBD 1, PSA 98.08, QED 0.71
Compound (SMILES): CC(C)(O)[C@H](F)CNC(=O)c1cnc(Nc2nc3ccnn3cc2F)cc1NC1CCC(F)(F)C1
InChIKey: LTLSSBFGELQHTO-PVQCJRHBSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 μL prepared from 15 μL additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 495.48, ALogP 3.45, HBA 8, HBD 4, PSA 116.47, QED 0.35
Compound (SMILES): C[C@H]1CC(=O)Nc2cccc(C(=O)Nc3ccccc3)c2O1
InChIKey: BJIRPCHZVXAWDW-NSHDSACASA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: BRD4 AlphaLisa Binding Assay: His/Flag epitope tagged BRD4 BD142-168 was cloned, expressed, and purified to. BRD4 binding and inhibition was assessed by monitoring the engagement of biotinylated H4-tetraacetyl peptide (New England Peptide, NEP2069-1/13) with the target using the AlphaLisa technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate BRD4(BD1) (30 nM final) was combined with peptide (200 nM final) in 40 mM HEPES (pH 7.0), 40 mM NaCl, 1 mM DTT, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 1.2% DMSO) or compound dilution series in DMSO. After 20 minutes incubation at room temperature Alpha streptavidin donor beads and AlphaLisa anti-Flag acceptor beads were added to a final concentration of 10 ug/mL each. After three hours equilibration plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 22137 nM (pChEMBL 4.659999847412109)
Descriptors: MW 296.33, ALogP 3.05, HBA 3, HBD 2, PSA 67.43, QED 0.89
Compound (SMILES): Cc1nc(-c2ccnn2C)c2c(n1)[C@H]1CCC[C@@H](C2)N1C(=O)c1cccc(Cl)c1Cl
InChIKey: QAKRFOLVXVFREV-SCLBCKFNSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q64663
Assay: Radioligand Binding: human or rat P2X7-1321N1 cells were collected and frozen @ −80° C. On the day of the experiment, cell membrane preparations were made according to standard published methods. The total assay volume was 100 μl: 10 μl compound (10×)+(b) 40 μl tracer (2.5×)+50 μl membrane (2×). The tracer used for the assay was tritiated A-804598. The compound can be prepared as described in the literature. (Donnelly-Roberts, D. Neuropharmacology 2008, 56 (1), 223-229.) Compounds, tracer and membranes were incubated for 1 hour @ 4° C. The assay was terminated by filtration (GF/B filters pre-soaked with 0.3% PEI) and washed with washing buffer (Tris-HCl 50 mM). The IC50 generated in the binding assay was corrected for tracer concentration and affinity of the tracer to derive at the affinity (Ki) of the test compounds. The data are presented in Table 2 under the headings: P2X7 human Ki (μM) and P2X7 rat Ki (μM). Data are analyzed and graphed on Graphpad Prism 5. For analysis, each concentration point is averaged from triplicate values and the averaged values are plotted on Graphpad Prism.
Measurement: IC50 = 1091 nM (pChEMBL 5.960000038146973)
Descriptors: MW 442.35, ALogP 4.78, HBA 5, HBD 0, PSA 63.91, QED 0.57
Compound (SMILES): COc1ccc(-c2cnc(N)c(-c3ccc(NC(=O)c4cn([C@@H](C)C5CCOCC5)cc(-c5ccc(C)cc5)c4=O)cc3)c2)cc1OC
InChIKey: YLSAFDQGYSUAFT-VWLOTQADSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: Cell-Free Mer Kinase Inhibitory Activity: A kinase dilution solution containing 20 ng/ml Mer (the 528th to 999th amino acids of the intracellular domain of human MER expressed as a fusion protein with glutathione transferase in a baculovirus expression system and purified by glutathione Sepharose chromatography and ion-exchange chromatography; Carna Biosciences, Inc., catalog No. 08-108) was prepared using a kinase reaction buffer solution (100 mM HEPES (pH 7.4), 0.003% Brij-35, 0.004% Tween-20, 1 mM DTT, and 10 mM MgCl2) and added at 19 μl/well to a 384-well plate.Next, each test compound was diluted with DMSO, and this dilution solution was added at 1 μl/well to the plate.After preincubation at room temperature for 20 minutes, a solution containing a substrate peptide (FL-Peptide 27 (5FAM-EFPIYDFLPAKKK-CONH2), Caliper Life Sciences, catalog No. 760424) and ATP at 5 mM was prepared and added at 5 μl/well to the plate to start kinase reaction. The plate was incubated at 28° C. for 45 minutes, and the reaction was terminated by the addition of a termination buffer solution (100 mM HEPES (pH 7.4), 0.015% Brij-35, 40 mM EDTA, and 0.1% Coating Reagent 3) at 40 μl/well.The substrate peptide and the phosphorylated peptide in the reaction mixture were separated and quantified using EZ Reader II (Caliper Life Sciences).The kinase reaction was evaluated on the basis of a product ratio (P/(P+S)) calculated from the peak height (S) of the substrate peptide and the peak height (P) of the phosphorylated peptide.The rate of inhibition (Inhibition) was determined according to the following expression (automatically calculated using the software of the EZ Reader II system).
Measurement: IC50 = 112 nM (pChEMBL 6.949999809265137)
Descriptors: MW 644.77, ALogP 7.39, HBA 8, HBD 2, PSA 117.7, QED 0.17
Compound (SMILES): CC(C)OC(=O)C(C)(C)C(O)(c1ccc(Nc2nn([C@@H](CC#N)C3CC3)c3cc[nH]c(=O)c23)cc1)C(F)(F)F
InChIKey: DZBUGZXBQABLFQ-SXMXNQBWSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Jak Biochemical HTRF Assay Protocol: The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer #PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at room temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading. After normalization to untreated controls, the percent inhibition of the HTRF signal at each compound concentration was calculated.
Measurement: IC50 = 10020 nM (pChEMBL 5)
Descriptors: MW 545.56, ALogP 5.06, HBA 8, HBD 3, PSA 133.03, QED 0.32
Compound (SMILES): CN[C@@H](C)C(=O)N[C@H]1CN(C(=O)CCCC(C)=O)c2ccccc2N(Cc2c(OC)ccc3cc(C(=O)OC)ccc23)C1=O
InChIKey: KEAUHXQFUGWZRT-LVXARBLLSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay for BIR2 and BIR3: Ten nanomolar of 6× Histidine-tagged BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(c-biotin)-OH 1:2 TFA, in the presence of 50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/mL bovine serum albumin (BSA). Following a 45 min. incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations. Percentage of inhibition at each concentration was determined to generate an IC50 value for each test compound.
Measurement: IC50 > 54800 nM (pChEMBL 4.261000156402588)
Descriptors: MW 602.69, ALogP 3.37, HBA 8, HBD 2, PSA 134.35, QED 0.32
Compound (SMILES): CS(=O)(=O)c1ccc(Cn2c(=O)c(O)c(C(=O)O)c3sccc32)cc1
InChIKey: FINSMKXMECPLRF-UHFFFAOYSA-N
Target: DNA excision repair protein ERCC-5 (SINGLE PROTEIN), Homo sapiens
UniProt: P28715
Assay: Enzymatic IC50 Assays: FEN1, EXO1 or XPG enzyme was incubated with compound or vehicle (DMSO) and the FAM-labeled DNA oligomer substrate in a microtiter plate. The stop buffer contains EDTA to stop the enzymatic reaction. The plate is read for fluorescence intensity. The high control (DMSO) with high fluorescence intensity represents no inhibition of enzymatic reaction while the low control (10 μM) with low fluorescence intensity represents full inhibition of enzymatic reaction.
Measurement: IC50 = 6125 nM (pChEMBL 5.210000038146973)
Descriptors: MW 379.42, ALogP 1.92, HBA 7, HBD 2, PSA 113.67, QED 0.72
Compound (SMILES): CC(=O)N1CCc2c(c(Nc3cc(C(=O)NC(C)C)cc(-c4cnn(C)c4)c3)nn2C)C1
InChIKey: SWFTUKKNWAHCOE-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: AlphaLisa Binding Assay: His/Flag epitope tagged BRD4 BD142-168 was cloned, expressed, and purified to. BRD4 binding and inhibition was assessed by monitoring the engagement of biotinylated H4-tetraacetyl peptide (New England Peptide, NEP2069-1/13) with the target using the AlphaLisa technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate BRD4(BD1) (30 nM final) was combined with peptide (200 nM final) in 40 mM HEPES (pH 7.0), 40 mM NaCl, 1 mM DTT, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 1.2% DMSO) or compound dilution series in DMSO. After 20 minutes incubation at room temperature Alpha streptavidin donor beads and AlphaLisa anti-Flag acceptor beads were added to a final concentration of 10 ug/mL each. After three hours equilibration plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 2930 nM (pChEMBL 5.53000020980835)
Descriptors: MW 435.53, ALogP 2.61, HBA 7, HBD 2, PSA 97.08, QED 0.64
Compound (SMILES): O=C(c1ccoc1)n1nc(-c2ccc[nH]c2=O)cc1NCc1ccc(Cl)s1
InChIKey: ZPZMUYKLUQEYBK-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: KLKB1 Activity Assay: Human KLKB1 protein was obtained from Enzyme Research Labs. The chromogenic substrate S-2302 was obtained from DiaPharma. KLKB1 was assayed in buffer containing 0.05 M Tris (pH 7.4), 0.01 M NaCl and 0.2% w/v PEG-8000. The final concentration of enzyme used was 3 nM KLKB1. The final concentration of substrate used was 250 μM S-2302 for KLKB1. All assays were performed in 96-well microtiter plates at room temperature (RT). The enzyme and inhibitor were pre-incubated for 10 minutes then substrate was added and read at 405 nm in a SpectraMax Plus Spectrophotometer (Molecular Devices). Inhibitor IC50 values were determined by adding test compound as ten point, three-fold serial dilutions in buffer solution, as known in the art. The plate was read at 10 minutes after substrate addition.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 400.85, ALogP 3.85, HBA 7, HBD 2, PSA 92.92, QED 0.53
Compound (SMILES): Cc1ncc(CC(=O)N[C@H]2CC[C@H](CCN3CCc4sc(OCC(F)F)nc4C3)CC2)s1
InChIKey: LKZZDSXSPJYUJI-WKILWMFISA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Binding Assay: 225 nL of the solutions of the non-specific ligand or the compounds of the present invention at each concentration (in case of vehicle, final concentration 0.3% DMSO) were added in each well of a 384-well white/clear bottom microplate (3706, Corning). Jump-In HEK Cell membranes (final reaction amount: 4 ug/well), SPA beads (final reaction amount: 0.2 mg/well) and Tris-HCl buffer were mixed and the mixed solution was left still for more than 60 min at 4° C. Then, 50 uL of the mixture was added to each well of the plate. In addition, 25 uL of 6 nM [3H]-Metylspiperone (final concentration: 2 nM) was added to each well. The plate was sealed by putting TopSeal-A 96/384 well (6050185, PerkinElmer) on the top of the plate, mixed using stirring deaerator (Welltornado, FK-62, Sakaki) and incubated for 120 min at 25° C. After incubation, the radioactivity of [3H]-Metylspiperone which was binded to D3 receptor was determined by liquid scintillation counter (1450 Microbeta, PerkinElmer) in each well. Non-specific binding was calculated based on the radioactivity of [3H]-Metylspiperone in the presence of 10 uM non-labeled Butaclamol. The total binding was calculated based on the radioactivity of [3H]-Metylspiperone in the absence of the compounds of the present invention (Vehicle). The Ki values were calculated from dose-response curves.
Measurement: Ki = 0.82 nM (pChEMBL 9.09000015258789)
Descriptors: MW 484.64, ALogP 4.22, HBA 7, HBD 1, PSA 67.35, QED 0.58
Compound (SMILES): O=C(NCc1cc2c(Cl)c[nH]c2cc1F)c1ccnc(Cc2ccc3ncc(C4CC4)cc3c2)c1
InChIKey: PUSXIKSJCXOULP-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: In Vitro Enzyme Inhibition: A 10 mM solution of the test compound was made in DMSO. This solution was serially diluted 1:5 in DMSO to yield 2000, 400, 80, 16, 3.2, 0.64, 0.128, 0.0256 and 0.00512 μM compound test solutions. A control tube containing only DMSO is included. 16 μL of each compound test solution was combined with 384 μL of assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.01% Triton X-100) to yield a 4× test compound buffer stock.Separately, a 40 nM solution of human Plasma Kallikrein (Abcam) and a 93.6 μM solution Pro-Phe-Arg-AMC (Bachem) were made using assay buffer. These solutions are hereby referred to as 4×hPK and 2×PFR-AMC, respectively.60 μL of each 4× test compound buffer stock was combined with 60 μL of 4×hPK to yield 120 μL of 2× test compound buffer stock/2×hPK. 50 μL was removed from this mixture and placed into duplicate wells on a Microfluor 1Black U-bottom microtiter plate (Thermo Scientific). This plate was incubated for 5 minutes at 37° C. To each well, 50 L of pre-warmed 2×PFR-AMC was added to start the enzymatic reaction. Cleavage of PFR-AMC was monitored in a Biotek Synergy H4 reader set at 37° C. Readings are taken every 43 seconds for 1 hour. The highest mean velocity over 20 reads (15 minutes) is used to calculate the IC50. The IC50 is calculated using the Gen5 (Biotek Instruments).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 484.96, ALogP 6.3, HBA 3, HBD 2, PSA 70.67, QED 0.3
Compound (SMILES): CNC1=C(C(=N)Nc2ccnc(-c3cnn(C)c3)c2)CN(C(C)=O)CC1
InChIKey: UEVONUKFMUKGKR-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: AlphaLisa Binding Assay: His/Flag epitope tagged BRD4 BD142-168 was cloned, expressed, and purified to. BRD4 binding and inhibition was assessed by monitoring the engagement of biotinylated H4-tetraacetyl peptide (New England Peptide, NEP2069-1/13) with the target using the AlphaLisa technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate BRD4(BD1) (30 nM final) was combined with peptide (200 nM final) in 40 mM HEPES (pH 7.0), 40 mM NaCl, 1 mM DTT, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 1.2% DMSO) or compound dilution series in DMSO. After 20 minutes incubation at room temperature Alpha streptavidin donor beads and AlphaLisa anti-Flag acceptor beads were added to a final concentration of 10 ug/mL each. After three hours equilibration plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 > 19740 nM (pChEMBL 4.704999923706055)
Descriptors: MW 353.43, ALogP 1.6, HBA 6, HBD 3, PSA 98.93, QED 0.57
Compound (SMILES): CNC(=O)c1cnc(OCc2cn(-c3ccc(F)c(C(F)(F)F)c3)nn2)nc1
InChIKey: OABQISRZJRIOGY-UHFFFAOYSA-N
Target: Retinoic acid receptor RXR-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P19793
Assay: NR2B standard assay: NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 149 mM NaCl, 4 mM KCl, 2 mM CaCl2, and 1.5 mM MgCl2, 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (149 mM NaCl (standard assay) or 150 mM (HTS assay), 4 mM KCl (standard assay) or 3 mM (HTS assay), 2 mM CaCl2, 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to ¿EC80 (standard assay) or EC40 (HTS assay) is used to maximize the assay's signal window or the ability to detect NMDA receptor antagonists and negative allosteric modulators, respectively. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 218 nM (pChEMBL 6.659999847412109)
Descriptors: MW 396.3, ALogP 2.15, HBA 7, HBD 1, PSA 94.82, QED 0.66
Compound (SMILES): Cc1cc(Nc2nn(C3(CC#N)CCN(C(=O)OC(C)(C)C)CC3)c3cc[nH]c(=O)c23)ccc1C(=O)N1CCSCC1
InChIKey: LHZRALJRBZXNPX-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biochemical HTRF Assay : The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer #PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at room temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading.
Measurement: IC50 = 0.32 nM (pChEMBL 9.489999771118164)
Descriptors: MW 591.74, ALogP 4.61, HBA 9, HBD 2, PSA 136.35, QED 0.44
Compound (SMILES): CCCN1Cc2[nH]c(-c3n[nH]c4cc(-c5cc(F)c(O)cc5CC)ccc34)nc2CC1C(=O)N1CC(N2CCCCC2)C1
InChIKey: XJYIAUXZPZHRGV-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Biochemical JAK Kinase Assays: 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 1 h. 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 1 h 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.
Measurement: Ki = 0.2 nM (pChEMBL 9.699999809265137)
Descriptors: MW 585.73, ALogP 4.86, HBA 6, HBD 3, PSA 104.38, QED 0.29