lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1nc(F)ccc1[C@H](Nc1cc(Cl)c2ncc(C#N)c(NCC(C)(C)C)c2c1)C1=CN(C2(C(F)(F)F)CC2)NN1
InChIKey: BVDKRMLBBATPQF-VWLOTQADSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: High Throughput Biochemical Assay: EGFR activity was measured using KinEASE (Cisbio), a time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay. In this assay, EGFR-catalyzes the phosphorylation of a universal Tyrosine kinase peptide substrate labeled with XL665. Europium conjugated phosphor-tyrosine specific antibody binds the resulting phosphorylated peptide. Formation of phosphorylated peptide is quantified by TR-FRET with Europium as the donor and XL665 the acceptor. The assay was performed in two main steps. The first step is the kinase reaction step and the second step is the detection step with TR-FRET reagents. In brief, test compounds 1:3 serially diluted in DMSO were delivered into Corning white, low volume, non-binding 384 well plates using the Echo 550 acoustic liquid dispenser (Labcyte®). EGFR enzyme (Human EGFR, cytoplasmic domain [669-1210] from Carna Biosciences Cat. No. 08-115) and substrates TK substrate-biotin (included in Cisbio HTRF KinEASE-TK kit Cat. No. 62TK0PEJ) were dispensed into assay plates using a Multi-Flo (Bio-Tek Instruments). The standard 10 μL reaction mixture contained 6 μM ATP (1×Km) or 12 μM ATP (2×Km), 1 μM biotinylated peptide, 0.3 nM EGFR (for 1×Km ATP) or 0.1 nM EGFR (for 2×Km ATP) in reaction buffer (10 mM MOPs, pH 7.0, 1.5% Glycerol, 0.5 mg/ml BSA, 10 mM Mg-Acetate, 1 mM DTT, 0.025% NP-40). After 60 min of incubation at room temperature, 10 μL of Stop and Detect Solution (1:400 Europium Cryptate labeled anti-phosphorylated peptide antibody solution and 125 nM strepavidin-XL665 Tracer in a 50 mM Hepes pH 7.0 detection buffer containing sufficient EDTA) was added. The plate was then further incubated for over 60 minutes at room temperature and read using an Envision 2103 Multilabeled reader (PerkinElmer) with excitation/emission/FRET emission at 340 nm/615 nm/665 nm, respectively.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 589.04, ALogP 6.47, HBA 8, HBD 4, PSA 100.93, QED 0.18
Compound (SMILES): Cc1cc(N)ncc1-c1ccc(CNc2ncc(C#N)cc2C(=O)N[C@@H](C)c2ccc(F)cc2)cc1
InChIKey: VMSDQDFRFAMPGR-SFHVURJKSA-N
Target: 3-phosphoinositide-dependent protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O15530
Assay: PDK1 kinase assay: PDK1 (amino acids 51-359) and AKT2 (amino acids 140-467 fused to PIFtide, amino acids EEQEMFRDFDYIADW) were expressed as N-terminally tagged GST fusion proteins in insect cells and purified to homogeneity. Enzyme activity was determined in a coupled PDK1/AKT/FAM-crosstide assay and phosphorylation of FAM-crosstide was determined by standard IMAP protocol (Molecular Devices). For inhibition studies, compounds were titrated 4-fold in DMSO and diluted 40-fold into assay buffer (10 mM Tris HCl pH7.2; 10 mM MgCl2; 0.01% Triton X-100; 1 mM DTT) containing PDK1, AKT2, and FAM-crosstide (final concentrations: 0.75 nM PDK1, 10 nM unphosphorylated AKT2, and 100 nM crosstide substrate). The kinase reaction was initiated by adding ATP to a final concentration of 40 μM of PDK1 and incubated at 25° C. for 30 min. To detect assay product, the kinase reaction was combined with Progressive Binding Solution (1:600 Progressive Binding Reagent, 50% Buffer A, 50% Buffer B, Molecular Devices) in a 1:3 ratio. The mixture was incubated for 2 hours at 25° C. and the plate was scanned on an Analyst AD with excitation at 485 nm and emission at 530 nm. The fluorescence polarization value P is defined by the equation below. The value mP is generated by multiplying the P value for each reaction well by a factor of 1000. The value ΔmP for each well is the mP value for that well minus the mP value for the average negative control. P=(Fpar−Fperp)/(Fpar+Fperp)  Eq.:Where par is fluorescence intensity parallel to the excitation plane; and perp is fluorescence intensity perpendicular to the excitation plane. ΔmP values were plotted as a function of compound concentration and the data were analyzed with a 4-parameter fit using GraphPad Prism software.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 480.55, ALogP 5.15, HBA 6, HBD 3, PSA 116.72, QED 0.34
Compound (SMILES): N#C/C(=C\c1cccc(-n2cc(-c3ccc(Oc4ccccc4)cc3)c3c(N)ncnc32)c1)C(=O)NCCO
InChIKey: VVDLREIJMXMLEQ-PXLXIMEGSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Assay: Btk (human, full length) was purchased (Invitrogen, catalog number PV3363) and used as specified in the product literature. Btk (2 nM final concentration) was pre-incubated with inhibitors (six or ten concentrations, in duplicate) for 30 minutes at room temperature. Kinase reactions were initiated by the addition of 0.16 μCi/μL of γ-32P-ATP (6000 Ci/mmol, NEN) and 0.2 mg/mL substrate (poly[Glu:Tyr], 4:1 Glu:Tyr) and incubated for 30 minutes at room temperature. Kinase activity was determined by spotting 6 μL of each reaction onto sheets of phosphocellulose. Each blot was washed once with 1% AcOH solution, twice with 0.1% H3PO4 solution, and once with MeOH (5-10 minutes per wash). Dried blots were exposed for 30 minutes to a storage phosphor screen and scanned by a Typhoon imager (GE Life Sciences).
Measurement: IC50 = 240 nM (pChEMBL 6.619999885559082)
Descriptors: MW 516.56, ALogP 4.48, HBA 8, HBD 3, PSA 139.08, QED 0.2
Compound (SMILES): Fc1ccc(OC(F)F)c(-c2ccnc3[nH]c(C4=CCNCC4)cc23)c1
InChIKey: KLHIKJKCGWAAII-UHFFFAOYSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P50750
Assay: LANCE ULight TR-FRET Kinase Assay: CDK9 enzyme activities were measured using LANCE ULight TR-FRET kinase assay reagents (PerkinElmer, Waltham, Mass.). Compounds were directly added in 100% DMSO to white low volume assay plates (Perkin Elmer Proxiplate 6008289) using a Labcyte Echo acoustic dispenser. Assay reagents in serine/threonine kinase assay buffer containing 20 mM HEPES, 10 mM MgCl2, 100 mM Na3VO4, and 0.0075% Triton X-100. were added for final reaction mixture concentrations of 1000 μM ATP, 100 nM U-light MBP peptide (Perkin Elmer TRF0109M) and reaction initiated with 4 nM CDK9/Cyclin T1 (Carna Biosciences 04-110). The kinase reaction was carried out for 30 minutes before addition of stopping buffer to a final of 20 mM EDTA and 0.5 nM of LANCE Ultra Europium anti-phospho-MBP antibody (PerkinElmer TRF0201M) in LANCE detection buffer (PerkinElmer CR97-100). The reaction was equilibrated for 1 hour and the signal read in the Perkin Elmer Envision in TR-FRET mode (excitation at 320 nm and emission at 615/665 nm).
Measurement: IC50 = 68 nM (pChEMBL 7.170000076293945)
Descriptors: MW 359.35, ALogP 4.35, HBA 3, HBD 2, PSA 49.94, QED 0.73
Compound (SMILES): CCOc1ccc(S(=O)(=O)N2C[C@@H](C)[C@@H](n3nc(CC(=O)O)c4ccccc43)C2)c(Br)c1
InChIKey: MTWXTPGCGMLSSX-VLIAUNLRSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Receptor Binding Inhibition Assay: Cell membrane was prepared from CRTH2-expressing K562 cell. The cell membrane (20 μg/well) was mixed with reaction buffer (50 mM Tris/HCl, pH 7.4, 10 mM MgCl2). Then, [3H]-PGD2 was added to the mixture and incubated for 60 min at room temperature. The concentration of [3H]-PGD2 was determined as equal to Kd calculated from Scatchard plot. Then, the mixture was filtrated with glass paper immediately. The filter paper was washed several times and radio activity on the filter was measured. Specific binding was defined as difference between total and non-specific binding (radio activity with 10 μM PGD2). Ki values for each compound were calculated from displacement curve.
Measurement: Ki < 100 nM (pChEMBL 7)
Descriptors: MW 522.42, ALogP 3.71, HBA 6, HBD 1, PSA 101.73, QED 0.51
Compound (SMILES): CC(C)(CCCS(=O)(=O)c1cccc(COc2ccc(-c3ccn[nH]3)nc2)c1)C(=O)O
InChIKey: JNDHYNIQOJQCTJ-UHFFFAOYSA-N
Target: Cytochrome P450 4F2 (SINGLE PROTEIN), Homo sapiens
UniProt: P78329
Assay: Inhibition Test for Each Compound of the Present Invention Against 20-HETE Producing Enzymes (CYP4F2 and CYP4A11): In the CYP4F2 inhibition test, the reaction solution containing each compound [final concentration of 50 mM, KPO4 (pH 7.4), 2.5 μM luciferine derivative, and 1 mM NADPH] was added to an Escherichia coli membrane fraction (100 μg/mL protein) in which human CYP4F2 had been expressed. In the CYP4A11 inhibition test, the reaction solution containing each compound [final concentration of 100 mM, Tris-HCl (pH 7.5), 60 μM luciferine derivative, 1.3 mM NADP+, 3.3 mM Glucose 6-Phosphate, 3.3 mM MgCl2, and 0.4 U/mL Glucose 6-Phosphate dehydrogenase] was added to an Escherichia coli membrane fraction (100 μg/mL protein) in which human CYP4A11 had been expressed. Following this, the membrane fraction was left to stand at room temperature for 60 minutes to perform an enzymatic reaction. After the reaction, a luciferine detection reagent was added, and the luminescence value was measured using a plate reader. By using that value, the percent inhibition of 20-HETE producing enzyme (%) was calculated according to the equation described below, and the 50% inhibitory concentration (IC50 value) for each compound was calculated.
Measurement: IC50 = 32 nM (pChEMBL 7.5)
Descriptors: MW 443.53, ALogP 3.72, HBA 6, HBD 2, PSA 122.24, QED 0.49
Compound (SMILES): N=C(CCCCC(=N)SC(=N)NC(=O)Cc1cccc(-c2cccc(F)c2)c1)SC(=N)NC(=O)Cc1cccc(-c2cccc(F)c2)c1
InChIKey: QXDQMSONNZXCTG-UHFFFAOYSA-N
Target: Glutaminase kidney isoform, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: O94925
Assay: Recombinant Enzyme Assay: No preinc (Column 4).
Measurement: IC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 684.84, ALogP 8.17, HBA 8, HBD 6, PSA 153.6, QED 0.05
Compound (SMILES): O=C(Nc1cc2cc(-c3cncc(OC4CNC4)n3)ccc2cn1)c1ccc(F)cc1
InChIKey: IYSRVCXRVJFAPP-UHFFFAOYSA-N
Target: Dual specificity tyrosine-phosphorylation-regulated kinase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: Q13627
Assay: Kinase Assay: The DYRK1A kinase assay was run using the Ser/Thr 18 peptide Z-lyte assay kit according to manufacturer's instructions (Life Technologiesa Division of Thermo-Fisher). This is a non-radioactive assay using fluorescence resonance energy transfer (FRET) between coumarin and fluorescein to detect kinase activity which is represented as a ratio of coumarin emission/fluorescein emission.Briefly, recombinant DYRK1A kinase, ATP and Ser/Thr peptide 18 were prepared in 1× Kinase buffer to final concentrations of 0.19 μg/mL, 30 μM, and 4 μM respectively. The mixture was allowed to incubate with the representative compounds for one hour at room temperature. All reactions were performed in duplicate. Unphosphorylated ("0% Control") and phosphorylated ("100% control") forms of Ser/Thr 18 served as control reactions. Additionally, an 11-point dose-response curve of Staurosporine (luM top) was run to serve as a positive compound control.
Measurement: EC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 415.43, ALogP 3.43, HBA 6, HBD 2, PSA 89.03, QED 0.52
Compound (SMILES): COC(=O)NCc1nc(NC(=O)N[C@H](C)c2ccccc2)cc2cnn(-c3ccc(F)cc3)c12
InChIKey: LFWBJFFMTVJBLC-OAHLLOKOSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 μM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 μL) was dispensed, followed by the addition of 15 μL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 μL kinase buffer containing 2.45 μM ERK2 IMAP substrate peptides and 75 μM ATP. The final reaction in each well of 25 μL consists of 0.5 nM hERK2, 900 nM unlabeled peptide, 80 nM labeled-peptide, and 30 μM ATP. Phosphorylation reactions were allowed to proceed for 60 minutes and were immediately quenched by the addition of 60 μL IMAP detection beads (1:1000 dilutions) in IMAP binding buffer (Molecular Devices) with 24 mM NaCl. Plates were read on EnVision reader after 60 minutes binding equilibration using Fluorescence Polarization protocol (Perkin Elmer).
Measurement: IC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 462.49, ALogP 4.3, HBA 6, HBD 3, PSA 110.17, QED 0.4
Compound (SMILES): N#Cc1ccccc1C1CCN(c2cnn3c(CC4CC4)nnc3c2Cl)CC1
InChIKey: IMPDESNOTYLKHN-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: MGluR2 cAMP Assay: Clonally expressed human mGluR2 in HEK293 background was glutamate starved overnight in media without glutamine (GIBCO MEM 12360), containing 10% FBS, 1% penicillin streptomycin. Compounds were added to white, standard volume 384 non-binding surface plates (Corning 3574). Cells were resuspended in stimulation buffer consisting of Hanks Balanced Salt Solution (14175-095) pH 7.0, 20 mM HEPES, 2.0 mM CaCl2, 5.0 mM MgCl2, and 1 mM IBMX (Sigma 15879), 1 Î¼M forskolin, and 1 Î¼M LY-341495, for 30 minutes. Buffer without forskolin was used as a negative control. Solutions of D2 and cryptate detection reagents from the CISBIO dynamic cAMP kit (62AM4PEJ) were diluted 1:40 in lysis buffer. Lysis buffer consisted of 50 mM Phosphate Buffer pH 7.0, 800 mM Potassium Fluoride, 0.2% BSA, and 1.0% Triton. Assay reaction was terminated by addition of detection reagents in lysis buffer. One hour later, plates were read on a PE Viewlux.
Measurement: EC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 392.89, ALogP 3.99, HBA 6, HBD 0, PSA 70.11, QED 0.67
Compound (SMILES): Cc1nc(CNC(C)C)ccc1-n1cc(-c2nc(N[C@H]3CCN(S(=O)(=O)c4cn(C)cn4)C[C@H]3C)ncc2C#N)cn1
InChIKey: ZJGYARBTFIIUBB-DVECYGJZSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24864
Assay: CDK2/Cyclin E1 HERE Enzyme Activity Assay: CDK2/Cyclin E1 enzyme activity assays utilize full-length human CDK2 co-expressed as N-terminal GST-tagged protein with FLAG-Cyclin E1 in a baculovirus expression system (Cama Product Number 04-165). Assays are conducted in white 384-well polystyrene plates in a final reaction volume of 8 μL. CDK2/Cyclin E1 (0.25 nM) is incubated with compounds (40 nL serially diluted in DMSO) in the presence of ATP (50 μM or 1 mM) and 50 nM ULight-labeled eIF4E-binding protein 1 (THR37/46) peptide (PerkinElmer) in assay buffer (containing 50 mM HEPES pH 7.5, 1 mM EGTA, 10 mM MgCl2, 2 mM DTT, 0.05 mg/ml BSA, and 0.01% Tween 20) for 60 minutes at room temperature. The reactions are stopped by the addition of EDTA and Europium-labeled anti-phospho-4E-BP1 antibody (PerkinElmer), for a final concentration of 15 mM and 1.5 nM, respectively. HTRF signals are read after 1 hour at room temperature on a PHERAstar FS plate reader (BMG Labtech). Data is analyzed with IDBS XLFit and GraphPad Prism 5.0 software using a three or four parameter dose response curve to determine IC50 for each compound.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 589.73, ALogP 2.65, HBA 12, HBD 2, PSA 159.54, QED 0.3
Compound (SMILES): Cc1ccc(NC(=O)c2cc(C3CC3)on2)cc1-c1cc(N2CCOCC2)nc(N2CC(O)C2)c1
InChIKey: XKGGRFGDCYJJJU-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 = 1.11 nM (pChEMBL 8.960000038146973)
Descriptors: MW 475.55, ALogP 3.19, HBA 8, HBD 2, PSA 103.96, QED 0.56
Compound (SMILES): COc1cn(Cc2cccc(F)c2)c(Nc2cc3c(cc2C)OCCO3)nc1=O
InChIKey: CAPBZKKMMMIJCT-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Evaluation of a Human P2X7 Receptor Inhibitory Activity: Stably expressing cell line (1321N1 cell transfected with the human P2X7 receptor gene (GenBank accession number NM_002562.5 including T606C and G952A SNP)) was used. The cells were seeded in a 384-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (10% fetal bovine serum, 25 mM HEPES, 1% penicillin and streptomycin in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. After replacing with 20 μL of the HBSS buffer (20 mM HEPES, 55.6 mM D-glucose, 1×HBSS(−), pH7.4-7.5), 15 μL of 17.3 μM Yo-Pro solution in the HBSS buffer was added. The plate was placed in high-throughput cellular screening system FLIPR TETRA (Molecullar Devices, LLC.) and 15 μL of 130 μM BzATP solution in the HBSS buffer was added. Measurement of fluorescence intensity by FLIPR TETRA was started. After eight minutes, 15 μL of DMSO solutions containing different concentrations of the compound of the present invention as prepared by dilution with the HBSS buffer were dispensed to each well through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 20 minutes. The maximum fluorescence intensity without the compound of the present invention is calculated as 0% inhibition and the maximum fluorescence intensity when the reference compound was added is calculated as 100% inhibition. Changing values of fluorescence intensity by the compound of the present invention were calculated by difference between maximum and minimum fluorescence intensity for 20 minutes. IC50 was calculated using logistic approximation.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 397.41, ALogP 3.26, HBA 7, HBD 1, PSA 74.61, QED 0.71
Compound (SMILES): Cc1cc(Nc2nn([C@@H]3CCCCC[C@H]3C#N)c3cc[nH]c(=O)c23)ccc1C(O)C(F)(F)F
InChIKey: ISRSDFMAQWVGFF-ZUIVQFGISA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: 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 = 380 nM (pChEMBL 6.420000076293945)
Descriptors: MW 459.47, ALogP 5.02, HBA 6, HBD 3, PSA 106.73, QED 0.48
Compound (SMILES): CC(C)NC(=O)Cn1nc(-c2ccc(CCN3C4CCC3COC4)cn2)nc1-c1ccc(F)c(Cl)c1
InChIKey: CYWFUIGIZZSEKZ-UHFFFAOYSA-N
Target: Vasopressin V1b receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P47901
Assay: Binding Test for V1b Receptor: Human V1b receptor was transiently expressed in 293FT cells (Invitrogen). The cells were collected and then homogenated in a 15 mmol/L tris-hydrochloric acid buffer (pH 7.4 and containing 2 mmol/L magnesium chloride, 0.3 mmol/L ethylenediaminetetracetic acid, and 1 mmol/L glycol ether diaminetetraacetic acid). The resulting homogenate was centrifuged at 50,000×g at 4° C. for 20 minutes. The precipitate was resuspended in a 75 mmol/L tris-hydrochloric acid buffer (pH 7.4 and containing 12.5 mmol/L magnesium chloride, 0.3 mmol/L ethylenediaminetetracetic acid, 1 mmol/L glycol ether diaminetetraacetic acid, and 250 mmol/L sucrose) to give a crude membrane preparation, which was stored at −80° C. until the binding test was initiated. In the binding test, the crude membrane preparation was diluted with a 50 mmol/L tris-hydrochloric acid buffer (pH 7.4 and containing 10 mmol/L magnesium chloride and 0.1% bovine serum albumin) and mixed with each test compound and [3H]AVP (final concentration: 0.4 to 1 nmol/L), followed by incubation at room temperature for 60 minutes. The test compound was serially diluted with DMSO so that it would have final concentrations of 0.01 nmol/L to 1 μmol/L at the time of mixing. After the incubation, the mixture was suction filtered through a GF/C filter that was preliminarily impregnated with 0.3% polyethyleneimine. The GF/C filter was dried and after adding a scintillator, the residual radioactivity on the filter was measured using TopCount (PerkinElmer Inc.). The radioactivity in the presence of unlabeled AVP at 10 mmol/L was defined as 0%, and the radioactivity in the absence of unlabeled AVP was defined as 100%. A dose-response curve was plotted from radio activities in the presence of a test compound at various concentrations, and the 50% inhibitory concentration (IC50 value) of the test compound was calculated.
Measurement: IC50 = 2.8 nM (pChEMBL 8.550000190734863)
Descriptors: MW 513.02, ALogP 3.73, HBA 7, HBD 1, PSA 85.17, QED 0.49
Compound (SMILES): N#Cc1ccc2nc(-c3cnnc(C(F)F)c3)n(C3CC3)c2n1
InChIKey: CSBQTXOGAACCIS-UHFFFAOYSA-N
Target: Cytochrome P450 11B2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P19099
Assay: Metalloenzyme Activity: V79-4 cells expressing recombinant andrenodoxin and andrenodoxin reductase with either recombinant human CYP11B2 or CYP11B1 were prepared according to methods previously described (LaSala et al 2009 Anal Bioch 394:56-61). An enzyme enriched microsomal fraction was prepared from cellular lysates and subsequently used as the enzyme source for determining inhibitor IC50s. The substrate Km values were experimentally determined for 11-deoxycorticosterone (CYP11B2 substrate) and 11-deoxycortisol (CYP11B1 substrate). Enzyme assays for inhibitor screening employed CYP11B2 and CYP11B1 enzyme enriched microsomes and were run at the Km of the respective substrates. Products of the enzyme reactions, aldosterone for CYP11B2 or cortisol for CYP11B1, were measured by LC-MS. Assays were run under conditions of less than 20% substrate turnover. Inhibitor IC50s were generated by determining the product formation in the absence or presence of inhibitor at various concentrations. In the absence of the test compound, the product formed (Pt) in each data set was defined as 100% activity. In the absence of enzyme, the product formed (Pb) in each data set was defined as 0% activity. The percent activity in the presence of each inhibitor was calculated according to the following equation: % activity=(P−Pb)/(Pt−Pb), where P=the product formed in the presence of the inhibitor. The IC50 value was defined as the inhibitor concentration causing a 50% decrease in activity relative to the no inhibitor control reaction.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 312.28, ALogP 3.03, HBA 6, HBD 0, PSA 80.28, QED 0.74
Compound (SMILES): Cc1ccc(C(=O)N2C[C@H](n3ncc(C(C)(C)O)n3)CC[C@H]2C)c(-n2nccn2)n1
InChIKey: NKCXIYOQQWLTEM-HUUCEWRRSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P56718
Assay: FLIPR Ca2+ Flux Assay: For intracellular calcium measurements, Chinese hamster ovary (CHO) cells expressing the rat orexin-1 receptor or the human orexin-2 receptor, are grown in Iscove's modified DMEM containing 2 mM L-glutamine, 0.5 g/ml G418, 1% hypoxanthine-thymidine supplement, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat-inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into Becton-Dickinson black 384-well clear bottom sterile plates coated with poly-D-lysine. All reagents were from GIBCO-Invitrogen Corp. The seeded plates are incubated overnight at 37° C. and 5% CO2. Ala-6,12 human orexin-A as the agonist is prepared as a 1 mM stock solution in 1% bovine serum albumin (BSA) and diluted in assay buffer (HBSS containing 20 mM HEPES, 0.1% BSA and 2.5 mM probenecid, pH7.4) for use in the assay at a final concentration of 70 pM. Test compounds are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates, first in DMSO, then assay buffer. On the day of the assay, cells are washed 3 times with 100 μl assay buffer and then incubated for 60 min (37° C., 5% CO2) in 60 μl assay buffer containing 1 μM Fluo-4AM ester, 0.02% pluronic acid, and 1% BSA. The dye loading solution is then aspirated and cells are washed 3 times with 100 μl assay buffer. 30 μl of that same buffer is left in each well. Within the Fluorescent Imaging Plate Reader (FLIPR, Molecular Devices), test compounds are added to the plate in a volume of 25 μl, incubated for 5 min and finally 25 μl of agonist is added. Fluorescence is measured for each well at 1 second intervals for 5 minutes and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 70 pM Ala-6,12 orexin-A with buffer in place of antagonist.
Measurement: IC50 = 222 nM (pChEMBL 6.650000095367432)
Descriptors: MW 410.48, ALogP 1.66, HBA 9, HBD 1, PSA 114.85, QED 0.7
Compound (SMILES): CCC1(N)CN(c2nc(N3CC4CCC(C3)N4)c3cnc(-c4cccc5cccc(Cl)c45)c(F)c3n2)C1
InChIKey: MGIBXLFAJPWNFZ-UHFFFAOYSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: KRas G12D Binding Assay: Table 2: The ability of a compound to bind to KRAS G12D was measured using a TR-FRET displacement assay. Biotinylated GDP-loaded recombinant human KRAS G12D (corresponding to amino acids 1:169, produced at Array BioPharma) was incubated with a custom-made Cy5 labelled tracer, europium labelled streptavidin and compound (2% DMSO final) in buffer (50 mM HEPES [pH 7.5], 5mM MgCl2, 0.005% Tween-20 & 1 mM DTT). After a 60 minute incubation at 22° C., the reaction was measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) calculated using a ratiometric emission factor. 100 POC is determined using no test compound and 0 POC is determined using a concentration of control compound that completely inhibits binding of the tracer to KRAS. The POC values were fit to a 4-parameter logistic curve and the IC50 value was determined as the concentration where the curve crosses 50 POC.
Measurement: IC50 = 501 nM (pChEMBL 6.300000190734863)
Descriptors: MW 518.04, ALogP 4.51, HBA 7, HBD 2, PSA 83.2, QED 0.41
Compound (SMILES): COC[C@H]1CCCN1C(=O)C1CCCN(S(=O)(=O)c2ccc(C)c(-c3cnc4c(N)nc(C(F)(F)F)cn34)c2)C1
InChIKey: WECOPMIJGIGEPG-QRWMCTBCSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Scintillation Proximity Assay: PI3K-γ:[γ-33P]ATP (10 mCi/mL) and Wheat Germ Agglutinin (WGA) YSi SPA Scintillation Beads was purchased from Perkin-Elmer (Waltham, Mass.). Lipid kinase substrate, D-myo-Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)D (+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho linked (PIP2), CAS 204858-53-7, was purchased from Echelon Biosciences (Salt Lake City, Utah). PI3Kγ (p110γ) Recombinant Human Protein was purchased from Life technology (Grand Island, N.Y.). ATP, MgCl2, DTT, EDTA, MOPS and CHAPS were purchased from Sigma-Aldrich (St. Louis, Mo.).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).
Measurement: IC50 < 2000 nM (pChEMBL 5.698999881744385)
Descriptors: MW 580.63, ALogP 3.34, HBA 8, HBD 1, PSA 123.13, QED 0.47
Compound (SMILES): Cn1cc(-c2ncc(NC(=O)[C@@H](NCCc3ccc(C#N)cc3)c3ccccc3)cn2)cn1
InChIKey: SHAJSBACPAWTQT-QHCPKHFHSA-N
Target: Histone acetyltransferase p300 (SINGLE PROTEIN), Homo sapiens
UniProt: Q09472
Assay: Biochemical Assay: The full length p300 SPA assay was run following the same protocol as p300 HAT SPA assay, but used 6 nM purified full length p300 (purchased from Active Motif) in place of the purified p300-HAT domain.Select compounds were also evaluated in a H3K18Ac MSD cellular assay that measures the ability of compounds to inhibit the the acetylation of chromatin at H3K18, a process catalyzed by p300 and CBP. In a typical experiment the p300 HAT inhibitory activity inside cells of the compounds described herein was determined in accordance with the following experimental method. 20 k HCT-116 cells per well are plated in 75 μL RPMI+10% FBS media the night before treatment. Compounds plated in DMSO at 4× final concentration are resuspended in 30 μL RPMI+10% FBS, then 25 μL is combined with corresponding wells containing cells. Treated cells are incubated for 2 hr at 37° C., then lysed in 500 μL final volume and frozen at −80° C. MSD plates (Meso Scale Discovery) are coated overnight at 4° C. with 60 μL 1:500 α-total histone antibody (Millipore MAB3422) in PBS. Plates are then blocked with 5% BSA in TBST shaking at RT for 1 hr, washed, and 30 μL lysate added to each well for 2 hr shaking at RT. Plates are washed and 25 μL 1:216 α-H3K18ac antibody (CST 9675) in PBS added, then incubated for 1 hr shaking at RT. Plates are washed again, then 25 μL 1:1000 Sulfo-Tag goat α-rabbit antibody (Meso Scale Discovery R32Ab-1) in PBS is added for 1 hr shaking at RT. Plates are washed once more, then 150 μL 1× Read Buffer (MSD #R92TD-3) is added to all wells and read on MSD SECTOR Imager 2400 using the conventional read setup.
Measurement: IC50 > 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 437.51, ALogP 3.26, HBA 7, HBD 2, PSA 108.52, QED 0.44
Compound (SMILES): CCC(C)[C@@H](CC(=O)O)NC(=O)C(CC)(CC)Cc1ccc(C(=O)Oc2ccc(C(=N)N)cc2F)s1.Cl
InChIKey: AHVCLHIIDXARTR-SAQPSMIMSA-N
Target: Enteropeptidase (SINGLE PROTEIN), Homo sapiens
UniProt: P98073
Assay: Inhibitory Activity Assay: Using a 96 well plate (#3915, Costar), a test compound (25 μL) was mixed with 20 μM fluorescence enzyme substrate (Boc-Phe-Ser-Arg-AMC, 50 μL) mixed with 200 mM Tris-HCl buffer (pH 8.0), and human trypsin (Sigma, 25 μL) was added. Using a fluorescence plate reader fmax (Molecular Devices, Inc.), the reaction rate was measured from the time-course changes at excitation wavelength 355 nm and fluorescence wavelength 460 nm. The Ki value was calculated from the concentration of the test compound, reciprocal of reaction rate, and Km value of the enzyme substrate, and by using Dixon plot.
Measurement: Ki = 3.26 nM (pChEMBL 8.489999771118164)
Descriptors: MW 519.64, ALogP 4.74, HBA 6, HBD 4, PSA 142.57, QED 0.13
Compound (SMILES): Cc1ncc(COc2cc(F)cc(Oc3ccc(-n4nc(C5CCCC5)c5ncnc(N)c54)cc3)c2)cn1
InChIKey: PBBBXUSXYRAQLS-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Inhibition Assay : Fluorescence polarization-based kinase assays were performed in 384 well-plate format using histidine tagged recombinant human full-length Bruton Agammaglobulinemia Tyrosine Kinase (Btk) and a modified protocol of the KinEASE FP Fluorescein Green Assay supplied from Millipore. Kinase reaction were performed at room temperature for 60 minutes in presence of 250 μM substrate, 10 μM ATP and variable test article concentrations. The reaction was stopped with EDTA/kinease detection reagents and the polarization measured on a Tecan 500 instrument. From the dose-response curve obtained, the IC50 was calculated using Graph Pad Prisms® using a non linear fit curve. The Km for ATP on each enzyme was experimentally determined and the Ki values calculated using the Cheng-Prusoff equation (see: Cheng Y, Prusoff W H. (1973) Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 percent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 22 (23): 3099-108).
Measurement: Ki < 100 nM (pChEMBL 7)
Descriptors: MW 511.56, ALogP 5.66, HBA 9, HBD 1, PSA 113.86, QED 0.3
Compound (SMILES): C[C@@]1(c2cccc(NC(=O)c3ccc(Cl)cn3)n2)CO[C@@](C)(C(F)(F)F)C(N)=N1
InChIKey: CKRKVUFHQBIJHR-DLBZAZTESA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: Inhibition Assay: Recombinant BACE-1 (extracellular domain, expressed in baculovirus and purified using standard methods) at 0.1 to 10 nM concentrations is incubated with the test compound at various concentrations for 1 hour at room temperature in 10 to 100 mM acetate buffer, pH 4.5, containing 0.1% CHAPS. Synthetic fluorescence-quenched peptide substrate, derived from the sequence of APP and containing a suitable fluorophore-quencher pair, is added to a final concentration of 1 to 5 μM, and the increase in fluorescence is recorded at a suitable excitation/emission wavelength in a microplate spectro-fluorimeter for 5 to 30 minutes in 1-minute intervals. IC50 values are calculated from percentage of inhibition of BACE-1 activity as a function of the test compound concentration.
Measurement: IC50 = 31 nM (pChEMBL 7.510000228881836)
Descriptors: MW 427.81, ALogP 3.31, HBA 6, HBD 2, PSA 102.49, QED 0.78
Compound (SMILES): NC(=O)c1ccc(-c2cc(N3CCOCC3)nc3c(-c4ccn[nH]4)nccc23)cc1
InChIKey: WKFBCIAVQXWORS-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Time-Resolved Fluorescence Energy Transfer (TR-FRET Tracer A): To determine of binding activity of the test compounds, full-length human ATR protein was expressed and purified together with ATRIP as described above. Furthermore, a fluorescently labelled compound (tracer A) was used as a tracer molecule. Detection of the binding event of the tracer was achieved by time-resolved fluorescence energy transfer (TR-FRET). We used an anti-GST-Terbium antibody (CisBio) that binds to the GST-tag at the N-terminus of ATR-kinase. Excitation of Terbium with 337 nm light results in emission of fluorescent light with 545 nm. In case a tetrameric complex has formed (antiGST-Tb+GST-ATR+Strp2-ATRIP+tracer), part of the energy will be transferred from the Terbium to the fluorophore that itself emits light of 570 nm. Displacement of the fluorescent tracer by a test compound leads to a reduction of the TR-FRET-signal.For the assay 50 nl of a 100-fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384well microtiter plate (MTP, Greiner Bio-One, Frickenhausen, Germany). To prepare the ATR-working solution, ATR/ATRIP stock solution was diluted in assay buffer [50 mM HEPES (pH 7.0), 10 mM MgCl2, 1 mM DTT, 0.01% (w/v) Igepal, 0.01% (w/v) BSA] to 4.2 nM protein concentration (concentration may vary from lot to lot of protein preparation). AntiGST-Tb antibody was diluted to 4.2 nM. The ATR-working solution was incubated for 30 min at 22° C. prior to dispensing to pre-form the complex of antiGST-Tb+GST-ATR+ATRIP. Then, 3 μl of the ATR-working solution were added to the test compound and the mixture was incubated for 10 min at 22° C. to allow pre-binding of the test compounds to ATR/ATRIP. Then, 2 μl of a 100 nM solution of either tracer A or B in assay buffer were added to the ATR-working solution. The resulting mixture was incubated for 30 min at 22° C. The measurement of the TR-FRET signal was performed in a standard HTRF-compatible MTP reader instrument (e.g. BMG Pherastar) by recording the fluorescence emissions at 545 nm and 570 nm after excitation at 337-350 nm. The ratio between emission at 570 nm divided by emission at 545 nm was calculated to give the well ratio. The experimental data (well ratios) were normalised by the following way: positive control contained ATR-working solution plus either tracer A or B solution (=0% inhibition), the negative control contained all components except GST-ATR/ATRIP (=100% inhibition).
Measurement: IC50 = 76.8 nM (pChEMBL 7.119999885559082)
Descriptors: MW 400.44, ALogP 2.62, HBA 6, HBD 2, PSA 110.02, QED 0.54
Compound (SMILES): N#Cc1ccc(-c2nc(N3CCC4(CCNC4)CC3)cc(=O)n2-c2ccc(C3CC3)cc2)cc1F
InChIKey: XKUQXIITJZVRCO-UHFFFAOYSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: n Vitro Enzyme Inhibition Assay LSD-1: This assay determines the ability of a test compound to inhibit LSD1 demethylase activity. E. coli expressed full-length human LSD1 (Accession number 060341) was purchased from Active Motif (Cat#31334).The enzymatic assay of LSD1 activity is based on Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) detection. The inhibitory properties of compounds to LSD1 were determined in 384-well plate format under the following reaction conditions: 0.1-0.5 nM LSD1, 50 nM H3K4me1-biotin labeled peptide (Anaspec cat #64355), 2 μM FAD in assay buffer of 50 mM HEPES, pH7.3, 10 mM NaCl, 0.005% Brij35, 0.5 mM TCEP, 0.2 mg/ml BSA. Reaction product was determined quantitatively by TR-FRET after the addition of detection reagent Phycolink Streptavidin-allophycocyanin (Prozyme) and Europium-anti-unmodified histone H3 lysine 4 (H3K4) antibody (PerkinElmer) in the presence of LSD1 inhibitor such as 1.8 mM of Tranylcypromine hydrochloride (2-PCPA) in LANCE detection buffer (PerkinElmer) to final concentration of 12.5 nM and 0.25 nM respectively.The assay reaction was performed according to the following procedure: 2 μL of the mixture of 150 nM H3K4me1-biotin labeled peptide with 2 μL of 11-point serial diluted test compound in 3% DMSO were added to each well of plate, followed by the addition of 2 μL of 0.3 nM LSD1 and 6 μM of FAD to initiate the reaction. The reaction mixture was then incubated at room temperature for one hour, and terminated by the addition of 6 μL of 1.8 mM 2-PCPA in LANCE detection buffer containing 25 nM Phycolink Streptavidin-allophycocyanin and 0.5 nM Europium-anti-unmodified H3K4 antibody. Enzymatic reaction is terminated within 15 minutes if 0.5 LSD1 enzyme is used in the plate. Plates were read by EnVision Multilabel Reader in TR-FRET mode (excitation at 320 nm, emission at 615 nm and 665 nm) after 1 hour incubation at room temperature. A ratio was calculated (665/615) for each well and fitted to determine inhibition constant (IC50).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 469.56, ALogP 4.37, HBA 6, HBD 1, PSA 73.95, QED 0.61
Compound (SMILES): CCN(CC)C(=N)/C(=N\S(=O)(=O)c1ccc(Cl)cc1)N1CC(c2ccccc2)C(c2ccc(Cl)cc2)=N1
InChIKey: XZHISHCABYOAHC-CTYQQEKESA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Mus musculus
UniProt: P47746
Assay: Radioligand Displacement Assay: Measuring its tissue levels 1 hour after administration of 10 mg/kg to mice, plasma levels were comparable after oral or i.p. administration (indicating good oral bioavailability), and brain tissue level was <2% of plasma level, indicating low brain penetrance/peripheral selectivity. 1 h after oral administration of compound 2 at 10 mg/kg dose in mice, the metabolite generation in plasma was monitored by LC-MS/MS. As expected, this compound underwent in vivo metabolism to liberate the amidine moiety and a metabolite (structure 41V in the FIG. 1). Besides CB1R antagonism, both intact compound and its metabolically cleaved amidine moiety were able to inhibit iNOS activity about 48% and 37% at 1 μM concentration in lung homogenates from LPS-treated mouse (FIG. 4). Mice with diet-induced obesity (DIO) mice were orally treated for 14 days with the compound 2 (10 mg/kg/day).
Measurement: Ki = 49 nM (pChEMBL 7.309999942779541)
Descriptors: MW 556.52, ALogP 5.9, HBA 4, HBD 1, PSA 89.19, QED 0.3
Compound (SMILES): C=CC(=O)N1CCC2CN(c3c(F)cc(C(N)=O)c4[nH]c(C)c(C)c34)CC21
InChIKey: CDCZNLRXQCCGJO-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Btk Enzyme Assay: To V-bottom 384-well plates were added test compounds, human recombinant Btk (1 nM, Invitrogen Corporation), fluoresceinated peptide (1.5 μM), ATP (20 μM), and assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij 35 surfactant and 4 mM DTT in 1.6% DMSO), with a final volume of 30 μL. After incubating at room temperature for 60 min., the reaction was 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 control reactions with no enzyme (for 100% inhibition) and controls with no inhibitor (for 0% inhibition). Dose response curves were generated to determine the concentration required for inhibiting 50% of Btk activity (IC50). Compounds were dissolved at 10 mM in DMSO and evaluated at eleven concentrations.
Measurement: IC50 = 0.37 nM (pChEMBL 9.430000305175781)
Descriptors: MW 370.43, ALogP 2.25, HBA 3, HBD 2, PSA 82.43, QED 0.81
Compound (SMILES): COc1cc2c(cc1OCCCN1CCCC1)nc(-c1ccc(C)o1)c1c(=O)n(CC3CCN(C)CC3)[nH]c12
InChIKey: UTEBOEXJOVLTEJ-UHFFFAOYSA-N
Target: DNA (cytosine-5)-methyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P26358
Assay: DNMT1 Enzyme Activity Assay: The biochemical assay to measure DNMT1 enzyme activity relies on time-resolved fluorescence energy transfer (TR-FRET) between lumi4-Tb (donor) and d2 (acceptor) using the EPlgeneous methyltransferase assay (CisBio Cat#62SAHPEB). TR-FRET is observed when antibody specific to S-adenosylhomocysteine labeled with Lumi4-Tb is incubated with d2-labeled S-adenosylhomocysteine. TR-FRET signal is inversely proportional to the concentration of SAH, product of DNMT1 enzyme activity, in the sample. The human DNMT1 was obtained from Reaction Biology Corp. (Cat# DMT-21-124).Enzyme activity assay was carried out in a white 384-well plate in a final volume of 20 μl, as follow:    4 μl of vehicle or studied compound 2.5× concentrated prepared in assay buffer (50 mM Tris-HCl, 1 mM EDTA, 1 mM DTT, 0.1% Triton X-100, 5% glycerol pH 7.5). Final percentage of DMSO was 0.5%.    2 μl of 1 nM DNMT1 enzyme diluted in assay buffer. Final concentration was 20 nM.    Start the reaction by adding 4 μl of substrate mixture containing 1 μM S-adenosylmethionine and 1 μM poly-deoxy inosine poly-deoxy cytosine (pdI-pdC) DNA.    Reaction was carried out during 15 minutes at 37° C.    Enzyme activity was stopped by adding 2 μl of buffer one of the EPIgeneous methyltransferase assay.    After 10 minutes at room temperature, it was added 4 μl of antibody specific to S-adenosylhomocysteine labeled with Lumi4-Tb 50× diluted in buffer two of the EPIgeneous methyltransferase assay.    Add 4 μl of d2-labeled S-adenosylhomocysteine 31× diluted in buffer two of the EPIgeneous methyltransferase assay.    Read the plate after 1 hour of incubation at room temperature.For each well, fluorescence was measured at 620 nm and 665 nm. A ratio (665 nm/620 nm) was then calculated in order to minimize medium interferences. Positive control was obtained in the presence of the vehicle of the compounds. Negative control was obtained in the absence of G9a enzyme activity. Calculated IC50 values were determined using GraphPrism using 4-parameters inhibition curve.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 533.67, ALogP 4.66, HBA 8, HBD 1, PSA 88.76, QED 0.31
Compound (SMILES): OC[C@H]1O[C@@H](n2nncc2-c2ccccc2)C[C@@H](n2cc(-c3cc(F)c(F)c(F)c3)nn2)[C@H]1O
InChIKey: ZNAUVNSNQHODCQ-JWUVWSEFSA-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 = 902 nM (pChEMBL 6.039999961853027)
Descriptors: MW 472.43, ALogP 2.5, HBA 9, HBD 2, PSA 111.11, QED 0.43