lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1cccc(CN(Cc2ccc(C(=O)O)cc2)C(=O)c2ccc(Oc3ccccc3F)cc2)c1
InChIKey: BETITZCZDCWBIT-UHFFFAOYSA-N
Target: Lysophosphatidic acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92633
Assay: In Vitro LPA1 Receptor Assay: Engagement of the LPA1 receptor by its ligand, oleoyl-L-α-lysophosphatidic acid (LPA), leads to the release of intracellular stores of calcium into the cytoplasm mediated through a Gq-driven increase in inositol triphosphate (IP3) levels. Gq is a heterotrimeric G protein subunit that activates phospholipase C.The ability of test compounds to prevent LPA-driven intracellular release of stored calcium from RH7777 cells expressing human LPA1 receptors was assayed by stimulating the cells with LPA in the presence of various test compounds to measure the test compounds' antagonism, i.e. activity in vitro. Approximately 10,000 cells in normal culture medium (Dulbecco's Modified Eagle Media, 10% Foetal Calf Serum) were dispensed per assay well in a black clear bottom collagen coated 384 well plate (Beckton Dickinson). Cells were allowed to settle for thirty minutes at room temperature before being incubated overnight at 37° C., 5% CO2. Sixteen to twenty hours after dispensing, the cells were loaded with a calcium-sensitive fluorescent dye by replacing the culture medium with assay buffer (1× Hanks buffered saline, 25 mM HEPES, 0.1% w/v fatty acid free BSA (bovine serum albumin), pH7.4) containing 1.25 mM probenecid and 1× Calcium 5 Reagent (Molecular Devices). Cells were incubated at 37° C. for one hour to allow for dye uptake. To test for antagonist activity, test compounds at a final concentration range between 0.32 nM-10 μM (diluted in assay buffer) were added to the assay wells and allowed to incubate for twenty five minutes. After incubation with test compounds the assay plate was placed in a FLIPR Tetra system (Molecular Devices) and LPA (diluted in assay buffer) was added to give a final concentration equivalent to its determined EC80 against LPA1 receptor. Ligand-dependent changes in intracellular calcium levels were determined by measuring changes in fluorescence of the dye over 515-575 nM following excitation over 470-495 nM.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 485.51, ALogP 6.17, HBA 4, HBD 1, PSA 76.07, QED 0.31
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3)ncnc2cc1OCc1csc(COC(=O)c2ccccc2)n1
InChIKey: SROSEDFOBYVYCH-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human N-terminal GST His6-tagged thrombin cleavage site-fused EGFR (H672 to A1210 residues) expressed in baculovirus using poly-EY as substrate after 210 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 567.45, ALogP 7.08, HBA 9, HBD 1, PSA 95.46, QED 0.19
Compound (SMILES): CC(C(=O)Nc1ccc(-c2ncc[nH]2)cc1)n1ccc(-c2cc(Cl)ccc2C#N)cc1=O
InChIKey: AYSNUYNCWUEWBG-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Measurement of FXIa Inhibition: To determine the factor XIa inhibition of the substances according to the invention, a biochemical test system is used which utilizes the reaction of a peptidic factor XIa substrate to determine the enzymatic activity of human factor XIa. Here, factor XIa cleaves from the peptic factor XIa substrate the C-terminal aminomethylcoumarin (AMC), the fluorescence of which is measured. The determinations are carried out in microtitre plates.Test substances are dissolved in dimethyl sulphoxide and serially diluted in dimethyl sulphoxide (3000 μM to 0.0078 μM; resulting final concentrations in the test: 50 μM to 0.00013 μM). In each case 1 μl of the diluted substance solutions are placed into the wells of white microtitre plates from Greiner (384 wells). 20 μl of assay buffer (50 mM of Tris/HCl pH 7.4; 100 mM of sodium chloride solution; 5 mM of calcium chloride solution; 0.1% of bovine serum albumin) and 20 μl of factor XIa from Kordia (0.45 nM in assay buffer) are then added successively. After 15 min of incubation, the enzyme reaction is started by addition of 20 μl of the factor XIa substrate Boc-Glu(OBzl)-Ala-Arg-AMC dissolved in assay buffer (10 μM in assay buffer) from Bachem, the mixture is incubated at room temperature (22° C.) for 30 min and fluorescence is then measured (excitation: 360 nm, emission: 460 nm). The measured emissions of the test batches with test substance are compared to those of control batches without test substance (only dimethyl sulphoxide instead of test substance in dimethyl sulphoxide), and the IC50 values are calculated from the concentration/activity relationships.
Measurement: IC50 = 590 nM (pChEMBL 6.230000019073486)
Descriptors: MW 443.89, ALogP 4.63, HBA 5, HBD 2, PSA 103.57, QED 0.47
Compound (SMILES): CC(=O)N1CCc2c(c(Nc3cccc(C)c3)nn2C2CC2)C1
InChIKey: QESLMEPTLHITHH-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 = 13600 nM (pChEMBL 4.869999885559082)
Descriptors: MW 310.4, ALogP 3.17, HBA 4, HBD 1, PSA 50.16, QED 0.95
Compound (SMILES): COc1cc(OC)cc(N(CC(O)C(F)(F)F)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: INWHLZUNIJSPHA-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 5370.32 nM (pChEMBL 5.269999980926514)
Descriptors: MW 473.46, ALogP 4.11, HBA 8, HBD 1, PSA 85.53, QED 0.43
Compound (SMILES): CC(C)Oc1cccc(-c2c[nH]c3ccc(-c4nnc(N5CC[C@@H](N)C5)s4)cc23)n1
InChIKey: NLOZLPYLNHZXJD-OAHLLOKOSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Enzyme Assay: The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction.Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 uM. A reference compound was included on each assay plate in order to validate that plate; on
Measurement: IC50 = 1.321 nM (pChEMBL 8.880000114440918)
Descriptors: MW 420.54, ALogP 4.07, HBA 7, HBD 2, PSA 92.95, QED 0.51
Compound (SMILES): CCn1cc(-c2cn3ncc(C(N)=O)c(N[C@H]4CN(c5ccc(C#N)cn5)C[C@H]4C)c3n2)cn1
InChIKey: YWZJISIXPFORET-KDOFPFPSSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Kinase Assay: The assay reactions 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 7.4, 10 mM MgCl2, 25 mM beta-glycerol phosphate, 0.015% Brij35 and 4 mM DTT). The reaction was initiated by the combination of GST-JAK1 enzyme with substrates and test compounds. The reactions were incubated at room temperature for 3 hours and terminated by adding 60 μl of 35 mM EDTA to each sample. Each 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 with no enzyme control reactions for 100% inhibition, and vehicle-only treated reactions for 0% inhibition. The final concentration of reagents in the assays was: ATP, 100 μM; fluoresceinated peptide, 1.5 μM; GST-JAK1, 12.5 nM; and DMSO, 1.6%.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 456.51, ALogP 1.92, HBA 10, HBD 2, PSA 143.05, QED 0.45
Compound (SMILES): CCOc1ccc(CC2CC(C(=O)NO)C2)cc1-c1nc2c(CC)nn(C)c2c(=O)[nH]1
InChIKey: WFSWUBOROARLDR-UHFFFAOYSA-N
Target: Histone deacetylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92769
Assay: Inhibition of full length recombinant human C-terminal His-tagged HDAC2 expressed in baculovirus infected Sf9 cells using fluorogenic HDAC substrate after 30 mins by fluorescence analysis
Measurement: IC50 = 490 nM (pChEMBL 6.309999942779541)
Descriptors: MW 425.49, ALogP 2.36, HBA 7, HBD 3, PSA 122.13, QED 0.39
Compound (SMILES): C#CCN(c1cc(OC)cc(OC)c1)c1ccc2ncc(-c3cnn(C)c3)nc2c1
InChIKey: UEOZGCZDSDHALE-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 7244.36 nM (pChEMBL 5.139999866485596)
Descriptors: MW 399.45, ALogP 3.82, HBA 7, HBD 0, PSA 65.3, QED 0.46
Compound (SMILES): CC1c2nnn(-c3cncnc3)c2CCN1C(=O)c1cccc(C(F)(F)F)c1Cl
InChIKey: NBUGIOFQQAFLCM-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q64663
Assay: FLIPR Assay: 1321 N1 cells expressing the recombinant human, rat or mouse P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 ul volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250Ã¿ the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 uL of the compound into 300 uL of assay buffer. A further 3x dilution occurred when transferring 50 uL/well of the compound plate to 100 uL/well in the cell plate. Cells were incubateÂ  with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 uL/well of BzATP (final concentration is 250 uM BzATP (human and rat) or 600 (mouse)). The fluorescence change was measured 180 seconds after adding the agonist.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 422.8, ALogP 3.49, HBA 6, HBD 0, PSA 76.8, QED 0.63
Compound (SMILES): CCOc1ccc(C[C@@H](NC(=O)c2ccc(C)cc2)C(=O)N[C@@H](Cc2ccccc2)C(=O)NCc2ccc(CN)cc2)cc1
InChIKey: PQYXYUJVSKCKFL-JHOUSYSJSA-N
Target: Coagulation factor XII (SINGLE PROTEIN), Homo sapiens
UniProt: P00748
Assay: Enzyme Assay: Human serine protease enzymes plasmin, thrombin, trypsin, Factor Xa and Factor XIIa were assayed for enzymatic activity using an appropriate fluorogenic substrate. Protease activity was measured by monitoring the accumulation of liberated fluorescence from the substrate over 5 minutes. The linear rate of fluorescence increase per minute was expressed as percentage (%) activity. The Km for the cleavage of each substrate was determined by standard transformation of the Michaelis-Menten equation.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 592.74, ALogP 4.24, HBA 5, HBD 4, PSA 122.55, QED 0.17
Compound (SMILES): c1cc2c(ncn3nnc([C@H]4CC[C@H](CN5CCCC5)CC4)c23)[nH]1
InChIKey: KQBISFOOFCOEHG-HDJSIYSDSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 324.43, ALogP 2.98, HBA 5, HBD 1, PSA 62.11, QED 0.8
Compound (SMILES): C[C@H]1CC[C@H](N)CN1c1ccncc1Nc1ncc2ccc(-c3c(F)cccc3F)nn12
InChIKey: DFKOCMIZZATJNQ-GJZGRUSLSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Inhibition of recombinant Pim1 (unknown origin) by electrochemiluminescence assay
Measurement: IC50 = 0.037 nM (pChEMBL 10.430000305175781)
Descriptors: MW 435.48, ALogP 4.13, HBA 7, HBD 2, PSA 84.37, QED 0.5
Compound (SMILES): COc1ccc(N2CCOc3cc(S(=O)(=O)Nc4ncns4)ccc32)cc1
InChIKey: VEZJEWXCMWVSCZ-UHFFFAOYSA-N
Target: Sodium channel protein type 5 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q14524
Assay: In Vitro Assay: HEK 293 cells stably transfected with either Nav 1.7 or with Nav 1.5 were recorded in population patch-clamp mode with the IonWorks® Quattro automated electrophysiology system in accordance with the manufacturer's specifications (Molecular Devices, LLC, Sunnyvale, Calif.). Sodium channel currents were measured in response to a train of depolarizations that induced successively greater inactivation. Cells were held at −110 mV for three seconds (Nav 1.7) or half a second (Nav 1.5) from a holding voltage of −15 mV, then put through a series of 26 pulses of 150 msec duration to −20 mV at a frequency of 5 Hz. Cells were then left unclamped for a period of 3 to 8 minutes while a single concentration of test compound was added. Cells were then reclamped and put through the same voltage protocol. Current at the end of the 26th pulse to −20 mV was subtracted from the peak current evoked by the 26th pulse to −20 mV to correct for leak current.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 404.47, ALogP 2.88, HBA 8, HBD 1, PSA 93.65, QED 0.7
Compound (SMILES): CNC(=O)[C@H]1CC[C@H](NC(=O)c2cnc(-n3ncc4cc(C#N)cnc43)cc2NC(C)C)CC1
InChIKey: XVNVVUWOJIUHSB-SAABIXHNSA-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 L prepared from 15 L 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% Brij 35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction mixture 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 = 1.7 nM (pChEMBL 8.770000457763672)
Descriptors: MW 460.54, ALogP 2.54, HBA 8, HBD 3, PSA 137.62, QED 0.51
Compound (SMILES): [C-]#[N+][C@@H]1CCCN1C(=O)CNC(=O)c1ccnc2cc(C)ccc12
InChIKey: QCKHSJAFAMATFO-INIZCTEOSA-N
Target: Dipeptidyl peptidase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86TI2
Assay: Enzymatic Assay: Enzyme activities were determined kinetically in a final volume of 200 μl for 10 minutes at 37° C. by measuring the initial velocities of pNA release (405 nm) from the substrate using a Spectramax plus microtiterplate reader (Molecular devices). One unit of enzyme activity was defined as the amount of enzyme that catalyzes the release of 1 μmol pNA from the substrate per minute under assay conditions. The chromogenic substrate Gly-Pro-p-nitroanilide (100 μmol/l) was used at pH 8.3 for DPP IV, Lys-Ala-p-nitroanilide (1 mmol/l) at pH 5.5 for DPP II, Ala-Pro-p-nitroanilide (300 μmol/l) at pH 7.4 for DPP9 and Ala-Pro-p-nitroanilide (2 mmol/l) at pH 7.4 for FAP activity measurement. To evaluate the endopeptidase activity of FAP and the influence of inhibitors thereon, Z-Gly-Pro-AMC and Z-Gly-Pro-p-nitroanilide were used at a final concentration of 300 and 100 μmol/l, respectively. The substrate concentrations were chosen around the Km value obtained under the assay conditions used. Buffer compositions for the DPP assays were reported before in the purification articles−vide supra. The FAP assay buffer consisted of 50 mM Tris pH7.4 containing 100 mmol/l NaCl and 0.1 mg/ml bovine serum albumin. The PREP activity was measured as described by Brandt et al. using the chromogenic substrate Z-Gly-Pro-p-nitroanilide (0.25 mmol/l) at pH 7.5 in the presence of 10 mmol/l DTT. Test compounds were dissolved and diluted in DMSO (final concentration DMSO during assay 5% v/v) except for FAP where dilution of the inhibitor was done in water. Inhibitors are pre-incubated with the enzyme for 15 min at 37° C. before starting the assay by the addition of substrate. The concentration of enzyme and of inhibitor during the preincubation is double of the final concentration during activity measurement.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 322.37, ALogP 2.14, HBA 3, HBD 1, PSA 66.66, QED 0.88
Compound (SMILES): O=C(O)CNC(=O)C1=C(O)CCN(Cc2ccc(Oc3ccc(Cl)cc3)nc2)C1=O
InChIKey: HMMHKGLPKAQOOH-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZT9
Assay: Inhibition of human PHD2 using FITC-HIF1-alpha as substrate after 10 mins in presence of 2-oxoglutarate by fluorescence polarization assay
Measurement: IC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 431.83, ALogP 2.27, HBA 6, HBD 3, PSA 129.06, QED 0.57
Compound (SMILES): COc1cccc(OC[C@@H]2CN(CC(=O)N3CCc4ccccc43)CCO2)c1
InChIKey: KJWABLDVLWKPKR-FQEVSTJZSA-N
Target: 5-hydroxytryptamine receptor 2A (SINGLE PROTEIN), Mus musculus
UniProt: P35363
Assay: Radioligand Binding Assay: The detailed experimental protocols for the radioligand and functional receptor assays are available on the NIMH PDSP website at http://pdsp.med.unc.edu/UNC-CH %20Protocol %20Book.pdf. A. Serotonin receptors: 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3, 5-HT5A, 5-HT6 and 5-HT7. Assay Buffer: Standard Binding Buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM EDTA, pH 7.4) Membrane Fraction Source: Transiently or stably transfected cell lines (e.g., HEK293, COS, CHO, NIH3T3). Protocol adapted from Roth et al. (1986), J. Pharmacol. Exp. Ther., 238(2): 480-485; and Roth et al. (1994), J. Pharmacol. Exp. Ther., 268(3): 1403-1410. Adrenergic Receptors: alpha1A, alpha1B, alpha2A, alpha2B, alpha2C, beta1, beta2, beta3 Assay Buffers: For alpha1 receptors, alpha1 Binding Buffer (20 mM Tris-HCl, 145 mM NaCl, pH 7.4); for alpha2 receptors, alpha2 Binding Buffer (50 mM Tris-HCl, 5 mM MgCl2, pH 7.7); for beta receptors.
Measurement: Ki = 1945 nM (pChEMBL 5.710000038146973)
Descriptors: MW 382.46, ALogP 2.36, HBA 5, HBD 0, PSA 51.24, QED 0.77
Compound (SMILES): O=C(O)CC[C@H]1CCc2cc(-c3cc(OC4CCC4)cnc3F)ccc2O1
InChIKey: HGDFRNBNPYHIOH-MRXNPFEDSA-N
Target: Free fatty acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14842
Assay: Agonist activity at human GPR40 expressed in HEK293 cells assessed as increase in IP1 accumulation after 60 mins by HTRF assay
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 371.41, ALogP 4.38, HBA 4, HBD 1, PSA 68.65, QED 0.76
Compound (SMILES): O=S(=O)(Nc1nccs1)c1ccc2c(c1)OCCN2c1ccc(C(F)(F)F)c(Cl)c1
InChIKey: PGQPVUNTRHYZJA-UHFFFAOYSA-N
Target: Sodium channel protein type 5 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q14524
Assay: In Vitro Assay: HEK 293 cells stably transfected with either Nav 1.7 or with Nav 1.5 were recorded in population patch-clamp mode with the IonWorks® Quattro automated electrophysiology system in accordance with the manufacturer's specifications (Molecular Devices, LLC, Sunnyvale, Calif.). Sodium channel currents were measured in response to a train of depolarizations that induced successively greater inactivation. Cells were held at −110 mV for three seconds (Nav 1.7) or half a second (Nav 1.5) from a holding voltage of −15 mV, then put through a series of 26 pulses of 150 msec duration to −20 mV at a frequency of 5 Hz. Cells were then left unclamped for a period of 3 to 8 minutes while a single concentration of test compound was added. Cells were then reclamped and put through the same voltage protocol. Current at the end of the 26th pulse to −20 mV was subtracted from the peak current evoked by the 26th pulse to −20 mV to correct for leak current.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 475.9, ALogP 5.15, HBA 6, HBD 1, PSA 71.53, QED 0.56
Compound (SMILES): FC(F)(F)c1cc(CN2CCC(c3nnn4cnc5[nH]ccc5c34)CC2)cc(C(F)(F)F)c1
InChIKey: IUCHEHNJQRLWBF-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS. From the plot of logarithm of a compound concentration and inhibitory activity, the IC50 was calculated.
Measurement: IC50 = 110 nM (pChEMBL 6.960000038146973)
Descriptors: MW 468.4, ALogP 5.02, HBA 5, HBD 1, PSA 62.11, QED 0.43
Compound (SMILES): N#Cc1cc(CN2CCC(c3nnn4cnc5[nH]ccc5c34)CC2)ccc1C(F)(F)F
InChIKey: VGADABIBZTUEFS-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 425.42, ALogP 3.88, HBA 6, HBD 1, PSA 85.9, QED 0.54
Compound (SMILES): FC(F)(F)c1ccc(CNC[C@H]2CC[C@H](c3nnn4cnc5[nH]ccc5c34)CC2)cc1
InChIKey: YURQDIZYRJQUTR-KOMQPUFPSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 428.46, ALogP 4.69, HBA 5, HBD 2, PSA 70.9, QED 0.48
Compound (SMILES): O=C1Nc2ccccc2C12Oc1ccccc1C1CC(c3ccccc3)=NN12
InChIKey: PLTMMVSHYQKBKL-UHFFFAOYSA-N
Target: Aldehyde dehydrogenase, dimeric NADP-preferring (SINGLE PROTEIN), Homo sapiens
UniProt: P30838
Assay: Inhibiton Assay: IC50 values were determined for CB29 and its analogs using propionaldehyde as the substrate for ALDH1A1 and ALDH2 or benzaldehyde as the substrate for ALDH3A1. The assays were performed on a Beckman DU-640 spectrophotometer at various concentrations of inhibitors ranging from 50 nM to 250 μM following a 1 minute pre-incubation. There was no pre-incubation time-dependence to the inhibition. All reactions were initiated by the addition of the aldehyde substrate. The inhibition curves were fit to the Logistic four parameter IC50 equation using the SigmaPlot (v11, StatSys). We characterized the mode of inhibition using steady-state kinetics through co-variation of inhibitor and substrate concentrations. The steady state kinetic measurements were performed in 100 mM Na2HPO4 buffer, pH 7.5. The reaction mixture contained 10 nM ALDH3A1, varied benzaldehyde (50 μM-800 μM; fixed NADP+, 1.5 mM) and varied inhibitor concentrations. In all cases−including the control reactions lacking inhibitors, the final reaction mixture contained 2% (v/v) DMSO. The reaction was initiated by addition of substrate and the initial rate of product formation was determined on a Beckman DU-640.
Measurement: IC50 = 38500 nM (pChEMBL 4.420000076293945)
Descriptors: MW 367.41, ALogP 4.04, HBA 4, HBD 1, PSA 53.93, QED 0.71
Compound (SMILES): C#CCN1CCCC(CN(CCOC)S(=O)(=O)c2ccc3ccccc3c2)C1
InChIKey: VHUHFVUKZMULIP-UHFFFAOYSA-N
Target: Amine oxidase [flavin-containing] A (SINGLE PROTEIN), Homo sapiens
UniProt: P21397
Assay: Inhibition of human recombinant microsomal MAO-A expressed in baculovirus-infected insect cells using p-tyramine as substrate preincubated for 15 mins followed by substrate addition measured for 20 mins by amplex red assay
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 400.54, ALogP 2.82, HBA 4, HBD 0, PSA 49.85, QED 0.64
Compound (SMILES): CC(C)N(C(=O)N[C@H]1CC[C@H](CCN2CCN(c3nsc4ccccc34)CC2)CC1)C(C)C
InChIKey: XOLGUSIUXDCCCM-HZCBDIJESA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Displacement of [3H]spiperone from recombinant human D2L receptor expressed in CHO cell membranes after 60 mins by scintillation counting method
Measurement: Ki = 0.72 nM (pChEMBL 9.140000343322754)
Descriptors: MW 471.72, ALogP 5.2, HBA 5, HBD 1, PSA 51.71, QED 0.6
Compound (SMILES): CC[C@H](C)[C@H](NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@@H](N)CCCCN)[C@@H](C)O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)O)[C@@H](C)O)[C@@H](C)CC)[C@@H](C)O)C(C)C)[C@@H](C)O
InChIKey: UHZRTPBKWLFDGJ-IWTRNUCXSA-N
Target: G protein-coupled receptor kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P34947
Assay: Inhibition of recombinant human GRK5 expressed in Sf9 insect cells assessed as decrease in phosphorylation of urea-washed bovine rod outer segments in presence of Gbetagamma subunits and [gamma-32P]-ATP after 5 to 60 mins by SDS-PAGE analysis
Measurement: IC50 = 61300 nM (pChEMBL 4.210000038146973)
Descriptors: MW 2459.88
Compound (SMILES): CCOc1ccc(C[C@@H](NC(=O)c2ccc(OC)cc2)C(=O)N[C@@H](Cc2cccnc2)C(=O)NCc2ccc(CN)cc2)cc1
InChIKey: AJYZUIMBRBUFPQ-AJQTZOPKSA-N
Target: Kallikrein-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P06870
Assay: In Vitro Inhibition Assay: KLK1 inhibitory activity in vitro was determined using standard published methods (see e.g. Johansen et al., Int. J. Tiss. Reac. 1986, 8, 185; Shori et al., Biochem. Pharmacol., 1992, 43, 1209; Stürzebecher et al., Biol. Chem. Hoppe-Seyler, 1992, 373, 1025). Human KLK1 (Callbiochem) was incubated at 37° C. with the fluorogenic substrate H-DVal-Leu-Arg-AFC and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in optical absorbance at 410 nm and the IC50 value for the test compound was determined.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 609.73, ALogP 3.33, HBA 7, HBD 4, PSA 144.67, QED 0.16
Compound (SMILES): C[C@H](Nc1ncnc(N)c1C#Cc1ccc(F)cn1)c1nc2cccc(C(F)F)c2c(=O)n1-c1cccc(C#N)c1
InChIKey: DRBLPZRLJYMQDX-INIZCTEOSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2xKm ATP (100-300 uM), 10 uM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 1% (v/v) DMSO at the following concentrations for each isoform: PI3K alpha, beta, delta, and gamma at 50 picomolar (pM) and PI3Kgamma at 2 nanomolar (nM). After an assay reaction time of 30 minutes at 25° C., reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 us delay and 500 us read window).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 552.52, ALogP 4.67, HBA 9, HBD 2, PSA 135.4, QED 0.3
Compound (SMILES): Cc1cccnc1-n1c(Cn2nc(I)c3c(N)ncnc32)cc2cccc(C)c2c1=O
InChIKey: QMJYFHMVZOBHFR-UHFFFAOYSA-N
Target: PI3-kinase p110-alpha/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P42336
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110alpha/untagged recombinant full length human p85alpha expressed in baculovirus infected Sf21 cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 523.34, ALogP 3.38, HBA 8, HBD 1, PSA 104.51, QED 0.36
Compound (SMILES): COc1cn(C(C)C(=O)Nc2ccc(C(=O)O)cc2)c(=O)cc1-c1cc(Cl)ccc1OC(F)F
InChIKey: YRKPZRMKWAQTNS-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Measurement of FXIa Inhibition: To determine the factor XIa inhibition of the substances according to the invention, a biochemical test system is used which utilizes the reaction of a peptidic factor XIa substrate to determine the enzymatic activity of human factor XIa. Here, factor XIa cleaves from the peptic factor XIa substrate the C-terminal aminomethylcoumarin (AMC), the fluorescence of which is measured. The determinations are carried out in microtitre plates.Test substances are dissolved in dimethyl sulphoxide and serially diluted in dimethyl sulphoxide (3000 μM to 0.0078 μM; resulting final concentrations in the test: 50 μM to 0.00013 μM). In each case 1 μl of the diluted substance solutions are placed into the wells of white microtitre plates from Greiner (384 wells). 20 μl of assay buffer (50 mM of Tris/HCl pH 7.4; 100 mM of sodium chloride solution; 5 mM of calcium chloride solution; 0.1% of bovine serum albumin) and 20 μl of factor XIa from Kordia (0.45 nM in assay buffer) are then added successively. After 15 min of incubation, the enzyme reaction is started by addition of 20 μl of the factor XIa substrate Boc-Glu(OBzl)-Ala-Arg-AMC dissolved in assay buffer (10 μM in assay buffer) from Bachem, the mixture is incubated at room temperature (22° C.) for 30 min and fluorescence is then measured (excitation: 360 nm, emission: 460 nm). The measured emissions of the test batches with test substance are compared to those of control batches without test substance (only dimethyl sulphoxide instead of test substance in dimethyl sulphoxide), and the IC50 values are calculated from the concentration/activity relationships.
Measurement: IC50 = 160 nM (pChEMBL 6.800000190734863)
Descriptors: MW 492.86, ALogP 4.68, HBA 6, HBD 2, PSA 106.86, QED 0.47
Compound (SMILES): Cn1nc2c(c1CC(=O)Nc1nc(-c3ccc(OC(F)(F)F)c(F)c3)cs1)c(=O)n(C)c(=O)n2C
InChIKey: NLOJJXVQKKKSMA-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NET8
Assay: Radiometric Calcium Assay: The inhibition of TRPA1 receptor activation was measured as inhibition of allylisothiocyanate (AITC) induced cellular uptake of radioactive calcium. Test compounds were dissolved in DMSO to prepare 10 mM stock solution and then diluted using plain medium with 0.1% BSA and 1.8 mM CaCl2 to get desired concentration. Final concentration of DMSO in the reaction was 0.5% (v/v). Human TRPA1 expressing CHO cells were grown in F-12 DMEM medium with 10% FBS, 1% penicillin-streptomycin solution, and 400 ug/ml of G-418. Cells were seeded 24 h prior to the assay in 96 well plates so as to get 50,000 cells per well on the day of experiment. Cells were treated with test compounds for 10 min followed by addition of AITC at a final concentration of 30 micromolar and 5 uCi/ml 45Ca+2 for 3 min at 25° C. Cells were washed and lysed using buffer containing 1% Triton X-100, 0.1% deoxycholate and 0.1% SDS. Radioactivity in the lysate was measured in Packard Top count.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 512.45, ALogP 2.31, HBA 10, HBD 1, PSA 113.04, QED 0.41
Compound (SMILES): Cc1ccc(F)cc1[C@@H]1C/C(=N\N=C(N)N)c2c(C)cccc2C1
InChIKey: UYWUYWVHLMJGJO-WYOZQYPVSA-N
Target: Sodium/hydrogen exchanger 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P48764
Assay: Inhibition of NHE-3 (unknown origin)
Measurement: IC50 = 11000 nM (pChEMBL 4.960000038146973)
Descriptors: MW 324.4, ALogP 3.15, HBA 2, HBD 2, PSA 76.76, QED 0.51
Compound (SMILES): Cc1cncc(-c2cc3c(cn2)cnn3-c2cccc(N3CCCN(C4COC4)CC3)n2)n1
InChIKey: YENXUYDPJBMALY-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 2.03 nM (pChEMBL 8.6899995803833)
Descriptors: MW 442.53, ALogP 2.49, HBA 9, HBD 0, PSA 85.09, QED 0.48
Compound (SMILES): COC1CCC2(C=C(c3cc(-c4ccc(Cl)c(Cl)c4)ccc3C)C(=O)N2)CC1
InChIKey: ICFCKZFXCSWFJG-UHFFFAOYSA-N
Target: Acetyl-CoA carboxylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13085
Assay: ACC1 Enzyme Assay B1: For the assay, 50 nl of a 100-times concentrated solution of the test substance in DMSO were pipetted into a white low-volume 384-well microtitre plate (Greiner Bio-One, Frickenhausen, Germany), 2.5 ul of a solution of hACC1 in assay buffer [50 mM HEPES/NaOH pH 7.5, 2 mM MgCl2, 2 mM potassium citrate, 12 mM NaHCO3, 2 mM dithiothreitol (DTT), 0.005% (w/v) bovine serum albumin (BSA)] were added and the mixture was incubated for 15 min to allow prebinding of the substances to the enzyme prior to the enzyme reaction. The enzyme reaction was then started by addition of 2.5 ul of a solution of adenosine triphosphate (ATP, 100 uM=>final concentration in 5 ul of assay volume: 50 uM) and acetyl-CoA (20 uM=>final concentration in 5 ul assay volume: 10 uM) in assay buffer, and the resulting mixture was incubated at 22° C. for the reaction time of 45 min. The concentration of the hACC1 was adapted to the respective activity of the enzyme and adjusted such that the assay operated in the linear range. Typical concentrations were in the range of 1.75 ng/ul. The reaction was stopped by addition of 2.5 ul of the "ADP-GLO reagent" (1:1.5-times diluted), and the resulting mixture was incubated at 22° C. for 1 h to convert the unreacted ATP completely into cAMP. 2.5 ul of the "kinase detection reagent" were then added (1.2-times more concentrated than rec mmended by the manufacturer), the resulting mixture was incubated at 22° C for 1 h.
Measurement: IC50 = 428 nM (pChEMBL 6.369999885559082)
Descriptors: MW 416.35, ALogP 5.81, HBA 2, HBD 1, PSA 38.33, QED 0.69
Compound (SMILES): FC(F)(F)Cn1cc(-c2cc3c(cn2)cnn3-c2cncc(N3CCCNCC3)n2)cn1
InChIKey: MYYHUGRZNUGCBD-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 1.35 nM (pChEMBL 8.869999885559082)
Descriptors: MW 443.44, ALogP 2.44, HBA 9, HBD 1, PSA 89.58, QED 0.52
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3)ncnc2cc1OCc1nc(N2CCN(C(=O)OC(C)(C)C)CC2)no1
InChIKey: VVDWMCFFRNYWGL-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human N-terminal GST His6-tagged thrombin cleavage site-fused EGFR (H672 to A1210 residues) expressed in baculovirus using poly-EY as substrate after 210 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 602.48, ALogP 5.71, HBA 11, HBD 1, PSA 127.97, QED 0.28
Compound (SMILES): Cc1cncc(-c2cc3c(cn2)cnn3-c2ccc3c(n2)CC(CN)CO3)n1
InChIKey: KKZJKNBXDCSEGW-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 0.796 nM (pChEMBL 9.100000381469727)
Descriptors: MW 373.42, ALogP 2.09, HBA 8, HBD 1, PSA 104.63, QED 0.59
Compound (SMILES): O=C(O)C1CN(c2cc3c(cc2F)-c2noc(-c4onc(-c5ccccc5)c4C(F)(F)F)c2CC3)C1
InChIKey: TTXDTJUDJNHFLR-UHFFFAOYSA-N
Target: Sphingosine 1-phosphate receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99500
Assay: GTPgammaS Binding Assay: Compounds were loaded in a 384 Falcon v-bottom plate (0.5 ul/well in a 11 point, 3-fold dilution). Membranes prepared from S1P1/CHO cells or EDG3-Ga15-bla HEK293T cells were added to the compound plate (40 ul/well, final protein 3 ug/well) with multidrop. [35S]GTP (1250 Ci/mmol, Perkin Elmer) was diluted in assay buffer: 20 mM HEPES, pH7.5, 10 mM MgCl2, 150 mM NaCl, 1 mM EGTA, 1 mM DTT, 10 uM GDP, 0.1% fatty acid free BSA, and 10 ug/ml Saponin to 0.4 nM. 40 ul of the [35S] GTP solution was added to the compound plate with a final concentration of 0.2 nM. The reaction was kept at room temperature for 45 min. At the end of incubation, all the mixtures in the compound plate were transferred to Millipore 384-well FB filter plates via the Velocity11 Vprep liquid handler. The filter plate was washed with water 4 times by using the manifold Embla plate washer and dried at 60° C. for 45 min. MicroScint 20 scintillation fluid (30 u) was added to each well.
Measurement: EC50 = 14700 nM (pChEMBL 4.829999923706055)
Descriptors: MW 499.42, ALogP 5.44, HBA 6, HBD 1, PSA 92.6, QED 0.37
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)c3cccc(F)c3)CC2)C1
InChIKey: YWETXYVWGHGDPH-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: In Vitro JAK Kinase Assay: Compounds herein were tested for inhibitory activity of JAK targets according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 484.54, ALogP 3.19, HBA 7, HBD 1, PSA 106.73, QED 0.47
Compound (SMILES): Cc1cccc2cc([C@H](C)Nc3ncnc4[nH]ncc34)n(-c3ccccc3)c(=O)c12
InChIKey: NSBUFITVVLLFGE-HNNXBMFYSA-N
Target: PI3-kinase p110-alpha/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P42336
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110alpha/untagged recombinant full length human p85alpha expressed in baculovirus infected Sf21 cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 396.45, ALogP 4.14, HBA 6, HBD 2, PSA 88.49, QED 0.48