lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Nc1nc(O[C@H](c2ccc(Cl)cc2C2=CCOCC2)C(F)(F)F)cc(C2=CCC(C[C@H](N)C(=O)O)CC2)n1
InChIKey: LAJBERCKZVTKEM-QIFMMXESSA-N
Target: Tryptophan 5-hydroxylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P17752
Assay: Inhibition of Tryptophan Hydroxylase Activity: In order to confirm the inhibitory effect of the compound represented by formula 1 according to the present invention on the tryptophan hydroxylase activity, it was measured using a TPH1 (tryptophan hydroxylase 1) inhibitor screening assay kit (BPS Bioscience, Catalog #72053). The TPH1 inhibitor screening assay kit was used according to the manufacturer's manual. The results are shown in table below.Particularly, the synthesized compound was dissolved in DMSO, which was added to a 96-well microplate (10 Figure US11407763-20220809-P00004/well), to which TPH1 enzyme was added (40 Figure US11407763-20220809-P00004/well). Then, TPH1 reaction solution was added to the microplate (50 Figure US11407763-20220809-P00004/well) and the microplate was shaded with aluminum foil. The microplate was transferred to 4° C. environment, shaken carefully and incubated for 4 hours. After adding a quench solution to the microplate (10 Figure US11407763-20220809-P00004/well), the TPH1 activity was measured by reading the degree of fluorescence color development with a Flexstation3 microplate reader. At this time, the excitation spectrum was 300 nm and the emission spectrum was 360 nm.
Measurement: IC50 = 940 nM (pChEMBL 6.03000020980835)
Descriptors: MW 552.98, ALogP 5.18, HBA 7, HBD 3, PSA 133.58, QED 0.41
Compound (SMILES): C/C=C/C(=O)N1CCC[C@@H](NC(=O)c2sc3nccc4c3c2NC(=O)N4c2cccc(-c3ccccc3)c2)C1
InChIKey: WNBDXVXSNRTYCQ-KUPRMMGYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Lanthascreen Binding Assay: A BTK kinase lanthascreen binding assay monitors compound binding to unphosphorylated-BTK kinase domain (UP-BTK), by competing with a fluorescent labeled tracer. UP-BTK, consisting of the kinase domain of non-phosphorylated BTK protein (389-659aa), was produced in a Baculovirus/insect cell expression system. Into a 384-well plate, 2 ng of GST-tagged human BTK (389-659aa) was incubated with compound, 50 nM of Tracer 236 and 2 nM anti-GST antibody for 60 minutes using an optimized Lanthascreen assay. After 60 minutes, plates were read at 340 nM and 615/665 nM in an Infinite F500 (Tecan).
Measurement: IC50 = 63.1 nM (pChEMBL 7.199999809265137)
Descriptors: MW 537.65, ALogP 5.94, HBA 5, HBD 2, PSA 94.64, QED 0.31
Compound (SMILES): CCCCOc1cccc(-c2[nH]ncc2Oc2ccc(C#N)cc2)c1
InChIKey: HENRRPKCXLZBHB-UHFFFAOYSA-N
Target: Cytochrome P450 11B2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P19099
Assay: Inhibition Assay: G-402 cells can be obtained from ATCC (CRL-1440) and were originally derived from a renal leiomyoblastoma.The expression plasmids contains the ORF for either human/cyno CYP11B1 or CYP11B2 under the control of a suitable promoter (CMV-promoter) and a suitable resistance marker (neomycin). Using standard techniques the expression plasmid is transfected into G-402 cells and these cells are then selected for expressing the given resistance markers. Individual cell-clones are then selected and assessed for displaying the desired enzymatic activity using 11-Deoxycorticosterone (Cyp11B2) or 11-Deoxycortisol (Cyp11B1) as a substrate.G-402 cells expressing CYP11 constructs were established as described above and maintained in McCoy's 5a Medium Modified, ATCC Catalog No. 30-2007 containing 10% FCS and 400 μg/ml G418 (Geneticin) at 37° C. under an atmosphere of 5% CO2/95% air. Cellular enzyme assays were performed in DMEM/F12 medium containing 2.5% charcoal treated FCS and appropriate concentration of substrate (0.3-10 uM 11-Deoxycorticosterone, 11-Deoxycortisol or Corticosterone). For assaying enzymatic activity, cells were plated onto 96 well plates and incubated for 16 h. An aliquot of the supernatant is then transferred and analyzed for the concentration of the expected product (Aldosterone for CYP11B2; Cortisol for CYP11B1). The concentrations of these steroids can be determined using HTRF assays from CisBio analyzing either Aldosterone or Cortisol.
Measurement: EC50 = 0.011 nM (pChEMBL 10.960000038146973)
Descriptors: MW 333.39, ALogP 4.92, HBA 4, HBD 1, PSA 70.93, QED 0.62
Compound (SMILES): CN(c1ncnc2[nH]ccc12)[C@H]1C[C@@H](CS(=O)(=O)C2CN(C3CC3)C2)C1
InChIKey: NAGOVPAHANTRPK-XBXGTLAGSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Caliper Enzyme Assay: Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK3 or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 375.5, ALogP 1.43, HBA 6, HBD 1, PSA 82.19, QED 0.82
Compound (SMILES): CCOc1cccc(-c2nnc(N)s2)c1
InChIKey: APEORNRUGDLVDP-UHFFFAOYSA-N
Target: Palmitoleoyl-protein carboxylesterase NOTUM (SINGLE PROTEIN), Homo sapiens
UniProt: Q6P988
Assay: Biochemical Notum Enzymatic Activity Assay: An Echo liquid handler was used to acoustically dispense 500 nL of compounds into dry Greiner 384-well plates (catalog #781076), followed by 25 μL of 2 μM trisodium 8-octanoyloxypyrene-1,3,6-trisulfonate (OPTS, Sigma #74875) solution in 50 mM Tris, 5 mM CaCl2), 0.5 mM MgCl2, pH 7.4 assay buffer, and 25 μL of 2.38 nM notum carboxyesterase enzyme solution in the same buffer to every well in the assay plate. After 1 hour incubation at room temperature, fluorescence was measured on a PheraSTAR FSX microplate reader with an excitation wavelength of 485 nm and emission wavelength of 520 nm.
Measurement: IC50 = 85900 nM (pChEMBL 4.070000171661377)
Descriptors: MW 221.28, ALogP 2.19, HBA 5, HBD 1, PSA 61.03, QED 0.86
Compound (SMILES): NC(=O)c1c(-c2ccc(Cl)c(Cl)c2)nn2c1CN(C(=O)Nc1cc(F)cc(C(F)(F)F)c1)CC2
InChIKey: KSHPWLMBYOSYRX-UHFFFAOYSA-N
Target: Casein kinase I isoform epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: P49674
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours 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 unphosphorylated substrate and phosphorylated product. Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 μM ATP, 1.5 μM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 516.28, ALogP 5.16, HBA 4, HBD 2, PSA 93.25, QED 0.47
Compound (SMILES): Cn1c(SCCCN2CC3CCN(c4ccccc4C(F)(F)F)C3C2)nnc1-c1ccc(=O)[nH]c1
InChIKey: WAFXOAGOOZIJGE-UHFFFAOYSA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Inhibition Assay: 3H]-Spiperone Binding Assay at hD3 and hD4 recombinant receptors CHO cells transiently transfected with human dopamine type 3 or 4 receptors (CHO-hD3 or CHO-hD4, respectively), were re-suspended in 20 mM HEPES, 2 mM EDTA (pH 7.4), homogenised and centrifuged at 40,000 g (20 min, 4° C.). After re-suspension, homogenization and centrifugation as above, the final pellet was re-suspended in 20 mM HEPES, 100 mM NaCl, 10 mM MgCl2, 1 mM EDTA (pH 7.4) and aliquots were kept at −80° C. [3H]-Spiperone Binding experiments were performed in 96 deep-well polypropylene plates in 50 mM Tris/HCl, 120 mM NaCl, 5 mM KCl, 5 mM MgCl2 (pH 7.4). Compounds of invention were serially diluted in DMSO at 100 fold final concentrations in the assay (1% DMSO final in the assay). Displacement was performed in the presence of 0.3 nM [3H]-Spiperone. The reaction was initiated by the addition of membrane suspension (4 μg and 12 μg of protein for CHO-hD3- and CHO-hD4 membranes, respectively) and lasted for 90 or 100 min (for hD3 or hD4 membranes, respectively) at 23° C. in a final volume of 500 μl. Non specific binding (NSB) was determined in the presence of 1 μM Spiperone. The binding reaction was stopped by rapid filtration through GF/B filterplates pre-soaked in 0.5% polyetylenimmine (PEI) using a Packard cell harvester. After washing with ice-cold 0.9% NaCl, the plate was left to dry before the addition of Microscint 20 (50 μl/well, PerkinElmer). Radioactivity was counted with a TopCount (PerkinElmer). Data were analysed by non-linear regression analysis using GraphPad Prism 5.0 (GraphPad Software). Saturation binding experiments were performed similar to the competition binding experiments using a radioligand concentrations ranging from 0.015 to 4.0 nM. Ref: Mackenzie R. G. et al. (1994). Characterization of the human dopamine D3 receptor expressed in transfected cell lines. Eur. J. Pharmacol. 266: 79-85.
Measurement: Ki = 7.94 nM (pChEMBL 8.100000381469727)
Descriptors: MW 504.58, ALogP 3.88, HBA 7, HBD 1, PSA 70.05, QED 0.39
Compound (SMILES): Cc1nc(Oc2ccccc2)ncc1N1C(=O)Nc2c(C(=O)N[C@@H]3CCNC3)sc3nccc1c23
InChIKey: VACYVSNZJLYNHQ-CQSZACIVSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: TBD: TBD
Measurement: IC50 = 501 nM (pChEMBL 6.300000190734863)
Descriptors: MW 487.55, ALogP 3.96, HBA 8, HBD 3, PSA 121.37, QED 0.39
Compound (SMILES): COc1ccc(-n2nc(C(C)C)cc2NC(=O)Nc2ccc(Oc3ccnc(Nc4ccc(C(=O)NCCN5CCOCC5)cc4OC)n3)c3ccccc23)cc1
InChIKey: PEDMIJDFAWDUDE-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16539
Assay: p38 MAPKalpaha Enzyme Inhibition 1: The inhibitory activities of test compounds against the p38 MAPKα isoform (MAPK14: Invitrogen), are evaluated indirectly by determining the level of activation/phosphorylation of the down-stream molecule, MAPKAP-K2. The p38 MAPKα protein (80 ng/mL, 2.5 μL) is mixed with the test compound (2.5 μL of either 4 μg/mL, 0.4 μg/mL, 0.04 μg/mL or 0.004 μg/mL) for 2 hr at RT. The mix solution (2.5 μL) of the p38a inactive target MAPKAP-K2 (Invitrogen, 600 ng/mL) and FRET peptide (8 μm; a phosphorylation target for MAPKAP-K2) is then added and the kinase reaction is initiated by adding ATP (40 μm, 2.5 μL). The mixture is incubated for 1 hr at RT. Development reagent (protease, 5 μL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan Flash, ThermoFisher Scientific).
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 771.88, ALogP 7.2, HBA 12, HBD 4, PSA 166.02, QED 0.09
Compound (SMILES): N=C(/C=C\N)c1ccc2ncnc(N[C@H]3CC[C@H](N4CCOCC4)CC3)c2c1
InChIKey: LKCBPKCTFGQFPP-OILXZFEESA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Kinase Assay: The kinase activity of IRAK4 is determined by its ability to catalyze the phosphorylation of a fluorescent polypeptide substrate. The extent of phosphorylation is measured using the IMAP technology (Molecular Devices) where the phosphorylated fluorescent substrate binds to the large M(III)-based nanoparticles which reduces the rotational speed of the substrate and thus increases its fluorescent polarization (FP).20 μL reaction mixture contains 10 mM TriHCl, pH 7.2, 0.5 nM GST tagged IRAK4 (SignalChem), 100 nM fluorescent peptide substrate (RP7030, Molecular Devices), 100 μM ATP, 1 mM DDT, 1 mM MgCl2, and 0.01% Tween 20. The reaction is initiated by the addition of ATP. After incubation for 30 minutes at 25° C., 60 μL of Progressive IMAP Reagent (Molecular Devices) is added to stop the reaction. Change in RP7030's FP is determined by a FP reader (Analyst HT, LJL BioSystems).
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 380.5, ALogP 2.53, HBA 7, HBD 3, PSA 100.15, QED 0.69
Compound (SMILES): CC(C)Oc1cc2[nH]c(=O)c([C@H](C)Nc3ccc(C#N)n(C)c3=O)cc2cc1Cl
InChIKey: HPOPOVVUDKGCAN-LBPRGKRZSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: In Vitro Biochemical Assay: Please see paper.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 412.88, ALogP 3.71, HBA 6, HBD 2, PSA 99.91, QED 0.66
Compound (SMILES): CCOC(=O)[C@H]1N[C@@H]2C[C@@H]1CN(c1ncc(Cl)c(Nc3ccc4[nH]ncc4c3)n1)C2
InChIKey: FHYXUDMAHFSPIM-MRRJBJDNSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: Determination of Compound's ROCK Inhibitory Activity In Vitro (Z'Lyte Assay): Recombinant ROCK1 (amino acids 1-535) and ROCK2 (amino acids 1-552) proteins were purchased from ThermoFisher Scientific. Compound's activities were measured by Z′-lyte kinase kit (ThermoFisher Scientific) and IC50 was calculated
Measurement: IC50 = 305 nM (pChEMBL 6.519999980926514)
Descriptors: MW 427.9, ALogP 2.48, HBA 8, HBD 3, PSA 108.06, QED 0.53
Compound (SMILES): CC(c1ccc(OC(C)(C)C#N)cc1)N1C[C@H](C)N(c2nc(=O)n(C)c3ccc(C#N)nc23)C[C@H]1C
InChIKey: KPVBXBDXGNBRKX-YTYFACEESA-N
Target: Diacylglycerol kinase alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P23743
Assay: In Vitro DGK Inhibition Assay: The reactions were carried out in 50 mM MOPS pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 μM CaCl2, and 1 mM DTT (assay buffer). The reactions using a detergent/lipid micelle substrate also contained 50 mM octyl B-D-glucopyranoside. The lipid substrate concentrations were 11 mM PS and 1 mM DAG for the detergent/lipid micelle reactions. The lipid substrate concentrations were 2 mM PS, 0.25 mM DAG, and 2.75 mM PC for the extruded liposome reactions (5 mM total lipid). The reactions were carried out in 150 μM ATP. The enzyme concentrations for the DGKα and DGKζ were 5 nM. The compound inhibition studies were carried out as follows: 25 nL (ADPGLO assay) or 50 nL (LIPGLO assay) droplets of each test compound (top concentration 10 mM with 11 point, 3-fold dilution series for each compound) solubilized in DMSO were transferred to wells of a white 1536 well plate (Corning 3725). A 5 mL enzyme/lipid substrate solution at 2× final reaction concentration was prepared by combining 2.5 mL 4× enzyme solution (20 nM DGKα or DGKζ (prepared as described below) in assay buffer) and 2.5 mL of either 4× liposome or 4× detergent/lipid micelle solution (compositions described below) and incubated at room temperature for 10 minutes. Next, 1 μL 2× enzyme/lipid substrate solution was added to wells containing the test compound and reactions were initiated with the addition of 1 μL 300 uM ATP. The reactions were allowed to proceed for 2 hr (ADPGLO assay) or 1 hr (LIPGLO assay), after which 2 μL Glo Reagent (Promega V9101) was added and incubated for 40 minutes. Next, 4 μL Kinase Detection Reagent was added and incubated for 30 minutes. Luminescence was recorded using an EnVision microplate reader. The percent inhibition was calculated from the ATP conversion generated by no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 485.59, ALogP 3.54, HBA 9, HBD 0, PSA 111.07, QED 0.54
Compound (SMILES): CC1(C)Cc2cc(NC(=O)c3coc(-c4ccncn4)n3)c(N3CCOCC3)cc2O1
InChIKey: DRHZGBJNWVTJIP-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Transcreener-Fluorecescence Polarization Assay: Kinase activities were assayed using the Transcreener-Fluorecescence polarization platform (BelBrook Labs, Madison, Wis., USA) that measures amounts of the reaction product, ADP. The IRAK4 reaction conditions were optimized using an IRAK1-derived peptide (sequence H-KKARFSRFAGSSPSQSSMVAR) to provide a linear reaction rate over the course of a 90 min incubation, which resulted in 10-12% conversion of the starting ATP to ADP. Final IRAK4 assay conditions were 1.25 nM IRAK4; 125 μM ATP; 10 μM MgCl2; 125 μM peptide in reaction buffer (25 mM HEPES (pH7.4); 2 mM Dithiothreitol; 0.015% Brij-35; and 0.5% dimethyl sulfoxide. The IRAK1 activity was optimized similarly, yielding final assay conditions of 1.5 nM IRAK1; 62.5 μM ATP; 5 μM MgCl2, and 62.5 μM IRAK1 peptide in reaction buffer for 60 min.
Measurement: Ki = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 421.46, ALogP 2.93, HBA 8, HBD 1, PSA 102.61, QED 0.69
Compound (SMILES): CN(C)C(=O)Cn1cc(NC(=O)c2cnn3cccnc23)c(-c2cc(CC(F)F)ccc2OC(F)F)n1
InChIKey: FSLVHQSSGGVVQZ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Enzyme Assays: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications. Ki values were then determined using the Morrison tight binding model (Morrison, J. F., Biochim. Biophys. Acta. 185:269-296 (1969); William, J. W. and Morrison, J. F., Meth. Enzymol., 63:437-467 (1979)) modified for ATP-competitive inhibition [Ki=Ki,app/(1+[ATP]/Km,app)].
Measurement: Ki = 0.52 nM (pChEMBL 9.279999732971191)
Descriptors: MW 519.46, ALogP 3.34, HBA 8, HBD 1, PSA 106.65, QED 0.34
Compound (SMILES): O=C(N[C@H](Cc1ccc(-c2cccc(Cl)c2)cc1)C[C@@H](O)C(=O)O)c1nnc(C(F)(F)F)[nH]1
InChIKey: KPKHMCWUWITJJJ-HZPDHXFCSA-N
Target: Neprilysin (SINGLE PROTEIN), Homo sapiens
UniProt: P08473
Assay: Enzyme Inhibition Assay: The assays were performed in 384-well white opaque plates at 37° C. using the fluorogenic peptide substrates at a concentration of 10 μM in Assay Buffer (NEP: 50 mM HEPES, pH 7.5, 100 mM NaCl, 0.01% polyethylene glycol sorbitan monolaurate (Tween-20), 10 μM ZnSO4; ACE: 50 mM HEPES, pH 7.5, 100 mM NaCl, 0.01% Tween-20, 1 μM ZnSO4). The respective enzymes were used at concentrations that resulted in quantitative proteolysis of 1 μM of substrate after 20 minutes at 37° C.Test compounds were assayed over the range of concentrations from 10 μM to pM. Test compounds were added to the enzymes and incubated for 30 minute at 37° C. prior to initiating the reaction by the addition of substrate. Reactions were terminated after 20 minutes of incubation at 37° C. by the addition of glacial acetic acid to a final concentration of 3.6% (v/v).
Measurement: Ki < 1 nM (pChEMBL 9)
Descriptors: MW 482.85, ALogP 3.32, HBA 5, HBD 4, PSA 128.2, QED 0.39
Compound (SMILES): COc1ncccc1-c1cc2c(n1C)[C@H](c1ccc(Cl)cc1)N(c1cc(C)c3nnc(C)n3c1)C2=O
InChIKey: UQMYMWRETIHSGQ-QHCPKHFHSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: TR-FRET In-Vitro Binding Assay: All assays were performed in 384-well microtiter plates. Each assay plate contained 8-point serial dilutions for 40 test compounds, plus 16 high- and 16 low controls. Liquid handling and incubation steps were done on an Innovadyne Nanodrop Express equipped with a robotic arm (Thermo CatX, Perkin Elmer/Caliper Twister II) and an incubator (Liconic STX40, Thermo Cytomat 2C450). The assay plates were prepared by addition of 50 nl per well of compound solution in 90% DMSO HummingBird nanodispenser (Zinsser Analytic). The assay was started by stepwise addition of 4.5 μL per well of bromo domain protein (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 45 nM His-Brd2(60-472) or 45 nM His-Brd3(20-477) or 45 nM His-Brd4(44-477) all proteins produced in-house) and 4.5 μL per well of peptide solution (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 60 nM acetyl-histone H4 (AcK 5, 8, 12, 16) (Biosyntan GmbH)). Reactions were incubated at 30° C. for 35 minutes. Subsequently 4.5 μL per well detection mix (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 3 nM Eu-labeled anti-His6 antibody, 21 nM streptavidin-allophycocyanin) were added. After 35 minutes incubation at 30° C., plates were measured in a Perkin Elmer EnVision multilabel reader. Concentrations causing 50% inhibition (1050 values) were determined from percent inhibition values at different compound concentrations by non-linear regression analysis.
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 498.97, ALogP 5.16, HBA 7, HBD 0, PSA 77.55, QED 0.34
Compound (SMILES): COc1cc(Cn2cnc3cc(OC4CN(C)C4)cnc32)cc2c1OC(c1ccc(C3CC3)nc1)CO2
InChIKey: INYRXTSFJRVVMK-UHFFFAOYSA-N
Target: Macrophage colony-stimulating factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P07333
Assay: In Vitro Studies: All assay reaction conditions for IC50 determinations were within the linear range with respect to time and enzyme concentration. In a 384 well polypropylene plate, c-FMS (0.14 nM, Carna 08-155) was pre-incubated in a 100 mM Hepes-NaOH pH 7.5 buffer containing 0.01% Triton X-100, 10 mM MgCl2, 0.1% BSA, 1 mM DTT, 10 μM sodium orthovanadate and 10 μM beta-glycerophosphate and compound with a concentration of 2.5% DMSO for 15 minutes at room temperature. The reaction was initiated with an equal volume of peptide substrate (Caliper Life Sciences catalog no. 760430) and ATP in the aforementioned buffer. The final concentrations in the reaction were 70 pM c-FMS, 1.5 μM peptide substrate and 500 μM ATP (ATP Km). The reaction was incubated at room temperature for 120 minutes and terminated with a buffer containing excess EDTA (100 mM Hepes-NaOH pH 7.5, 0.02% Brij, 0.1% CR-3, 0.36% DMSO and 100 mM EDTA). The plate was run for one cycle on a LabChip 3000 (Caliper Life Sciences, Hopkinton, Mass.) in an off-chip mobility shift type assay with an upstream voltage of −2250 volts, a downstream voltage of −500 volts and a vacuum pressure of −1.6 psi.
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 499.57, ALogP 3.97, HBA 9, HBD 0, PSA 83.76, QED 0.38
Compound (SMILES): O=C(Nc1cccc(C2CN(c3ncnc4[nH]ccc34)CCN2CCO)c1)Nc1c(F)cccc1N1CCCC1
InChIKey: ZJSNZHATZAWKIQ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: In Vitro BTK Kinase Assay: The purpose of the BTK in vitro assay is to determine compound potency against BTK through the measurement of IC50. Compound inhibition is measured after monitoring the amount of phosphorylation of a fluorescein-labeled polyGAT peptide (Invitrogen PV3611) in the presence of active BTK enzyme (Upstate 14-552), ATP, and inhibitor. The BTK kinase reaction was done in a black 96 well plate (costar 3694). For a typical assay, a 24 μL aliquot of a ATP/peptide master mix (final concentration; ATP 10 μM, polyGAT 100 nM) in kinase buffer (10 mM Tris-HCl pH 7.5, 10 mM MgCl2, 200 μM Na3PO4, 5 mM DTT, 0.01% Triton X-100, and 0.2 mg/ml casein) is added to each well. Next, 1 μL of a 4-fold, 40× compound titration in 100% DMSO solvent is added, followed by adding 15 uL of BTK enzyme mix in 1× kinase buffer (with a final concentration of 0.25 nM). The assay is incubated for 30 minutes before being stopped with 28 μL of a 50 mM EDTA solution. Aliquots (5 μL) of the kinase reaction are transferred to a low volume white 384 well plate (Corning 3674), and 5 μL of a 2× detection buffer (Invitrogen PV3574, with 4 nM Tb-PY20 antibody, Invitrogen PV3552) is added. The plate is covered and incubated for 45 minutes at room temperature. Time resolved fluorescence (TRF) on Molecular Devices M5 (332 nm excitation; 488 nm emission; 518 nm fluorescein emission) is measured. IC50 values are calculated using a four parameter fit with 100% enzyme activity determined from the DMSO control and 0% activity from the EDTA control.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 544.64, ALogP 4.2, HBA 7, HBD 4, PSA 112.65, QED 0.28
Compound (SMILES): CCC[C@@H](CCO)Nc1nc(N)nc(C)c1Cc1ccc(OCCCN(CC)CCC(=O)O)cc1OC
InChIKey: VLBOFYFPALJWKB-NRFANRHFSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: Human TLR7 Assay: Recombinant human TLR7 was stably expressed in a HEK293 cell line already stably expressing the pNiFty2-SEAP reporter plasmid; integration of the reporter gene was maintained by selection with the antibiotic zeocin. The most common variant sequence of human TLR7 (represented by the EMBL sequence AF240467) was cloned into the mammalian cell expression vector pUNO and transfected into this reporter cell-line. Transfectants with stable expression were selected using the antibiotic blasticidin. In this reporter cell-line, expression of secreted alkaline phosphatase (SEAP) is controlled by an NFkB/ELAM-1 composite promoter comprising five NFkB sites combined with the proximal ELAM-1 promoter. TLR signaling leads to the translocation of NFkB and activation of the promoter results in expression of the SEAP gene. TLR7-specific activation was assessed by determining the level of SEAP produced following overnight incubation of the cells at 37° C. with the standard compound in the presence of 0.1% (v/v) dimethylsulfoxide (DMSO). Concentration dependent induction of SEAP production by compounds was expressed as the concentration of compound which produced half of the maximal level of SEAP induction for that compound (EC50).
Measurement: EC50 = 251 nM (pChEMBL 6.599999904632568)
Descriptors: MW 517.67, ALogP 3.5, HBA 9, HBD 4, PSA 143.06, QED 0.22
Compound (SMILES): Cc1c(-c2[nH]c3ccc(C4CC(N)C4)nc3c2C(C)C)cn2ncnc2c1C
InChIKey: SJMBBMOJKZFJSH-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assays: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 2219 nM (pChEMBL 5.650000095367432)
Descriptors: MW 374.49, ALogP 4.22, HBA 5, HBD 2, PSA 84.89, QED 0.56
Compound (SMILES): Cc1cc(-c2cc3nc([C@@H](N)C(C)C)[nH]c(=O)c3s2)ccn1
InChIKey: GZFWZLVDZVGSHA-ZDUSSCGKSA-N
Target: CDC7/DBF4 (Cell division cycle 7-related protein kinase/Activator of S phase kinase) (PROTEIN COMPLEX), Homo sapiens
UniProt: Q9UBU7
Assay: Biochemical Assay: Biochemical assay was tested in the U-shaped bottom 384 plate (corning, #4514) with a final volume of 20 ul at 27° C. The concentration of CDC7 was optimized by the enzyme titration experiment. CDC7 kinase was diluted in assay buffer (40 mM Tris.HCl pH 7.25, 100 ug/mL BSA, and 20 mM MgCl2) to get 2.4× enzyme solutions. Compounds were dissolved in 10 mM DMSO, and series diluted in DMSO from 0.3 mM to 0.3 nM (5 concentration pts); All dilutions were diluted 30× in assay buffer to get 6× compounds solutions. MCM2 Substrate and ATP were diluted to 2.4× mixed solution. Add 2 ul test compound solution to 384 assay plate, then add 5 ul substrate/ATP mixed solution, at last add 5 ul enzyme solution, incubate at 27° C. for 180 mins. Transfer 5 ul reaction solution to another 384 assay plate, then add 5 ul ADP-Glo Reagent (Promega) to each well and incubate at 27° C. for 40 mins; Add 10 ul Kinase Detection Reagent (Promega) to each well and incubate at 27° C. for 30 mins. 10 uM CDC7-3 compound was used as 100% inhibition while 100% DMSO control was used as 0% inhibition. Each test has three replications at least.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 314.41, ALogP 3.01, HBA 5, HBD 2, PSA 84.66, QED 0.78
Compound (SMILES): Bc1ccc(F)c2c1CC[C@H]2Nc1ccc([C@H]2C[C@@H]2C(=O)O)nc1
InChIKey: MMVVWLUTHGUYCG-MQIPJXDCSA-N
Target: Cytochrome P450 2C8 (SINGLE PROTEIN), Homo sapiens
UniProt: P10632
Assay: Inhibition of CYP-2C8: The inhibition of cytochrome P450 2C8-isoenzyme catalyzed deethylation of amodiaquine by the test compound is assayed at 37° C. with human liver microsomes. All assays are carried out on a robotic system in 96 well plates. The final incubation volume contains TRIS buffer (0.1 M), MgCl2 (5 mM), human liver microsomes (0.05 mg/mL), amodiaquine (1 μM) and the test compound at five different concentrations or no compound (high control) in duplicate (e.g. highest concentration 10-50 μM with subsequent serial 1:4 dilutions). Following a short preincubation period, reactions are started with the cofactor (NADPH, 1 mM) and stopped by cooling the incubation down to 8° C. and subsequently by addition of one volume of acetonitrile. An internal standard solution¿the stable isotope d5-desethylamodiaquine is added after quenching of incubations. Peak area analyte (=metabolite formed) and internal standard is determined by LC-MS/MS. The resulting peak area ratio analyte to internal standard in these incubations is compared to a control activity containing no test compound. Within each of the assay runs, the IC50 of a positive control inhibitor (Montelukast) is determined. Experimental IC50 values are calculated by least square regression according to the following equation: % control activity=(100% control activity/(1+(I/IC 50)S))−B with I=inhibitor concentration; S=slope factor; B=background activity (lower plateau of the inhibition curve).
Measurement: IC50 = 40000 nM (pChEMBL 4.400000095367432)
Descriptors: MW 324.16
Compound (SMILES): COCCN1C[C@@H](NC(=O)Nc2c(Br)c(COC)nn2-c2ccccc2)[C@H](c2cc(F)cc(F)c2)C1
InChIKey: LJSPXVIVLVNZQF-LEWJYISDSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: TrkA Omnia Assay: Trk enzymatic selectivity was assessed using Omnia Kinase Assay reagents from Invitrogen Corp. Enzyme (TrkA from Invitrogen Corp.) and test compound (various concentrations) were incubated for 10 minutes at ambient temperature in a 384-well white polypropylene plate (Nunc catalog#267462). Omnia Tyr Peptide #4, as well as ATP, were then added to the plate. Final concentrations were as follows: 20 nM enzyme, 500 μM of ATP, 10 μM peptide substrate. The assay buffer consisted of 25 mM MOPS pH 7.5, 0.005% (v/v) Triton X-100 and 5 mM MgCl2. The production of phosphorylated peptide was monitored continuously for 70 minutes using a Molecular Devices FlexStation II384 microplate reader (excitation=360 nm; emission=485 nm). Initial rates were calculated from the progress curves. IC50 values were calculated from these rates using either a 4 or 5-parameter logistic curve fit.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 564.43, ALogP 4.3, HBA 6, HBD 2, PSA 80.65, QED 0.41
Compound (SMILES): C=CC(=O)N1CCC(Oc2cc3c(Nc4ccc(Cl)cc4Cl)ncnc3cn2)CC1
InChIKey: UULQZGZMEOIOFK-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Cell Viability Assay and IC50 Estimation: Cell viability was determined using the Cell Titer Glo assay (Promega) as previously described. Briefly, 2000-3000 cells per well were plated in 384-well plates (Greiner Bio-One) in technical triplicate. Cells were treated with seven different concentrations of tyrosine kinase inhibitors or vehicle alone at a final volume of 40 μL per well. After 3 days hours, 11 μL of Cell Titer Glo was added to each well. Plates were shaken for 15 minutes, and bioluminescence was determined using a FLUOstar OPTIMA multi-mode micro-plate reader (BMG LABTECH). Bioluminescence values were normalized to DMSO treated cells, and normalized values were plotted in GraphPad Prism using non-linear regression fit to normalized data with a variable slope. IC50 values were calculated by GraphPad Prism at 50% inhibition.
Measurement: IC50 = 3400 nM (pChEMBL 5.46999979019165)
Descriptors: MW 444.32, ALogP 4.63, HBA 6, HBD 1, PSA 80.24, QED 0.58
Compound (SMILES): CC(O)(c1cc(F)c2c(c1)C(=O)N(Cc1ccc(Cl)cn1)[C@@]2(OCC1(O)CC1)c1ccc(Cl)cc1)c1ncccn1
InChIKey: ZDTDRSMIUYSHRA-FUJFQHJFSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: Binding Assay (ELISA): The ELISA assay was performed in streptavidin coated plates which were preincubated with 200 ul per well of 1 μg ml−1 biotinylated IP3 peptide. The plates were ready to use for MDM2 binding after washing the plate with PBS.Compounds and control solutions in DMSO aliquoted in 96-well plates were pre-incubated in a final 2.5-5% (v/v) DMSO concentration at room temperature (for example 20C.) for 20 min with 190 ul aliquots of optimized concentrations of in vitro translated MDM2, before transfer of the MDM2-compound mixture to the b-IP3 streptavidin plates, and incubation at 4C. for 90 min. After washing three times with PBS to remove unbound MDM2, each well was incubated at 20C. for 1 hour with a TBS-Tween (50 mM Tris pH7.5; 150 mM NaCl; 0.05% Tween 20 nonionic detergent) buffered solution of primary mouse monoclonal anti-MDM2 antibody (Ab-5, Calbiochem, used at a 1/10000 or 1/200 dilution depending on the antibody stock solution used), then washed three times with TBS-Tween before incubation for 45 mins at 20C. with a TBS-Tween buffered solution of a goat-anti-mouse horseradish peroxidase (HRP) conjugated secondary antibody (used at 1/20000 or 1/2000 depending on the antibody stock solution). The unbound secondary antibody was removed by washing three times with TBS-Tween. The bound HRP activity was measured by enhanced chemiluminesence (ECL, Amersham Biosciences) using the oxidation of the diacylhydrazide substrate, luminol, to generate a quantifiable light signal. The percentage of MDM2 inhibition at a given concentration is calculated as the [1−(RLU detected in the compound treated sample−RLU negative DMSO control)÷(RLU of DMSO positive and negative controls)]x100 or as the (RLU detected in the compound treated sample ÷ RLU of DMSO controls)x100.
Measurement: IC50 = 4.4 nM (pChEMBL 8.359999656677246)
Descriptors: MW 595.46, ALogP 4.97, HBA 7, HBD 2, PSA 108.67, QED 0.3
Compound (SMILES): O=C(O)c1ccc(-c2[nH]c([C@@H]3CCc4cc(-c5cc(Cl)ccc5-n5cnnn5)cc(=O)n43)nc2F)s1
InChIKey: YRMPVVCGIICVEN-INIZCTEOSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Enzyme Activity Assay: Human Factor XIa (Haematologic Technologies Inc.) activity was measured at an enzyme concentration of 0.1 U/mL in 150 mM NaCl, 5 mM KCl, 1 mg/mL PEG6000, 50 mM HEPES-NaOH (pH7.4) with 300 μM S-2366 (pyroGlu-Pro-Arg-pNA, Chromogenix).
Measurement: IC50 = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 523.94, ALogP 3.97, HBA 9, HBD 2, PSA 131.58, QED 0.36
Compound (SMILES): COc1cc(-n2cnnc2)cc(Cl)c1C(=O)NCC(c1ccccc1)c1nc2cc(C)c(C)cc2[nH]1
InChIKey: HQBXONCZYNQXOV-UHFFFAOYSA-N
Target: Coagulation factor IX (SINGLE PROTEIN), Homo sapiens
UniProt: P00740
Assay: Determination of Inhibitory Activity Against Factor IXa: Formation of a clot to stem bleeding at a site of blood vessel injury involves the coordinated activity of a group of plasma proteins that initiate and propagate fibrin formation and subsequently protect fibrin from premature degradation. Factor IX is a key component of the plasma system that forms a fibrin clot at a site of vascular injury. The activity of Factor IXa is measured by monitoring the cleavage of the fluorescent peptide, CH3SO2-D-CHG-Gly-Arg-AFC.AcOH (CHG is cyclohexyl-glycine and AFC is trifluoro aminomethyl coumarin). Factor IXa cleaves the amide bond between Arg and AFC, thereby releasing the AFC fluorophore. The free AFC can be detected with a fluorescence detector at an excitation wavelength of 405 nM and emission wavelength of 510 nM.
Measurement: IC50 = 14.8 nM (pChEMBL 7.829999923706055)
Descriptors: MW 500.99, ALogP 4.98, HBA 6, HBD 2, PSA 97.72, QED 0.33
Compound (SMILES): CN(C)[C@H](CNC(=O)C[C@H](c1ccccc1)C1(C(F)(F)F)CC1)Cc1ccc2c(c1)CC(=O)N2
InChIKey: XCJHFRBQNKLNPA-LEWJYISDSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: MOR cAMP Agonist and Antagonist Assays: CHO Tag-lite human mOR stable cell line from Cisbio (NCBI accession number: NM_000914.3, Bedford, Mass.) were seeded and grown to approximately 80% confluence in Ham F-12 with 10% FBS, 50 U/ml penicillin, 50 μg/ml streptomycin, 2 mM Hepes, and 1 mg/ml geneticin (Invitrogen, Carlsbad, Calif.). Cells were then harvested using accutase (Corning. Coming, N.Y.), centrifuged at 1300 RPM for 5 min, and plated at 5000 cells per 5 μL per well in a 5× dilution of stimulation buffer consisting of the HTRF cAMP Gi kit, water and IBMX at 0.5 mM (Cisbio, Bedford, Mass.) in white HTRF low volume 384 well plates (Cisbio, Bedford, Mass.). Plates were then incubated at 37° C. in 5% CO2 for 10 minutes. For the agonist assay, forskolin was added to a final concentration of 4 μM. For the antagonist assay, forskolin (4 μM) and DAMGO at EC90 final concentration were added. Test compounds were dissolved in DMSO and water, and then serially diluted to working concentrations such that the concentration of DMSO was less than 0.1%. Diluted test compounds were added at 2.5 μL per well, and plates were incubated at 37° C., and 5% CO2 for 15 minutes, and then at room temperature for 15 minutes. Next 5 μl/well of cAMP Eu-cryptate and 5 μl/well of anti-cAMP-d2 (both diluted 1:20 in lysis buffer) were added, and plates were incubated at room temperature for 1 hour. Following incubation, plates were read in a Synergy Neo2 multi-mode reader (Biotek, Winooski, Vt.). Plate reader settings were set to time resolved fluorescence with excitation at 330 nm and emissions of 620 nm and 665 nm. Emission fluorescence was normalized (665/620 nm signal×1000). For the agonist assay, data was normalized using the maximal DAMGO response. Measurements were performed in triplicate and the dose response was fit using nonlinear regression.
Measurement: IC50 > 3000 nM (pChEMBL 5.5229997634887695)
Descriptors: MW 473.54, ALogP 4.29, HBA 3, HBD 2, PSA 61.44, QED 0.57
Compound (SMILES): COc1cc(C(=O)N2CC3CCC2[C@@H]3N)cc2nc(-c3cc4ccc(-c5cccc6c5CNC6=O)nc4n3CC3CC3)n(C)c12
InChIKey: JNSLXLJZKMOIDF-KTBVXVCESA-N
Target: Protein-arginine deiminase type-4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM07
Assay: Rapid-Fire Mass Spectrum (RFMS) Assay: Compounds were solubilized in 100% DMSO to achieve a 10 mM compound concentration. Compound stock solutions were stored at RT. A series of dilutions were prepared in DMSO and mixed 8 times with 20 μL mixing volume. Final top concentration of compound in the assay is 50 μM. Final assay conditions were as follows:Reaction volume: 26 μlAssay buffer: 25 mM hepes, pH 7.5, 5 mM NaCl, 1 mM DTT, 0.2 mg/ml BSA, 0.01% CHAPS, 50 μM Calcium, and 5 μM TPENFinal concentrations: 5 nM hPAD4 enzyme, 250 μM BAEE, and 0.5% DMSOTotal incubation time: 30 mins compound and enzyme preincubation at 37° C., 90 min enzyme/substrate reaction, 30 min reaction with phenyl glyoxal at 37° C.Stop solution: 40 μl 5% TCA in ACN
Measurement: IC50 = 27 nM (pChEMBL 7.570000171661377)
Descriptors: MW 601.71, ALogP 4.48, HBA 8, HBD 2, PSA 120.3, QED 0.3
Compound (SMILES): Cn1cc(C(F)(F)F)nc1CN(Cc1ccc(F)cc1)Cc1ccc(OCc2ccccc2)cc1
InChIKey: XADBBDLUHAUGSV-UHFFFAOYSA-N
Target: Collagenase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P45452
Assay: Activity Assay: Stock solution of substrate (Mca-Lys-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2) was prepared in DMSO at a concentration of 6 mM. Assays were performed in an assay buffer (50 mM Tris pH 7.5, 300 mM NaCl, 10 uM ZnSO4, 5 mM CaCl2, 0.005% Brij-35). The final DMSO concentration in all assays was 1.0%. The used substrate concentration was 3.33 uM. The assays were carried out at 37° C. The fluorescence changes, resulting from the substrate cleavage, were detected using excitation at 340 nm and emission wavelength of 405 nm using a Wallac 1420 Microplate Reader. The reaction mixtures were preincubated with the inhibitors for 30 minutes. The reactions were started by the addition of MMP substrate, and the fluorescence intensity changes were measured after 60 minutes. The obtained fluorescence values were normalized and % inhibition values calculated. Dose response curves were determined from 6 concentration points using three fold dilution steps, each concentration point was determined in dupllicate. IC50 values were calculated from the dose response curves. FIGS. 1-4 show the dose response curves of Compound 1 and ilomastat on on MMP1, -2, -9, -13 activities respectively.
Measurement: IC50 = 34000 nM (pChEMBL 4.46999979019165)
Descriptors: MW 483.51, ALogP 6.36, HBA 4, HBD 0, PSA 30.29, QED 0.26
Compound (SMILES): CC(C)S(=O)(=O)NCc1c2c(nn1CCC1CC1)C[C@]1(CCc3cc(OCC(F)(F)F)ccc31)NC2=O
InChIKey: WLMQLUFYZOGYPE-DEOSSOPVSA-N
Target: Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q10469
Assay: Inhibitory Activity: Solutions of the compounds of the present invention in DMSO were each aliquoted into 0.2-μL portions in a 384-well polystyrene microplate produced by Corning Incorporated, and 5 μL of an enzyme solution prepared with an assay buffer (100 mmol/L phosphate buffer (pH 7.4) containing 2 mmol/L DTT) and 5 μL of a substrate solution (100 mmol/L phosphate buffer (pH 7.4), 30 μmol/L 2-Oleoylglycerol, 10 μmol/L Oleoyl-CoA) were added thereto, and the resultant was stirred and centrifuged, and incubated in a moist chamber at room temperature for 1 hour. After enzymatic reaction, 50 μL of a quenching solution (containing 0.2 μmol/L Diolein-d5, 0.4% formic acid, and 50% isopropanol) containing Internal Standard (IS) was added to terminate the reaction, and the resultant was sealed in a plate produced by Shimadzu GLC Ltd., and then stirred and centrifuged, and measurement was performed by using an electrospray ionization method with a RapidFire360 and Agilent 6550 Q-TOF mass spectrometer. Diolein as a reaction product (P) of 2-Oleoylglycerol as the substrate and an ammonium adduct ion of the IS were detected, and the peak intensity ratio, P/IS, was calculated from the peak heights to evaluate the inhibitory activity. Inhibitory activities with/without addition of enzyme were defined as Control (+)/Control (−), respectively, and the respective % inhibitions were defined as 0% inhibition and 100% inhibition. The inhibitory activity was calculated from formula below with TIBCO Spotfire (produced by TIBCO Software Inc.):Inhibitory activity (%)=[1−{Sample−Control(−)}/{Control(+)−Control(−)}]*100where Sample indicates a peak intensity ratio: P/IS, when the compound of the present invention was added.
Measurement: IC50 = 430 nM (pChEMBL 6.369999885559082)
Descriptors: MW 540.61, ALogP 3.58, HBA 6, HBD 2, PSA 102.32, QED 0.51
Compound (SMILES): CNc1cc(-c2cn([C@H]3CC(=O)N(C4CCOCC4)C3)c3ncccc23)nc2c(C(=O)N[C@H]3CC[C@@H]3OC)cnn12
InChIKey: OEIAXAMFCQVCLT-OEOAZWSVSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: TYK2 JH2 Domain Binding Assay: The binding reaction was assembled by combining 16 μl of DNA-tagged kinase extract, 3.8 μl liganded affinity beads, and 0.18 μl test compound (PBS/0.05% Tween 20/10 mM DTT/0.1% BSA/2 μg/ml sonicated salmon sperm DNA)]. Extracts were used directly in binding assays without any enzyme purification steps at a ≥10,000-fold overall stock dilution (final DNA-tagged enzyme concentration <0.1 nM). Extracts were loaded with DNA-tag and diluted into the binding reaction in a two step process. First extracts were diluted 1:100 in 1× binding buffer (PBS/0.05% Tween 20/10 mM DTT/0.1% BSA/2 μg/ml sonicated salmon sperm DNA) containing 10 nM DNA-tag. This dilution was allowed to equilibrate at room temperature for 15 minutes and then subsequently diluted 1:100 in 1× binding buffer. Test compounds were prepared as 111× stocks in 100% DMSO. Kds were determined using an 11-point 3-fold compound dilution series with three DMSO control points. All compounds for Kd measurements are distributed by acoustic transfer (non-contact dispensing) in 100% DMSO
Measurement: Kd < 0.2 nM (pChEMBL 9.699000358581543)
Descriptors: MW 558.64, ALogP 2.65, HBA 10, HBD 2, PSA 127.91, QED 0.35