lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): C[C@@H]1Cc2c([nH]c3ccccc23)[C@@H](c2ccnc(OCCNCCCF)c2Cl)N1CC(F)(F)CO
InChIKey: JAXCPQCVOJTKRR-WAIKUNEKSA-N
Target: Estrogen receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P03372
Assay: ERalpha Binding Assay: The ability of compounds to bind to isolated Estrogen Receptor Alpha Ligand binding domain (ER alphaLBD (GST)) was assessed in competition assays using a LanthaScreen Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) detection end-point. For the LanthaScreen TR-FRET endpoint, a suitable fluorophore (Fluormone ES2, ThermoFisher, Product code P2645) and recombinant human Estrogen Receptor alpha ligand binding domain, residues 307-554 (expressed and purified in-house) were used to measure compound binding. The assay principle is that ER alpha-LBD (GST) is added to a fluorescent ligand to form a receptor/fluorophore complex. A terbium-labelled anti-GST antibody (Product code PV3551) is used to indirectly label the receptor by binding to its GST tag, and competitive binding is detected by a test compound's ability to displace the fluorescent ligand, resulting in a loss of TR-FRET signal between the Tb-anti-GST antibody and the tracer. The assay was performed as follows with all reagent additions carried out using the Beckman Coulter BioRAPTR FRD microfluidic workstation:1. Acoustic dispense 120 nL of the test compound into a black low volume 384 well assay plates.2. Prepare 1× ER alpha-LBD/Tb-antiGST Ab in ES2 screening buffer and incubate for 15 minutes.3. Dispense 6 μL of the 1× AR-LBD/Tb-anti-GST Ab reagent into each well of the assay plate followed by 6 μL of Fluorophore reagent into each well of the assay plate4. Cover the assay plate to protect the reagents from light and evaporation, and incubate at room temperature for 4 hours.5. Excite at 337 nm and measure the fluorescent emission signal of each well at 490 nm and 520 nm using the BMG PheraSTAR.Compounds were dosed directly from a compound source microplate containing serially diluted compound (4 wells containing 10 mM, 0.1 mM, 1 μM and 10 nM final compound respectively) to an assay microplate using the Labcyte Echo 550. The Echo 550 is a liquid handler that uses acoustic technology to perform direct microplate-to-microplate transfers of DMSO compound solutions and the system can be programmed to transfer multiple small nL volumes of compound from the different source plate wells to give the desired serial dilution of compound in the assay which is then back-filled to normalise the DMSO concentration across the dilution range.
Measurement: IC50 = 0.72 nM (pChEMBL 9.140000343322754)
Descriptors: MW 510.99, ALogP 4.51, HBA 5, HBD 3, PSA 73.41, QED 0.33
Compound (SMILES): CCN1CCC(NC(=O)c2n[nH]c3ccc(-c4cnc5ccccc5c4)cc23)CC1
InChIKey: GQPCNEBNKOBBDY-UHFFFAOYSA-N
Target: Proto-oncogene Wnt-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P04628
Assay: Wnt activity assay: Reporter cell lines were generated by stably transducing cells of cancer cell lines (e.g., colon cancer) with a lentiviral construct that include a Wnt-responsive promoter driving expression of the firefly luciferase gene.Lentiviral constructs were made in which the SP5 promoter, a promoter having eight TCF/LEF binding sites derived from the SP5 promoter, was linked upstream of the firefly luciferase gene. The lentiviral constructs included a hygromycin resistance gene as a selectable marker. The SP5 promoter construct was used to transduce SW480 cells, a colon cancer cell line having a mutated APC gene that generates a truncated APC protein, leading to de-regulated accumulation of β-catenin. A control cell line was generated using another lentiviral construct containing the luciferase gene under the control of the SV40 promoter which does not require β-catenin for activation.Cultured SW480 cells bearing a reporter construct were distributed at approximately 10,000 cells per well into 384 well multiwell plates. Compounds were then added to the wells in half-log dilutions using a three micromolar top concentration. A series of control wells for each cell type received only buffer and compound solvent. Twenty-four hours after the addition of compound, reporter activity for luciferases was assayed, for example, by addition of the BrightGlo luminescence reagent (Promega) and the Victor3 plate reader (Perkin Elmer). Readings were normalized to DMSO only treated cells, and normalized activities were then used for the IC50 calculations.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 399.5, ALogP 3.99, HBA 4, HBD 2, PSA 73.91, QED 0.54
Compound (SMILES): CC(NC(=O)OCc1ccccc1)C(=O)N[C@@H](C)C(=O)N[C@@H]1C(=O)N[C@@H](C2CC2)C(=O)N[C@H]2CC[C@@H](O)N(C2=O)[C@@H](CCCNC(N)=O)C(=O)N(C)[C@@H](Cc2ccccc2)C(=O)N[C@@H](C(C)C)C(=O)O[C@@H]1C
InChIKey: HWDRMEYUOBQZQY-BHASSOBKSA-N
Target: Serine protease HTRA1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92743
Assay: Chromogenic Enzyme Activity Assays: In general, proteolytic activity was tested by monitoring the cleavage of the specific chromogenic substrate at 405 nm wavelength for 60 or 120 minutes in a Tecan Spark M10 or Tecan Genios Pro plate reader at 37° C. After optimization of assay conditions, every enzyme was tested in 100 μL final volume with 500 μM chromogenic substrate. Where applicable, inhibitor and enzyme were pre-incubated for 10 minutes in the buffer at 37° C. before substrate addition. Compounds were pre-solved in DMSO and used in the assays with a final DMSO concentration of 3% (v/v). n order to determine KM values, the assays were performed as described but with differing substrate concentrations (10 μM to 4 mM). Specific activity was calculated and plotted against substrate concentrations in GraphPad Prism 5 software. Michaelis-Menten (software built-in) analysis was used to calculate the KM values. In order to determine IC50 and Ki values, enzyme assays were performed with differing inhibitor concentrations (2.5 nM to 20/200 μM), specific activities were calculated, and plotted against log inhibitor concentrations in GraphPad Prism 5 software. One siteFit Ki and One siteFit log IC50 (software built-in) analysis were used to calculate IC50 and Ki values.
Measurement: Ki = 700 nM (pChEMBL 6.159999847412109)
Descriptors: MW 1005.14
Compound (SMILES): COc1cc(OC)c(-c2ccc(F)c(CNc3ccc([C@H]4C[C@@H]4C(=O)O)nc3)c2)c(OC)c1
InChIKey: DKZYHIDJMINCFF-OALUTQOASA-N
Target: Free fatty acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14842
Assay: IPOne assay: IP1 accumulation measurements using the IPOne assay system1321N1 cells stably expressing human GPR40 receptor (Euroscreen, Belgium) are seeded 24 h before the assay in white 384-well plates in culture medium containing 10% FCS, 1% Na-Pyruvate and 400 μg/mL G418. IP1 is assayed according to the manufacturer's description (Cisbio Bioassays, France). In brief, the assay is started by substitution of the culture medium by stimulation buffer (Hepes 10 mM, CaCl2 1 mM, MgCl2 0.5 mM, KCl 4.2 mM, NaCl 146 mM, glucose 5.5 mM and LiCl 50 mM, pH 7.4). Cells are stimulated for 1 h at 37° C., 5% CO2 by addition of the compounds that are diluted in stimulation buffer containing LiCl. Assays are stopped by adding HTRF-conjugates (IP1-d2 and Anti-IP1 cryptate Tb) and lysis buffer, provided by the manufacturer. After an incubation time of 1 h at room temperature plates are measured using an EnVision, Perkin Elmer. The obtained fluorescence ratios at 665/615 nM are then used to calculate the pEC50 values using Assay Explorer 3.3 Software (Accelrys, Inc.) by interpolation using an IP1 reference curve and subsequent sigmoidal curve fitting allowing for a variable hill slope.
Measurement: EC50 = 453 nM (pChEMBL 6.340000152587891)
Descriptors: MW 452.48, ALogP 4.71, HBA 6, HBD 2, PSA 89.91, QED 0.49
Compound (SMILES): Nc1cc(Cl)cnc1N1CCN(C(=O)C2C[C@@H]2C(=O)O)CC1c1ccc(Cl)cc1
InChIKey: UJEBKBZZUZSGCC-CKDBGZEDSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: Fluorescence Polarization (FP) Binding Assay: The compounds binding to MDM2 were determined by a quantitative fluorescence polarization binding assay using a recombinant MDM2 protein and fluorescently labeled peptide probes using a BioTek Cytation 5 machine. Compounds were tested in 10% DMSO, 100 mM potassium phosohate pH 7.2, 100 ug/ml bovine γ-globulin, 0.02% (w/v) sodium azideand 0.01% (v/v) triton X-100. KI values of tested compounds were determined in a dose-dependent competitive binding experiment by fitting the sigmoidal dose-dependent FP increases as a function of protein concentrations using Graphpad Prism software.
Measurement: Ki = 34000 nM (pChEMBL 4.46999979019165)
Descriptors: MW 435.31, ALogP 3.08, HBA 5, HBD 2, PSA 99.76, QED 0.77
Compound (SMILES): CC(=O)N[C@@H](C)CO[C@H]1C[C@H](COc2ncnc3c2ccn3CCC2CC2)C1
InChIKey: CFKYVJGUQPBHQG-WBAXXEDZSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Inhibitory Activity on Human ACC1 and the ACC2: Recombinant human ACC1 and recombinant human ACC2, which were prepared by the method mentioned above, were preincubated with assay buffer solution (50 mM HEPES-KOH (pH 7.4), 10 mM magnesium chloride, 6-10 mM potassium citrate, 4 mM reduced form of glutathione, 1.5 mg/ml bovine serum albumin) for one hour. Then, 0.2 μL of each this invention compound solution (in DMSO) were dispensed to 384-well microplate, 5 μL of the preincubated enzyme solution and 5 μL of substrate solution (50 mM HEPES-KOH (pH 7.4), 1 mM ATP, 0.8 mM acetyl CoA and 25-50 mM potassium bicarbonate) were added to microplate. After centrifugation and shaking, the reaction mixtures were incubated in a humidified box at room temperature for 1 to 3 hours. After the incubation, the enzyme reactions were stopped by the addition of EDTA. Then, after the samples were cocrystallized with CHCA (α-cyano-4-hydroxy cinnamic acid) matrices on MALDI target plate, by using the matrix assist laser deionization time-of-flight mass spectrometer (MALDI-TOF MS), samples were measured in reflector negative mode. Deprotonated ions of acetyl CoA (AcCoA) of substrate and malonyl CoA (MalCoA) of the reaction product were detected, then, the conversion rates of acetyl CoA to malonyl CoA was calculated by the intensity of [MalCoA-H]−/(Intensity of [MalCoA-H]−+Intensity of [AcCoA-H]−) using each signal strength. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate of the enzymatic reaction at each concentration of the compounds. In addition, potassium citrate concentrations in assay buffer solution, potassium hydrogen carbonate concentrations in substrate solution and incubation time were adjusted by each lot of enzyme.
Measurement: IC50 = 51 nM (pChEMBL 7.289999961853027)
Descriptors: MW 386.5, ALogP 2.93, HBA 6, HBD 1, PSA 78.27, QED 0.68
Compound (SMILES): C=CC(=O)Nc1ccc(F)c(Oc2nc(Nc3ccc(-n4nc(C)cc4C)cc3)nn3cccc23)c1
InChIKey: UZTZORWJUIMIIM-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Inhibitory Activity Assay: Specifically, the inhibitory activities of JAK3 and BTK kinase were measured using a JAK3 kinase assay kit (Promega, V9441) and a BTK kinase assay kit (Promega, V9071) which were purchased from Promega. Recombinant purified human JAK3 and BTK were diluted with 1× kinase reaction buffer (JAK3: 40 mM Tris-Cl, pH 7.5, 20 mM MgCl2, 0.1 mg/mL BSA and 50 uM DTT/BTK: 40 mM Tris-Cl, pH 7.5, 20 mM MgCl2, 0.1 mg/mL BSA, 2 mM MnCl2 and 50 uM DTT) and added to a 96 well plate (JAK3: final concentration of 4 ng per reaction/BTK: final concentration of 8 ng per reaction). The compounds were treated so as to be finally a 1% DMSO aqueous solution, and a substrate cocktail containing ATP (JAK3: final concentration of 5 uM/BTK: final concentration of 10 uM) and 0.2 ug/uL of Poly(Glu4, Tyr1)peptide (JAK3 and BTK final concentration) in the total 25 uL reactants was added to a 96-well plate to initiate enzymatic reaction. After incubation (30° C.) for 1 hour, equivalent volume (25 uL per reaction) of ADP Glo was added and incubated (30° C.) for 40 minutes at room temperature. Then, a kinase detection reagent (50 uL per reaction) was added and incubated (30° C.) for 30 minutes at room temperature.
Measurement: IC50 = 23.5 nM (pChEMBL 7.630000114440918)
Descriptors: MW 483.51, ALogP 5.33, HBA 8, HBD 2, PSA 98.37, QED 0.3
Compound (SMILES): C[C@@H]1C[C@H](n2c(Cn3cncn3)nc3cnc4ccc(C(F)(F)F)cc4c32)CCO1
InChIKey: PYFVAVZYKLONHU-TZMCWYRMSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LRRK2 Assay: LRRK2 kinase activity was measured using Lantha Screen technology from Invitrogen. GST-tagged truncated LRRK2 from Invitrogen (Cat #PV4874) was incubated with a fluorescein-labeled peptide substrate based upon ezrin/radixin/moesin (ERM), also known as LRRKtide (Invitrogen cat #PR8976A), in the presence of a dose response of compound. Upon completion, the assay was stopped and detected with a terbium labeled anti-phospho-ERM antibody (Invitrogen, cat #PR8975A). The assay was carried out under the following protocol: The compound dose response was prepared by diluting compound to a top concentration of 0.3 mM in 100% DMSO and serial diluted by half-log in DMSO to give an 11 point curve, 100× final assay concentration. Using Echo acoustic dispensing, 60 nL of compound was transferred to a low volume Corning 384-well assay plate. 3 μL of a working solution of substrate (200 nM LRRKtide, 2 mM ATP) prepared in assay buffer (50 mM HEPES, pH 7.5, 3 mM MgCl2, with 2 mM DTT and 0.01% Brij35 added fresh) was added to the 60 nL compound assay plate. The kinase reaction was started with 3 μL of a working solution of LRRK2 enzyme at a concentration of 4 μg/m L. The final reaction concentrations were 100 nM LRRKtide, 1 mM ATP, 2 μg/mL LRRK2 enzyme and a compound dose response with a top dose of 3 μM. The reaction was allowed to progress at room temperature for 30 minutes and then stopped with the addition of 6 μL of detection buffer (20 mM Tris pH 7.6, 0.01% NP-40, 6 mM EDTA with 2 nM terbium labeled antiphospho-ERM). After an incubation of 1 hour at room temperature, the plate was read on an Envision with an excitation wavelength of 340 nm and a reading emission at both 520 nm and 495 nm. The ratio of the 520 nm and 495 nm emission was used to analyze the data. Inhibition of mutant G2019S LRRK2 (Invitrogen cat #PV4881) was measured in the exact same method. All final concentrations of substrate ATP and enzyme were the same.
Measurement: IC50 = 8.32 nM (pChEMBL 8.079999923706055)
Descriptors: MW 416.41, ALogP 3.98, HBA 7, HBD 0, PSA 70.65, QED 0.51
Compound (SMILES): O=C(NCC1(C(=O)O)CCOCC1)c1cccc(COc2ccc(-c3ccn[nH]3)nc2)c1
InChIKey: WGBICTNDPVTQIN-UHFFFAOYSA-N
Target: Cytochrome P450 4F2 (SINGLE PROTEIN), Homo sapiens
UniProt: P78329
Assay: Inhibition Test for Each Compound of the Present Invention Against 20-HETE Producing Enzymes (CYP4F2 and CYP4A11): In the CYP4F2 inhibition test, the reaction solution containing each compound [final concentration of 50 mM, KPO4 (pH 7.4), 2.5 μM luciferine derivative, and 1 mM NADPH] was added to an Escherichia coli membrane fraction (100 μg/mL protein) in which human CYP4F2 had been expressed. In the CYP4A11 inhibition test, the reaction solution containing each compound [final concentration of 100 mM, Tris-HCl (pH 7.5), 60 μM luciferine derivative, 1.3 mM NADP+, 3.3 mM Glucose 6-Phosphate, 3.3 mM MgCl2, and 0.4 U/mL Glucose 6-Phosphate dehydrogenase] was added to an Escherichia coli membrane fraction (100 μg/mL protein) in which human CYP4A11 had been expressed. Following this, the membrane fraction was left to stand at room temperature for 60 minutes to perform an enzymatic reaction. After the reaction, a luciferine detection reagent was added, and the luminescence value was measured using a plate reader. By using that value, the percent inhibition of 20-HETE producing enzyme (%) was calculated according to the equation described below, and the 50% inhibitory concentration (IC50 value) for each compound was calculated.
Measurement: IC50 = 4500 nM (pChEMBL 5.349999904632568)
Descriptors: MW 436.47, ALogP 2.66, HBA 6, HBD 3, PSA 126.43, QED 0.5
Compound (SMILES): O=P(O)(O)C[C@H]1CC[C@H](c2ccc(-n3cnc4c(Cl)ncnc43)cc2)CC1
InChIKey: JQNDCIFORLMANV-JOCQHMNTSA-N
Target: Diacylglycerol O-acyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75907
Assay: Inhibition Assay: The inhibition of DGAT-1 activity can be assessed in intestinal microsome preparations by monitoring the incorporation of radiolabeled fatty acyl-CoA into DAG.Methods: Commercial microsomal preparations containing 60 ug of protein are incubated in assay buffer [20 uM 1,2-didecanloyl glycerol, 5 uM 14C decanoyl-CoA, 5 mM MgCl2, 0.4% BSA, 0.1% dimethyl sulfoxide (DMSO), 50 mM HEPES-pH 7.5] in the presence of varying concentrations of inhibitors that are dispensed from 100% DMSO stock solutions. Final assay volumes are 200 uL. Reactions are carried out for 45 minutes in 96-well polystyrene microtiter plates at ambient temperature. Following the ambient temperature incubation, assay mixtures are applied to a 96-well filter plate (Catalog # MSHVN4510, Millipore Inc.; Billerica, Mass.) under vacuum. The filter plate is pre-equilibrated with 100 uL of 70% ethanol followed by 200 uL of assay buffer. Filters are dried, removed and placed in scintillation vials with 4 mL of scintillation cocktail (Catalog #6013329; PerkinElmer Inc.; Waltham, Mass.). De novo 14C-TAG formed in the assay and trapped on the filters is quantified with use of a liquid scintillation counter (Model # LS6500 Beckman Coulter, Inc.; Fullerton, Calif.). The IC50 is defined as the concentration of compound that results in a 50% reduction in TAG synthesis.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 406.81, ALogP 3.92, HBA 5, HBD 2, PSA 101.13, QED 0.5
Compound (SMILES): COC1(C(C)(C)O)CCN(c2cc(-n3ncc4ccc([C@@]5(C#N)CC56CC6)cc43)ncn2)C1
InChIKey: UZGAFZZZTKPCDB-SKCDSABHSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LanthaScreen Assay: The LRRK2 kinase activity reported herein as IC50 values was determined with LanthaScreen technology from Life Technologies Corporation (Carlsbad, Calif.) using GST-tagged truncated human mutant G2019S LRRK2 in the presence of the fluorescein-labeled peptide substrate LRRKtide, also from Life Technologies. The data presented for the Km ATP LanthaScreen Assay represents mean IC50 values based on several test results and may have reasonable deviations depending on the specific conditions and reagents used. Assays were performed in the presence of 134 μM ATP (Km ATP). Upon completion, the assay was stopped and phosphorylated substrate detected with a terbium (Tb)-labeled anti-pERM antibody (cat. no. PV4898). The compound dose response was prepared by diluting a 10 mM stock of compound to a maximum concentration of 9.99 M in 100% dimethylsulfoxide followed by custom fold serial dilution in dimethylsulfoxide nine times. Twenty nanoliters of each dilution was spotted via a Labcyte Echo onto a 384-well black-sided plate (Corning 3575) followed by 15 μl of a 1.25 nM enzyme solution in 1× assay buffer (50 mM Tris pH 8.5, 10 mM MgCl2, 0.01% Brij-35, 1 mM EGTA, 2 mM dithiothreitol, 0.05 mM sodium orthovanadate). Following a 15-minute incubation at room temperature, the kinase reaction was started with the addition of 5 μl of 400 nM fluorescein-labeled LRRKtide peptide substrate and 134 μM ATP solution in 1× assay buffer. The reaction was allowed to progress at ambient temperature for 90 minutes. The reaction was then stopped by the addition of 20 μl of TR-FRET Dilution Buffer (Life Technologies, Carlsbad, Calif.) containing 2 nM Tb-labeled anti-phospho LRRKtide antibody and 10 mM EDTA (Life Technologies, Carlsbad, Calif.). After an incubation of 1 hour at room temperature, the plate was read on an EnVision multimode plate reader (Perkin Elmer, Waltham, Mass.) with an excitation wavelength of 337 nm (Laser) and a reading emission at both 520 and 495 nm. Compound IC50s were interpolated from nonlinear regression best fits of the log of the final compound concentration, plotted as a function of the 520/495-nm emission ratio using Activity base. Abase uses a 4 parameter (4P) logistic fit based on the Levenberg-Marquardt algorithm.
Measurement: IC50 = 15.1 nM (pChEMBL 7.820000171661377)
Descriptors: MW 444.54, ALogP 3.13, HBA 8, HBD 1, PSA 100.09, QED 0.65
Compound (SMILES): COCCC(C(=O)Nc1ccc(C(=O)O)cc1)n1cc(OC)c(-c2cc(Cl)ccc2-n2cnc(Cl)c2)cc1=O
InChIKey: ATCFLROFGUZFNA-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: The factor XIa inhibition of the substances according to the invention is determined using a biochemical test system 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 is 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; 5 mM of calcium chloride; 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).
Measurement: IC50 = 1.7 nM (pChEMBL 8.770000457763672)
Descriptors: MW 571.42, ALogP 4.93, HBA 8, HBD 2, PSA 124.68, QED 0.28
Compound (SMILES): O=C(CSc1nc2cnc(Cl)cc2[nH]1)Nc1cccc(O)c1
InChIKey: LMZXMYBQERYAME-UHFFFAOYSA-N
Target: Ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P22413
Assay: Inhibition Assay: Materials:    Assay Buffer: 1 mM CaCl2, 0.2 mM ZnCl2, 50 mM Tris, pH 9.0    Substrate: 8 mM Thymidine 5′-monophosphate p-nitrophenol ester sodium salt (Sigma Cat # T4510)    Enzyme: 5 ng/μL Recombinant Human ENPP-1 Protein (R&D Cat #6136-EN-010) DMSO    96-well clear assay platesMethods:An eight point serial dilution of drugs was prepared in 10× in assay buffer with the final assay concentrations starting at 10 μM, 3 μM, 1 μM, 0.3 μM . . . 0 μM. A dilution of DMSO was included as a control. The assay plate was set up as follows with each well in duplicate: 81 μL assay buffer+10 μL ENPP1 inhibitor or DMSO+5 μL Substrate+4 μL Enzyme. Both the enzyme and substrate were added to opposite sides of the well to ensure that there was no interaction until all wells had both components. The plate was then centrifuged gently for 10 seconds, followed by an incubation at 37° C. for 45 minutes. The reaction was quantified by measuring absorbance at 405 nm using the Envision.
Measurement: IC50 = 7932 nM (pChEMBL 5.099999904632568)
Descriptors: MW 334.79, ALogP 3.05, HBA 5, HBD 3, PSA 90.9, QED 0.5
Compound (SMILES): O=C(NCc1ccc(Cl)cc1Cl)c1ccc(CN2CCN3C(=O)CCC3C2)s1
InChIKey: XHLYSPFHJSLMDU-UHFFFAOYSA-N
Target: Bifunctional epoxide hydrolase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34913
Assay: Biochemical Screening Assay: Compounds were tested in a biochemical screening assay using recombinant sEH purified from Sf9 insect cells and an artificial substrate, (3-phenyl-oxiranyl)-acetic acid cyano-(6-methoxy-naphtalen-2-yl)-methyl ester, Phome. The biochemical assay was performed in analogy to a fluorometric described in the literature (P. D. Jones et al., Anal. Biochem. 2005, 343, 66-75). The assay principle bases on the sEH-catalyzed hydrolysis of an artificial α-cyano-ester substrate. O-deacetylation liberates a cyanohydrin intermediate that rapidly decomposes to the highly fluorescent 6-methoxy-2-naphtaldehyde. To discriminate sample autofluorescence, the assay was carried out as a kinetic measurement with two time points. The first measurement is performed immediately before addition of the substrate and the 15 second measurement is done after completion of the assay. The assay format is either in 96- or in 384-well format.Details of the assay using a 96-well plate format are described below. 40 μl recombinant sEH enzyme and 5 μl test compound are pre-incubated for 15 minutes at 30° C. Following pre-incubation, the reaction is started by addition of 5 μl Phome. The assay mixture containing 2 nM final sEH concentration, test compound ranging in a concentration from 0.0001-10 μM and 5 μM Phome is incubated for 60 minutes at 30° C.
Measurement: IC50 = 1.67 nM (pChEMBL 8.779999732971191)
Descriptors: MW 438.38, ALogP 3.79, HBA 4, HBD 1, PSA 52.65, QED 0.77
Compound (SMILES): COc1ccc(C[C@@H](C(=O)O)N2CCN(C(=O)[C@@H]3CN(C(C)(C)C)C[C@H]3c3ccc(Cl)cc3F)[C@@H](C)C2)c(F)c1
InChIKey: MHDZXNALJQBUON-VSNQPBSISA-N
Target: Melanocortin receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P32245
Assay: Test for Evaluating Human MC Receptor Activation, Using Cells Expressing Human MC4, MC1, or MC3 Receptor: Experiment Method(1) Construction of Human MC Receptor-Expressing VectorA human MC4 receptor gene (GenBank Accession No.: NM_005912.2), a human MC1 receptor gene (GenBank Accession No.: NM_002386.3), or a human MC3 receptor gene (GenBank Accession No.: NM_019888.3) was introduced into an expression vector pcDNA 3.1/V5-His TOPO (registered trademark) (Thermo Fisher Scientific Inc.).(2) Construction of Cells Transiently Expressing Human MC ReceptorAn expression vector for a human MC4, MC1, or MC3 receptor was introduced into FreeStyle 293-F cell (Thermo Fisher Scientific Inc., product number: R790-07). For the introduction, electroporation was employed. That is, 1×107 FreeStyle 293-F cell were suspended in 80 μL of an electroporation buffer (Thermo Fisher Scientific Inc., product number: B201-100), and 20 μg of the expression vector was added thereto. The resultant was put into a cuvette (OC-100 Processing Assembly, MaxCyte, Inc.) and electroporated with MaxCyte SIX (registered trademark) (MaxCyte, Inc.). The cells were cultured over one day, suspended in a Cell Banker (registered trademark) 1 (JUJI FIELD Inc.), product number: BLC-1), and stored frozen until their use.(3) Measurement of Amount of cAMP ProducedMeasurement was carried out by using a LANCE (registered trademark) Ultra cAMP Kit (PerkinElmer, Inc.) in accordance with the attached instructions. That is, after dissolution in DMSO, the test compound (a final concentration of 1 pM to 30 μM) diluted with an assay buffer (Hank's balanced salt solution, 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.1% bovine serum albumin, pH 7.4), or α-MSH (Bachem Inc., a final concentration of 1 pM to 30 μM) was added to OptiPlate-384 (PerkinElmer, Inc.). Furthermore, a suspension of cells transiently expressing the human MC4, MC1, or MC3 receptor prepared by using the assay buffer was added thereto at 1,000 cells/well, followed by being left to stand at room temperature for about 1 hour. Thereafter, an Eu-cAMP tracer solution and an ULight-anti-cAMP solution were added thereto, followed by being left to stand at room temperature for about 1 hour. The amount of cAMP was calculated using EnVision (registered trademark) (PerkinElmer Inc.).For the agonistic activity, an efficacy (EC50 (μM)) was calculated by a non-linear regression method with a Sigmoid-Emax model, by defining the maximum reaction with α-MSH as 100% and the reaction with the vehicle alone as 0%, respectively.
Measurement: EC50 = 5.1 nM (pChEMBL 8.289999961853027)
Descriptors: MW 578.1, ALogP 4.67, HBA 5, HBD 1, PSA 73.32, QED 0.52
Compound (SMILES): O=C(CN1CCN(C(=O)Cn2cc3cc(NC(=O)c4cccc(C(F)(F)F)n4)ccc3n2)CC1)NC1CC1
InChIKey: MFOIUVAOBPOQLT-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Kinase Assay: For the assay, 11 different concentrations in the range from 20 μM to 0.073 nM were prepared from a 2 mM solution of the test substance in DMSO. For the assay, 50 nl of the respective solution were pipetted into a black low-volume 384-well microtitre plate (Greiner Bio-One, Frickenhausen, Germany), 2 μl of a solution of Irak4 in assay buffer [50 mM HEPES pH 7.5, 5 mM MgCl2, 1.0 mM dithiothreitol, 30 μM activated sodium orthovanadate, 0.1% (w/v) of bovine gamma-globulin (BGG) 0.04% (v/v) nonidet-P40 (Sigma)] were added and the mixture was incubated for 15 min to allow prebinding of the substances to the enzyme prior to the kinase reaction. The kinase reaction was then started by addition of 3 μl of a solution of adenosine triphosphate (ATP, 1.67 mM=final concentration in 5 μl of assay volume: 1 μM) and peptide substrate (0.83 μM=final concentration in 5 μl assay volume: 0.5 μM) in assay buffer, and the resulting mixture was incubated at 22° C. for the reaction time of 45 min. The concentration of the Irak4 was adjusted to the respective activity of the enzyme and set such that the assay was carried out in the linear range. Typical concentrations were in the order of about 0.2 nM. The reaction was stopped by addition of 5 μl of a solution of TR-FRET detection reagents [0.1 μM streptavidin-XL665 (Cisbio Bioassays; France, catalogue No. 610SAXLG) and 1.5 nM anti-phosphoserin antibody [Merck Millipore, STK Antibody, catalogue No. 35-002] and 0.6 nM LANCE EU-W1024-labelled anti-mouse-IgG antibody (Perkin-Elmer, product No. AD0077, alternatively it is possible to use a terbium cryptate-labelled anti-mouse-IgG antibody from Cisbio Bioassays) in aqueous EDTA solution (100 mM EDTA, 0.4% [w/v] bovine serum albumin [BSA] in 25 mM HEPES pH 7.5).
Measurement: IC50 = 511 nM (pChEMBL 6.289999961853027)
Descriptors: MW 529.52, ALogP 2.13, HBA 7, HBD 2, PSA 112.46, QED 0.49
Compound (SMILES): Cc1cn(C(=O)c2c(Cl)ccc(C(=O)N3CCC(C(=O)O)CC3)c2Cl)c2c(C)cc(C(F)(F)F)cc12
InChIKey: FOPRDSZNQDJPIG-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: transient transactivation assay: The primary screen was performed by transient transactivation assays. These cell-based assays were carried out using Cos-7 cells transfected with a chimeric human RORγ-Gal4 receptor expression plasmid and a 5Gal4 pGL3 TK Luc reporter plasmid. Transfections were performed by a chemical agent (Jet PEI). Transfected cells were distributed in 384-wells plates and were allowed to recover for 24 h. The culture medium was then removed and fresh medium containing the compounds to be tested was added (final concentration ranging from 10−4 M to 3 10−10 M). After an overnight incubation, luciferase expression was measured by adding SteadyGlo according to the manufacturer's instructions (Promega). T0901317 at 10−5 M was used as reference. Results were expressed as fold induction compared to basal level or as percentage activity compared to references taken as 100% (basal) or 0% (T0901317). Dose-effect curves and IC50 were calculated using the software Assay Explorer (MDL).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 541.35, ALogP 6.21, HBA 4, HBD 1, PSA 79.61, QED 0.43
Compound (SMILES): COc1cc(C(=O)N2CC3CCC2[C@@H]3N)cc2nc(-c3cc4ccc(-c5c(C)nn(C)c5C)nc4n3CC3CC3)n(C)c12
InChIKey: IFXVSEZMBAULFI-CXBZOWBPSA-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 = 35 nM (pChEMBL 7.460000038146973)
Descriptors: MW 578.72, ALogP 4.59, HBA 9, HBD 1, PSA 109.02, QED 0.32
Compound (SMILES): CCN1CCN(C(=O)c2ccc(-n3ncc(-c4cc(F)c(C#N)cc4C)c3O)nc2)C[C@H]1C
InChIKey: KAOROSNDUNJDEV-MRXNPFEDSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZT9
Assay: Inhibition Assay: The IC50 values for the PHD2 enzyme (residues 181-417) were determined by mixing increasing amounts of inhibitor with a fixed amount of enzyme (5 nM, final concentration) and Biotin labeled peptide (Biotin-Asp-Leu-Glu-Met-Leu-Ala-Pro-Tyr-Ile-Pro-Met-Asp-Asp-Asp-Phe-Gln-Leu, 1 uM final concentration) and 2-Oxyglutarate (2 uM final concentration) in 50 mM HEPES, 50 mM KCl, 0.5 mM TCEP, 2 uM FeCl2, 0.1 mg/ml BSA, at pH 7.3. The reaction was conducted by pre-incubating the enzyme in the presence of inhibitor for 60 min at room temperature. The activity of the free enzyme was measured by adding the peptide, the 2-Oxoglutarate (see above for final concentrations), and Ascorbic Acid (1 mM final concentration). The enzymatic activity was quenched after 60 min by adding an excess of a tight binding inhibitor to the assay mixture. The amount of product released was measured by using a LC/MS system (Agilent HPLC with Applied Biosystems API3000 Mass Spectrometer).
Measurement: IC50 = 3.31 nM (pChEMBL 8.479999542236328)
Descriptors: MW 448.5, ALogP 3.13, HBA 7, HBD 1, PSA 98.28, QED 0.66
Compound (SMILES): Cc1ccc2c(c1)c(C(N)=O)c1n2C[C@@](NC(=O)c2ccc(-n3cnc(C)n3)cc2Cl)(c2ccccc2)CC1
InChIKey: SXZJLEXYVSTFDU-SSEXGKCCSA-N
Target: Coagulation factor IX (SINGLE PROTEIN), Homo sapiens
UniProt: P00740
Assay: TBD: TBD
Measurement: IC50 = 2.99 nM (pChEMBL 8.520000457763672)
Descriptors: MW 539.04, ALogP 4.86, HBA 6, HBD 2, PSA 107.83, QED 0.33
Compound (SMILES): CC1(C)C(=O)N(c2ccc(C#N)c(C(F)(F)F)c2)C(=O)N1CCOc1ccc(-c2cc[n+]([O-])cc2)cc1
InChIKey: WLCPAHOLUKGJLC-UHFFFAOYSA-N
Target: Androgen receptor (SINGLE PROTEIN), Mus musculus
UniProt: P19091
Assay: Androgen Receptor Assay: Androgen binding is measured using the hydroxylapatite (HAP) assay. In brief, the radioactive steroid [3H]R1881 solubilized in ethanol is diluted with buffer B (10 mM Tris-HCl, 1.5 mM EDTA disodium salt, 10 mM α-monothioglycerol, pH 7.4). Aliquots of the cell or prostate cytosol preparation (0.1 ml) are then incubated with 5 nM [3H]R1881 (0.1 ml, ¿100 000 cpm) in the presence or absence of the indicated concentrations of unlabeled compounds (0.1 ml, prepared in buffer B containing 30% ethanol) for 16-18 h at 0-4° C. Triamcinolone acetonide (TAC; 100 nM) is added to mask progesterone receptors. Unbound steroids are separated by incubation for 40 min at 0-4° C. with 0.3 ml HAP prepared in buffer P (50 mM Tris-HCl, 10 mM KH2PO4, pH 7.4). After incubation with HAP and 10 min of centrifugation at 1000×g, the pellet is washed 3 times with 1 ml of buffer P. Thereafter, the radioactivity is extracted from the pellet by incubation at room temperature for 60 min with 1 ml of ethanol. After centrifugation, the supernatant is decanted into a scintillation vial and the pellet is extracted again with ethanol. After the addition of scintillation liquid, the radioactivity is measured in a liquid scintillation counter.
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 510.47, ALogP 4.5, HBA 5, HBD 0, PSA 100.58, QED 0.28
Compound (SMILES): Nc1ncnc2c1c(-c1ccc3nc(OC4CC4)ccc3c1)nn2CC1(O)CCNCC1
InChIKey: OCBNPAIXXHWADI-UHFFFAOYSA-N
Target: Calmodulin-domain protein kinase 1 (SINGLE PROTEIN), Toxoplasma gondii
UniProt: Q9BJF5
Assay: Enzymatic Inhibition Assay: Inhibitors were evaluated in triplicate in eight-point dilutions (3-fold dilutions) during the enzymatic reactions. TgCDPK1 enzymatic inhibition was determined with a coupled luciferase assay (Kinaseglo®). 2.1 nM TgCDPK1 and 20 μM BioSyntide-2 (American Peptide Company, Inc. Sunnyvale, Calif.)) were incubated in 25 μL of buffer containing 1 mM EGTA (pH 7.2), 10 mM MgCl2, 20 mM HEPES, pH 7.5 (KOH), 0.1% BSA, and 2 mM CaCl2. The reaction was initiated with the addition of ATP at a 10 μM final concentration. After incubating at 30° C. for 90 min., changes in ATP concentration were determined by adding Kinaseglo® luciferase reagent (Promega, Madison, Wis.) and measuring luminescence with a MicroBeta2 multi-label plate reader (Perkin Elmer, Waltham, Mass.). Results were converted to percent inhibition, and IC50 values were calculated using nonlinear regression analysis in GraphPad Prism.
Measurement: IC50 = 0.7 nM (pChEMBL 9.149999618530273)
Descriptors: MW 431.5, ALogP 2.28, HBA 9, HBD 3, PSA 124, QED 0.44
Compound (SMILES): O=c1c2cn[nH]c2c2cnc(-c3ccccc3C(F)(F)F)cc2n1CC(F)(F)F
InChIKey: AEPCXOGUHSYOCC-UHFFFAOYSA-N
Target: Methionine aminopeptidase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P50579
Assay: Enzyme Assay: For the enzymatic test, human MetAP2 protein was obtained from a culture supernatant of insect cells (sf9) infected with MetAP2 recombinant baculovirus.Before performing the experiment, dialysis of the MetAP2 supernatant was performed over 24 hours at 4° C. in a buffer (10 mM Hepes, 100 mM KCl, 10% glycerol, pH 7.4) in the presence of EDTA (1 mM) over the first 12 hours.The dialysis supernatant was recovered and manganese, used as cofactor, was added to a final concentration of 300 μM.The enzymatic test is a two steps procedure.In a first step, it consists in placing in contact the compound according to the invention, the dialysed MetAP2 protein and the substrate (Met-Pro-Arg-pNa peptide synthesized by Neosystem), the N-terminal methionine of which can be cleaved with MetAP2, and which bears at the C-terminal end a para-nitroaniline (pNa) chromophore, which can itself be released by another peptidase only when the N-terminal methionine has been cleaved beforehand.Consequently, the second step consists in reacting the peptides cleaved in the preceding step with a second peptidase in order to release the chromophore. The peptidase used in this second step is cathepsin, which comes from the TagZyme DAPase kit (Quiagen, 34366).The MetAP2 activity is proportional to the amount of para-nitroaniline released, which is measured by absorbance at 405 nm.
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 412.29, ALogP 4.52, HBA 4, HBD 1, PSA 63.57, QED 0.49
Compound (SMILES): COc1ccccc1C(Cn1c(=O)n(C2CN(C(=O)N3CCOCC3)C2)c(=O)c2c(C)c(-c3ncco3)sc21)OC1CCOCC1
InChIKey: YXSKGGGOAINTSB-UHFFFAOYSA-N
Target: Acetyl-CoA carboxylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13085
Assay: ACC1 Enzyme Activity Assay: 4.5 μL of 2.2×ACC1 enzyme (2 nM) working solution was added to a 384-well plate; and then 0.5 μL of different concentrations of compound were added. The mixture was incubated at room temperature for 15 min.2× Substrate (40 μM ATP, 20 μM acetyl CoA, 60 mM NaHCO3) was prepared using the buffer prepared in 2.1; 5 μL of 2× substrate was added to the 384-well plate, and the mixture was incubated at room temperature for 30 min; 10 μL ADP-Glo reagent was added, the mixture was incubated at room temperature for 40 min, and then the reaction was terminated; finally, 20 μL of the enzyme detection reagent was added, the mixture was incubated at room temperature for 40 min, and an Envision 2104 instrument was used to read the relative light units (RLU).
Measurement: IC50 = 1.22 nM (pChEMBL 8.90999984741211)
Descriptors: MW 651.74, ALogP 3.44, HBA 12, HBD 0, PSA 130.5, QED 0.28
Compound (SMILES): CCN1CCC(c2ccc(Cn3c(-c4cccnc4)nc4c(C)nc(OC)nc43)c(F)c2)CC1
InChIKey: AGYICJMUUVAHGP-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: Inhibition of the Activation Assay: As human TLR7 expressing cell line, HEK293 cell line was bought from IMGENEX Corporation (TLR7/NF-ηB/SEAPorter HEK293 cell), which is human embryonic kidney cell line and stably expresses full-length human TLR7 gene and secreted alkaline phosphatase (SEAP) reporter gene under the transcriptional control of an NE-ηB response element. The TLR7/NF-ηB/SEAPorter HEK293 cell was cultivated with DMEM containing 10% fetal bovine serum (FBS) and 10 μg/mL blasticidin S at 37° C. in the presence of 5% CO2. The TLR7/NF-ηB/SEAPorter HEK293 cell was seeded into 96-well microtiter plate at 5×104 cell/90 μL/well, and the place was cultivated at 37° C. in a CO2 incubator overnight. Each test compound that was diluted with the medium was added to the wells (10 μL/well), wherein each final concentration was adjusted to 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 μmol/L, or 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 μmol/L. After 0.5 hour, R-848 that is TLR7/8 ligand was added to each well (10 μL/well), wherein each final concentration was adjusted to 200 nmol/L. The total volume was adjusted to 110 μL/well, the test samples were incubated in CO2 incubator for 20±1 hours, and then the SEAP activity was measured as activation of TLR7. The SEAP activity was evaluated as follows: p-nitro-phenyl phosphate (pNPP) (Invitrogen) was added to the incubated sample (50 μL/well); after 15 minutes, 4 mol/L sodium hydroxide solution (nacalai tesque) was added thereto (50 μL/well) to quench the reaction; and the absorbance of each sample was measured at 405 nm with microplate reader Elx808 (BioTek). The 50% inhibitory concentration (IC50 value) of each sample compound was calculated based on 100% of the SEAP activity wherein the test sample comprises no sample compound.
Measurement: IC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 460.56, ALogP 4.59, HBA 7, HBD 0, PSA 68.96, QED 0.42
Compound (SMILES): CC(C)n1cnnc1-c1cccc(N2Cc3ccc(-c4cc(C(=O)O)ccc4C#N)cc3C2=O)n1
InChIKey: XLMRJTYKAWSBKK-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99683
Assay: Kinase Activity Inhibition Assay: Inhibition of ASK1 kinase activity was determined radiometrically using 33P substrate incorporation (Reaction Biology Corp., Malvern, Pa.). Briefly, recombinant human ASK1 protein, 20 μM substrate (myelin basic protein (MBP)), and compound (diluted from DMSO stock to give 1% final DMSO) were incubated in Base Reaction buffer (20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO) at room temperature for 20 minutes. The reaction was initiated by addition of 10 μM 33P-ATP (specific activity 10 μCi/4l). The reaction was then allowed to proceed for 2 hours at room temperature. Upon reaction termination, the mixture was spotted on P81 exchange paper and kinase activity by incorporation of 33P into MBP substrate was detected by filter-binding method.
Measurement: IC50 = 15.6 nM (pChEMBL 7.809999942779541)
Descriptors: MW 464.49, ALogP 4.32, HBA 7, HBD 1, PSA 125, QED 0.47
Compound (SMILES): CCNc1nc(Nc2cn([C@H]3C[C@H](c4cocn4)C3)nc2C)ncc1C(F)(F)F
InChIKey: LOBBYCBRFZXCNJ-HAQNSBGRSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Biochemical Assay : 50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Brij-35, 2 mM DTT, 5 nM LRRK2, 134 μM ATP, 60 minute reaction time, 23° C. reaction temperature, and 10 μL total reaction volume.Detection Reaction Conditions:1×TR-FRET dilution buffer, 10 mM EDTA, 2 nM antibody, 23° C. reaction temperature, and 10 μL total reaction volume.Compound solutions were prepared by initially diluting to 1 mM with DMSO. 35 μL of reference compound solution, 35 μL of test compound solution, and 35 μL HPE were successively added to the source plate (384-well assay plate, Labcyte). The plates were centrifuged at 2500 rpm for 1 minute and sealed in foil. POD was used to perform a 3.162 fold serial dilution and 100 nL of reference compound solution, test compound solution, HPE and ZPE were transferred to assay plates. The assay plate was centrifuged at 2500 rpm for 1 minute, and sealed with foil.
Measurement: IC50 > 60 nM (pChEMBL 7.2220001220703125)
Descriptors: MW 407.4, ALogP 4.28, HBA 8, HBD 2, PSA 93.69, QED 0.63
Compound (SMILES): O=C(c1ccc2c(c1)N(c1ncc(-c3ccccc3F)cn1)CC21CC1)N1CCC(CO)CC1
InChIKey: BLCMXCFUVBPCRY-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4B (SINGLE PROTEIN), Homo sapiens
UniProt: Q07343
Assay: cAMP HTRF Assay to Determine the Activity of hPDE4B1: The inhibiting effect of the compounds on the enzyme activity of human PDE4B1 was measured by the quantification of 5′-adenosine monophosphate (5′-AMP), which is formed from 3′,5′-cyclic adenosine monophosphate (cAMP). Human recombinant enzyme, expressed in Sf9 cells, and the HTRF (homogeneous time-resolved fluorescence) detection method were used in the assay.The test compound or water (control) was mixed with the human recombinant PDE4B1 enzyme (4.8 U) in a buffer consisting of 44.4 mM tris-HCl, 5.28 mM MgCl2, 2.64 mM DTT and 0.044% Tween 20 (pH 7.8). After adding the cAMP enzyme substrate (final concentration 40 nM), the mixture was incubated for 30 minutes at room temperature. Then a fluorescence acceptor (Dye2 marked with cAMP), a fluorescence donor (anti-cAMP antibody marked with a europium cryptate) and the non-specific phosphodiesterase inhibitor IBMX (3-isobutyl-1-methylxanthine; final concentration 1 mM) were added. After 60 min the fluorescence transfer, which correlates with the amount of remaining cAMP, was measured with a microplate reader (Rubystar, BMG) at λex=337 nm, λem=620 nm and λem=665 nm. The enzyme activity was calculated from the quotient formed from the measured signal at 665 nm and that at 620 nm. The result was expressed as the percentage inhibition of enzyme activity of the control (without PDE4 inhibitor). The enzyme was omitted for measurement of the basal control.[N. Saldou et al., Comparison of recombinant human PDE4 isoforms: interaction with substrate and inhibitors, Cell. Signal. Vol. 10, No. 6, 427-440, 1998].
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 458.54, ALogP 4.31, HBA 5, HBD 1, PSA 69.56, QED 0.63
Compound (SMILES): Cc1c(-c2[nH]c3ccc(N[C@@H]4C[C@H](C)N(CC#N)C4)nc3c2C(C)C)cn2ncnc2c1C
InChIKey: JLAOGQKGFFXIMM-MAUKXSAKSA-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 = 0.86 nM (pChEMBL 9.069999694824219)
Descriptors: MW 442.57, ALogP 4.41, HBA 7, HBD 2, PSA 97.93, QED 0.44