lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1cccc(C(=O)N2C3CCC2C(Nc2ccc(C#N)cn2)C3)c1-c1ncco1
InChIKey: LJIWEDLNXOPGTL-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P56718
Assay: Rat and Human Orexin 1 Receptor Radioligand Binding Assay: Human Embryonic Kidney 293 cells (HEK293) stably expressing rat orexin 1 receptor (Genebank accession number NM_001525) or Chinese ovary cells (CHO) stably expressing human orexin 1 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM (Hyclone, cat #SH30022), 10% FBS, 1× Pen/Strep, 1× sodium pyruvate, 10 mM HEPES, 600 μg/mL G418 and DMEM/F12 (Gibco, Cat #11039), 10% FBS, 1× Pen/Strep, 600 μg/mL G418 media, respectively on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1× with Calcium and Magnesium, Cat #SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2K×G, 5 min at 4° C.), the supernatant was aspirated and the pellets frozen and stored at −800 C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and homogenized for 45 sec prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H]-(1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone) (Moraveck Corporation, specific activity=35.3 Ci/mmol), diluted to a 10 nM concentration in PBS (4 nM final). Compounds were solubilized in 100% DMSO (Acros Organics, Cat. #61042-1000) and tested over a range of 7 concentrations (from 0.1 nM to 10 μM). The final concentration of DMSO in the reactions is equal to or less than 0.1%. Total and nonspecific binding was determined in the absence and presence of 10 μM almorexant. The total volume of each reaction is 200 μL (20 μL of diluted compounds, 80 μL of [3H]-(1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone) diluted in PBS and 100 μL of the cell suspension). Reactions were run for 60 min at room temperature and terminated by filtration through GF/C filter plates (PerkinElmer, Cat. #6005174) presoaked in 0.3% polyethylenimine using the cell harvester (PerkinElmer Filtermate). The plates were washed 3 times by aspirating 30 ml PBS through the plates. Plates were dried in 55° C. oven for 60 min, scintillation fluid was added, and the radioactivity was counted on a Topcount (Packard).
Measurement: Ki = 187 nM (pChEMBL 6.730000019073486)
Descriptors: MW 399.45, ALogP 3.77, HBA 6, HBD 1, PSA 95.05, QED 0.72
Compound (SMILES): Cc1cc(N)nc(C)c1CNC(=O)c1cnn(Cc2ccc3ncc(Cl)cc3c2)c1C#N
InChIKey: JDHSXDQGXRVALU-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Enzyme Inhibition: A 10 mM solution of the test compound was made in DMSO. This solution was serially diluted 1:5 in DMSO to yield 2000, 400, 80, 16, 3.2, 0.64, 0.128, 0.0256 and 0.00512 μM compound test solutions. A control tube containing only DMSO is included. 16 μL of each compound test solution was combined with 384 μL of assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.01% Triton X-100) to yield a 4× test compound buffer stock.Separately, a 40 nM solution of human Plasma Kallikrein (Abcam) and a 93.6 μM solution Pro-Phe-Arg-AMC (Bachem) were made using assay buffer. These solutions are hereby referred to as 4×hPK and 2×PFR-AMC, respectively.60 μL of each 4× test compound buffer stock was combined with 60 μL of 4×hPK to yield 120 μL of 2× test compound buffer stock/2×hPK. 50 μL was removed from this mixture and placed into duplicate wells on a Microfluor 1Black U-bottom microtiter plate (Thermo Scientific). This plate was incubated for 5 minutes at 37° C. To each well, 50 μL of pre-warmed 2×PFR-AMC was added to start the enzymatic reaction. Cleavage of PFR-AMC was monitored in a Biotek Synergy H4 reader set at 37° C. Readings are taken every 43 seconds for 1 hour. The highest mean velocity over 20 reads (15 minutes) is used to calculate the IC50. The IC50 is calculated using the Gen5 (Biotek Instruments).
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 445.91, ALogP 3.53, HBA 7, HBD 2, PSA 122.51, QED 0.48
Compound (SMILES): CCNC(=O)c1cc(Nc2nc(-c3cnc4c(c3)N(C3CC(C)(N5CCCC6(CC6)C5)C3)C(=O)C4(C)C)cc3ncn(C(C)C)c23)c(F)c(F)c1C
InChIKey: VHAXLFVPMAAEEN-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: TBHPK1 IC50 Assay: Ten nanoliters of test compounds dissolved in DMSO at various concentrations were dispensed into a 384-well ProxiPlate (PerkinElmer #6008289). Five microliters of a solution of recombinant HPK1 diluted in HPK1 kinase assay buffer (50 mM BES [N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid], pH 7.0; 10 mM magnesium chloride; 0.01% Triton X-100; 1 mM dithiothreitol; 0.01% bovine serum albumin; 0.1 mM sodium orthovanadate) was added to the compound-containing plate and was incubated for 15 minutes at 25° C. Five microliters of a mixture of ATP (Sigma-Aldrich #A6559) and peptide substrate STK S1 (Cisbio #61ST1BLC) diluted in HPK1 kinase assay buffer was then added to start the reaction. Final concentrations were 0.15 nM for HPK1, 10 μM for ATP, and 1 μM for the STK S1 peptide substrate. The reaction mixture was incubated at 25° C. for 3 hours and was stopped with the addition of 10 μl of an EDTA (Ethylenediaminetetraacetic acid)-containing detection buffer (Cisbio #62SDBRDF) supplemented with Europium cryptate-labeled anti-phospho-serine/threonine antibodies (Cisbio #62ST1PEJ) and XL665-labeled streptavidin (Cisbio #610SAXLG). The mixture was incubated for 16 hours at room temperature and peptide phosphorylation was measured by time-resolved fluorescence energy transfer (665 nm/620 nm) on an Envision plate reader (PerkinElmer).
Measurement: IC50 = 0.1 nM (pChEMBL 10)
Descriptors: MW 710.87, ALogP 7.58, HBA 8, HBD 2, PSA 108.28, QED 0.19
Compound (SMILES): CCc1cc(CN2CC3(C2)CN(S(C)(=O)=O)C3)c(F)cc1-c1ccc(C(O)(C(F)(F)F)C(F)(F)F)cc1
InChIKey: SWDYXNSGLCCVEP-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Gal4 Ligand Binding Assay: Compounds of the present invention were tested in a human RORγ ligand binding assay using a commercially available cell based ligand binding reporter assay in 96-well format (Cat #1304001, INDIGO Biosciences, State College, Pa.). The N-terminal DNA binding domains (DBD) of the native RORγ and RORγt receptors have been substituted with that of the yeast GAL4-DBD and stably transfected in HEK293T cells that also stably express luciferase under the regulation by upstream activation sequence of yeast Gal4. These cells constitutively express high level RORγ activity due to binding of endogenous co-factors. Both agonist and inverse agonist activity can be detected. The assay was performed according to kit manufacturer's instructions as follows. 10 mM compound stocks were diluted serially 1:3 with DMSO and further diluted with provided media to generate 10 titration points from 60 μM to 3 nM. These treatment conditions were added to the plates as 2× media in 100 μL volume. Each plate includes a positive control with 10 titration points as well as 6 negative control wells with vehicle only, with final DMSO concentration of 0.2%. RORγ reporter cells were rapidly thawed and added to the plates in 100 μL volume. The plates were incubated for 24 h in a 37° C. humidified 5% CO2 incubator. Media was removed before the addition of room temperature luciferous detection substrate. After 5 minute incubation, relative light units (RLUs) were quantified using a plate reading luminometer. Data was normalized to positive control wells with only 0.2%0DMSO. Before establishing internal controls, ursolic acid was used as control.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 554.53, ALogP 4.44, HBA 4, HBD 1, PSA 60.85, QED 0.54
Compound (SMILES): O=C(O)c1cnn(-c2nc3cc(Oc4ccccc4)c(C(F)(F)F)cc3[nH]2)c1
InChIKey: YJCUECDMBKMFIJ-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZT9
Assay: Enzyme Activity Assay: The PHD2181-417 polypeptide (3 μg) was pre-incubated for 30 minutes with test compound prior to assessing the enzymatic activity of the polypeptide. The PHD enzymatic assay was then performed by transferring the purified PHD2181-417 polypeptide (3 μg) mixture with compound to 0.5 ml of reaction mixture containing the following: synthetic HIF-1α peptide comprising residues [KNPFSTGDTDLDLEMLAPYIPMDDDFQLRSFDQLS] (10 μM, California Peptide Research Inc., Napa, Calif.), and [5-14C]-2-oxoglutaric acid (50 mCi/mmol, Moravek Chemicals, Brea, Calif.) in reaction buffer (40 mM Tris-HCl, pH 7.5, 0.4 mg/ml catalase, 0.5 mM DTT, 1 mM ascorbate) for 10 minutes in the presence of compound. The reaction was stopped by addition of 50 μl of 70 mM H3PO4 and 50 μl of 500 mM NaH2PO4, pH 3.2. Detection of [14C]-succinic acid was achieved by separating from [5-14C]-2-oxoglutaric acid by incubating the reaction mixture with 100 μl of 0.16 M DNP prepared in 30% perchloric acid. Next, 50 μl of unlabeled 20 mM 2-oxoglutaric acid/20 mM succinic acid, serving as carrier for the radioactivity, was added to the mixture, and was allowed to proceed for 30 minutes at room temperature. The reaction was then incubated with 50 μl of 1 M 2-oxoglutaric acid for 30 additional minutes at room temperature to precipitate the excess DNP. The reaction was then centrifuged at 2800×g for 10 minutes at room temperature to separate [14C]-succinic acid in the supernatant from the precipitated [14C]-dinitrophenylhydrazone. Fractions of the supernatant (400 μl) were counted using a beta counter (Beckman Coulter, Fullerton, Calif.). Inhibition of PHD2181-417 activity was measured as a decrease in [14C]-succinic acid production. The IC50 values were estimated by fitting the data to a three-parameter logistic function using GraphPad Prism, version 4.02 (Graph Pad Software, San Diego, Calif.). IC50 values up to 10 μM were quantified otherwise were noted as >10 μM. All compounds were diluted at 10 mM in 100% DMSO (w/v) and tested from 10 μM to 3 nM at half-log serial dilutions, with a final concentration of 2% DMSO (w/v) in the assay.
Measurement: IC50 = 63.1 nM (pChEMBL 7.199999809265137)
Descriptors: MW 388.31, ALogP 4.26, HBA 5, HBD 2, PSA 93.03, QED 0.55
Compound (SMILES): O=C(Nc1ccc2c(OCC3COCCO3)ncc(F)c2c1)c1ncccc1O
InChIKey: AVCHEXLXHCWRDN-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14833
Assay: Metabotropic Glutamate Receptor Activity: For human mGluR4 assays, a fluorescence baseline was established for 3-5 seconds, the disclosed compounds were then added to the cells, and the response in cells was measured. Approximately two and a half minutes later, a concentration of mGluR4 orthosteric agonist (e.g. glutamate, L-AP4, or L-SOP) eliciting approximately 20% (EC20) of the maximal agonist response was added to the cells, and the response was measured. Two minutes later, a concentration of mGluR4 agonist (e.g. glutamate, L-AP4, or L-SOP) eliciting 80% (EC80) of the maximal agonist response was added to the cells, and the response was measured. For rat mGluR4/GIRK experiments, a baseline was established for approximately five seconds, disclosed compounds were added, and either an EC20 or EC80 concentration of agonist was added approximately two and one half minutes later.
Measurement: EC50 = 710 nM (pChEMBL 6.150000095367432)
Descriptors: MW 399.38, ALogP 2.52, HBA 7, HBD 2, PSA 102.8, QED 0.68
Compound (SMILES): COc1nc2ccc([C@](O)(c3cnnn3C)c3cnc(C)n3C)cc2c(Cl)c1C1CCCC1
InChIKey: FXEFDYYQSAXYEF-XMMPIXPASA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor Assay: ThermoFluor is a fluorescence based assay that estimates ligand binding affinities by measuring the effect of a ligand on protein thermal stability (Pantoliano, M. W., Petrella, E. C., Kwasnoski, J. D., Lobanov, V. S., Myslik, J., Graf, E., Carver, T., Asel, E., Springer, B. A., Lane, P., and Salemme, F. R. (2001) High-density miniaturized thermal shift assays as a general strategy for drug discovery. J Biomol Screen 6, 429-40, and Matulis, D., Kranz, J. K., Salemme, F. R., and Todd, M. J. (2005) Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor. Biochemistry 44, 5258-66). This approach is applicable to a wide variety of systems, and rigorous in theoretical interpretation through quantitation of equilibrium binding constants (KD).In a ThermoFluor experiment where protein stability is monitored as the temperature is steadily increased, an equilibrium binding ligand causes the midpoint of an unfolding transition (Tm) to occur at a higher temperature. The shift in the melting point described as a ΔTm is proportional to the concentration and affinity of the ligand. The compound potency may be compared as a rank order of either ΔTm values at a single compound concentration or in terms of KD values, estimated from concentration response curves.
Measurement: Kd = 1.2 nM (pChEMBL 8.920000076293945)
Descriptors: MW 466.97, ALogP 4.01, HBA 8, HBD 1, PSA 90.88, QED 0.48
Compound (SMILES): Cc1ccc(-c2cnn(C)c2C(=O)NC2CCn3nc(-c4ccccc4)nc3C2)nc1
InChIKey: PGNLNLRLDQZYIU-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Phosphodiersterase Evaluation Assay: IMAP TR-FRET Phosphodiersterase Evaluation Assay Kit (Molecular Device) was used for the measurement. Ten μL of a diluted test compound having each concentration and 5 μL of the enzyme PDE10 (BPA Bioscience) that had been diluted to 2 ng/mL with 1×IMAP Reaction Buffer (prepared with 5×included with the kit, 10 mM Tris-HCl, pH=7.2, 10 mM MgCl2, 0.05% NaN3, and 0.1% BSA) containing 0.1% BSA were added to a 384-well plate (Corning). The obtained mixture was then pre-incubated at room temperature for 5 minutes. Thereafter, 5 μL of cAMP substrate solution which was included with the kit and diluted to 400 nM with 1×IMAP Reaction Buffer containing 0.1% BSA, was added to the reaction solution, and the obtained mixture was reacted at room temperature for 60 minutes. Thereafter, 60 μL of an IMAP TR-FRET Binding solution included with the kit was further added to the reaction solution, and the obtained mixture was then left for 3 hours or more. Subsequently, using ARVO SX (PerkinElmer), the fluorescent intensity of Terbium (Emission=490 nm) and TR-FRET (Emission=520 nm) in the reaction solution were measured at an excitation wavelength of 340 nm, so as to calculate the amount of the generated 5′-AMP. The count of a well to which a solvent had been added instead of the test compound was set at 0%, and the count of a well to which the enzyme PDE10 had not been added was set at 100%. Thus, the inhibitory activity of each test compound was calculated.
Measurement: IC50 = 52 nM (pChEMBL 7.28000020980835)
Descriptors: MW 413.49, ALogP 2.79, HBA 7, HBD 1, PSA 90.52, QED 0.56
Compound (SMILES): C[C@@H]1CN(Cc2cc(F)cc(Cl)c2)CCN1C(=O)Oc1cncc(C(N)=O)c1
InChIKey: SLKCLNFOVDYIHW-GFCCVEGCSA-N
Target: Fatty-acid amide hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O00519
Assay: In Vitro Competitive Activity-Based Protein Assay: Proteomes (mouse brain membrane fraction or cell lysates for mouse assays; human prefrontal cortex or cell membrane fractions for human assays) (50 μL, 1.0 mg/mL total protein concentration) were preincubated with varying concentrations of inhibitors at 37° C. After 30 min, FPRh or HT-01 (1.0 μL, 50 μM in DMSO) was added and the mixture was incubated for another 30 min at 37° C. Reactions were quenched with SDS loading buffer (15 μL-4×) and run on SDS-PAGE. Following gel imaging, serine hydrolase activity was determined by measuring fluorescent intensity of gel bands corresponding to MAGL and FAAH using ImageJ 1.43u software.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 406.85, ALogP 2.68, HBA 5, HBD 1, PSA 88.76, QED 0.84
Compound (SMILES): CNC(=O)c1cncc(NS(=O)(=O)c2cc(Cl)ccc2OC(F)(F)F)c1
InChIKey: KVOLRXYWZDBNOW-UHFFFAOYSA-N
Target: Alkaline phosphatase, tissue-nonspecific isozyme (SINGLE PROTEIN), Homo sapiens
UniProt: P05186
Assay: Enzyme Kinetic Assay: To determine the inhibition selectivity for inhibitor candidates, human TNAP, PLAP or IAP were added to microtiter plates followed by addition of the substrate pNPP (0.5 mM) and activity was measured in 1 M DEA-HCl buffer, pH 9.8 or in 1 M Tris-HCl buffer, pH 7.5, containing 1 mM MgCl2 and 20 μM ZnCl2, in the presence of potential inhibitors (0-30 μM). TNAP, PLAP and IAP activities were adjusted to an approximate λA405 nm, equivalent to 1, measured after 30 min. Residual AP activity in the presence of inhibitors was expressed as percentage of the control activity. To investigate the mechanism of inhibition, double reciprocal plots of enzyme activity (expressed as mA405 nm min−1) vs. substrate concentration were constructed, in the presence of various concentrations of added inhibitors (0-30 μM). The y-axis intercepts of the 1/v vs. 1/[S] plots, were then plotted vs. [I] to graphically extract Ki values as the x-intercept in this plot. The numerical values from y- and x-intercepts were derived via linear regression analysis, using software Prism 3.02 (GraphPad Software, CA). These analyses were performed, using pNPP as a substrate in 1 M DEA-HCl buffer, pH 9.8, as well as in 1 M Tris-HCl buffer, pH 7.5, to determine Ki at optimal and physiological pH respectively. Inhibitors were further tested and sorted based on their kinetic properties at pH 7.4 using PPi, the relevant natural substrate of TNAP. In this part of the study, pyrophosphate sodium salt (99% ACS reagent, Sigma-Aldrich, St Louis, Mo.) was used as a substrate. Amounts of released phosphate were measured using the Biomol Green Reagent (Biomol Research Laboratories, Inc., Plymouth Meeting, Pa.). Finally, to document the potency of selected inhibitors in physiological media, TNAP inhibition by compounds of Formula I-IV (0-30 μM) was studied at pH 7.4, during catalysis of 0.1 mM pNPP, in the presence of increasing concentrations of Na2HPO4 (0-10 mM) and pyrophosphate (0-40 mM).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 409.77, ALogP 2.79, HBA 5, HBD 2, PSA 97.39, QED 0.79
Compound (SMILES): CC[C@@H](N)c1nc(N(C)C)cc2c1CN(c1cccc(-c3nnc4n3[C@@H](C)CC4)n1)C2=O
InChIKey: RHAFSJIWKGMHDN-XJKSGUPXSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: Biochemical Selectivity Kinase Assay: PRKD2 (PKD2) and RPS6KA2 (RSK3) kinase assays were performed with Km-levels of ATP at the Thermo Fisher Scientific, Inc. (Madison, Wis.), using their Invitrogen SelectScreen fluorescence resonance energy transfer (FRET) Z′-LYTE technology, based on the differential sensitivity of phosphorylated and nonphosphorylated peptides to proteolytic cleavage. Human recombinant full-length GST-tagged PRKD2 (PKD2) and RPS6KA2 (RSK3) were produced by Thermo Fisher Scientific, Inc. (Madison, Wis.). The kinase assays were conducted in 10-μL reactions. PRKD2 (PKD2) reactions contained 0.64-5.84 ng enzyme, 25 μM ATP (Km), 2 μM of Ser/Thr 17 (Z′-LYTE peptide substrate), 10 mM MgCl2, 0.01% BRIJ-35, 1 mM EGTA in 50 mM HEPES, pH 7.5. RPS6KA2 (RSK3) reactions were conducted similarly, and contained 0.5-9 ng enzyme, or 10 μM ATP (Km) and 2 μM Ser/Thr 06 peptide. After the 1-hour kinase reaction incubation, 5 μL of a 1:256 and 1:4096 dilution of Z′-LYTE Development Reagent A was added to the PRKD2 (PKD2) and RPS6KA2 (RSK3) reactions, respectively. The extent of kinase reactions, resulting in a change of FRET signal of the peptide substrate, was measured, and inhibition for each kinase was measured with respect to DMSO control and reported as an average of duplicate measurements.
Measurement: Ki < 0.045 nM (pChEMBL 10.347000122070312)
Descriptors: MW 432.53, ALogP 2.88, HBA 8, HBD 1, PSA 106.06, QED 0.66
Compound (SMILES): Cn1cncc1-c1ccc2cnc(NC(=O)C3CCN(S(C)(=O)=O)CC3)cc2c1
InChIKey: HYQBHOYZVXVDNS-UHFFFAOYSA-N
Target: Proto-oncogene Wnt-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P04628
Assay: The screening assay for Wnt activity: SW480 colon carcinoma cells were transduced with a lentiviral vector expressing luciferase with a human Sp5 promoter consisting of a sequence of eight TCF/LEF binding sites. SW480 cells stably expressing the Sp5-Luc reporter gene and a hygromycin resistance gene were selected by treatment with 150 μg/mL of hygromycin for 7 days. These stably transduced SW480 cells were expanded in cell culture and used for all further screening activities. Each compound was dissolved in DMSO as a 10 mM stock and used to prepare compound source plates. Serial dilution (1:3, 10-point dose-response curves starting from 10 μM) and compound transfer was performed using the ECHO 550 (Labcyte, Sunnyvale, Calif.) into 384-well white solid bottom assay plates (Greiner Bio-One) with appropriate DMSO backfill for a final DMSO concentration of 0.1%. For Sp5-Luc reporter gene assays, the cells were plated at 4,000 cells/well in 384-well plates with a DMEM medium containing 1% fetal bovine serum, and 1% Penicillin-Streptomycin and incubated for 36 to 48 hours at 37° C. and 5% CO2. Following incubation, 15 μl of BriteLite Plus luminescence reagent (Perkin Elmer) was added to each well of the 384-well assay plates. The plates were placed on an orbital shaker for 2 min and then luminescence was quantified using the Envision (Perkin Elmer) plate reader. Readings were normalized to DMSO only treated cells, and normalized activities were utilized for EC50 calculations using the dose-response log (inhibitor) vs. response-variable slope (four parameters) nonlinear regression feature available in GraphPad Prism 5.0 (or Dotmatics).
Measurement: EC50 = 1456 nM (pChEMBL 5.840000152587891)
Descriptors: MW 413.5, ALogP 2.25, HBA 6, HBD 1, PSA 97.19, QED 0.71
Compound (SMILES): CC(=O)N1CCc2nc(-c3cccc(OCC(=O)NCC(C)C)c3)nc(Nc3ccc(-c4cn[nH]c4)cc3)c2C1
InChIKey: MKJGVJNQVUHANM-UHFFFAOYSA-N
Target: Hexose transporter 1 (SINGLE PROTEIN), Plasmodium falciparum
UniProt: O97467
Assay: Lactate Excretion Assays: Parasite cultures were seeded in 96-well plates (1% hematocrit, 1-2% starting parasitemia, 100 μL total volume) with varying concentrations of the test compounds in a final concentration of 0.1% DMSO. Plates were cultured for 48 hours before harvesting the culture medium for LC-MS analysis. At harvest, thin smears were taken and examined microscopically to observe any alterations in parasite morphology.
Measurement: IC50 = 175 nM (pChEMBL 6.760000228881836)
Descriptors: MW 539.64, ALogP 4.33, HBA 7, HBD 3, PSA 125.13, QED 0.29
Compound (SMILES): Cc1ncc(-c2cnc3[nH]cc(C(=O)c4cccc(NS(=O)(=O)N(C)CC5CC5)c4F)c3c2)cn1
InChIKey: CGSIFJGXYUUSOI-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Biochemical activity: Assays for biochemical and cell based activity are known in the art, for example, as described in PCT publication WO 2007/002433, the disclosure of which is hereby incorporated by reference as it relates to such assays. For example, the biochemical activity IC50 values are determined with respect to inhibition of B-Raf V600E kinase activity or p-Erk kinase activity, where inhibition of phosphorylation of a peptide substrate is measured as a function of compound concentration. Compounds to be tested are diluted in dimethyl sulfoxide to a concentration of 0.1 mM. These are serially diluted 15 uL into 30 uL of dimethyl sulfoxide seven times in 96 well plates for a total of 8 dilution points, and for each dilution point 1 uL is added to a well of an assay plate. Plates are prepared such that each well in a 384 well plate contains 1 uL of compound in 10 uL volume with 0.1 ng Raf enzyme (i.e. any of B-Raf, c-Raf-1 or B-Raf V600E, Upstate Biotechnology or prepared by methods known to one of skill in the art), 50 mM HEPES, pH 7.0, 50 mM NaCl, 2 mM MgCl2, 1 mM MnCl2, 0.01% Tween-20, 1 mM DTT, and 100 nM biotin-MEK1 as substrate. The reaction is started with addition of 10 uL of 200 uM ATP (i.e. final 100 uM ATP). After incubation of the kinase reaction for 45 minutes at room temperature, 5 uL/well of Stop Solution is added (25 mM Hepes pH 7.5, 100 mM EDTA, 0.01% BSA with donor beads (Streptavidin coated beads, Perkin Elmer), acceptor beads (Protein A coated, Perkin Elmer), and anti phosphor MEK1/2 antibody (CellSignal), each at final concentration 10 ug/mL).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 494.55, ALogP 3.7, HBA 6, HBD 2, PSA 120.94, QED 0.36
Compound (SMILES): Clc1cc(Cl)cc(-c2cc(NCCCC3CCCNC3)c3ccccc3n2)c1
InChIKey: SVDUBWCVZHDDRX-UHFFFAOYSA-N
Target: Low molecular weight phosphotyrosine protein phosphatase (SINGLE PROTEIN), Homo sapiens
UniProt: P24666
Assay: LMPTP Primary Screening Protocol: A listing of materials is provided:    Item, source, catalog no.    LMPTP-A Enzyme Stock Solution (4.22 mg/ml or 206.8 μM), SBMRI Protein Facility,    N/A    OMFP, Sigma, M2629-100 MG    Bis-Tris pH 6.0, Fisher Sci, BP301-100    Triton-X 100, Sigma, T9284    DTT, Sigma, D9779    Mol. Grade Water, Mediatech, Inc., 46-000-CM    1536-well black High base opaque bottom plate, Nexus Biosystems, 00019120Final assay conditions are:    0.625 nM LMPTP-A Enzyme    40 μM OMFP    50 mM Bis-Tris pH 6.0    1 mM DTT    0.01% Triton-X 100    20 μM test compound    3% DMSO (2% from substrate and 1% from compounds)    6 μL reaction volume    50 minutes incubation at room tempAssay Procedure    1. Prepare Reagents as described in section F. Recipe.    2. Using LabCyte Echo, transfer 60 nL from 2 mM test compound source plate into assay plate Col. 5-48 (final concentration of test compounds is 20 μM). 60 nL of DMSO should be transferred to col. 1-4 for positive and negative control wells.    3. Spin plates at 1000 rpm for 1 minute in centrifuge.    4. Using the Beckman Coulter Bioraptr, add 3 μL/well of control buffer to columns 1 and 2.    5. Using the Bioraptr, add 3 μL/well of enzyme solution to col. 3-48.    6. Using the Bioraptr, add 3 μL/well of substrate solution to col. 1-48.    7. Spin plates at 1000 rpm for 1 minute in centrifuge.    8. Incubate plates in the dark at room temperature for 50 minutes.    9. Read plates on PerkinElmer Viewlux using a FI protocol.
Measurement: IC50 = 2900 nM (pChEMBL 5.539999961853027)
Descriptors: MW 414.38, ALogP 6.4, HBA 3, HBD 2, PSA 36.95, QED 0.46
Compound (SMILES): Oc1cc(-c2ccc3ncnc(N[C@H]4CC[C@H](N5CCC(F)CC5)CC4)c3c2)cnc1F
InChIKey: MBPDMDMYRMNMQF-WGSAOQKQSA-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 < 0.5 nM (pChEMBL 9.300999641418457)
Descriptors: MW 439.51, ALogP 4.69, HBA 6, HBD 2, PSA 74.17, QED 0.57
Compound (SMILES): COc1cc2nc(NCCCCNC(=O)c3ccc(C(C)(C)C)cc3)c3nnc(C)n3c2cc1OC
InChIKey: XMXGGHGXSDTFDY-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Evaluation of BRD4 binding inhibition ability: The following experiment was performed to evaluate the ability of [1,2,4]triazolo[4,3-a]quinoxaline derivative of the present invention to inhibit the interaction between BRD4 (BD1+BD2) bromodomain, one of BET protein family, and tetraacetylated histone H4 peptide.The compound was serially diluted at the ratio of 1:5 in assay buffer from 10 mM stock in DMSO (initial concentration: 100 μM) on white OptiPlate-384 (PerkinElmer). A mixture comprising 100 nM GST-BRD4 (BD1+BD2) and 100 nM biotinylated acetyl-histone H4 (Lys5,8,12,16) peptide was prepared in assay buffer (50 mM HEPES pH 7.4; 25 mM NaCl; 0.05% Tween 20; 0.1% bovine serum albumin (BSA); 10 mM dithiothreitol (DTT)). After adding 6 μl of the mixture to the diluent, 6 μl of the pre-mixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads (PerkinElmer, 10 μg/ml in assay buffer, respectively) was added thereto. The samples were incubated in the dark at room temperature for 30 minutes (shaking at 300 rpm). Then, the signals were measured with PerkinElmer Envision HTS Multilabel Reader using an alpha screen protocol of PerkinElmer. Each plate contained the negative control in which biotinylated acetyl-histone H4 peptide and GST-BRD4 (BD1+BD2) were replaced by assay buffer. In the case of using the software GraphPad Prism for calculation, the negative control point was input as a low standard value. The positive control (probe molecule l-BET762 containing protein/peptide mixture) proceeded to pipetting. IC50 value was determined by using GraphPad Prism 3.03 software (or an updated version thereof).
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 490.61, ALogP 4.52, HBA 8, HBD 2, PSA 102.67, QED 0.33
Compound (SMILES): CCC(=O)N1CCC(Cc2ncnc3c2nc(-c2cnn(CC)c2C)n3C)C1
InChIKey: LYTDGSQZQNZFER-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Biochemical Assay: The PI3-Kinase biochemical assay was optimized using the HTRF kit provided by Upstate (Millipore). The assay kit contains six reagents: 1) 4× Reaction Buffer; 2) native PIP2 (substrate); 3) Stop (EDTA); 4) Detection Mix A (Streptavidin-APC); 5) Detection Mix B (Eu-labeled Anti-GST plus GST-tagged PH-domain); 6) Detection Mix C. In addition, the following items were obtained or purchased; PI3Kinase (alpha 14-602, beta 14-603, gamma 14-558 and delta 14-604 from Upstate; Millipore), dithiothreitol (Sigma, D-5545), Adenosine-5′ triphosphate (InVitrogen, Cat#AS001A), native PIP3 (PI(3,4,5)P3, diC8, H+, CELLSIGNALS, INC. Cat #907) DMSO (Sigma, 472301).PI3Kinase Reaction Buffer is prepared by dilution the stock 1:4 with de-ionized water. DTT, PIP2 and Biotin-PIP3 were added to 1536 assay plate at a final concentration of 5 mM, 5 mM and 25 nM on the day of use. Enzyme addition and compound pre-incubation are initiated by the addition of 1.25 ul of PI3K (at twice its final concentration) in the 1× reaction buffer to all wells using a BioRaptor. Plates are incubated at room temperature for 15 minutes. Reactions are initiated by addition of 1.25 ul of 2× substrate solution (PIP2 and ATP in 1× reaction buffer) using BioRaptor. Plates are incubated in humidified chamber at room temperature for one hour. Reactions are quenched by addition of 0.625 uL of stop solution to all wells using the BioRaptor. The quenched reactions are then processed to detect product formation by adding 0.625 uL of Detection Solution to all wells using the BioRaptor (Detection mix C, Detection Mix A, and Detection Mix B combined together in an 18:1:1 ratio prepared 2 hours prior to use). Following a one hour incubation in the dark, the HTRF signal is measured on the Envision plate reader set for 330 nm excitation and dual emission detection at 620 nM (Eu) and 665 nM (APC).
Measurement: IC50 = 250 nM (pChEMBL 6.599999904632568)
Descriptors: MW 381.48, ALogP 2.36, HBA 7, HBD 0, PSA 81.73, QED 0.68
Compound (SMILES): COc1ncccc1-c1ccncc1N(CC(F)(F)F)C(=O)c1cc(F)cc(C(F)(F)F)c1
InChIKey: QTCVGOWZLZDNHC-UHFFFAOYSA-N
Target: G-protein coupled bile acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TDU6
Assay: cAMP Assay: CHO-dhfr(minus) cells expressing human GPBAR1 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), lx HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min Compounds were added to a final assay volume of 100 μl and incubated for 30 min at 30° C. The assay was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaked for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH, Hamburg Germany), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The measured signal at 730 nm has to be corrected for the ruthenium background, the direct excitation of Alexa and the buffer control. The FRET signal is calculated as follows: FRET=T730−Alexa730−P(T645−B645) with P=Ru730−B730/Ru645−B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.
Measurement: EC50 = 1663 nM (pChEMBL 5.78000020980835)
Descriptors: MW 473.35, ALogP 5.52, HBA 4, HBD 0, PSA 55.32, QED 0.46
Compound (SMILES): CC(C)C[C@H](NC(=O)Nc1ccc(Br)cc1)C(=O)N[C@H](C(N)=O)C(C)C
InChIKey: YLBUEARKTSANET-GJZGRUSLSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: FLIPR: HEK-Gα16 cells stably expressing the human FPR2 receptor was utilized. Cells were plated into 384-well poly-D-lysine coated plates at a density of 18,000 cells per well one day prior to use. The growth media was DMEM medium supplemented with 10% fetal bovine serum (FBS), 1% antibiotic-antimycotic, 50 μg/ml hygromycin, and 400 μg/ml geneticin. On the day of the experiment, the cells were washed twice with Hank's Balanced Salt Solution supplemented with 20 mM HEPES (HBSS/hepes buffer). The cells were then dye loaded with 2 μM Fluo-4 diluted in the HBSS/Hepes buffer and incubated at 37° C. for 40 minutes. Extracellular dye was removed by washing the cell plates four times prior to placing the plates in the FLIPR (Fluorometric Imaging Plate Reader, Molecular Devices). Ligands were diluted in HBSS/Hepes buffer and prepared in 384-well microplates.
Measurement: EC50 = 5800 nM (pChEMBL 5.239999771118164)
Descriptors: MW 427.34, ALogP 2.61, HBA 3, HBD 4, PSA 113.32, QED 0.51
Compound (SMILES): Cc1ccc(NC(=O)c2cnnc(C(F)(F)F)c2)cc1-c1cc(N2CCOCC2)c(=O)n(C)c1
InChIKey: HJZOLMWTAJVUIP-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Raf Activity Assay: Raf and biotinylated Mek, kinase dead, were combined at 2x final concentrations in assay buffer (50 mM Tris, pH 7.5, 15 mM MgCl2, 0.01% BSA and 1 mM DTT) and dispensed 5 ml per well in assay plates (Greiner white 384 well assay plates #781207) containing 0.25 ml of 40x of a Raf kinase inhibitor test compound diluted in 100% DMSO. The plate was incubated for 60 minutes at room temperature. The Raf kinase activity reaction was started by the addition of 5 mL per well of 2xATP diluted in assay buffer. After 3 hours (b-Raf(V600E)) or 1 hour (c-Raf). The reactions were stopped and the phosphorylated product was measured using a rabbit anti-p-MEK (Cell Signaling, #9121) antibody and the Alpha Screen IgG (ProteinA) detection Kit (PerkinElmer #6760617R), by the addition of 10 mL to the well of a mixture of the antibody (1:2000 dilution) and detection beads (1:2000 dilution of both beads) in Stop/bead buffer (25 mM EDTA, 50 mM Tris, pH 7.5, 0.01% Tween20). The additions were carried out under dark conditions to protect the detection beads from light. A lid was placed on top of the plate and incubated for 1 hour at room temperature, then the luminescence was read on a PerkinElmer Envision instrument. The concentration of each compound for 50% inhibition (IC50) was calculated by non-linear regression using XL Fit data analysis software.
Measurement: IC50 = 2.8 nM (pChEMBL 8.550000190734863)
Descriptors: MW 473.46, ALogP 3.26, HBA 7, HBD 1, PSA 89.35, QED 0.63
Compound (SMILES): CNC(=O)CN1CCC(c2ccc3[nH]c(-c4cc(C)c5ncnn5c4)c(CC(F)F)c3c2)CC1
InChIKey: MUWHDZKWYGDPQV-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assay: 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 = 1.5 nM (pChEMBL 8.819999694824219)
Descriptors: MW 466.54, ALogP 3.92, HBA 5, HBD 2, PSA 78.32, QED 0.45
Compound (SMILES): CC(c1cccc(C(F)(F)F)c1)N1CCC[C@H]([C@@H]2CC(Br)=NO2)C1
InChIKey: PGQLBVVCGRCKBG-MONRWBKMSA-N
Target: Transcriptional enhancer factor TEF-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28347
Assay: TEAD Inhibition Assay: TEAD inhibition can be assayed using Hippo Pathway TEAD ReporterMCF7 Cell Line (BPS Bioscience, Catalog #: 60618).
Measurement: EC50 > 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 405.26, ALogP 4.98, HBA 3, HBD 0, PSA 24.83, QED 0.71
Compound (SMILES): CC(C)(OC(=O)Nc1ccc2c(c1)N(S(=O)(=O)c1ccc(F)cc1)C[C@H](CNS(=O)(=O)C1(C)CC1)O2)C(F)(F)F
InChIKey: RYQKDWCFYGSODT-KRWDZBQOSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Biochemical TR-FRET assay: The compounds of the invention inhibit RORgammaT activity. Activation of RORgammaT activity can be measured using, e.g., a biochemical TR-FRET assay. In such an assay, interaction of cofactor-derived peptides with human RORgammaT-Ligand Binding Domain (LBD) can be measured. The TR-FRET technique is a sensitive biochemical proximity assay that will give information concerning the interaction of a ligand with the LBD, in the presence of cofactor-derived peptides (Zhou et al., Methods 25:54-61, 2001).To identify novel antagonists of RORgammaT, an assay was developed which employs the interaction of RORgammaT with its co-activator peptide SRC1_2. This peptide mimics the recruitment of co-activators to RORgammaT through its interaction with the LXXLL (SEQ ID NO:1) (e.g., NR box) motifs (Xie et al., J. Immunol. 175: 3800-09, 2005; Kurebayashi et al., Biochem. Biophys. Res. Commun. 315: 919-27, 2004; Jin et al., Mol. Endocrinology 24:923-29, 2010). The RORγ-Ligand Binding Domain TR-FRET Assay was run according to the following protocol.HIS-tagged RORγ-LBD protein was recombinantly expressed in Escherichia coli. The RORγ-LBD protein was purified by Ni2+-affinity resin. Purified protein was then diluted in assay buffer (50 mM Tris pH 7.0, 50 mM KCl, 1 mM EDTA, 0.1 mM DTT, 100 μg/mL bovine serum albumin, delipidated) to obtain a RORγ-LBD final concentration of 3 nM. Europium tagged anti-HIS antibody was also added to this solution (1.25 nM). Separately, SF9 cells not expressing any recombinant protein were lysed (32,000 cells per μl in 25 mM Tris, 50 mM NaCl) and the previously frozen lysate was added to the diluted RORγ-LBD solution at a ratio of 0.75 μl SF9 lysate per 15 μl of diluted RORγ-LBD.
Measurement: IC50 = 6.6 nM (pChEMBL 8.180000305175781)
Descriptors: MW 609.62, ALogP 4.14, HBA 7, HBD 2, PSA 131.11, QED 0.43
Compound (SMILES): CC(C)N1CCC(S(=O)(=O)c2cc(-c3cnc(N)nc3)cc(N3CCOCC3)c2)CC1
InChIKey: YWBOOFIYFHDXJW-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Inhibition of PI3K alpha: Quantification of ATP to ADP conversion as a measure of PI3Kα activity. Active PI3Kα (Life Technologies), in the presence or absence of PI3Kα inhibitor, was reacted with PIP2:PS (Life Technologies), a substrate specifically optimized for use with Class I PI3 kinases, and ultrapure ATP (Promega). The conversion of ATP to ADP by PI3Kα was measured as luminescence signal via Promega ADP-Glo kinase activity assay. Assay was validated using published PI3Kα inhibitors LY294002, PI-103, BYL719, GDC0198 and also DMSO vehicle control.Compounds were prepared at 100× final concentration as a 12-point, 1:3 serial-dilution in DMSO series, with DMSO control as 12th point. Compound was then diluted in (25 mM HEPES pH 7.5, 1 mM EGTA, 0.3% CHAPS) prior to addition to PI3Kα. Active PI3Kα diluted to 0.24 ng/μL (1.1 nM) in (50 mM HEPES pH 7.5, 6 mM MgCl2, 1 mM EGTA, 200 mM NaCl, 0.03% CHAPS, 8 mM DTT) was incubated with compound for 0 hr and 3 hr prior to the start of the reaction. 25 μM PIP2:PS and 60 μM ATP were diluted from stock in (25 mM HEPES pH 7.5, 1 mM EGTA, 0.3% CHAPS) and added to initiate the PI3Kα reaction. Reaction time was 30 minutes. ATP to ADP conversion was measured in Luminescence Counts on DTX880 Plate Reader (Beckman Coulter). Compound IC50s were reported using GraphPad Prism software. Analytical method was non-linear regression, 4-parameter curve fit with bottom fit to validated PI3Kα inhibitor reference controls and no top fit (floating top).
Measurement: IC50 = 7220 nM (pChEMBL 5.139999866485596)
Descriptors: MW 445.59, ALogP 2.21, HBA 8, HBD 1, PSA 101.65, QED 0.75
Compound (SMILES): C=CC(=O)N1C[C@H](C)N(c2nc(=O)n(-c3c(C(C)C)ccnc3C(C)C)c3nc(-c4ccccc4C)c(Cl)cc23)C[C@H]1C
InChIKey: IOMOSRBKUOKBBA-PKTZIBPZSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Coupled Nucleotide Exchange Assay: Purified GDP-bound KRAS protein (aa 1-169), containing both G12C and C118A amino acid substitutions and an N-terminal His-tag, was pre-incubated in assay buffer (25 mM HEPES pH 7.4, 10 mM MgCl2, and 0.01% Triton X-100) with a compound dose-response titration for either 5 min or 2 hours (see Table 15). Following compound pre-incubation, purified SOS protein (aa 564-1049) and GTP (Roche 10106399001) were added to the assay wells and incubated for an additional 30 min (for 5 min compound pre-incubation) or 1 hour (for 2 hour compound pre-incubation). To determine the extent of inhibition of SOS-mediated nucleotide exchange, purified GST-tagged cRAF (aa 1-149), nickel chelate AlphaLISA acceptor beads (PerkinElmer AL108R), and AlphaScreen glutathione donor beads (PerkinElmer 6765302) were added to the assay wells and incubated for 10 minutes. The assay plates were then read on a PerkinElmer EnVision Multilabel Reader, using AlphaScreen® technology, and data were analyzed using a 4-parameter logistic model to calculate IC50 values.
Measurement: IC50 = 33 nM (pChEMBL 7.480000019073486)
Descriptors: MW 599.18, ALogP 6.66, HBA 7, HBD 0, PSA 84.22, QED 0.23
Compound (SMILES): O=C(c1ccc2c(c1)N(c1ncc(-c3ccccc3F)cn1)CC21CC1)N1CCCCC12COC2
InChIKey: FSRTUIOZHPBKOS-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 470.55, ALogP 4.86, HBA 5, HBD 0, PSA 58.56, QED 0.55
Compound (SMILES): CCCCCCCCCCCc1ccc(-c2noc(C3(NC(=N)N)CC3)n2)cc1
InChIKey: CDECMEBTSBYJHQ-UHFFFAOYSA-N
Target: Sphingosine kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYA1
Assay: Scintillation Proximity Assay: SphK activity was measured by a scintillation proximity assay as previously described [1], the disclosure of which is incorporated herein. Briefly, recombinant SphK1 or SphK2 were expressed in Sf9 insect cells, crude homogenates were prepared and incubated in 96 well FlashPlates (Perkin-Elmer) in a buffer containing D-erythro-sphingosine and γ-[33P]ATP. The [33P]S1P product, which adheres to the plate wall, was quantified by scintillation counting. To assay ceramide kinase or diacylglycerol kinases, the recombinant proteins were incubated with γ-[32P]ATP and substrate (C6 ceramide or 1-Ohexadecyl-2-acetyl-sn-glycerol, respectively) and the lipid product, after recovery by organic extraction, was resolved by thin layer chromatography, detected by autoradiography, and quantified by liquid scintillation counting. These assays were performed with and without a fixed concentration of inhibitor and its effect on KM and Vmax determined.
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 397.57, ALogP 5.28, HBA 4, HBD 3, PSA 100.82, QED 0.24