lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(C)c1c(-c2cnn(C(C)(C)C)c2)[nH]c2ncnn2c1=O
InChIKey: BYTQWDUCKBMJMU-UHFFFAOYSA-N
Target: Lysine-specific demethylase 5B (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UGL1
Assay: TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) Assay: In this assay, the potency (IC50) of each compound was determined from a ten point (1:3 serial dilution; final compound concentration range in assay from 100000 nM to 5.08 nM) titration curve using the following outlined procedure. To each well of a white Greiner 1536 Lumitrac 1536 well-plate, 50 nL of compound (100 fold dilution in final assay volume of 5 μL) was dispensed, followed by the addition of 4 μL of 1× assay buffer (50 mM Hepes 7.3, 0.5 mM TCEP, 0.005% Brij-35, 0.02% BSA, 50 μM Na-L-Ascorbate and 2 μM AmFe(II)Sulfate) containing 5 nM of Full-length KDM5B enzyme (recombinant protein from baculovirus-transfected Sf21 cells: full-length KDM5B; MW=176.825 kDa). Following a 30 minutes compound and enzyme incubation in a humidified chamber, each reaction was initiated by the addition of 1 μL 1× assay buffer containing 50 nM biotinylated H3K4Me3 peptide, and 500 nM α-ketoglutarate. The final reaction in each well of 5 μL consists of 5 nM KDM5B, 500 nM biotinylated-peptide, and 500 nM α-ketoglutarate. De-methylation reactions were allowed to proceed for 60 minutes. Reactions were immediately quenched by the addition of 5 uL of 2× Lance Detection Buffer (PerkinElmer) with 0.5 mM EDTA and 2 nM of Eu-anti-H3K4Me1-2 antibody, and 30 nM of Streptavidin-conjugated Dylight 650 detection reagents. After 60 minutes incubation with detection reagents, reaction plates were read on a PerkinElmer EnVision plate reader using standard TR-FRET protocol. Briefly, excitation of donor molecules (Eu-chelate-anti-H3K4Me1-2-antibody) with a laser light source at 337 nm produces energy that can be transferred to Dylight-650 acceptor molecules if this donor:acceptor pair is within close proximity. Fluorescence intensity at both 665 nm (acceptor) and 615 nm (donor) are measured and a TR-FRET ratio calculated for each well (acceptor intensity/donor intensity).
Measurement: IC50 = 5.08 nM (pChEMBL 8.289999961853027)
Descriptors: MW 300.37, ALogP 2.16, HBA 6, HBD 1, PSA 80.87, QED 0.79
Compound (SMILES): COC(=O)N1CC=C(c2cc3c(N4CC(C(=O)Nc5ccc(C#N)cc5)C4)ncnn3c2)CC1
InChIKey: UDFYPFGVAKGFKG-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Kit Wild Type Assay: In each well of a 384-well plate, 0.2 ng/ul final (2 nM) of wild type Kit (Carna Bioscience 08-156) was incubated in a total of 12.5 ul of buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 10 mM MgCl2, 1 mM DTT) with 1 uM Srctide (5-FAM-GEEPLYWSFPAKKK-NH2) and 400 uM ATP at 25 C for 90 minutes in the presence or absence of a dosed concentration series of compound (1% DMSO final concentration). The reaction was stopped by the addition of 70 ul of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 35 mM EDTA and 0.2% of Coating Reagent 3 (Caliper Lifesciences)). The plate was then read on a Caliper EZReader 2 (protocol settings: −1.9 psi, upstream voltage −700, downstream voltage −3000, post sample sip 35s).
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 457.49, ALogP 2.53, HBA 8, HBD 1, PSA 115.86, QED 0.64
Compound (SMILES): Cc1ccc(NC(=O)Nc2cc(-c3cccc(C(=O)O)c3)ccc2Oc2cccc(Cl)c2)cc1
InChIKey: CWYGMEGJIBVEDD-UHFFFAOYSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: IDO Kynurenine Assay: Human IDO1/HEK293 cells were seeded at 10,000 cells per 50 uL per well with RPMI/phenol red free media contains 10% FBS in a 384-well black wall clear bottom tissue culture plate (Matrix Technologies LLC) 125 nL of certain concentration of compound was then added to each well using ECHO liquid handling systems. The cells were incubated for 20 hours in 37° C. incubator with 5% CO2.The compound treatments were stopped by adding Trichloroacetic Acid (Sigma-Aldrich) to a final concentration at 0.2%. The cell plate was further incubated at 50° C. for 30 minute. The equal volume supernatant (20 uL) and 0.2% (w/v) Ehrlich reagent (4-dimethylaminobenzaldehyde, Sigma-Aldrich) in glacial acetic acid were mixed in a new clear bottom 384-well plate. This plate was then incubated at room temperature for 30 minute. The absorbance at 490 nm was measured on Envision plate reader.Compound IC50 values were calculated using the counts of 500 nM of a reference standard treatment as one hundred percent inhibition, and counts of no compound but DMSO treatment as zero percent inhibition.
Measurement: IC50 = 6580 nM (pChEMBL 5.179999828338623)
Descriptors: MW 472.93, ALogP 7.45, HBA 3, HBD 3, PSA 87.66, QED 0.27
Compound (SMILES): CCCCc1nc(O)c(-c2nnc(Cc3ccc(F)cc3)o2)c(O)c1-c1c(OC)cccc1OC
InChIKey: BQAAGVORUABYQQ-UHFFFAOYSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: Intracellular cAMP Accumulation Assay: HEK293 cells stably expressing human APJ receptor were used to assess the activity of compounds. Cultured cells were detached and resuspended in the cAMP Homogeneous Time-Resolved Fluorescence (HTRF) assay buffer (Cisbio cat; #62AM4PEJ). The assay was performed in 384-well assay plates (Perkin-Elmer; cat #6008289) according to assay protocol provided by the manufacturer. Serial dilutions of a compound together with assay buffer containing 0.2 nM IBMX and 2 μM forskolin were added to each well containing 5,000 cells and incubated for 30 minutes at room temperature. Subsequently, cAMP D2 reagent was added in the lysis buffer followed by the EuK antibody (Cisbio; cat #62AM4PEJ) and incubated for 60 min. The fluorescence emission ratio was measured using fluorometer. The intracellular cAMP concentrations (compound-stimulated inhibition of forskolin-mediated cAMP production) were calculated by extrapolation from a standard curve using known cAMP concentrations. The EC50 values were obtained by fitting the data to a sigmoidal concentration-response curve with variable slope. The maximal achievable inhibition of forskolin-induced cAMP levels (Ymax) for each compound was expressed as relative percentage of inhibition attained using pyroglutamated apelin-13 ((Pyr1)apelin-13) peptide, which was set to 100%.The examples disclosed below were tested in the APJ in vitro assays described above and were found having human APJ cyclic AMP (hcAMP) activity. The EC50 value of each compound is presented at the end of the example description.
Measurement: EC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 479.51, ALogP 5.3, HBA 8, HBD 2, PSA 110.73, QED 0.33
Compound (SMILES): CCN[C@H]1CCCC[C@@H]1N(CC)c1ccc2c(c1)CN(C1CCC(=O)NC1=O)C2=O
InChIKey: NXPINYXVXVKKSQ-NFBCFJMWSA-N
Target: DNA-binding protein Ikaros (SINGLE PROTEIN), Homo sapiens
UniProt: Q13422
Assay: Prolabel Quantification of IKZF1, IKZF2, or GSPT1 Protein Levels in 293GT Cells: The Prolabel system from DiscoverX was used to develop high-throughput and quantitative assays to measure changes in IKZF1, IKZF2, and GSPT1 protein levels in response to compounds. The prolabel tag is derived from the alpha fragment of beta galactosidase and has the following protein sequence: mssnslavvlqrrdwenpgvtqlnrlaahppfaswrnseeartdrpsqqlrslnge. The complementary fragment of beta-galactosidase (from DiscoverX), is added to the prolabel tag to form an active beta galactosidase enzyme whose activity can be precisely measured. In this way, the levels of a fusion protein with the prolabel tag can be quantified in cell lysates.Lentiviral vectors, based on the Invitrogen pLenti6.2/V5 DEST backbone, were constructed that placed the prolabel tag upstream of IKZF1, IKZF2, or GSPT1 and expressed the fusion protein from a CMV promoter.To ensure moderate and consistent expression of the prolabel fusion proteins across all cells in the population, stable cell lines were constructed from cells expressing a single copy of the construct. Lentivirus packaged with the constructs was made using the Virapower kit from Invitrogen. Strongly adherent 293GT cell, GripTite 293 MSR cells from Thermo Fisher Scientific (Catalog number: R79507), were infected with the virus at low multiplicity of infection and selected by 5 μg/mL blasticidin for 2 weeks.
Measurement: EC50 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 412.53, ALogP 2.19, HBA 5, HBD 2, PSA 81.75, QED 0.7
Compound (SMILES): Cn1cc(-c2nc(N3CCOCC3)c3nc(C(=O)N4CCN(C(=O)Cc5cnccn5)CC4)[nH]c3n2)cn1
InChIKey: LYGAOBRONOIVGO-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: In Vitro Enzyme PI3K Assay: The Kinase-Glo luminescent kinase assay kit (from Promega) was used to measure kinase activity. In this assay, amount of ATP remaining in solution following a kinase reaction is measured. The effect of compounds on PI3Kδ inhibition was carried out by adding 2.29 μg/ml of recombinant PI3K δ enzyme (Proteros, Germany) to reaction mixture containing assay buffer (50 mM HEPES, pH 7.4, 50 mM NaCl, 0.05% CHAPS) supplemented with 10 mM MgCl2, 5 mM DTT, 60 μM Phosphatidyl inositol bisphosphate (PIP2) and 10 μM ATP in the absence or presence of different concentrations of the compounds in a final volume of 15 μl/well, in a 384 well plate. The reaction mixture was incubated for 2 hours at room temperature. At the end of incubation period the equal volume of Kinase-Glo plus (Promega, V3772), was added per well and the luminescence was measured after incubating for 10 minutes at room temperature in dark. Results were calculated by measuring the luminescence units of test samples to blanks containing no enzyme.In certain embodiments, the compounds showed IC50 of less than 1000 nM, in another embodiment, the IC50 values range from about 100 nM to 500 nM, and in yet another embodiment, it is even less than 30 nM for PI3Kδ as shown in Table 3 and 4.The compounds of the present invention were tested for their selectivity for PI3Kδ over PI3Kα, PI3Kβ and PI3Kγ following the above assay using specific recombinant enzymes (Proteros, Germany) for each kinase. Assay condition of kinase assay (PI3Kα, PI3Kβ, PI3Kγ and PI3Kδ) was as follows. Enzyme: 2.29 μg/mL; ATP: 10 μM; PIP2 substrate: 60 μM and Reaction Time: 2 hours.
Measurement: IC50 = 140 nM (pChEMBL 6.849999904632568)
Descriptors: MW 517.55, ALogP -0.09, HBA 11, HBD 1, PSA 151.15, QED 0.38
Compound (SMILES): Cc1cnc2cc(-c3ccccc3)nn2c1N[C@@H]1C[C@H](COS(N)(=O)=O)[C@@H](O)[C@H]1O
InChIKey: UPUSYLJKPLQMMM-UWIFEVILSA-N
Target: Ubiquitin-like modifier-activating enzyme 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P22314
Assay: Ubiquitin Activating Enzyme (UAE) HTRF Assay: The UAE enzymatic reaction totals 50 μL and contains 50 mmol HEPES (pH 7.5), 0.05% BSA, 2.5 mM MgCl2, 0.1 uM ATP, 8 nM GST-Ubc-2, 35 nM flag-ubiquitin, 1 nM recombinant human UAE or mouse UAE. Compounds for this hUAE IC50 assay are tested at 10 point 3-fold dilution. The top concentration for this assay is 1 μM. Each compound is ordered in duplicate on the same plate. The enzymatic reaction mixture is incubated for 90 minutes at room temperature (24 degrees C.) in a 384 well plate prior to termination with a stop solution (0.1 M HEPES/0.05% Tween20, 20 mmol EDTA, 410 mM KF, 0.5 nM Eu Cryptate anti-FLAG M2-K antibody (Cis-bio International), 8 ug/mL Anti-GST XL-APC (Prozyme)). After incubation for 120 minutes, quantification of the FRET is performed on the Pherostar (BMG).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 433.49, ALogP 0.45, HBA 9, HBD 4, PSA 152.07, QED 0.44
Compound (SMILES): Cc1ccc(CNC(=O)c2nnc(CCCCn3cc(C(=O)NCc4ccccn4)nn3)s2)cn1
InChIKey: WYLDFPUAWCTHTB-UHFFFAOYSA-N
Target: Glutaminase kidney isoform, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: O94925
Assay: Enzymatic Activity Assay: The inhibition of purified recombinant human GAC by varying concentrations of inhibitors is assessed via a dual-coupled enzymatic assay. The glutamate produced by the glutaminase reaction is used by glutamate oxidase to produce α-ketoglutarate, ammonia, and hydrogen peroxide, with this hydrogen peroxide subsequently being used by horseradish peroxidase to produce resorufin in the presence of Amplex UltraRed. The assay buffer consisted of 50 mM Hepes (pH 7.4), 0.25 mM EDTA and 0.1 mM Triton X-100. GAC was incubated with potassium phosphate (10 minutes at room temperature) prior to incubation with inhibitor (10 minutes at room temperature). The final reaction conditions were as follows: 2 nM GAC, 50 mM potassium phosphate, 100 mU/mL glutamate oxidase (Sigma), 1 mM glutamine (Sigma), 100 mU/mL horseradish peroxidase (Sigma), 75 μM Amplex UltraRed (Life Technologies), and 1% (v/v) DMSO. The production of resorufin was monitored on a Perkin Elmer Envision plate reader (excitation 530 nm, emission 590 nm) either in a kinetics or endpoint mode (at 20 minutes). IC50 values were calculated using a four-parameter logistic curve fit.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 491.58, ALogP 2.11, HBA 10, HBD 2, PSA 140.47, QED 0.3
Compound (SMILES): CC(C)(NC(=O)[C@H]1[C@@H]2CNC[C@@H]21)c1ncc2c(OC(F)(F)F)cccn12
InChIKey: FXPCDLZHUXTNEN-IWIIMEHWSA-N
Target: Somatostatin receptor type 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P31391
Assay: AMP Assay with Human Somatostatin 4 Receptor: The activation of the SSTR4 receptor (Gi coupled) causes an inhibition of intracellular cAMP after stimulation with Forskolin, which can be quantifiable by use of a suitable assay Kit and an adequate plate reader. This technique is used to characterize pharmacological effects of the SSTR4 receptor agonists by use of hSSTR4 expressing H4 cells. Compounds are dissolved and diluted in DMSO. The final test solution contains 1% DMSO. The cAMP standard (Lance cAMP 384 Kit; PerkinElmer, Cat# AD0264) is prepared in assay buffer (HBSS with 0.1% BSA, 5 mM HEPES, 0.5 M IBMX, pH 7.4) containing 1% DMSO and the cAMP standard curve is included at least on one plate. Cells are centrifuged and suspended in assay buffer (incl. 1:100 diluted Alexa antibody).
Measurement: EC50 = 26.1 nM (pChEMBL 7.579999923706055)
Descriptors: MW 368.36, ALogP 2.05, HBA 5, HBD 2, PSA 67.66, QED 0.87
Compound (SMILES): O=C(Nc1ccc(S(=O)(=O)N2CCOCC2)cc1)c1cnc(NC2CCOCC2)nc1NC1CCC(O)CC1
InChIKey: QEGOHWRXPWNJLM-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: Activity Assay: Briefly, activity assays were performed in a 384 well, polypropylene microplate in a final volume of 50 μL of 50 mM Hepes, Ph 7.4 containing 10 mM MgCl2, 1.0 mM DTT, 0.01% Triton X-100, 0.1% Bovine Serum Albumin (BSA), containing 1.0 μM fluorescent substrate and ATP at the Km for each enzyme. All reactions were terminated by addition of 20 μL of 70 mM EDTA. After a 180 min incubation, phosphorylated and unphosphorylated substrate peptides were separated in buffer supplemented with 1×CR-8 on a LabChip EZ Reader equipped with a 12-sipper chip. Data were analyzed using EZ Reader software. Assay conditions for MCE assays
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 560.68, ALogP 2.06, HBA 10, HBD 4, PSA 155.01, QED 0.38
Compound (SMILES): CC(C)O[C@H]1CC[C@H](C(=O)N2Cc3cccnc3Nc3ccc(C4CCOCC4)cc32)OC1
InChIKey: YAXSEEBTOAIRNS-XUZZJYLKSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Enzymatic Assay: Each test compound (10 mM stock in DMSO) is diluted in DMSO to make a 10-point, 3-fold dilution series. 125 nL of each dilution or DMSO alone is dispensed to a 384-well Greiner Lumitrac 200 assay plate using an Echo® Liquid Handler. To each well of the plate is added 20 uL of enzyme in assay buffer or assay buffer alone. Assay buffer consists of 50 mM sodium phosphate, pH 7.0, 50 mM magnesium chloride, 50 mM sodium chloride, and 0.01% (w/v) bovine serum albumin. When present, the R132H mutant IDH1 enzyme is at a working concentration of 1.875 nM (final concentration in assay of 1.5 nM). The assay plate is allowed to incubate for 30 minutes at room temperature and 5 uL of 5× substrate mixture (2.5 uM nicotinamide adenine dinucleotide phosphate, 100 uM adenosine diphosphate, 7.5 mM glyceraldehyde-3-phosphate, 7.5 ug/mL of spinach glyceraldehyde-3-phosphate dehydrogenase, 25 nM phosphoglycerate kinase, and 5 mM alpha-ketoglutarate in assay buffer) is added to all wells. The reaction plate is incubated for 60 minutes followed by addition of 25 uL of Promega Kinase-GLO reagent to all wells and 10-minute incubation.Luminescence is measured using a PerkinElmer Envision plate reader. The percent activity of each dilution is determined as the ratio of background corrected signal to the background corrected signal of wells receiving only DMSO. IC50 values are determined by fitting percent activity data to a four-parameter logistic dose response equation.
Measurement: IC50 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 451.57, ALogP 4.54, HBA 6, HBD 1, PSA 72.92, QED 0.74
Compound (SMILES): C[C@@H](Nc1nc(-c2noc(=O)[nH]2)nc2nc(N3CCCC3C(C)(C)C)n(C[C@H]3CC[C@H](C)CC3)c12)C1CCC1
InChIKey: JGWCBPNRZQUGSQ-OCQFDOBUSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: FRET Assay: An HDM2 FRET assay was developed to assess the compounds' inhibitory activity towards binding of p53 protein. A truncated version of HDM2 with residues 17 to 125 (containing p53 binding surface, Science (1994) 265, 346-355), with N-terminal His and Thioredoxin tag was generated in pET32a expression vector and expressed in E. coli strain BL21(DE3)Rosetta. Protein was purified using Ni-affinity chromatography, followed by size exclusion chromatography using Superdex 75 26/60 column. To assess inhibition of p53 binding to HDM2, a FITC labeled 8-mer peptide (sequence: Ac-Phe-Arg-Dpr-Ac6c-(6-Br)Trp-Glu-Glu-Leu-NH2; Anal Biochem. 2004 Aug. 1; 331(1):138-46) with strong affinity towards p53 binding pocket of HDM2 was used. The HDM2 assay buffer contained 1× Phosphate Buffered Saline (Invitrogen, Cat#14190), 0.01% BSA (Jackson ImmunoResearch, Cat#001-000-162), 0.01% Tween-20. In the 1× assay buffer recombinant HDM2 protein, peptide and Lumi4-Tb Cryptate-conjugate mouse anti-6×His antibody (cisbio, Cat# Tb61HISTLB) were added and transferred to ProxiPlate PLUS (PerkinElmer, Cat#6008269), containing compounds so that final DMSO concentration is 0.1%. Final concentrations of reagents in the assay wells are 0.5 nM HDM2, 0.25 nM anti HIS (Tb label) antibody and 3 nM peptide. After two hour incubation at room temperature in a humidified chamber plates were read on EnVision plate reader with the following settings: excitation: UV, 340 nM, two emission filters: 520 nm and 495 nm respectively. Ratio of em520/em495 was used to calculate % inhibition and to obtain IC50 with 4-parameter logistic equation.
Measurement: IC50 = 3.931 nM (pChEMBL 8.399999618530273)
Descriptors: MW 536.73, ALogP 5.61, HBA 9, HBD 2, PSA 117.76, QED 0.4
Compound (SMILES): C=CC(=O)N1CCC[C@@H](n2cc(C(N)=O)c(-c3ccc(Oc4ccc(C#N)cc4)cc3)n2)C1
InChIKey: CDBHUBBNPNBKSV-LJQANCHMSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: BTK Assay: TBDAn HTRF assay (Cisbio KinEASE-TK cat #62TKOPEC) was performed to quantitate the ability of test compounds to inhibit BTK mediated phosphorylation of substrate. Assays were assembled in 384 well plates where 6 nM of full-length human His-tagged BTK (Life Technologies cat # PV3587) and test compound at varying concentrations were preincubated for 15 minutes at 28° C. Then, 1 uM of TK substrate-biotin and 30 uM ATP were added and incubated for an additional 30 minutes at 28° C. Phospohrylation was detected by adding 62.5 nM Streptavidin-XL665 and TK-Antibody Cryptate diluted 1:100 in HTRF detection buffer (Cisbio cat #62SDBRDF) and incubated for 60 minutes at RT. The plate was read on an Envision plate reader and the fluorescence is measured at 620 nm (cryptate) and 665 nm (XL665). A ratio is calculated (665/620) and converted to POC relative to control and blank wells.
Measurement: IC50 = 6600 nM (pChEMBL 5.179999828338623)
Descriptors: MW 441.49, ALogP 3.66, HBA 6, HBD 1, PSA 114.24, QED 0.59
Compound (SMILES): Cn1nnn(-c2cc(Nc3ncc(Cl)c(N[C@@H]4C[C@@H]5CCCN5C(C)(C)C4)n3)c(F)cc2O)c1=O
InChIKey: MRPPCAXBPQTACM-OLZOCXBDSA-N
Target: Interleukin-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P60568
Assay: IL-2 ELISA Assay: Primary T cell stimulation and IL2 ELISA: Human primary T cells (100,000 cells per well) were pre-incubated with or without test compound in Yssel's medium for 1 hour at 37° C. Cells were then stimulated by transferring them to round-bottom 96-well plates pre-coated with 1 μm/ml αCD3 and 5 μg/ml αCD28. For counter assay, cells were instead stimulated by adding 8× stock solutions of PMA and ionomycin in Yssels (for final concentrations of 0.5 ng/ml PMA and 0.1 uM ionomycin, both from Calbiochem). Cells were incubated at 37° C. for 24 hours before 100 μL supernatants were harvested for quantification of IL-2 by ELISA using Human IL-2 Duoset ELISA Kit from R and D Systems, Cat. # DY202E.
Measurement: EC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 503.97, ALogP 2.82, HBA 11, HBD 3, PSA 126.02, QED 0.45
Compound (SMILES): C[C@@H]1CC(F)(F)CN(C(=O)c2nn(C)cc2-c2ncc(F)cn2)[C@@H]1COc1cnc(C(F)(F)F)cn1
InChIKey: AXQODOJGNSQRMR-BXUZGUMPSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Orexin Receptor Cell-Based Functional Assay: Measurement of [Ca2+]i using a FLIPR: CHOOX1 or CHOOX2 cells were seeded into black-walled clear-base 384-well plates (Corning, catalog #3712) at a density of 20,000 cells per well in F12-K medium supplemented with 10% FBS and then incubated in a 5% CO2, 37° C. incubator overnight to reach 90% confluency. The cells were incubated with equal volume of calcium 6 loading buffer (Molecular Devices, Inc.) containing 2.5 mM probenecid at 37° C. for 2 h, followed by test compounds (dose-range 0.1 nM-10 μM) for another 30 min. The plates were then placed into a FLIPR (Molecular Devices, Inc.) to monitor fluorescence (λ excitation 488 nm, λ emission 540 nm) before and after the addition of EC90 of [OX].
Measurement: IC50 > 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 515.42, ALogP 3.39, HBA 8, HBD 0, PSA 98.92, QED 0.48
Compound (SMILES): NS(=O)(=O)c1c(C(F)(F)F)ccc(-c2ccc(C3CNC3)cc2)c1-c1nn[nH]n1
InChIKey: SKBNRLXMOUEXLT-UHFFFAOYSA-N
Target: Metallo-beta-lactamase type 2 (SINGLE PROTEIN), Bacteria
UniProt: C7C422
Assay: Class B Enzyme Assay: The Class B enzyme activities were measured in the presence of the test inhibitor in a fluorescence assay against a commercially available substrate consisting of a cephalosporin core linking 7-hydroxycoumarin to fluorescein (CCF2-FA). The enzyme (NDM-1, IMP-1 or VIM-1) and the substrate were diluted in 100 mM KH2PO4 buffer (pH 7) containing 0.005% Tween-20 and 10 μM ZnSO4. In the assay, the final concentration of enzyme was 1 pM, 2 pM and 30 pM for NDM-1, IMP-1 and VIM-1, respectively, and the final concentration of CCF2-FA was 1.25 μM. The test inhibitor was dissolved in dimethylsulfoxide and diluted 1:50 in the assay, resulting in a final concentration range of 20 uM to 0.00063 uM. In a 384-well microplate, the test inhibitor was incubated with the metallo-β-lactamase enzyme and the substrate for 2 hours at 25° C. Fluorescence at 460 nm following excitation at 405 nm was measured. The IC50 value was determined from semi-logarithmic plots of enzyme inhibition versus inhibitor concentration, with a curve generated using a 4-parameter fit.
Measurement: IC50 = 0.875 nM (pChEMBL 9.0600004196167)
Descriptors: MW 424.41, ALogP 1.89, HBA 6, HBD 3, PSA 126.65, QED 0.59
Compound (SMILES): Nc1nccc(-n2ccc3c(OCc4cccc(Cl)c4)cccc32)n1
InChIKey: NOGISNUMCISADM-UHFFFAOYSA-N
Target: Rho-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13464
Assay: Z-lyte FRET Assay: The inventors undertook a screen of the NCI Diversity set using a Z-lyte FRET assay (Kang et al. (2009); Wu et al. (2010)) to measure kinase inhibition.
Measurement: IC50 = 34600 nM (pChEMBL 4.460000038146973)
Descriptors: MW 350.81, ALogP 4.24, HBA 5, HBD 1, PSA 65.96, QED 0.6
Compound (SMILES): CC(C)(C(=O)O)c1cccc(CCCOc2ccc(-c3ccn[nH]3)nc2)c1
InChIKey: TXTNYSMRKKUSMS-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 = 1300 nM (pChEMBL 5.889999866485596)
Descriptors: MW 365.43, ALogP 3.85, HBA 4, HBD 2, PSA 88.1, QED 0.59
Compound (SMILES): CCS(=O)(=O)c1ccc(Cl)cc1Cn1cnc2c(Cl)c(CN3CC[C@@H](N)C3)c(OC(F)(F)F)cc2c1=O
InChIKey: YLTZEXUOZJLEQX-OAHLLOKOSA-N
Target: Epithelial discoidin domain-containing receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q08345
Assay: LanthaScreen Assay: DDR1 binding activity was measured using the LanthaScreen (Registered trademark) Eu Kinase Binding Assay (manufactured by Life Technologies Corporation). The test compound and the Alexa Fluor® 647-labeled Kinase Tracer 178 (manufactured by Life Technologies Corporation) were added to a mixture of DDR1 and the LanthaScreen (Registered trademark) Eu-anti-GST antibody. After one-hour reaction at room temperature, the fluorescence resonance energy transfer was measured. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate relative to the test compound-free control.
Measurement: IC50 = 26 nM (pChEMBL 7.579999923706055)
Descriptors: MW 579.43, ALogP 3.98, HBA 8, HBD 1, PSA 107.52, QED 0.45
Compound (SMILES): CC(C)OCCCNC(=O)[C@@H]1CN(S(=O)(=O)c2cn(C)cn2)C[C@H]1c1ccc(F)cc1
InChIKey: XJGQAOAVAUVCKM-RBUKOAKNSA-N
Target: Alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P26572
Assay: Radioligand Binding Assay: Radioligand binding assays using recombinant CHO cell membranes expressing human GlyT1: Radioligand binding to human GlyT1c transporter-expressing membranes was determined as described in Mezler et al., Molecular Pharmacology 74:1705-1715, 2008.
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 452.55, ALogP 1.89, HBA 6, HBD 1, PSA 93.53, QED 0.59
Compound (SMILES): CS(=O)(=O)N1CC(n2cc(-c3cc(NC(=O)C4CC4)nc4[nH]ccc34)cn2)C1
InChIKey: BMDLGTPDZUGUKW-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: ADP-Glo Kinase Assay: A control material and a test material were prepared for each concentration, in such a way that they were diluted by means of DMSO. At the same time, ATP (250 uM) and JAK substrate (JAK1, IRS-itide 40 ng/mL) were prepared, in such a way that they were diluted by means of kinase buffer (40 mM Tris-HCl pH 7.5, 20 mM MgCl2, 0.5 mg/mL BSA, 50 uM DTT).A test drug for each concentration, substrate, ATP and JAK enzyme were mixed in an eppendorf tube, and reacted in a 30° C. incubator for 40 minutes.ADP-Glo reagent contained in ADP-Glo Kinase Enzyme System (Promega, USA, V9571) was added into each eppendorf tube, and reacted in a 30° C. incubator for 40 minutes. A kinase detection reagent contained in ADP-Go Kinase Enzyme System was inserted into the eppendorf tube, then an integration time was set to 1 second by using Wallac Victor 2, then luminescence was measured, and then JAKs phosphorylation inhibition ability of the test material was analyzed. The concentration of the compound, which causes 50% of the JAK enzyme activity inhibition in comparison With the control group, was determined as IC50 (nM) of an inhibitor, wherein the results thereof are shown in a following table 1.
Measurement: IC50 = 110 nM (pChEMBL 6.960000038146973)
Descriptors: MW 400.46, ALogP 1.59, HBA 6, HBD 2, PSA 112.98, QED 0.68
Compound (SMILES): C[C@H](c1ccc(C(F)(F)F)nc1)n1nc(C#N)c2c(=O)[nH]c([C@H]3CC[C@@H]3c3ncccn3)nc21
InChIKey: AGJXXEHLPVMHRJ-XBFCOCLRSA-N
Target: High affinity cGMP-specific 3',5'-cyclic phosphodiesterase 9A (SINGLE PROTEIN), Homo sapiens
UniProt: O76083
Assay: Enzyme Assay: Assays for PDE 1 through 11 were performed in parallel at room temperature in 384-well microtiter plates with an incubation volume of 20.2 μL. Solutions of test compounds were prepared in DMSO and serially diluted with DMSO to yield 30 μL of each of ten solutions differing by 3-fold in concentration, at 32 serial dilutions per plate. 100% inhibition was determined by adding buffer in place of the enzyme and 0% inhibition is determined by using DMSO (1% final concentrations). A Labcyte POD 810 (Labcyte, Sunnyvale, Calif.) was used to dispense 200 nL from each well of the titration plate to make eleven copies of the assay plate for each titration, one copy for each PDE enzyme. A solution of each enzyme (dilution from aliquots, sufficient to produce 20% substrate conversion) and a separate solution of FAM-labeled cAMP or FAM-labeled cGMP from Molecular Devices (Sunnyvale, Calif., product # R7506 or cGMP#R7508), at a final concentration of 50 nM were made in the assay buffer (10 mM Tris HCl, pH 7.2, 10 mM MgCl2, 0.05% NaN3 0.01% Tween-20, and 1 mM DTT). Note that the substrate for PDE2 is 50 nM FAM cAMP containing 1000 nM of cGMP. The enzyme and the substrate were then added to the assay plates in two consecutive additions of 10 μL and then shaken to mix. The reaction was allowed to proceed at room temperature for 60 minutes. A binding solution was then made from the kit components, comprised of 80% Solution A, 20% Solution B and binding reagent at a volume of 1/600 the total binding solution. The enzymatic reaction was stopped by addition of 60 μL of the binding solution to each well of the assay plate. The plates were sealed and shaken for 10 seconds
Measurement: Ki = 4.7 nM (pChEMBL 8.329999923706055)
Descriptors: MW 466.43, ALogP 3.47, HBA 8, HBD 1, PSA 126.03, QED 0.49
Compound (SMILES): CC1CC(=O)Nc2cccc(C(=O)Nc3cccc(-c4nncn4C)c3)c2N1
InChIKey: DSPRUHNYLWDVCX-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: CBP TR-FRET Binding Assay: His/Flag epitope tagged CBP was cloned, expressed, and purified to homogeneity. CBP binding and inhibition was assessed by monitoring the engagement of a biotinylated small molecule compound with the target using the TR-FRET assay technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate CBP (4 nM final) was combined with biotin-ligand (60 nM final) in 50 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.2% DMSO) or compound dilution series in DMSO. After 10 minutes incubation at room temperature, a mixture Eu-W1024 Anti-6×His antibody (Perkin Elmer ADO 110) and SureLight Allophycocyanin-Streptavidin (APC-SA, Perkin Elmer CR130-100) were added to a final concentrations of 0.2 nMolar antibody and 50 nMolar APC-SA, respectively. After twenty minutes of equilibration, the plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 403 nM (pChEMBL 6.389999866485596)
Descriptors: MW 376.42, ALogP 2.88, HBA 6, HBD 3, PSA 100.94, QED 0.65
Compound (SMILES): Cc1c(-c2[nH]c3ccc(C4CCN(C(=O)CNC5CC5)CC4)cc3c2C(C)C)cn2ncnc2c1C
InChIKey: LJBNVOFLHDDNPO-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 nM (pChEMBL 9)
Descriptors: MW 484.65, ALogP 5.08, HBA 5, HBD 2, PSA 78.32, QED 0.4
Compound (SMILES): C[C@H](Nc1nc(C(=O)N2CC(NC(=O)C3CC3)C2)c2sccc2n1)c1cncc(F)c1
InChIKey: FNUFNFZDEXZSLH-NSHDSACASA-N
Target: Adenosine receptor A2b (SINGLE PROTEIN), Homo sapiens
UniProt: P29275
Assay: Activity Assay: Compounds (Adenosine Receptor modulators) and activity assay.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 440.5, ALogP 2.75, HBA 7, HBD 2, PSA 100.11, QED 0.61
Compound (SMILES): CC(C)(C)c1cccc(NC(=O)Nc2cccc(C3CCCN(c4ncnc5[nH]ccc45)C3)c2)c1
InChIKey: XIZARNQSMWJZSR-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: In Vitro BTK Kinase Assay: The purpose of the BTK in vitro assay is to determine compound potency against BTK through the measurement of IC50. Compound inhibition is measured after monitoring the amount of phosphorylation of a fluorescein-labeled polyGAT peptide (Invitrogen PV3611) in the presence of active BTK enzyme (Upstate 14-552), ATP, and inhibitor. The BTK kinase reaction was done in a black 96 well plate (costar 3694). For a typical assay, a 24 μL aliquot of a ATP/peptide master mix (final concentration; ATP 10 μM, polyGAT 100 nM) in kinase buffer (10 mM Tris-HCl pH 7.5, 10 mM MgCl2, 200 μM Na3PO4, 5 mM DTT, 0.01% Triton X-100, and 0.2 mg/ml casein) is added to each well. Next, 1 μL of a 4-fold, 40× compound titration in 100% DMSO solvent is added, followed by adding 15 uL of BTK enzyme mix in 1× kinase buffer (with a final concentration of 0.25 nM). The assay is incubated for 30 minutes before being stopped with 28 μL of a 50 mM EDTA solution. Aliquots (5 μL) of the kinase reaction are transferred to a low volume white 384 well plate (Corning 3674), and 5 μL of a 2× detection buffer (Invitrogen PV3574, with 4 nM Tb-PY20 antibody, Invitrogen PV3552) is added. The plate is covered and incubated for 45 minutes at room temperature. Time resolved fluorescence (TRF) on Molecular Devices M5 (332 nm excitation; 488 nm emission; 518 nm fluorescein emission) is measured. IC50 values are calculated using a four parameter fit with 100% enzyme activity determined from the DMSO control and 0% activity from the EDTA control.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 468.61, ALogP 6.28, HBA 4, HBD 3, PSA 85.94, QED 0.33
Compound (SMILES): Cc1noc(C)c1-c1cnc2c(-c3cnn(CC(F)(F)F)c3)cn(CC3CC(F)(F)C3)c2c1
InChIKey: ZVROERWTIKLVHP-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Alphascreen Binding Assay: Binding of compounds of formula (I) with bromodomain 2, 3 4 was assessed using Alphascreen binding assay. The inhibition of the interaction between bromodomain and its acetylated target protein (Filippakopoulos P et al. 2012) was measured quantitatively using recombinant BRD proteins, an acetylated Histone 4 peptide and AlphaScreen technology. In absence of inhibition the BRD protein bound to AlphaScreen nickel chelate acceptor beads can interact with the acetylated Histone 4 peptide which is immobilized by the AlphaScreen Streptavidin coated beads. This interaction brings donor and acceptor beads in proximity. The close proximity allows the singlet oxygen produced by laser excitation of the donor beads to reach the acceptor beads and generate a luminescence signal. BRD inhibitors result in a decrease in the proximity signal through an inhibition of the BRD-acetylated peptide interaction.Recombinant human bromodomains containing the N-terminal bromodomain (BRD2-BD1 (71-194), BRD3-BD1 (24-144) and BRD4-BD1 (44-164)) or dual bromodomains (BRD4-BD12 (1-477), BRD4-BD12 (1-472)) were prepared and purified as described in protein expression and purification session.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 465.43, ALogP 5.78, HBA 6, HBD 0, PSA 61.67, QED 0.35
Compound (SMILES): Cc1ccc(C[C@@H](C(=O)O)N2CCN(C(=O)C3CN(C4(C)CCOCC4)CC3c3ccc(F)cc3F)[C@@H](C)C2)c(F)c1
InChIKey: DXRNAZVQZBKAKG-PKISZUCKSA-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 = 18 nM (pChEMBL 7.75)
Descriptors: MW 587.68, ALogP 4.23, HBA 5, HBD 1, PSA 73.32, QED 0.52
Compound (SMILES): Cc1nc(C2CCN(c3ncnc4[nH]c(-c5ccc(F)cc5)cc34)CC2)[nH]c1-c1ccccc1
InChIKey: ZDKDTUKRVULWGY-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Biochemical Assay: Assays for biochemical cell-based activity of c-kit kinase are known in the art, for example, as described in U.S. Pat. Nos. 7,498,342 and 7,846,941, the disclosures of which are hereby incorporated by reference as it relates to such assays. The c-kit (or kinase domain thereof) is an active kinase in AlphaScreen. IC50 values are determined with respect to inhibition of c-Kit kinase activity, where inhibition of phosphorylation of a peptide substrate is measured as a function of compound concentration. Compounds to be tested were dissolved in DMSO to a concentration of 30 mM. These were diluted 30 μl into 130 μl of DMSO (4 mM) and 1 μl was added to an assay plate. These were then serially diluted 1:3 (50 μl to 100 μl DMSO) for a total of 8 points. Plates were prepared such that each kinase reaction is 30 μl in 1× kinase buffer (50 mM HEPES, pH 7.2, 5 mM MgCl2, 5 mM MnCl2, 0.01% NP-40, 0.2% BSA), 5% DMSO and 10 μM ATP. Substrate was 100 nM biotin-(E4Y)3 (Open Source Biotech, Inc.). C-kit kinase was at 0.1 ng per sample. After incubation of the kinase reaction for 1 hour at room temperature, 5 μl of donor beads (Streptavidin coated beads (Perkin Elmer Life Science) final concentration 1 μg/ml) in stop buffer (50 mM EDTA in 1× kinase buffer) was added, the sample was mixed and incubated for 30 minutes at room temperature before adding 5 ml of acceptor beads (PY30 coated beads (Perkin Elmer Life Science) final concentration 1 μg/ml) in stop buffer. The samples were incubated for 60 minutes at room temperature and the signal per well was read on AlphaQuest reader. Phosphorylated substrate results in binding of the PY30 antibody and association of the donor and acceptor beads such that signal correlates with kinase activity.
Measurement: Ki = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 452.54, ALogP 5.85, HBA 4, HBD 2, PSA 73.49, QED 0.36
Compound (SMILES): N#Cc1cc(NC(=O)c2cnn(-c3ncnn4cccc34)c2C(F)(F)F)cnc1-n1nccn1
InChIKey: GZUFBGXIUYFHGZ-UHFFFAOYSA-N
Target: Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UDY8
Assay: In Vitro Assay: MALT1 protease activity was assessed in an in vitro assay using a tetrapeptide as substrate and full-length MALT1 protein (Strep-MALT1(1-824)-His) purified from baculovirus-infected insect cells. The tetrapeptide LRSR is coupled to AMC (7-amino-4-methylcoumarin) and provides a quenched, fluorescent substrate for the MALT1 protease (SM Biochemicals). Cleavage of AMC from the Arginine residue results in an increase in coumarin fluorescence measured at 460 nm (excitation 355 nm). The final assay buffer consisted of 10 nM FL MALT1 protein, 200 μM Ac-LRSR-AMC, 50 mM Tris pH 7.5, 0.6 M Citrate, 1 mM DTT, 1 mM EDTA, 0.05% BSA and 1.5% DMSO. Test compounds were spotted at 50 nL in 100% DMSO per well of a black 384-Proxiplate (Perkin Elmer). Test compound concentrations ranged from 30 μM to 0.5 nM using 11 dilution steps (1:3). Background signal was measured from control wells containing assay buffer without enzyme which functions as low control (LC). High control (HC) values were generated using the reaction with enzyme but no compound treatment. Compounds were pre-incubated with MALT1 enzyme for 50 minutes at RT. Substrate was added subsequently and fluorescence was measured in Labsystems fluoroskan at excitation 355 nm and emission 460 nm to determine time 0. The reaction was subsequently incubated for 4 h at RT and fluorescence was measured.
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 465.36, ALogP 2.03, HBA 11, HBD 1, PSA 144.5, QED 0.42
Compound (SMILES): N#CCC1(CS(=O)(=O)NC2CC=C(c3cc(NC(=O)C4CC4)nc4[nH]ccc34)CC2)CC1
InChIKey: PJEKKDPNWXTZPG-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: ADP-Glo Kinase Assay: A control material and a test material were prepared for each concentration, in such a way that they were diluted by means of DMSO. At the same time, ATP (250 uM) and JAK substrate (JAK1, IRS-itide 40 ng/mL) were prepared, in such a way that they were diluted by means of kinase buffer (40 mM Tris-HCl pH 7.5, 20 mM MgCl2, 0.5 mg/mL BSA, 50 uM DTT).A test drug for each concentration, substrate, ATP and JAK enzyme were mixed in an eppendorf tube, and reacted in a 30° C. incubator for 40 minutes.ADP-Glo reagent contained in ADP-Glo Kinase Enzyme System (Promega, USA, V9571) was added into each eppendorf tube, and reacted in a 30° C. incubator for 40 minutes. A kinase detection reagent contained in ADP-Go Kinase Enzyme System was inserted into the eppendorf tube, then an integration time was set to 1 second by using Wallac Victor 2, then luminescence was measured, and then JAKs phosphorylation inhibition ability of the test material was analyzed. The concentration of the compound, which causes 50% of the JAK enzyme activity inhibition in comparison With the control group, was determined as IC50 (nM) of an inhibitor, wherein the results thereof are shown in a following table 1.
Measurement: IC50 < 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 453.57, ALogP 3.46, HBA 5, HBD 3, PSA 127.74, QED 0.56
Compound (SMILES): Cc1ccc2nc(-c3ccc(O)c4c3C(C)(C)CN4c3ccccc3NC(=O)Nc3ccc(OC(F)(F)F)cc3)oc2c1
InChIKey: CQYQLYINFJRGBM-UHFFFAOYSA-N
Target: P2Y purinoceptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P47900
Assay: Scintillation Proximity Assay (SPA): A SPA membrane binding assay was used to identify inhibitors of [33P] 2MeS-ADP binding to cloned human P2Y1 receptors (The P2Y1 receptor membranes were provided by Biology and the cloning of the receptor and P2Y1 receptor membrane preparation is same as described by Biology). Binding reactions were performed in 384-well OptiPlates (PerkinElmer Life Sciences, Cat #6007299) in a volume of 50 μL containing 15 fmol of P2Y1 receptor (1.7 μg of total protein), 0.3 nM [33P] 2MeS-ADP (PerkinElmer; 2,000 Ci/mmol), various concentrations of the test compound (usually between 10 μM and 160 pM) in Buffer B containing 1% DMSO in assay buffer (15 mM, HEPES, 145 mM potassium chloride, 5 mM sodium chloride, 5 mM EDTA, 0.1 mM MgCl2, pH 7.4) and 100 μg of SPA bead (WGA polystyrene Image beads, #RPNQ 0260V, Amersham). Reactions were allowed to proceed to completion at room temperature for 1 hour followed by centrifugation of the plate for 5 min. About 40 μL of the aqueous solution was aspirated. Plates were sealed and the [33P] 2MeS-ADP bound to the P2Y1 receptor membranes that were bound to the SPA bead were determined in a Gen 4 LEADSEEKER (Amersham) Image Reader.
Measurement: Ki = 33 nM (pChEMBL 7.480000019073486)
Descriptors: MW 588.59, ALogP 8.48, HBA 6, HBD 3, PSA 99.86, QED 0.19