lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCC(CC)n1cc(-c2cnc(N)c(-c3cc(-c4cccc(C(C)(C)N)c4)no3)n2)ccc1=O
InChIKey: FAFYGQMJDFRXNJ-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Inhibition Assay: Compounds were screened for their ability to inhibit ATR kinase using a radioactive-phosphate incorporation assay. Assays were carried out in a mixture of 50 mM Tris/HCl (pH 7.5), 10 mM MgCl2 and 1 mM DTT. Final substrate concentrations were 10 μM [γ-33P]ATP (3 mCi 33P ATP/mmol ATP, Perkin Elmer) and 800 μM target peptide (ASELPASQPQPFSAKKK).Assays were carried out at 25° C. in the presence of 5 nM full-length ATR. An assay stock buffer solution was prepared containing all of the reagents listed above, with the exception of ATP and the test compound of interest. 13.5 μL of the stock solution was placed in a 96 well plate followed by addition of 2 μL of DMSO stock containing serial dilutions of the test compound (typically starting from a final concentration of 15 μM with 3-fold serial dilutions) in duplicate (final DMSO concentration 7%). The plate was pre-incubated for 10 minutes at 25° C. and the reaction initiated by addition of 15 μL [γ-33P]ATP (final concentration 10 μM).The reaction was stopped after 24 hours by the addition of 30 μL 0.1 M phosphoric acid containing 2 mM ATP. A multiscreen phosphocellulose filter 96-well plate (Millipore, Cat no. MAPHNOB50) was pretreated with 100 μL 0.2M phosphoric acid prior to the addition of 45 μL of the stopped assay mixture. The plate was washed with 5×200 μL 0.2M phosphoric acid. After drying, 100 μL Optiphase SuperMix liquid scintillation cocktail (Perkin Elmer) was added to the well prior to scintillation counting (1450 Microbeta Liquid Scintillation Counter, Wallac).
Measurement: Ki < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 458.57, ALogP 4.76, HBA 8, HBD 2, PSA 125.85, QED 0.41
Compound (SMILES): Cc1cc(Nc2ncnc3sc(C(=O)N4CC(C#N)C4)cc23)c(=O)n2c1C(=O)NC21CCCC1
InChIKey: AVTZSVWIAFBHGI-UHFFFAOYSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BUB5
Assay: MKNK1 Kinase High ATP Assay: MKNK1-inhibitory activity at high ATP of compounds of the present invention after their preincubation with MKNK1 was quantified employing the TR-FRET-based MKNK1 high ATP assay as described in the following paragraphs.A recombinant fusion protein of Glutathione-S-Transferase (GST, N-terminally) and human full-length MKNK1 (amino acids 1-424 and T344D of accession number BAA 19885.1), expressed in insect cells using baculovirus expression system and purified via glutathione sepharose affinity chromatography, was purchased from Carna Biosciences (product no 02-145) and used as enzyme. As substrate for the kinase reaction the biotinylated peptide biotin-Ahx-IKKRKLTRRKSLKG (SEQ ID NO: 1) (C-terminus in amide form) was used, which can be purchased e.g. from the company Biosyntan (Berlin-Buch, Germany)For the assay 50 nL of a 100 fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 μL of a solution of MKNK1 in aqueous assay buffer [50 mM HEPES pH 7.5, 5 mM MgCl2, 1.0 mM dithiothreitol, 0.005% (v/v) Nonidet-P40 (Sigma)] was added and the mixture was incubated for 15 mM at 22° C. to allow pre-binding of the test compounds to the enzyme before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 μL of a solution of adenosine-tri-phosphate (ATP, 3.3 mM=>final conc. in the 5 μL assay volume is 2 mM) and substrate (0.1 μM=>final conc. in the 5 μL assay volume is 0.06 μM) in assay buffer and the resulting mixture was incubated for a reaction time of 30 min at 22° C. The concentration of MKNK1 was adjusted depending of the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical concentrations were in the range of 0.003 μg/mL. The reaction was stopped by the addition of 5 μL of a solution of TR-FRET detection reagents (5 nM streptavidine-XL665 [Cisbio Bioassays, Codolet, France] and 1 nM anti-ribosomal protein S6 (pSer236)-antibody from Invitrogen [#44921G] and 1 nM LANCE EU-W1024 labeled ProteinG [Perkin-Elmer, product no. AD0071]) in an aqueous EDTA-solution (100 mM EDTA, 0.1% (w/v) bovine serum albumin in 50 mM HEPES pH 7.5).
Measurement: IC50 = 5.9 nM (pChEMBL 8.229999542236328)
Descriptors: MW 475.53, ALogP 2.47, HBA 9, HBD 2, PSA 133.01, QED 0.59
Compound (SMILES): CC(C)(NC(=O)[C@H]1[C@@H]2CNC[C@@H]21)c1cnn2c1CCCC2
InChIKey: NRCSPKPQUQHJKO-YABSGUDNSA-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 = 47.3 nM (pChEMBL 7.329999923706055)
Descriptors: MW 288.4, ALogP 1.04, HBA 4, HBD 2, PSA 58.95, QED 0.87
Compound (SMILES): C[C@H]1CCCOc2ccc(F)cc2C(=O)Nc2cccc(n2)-c2nncn21
InChIKey: CBPOLKLECMHSOW-LBPRGKRZSA-N
Target: Mitogen-activated protein kinase kinase kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99683
Assay: TR-FRET Assay: Table 2: Alternatively, the protein kinase activity of the compounds described herein were tested using the ASK1/MAP3K5 assay by Reaction Biology Corp. The assay procedure can be found on Reaction Biology Corp.website at http://www.reactionbiology.com/webapps/site/KinasePDFs/ASK1_MAP3K5.pdf. ASK1/MAP3K5 was used as the protein kinase and 20 μM of myelin basic protein (MBP) and 10 μM ATP were used as the substrate.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 367.38, ALogP 3.47, HBA 6, HBD 1, PSA 81.93, QED 0.66
Compound (SMILES): CC(C)C(Nc1cc(-c2cnc(NC(=O)C3CC3)cn2)ccc1C#N)C(=O)N(C)C
InChIKey: DFWTVNIOPWJYRQ-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: Inhibition Assay: TBK1 inhibition is determined using a 384 well μlate format in buffer containing 20 mM Hepes, pH 7.4, 10 mM MgCl2, 1 mM EDTA, 0.01% Brij-L23, 1 mM DTT. Each test compound is prepared in DMSO using 2.5-fold serial dilutions for 11 data points, which are added to the buffer so that each dilution contains 1% DMSO. To each well is added 2 μL of 1 μM 5FAM-DRHDSGLDSMKDE-NH2 (in buffer), 2 μL of diluted test compound (1% DMSO in buffer), and 5 μL of 3 nM TBK1 and 25 μM ATP (in buffer). The reaction mixture is incubated at RT for 60 min, and quenched by adding 20 mM Hepes, pH 7.4, 10 mM MgCl2, 1 mM EDTA, 0.01% Brij-L23, 1 mM DTT+25 mM EDTA. To quantify the fluorescent-labeled substrate and product following reaction, the test plate is loaded on a Caliper LC-3000, which measures percent of conversion by microfluidic-based separation.
Measurement: IC50 = 50.1 nM (pChEMBL 7.300000190734863)
Descriptors: MW 406.49, ALogP 2.89, HBA 6, HBD 2, PSA 111.01, QED 0.73
Compound (SMILES): CNC(=O)c1cc(OC)c(Nc2nc(OC3CCCC3)c3c(-c4ccc5nc(C)oc5c4)c[nH]c3n2)cc1F
InChIKey: FVXILEFBMIKZCO-UHFFFAOYSA-N
Target: Dual specificity protein kinase TTK (SINGLE PROTEIN), Homo sapiens
UniProt: P33981
Assay: Radioactivity Based Kinase Assay: The kinase activity is measured using a radioactivity based kinase assay, which measures the incorporation of a radioactively labeled phosphate moiety (for example, 33P labeled phosphate) into a substrate, for example, a peptide substrate. Reduced levels of radioactively labeled phosphate incorporation into the substrate indicates inhibition of kinase activity. In another embodiment, the kinase activity is measured using a time-resolved-fluorescence resonance energy transfer (TR-FRET) based kinase assay, which measures loss of fluorescence as a result of substrate phosphorylation, for example, a peptide substrate (see for example Invitrogen Z'-Lyte Assay®). Increased levels of fluorescence indicates inhibition of kinase activity. In another embodiment, the kinase activity is measured using a competitive tracer binding assay (for example, Invitrogen Lanthascreen® Eu binding assay), which measures fluorescence as a result of tracer binding (for example ATP site binding). Reduced fluorescence indicates displacement of tracer binding, which indicates inhibition of kinase activity.
Measurement: IC50 = 8.06 nM (pChEMBL 8.09000015258789)
Descriptors: MW 530.56, ALogP 5.65, HBA 8, HBD 3, PSA 127.19, QED 0.25
Compound (SMILES): CN(C(=O)OC(C)(C)C)[C@H]1[C@H]2OC[C@](CO)(O2)[C@H](O)[C@@H]1O
InChIKey: LGLYGISPBZZYBP-LGMRYKSHSA-N
Target: Asialoglycoprotein receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P07306
Assay: SPR Binding Measurements: All SPR measurements with compounds were performed using a Biacore 3000 (GE Healthcare) at 25° C. Biotinylated ASGPR was immobilized typically at 2000-3000 resonance units (Ru) using either SA sensor chips (GE Healthcare) or custom sensor chips with Neutravidin (Pierce Biochemical) immobilized by standard amine coupling to CM5 sensor chips (GE Healthcare). The running buffer was HBS (10 mM HEPES, 150 mM NaCl), 20 mM CaCl2, 0.01% p20, 3% DMSO or 50 mM tris, 150 mM NaCl, 50 mM CaCl2), 0.01% p20, 3% DMSO pH 7.5. Compounds were diluted into running buffer at a concentration of 900 uM and serially diluted 3 fold to 3.7 uM. Compound solutions were injected at 50 ul/min for 1 min followed by a 1 min dissociation in duplicate for each concentration. For the multimeric conjugates (dimers, trimers), the conjugates were diluted in running buffer to concentrations of 100 nM or 10 nM and serially diluted. Conjugates were injected for 2 min and off rates were detected for 300 or 600 sec. After completion of off phase data the compounds were displaced using an injection of 900 uM GalNAc returning the receptor surface to the free state. All data was processed using Scrubber2 (Biologic Software, Inc.) to zero, align, reference and correct for excluded volume effects. KDs were determined by fitting the steady state binding responses for the compounds and single conjugated molecules in Scrubber2. KD for multimeric conjugates showing kinetic responses were processed in Scrubber2 and fit in BiaEval (GE Healthcare) to extract the on and off rate parameters in order to calculate KD. Values reflect standard deviations from multiple experiments.
Measurement: Kd = 12300 nM (pChEMBL 4.909999847412109)
Descriptors: MW 305.33, ALogP -0.94, HBA 7, HBD 3, PSA 108.69, QED 0.6
Compound (SMILES): Cc1noc(C)c1-c1cnc2c3ccc(C(C)(C)O)cc3n(Cc3ccccc3)c2c1
InChIKey: CXXAVJTXGKXXEX-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: TR-FRET Assay: All assay components were dissolved in buffer composition 20 mM Hepes pH 7.5, 150 mM NaCl, 5 mM DTT, 0.005% Tween 20, and 100 ug/ml BSA for BRD4 (1-477 and 44-460). The final concentrations of the bromodomain proteins are 1.6 nM BRD4(44-168), 1 nM BRD4(333-460), and 1 nM BRD4(1-477 or 44-460), and the fluorescent probe molecule is 100 nM, 50 nM, and 7.5 nM respectively. All proteins were biotinylated. A streptavidin labeled with terbium cryptate (Cisbio SA-Tb) was used as detection, and pre-mixed with the bromodomain protein at a final concentration of 0.2 nM. In some instances for BRD4 (44-460), anti-His terbium cryptate was used as a detection. 7.5 nl of dose-responsed test compound or dmso vehicle (0.0375%) was pre-spotted in a black Corning 384 well plate and 10 ul each of bromodomain/detection reagent and fluorescent small molecule solution were added to the plate, and the reaction incubated for 60 min at room temperature. Plates were then read on EnVision plate reader, (λex=340 nm, acceptor λEm=520 nm, and donor λEm=615 nm, LANCE D400 mirror). Time resolved fluorescence intensity measurements were made at both emissions, and the ratio of acceptor/donor was calculated and used for data analysis.
Measurement: IC50 < 1500 nM (pChEMBL 5.823999881744385)
Descriptors: MW 411.51, ALogP 5.74, HBA 5, HBD 1, PSA 64.08, QED 0.41
Compound (SMILES): C[C@@H](CO[C@H]1C[C@H](COc2ccc3nc(OCC(F)F)sc3n2)C1)NC(N)=O
InChIKey: KGBFRGAXMSNDIH-DCAQKATOSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Inhibitory Activity on Human ACC1 and the ACC2: Recombinant human ACC1 and recombinant human ACC2, which were prepared by the method mentioned above, were preincubated with assay buffer solution (50 mM HEPES-KOH (pH 7.4), 10 mM magnesium chloride, 6-10 mM potassium citrate, 4 mM reduced form of glutathione, 1.5 mg/ml bovine serum albumin) for one hour. Then, 0.2 μL of each this invention compound solution (in DMSO) were dispensed to 384-well microplate, 5 μL of the preincubated enzyme solution and 5 μL of substrate solution (50 mM HEPES-KOH (pH 7.4), 1 mM ATP, 0.8 mM acetyl CoA and 25-50 mM potassium bicarbonate) were added to microplate. After centrifugation and shaking, the reaction mixtures were incubated in a humidified box at room temperature for 1 to 3 hours. After the incubation, the enzyme reactions were stopped by the addition of EDTA. Then, after the samples were cocrystallized with CHCA (α-cyano-4-hydroxy cinnamic acid) matrices on MALDI target plate, by using the matrix assist laser deionization time-of-flight mass spectrometer (MALDI-TOF MS), samples were measured in reflector negative mode. Deprotonated ions of acetyl CoA (AcCoA) of substrate and malonyl CoA (MalCoA) of the reaction product were detected, then, the conversion rates of acetyl CoA to malonyl CoA was calculated by the intensity of [MalCoA-H]−/(Intensity of [MalCoA-H]−+Intensity of [AcCoA-H]−) using each signal strength. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate of the enzymatic reaction at each concentration of the compounds. In addition, potassium citrate concentrations in assay buffer solution, potassium hydrogen carbonate concentrations in substrate solution and incubation time were adjusted by each lot of enzyme.
Measurement: IC50 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 416.45, ALogP 2.57, HBA 7, HBD 2, PSA 108.59, QED 0.62
Compound (SMILES): C[C@@H]1CCCCN1[C@H]1Cc2c(Cl)cc(Cl)cc2[C@@H]1Oc1ccc(S(=O)(=O)NCCOCCOCCNC(=O)NCCCCNC(=O)NCCOCCOCCNS(=O)(=O)c2ccc(O[C@H]3c4cc(Cl)cc(Cl)c4C[C@@H]3N3CCCC[C@H]3C)cc2)cc1
InChIKey: YXPVJXZWBVFMGQ-WVYBSBGMSA-N
Target: Sodium/hydrogen exchanger 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P48764
Assay: Cell-Based Assay of NHE-3 Activity (Pre-Incubation Inhibition): Rat and human NHE-3-mediated Na+-dependent H+ antiport was measured using a modification of the pH sensitive dye method originally reported by Paradiso (Proc. Natl. Acad. Sci. USA. (1984) 81(23): 7436-7440). PS120 fibroblasts stably expressing human NHE3 and NHERF2 were obtained from Mark Donowitz (Baltimore, Md.). Opossum kidney (OK) cells were obtained from the ATCC and propagated per their instructions. The rat NHE-3 gene (GenBank M85300) was introduced into OK cells via electroporation, and cells were seeded into 96 well plates and grown overnight. Medium was aspirated from the wells then incubated for 30 min at 37° C. with NH4Cl-HEPES buffer (20 mM NH4Cl, 80 mM NaCl, 50 mM HEPES, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4) containing 5 μM BCECF-AM. Cells were washed once with Ammonium free, Na+-free HEPES (100 mM choline, 50 mM HEPES, 10 mM glucose, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4) and incubated in the same buffer for 10 minutes at room temperature to lower intracellular pH with 0-30 μM test compound. After incubation, NHE-3-mediated recovery of neutral intracellular pH was initiated by addition of Na-HEPES buffer containing 0.4 μM ethyl isopropyl amiloride (EIPA, a selective antagonist of NHE-1 activity that does not inhibit NHE-3). Changes in intracellular pH were monitored using a FLIPR Tetra® (Molecular Devices, Sunnyvale, Calif.) by excitation at λex 439 to 505 nm, and measuring BCECF fluorescence at λem 538 nm. The initial rate of the fluorescence ratio change was used as a measure of NHE-mediated Na+/H+ activity, and reported as the change in fluorescence ratio per minute. Initial rates were plotted as the average of 2 or more replicates, and pIC50 values were estimated using GraphPad Prism.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 1313.31
Compound (SMILES): COc1c(Nc2cc(Nc3cnc(N4CCOCC4)c(C#N)n3)nc3[nH]c(C)nc23)cccc1-c1nnn(C)n1
InChIKey: KFCGBTZIMHDRFQ-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Binding Assay: Binding constants for compounds of the present invention against the JH2 domain were determined by the following protocol for a KINOMEscan® assay (DiscoveRx). A fusion protein of a partial length construct of human TYK2 (JH2domain-pseudokinase) (amino acids G556 to D888 based on reference sequence NP_003322.3) and the DNA binding domain of NFkB was expressed in transiently transfected HEK293 cells. From these HEK 293 cells, extracts were prepared in M-PER extraction buffer (Pierce) in the presence of Protease Inhibitor Cocktail Complete (Roche) and Phosphatase Inhibitor Cocktail Set II (Merck) per manufacturers' instructions. The TYK2 (JH2domain-pseudokinase) fusion protein was labeled with a chimeric double-stranded DNA tag containing the NFkB binding site (5′-GGGAATTCCC-3′) fused to an amplicon for qPCR readout, which was added directly to the expression extract (the final concentration of DNA-tag in the binding reaction is 0.1 nM).
Measurement: Kd = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 539.56, ALogP 2.45, HBA 14, HBD 3, PSA 180.5, QED 0.27
Compound (SMILES): Cc1nc(C(=O)N2C(COc3cc4ccccc4cn3)CC3CC2C3)c(-c2ccccc2)s1
InChIKey: MRYWAOSAXXOEPY-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Scintillation Proximity Assay (SPA) Binding Assay: CHO cells stably transfected with human Orexin type 1 receptors (CHO-hOX1) or HEK-293 cells transiently transfected with human Orexin type 2 receptors (HEK-hOX2) were collected after 16 h induction with 5 mM sodium butyrate. The cell pellets were re-suspended, homogenized in 15 mM Tris/HCl pH=7.5, 1 mM EGTA, 0.3 mM EDTA, 2 mM MgCl2, protease inhibitors and centrifuged at 40,000 g (20 min, 4° C.). After re-suspension, homogenization and centrifugation as above, the final pellets were re-suspended in 75 mM Tris/HCl pH=7.5, 1 mM EGTA, 0.3 mM EDTA, 12.5 mM MgCl2, 250 mM Sucrose, protease inhibitors, divided into aliquots and frozen down at −80° C.Compounds of invention were serially diluted in neat DMSO at 100-fold concentrations (1% DMSO final in the assay) and 2 μl/well were plated into 96-well Isoplates (Perkin Elmer).
Measurement: Ki = 24.5 nM (pChEMBL 7.610000133514404)
Descriptors: MW 455.58, ALogP 5.74, HBA 5, HBD 0, PSA 55.32, QED 0.39
Compound (SMILES): N#Cc1ccc(C(=O)NOCC2CC2)c(Nc2ccc(I)cc2F)c1F
InChIKey: PJNNUIQYQNFWEL-UHFFFAOYSA-N
Target: Dual specificity mitogen-activated protein kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q02750
Assay: MEK Inhibition Assay: Reaction buffer: 20mM Hepes (pH 7.5), 10mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02mg/mL BSA, 0.1 mM, Na3VO4, 2mM DTT, 1% DMSO Enzyme: MEK1, Invitrogen cat #PV3303 N-terminal His-tagged recombinant human full length protein, expressed in insect cells, Activated in vitro by RAF1. MW=49.2 kDa, GenBank Accession No. NP_002746. Substrate: 5uM ERK2 (K52R). Kinase-dead mutant (GenBank Acession No NM_0011949), aa2-358 with N-terminal His6 tag, MW=43.63 kDa, expressed in E.coli.
Measurement: IC50 < 150 nM (pChEMBL 6.823999881744385)
Descriptors: MW 469.23, ALogP 4.26, HBA 4, HBD 2, PSA 74.15, QED 0.49
Compound (SMILES): CN(C)C(=O)[C@@H]1CN(C2CCC(F)(F)CC2)CC[C@H]1NC(=O)c1cc(-c2ccc(F)cc2F)on1
InChIKey: FKWMETIYRSYUAN-IEBWSBKVSA-N
Target: Atypical chemokine receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P25106
Assay: In Vitro Assay: Tango CXCR7-bla U2OS cells are detached from culture dishes with 0.05% trypsin-EDTA and collected in growing medium (McCoy's 5A 90% (v/v), dialyzed FCS 10% (v/v), 0.1 mM NEAA, 25 mM HEPES (pH7.3), 1 mM sodium pyruvate, P/S 1% (v/v) 50 μg/ml Hygromycin, 100 μg/ml Geneticin, 200 μg/ml Zeocin), spinned down and resuspended in assay medium (McCoy's 5A 90% (v/v), dialyzed FCS 1% (v/v), 0.1 mM NEAA, 25 mM HEPES (pH7.3), P/S 1% (v/v)). 10′000 cells per well (in 30 μl) are seeded in a 384 well plate (black-walled, clear bottom). The plate is incubated at 37° C./5% CO2 for 24 hours. Test compounds are dissolved to 10 mM in DMSO and serially diluted in DMSO to 500× of the final concentration for dose response curves. Compounds are then diluted 1:100 in assay medium to 5× of the final concentration. 10 μl/well of diluted compounds are added to the assay plate and incubated for 15 minutes at 37° C. Thereafter CXCL12/SDF1-α is diluted in assay medium to 5× of the final concentration (its EC80 value for receptor activation) and 10 μl/well are added to the assay plate. The agonist leads to activation of the receptor and therefore to b-arrestin recruitment. Compounds acting as antagonists reduce this activation. The plate is incubated for 22 hrs at 37° C. 10 μl/well of detection reagent (LiveBLAzer-FRET BIG (CCF4-AM) substrate) is transferred to the assay plate and the plate is incubated for 2 hours at room temperature protected from light. Fluorescent counts are determined (Scan1: Ex 409/20 nm, Em 460/30 nm, Scan 2: Ex 409/20 nm, Em 530/30 nm).
Measurement: IC50 = 745 nM (pChEMBL 6.130000114440918)
Descriptors: MW 496.51, ALogP 3.71, HBA 5, HBD 1, PSA 78.68, QED 0.64
Compound (SMILES): COc1ccc(-c2cc(F)ccc2NC(=O)OC[C@@H]2CCCN2C)cc1OC
InChIKey: OTXYRGASWCDYKH-INIZCTEOSA-N
Target: Muscarinic acetylcholine receptor M3 (SINGLE PROTEIN), Homo sapiens
UniProt: P20309
Assay: Binding Assay: Cell membrane proteins (Perkin Elmer) wherein human muscarinic M3 receptor was overexpressed, [3H]-methyl scopolamine and test compounds in various concentration were cultured in 0.2 ml of Tris-HCl buffer at 25° C. for 120 minutes. The same was filtered under suction through glass fiber filter (Whatman GF/B), and then the filter was washed 5 times with 1 ml of Tris-HCl buffer. The radioactivity of [3H]-methyl scopolamine adsorbed on the filter was measured by a liquid scintillation counter. Non-specific binding was evaluated under existence of 5 μM of atropine. Affinity of the compound of the present invention to muscarinic M3 receptor was calculated as the dissociation constant (Ki), which can be calculated from concentration (IC50) of test compounds inhibiting 50% of binding of [3H]-methyl scopolamine (i.e. labeled ligand) according to Cheng and Prusoff [Cheng and Prusoff, Biochem. Pharmacol., 22, 3099, 1973]. In following Table, compounds having stronger binding affinity to human muscarinic M3 receptor have lower dissociation constant (Ki).
Measurement: Ki > 1000 nM (pChEMBL 6)
Descriptors: MW 388.44, ALogP 4.15, HBA 5, HBD 1, PSA 60.03, QED 0.81
Compound (SMILES): O=C(NCc1ccc(Cl)c(Cl)c1)c1cc(Cn2ccnc2)cc(-c2ccncc2Cl)c1
InChIKey: LQJZKPUTDJTISG-UHFFFAOYSA-N
Target: WD repeat-containing protein 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P61964
Assay: FPA Binding Assay: FPA Assay protocol adopted from Karatas et al. (J. Med. Chem. 2010, 5179.; J. Amer. Chem. Soc. 2013, 669.): WDR5 (Δ23, residues 24-334), is expressed and purified in sufficient quantities for screening. FITC-MLL peptide (FITC-GSARAEVHLRKS) and 10mer-Thr-FAM (ARTEVHLRKS-(Ahx-Ahx)(Lys-(5-FAM))) were purchased from GeneScript and used without additional purification. FITC-MLL peptide is used at 50 nM, while WDR5 is added at the Ki value of the protein:peptide interaction (WDR5-WIN Ki=2.5 μM). 10mer-Thr-FAM peptide is used at 4 nM, while WDR5 is added at the Ki value of the protein:peptide interaction (WDR5-10mer-Thr Ki=4 nM).Stock compounds are dispensed in barcoded 384-well plates as 30 mM solutions in DMSO. This plate is used as the source plate for the Echo Liquid Handler, which distributes the compounds to the assay plate (black, flat-bottom; Greiner) in a 10-point, 3-fold dilution scheme with a top concentration of 100 μM (5 nM low concentration) in a final volume of 50 μL. Both the top concentration and the dilution scheme can be adjusted to fit the anticipated potency of the compounds.For the FITC-MLL assay, 2.5 μM WDR5 and 50 nM FITC-MLL peptide in assay buffer (1× Phosphate Buffered Saline, pH 6.0, 300 mM NaCl, 0.5 mM TCEP, 0.1% CHAPS) is added to all compound-containing wells and to columns 2, 24 (negative control, 0% inhibition). 2 μL of 50 nM FITC-MLL peptide alone in assay buffer is added to columns 1, 23 (positive control, 100% inhibition). For the 10mer-Thr-FAM assay, a similar addition protocol is performed, using 4 nM WDR5 and 4 nM 10mer-Thr-FAM peptide in assay buffer (1× Phosphate Buffered Saline pH 6.0, 300 mM NaCl, 0.5 mM TCEP, 0.1% CHAPS).The plate is covered, shielded from light, and incubated for 60 min at room temperature, with rocking. Anisotropy is measured at excitation wavelength 480 nm and emission wavelength 535 nm using an EnVision Multi-label plate reader (PerkinElmer, Wellesley, Mass., USA) or a BioTek Cytation 3 (BioTek, Winooski, Vt., USA). Fluorescence anisotropy is plotted against compound concentration to generate an IC50 (inhibitor concentration at which 50% of bound peptide is displaced) by fitting the data to a 4-parameter logistic model using XLFit software (Guildford, Surrey, UK). IC50 is converted to a binding dissociation constant (Ki value) according to the formula of Wang Z. FEBS Lett (1996) 3, 245.K i=[I]50/([L]50 /K d+[P]0 /K d+1)where [I]50 is the concentration of the free inhibitor at 50% inhibition, [L]50 is the concentration of the free labeled ligand at 50% inhibition, [P]0 is the concentration of the free protein at 0% inhibition, Kd represents the dissociation constant of the FITC-MLL or 10mer-Thr-FAM probe for WDR5. Total fluorescence is also measured, to rule out compounds that are inherently fluorescent or able to act as quenchers in the assay.
Measurement: Ki = 1.9 nM (pChEMBL 8.720000267028809)
Descriptors: MW 471.78, ALogP 5.88, HBA 4, HBD 1, PSA 59.81, QED 0.38
Compound (SMILES): CCn1nc(C(=O)NC[C@H]2CC[C@H](S(C)(=O)=O)CC2)c(Cl)c1-c1ccc(CCC(F)(F)F)cc1C(F)(F)F
InChIKey: LYCKHZXTXAIHJL-WKILWMFISA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor Assay: ThermoFluor® experiments were carried out using instruments owned by Janssen Research and Development, L.L.C. through its acquisition of 3-Dimensional Pharmaceuticals, Inc. 1,8-ANS (Invitrogen) was used as a fluorescent dye. Protein and compound solutions are dispensed into black 384-well polypropylene PCR microplates (Abgene) and overlayed with silicone oil (1 μL, Fluka, type DC 200) to prevent evaporation. Bar-coded assay plates are robotically loaded onto a thermostatically controlled PCR-type thermal block and then heated at a typical ramp-rate of 1° C./min for all experiments. Fluorescence was measured by continuous illumination with UV light (Hamamatsu LC6) supplied via fiber optic and filtered through a band-pass filter (380-400 nm; >6 OD cutoff). Fluorescence emission of the entire 384-well plate was detected by measuring light intensity using a CCD camera (Sensys, Roper Scientific) filtered to detect 500±25 nm, resulting in simultaneous and independent readings of all 384 wells. Images were collected at each temperature, and the sum of the pixel intensity in a given area of the assay plate was recorded versus temperature.
Measurement: Kd = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 588.01, ALogP 6.07, HBA 5, HBD 1, PSA 81.06, QED 0.38
Compound (SMILES): CCN1CCN(Cc2ccc(Nc3ncc(F)c(-c4ncc5nc(C(=O)N(C)C)n(C)c5n4)n3)nc2)CC1
InChIKey: JEKCNQGMTAKGNP-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P11802
Assay: Caliper Mobility Shift Assay: CDK4: The test compound was dissolved in DMSO and diluted with a kinase buffer solution (20 mM HEPES-pH 7.5, 0.01% Triton X-100, 10 mM MgCl2, 2 mM DTT) and 5 μL of compound at 5-fold final concentration of reaction dissolved in 10% DMSO was added in a 384-well plate. The compound-free control well was 5 μL of 10% DMSO, and the no-activity control well was 5 μL of kinase buffer. 10 μL of a 2.5-fold diluted CDK4 enzyme solution (GST-CDK4(1-303 end)) was added and incubated at room temperature for 10 min, and then 10 μL of the 2.5-fold diluted substrate solution Peptide FAM-P8 was added. The reaction was stopped by adding 25 μL of stop solution after incubation at 28° C. for 3 h, and the conversion rate data was read on a Caliper EZ Reader II (Caliper Life Sciences) and the conversion rate was converted to inhibition rate data according to above method.
Measurement: IC50 < 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 519.59, ALogP 1.94, HBA 11, HBD 1, PSA 121.09, QED 0.39
Compound (SMILES): NCC1CC(NC2CC23CCc2ccc(Cl)cc23)C1
InChIKey: TXQXYHPWILCAGG-UHFFFAOYSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: Biochemical Assay for LSD1 Activity (15 minutes): LSD1 demethylase reactions were carried out in 50 mM HEPES pH 7.4, 100 mM NaCl, 1 mM DTT, 0.01% Tween-20, and 0.1 mg/mL BSA. All enzymatic reactions were performed for either 15 minutes at room temperature in a 10-μL volume. Five microliters of 800 nM biotinylated H3K4me1 peptide solution was added to each well of a black 384 well Proxiplate containing 80 nL compound (final concentration of 0.8% DMSO). Reactions were initiated by the addition of a mixture containing 20 nM LSD1 and 20 nM FAD (5 μL). Enzyme activity was stopped by the addition of 5 μL of high salt buffer consisting of 50 mM HEPES pH 7.4, 1.5 M NaCl, 1 mM DTT, 0.01% Tween-20, and 0.1 mg/mL BSA. Capture of the product peptide by the anti-H3K4me0 antibody and Streptavidin APC was allowed to proceed for 60 mM at room temperature before measuring the TR-FRET signal. Europium-labeled antibody and Streptavidin APC were used at final concentrations of 0.003 nM and 100 nM, respectively (total assay volume of 20 μL). Plates were read on a Perkin Elmer EnVision. Percent inhibition was calculated using Max (no inhibitor) and Min (quenched with stop buffer) controls and inhibition curves plotted to determine IC50 values.
Measurement: IC50 = 48.3 nM (pChEMBL 7.320000171661377)
Descriptors: MW 276.81, ALogP 2.62, HBA 2, HBD 2, PSA 38.05, QED 0.89
Compound (SMILES): C[C@@H]1CN(S(=O)(=O)c2cn(C)cn2)CC[C@@H]1Nc1ncc(C(F)(F)F)c(-c2cnn(CC(C)(C)O)c2)n1
InChIKey: UMPJPBWAEWLNBM-PBHICJAKSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24864
Assay: CDK2/Cyclin E1 HERE Enzyme Activity Assay: CDK2/Cyclin E1 enzyme activity assays utilize full-length human CDK2 co-expressed as N-terminal GST-tagged protein with FLAG-Cyclin E1 in a baculovirus expression system (Cama Product Number 04-165). Assays are conducted in white 384-well polystyrene plates in a final reaction volume of 8 μL. CDK2/Cyclin E1 (0.25 nM) is incubated with compounds (40 nL serially diluted in DMSO) in the presence of ATP (50 μM or 1 mM) and 50 nM ULight-labeled eIF4E-binding protein 1 (THR37/46) peptide (PerkinElmer) in assay buffer (containing 50 mM HEPES pH 7.5, 1 mM EGTA, 10 mM MgCl2, 2 mM DTT, 0.05 mg/ml BSA, and 0.01% Tween 20) for 60 minutes at room temperature. The reactions are stopped by the addition of EDTA and Europium-labeled anti-phospho-4E-BP1 antibody (PerkinElmer), for a final concentration of 15 mM and 1.5 nM, respectively. HTRF signals are read after 1 hour at room temperature on a PHERAstar FS plate reader (BMG Labtech). Data is analyzed with IDBS XLFit and GraphPad Prism 5.0 software using a three or four parameter dose response curve to determine IC50 for each compound.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 542.59, ALogP 2.37, HBA 10, HBD 2, PSA 131.06, QED 0.47
Compound (SMILES): C=CC(=O)N1CC=CN(n2nc(C(=O)Nc3ccc(OC(=O)N(C)c4ccccc4)cc3C)c3c(N)ncnc32)C1
InChIKey: UHTLWSJRYBKFIF-UHFFFAOYSA-N
Target: Receptor tyrosine-protein kinase erbB-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P04626
Assay: In Vitro Inhibitory Activity Assay: For the inhibitory activity measurement of each compound, the compound of the present invention or staurosporine was first serially diluted with dimethyl sulfoxide (DMSO). Next, the HER2 protein, the substrate peptide (final concentration: 0.5 uM), manganese chloride (final concentration: 10 mM), ATP (final concentration: 6 uM), and the solution of the compound of the present invention in DMSO (final concentration of DMSO: 5%) were added into a buffer solution for kinase reaction (15 mM Tris (pH 7.5), 2 mM dithiothreitol, and 0.01% Tween 20), and the mixture was incubated at 25° C. for 40 minutes for kinase reaction. The reaction was terminated by adding EDTA (final concentration: 30 mM) thereto. Finally, the unphosphorylated substrate peptide (S) and the phosphorylated peptide (P) were separated and detected by microcapillary electrophoresis using LabChip® EZ Reader II (PerkinElmer Inc.).
Measurement: IC50 = 7.7 nM (pChEMBL 8.109999656677246)
Descriptors: MW 553.58, ALogP 3.04, HBA 10, HBD 2, PSA 151.81, QED 0.34
Compound (SMILES): Cn1cc(C(F)(F)F)nc1-c1ccc(CN2C(=O)Cc3cnc(-c4cccc5c4CC(F)(F)C5)nc32)cc1
InChIKey: POENOCFYIVQKPC-UHFFFAOYSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O94782
Assay: HTS Assay: The HTS assay was performed in a final volume of 20 μL in assay buffer containing 20 mM Tris-HCl (pH 8.0, (1M Tris-HCl, pH 8.0 solution; Corning 46-031-CM)), 2 mM CaCl2) (1M Calcium Chloride solution; Sigma #21114) 1 mM GSH (L-Glutathione reduced; Sigma #G4251), 0.01% Prionex (0.22 μM filtered, Sigma #G-0411), and 0.01% Triton X-100. Stock compound solutions were stored at −20° C. as 10 mM in DMSO. Up to 1 month prior to the assay, 2 mM test compounds were pre-dispensed into assay plates (Black, low volume; Corning #3820) and frozen at −20° C. Prestamped assay plates were allowed to come to room temperature on the day of the assay. For the screen, 100 nL of 2 mM was pre-dispensed for a final screening concentration of 10 μM (DMSO(fc)=0.5%). The final concentration of the enzyme (USP1, construct USP1 (I-785, GG670, 671AA)/UAF1 (I-677)-Flag; Viva) in the assay was 100 μM. Final substrate (Ub-Rh110; Ubiquitin-Rhodamine 110, R&D Systems #U-555) concentration was 25 nM with [Ub-Rh110]<<Km. 10 μL of 2× enzyme was added to assay plates (pre-stamped with compound) either simultaneously with 2×Ub-Rh110 or preincubated with USP1 40 minutes prior to the addition of 10 μL of 2×Ub-Rh110 to compound plates. Plates were incubated stacked for 45 minutes at room temperature before fluorescence was read on the Envision (Excitation at 485 nm and Emission at 535 nm; Perkin Elmer) or on the PheraSTAR (Excitation at 485 nm and Emission at 535 nm; BMG Labtech).
Measurement: IC50 = 1100 nM (pChEMBL 5.960000038146973)
Descriptors: MW 525.48, ALogP 5.39, HBA 5, HBD 0, PSA 63.91, QED 0.34
Compound (SMILES): CC(C)[C@@H]1c2ncc(C(=O)NCc3ccc(S(C)(=O)=O)cc3)cc2CN1Cc1ccc(Cl)cc1
InChIKey: DJWHQLRAEOQUPG-RUZDIDTESA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Binding Assay: Compounds of the present invention were tested for ability to bind to RORγ in a cell-free competition assay with commercially available radio-ligand (RL), 25-hydroxy [26,27-3H]-cholesterol (PerkinElmer, Cat. #NET674250UC), for a ligand binding site on a recombinant RORγ Ligand Binding Domain (LBD) protein expressed as a 6×His-Glutathione-S-Transferase (GST) fusion. The assay was performed in 96-well SPA plates (PerkinElmer, Cat. #1450-401) in 50 mM HEPES buffer, pH 7.4, containing 150 mM NaCl, 5 mM MgCl2, 10% (v/v) glycerol, 2 mM CHAPS, 0.5 mM β-octylglucopyranoside and 5 mM DTT. Tested compounds were dissolved in DMSO, and semi-log (3.162×) serial dilutions of the compounds were prepared in the same solvent. Two μL of the DMSO solutions were mixed with 28 μL of 8.6 nM 25-hydroxy [26,27-3H]-cholesterol and 50 μL of 24 nM RORγ LBD. The plate was shaken at 700 rpm for 20 min and incubated for 10 min at rt, after which 40 μL of poly-Lys YSi SPA beads (PerkinElmer, Cat. #RPNQ0010) were added to achieve 50 μg of the beads per well. The plate was incubated on an orbital shaker for 20 min and then for 10 min without agitation at rt. SPA signal for tritium beta radiation was registered on PerkinElmer Microbeta plate reader.
Measurement: Ki > 1000 nM (pChEMBL 6)
Descriptors: MW 498.05, ALogP 4.78, HBA 5, HBD 1, PSA 79.37, QED 0.51
Compound (SMILES): Nc1ccc(-c2ccc(F)cc2)cc1NC(=O)N1CCCC1
InChIKey: PZPZSJUSIRGINC-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: Inhibition of Histone Deacetylase Enzymatic Activity Assay: HDAC1: The following trypsin-coupled protocol and Caliper protocol described Herein was used to assay the compounds of the invention. Trypsin Coupled Assay: HDAC1-Kinetic IC50 3Hr PreIncubation. Working Stocks: 1xHDAC Assay Buffer: 50 mM HEPES (GIBCO 15630-114), 100mM KCl, 0.001% Tweem-20 (Zymed 00-3005), 0.05% BSA (Invitrogen, P2489), pH 7.4, 1.5xHDAC+1.5xTrypsin: 225nM Trypsin(Worthinton)+0.18 ug/ml HDAC1 (BPS Inc) in assay buffer 300x Trypsin stock is made up in HDAC Assay Buffer, aliquoted and stored at -80C. 3xSubstrate: 18uM Substrate in assay buffer BPS Enzyme and Substrate amounts (per well).
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 299.35, ALogP 3.7, HBA 2, HBD 2, PSA 58.36, QED 0.83
Compound (SMILES): Oc1cc(-c2ncc3c(N4CC5CCC(C4)N5)nc(OCC4(C(F)(F)F)CCCC4)nc3c2F)c2ccccc2c1
InChIKey: IDPOQTSXOSKFIP-UHFFFAOYSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: KRas G12D Surface Plasmon Resonance (SPR) Binding Assay: Table 1: Briefly, 1 L of 1.05× HBS-Mg buffer (262.5 mM Bioultra Hepes, pH 7.5, 157.5 mM NaCl, 105 mM MgCl2, 0.525 mM TCEP, 0.0305% Brij-35) was prepared and filter sterilized using a 0.22 μm bottle top filter. Approximately 50 mL of 1.05× HBS-Mg buffer was removed and saved for future dilutions. A 50 mL aliquot of DMSO (Sigma Aldrich DMSO Lot. #SHBK2079) was added and continued to stir for 10 minutes, creating the final 1.0× HBS-Mg buffer (250 mM Bioultra Hepes pH 7.5, 150 mM NaCl, 100 mM MgCl2, 0.5mM TCEP, 0.03% Brij-35).
Measurement: Kd = 798 nM (pChEMBL 6.099999904632568)
Descriptors: MW 567.59, ALogP 6.13, HBA 7, HBD 2, PSA 83.4, QED 0.28
Compound (SMILES): Cc1cc(C#N)cnc1C(=O)Nc1ccc2[nH]nc(-c3cncc(N4CCOCC4)c3)c2c1
InChIKey: WBNHQECJGWYWNI-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LanthaScreen Kinase Activity Assay: The assay was carried out under the following protocol conditions: 1 mM compound in DMSO was serially diluted 1:3, 11 points in DMSO with a Biomek FX and 0.1 μL of the diluted compound was subsequently stamped into the assay plate (384-well format Lumitrac 200, Greiner, 781075) with an Echo Labcyte such that the final compound concentration in the assay was 10 μM to 169 μM. Subsequently, 5 μL of 2× kinase solution (2.9 nM final concentration) was added to the assay plate in assay buffer composed of 50 mM Tris pH 8.5 (Sigma, T6791), 5 mM MgCl2 (Fluka, 63020), 1 mM EGTA (Sigma, E3889), 0.01% BRIJ-35 (Sigma, P1254) and 2 mM DTT. The reaction was started by addition of 2×ATP/LRRKtide solution in assay buffer such that the final concentration was 400 nM LRRKtide and 25 μM ATP. After 60 min incubation at room temperature, the reaction was stopped by addition of 10 μL of 2× stop solution containing a final concentration of 2 nM anti-pERM antibody and 10 mM EDTA. After a 30 min incubation at RT, the TR-FRET signal was measured on a Wallac 2104 EnVision multilabel reader at an excitation wavelength of 340 nm and reading emission at 520 nm and 495 nm. The ratio of the 520 nm and 495 nm emission was used to analyze the data.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 439.48, ALogP 3.29, HBA 7, HBD 2, PSA 119.82, QED 0.5
Compound (SMILES): Cn1nccc1-c1ccnc(-c2ncc(-c3ccccc3)[nH]2)c1
InChIKey: NNEMQQMVSKBECS-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: PI3K-γ Scintillation Proximity Assay: The kinase reaction was conducted in polystyrene 384-well Greiner Bio-one white plate from Thermo Fisher Scientific in a final volume of 25 μL. Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 2%. The PI3Kγ assay was carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 μM PIP2, 2 μM ATP, 0.5 μCi [γ-33P] ATP, 13 nM PI3Kγ. Reactions were incubated for 120 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 163 mM potassium phosphate pH 7.8, 20% glycerol, 25 mM EDTA. The final concentration of SPA beads is 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1500 rpm for 10 min, the radioactivity of the product was determined by scintillation counting on Topcount (Perkin-Elmer). IC50 determination was performed by fitting the curve of percent of the solvent control activity versus the log of the inhibitor concentration using the GraphPad Prism 6.0 software.
Measurement: IC50 > 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 301.35, ALogP 3.54, HBA 4, HBD 1, PSA 59.39, QED 0.63
Compound (SMILES): NCCN(Cc1ccc(Cl)cc1)C(=S)Nc1ccc2cnccc2c1
InChIKey: WJLLWHYRDKWYIG-UHFFFAOYSA-N
Target: Rho-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13464
Assay: ELISA Assay: ROCK Substrate Coated Plate (Part No 241601): One strip well 96-well plate pre-coated with recombinant MYPT1.Buffer: 25 mM Tris, pH 7.5, 10 mM MgCl2, 5 mM Glycerol-2-Phosphate, 0.1 mM Na3VO4; 1% DMSO; 2.5 mM DTT; (Enzyme: ROCK active-II) (Cell Biolabs, Catalog #STA-406, Part No. 241505) 0.1 ng/μl.ATP Solution (Part No. 241604): 100 mM ATP. Final concentration of ATP in reaction mixture: 250 μM.Anti-phospho-MYPT1 (Thr696) (Part No. 241603).Secondary Antibody, HRP Conjugate (Part No. 231003).Biotinylated substrate, diluted to 0.25 μM with buffer described above (without ATP).Steps:1. Purified kinase or cell lysate sample can be used directly in the kinase assay or further diluted with 1× Kinase Buffer. Each sample should be assayed in duplicate.2. Add 90 μL of the diluted active ROCK-II positive control or unknown ROCK samples to the wells of the substrate plate.3. Initiate the kinase reaction by adding 10 μL of the 10× Kinase Reaction Buffer containing DTT and ATP. Mix well.4. Cover with a plate cover and incubate the wells at 30° C. for 30-60 minutes with gentle agitation.5. Stop kinase reaction by flicking out the content or by adding 50 μL of 0.5M EDTA, pH 8.0, to each well.6. Remove the plate cover and empty wells. Wash microwell strips 3 times with 250 μL 1× Wash Buffer per well with thorough aspiration between each wash. After the last wash, empty wells and tap microwell strips on absorbent pad or paper towel to remove excess 1× Wash Buffer.7. Add 100 μL of the diluted anti-phospho-MYPT1 (Thr696) antibody to each well.8. Cover with the plate cover and incubate at room temperature for 1 hour on an orbital shaker.9. Remove the plate cover and empty wells. Wash the strip wells 3 times according to step 6 above.10. Add 100 μL of the diluted HRP-conjugated secondary antibody to each well.11. Cover with the plate cover and incubate at room temperature for 1 hour on an orbital shaker.12. Remove the plate cover and empty wells. Wash microwell strips 3 times according to step 6 above. Proceed immediately to the next step.13. Warm Substrate Solution to room temperature. Add 100 μL of Substrate Solution to each well, including the blank wells. Incubate at room temperature for 5-20 minutes on an orbital shaker.14. Stop the enzyme reaction by adding 100 μL of Stop Solution into each well, including the blank wells. Results should be read immediately (color will fade over time).15. Read absorbance of each microwell on a spectrophotometer using 450 nm as the primary wave length.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 370.91, ALogP 4.05, HBA 3, HBD 2, PSA 54.18, QED 0.66
Compound (SMILES): CN(C)S(=O)(=O)c1ccc(Nc2nn([C@H]3COCC[C@@H]3C#N)c3cc[nH]c(=O)c23)cc1
InChIKey: SJDCORZYFJDIGG-DYVFJYSZSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Biochemical HTRF Assay: The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and TYK2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, TYK2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at room temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading. After normalization to untreated controls, the percent inhibition of the HTRF signal at each compound concentration was calculated.
Measurement: IC50 = 70 nM (pChEMBL 7.159999847412109)
Descriptors: MW 442.5, ALogP 1.82, HBA 8, HBD 2, PSA 133.11, QED 0.62