lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCOC[C@@]12CO[C@@H](O1)[C@H](NC(C)=O)[C@@H](O)[C@H]2O
InChIKey: NIZNXVQMJWWUHG-APLZJWDSSA-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 = 5300 nM (pChEMBL 5.28000020980835)
Descriptors: MW 261.27, ALogP -1.63, HBA 6, HBD 3, PSA 97.25, QED 0.56
Compound (SMILES): C=CCN=S(=O)(c1ccc(Oc2cc(F)cc(C#N)c2)c2c1[C@H](O)C(F)(F)C2)C(F)(F)F
InChIKey: KOWICVJSMQCNGQ-DVYMHAMMSA-N
Target: Endothelial PAS domain-containing protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99814
Assay: Scintillation Proximity Assay (SPA): HIF-2a: The total assay volume was about 100 uL in the following configuration: 2 uL compound in 100% DMSO, 88 uL buffer with protein and probe and 10 uL of SPA beads. The compound was diluted in a master plate consisting of a 10-point dose response with a 3-fold compound dilution from 100 nM to 5 nM. Assays were run on a 96-well plate in which one column, designated as the high signal control, contained DMSO with no compound and another column, designated as the low signal control, contained no protein. Prior to plating out of compound, a buffer solution, consisting of 25 mM TRIS pH 7.5 (Sigma), 150 mM NaCl (Sigma), 15% Glycerol (Sigma), 0.15% BSA (Sigma), 0.001% Tween-20 (Sigma), 150 nM Compound 183 and 100 nM HIF-2Î± HIS TAG-PASB Domain, was made and allowed to equilibrate for 30 minutes. Compounds that were to be tested were then plated in to a 96-well white clear bottom Isoplate-96 SPA plate (Perkin Elmer). To the compounds, 88 uL of the buffer solution was then added, the plate was covered with a plastic cover and then aluminum foil, placed onto a shaker and equilibrated for 1 hour. After equilibration, 10 uL of a 2 mg/mL solution of YSi Cu His tagged SPA beads (Perkin Elmer) were then added to each well of the plate, covered and equilibrated for another 2 hours. The plates were then removed from the shaker, placed into a 1450 LSC and luminescence counter MicroBeta Trilux (Perkin Elmer) to measure the extent of probe displacement.
Measurement: IC50 = 370 nM (pChEMBL 6.429999828338623)
Descriptors: MW 476.4, ALogP 5.25, HBA 5, HBD 1, PSA 82.68, QED 0.47
Compound (SMILES): CCCCc1nc(O)c(-c2nnc(Cc3nnc(-c4cccc(Cl)c4)o3)o2)c(O)c1-c1c(OC)cccc1OC
InChIKey: JNBVROJQKRAEHP-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 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 564, ALogP 5.86, HBA 11, HBD 2, PSA 149.65, QED 0.21
Compound (SMILES): Cc1cc(Nc2ncnc(N)n2)c(=O)n2c1C(=O)NC21CCC(O)(C(F)(F)F)CC1
InChIKey: HRTPGAXURJTPGQ-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 = 71.2 nM (pChEMBL 7.150000095367432)
Descriptors: MW 425.37, ALogP 0.93, HBA 9, HBD 4, PSA 148.05, QED 0.56
Compound (SMILES): CC(C)(C)NC(=O)c1cc(NC(=O)Cc2ccccc2Cl)ccn1
InChIKey: JAXMNSMBOXGRND-UHFFFAOYSA-N
Target: Anoctamin-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5XXA6
Assay: Automated Whole-Cell Patch Clamp Assay to Detect TMEM16A Activity in Recombinant Cells: Compound activity was quantified by measuring the increase in current upon compound addition and expressing this as a percentage increase of baseline TMEM16A current level. Percentage increases in current were determined for each concentration and the data plotted as a function of concentration using either the Qpatch software or Graphpad Prism v6.05 providing the concentration which gave 50% of its maximal effect (EC50) and maximum efficacy (percentage of baseline increase). The method of calculating the results is illustrated in FIG. 1, which shows an example trace from the Qpatch TMEM16A assay. In FIG. 1, IBL equals baseline current, I[#1] equals the peak current during test compound concentration 1 incubation period and so on.
Measurement: EC50 = 570 nM (pChEMBL 6.239999771118164)
Descriptors: MW 345.83, ALogP 3.44, HBA 3, HBD 2, PSA 71.09, QED 0.89
Compound (SMILES): CC(C)Oc1cccc2cc(C(=O)N[C@@H]3C4CCN(CC4)C34CC4)sc12
InChIKey: UQSXOKQEMNCJCE-LJQANCHMSA-N
Target: Neuronal acetylcholine receptor subunit alpha-7 (SINGLE PROTEIN), Homo sapiens
UniProt: P36544
Assay: Human alpha7 nAChR Binding Assay: The ability of compounds to displace binding of radioactive ligands from human α7 nAChR was determined, as a measure of the affinity of the compounds for these ligand-gated ion channels. The [125I]-αBungarotoxin competition binding assay was performed under contract by Cerep Poitiers, France following published the methods (Sharpies et al., J Neurosci. 2000; 20(8):2783-91). SH-SY5Y cells stably expressing human α7 nicotinic acetylcholine receptors, grown to confluency in 175 cm2 flasks, were washed briefly with warm PBS containing (in mm): (150 NaCl, 8 K2HPO4, 2 KH2PO4, pH 7.4, 37° C.) and scraped into cold phosphate buffer. Cells were washed by centrifugation for 3 min at 500×g and resuspended in 10 mL of ice-cold phosphate buffer. The suspension was homogenized for 10 sec using an Ultraturax and centrifuged for 30 min at 45,000×g. The pellet was resuspended in phosphate buffer (0.5 mL per original flask). SH-SY5Y membranes (30 μg protein) were incubated in a total volume of 2 mL in 50 mM phosphate buffer with 0.05 nM [125I]-αBgt and serial dilutions of test compound. Nonspecific binding was determined in the presence of α-bungarotoxin (1 μM). Samples were incubated for 120 min at 37° C. The reaction was terminated by filtration through Whatman GFA/E filter paper (presoaked overnight in 0.3% polyethyleneimine in PBS), using a Brandel Cell Harvester. Each condition was measured in duplicate. Filters were counted for radioactivity using a scintillation counter. The results were expressed as a percent inhibition of control specific binding obtained in the presence of the test compounds where Inhibition (%)=100−[(measured specific binding/control specific binding)×100].
Measurement: Ki = 220 nM (pChEMBL 6.659999847412109)
Descriptors: MW 370.52, ALogP 4.05, HBA 4, HBD 1, PSA 41.57, QED 0.88
Compound (SMILES): C[C@@H]1CN(c2ccc(-c3cc(NC(=O)c4c[nH]c(=O)cc4C(F)(F)F)c(N4CCN(C)[C@@H](C)C4)cc3F)cn2)C[C@H](C)O1
InChIKey: JNRWYOAGDOATQP-GBESFXJTSA-N
Target: WD repeat-containing protein 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P61964
Assay: Surface Plasmon Resonance (SPR) Assay: Exemplary compounds of the application were dissolved in 100% DMSO at 10 mM, assayed fresh, and then stored at −20° C. for repeat studies and other experiments. Full length WDR5 with an N-terminal His tag and C-terminal AviTag (Avidity Inc.) was expressed in E. coli with coexpression of BirA to biotin labelled protein in vivo. Purification of the protein was performed using Ni-NTA. The purified WDR5 protein has a molecular weight of 41976 Da.SPR studies were performed using a Biacore T200 instrument (GE Health Sciences Inc.). Biotinylated WDR5 protein (approximately 3000 RU) was stably captured to streptavidin coupled SA chips according to the manufacture's protocol (GE Health Sciences Inc.). The running buffer used was HBS-EP (20 mM Hepes pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P-20) plus 5% DMSO with a flow rate of 40 μl/min. For SPR analysis, 5 different concentrations of each exemplary compound of the application were sprayed into 96 or 384 well plates using an HP D300 digital dispenser. The concentration ranged from about 195 nM to about 12 nM in a two-fold series. Concentration ranges were adjusted higher or lower for weaker or more potent compounds respectively when necessary. For the KD determinations, single cycle kinetic analysis was performed with an on time of 60 seconds, and an off time of 300 or 600 seconds. Curve fitting and KD calculations were performed with the Biacore T200 Evaluation software (GE Health Sciences Inc).
Measurement: Kd = 1.97 nM (pChEMBL 8.710000038146973)
Descriptors: MW 602.63, ALogP 4.6, HBA 7, HBD 2, PSA 93.8, QED 0.41
Compound (SMILES): NC(=O)c1nn(CC(=O)N2C[C@H](F)C[C@H]2C(=O)Nc2cncc(C(F)(F)F)n2)c2ccc(C#Cc3ncc(F)cn3)cc12
InChIKey: KDYRBVUWSDVKGA-KDOFPFPSSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Inhibition Assay: Human factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration is incubated with test compound at various concentrations for 5 minutes at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBzl and DTNB (Ellman's reagent) are added to final concentrations of 100 μM each. The increase in color is recorded at OD405 nm in a microplate in kinetic mode over 30 minutes with 30 second time points in a spectrofluorimeter. IC50 values are calculated by non-linear regression from the percentage of inhibition of complement factor D activity as a function of test compound concentration.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 599.48, ALogP 1.85, HBA 9, HBD 2, PSA 161.88, QED 0.26
Compound (SMILES): COc1ccc(C(=O)Nc2cnc3c(c([C@@H]4CCN(C(=O)[C@H](C)C(F)(F)F)[C@@H](C)C4)cn3C)c2C)cc1C#N
InChIKey: FHECTQTXNDHRBR-RYQLBKOJSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Assay of Co-Activator Recruitment by TR-FRET: Specifically, in one embodiment the aforementioned assay was performed as outlined below. The assay was carried out in black polystyrene, 384-well plates in a total assay volume of 50.5 μL. The assay buffer contained 50 mM TRIS¿HCL pH 7.5, 1 mM NaCl, 2 mM MgCl2, 0.5 mg/mL bovine serum albumin, and 5 mM dithiothreitol. The final concentration of reagents was 6.3 nM RORC2 LBD, 200 nM SRC1-2, 50 nM streptavidin APC, 1 nM Europium-labeled anti-His antibody, and varying concentrations of compounds such that final concentration of DMSO is 1% (v/v). The assay steps were: (1) dispensing 500 μL compound at 100× final concentration in DMSO (test wells) or DMSO only (control wells for no inhibition); and (2) dispensing 50 μL mixture of the other assay components including receptor (test wells) or excluding receptor (control wells for maximal inhibition). Assay mixtures were incubated are room temperature for 3 hr and read in EnVision 2100 Multilabel Reader (PerkinElmer Life Sciences) at Excitation Filter 320, Emission Europium Filter 615, Emission APC Filter 665, Dichroic Mirror D400/D630. TR-FRET signal was determined by calculating the ratio of 665 nm by 615 nm and IC50 values of compounds of the invention (Table 1) were determined by the non-linear regression analysis of dose response curves.
Measurement: IC50 = 33 nM (pChEMBL 7.480000019073486)
Descriptors: MW 541.57, ALogP 5.31, HBA 6, HBD 1, PSA 100.25, QED 0.47
Compound (SMILES): COc1cnc(CN2C3CC2CN(c2ccc(-c4cc(OCC(C)(C)O)cn5ncc(C#N)c45)cn2)C3)c(F)c1
InChIKey: CPWKGJKZZVMDMQ-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of Formula I were screened for their ability to inhibit wildtype and V804M mutant RET kinase using CisBio's HTRF® KinEASE¿-TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Catalog No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Catalog No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 77.2 nM (pChEMBL 7.110000133514404)
Descriptors: MW 543.6, ALogP 3.42, HBA 10, HBD 1, PSA 112.04, QED 0.36
Compound (SMILES): N#Cc1c(N)nc(N)nc1N1CC[C@H]1c1nc2cccc(Cl)c2c(=O)n1-c1ccccc1
InChIKey: OKIQNDSPZILKID-INIZCTEOSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: TR-FRET assay : TR-FRET monitored the formation of 3,4,5-inositol triphosphate molecule that competed with fluorescently labeled PIP3 for binding to the GRP-1 pleckstrin homology domain protein. An increase in phosphatidylinositide 3-phosphate product resulted in a decrease in TR-FRET signal as the labeled fluorophore was displaced from the GRP-1 protein binding site. Class I PI3K isoforms were expressed and purified as heterodimeric recombinant proteins. All assay reagents and buffers for the TR-FRET assay were purchased from Millipore. PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2× Km ATP (75-500 μM), 2 μM PIP2, 5% glycerol 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, and 1% (v/v) DMSO at the following concentrations for each isoform: PI3Kα, PI3Kβ, and PI3Kδ between 25 and 50 pM, and PI3Kγ at 2 nM. The compounds of Table 1, Compound X ((S)-2-(1-((9H-purin-6-yl)amino)ethyl)-5-chloro-3-phenylquinazolin-4(3H)-one) and Compound Y ((S)-4-amino-6-((1-(5-chloro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)ethyl)amino)pyrimidine-5-carbonitrile), were added to the assay solution and incubated for 30 minutes at 25° C. Additionally, compounds 19 to 116 were added to the assay solution and incubated for 30 minutes at 25° C. The reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 500 μs read window).
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 444.89, ALogP 2.82, HBA 9, HBD 2, PSA 139.74, QED 0.49
Compound (SMILES): CCc1cc(CN2CC3(CC[S+]([O-])C3)C2)ccc1-c1ccc(C(O)(C(F)(F)F)C(F)(F)F)cc1
InChIKey: KKDUTDFFMLRFMS-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Gal4 Ligand Binding Assay: Compounds of the present invention were tested in a human RORγ ligand binding assay using a commercially available cell based ligand binding reporter assay in 96-well format (Cat #1304001, INDIGO Biosciences, State College, Pa.). The N-terminal DNA binding domains (DBD) of the native RORγ and RORγt receptors have been substituted with that of the yeast GAL4-DBD and stably transfected in HEK293T cells that also stably express luciferase under the regulation by upstream activation sequence of yeast Gal4. These cells constitutively express high level RORγ activity due to binding of endogenous co-factors. Both agonist and inverse agonist activity can be detected. The assay was performed according to kit manufacturer's instructions as follows. 10 mM compound stocks were diluted serially 1:3 with DMSO and further diluted with provided media to generate 10 titration points from 60 μM to 3 nM. These treatment conditions were added to the plates as 2× media in 100 μL volume. Each plate includes a positive control with 10 titration points as well as 6 negative control wells with vehicle only, with final DMSO concentration of 0.2%. RORγ reporter cells were rapidly thawed and added to the plates in 100 μL volume. The plates were incubated for 24 h in a 37° C. humidified 5% CO2 incubator. Media was removed before the addition of room temperature luciferous detection substrate. After 5 minute incubation, relative light units (RLUs) were quantified using a plate reading luminometer. Data was normalized to positive control wells with only 0.2%0DMSO. Before establishing internal controls, ursolic acid was used as control.
Measurement: IC50 > 250 nM (pChEMBL 6.6020002365112305)
Descriptors: MW 505.52, ALogP 5.18, HBA 3, HBD 1, PSA 46.53, QED 0.45
Compound (SMILES): N#Cc1ccc2[nH]cc(C(O)CCCN3CCN(c4ccc(-n5ccccc5=O)cc4)CC3)c2c1
InChIKey: PRGBGERIWBHFMH-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 1A (SINGLE PROTEIN), Rattus norvegicus
UniProt: P19327
Assay: Rat Synaptosome Binding Assay: The synaptosomes (150 μg) prepared from a rat brain were incubated at 37° C. for 15 minutes with 0.1 μCi [3H]5-HT in the absence or presence of the test compound or the reference compound in a buffer solution containing 106.2 mM NaCl, 4.5 mM KCl, 2.25 mM MgSO4, 1.08 mM NaH2PO4, 22.5 mM NaHCO3, 9.9 mM glucose, 9 μM EGTA and 45 μM ascorbic acid (pH 7.4).The basal control activity was determined by incubating the same mixture at 4° C. for 15 minutes in the presence of 10 μM imipramine to block the uptake of 5-HT, which was taken as the standard reference compound and tested in each experiment at several concentrations to obtain an inhibition curve.Following the incubation, the samples were filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) and rinsed twice with an ice-cold incubation buffer using a 96-sample cell harvester (Unifilter, Packard) to eliminate free [3H]5-HT. The filters were dried and the retained radioactivity was measured in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard). The experimental results were expressed as a percent inhibition of the control uptake of [3H]5-HT.Data AnalysisInhibition of serotonin transporter in rat synaptosome was measured by the concentrations of [3H]5-HT. The test compounds were required to be tested at least twice in the case of the concentration thereof being greater than 6 log, and the obtained data were subjected to a nonlinear regression analysis via a curve of Hill equation, to obtain IC50 value.
Measurement: IC50 = 330 nM (pChEMBL 6.480000019073486)
Descriptors: MW 467.57, ALogP 3.83, HBA 6, HBD 2, PSA 88.29, QED 0.43
Compound (SMILES): O=C(Nc1ccc(F)c(C(=O)c2ccc3ncc(N4CCOCC4)nc3c2)c1)Nc1ccc(Cl)c(C(F)(F)F)c1
InChIKey: WFPUMFOZJCPOQV-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibitory Activity Assay: BRAF and BRAF V600E: Inhibitory activities Inhibitory activities of compounds against BRAF, and BRAF V600E were measured by Invitrogen using Z′-LYTE Method as briefly described in the following. 4× Test compounds are dissolved in 1% DMSO. Kinase reaction mixture consists of 0.09-0.34 ng B-Raf (or 0.002-0.006 ng BRAF V600E), 1× inactive MAP2K1 (MEK1)/inactive MAPK1 (ERK2), and 2 μM Ser/Thr 03 in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA. ATP solutions are diluted to a 4×ATP working concentration in Kinase Buffer (50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA). Reaction started by 30-second shaking of mixture consisting of 2.5 μL 4× test compound, 2.5 μL 2× kinase reaction mixture and 2.5 μL 4×ATP Solution on Bar-coded Corning, low volume NBS, black 384-well plate (Corning Cat. #3676). Then the mixture was incubated for 60-minute at room temperature for the kinase reaction, followed by addition of 5 μL of a 1:1024 dilution of development reagent A and 30-second plate shake. The mixture was then incubated for another 60-minute at room temperature for development reaction. Fluorescence was read by plate reader.
Measurement: IC50 > 100 nM (pChEMBL 7)
Descriptors: MW 573.93, ALogP 6.15, HBA 6, HBD 2, PSA 96.45, QED 0.22
Compound (SMILES): Fc1ccc(-n2cc(COc3ncc(C(F)(F)F)cn3)nn2)cc1OC(F)F
InChIKey: DABZUJAFCKHBLJ-UHFFFAOYSA-N
Target: Retinoic acid receptor RXR-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P19793
Assay: NR2B standard assay: NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 149 mM NaCl, 4 mM KCl, 2 mM CaCl2, and 1.5 mM MgCl2, 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (149 mM NaCl (standard assay) or 150 mM (HTS assay), 4 mM KCl (standard assay) or 3 mM (HTS assay), 2 mM CaCl2, 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to ¿EC80 (standard assay) or EC40 (HTS assay) is used to maximize the assay's signal window or the ability to detect NMDA receptor antagonists and negative allosteric modulators, respectively. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 405.26, ALogP 3.4, HBA 7, HBD 0, PSA 74.95, QED 0.59
Compound (SMILES): CC1(C)CCCC(NC(=O)[C@@H]2C[C@@H](F)CN2C(=O)Cn2nc(C(N)=O)c3cc(C#Cc4ncccn4)ccc32)C1
InChIKey: TXYRZVZUOJPDNH-HUJVSABWSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Inhibition Assay: Human factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration is incubated with test compound at various concentrations for 5 minutes at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBzl and DTNB (Ellman's reagent) are added to final concentrations of 100 μM each. The increase in color is recorded at OD405 nm in a microplate in kinetic mode over 30 minutes with 30 second time points in a spectrofluorimeter. IC50 values are calculated by non-linear regression from the percentage of inhibition of complement factor D activity as a function of test compound concentration.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 545.62, ALogP 2.35, HBA 7, HBD 2, PSA 136.1, QED 0.47
Compound (SMILES): O=C(CC1CC1)NCC1(O)CCN(c2nc(Oc3cccc(F)c3)cc(C(F)(F)F)n2)CC1
InChIKey: TWCQYNMGGXBODD-UHFFFAOYSA-N
Target: Sodium-dependent dopamine transporter (SINGLE PROTEIN), Homo sapiens
UniProt: Q01959
Assay: Competition Binding Assay: The affinity of the compounds of the invention to the human DAT or NET or SERT transporters is assessed by using the [3H]WIN-35,428 or [3H]nisoxetine or [3H]citalopram binding assays in recombinant human DAT, NET and SERT membranes with the SPA technology. The final assay volume is 50 μL in 384 well plates.Briefly, 0.5 μL of test compound in neat DMSO or 0.5 μL of DMSO for total binding (TB) or 0.5 μL of indatraline 1 mM (10 μM final concentration) for non specific binding (NSB) are added to the assay plate. 50 μL of the SPA mixture is added to each well, containing: 30 μg/mL or 10 μg/mL or 25 μg/mL DAT, NET, SERT membranes, respectively; 5 nM [3H]WIN-35,428 or 5 nM [3H]nisoxetine or 1 nM [3H]citalopram, for DAT, NET, SERT assay, respectively; 2.5 mg/mL or 1 mg/mL or 4 mg/mL WGA-PVT SPA beads (PerkinElmer RPNQ0001, for DAT, NET, SERT assay, respectively. All components are added to Assay Buffer (20 mM HEPES pH 7.4, 145 mM NaCl, 5 mM KCl, 0.01% Pluronic F-127). 0.02% BSA was used for DAT binding only. Plates are sealed with Topseal A and centrifuged 1 min, 800 rpm. Plates are loaded into a 1450 Microbeta TriLux (Perkin-Elmer) plate reader and the radioactivity counted after at least 4 hrs or overnight incubation at room temperature. Curve fitting and IC50 estimations are performed using a four parameter model in XLfit (IDBS, Guilford, UK) for Microdoft Excel (Microsoft, Redmond, Wash.).
Measurement: IC50 = 26.3 nM (pChEMBL 7.579999923706055)
Descriptors: MW 468.45, ALogP 3.67, HBA 6, HBD 2, PSA 87.58, QED 0.6
Compound (SMILES): CCC(=O)N[C@H]1CC[C@@H](N(C)C(=O)c2ncc(-c3ccc4c(ccn4C)c3)cn2)C1
InChIKey: BTKUHKXXHAXIML-RBUKOAKNSA-N
Target: Fatty acid synthase (SINGLE PROTEIN), Homo sapiens
UniProt: P49327
Assay: Biochemical Assay of FASN: The biochemical FASN activity testing was performed as 384 well two-time-point kinetic fluorescence intensity assay format in Greiner low volume medium binding 384-well black microtiter plates in a total assay volume of 8 μl and was used for high throughput screen. In each well 3 μl 40 nM human recombinant full-length fatty acid synthase (produced in-house in SF9 cells) were dispensed in the following assay buffer: 50 mM potassium phosphate buffer pH 7.0, 0.005% (w/v) BSA, 2 mM Glutathione, 0.02% Tween-20. Then 2 μl of 200 μM NADPH in assay buffer were added, followed by the addition of the test compounds in 10 dilution concentrations starting with 30 μM (final concentration) to get a final DMSO content of 1% (v/v). The mixture was incubated for at least 15 min at room temperature. After the pre-incubation the enzymatic reaction was started by the addition of 2 μl substrate solution (80 μM acetyl-CoA, 240 μM malonyl-CoA). A first fluorescence intensity measurement (time point one) was performed with an Envision multimode reader (Perkin Elmer LAS Germany GmbH) at excitation wavelength 340 nm (lamp mode) and emission wavelength 460 nm. The reaction was incubated for 30 minutes at room temperature. After this the fluorescence intensity was measured again in the Envision using the same parameters as described above (second time point measurement). The data were analysed by subtracting the first time point measurement value from the second time point measurement value (after the enzymatic reaction). The differences of the emission signals were determined. These reflect directly the conversion rate of NADPH. The full value used was the inhibitor-free reaction. A pharmacological zero value was used like GSK837149A (Sigma-Aldrich) in a final concentration of 5-10 μM. The inhibitory values (IC50) were determined using either the program Symyx Assay Explorer® or Condosseo® from GeneData.
Measurement: IC50 < 300 nM (pChEMBL 6.5229997634887695)
Descriptors: MW 405.5, ALogP 3.15, HBA 5, HBD 1, PSA 80.12, QED 0.71
Compound (SMILES): Cc1cc(C)n(-c2cccc(C(=O)N[C@@H](CCN3CC[C@@H](OCc4ccc5c(n4)NCCC5)C3)C(=O)O)c2)n1
InChIKey: LNXJMMJAPARCBA-FTJBHMTQSA-N
Target: Integrin alpha-V/beta-6 (PROTEIN COMPLEX), Homo sapiens
UniProt: P06756
Assay: Binding Assay: Microplates were coated with recombinant human integrin αvβ6 (2 ug/ml) in PBS (100 ul/well 25° C., overnight). The coating solution was removed, washed with wash buffer (0.05% Tween 20; 0.5 mM MnCl2; in 1×TBS). Plate was blocked with 200 ul/well of Block Buffer (1% BSA; 5% sucrose; 0.5 mM MnCl2; in 1×TBS) at 37° C. for 2 h. Dilutions of testing compounds and recombinant TGFβ1 LAP (0.67 ug/ml) in binding buffer (0.05% BSA; 2.5% sucrose; 0.5 mM MnCl2; in 1×TBS) were added. The plate was incubated for 2 hours at 25° C., washed, and incubated for 1 hour with Biotin-Anti-hLAP. Bound antibody was detected by peroxidase-conjugated streptavidin. The IC50 values for testing compounds were calculated by a four-parameter logistic regression.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 532.65, ALogP 3.11, HBA 8, HBD 3, PSA 121.61, QED 0.36
Compound (SMILES): Cc1[nH]c(C2CCN(c3ncnc4[nH]c(-c5ccncc5)cc34)CC2)nc1-c1ccccc1
InChIKey: HIHHRUJHUCQLBR-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 435.54, ALogP 5.1, HBA 5, HBD 2, PSA 86.38, QED 0.41
Compound (SMILES): COc1cc(CCNC[C@H]2CN(C(=O)C3CC3)CCO2)c(Cl)cc1NC(=O)Nc1cnc(C#N)cn1
InChIKey: RYDRBWBCEGEWIS-SFHVURJKSA-N
Target: Serine/threonine-protein kinase Chk1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14757
Assay: Kinase Inhibition Asssay: Reaction Buffer:Base Reaction buffer: 20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSORequired cofactors are added individually to each kinase reactionReaction Procedure:(i) Prepare indicated substrate in freshly prepared Base Reaction Buffer(ii) Deliver any required cofactors to the substrate solution above(iii) Deliver indicated kinase into the substrate solution and gently mix(iv) Deliver compounds in DMSO into the kinase reaction mixture(v) Deliver 33P-ATP (specific activity 0.01 μCi/μl final) into the reaction mixture to initiate the reaction.(vi) Incubate kinase reaction for 120 minutes at room temperature(vii) Reactions are spotted onto P81 ion exchange paper (Whatman #3698-915)(viii) Wash filters extensively in 0.1% phosphoric acid.(ix) Dry filters and measure counts in scintillation counterKinase Information:CHK-1 Genbank Accession # AF016582Recombinant full length construct, N-terminal GST tagged, purified from insect cells.No special measures were taken to activate this kinase.Final concentration in assay=0.5 nMSubstrate: CHKtidePeptide sequence: [KKKVSRSGLYRSPSMPENLNRPR]Final concentration in assay=20 μM
Measurement: IC50 = 0.4 nM (pChEMBL 9.399999618530273)
Descriptors: MW 513.99, ALogP 2.42, HBA 8, HBD 3, PSA 141.5, QED 0.43
Compound (SMILES): CCOc1ccc(-c2c(-c3ccc(C#N)cc3)nc(N3CCC(N(C)C)CC3)c3cncn23)cn1
InChIKey: ZNVLNNNZZQQQOO-UHFFFAOYSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: Demethylase Biochemical Assay: LANCE LSD1/KDM1A demethylase assay¿10 uL of 1 nM LSD-1 enzyme (ENZO BML-SE544-0050) in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 25 mM NaCl, 5 mM DTT) were preincubated for 1 hour at 25° C. with 0.8 uL compound/DMSO dotted in black 384 well polystyrene plates. Reactions were started by addition of 10 uL of assay buffer containing 0.4 uM Biotin-labeled Histone H3 peptide substrate: ART-K(Me1)-QTARKSTGGKAPRKQLA-GGK(Biotin) SEQ ID NO:1 (AnaSpec 64355) and incubated for 1 hour at 25° C. Reactions were stopped by addition of 10 uL 1x LANCE Detection Buffer (PerkinElmer CR97-100) supplemented with 1.5 nM Eu-anti-unmodified H3K4 Antibody (PerkinElmer TRF0404), and 225 nM LANCE Ultra Streptavidin (PerkinElmer TRF102) along with 0.9 mM Tranylcypromine-HCl (Millipore 616431). After stopping the reactions plates were incubated for 30 minutes and read on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 467.58, ALogP 4.26, HBA 8, HBD 0, PSA 82.58, QED 0.42
Compound (SMILES): Cc1cc(C2CCCN(Cc3cc4ccccc4s3)C2)n2ncnc2n1
InChIKey: HFBFLDFOJDULCD-UHFFFAOYSA-N
Target: cGMP-dependent 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O00408
Assay: Enzymatic Assay: An IMAP TR-FRET-based phosphodiesterase assay was developed using the PDE2A isoform. IMAP technology is based on high-affinity binding of phosphate by immobilized metal (MIII) coordination complexes on nanoparticles. The IMAP binding reagent recognizes phosphate groups on AMP or GMP generated from cAMP or cGMP in a PDE reaction. Cyclic nucleotides that carry a phosphodiester bond and not a free phosphate are not recognized by the binding reagent. The time resolved fluorescence resonance energy transfer (TR-FRET) is afforded by a Terbium (Tb)-Donor pre-bound to the nanoparticles. FRET occurs when the fluorescent-labeled AMP or GMP product of a PDE reaction binds and comes into close proximity to the Tb-Donor complex. Due to the long lifetime of Tb fluorescence, detection can be run in time-resolved mode to reduce or eliminate interference from auto-fluorescent compounds.The IMAP TR-FRET PDE2A assay was performed in 1536-well white plates. A total of 250 pg per well of FLAG-tagged PDE2A1 (amino acids 2-941) was dispensed in 2.5 μL IMAP assay buffer consisting of 10 mM Tris pH 7.2, 10 mM MgCl2, 1 mM DTT, and 0.1% fatty acid free BSA. 30 nL of compound was then added from 1 mM stocks in DMSO using a Kalypsys Pintool. Plates were incubated for 5 min at room temperature before dispensing 1.5 μL of 533 nM FAM-cAMP substrate for a final concentration of 200 nM. Following a brief centrifugation, plates were incubated for 30 min at room temperature. The assay was terminated by adding 5 μL IMAP binding reagent Tb complex to each well which was prepared according to manufacturer's recommendations (Molecular Devices). Plates were incubated an additional 90 minutes at room temperature and read on a Viewlux plate reader. All compounds were solvated at a concentration of 10 mM in DMSO and tested in 11-point half-log dose-response. Curve fitting and IC50 values were determined using a standard four parameter fit.PDE2
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 363.49, ALogP 4.03, HBA 6, HBD 0, PSA 46.32, QED 0.55
Compound (SMILES): COc1ncc(-c2cc(C(F)(F)F)c3c(N)ncnn23)cc1C(=O)NC1CCC(O)(c2ccccc2)C1
InChIKey: BZEGCNIKMKNCKN-UHFFFAOYSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: RIPK1 HTRF Binding Assay: A solution was prepared containing 0.2 nM Anti GST-Tb (Cisbio, 61GSTTLB), 90.6 nM probe and 1 nM His-GST-TVMV-hRIPK1(1-324) in FRET Buffer (20 mM HEPES, 10 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 0.05 mg/mL BSA). Using Formulatrix Tempest, the detection antibody/enzyme/probe solution (2 mL) was dispensed into wells of a 1536 plate (Black Low Binding Polystyrene 1536 Plate (Corning, 3724)) containing 10 nL of compounds of interest at appropriate concentration in DMSO. The plate was incubated at rt for 1 h. FRET was measured using the EnVision plate reader (Excitation: 340 nM, Emission: 520 nM/495 nM). Total signal (0% inhibition) was calculated from wells containing 10 nL DMSO only. Blank signal (100% inhibition) calculated from wells containing 10 nL of 15 nM staurosporine and internal controls.
Measurement: IC50 = 28000 nM (pChEMBL 4.550000190734863)
Descriptors: MW 512.49, ALogP 3.57, HBA 8, HBD 3, PSA 127.66, QED 0.37
Compound (SMILES): C=CC(=O)N[C@@H]1CCC[C@@H]1NC(=O)c1sc2nccc3c2c1NC(=O)N3c1ccc(Oc2ccccc2)cc1
InChIKey: GSPSFBBIEJYSFS-RTWAWAEBSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Lanthascreen Binding Assay: A BTK kinase lanthascreen binding assay monitors compound binding to unphosphorylated-BTK kinase domain (UP-BTK), by competing with a fluorescent labeled tracer. UP-BTK, consisting of the kinase domain of non-phosphorylated BTK protein (389-659aa), was produced in a Baculovirus/insect cell expression system. Into a 384-well plate, 2 ng of GST-tagged human BTK (389-659aa) was incubated with compound, 50 nM of Tracer 236 and 2 nM anti-GST antibody for 60 minutes using an optimized Lanthascreen assay. After 60 minutes, plates were read at 340 nM and 615/665 nM in an Infinite F500 (Tecan).
Measurement: IC50 = 275 nM (pChEMBL 6.559999942779541)
Descriptors: MW 539.62, ALogP 5.73, HBA 6, HBD 3, PSA 112.66, QED 0.26
Compound (SMILES): CC[C@H](C)NC(=O)O[C@@H]1CC[C@H](c2cc(NC(=O)Cc3c(C)noc3C)n[nH]2)C1
InChIKey: JTCUFHHWKIXBFJ-TUKIKUTGSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24864
Assay: Biochemical Assay: The purpose of CDK2/Cyclin E1 assay is to evaluate the inhibition (% inhibition, Kiapp and Ki values) of small molecule inhibitors by using a fluorescence-based microfluidic mobility shift assay. CDK2/Cyclin E1 full length catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide FL-Peptide-18 (5-FAM-QSPKKG-CONH2, CPC Scientific, Sunnyvale, Calif.) (SEQ ID NO:1). The mobility shift assay electrophoretically separates the fluorescently labeled peptides (substrate and phosphorylated product) following the kinase reaction. Both substrate and product are measured and the ratio of these values is used to generate % conversion of substrate to product by the LabChip EZ Reader. Wild-type CDK2/wild-type full length Cyclin E1 enzyme complex was produced in-house (baculoviral expression, LJIC-2080/LJIC-2103) and phosphorylated by CDK7/Cyclin H1/Matl enzyme complex with CDK2:CDK7 ratio of 50:1 (concentration mg/mL) in the presence of 10 mM MgCl2 and 5 mM ATP at room temperature for one hour. Typical reaction solutions (50 μL final reaction volume) contained 2% DMSO (±inhibitor), 4 mM MgCl2, 1 mM DTT, 150 μM ATP (ATP Km=67.4 μM), 0.005% Tween-20, 3 μM FL-Peptide-18, and 0.36 nM (catalytically competent active site) phosphorylated wild-type full length CDK2/Cyclin E1 enzyme complex in 25 mM HEPES buffer at pH 7.15. The assay was initiated with the addition of ATP, following a fifteen minutes pre-incubation of enzyme and inhibitor at room temperature in the reaction mixture. The reaction was stopped after 45 minutes at room temperature by the addition of 50 μL of 80 mM EDTA.
Measurement: Ki = 2.24 nM (pChEMBL 8.649999618530273)
Descriptors: MW 403.48, ALogP 3.36, HBA 6, HBD 3, PSA 122.14, QED 0.65
Compound (SMILES): CO[C@H]1C[C@@H](C(C)(C)C)O[C@@H](c2onc(C(=O)NC3=C(C)N(C)N(C4CCCCC4)C3C)c2C)C1
InChIKey: JQATZKIUVQXNHA-LYIIJDOLSA-N
Target: E3 ubiquitin-protein ligase SMURF1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HCE7
Assay: Pharmaceutical Assay: To determine the HECT E3 ligase selectivity of the compounds, a panel of biochemical HECT E3 ligase autoubiquitinylation assays was employed (Smurf-1, Smurf-2, WWP1, WWP2, ITCH, Nedd4, Nedd4L and E6AP). The conjugation of ubiquitin to a protein substrate is a multistep process. In an initial ATP-requiring step, a thioester bond is formed between the carboxyl terminus of ubiquitin and an internal cystein residue of the ubiquitin-activating enzyme (E1). Activated ubiquitin is then transferred to a specific cystein residue of an ubiquitin-conjugating enzyme (E2). E2s donate ubiquitin to a HECT E3 ligase (E3) from which it is transferred to the substrate protein. HECT E3 ligases can auto-ubiquitinylate. This event is detected in the TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) assay used in this panel. The reaction mix contains E1, E2, tagged-E3, biotin-conjugated ubiquitin, the compound and ATP in a suitable buffer and is incubated for 45 minutes to allow auto-ubiquitinylation of the E3 ligase. To measure the extent of ubiquitinylated E3 ligase by TR-FRET, the donor fluorophore Europium cryptate (Eu3+ cryptate), conjugated to streptavidin which subsequently binds to biotinylated ubiquitin, and the modified allophycocyanin XL665 (HTRF primary acceptor fluorophore) coupled to a tag-specific antibody (HA, His or GST), which recognizes the respective E3 ligase fusion proteins, are added after the reaction is complete. When these two fluorophores are brought together by a biomolecular interaction (in this case ubiquitinylation of the E3 ligase), a portion of the energy captured by the Cryptate during excitation is released through fluorescence emission at 620 nm, while the remaining energy is transferred to XL665. This energy is then released by XL665 as specific fluorescence at 665 nm. Light at 665 nm is emitted only through FRET with Europium. Because Europium Cryptate is present in the assay, light at 620 nm is detected even when the biomolecular interaction does not bring XL665 within close proximity.
Measurement: IC50 = 640 nM (pChEMBL 6.190000057220459)
Descriptors: MW 488.67, ALogP 5.11, HBA 7, HBD 1, PSA 80.07, QED 0.62