lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CN(C)C(=O)C1CN(c2nc(=O)n(-c3ccccc3)c3cc(C(F)(F)F)ccc23)C1
InChIKey: QHQDJJLFIIOVHJ-UHFFFAOYSA-N
Target: S-adenosylmethionine synthase isoform type-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P31153
Assay: Phosphate Sensor Fluorescence Assay : MAT2A enzyme is incubated with a test compound in DMSO or DMSO and its substrates (L-methionine and ATP) in a microtiter plate. The enzymatic reaction is stopped by the addition of Working Phosphate Sensor Mixture. The plate is analyzed for fluorescence at 450 nm. The high control (DMSO with enzyme and its substrates) gives high fluorescence which represents no inhibition of enzymatic activity while the low control (DMSO with MAT2A substrates and no enzyme) gives low fluorescence which represents full inhibition of enzymatic activity.Materials:Human MAT2A: Cepter, amino acids 1-395Tris, pH 7.5: Invitrogen cat #15567-027KCl: Ambion cat #AM9640GMgCl2: Ambion cat #AM9530GBrij-35: Sigma cat B4184-10MLDTT: Goldbio cat #DTT100BGG: Sigma cat #G5009-25GPNP: Novus Biologicals cat #NBP1-508727-MEG: Cayman Chemical cat #15988L-Methionine: Alfa Aesar cat #J61904ATP: Alfa Aesar cat #J60336Phosphate Sensor: Thermo Fisher cat #PV4407EDTA: Life Tech cat #15575-038Assay plate: 384-well black polypropylene plate: Thomas Scientific cat #1149Q35Final Assay Conditions:Assay Buffer: 50 mM Tris, pH 7.5/50 mM KCl/10 mM MgCl2/0.01% Brij-35/1 mM DTT/0.1% BGG/40 nM PNP/6 uM 7-MEGMAT2A: 10 nM for Cepter clone ID 329, lot 00023-123 before the addition of Working Phosphate Sensor Mixture    5 nM for Cepter clone ID 334, lot 00023-148 before the addition of Working Phosphate Sensor MixtureL-methionine: 500 uM before the addition of Working Phosphate Sensor MixtureATP: 500 uM before the addition of Working Phosphate Sensor MixtureProcedure:For the assay, a mixture of 1 mM L-methionine/1 mM ATP (2× final pre-stopped concentration) in assay buffer; MAT2A (2× final pre-stopped concentration) in Assay Buffer and Working Phosphate Sensor Mixture (1.5 uM Phosphate Sensor/30 mM EDTA in Assay Buffer, which is 3× final concentrations) were prepared. Test compounds or DMSO were added to the appropriate well suing D300e digital dispenser. 5 μl/well of Assay Buffer was added to the wells corresponding to the negative control and 5 μl/well of MAT2A was added to all the wells except for those corresponding to the negative control. After incubating the plate at room temperature for 15 minutes, 5 μl/well of the 1 mM L-methionine/1 mM ATP mixture was added to all wells. The plate was centrifuged at 1000 rpm for 1 minute and then incubated at room temperature for 1 hour. 5 μl of the Working Phosphate Sensor Mixture was added to all wells and the plate was centrifuged at 1000 rpm for 1 minute. The plate was read for fluorescence at 450 nm after exciting at 430 nm.
Measurement: IC50 < 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 416.4, ALogP 2.93, HBA 5, HBD 0, PSA 58.44, QED 0.66
Compound (SMILES): C[C@H]1CCN1c1nc(N2C[C@@H]3[C@H](CC(=O)O)[C@@H]3C2)cc(C(F)(F)F)n1
InChIKey: MDUYWDNWFXSMJJ-MMWGEVLESA-N
Target: Ketohexokinase (SINGLE PROTEIN), Homo sapiens
UniProt: P50053
Assay: KHK-C Assay A: A 384-well format on a Corning 3653 assay plate is used, and monitored by UV-vis spectroscopy in continuous mode at rt. Compounds were prepared in DMSO as 4 mM stocks, diluted using an 11-point half-log scheme on a Biomek FX (Beckman Coulter), and incubated at rt for 30 minutes with the reaction mixture containing 50 mM HEPES, pH 7.4, 140 mM KCl, 3.5 mM MgCl2, 0.8 mM fructose, 2 mM TCEP, 0.8 mM PEP, 0.7 mM NADH, 0.01% Triton X-100, 30 U/mL pyruvate kinase-lactate dehydrogenase, and 10 nM purified KHK-C. The compound concentration in each well ranged from 1 nM to 100 μM. The reaction was initiated with the addition of 0.2 mM ATP. The absorbance was measured for 30 minutes on a SpectraMax reader (Molecular Devices) after ATP was added. The concentrations provided are based on the final mixture volume of 40 μL (referred to as the final concentration). Controls: N8-(cyclopropylmethyl)-N4-(2-(methylthio)phenyl)-2-(piperazin-1-yl)pyrimido[5,4-d]pyrimidine-4,8-diamine at 2 μM final concentration was used as high percent effect (HPE) control, and 2.5% DMSO which was present in all reaction wells was used as zero percent effect (ZPE) control. Reaction rates were obtained for 300-1800 seconds time window in units of 1000*AU/min (absorbance unit per minute), and average values for ZPE and HPE controls from 16 wells each were calculated, AveZPE and AveHPE, respectively.
Measurement: IC50 = 2084 nM (pChEMBL 5.679999828338623)
Descriptors: MW 356.35, ALogP 2.25, HBA 5, HBD 1, PSA 69.56, QED 0.89
Compound (SMILES): Cn1cc(C(C)(O)c2cc(F)c3c(c2)C(=O)N(Cc2ccc(C#N)cn2)[C@@]3(OCC2(CO)CC2)c2ccc(Cl)cc2)cn1
InChIKey: GCKLTVVEUHWDDO-BDJZEIKTSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: Binding Assay (ELISA): The ELISA assay was performed in streptavidin coated plates which were preincubated with 200 ul per well of 1 μg ml−1 biotinylated IP3 peptide. The plates were ready to use for MDM2 binding after washing the plate with PBS.Compounds and control solutions in DMSO aliquoted in 96-well plates were pre-incubated in a final 2.5-5% (v/v) DMSO concentration at room temperature (for example 20C.) for 20 min with 190 ul aliquots of optimized concentrations of in vitro translated MDM2, before transfer of the MDM2-compound mixture to the b-IP3 streptavidin plates, and incubation at 4C. for 90 min. After washing three times with PBS to remove unbound MDM2, each well was incubated at 20C. for 1 hour with a TBS-Tween (50 mM Tris pH7.5; 150 mM NaCl; 0.05% Tween 20 nonionic detergent) buffered solution of primary mouse monoclonal anti-MDM2 antibody (Ab-5, Calbiochem, used at a 1/10000 or 1/200 dilution depending on the antibody stock solution used), then washed three times with TBS-Tween before incubation for 45 mins at 20C. with a TBS-Tween buffered solution of a goat-anti-mouse horseradish peroxidase (HRP) conjugated secondary antibody (used at 1/20000 or 1/2000 depending on the antibody stock solution). The unbound secondary antibody was removed by washing three times with TBS-Tween. The bound HRP activity was measured by enhanced chemiluminesence (ECL, Amersham Biosciences) using the oxidation of the diacylhydrazide substrate, luminol, to generate a quantifiable light signal. The percentage of MDM2 inhibition at a given concentration is calculated as the [1−(RLU detected in the compound treated sample−RLU negative DMSO control)÷(RLU of DMSO positive and negative controls)]x100 or as the (RLU detected in the compound treated sample ÷ RLU of DMSO controls)x100.
Measurement: IC50 = 7.2 nM (pChEMBL 8.140000343322754)
Descriptors: MW 602.07, ALogP 4.38, HBA 8, HBD 2, PSA 124.5, QED 0.29
Compound (SMILES): O=C(c1cc(F)ccc1-c1ncc(F)cn1)N1C(CO)C2CCC1C(Oc1ccc(C(F)(F)F)cn1)C2
InChIKey: NLZFFZJXRVUYPY-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P56718
Assay: Receptor Radioligand Binding Assay: Human Embryonic Kidney 293 cells (HEK293) stably expressing rat orexin 1 receptor (Genebank accession number NM_001525) or Chinese ovary cells (CHO) stably expressing human orexin 1 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1× Pen/Strep, 1× sodium pyruvate, 10 mM HEPES, 600 μg/mL G418 and DMEM/F12 (Gibco, Cat #11039), 10% FBS, 1× Pen/Strep, 600 μg/mL G418 media, respectively on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1× with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2K×G, 5 min at 4° C.), the supernatant was aspirated and the pellets frozen and stored at −80° C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and homogenized for 45 sec prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H] (1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone) (Moraveck Corporation, specific activity=35.3 Ci/mmol), diluted to a 10 nM concentration in PBS (4 nM final). Compounds were solubilized in 100% DMSO (Acros Organics, Cat. #61042-1000) and tested over a range of 7 concentrations (from 0.1 nM to 10 μM). The final concentration of DMSO in the reactions is equal to or less than 0.1%. Total and nonspecific binding was determined in the absence and presence of 10 μM almorexant. The total volume of each reaction is 200 μL (20 μL of diluted compounds, 80 μL of [3H]¿ (1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone) diluted in PBS and 100 μL of the cell suspension). Reactions were run for 60 min at room temperature and terminated by filtration through GF/C filter plates (PerkinElmer, Cat. #6005174) presoaked in 0.3% polyethylenimine using the cell harvester (PerkinElmer Filtermate).
Measurement: Ki = 1100 nM (pChEMBL 5.960000038146973)
Descriptors: MW 520.46, ALogP 4.27, HBA 6, HBD 1, PSA 88.44, QED 0.51
Compound (SMILES): C[C@H]1C[C@@H](n2c(Nc3ccc(OC(F)(F)F)cc3)nc3ccc(C(=O)NS(C)(=O)=O)cc32)CC(C)(C)C1
InChIKey: HGQNWNQKDXQVQR-MAUKXSAKSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Mutant IDH1 R132H Biochemical Assay: mIDH1 catalyzes the NADPH-dependent reduction of alpha-ketoglutarate (α-KG) to (2R)-2-hydroxyglutarate (2-HG). NADPH consumption was measured by luminescent readout.The biochemical reactions were performed at 32° C. in 384-well plates using a reaction volume of 41 μl and the following assay buffer conditions: 50 mM Tris pH 7.5, 100 mM NaCl, 20 mM MgCl2, 0.05% BSA, 0.01% Brij, 1 μM NADPH, and 250 μM α-KG. The IDH1 R132H enzyme was used in a final concentration of 1.5 nM. Test compounds were used in a concentration range between 0.002 and 10 μM. The final DMSO concentration was 2.4%.The reaction was incubated for 30 minutes, then 40 μl of detection mix (0.75 μg/ml Luciferase, 0.02 U/ml Oxidoreductase, 4 μg/ml FMN, 2 μl/ml Decanal/Ethanol, 50 mM Tris pH 7.5, 0.5% Glycerin, 0.01% Tween-20, 0.05% BSA) was added. Luminescence was measured on a luminescent reader (10 seconds measuring time, 1 second integration period, 30% sensitivity). The decrease in luminescence is proportional to mIDH1 activity. IC50 values are determined by interpolation from plots of relative luminescence versus inhibitor concentration.
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 538.59, ALogP 5.76, HBA 7, HBD 2, PSA 102.32, QED 0.41
Compound (SMILES): C=CC(=O)N1CCC(c2nc(-c3ccc(Oc4ccccc4)cc3)c(C(N)=O)s2)CC1
InChIKey: AURKZZOJYVVJIF-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: ADP-Glo Kinase Assay: 1× and 2× assay buffer were made at first. BTK kinase was diluted with 1× assay buffer but substrate was diluted with 2× assay buffer. 1 ul of diluted compound was transferred into 384-well assay plate, and then 2.0 ul of enzyme solution was added, and spun at 2000 rpm for 1 min. This mixture was incubated at 24° C. for 30 mins. 2 ul of peptide substrate/ATP mixture was added into the assay plate to start the reaction. The mixture was mixed thoroughly and then the 384-well plate was spun and incubated at 24° C. for 60 mins. 5.0 ul of ADP-Glo Reagent was added to stop the kinase activity and deplete the ATP unconsumed, and the plate was mixed thoroughly and incubated at 24° C. for 40 min. Then, 10.0 ul of Kinase Detection reagent was added, and the plate was centrifuged and then kept at 24° C. for 30 min. The luminescence signal was read on Envision.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 433.53, ALogP 4.59, HBA 5, HBD 1, PSA 85.52, QED 0.58
Compound (SMILES): CSc1cnc(NC[C@H](C)CNc2nc3ccc(C(=O)NCc4n[nH]c(=O)[nH]4)cc3s2)nc1
InChIKey: GABGFTWAGCHLEH-NSHDSACASA-N
Target: Proprotein convertase subtilisin/kexin type 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NBP7
Assay: Surface Plasmon Resonance (SPR) Assay: Surface plasmon resonance data was collected on a Biacore T200 or 3000 system (GE Healthcare) at 25° C. Streptavidin was immobilized on a CMS (GE Healthcare) or CMD500d sensor chip (XanTec Bioanalytics) using standard amine-coupling chemistry at 25° C. with HBS-N (10 mM HEPES, 0.15 M NaCl, pH 7.4) as the running buffer. Briefly, the carboxymethyl dextran surface was activated with a 12 min injection of a 1:1 ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/0.1 M N-hydroxy succinimide (NHS) at a flow rate of 10 μL/min. For capture of streptavidin, protein was diluted to 0.2 mg/mL in 10 mM sodium acetate (pH 4.5) and captured by injecting 100 μL onto the activated chip surface. Residual activated groups were blocked with a 7 min injection of 1 M ethanolamine (pH 8.5). Avi-tagged PCSK9 protein was captured on the streptavidin surface by injection of 150 μL of protein diluted to 16 μg/mL in HBS-N, 0.05% tween-20, 0.1 mM CaCl2. Typical surface densities obtained were 8000-10000 RU. SPR binding data were obtained using an appropriate dilution series of each compound at a flow rate of 30 μL/ min, with a capture time of 100 s and dissociation times of 300 s. Running buffer for compound binding studies was HBS-N, 0.05% tween-20, 0.1 mM CaCl2, 4% DMSO. Data were corrected for DMSO excluded volume effects. All data were double-referenced for blank injections and reference surface using standard processing procedures and data processing and kinetic fitting were performed using Scrubber software, version 2.0c (BioLogic Software).
Measurement: Kd < 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 485.6, ALogP 2.31, HBA 10, HBD 5, PSA 153.37, QED 0.21
Compound (SMILES): COc1ccc(F)cc1-c1ccnc2[nH]c(C3=CCN(S(=O)(=O)NC(=O)OCCOC(C)=O)CC3)cc12
InChIKey: WQKJWWPFGYZLQE-UHFFFAOYSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P50750
Assay: LANCE ULight TR-FRET Kinase Assay: CDK9 enzyme activities were measured using LANCE ULight TR-FRET kinase assay reagents (PerkinElmer, Waltham, Mass.). Compounds were directly added in 100% DMSO to white low volume assay plates (Perkin Elmer Proxiplate 6008289) using a Labcyte Echo acoustic dispenser. Assay reagents in serine/threonine kinase assay buffer containing 20 mM HEPES, 10 mM MgCl2, 100 mM Na3VO4, and 0.0075% Triton X-100. were added for final reaction mixture concentrations of 1000 μM ATP, 100 nM U-light MBP peptide (Perkin Elmer TRF0109M) and reaction initiated with 4 nM CDK9/Cyclin T1 (Carna Biosciences 04-110). The kinase reaction was carried out for 30 minutes before addition of stopping buffer to a final of 20 mM EDTA and 0.5 nM of LANCE Ultra Europium anti-phospho-MBP antibody (PerkinElmer TRF0201M) in LANCE detection buffer (PerkinElmer CR97-100). The reaction was equilibrated for 1 hour and the signal read in the Perkin Elmer Envision in TR-FRET mode (excitation at 320 nm and emission at 615/665 nm).
Measurement: IC50 = 54 nM (pChEMBL 7.269999980926514)
Descriptors: MW 532.55, ALogP 3, HBA 8, HBD 2, PSA 139.92, QED 0.33
Compound (SMILES): Cc1cnc(OCC(C)(C)C(=O)N[C@H]2CCN(C)C[C@@H]2C)c(C)c1
InChIKey: OKSHSYZANJEHNU-HOTGVXAUSA-N
Target: Somatostatin receptor type 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P31391
Assay: Inhibition of Forskolin Stimulated cAMP in Cells Overexpressing SSTR4: This cell-based assay measures the ability of compounds to inhibit forskolin stimulated cAMP in CHO-K1 cells overexpressing SSTR4. CHO-K1 cells overexpressing SSTR4 (CHO-SSTR4) are purchased from DiscoveRx (product code 95-0059C2). The CHO-SSTR4 cells are maintained in F12K media with 10% Fetal Bovine Serum (Hyclone), 1% Pen/Strep (Life Technologies), and 800 μg/mL G418 (Life Technologies). To perform the assay, 3000 cells are plated per well in white 384-well plate (Corning 3570) in 50 μL complete media and the cells are allowed to attach for 16 hours in a 37° C., 5% CO2 incubator. The next day, the culture media is removed from the cells and the cells are washed (added then removed) with Kreb's Ringer Buffer (ZenBio, KRB-1000 mL). Test compounds are suspended in DMSO and diluted in stimulation buffer: Kreb's Ringer Buffer plus 0.5% BSA (Roche), 300 μM IBMX (Sigma), and 350 nM forskolin (Sigma). The cells are incubated in L compound/stimulation buffer for 30 minutes at room temperature. Cellular cAMP levels are detected with a HTRF LANCE Ultra cAMP kit (Perkin Elmer, catalog number TRF0264).The assay is performed in accordance with the manufacturer's instructions. Five μL of diluted Eu-W8044 labeled streptavidin (dilution: 1:50 in cAMP Detection Buffer) is added to each well. Then 5 μL of diluted biotin cAMP (dilution: 1:150 in cAMP Detection Buffer) is added to each well. The plates are covered and allowed to incubate for 60 minutes at room temperature on a shaker. HTRF (665 nm/615 nm) is read on a Perkin Elmer ENVISION plate reader. The pEC50 values are generated using Activity Base for Screening Data Management.
Measurement: EC50 = 25.1 nM (pChEMBL 7.599999904632568)
Descriptors: MW 333.48, ALogP 2.56, HBA 4, HBD 1, PSA 54.46, QED 0.9
Compound (SMILES): C[C@@H]1Cc2ccccc2CN1C(=O)c1cc2c(cc1-c1cc(C(=O)N(C)c3ccc(O)cc3)c3n1CCCC3)CCN(C(=O)N(C)c1ccccc1)C2
InChIKey: GJBPWMFUEGGZCD-GDLZYMKVSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Fluorescence Polarisation Assay: The fluorescence polarisation tests were carried out on microplates (384 wells). The Bcl-2 protein, labelled (histag-Bcl-2 such that Bcl-2 corresponds to the UniProtKB® primary accession number: P10415), at a final concentration of 2.50×10−8 M, is mixed with a fluorescent peptide (Fluorescein-REIGAQLRRMADDLNAQY), at a final concentration of 1.00×10−8 M in a buffer solution (Hepes 10 mM, NaCl 150 mM, Tween20 0.05%, pH 7.4), in the presence or in the absence of increasing concentrations of test compounds. After incubation for 2 hours, the fluorescence polarisation is measured.
Measurement: IC50 = 6.1 nM (pChEMBL 8.210000038146973)
Descriptors: MW 707.87, ALogP 7.68, HBA 5, HBD 1, PSA 89.33, QED 0.2
Compound (SMILES): CNC(=O)C(=N)c1c(N)cc(-c2cc(F)c(O)cc2CC(F)(F)F)nc1NCc1c(F)cccc1N(C)S(C)(=O)=O
InChIKey: GZSSSGDNHNAIFG-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Caliper Enzyme Assay at 1 mM ATP: Test article was solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. An 11-point half log dilution series was created in DMSO with a top concentration of 600 μM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 resulting in a top final assay compound concentration of 10 μM and a 2% DMSO concentration.Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK3 or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 600.57, ALogP 3.54, HBA 8, HBD 5, PSA 161.5, QED 0.19
Compound (SMILES): C[C@H]1C[C@H](C)N1C(=O)O[C@@H]1CC[C@H](c2cc(NC(=O)Cc3cnn(C)c3)n[nH]2)C1
InChIKey: AMFNSEGCJHUABD-DARAHFNDSA-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 = 0.32 nM (pChEMBL 9.489999771118164)
Descriptors: MW 400.48, ALogP 2.58, HBA 6, HBD 2, PSA 105.14, QED 0.8
Compound (SMILES): COc1cccc2c1NCC[C@@H]2C(=O)n1nc2c(c1N)CCN(S(C)(=O)=O)C2
InChIKey: VENDHXBGIRNKHA-LBPRGKRZSA-N
Target: Coagulation factor XII (SINGLE PROTEIN), Homo sapiens
UniProt: P00748
Assay: Factor XIIa (FXIIa) Inhibitory Activity: In a 96-well clear bottom plate, 80 μl of assay buffer was added to each well. Assay buffer consists of 0.5× Hank's Balanced Salt Solution (Invitrogen), buffered with 25 mM HEPES pH 7.4 (Invitrogen) and 0.5× Tris-buffered saline with Tween-20 0.05% (Santa Cruz Biotechnology). Test compounds were first dissolved in DMSO (Sigma) and then 4 μl were added to test wells containing assay buffer. Serial dilutions using an automated multi-channel pipette were used to generate a concentration range of approximately 1-100 μM. Human FXIIa (Enzyme Research Labs) was diluted in assay buffer to a final concentration of 12.5 nM. 80 μl of this enzyme solution was added to the assay wells. The enzyme/compound mixtures were incubated for 10 minutes at room temperature. Chromogenic substrate (Pefachrome XIIa; Enzyme Research Labs) was added to assay wells at a final concentration of 400 μM. The assay plate was spun for 1 minute at 1500 g and then read at 37° C. in a SpectraMax M2 plate reader (λ=405 nm). Activity was quantified as the rate of change in absorbance, which corresponds to the rate of substrate cleavage. IC50 values were determined as the concentration of inhibitor that produced 50% of the rate of change of control wells without any inhibitor. For compounds with activity <1 μM, the assay was repeated with a lower concentration range, typically from 10-1000 nM.
Measurement: IC50 = 140 nM (pChEMBL 6.849999904632568)
Descriptors: MW 405.48, ALogP 1.03, HBA 8, HBD 2, PSA 119.55, QED 0.78
Compound (SMILES): Cc1ccncc1-c1cc2cc(Nc3cnn([C@@H]4CCN(C5COC5)C[C@H]4O)c3)ncc2c(N)n1
InChIKey: GDNLJEKGVVKKDA-DHIUTWEWSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: Binding Assay: Lanth: For the binding assay, 4 ul 2×HPK1 and Eu-anti-GST antibody were added to each well of the assay plate using a Multidrop reagent dispenser. The solutions were incubated in a 23 C incubator for 1 h. To each well of the assay plate was added 4 ul 2× Tracer-222 using a Multidrop reagent dispenser. The solutions were again incubated in a 23 C incubator for 1 h. The results of the assay were read using an Envision plate reader with the following parameters: TR_FRET, 340ex/615 and 665em; 100 usec Delay; and 200 usec integration.
Measurement: Ki = 0.592 nM (pChEMBL 9.229999542236328)
Descriptors: MW 472.55, ALogP 2.53, HBA 10, HBD 3, PSA 127.24, QED 0.4
Compound (SMILES): Cc1ccc2c(c1)nc1n2CC(NC(=O)c2ccc(-n3cnc(C)n3)cc2Cl)(c2cc(F)cc(F)c2)CC1
InChIKey: VEAKHWBGINAGRU-UHFFFAOYSA-N
Target: Coagulation factor IX (SINGLE PROTEIN), Homo sapiens
UniProt: P00740
Assay: TBD: TBD
Measurement: IC50 = 11.6 nM (pChEMBL 7.940000057220459)
Descriptors: MW 532.98, ALogP 5.44, HBA 6, HBD 1, PSA 77.63, QED 0.33
Compound (SMILES): COc1nc2ccc([C@@](O)(c3ccnc(C(F)(F)F)c3)c3cncn3C)cc2c(C#N)c1CC(F)(F)F
InChIKey: IDGZKXWHIKXDOW-HSZRJFAPSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor Assay: ThermoFluor is a fluorescence based assay that estimates ligand binding affinities by measuring the effect of a ligand on protein thermal stability (Pantoliano, M. W., Petrella, E. C., Kwasnoski, J. D., Lobanov, V. S., Myslik, J., Graf, E., Carver, T., Asel, E., Springer, B. A., Lane, P., and Salemme, F. R. (2001) High-density miniaturized thermal shift assays as a general strategy for drug discovery. J Biomol Screen 6, 429-40, and Matulis, D., Kranz, J. K., Salemme, F. R., and Todd, M. J. (2005) Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor. Biochemistry 44, 5258-66). This approach is applicable to a wide variety of systems, and rigorous in theoretical interpretation through quantitation of equilibrium binding constants (KD).In a ThermoFluor experiment where protein stability is monitored as the temperature is steadily increased, an equilibrium binding ligand causes the midpoint of an unfolding transition (Tm) to occur at a higher temperature. The shift in the melting point described as a ΔTm is proportional to the concentration and affinity of the ligand. The compound potency may be compared as a rank order of either ΔTm values at a single compound concentration or in terms of KD values, estimated from concentration response curves.
Measurement: Kd = 3.2 nM (pChEMBL 8.489999771118164)
Descriptors: MW 521.42, ALogP 4.65, HBA 7, HBD 1, PSA 96.85, QED 0.38
Compound (SMILES): Nc1cc(Cc2cnn(Cc3ccc4c(c3)CCNC4)c2)c2nn[nH]c2n1
InChIKey: QALYNGDUFROUBQ-UHFFFAOYSA-N
Target: Myeloperoxidase (SINGLE PROTEIN), Homo sapiens
UniProt: P05164
Assay: EPX Bromination Assay : EPX bromination activity was measured in 100 mM KPi (pH 7.4) by monitoring the H2O2 catalyzed formation of 3-bromo tyrosine from tyrosine and potassium bromide. A 50 μl mixture of 0.6 μM EPX (Lee Biosolutions Cat. #342-60) was added to 100 nL inhibitor in 100% DMSO in a 384 well REMP plate. Enzyme and compound were preincubated for ten minutes at room temperature.After the ten minute preincubation of enzyme and inhibitor, 25 μL of a mixture containing 400 μM tyrosine and 1200 μM potassium bromide was added to the plate containing enzyme and inhibitor, followed by the addition of 25 μl of 20 μM H2O2. The reaction was allowed to proceed for 15 minutes, at which time it was quenched with 10 μL of 20% TCA. The final concentrations of all components were 0.3 μM EPX, 100 μM tyrosine, 400 μM potassium bromide, 5 μM H2O2, 0.1% DMSO, 2.0% TCA.
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 360.43, ALogP 1.42, HBA 7, HBD 3, PSA 110.33, QED 0.51
Compound (SMILES): CCc1ccncc1-c1cc2cc(NC(=O)CC#N)ncc2c(N)n1
InChIKey: XARSNAIYSAGJQG-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: Binding Assay: Lanth: For the binding assay, 4 ul 2×HPK1 and Eu-anti-GST antibody were added to each well of the assay plate using a Multidrop reagent dispenser. The solutions were incubated in a 23 C incubator for 1 h. To each well of the assay plate was added 4 ul 2× Tracer-222 using a Multidrop reagent dispenser. The solutions were again incubated in a 23 C incubator for 1 h. The results of the assay were read using an Envision plate reader with the following parameters: TR_FRET, 340ex/615 and 665em; 100 usec Delay; and 200 usec integration.
Measurement: Ki = 26.4 nM (pChEMBL 7.579999923706055)
Descriptors: MW 332.37, ALogP 2.69, HBA 6, HBD 2, PSA 117.58, QED 0.76
Compound (SMILES): CCS(=O)(=O)NC(=O)c1cc(C2CC2)c(OCC2CCN([C@@H](c3cc(Cl)cc(Cl)c3)C(C)(C)OC)CC2)cc1F
InChIKey: QMIHBNPMTMLUMI-MHZLTWQESA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Electrophysiological Assay (EP) (In vitro Assay): Patch voltage clamp electrophysiology allows for the direct measurement and quantification of block of voltage-gated sodium channels (NaV's), and allows the determination of the time- and voltage-dependence of block which has been interpreted as differential binding to the resting, open, and inactivated states of the sodium channel (Hille, B., Journal of General Physiology (1977), 69: 497-515).The following patch voltage clamp electrophysiology studies were performed on representative compounds of the invention using human embryonic kidney cells (HEK), permanently transfected with an expression vector containing the full-length cDNA coding for the desired human sodium channel α-subunit, grown in culture media containing 10% FBS, 1% PSG, and 0.5 mg/mL G418 at 37° C. with 5% CO2. HEK cells used for the electrophysiology (EP) recordings had a passage number of less than 40 for all studies and were used within three days from the time of plating, NaV1.7 and NaV1.5 cDNAs (NM_002977 and AC137587; SCN5A, respectively) were stably expressed in HEK-293 cells. The β1 subunit was coexpressed in both the NaV1.7 and NaV1.5 cell lines.Sodium currents were measured rising the patch clamp technique in the whole-cell configuration using either a PatchXpress automated voltage clamp or manually using an Axopatch 200B (Axon Instruments) or Model 2400 (A-M systems) amplifier. The manual voltage clamp protocol was as follows: Borosilicate glass micropipettes were fire-polished to a tip diameter yielding a resistance of 2-4 Mohms in the working solutions. The pipette was filled with a solution comprised of: 5 mM NaCl, 10 mM CsCl, 120 mM CsF, 0.1 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, 10 mM EGTA; and adjusted to pH 7.2 with CsOH. The external solution had the following composition: 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES; and adjusted to pH 7.4 with NaOH. In some studies, the external sodium was reduced by equimolar replacement with choline. Osmolality in the CsF internal and NaCl external solutions was adjusted to 300 mOsm/kg and 310 mOsm/kg with glucose, respectively. All recordings were performed at ambient temperature in a bath chamber with a volume of 150 μL. Control sodium currents were measured in 0.5% DMSO.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 615.6, ALogP 6.35, HBA 6, HBD 1, PSA 84.94, QED 0.32
Compound (SMILES): O=C(NCCC1CCN(Cc2ccccc2)CC1)N1CCN(c2cccc(Cl)c2)CC1
InChIKey: NCDHTVNTYJIWMF-UHFFFAOYSA-N
Target: Muscarinic acetylcholine receptor M4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08173
Assay: Binding Assay: Binding affinity (Ki) of compounds was measured by inhibition of radioligand binding to membranes from CHO cells expressing human M1, M2, M3, M4 and M5 receptors. Membranes were prepared by nitrogen cavitation and differential centrifugation as previously described (Hoare et al., Mol. Pharmacol. 2003 March; 63(3): 751-65). The radioligand employed was tritiated N-methylscopolamine, used at a concentration of 1.5 nM. A dose-response of twelve concentrations of compound was used, ranging from 10 Î¼M to 32 Î¼M. The assay buffer was 50 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 1 mM ethylenediaminetetraacetic acid, pH-adjusted to pH 7.4.
Measurement: Ki = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 441.02, ALogP 4.47, HBA 3, HBD 1, PSA 38.82, QED 0.72
Compound (SMILES): COCCN1C[C@@H](NC(=O)Nc2c(C)c(OCC(C)(C)O)nn2-c2ccccc2)[C@H](c2ccc(F)c(F)c2)O1
InChIKey: CMAZYHYFWZMENZ-PKTZIBPZSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Inhibition Assay: Seven point five μL per well of human TrkA (PV3144, Lifetechnologies, final concentration: 1 nmol/L) suspended in the assay buffer (100 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 10 mmol/L magnecium chloride, 0.003 vol % Brij-35, 0.004 vol % Tween20 and 1 mmol/L dithiothreitol (DTT)) was applied in a 384 well plate and the plate was pre-incubated for 15 min at room temperature with 0.4 μL of each compound (final concentration: 200 μmol/L-1 pmol/L) dissolved in DMSO. Then, fluorescent substrate (FL-peptide 27, 760424, PerkinElmer, final concentration: 1.5 μmol/L) and ATP (final concentration 500 μmol/L) dissolved in the assay buffer was added in each well. After the incubation of 120 min at 37° C., fifteen μL of termination buffer (100 mmol/L HEPES, 40 mmol/L ethylenediaminetetraacetic acid (EDTA), 10 mmol/L magnecium chloride, 0.003 vol % Brij-35, 0.004 vol % Tween20, 1 mmol/L DTT and 0.16 vol % Coating Reagent 3) was added in each well to stop the enzyme reaction. Fluorescent intensities (FI) of phosphorylated and non-phosphorylated fluorescent substrates were measured by LabChip EZReader II (Caliper LifeSciences, Inc.), and conversion ratio (CR) was calculated by the following formula-1. The CR in the well applied with DMSO alone was used as a negative control and the CR in the well without applying TrkA was used as a positive control.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 545.59, ALogP 3.73, HBA 8, HBD 3, PSA 110.11, QED 0.36
Compound (SMILES): CC(C)Oc1ccc(-c2cc(CN/C(N)=N/S(=O)(=O)c3ccccc3)cnc2-c2ccc(Cl)cc2)cc1
InChIKey: WBXSCDKSPDPAFJ-UHFFFAOYSA-N
Target: Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UDY8
Assay: Inhibition of Protease Activity Assay: Full-length cDNA of human MALT1 gene (GenBank accession No: AB026118.1) amplified by PCR was inserted in flame to a SalI site located downstream of GST gene in a pGEX6P3 vector (GE Healthcare Japan Corp.) to prepare a vector (hereinafter, referred to as a pGEX6P3-MALT1 vector). Subsequently, E. coli for protein expression (BL21-RIL-codon plus-DE3, Agilent Technologies, Inc.) was transformed with the pGEX6P3-MALT1 vector and then analyzed by ampicillin resistance screening and colony PCR to obtain an E. coli strain expressing recombinant GST fusion MALT1. Protein expression was induced with isopropyl-(3-thiogalactopyranoside. After the expression induction, E. coli precipitates were recovered by centrifugation from the E. coli culture solution, and the E. coli precipitates were homogenized and then centrifuged to obtain a supernatant. The supernatant was purified using GSTrap FF column (GE Healthcare Japan Corp.) to obtain recombinant GST fusion MALT1.B) Evaluation of Inhibition of Protease Activity of MALT1:To 89 μL of an enzyme solution (4.8 g/mL GST fusion MALT1, 50 mmol/L MES, 150 mmol/L NaCl, 10% sucrose, 0.1% CHAPS, 10 mmol/L dithiothreitol, and 1 mol/L tri-ammonium citrate) per specimen, 1 μL of a test compound (DMSO-diluted solution) of each concentration was added to prepare a mixed solution. The mixed solution was incubated at room temperature for 30 minutes, followed by the measurement of the fluorescence value of the mixed solution (fluorescence value of the first measurement) (Ex: 380 nm, Em: 460 nm; Envision (Perkin Elmer Inc.)). Next, 10 μL of 200 μmol/L substrate (Ac-LRSR-AMC, SM Biochemicals LLC) was added (final concentration: 20 μmol/L) to the mixed solution, and the mixture was reacted by incubation at 30° C. for 80 minutes, followed by the measurement of the fluorescence value of the reaction solution (fluorescence value of the second measurement) (Ex: 380 nm, Em: 460 nm; Envision (Perkin Elmer Inc.)).
Measurement: IC50 = 1725 nM (pChEMBL 5.760000228881836)
Descriptors: MW 535.07, ALogP 5.65, HBA 4, HBD 2, PSA 106.67, QED 0.22
Compound (SMILES): Cc1ccccc1-c1ccncc1N(C)C(=O)c1cc(C(F)(F)F)nc(C(F)(F)F)c1
InChIKey: NVFXPYYVZFUJSV-UHFFFAOYSA-N
Target: G-protein coupled bile acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TDU6
Assay: cAMP Assay: CHO-dhfr(minus) cells expressing human GPBAR1 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), lx HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min Compounds were added to a final assay volume of 100 μl and incubated for 30 min at 30° C. The assay was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaked for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH, Hamburg Germany), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The measured signal at 730 nm has to be corrected for the ruthenium background, the direct excitation of Alexa and the buffer control. The FRET signal is calculated as follows: FRET=T730−Alexa730−P(T645−B645) with P=Ru730−B730/Ru645−B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.
Measurement: EC50 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 439.36, ALogP 5.77, HBA 3, HBD 0, PSA 46.09, QED 0.49
Compound (SMILES): C=Cc1nc2nc(NC)ncc2cc1-c1cc(NC(=O)c2ccnc(C(F)(F)F)c2)ccc1C
InChIKey: WJSRNHCWEYXQPY-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Kinase Assay: Protein kinase assay: Assay platform was used to measure kinase/inhibitor interactions as described previously (Anastassiadis et al., 2011). In brief, for each reaction, kinase and substrate were mixed in a buffer containing 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, and 1% DMSO. All compounds were solubilized in DMSO. Compounds were then added to each reaction mixture via acoustic dispense using an ECHO 550 nanoliter dispenser. For human RAF1 testing, human MEK1 (K97R) was used as a substrate at a concentration of 3 micromolar, with a final ATP concentration of 10 micromolar. For human BRAF testing, human MEK1 (K97R) was used as a substrate at 1 micromolar concentration, with a final ATP concentration of 25 micromolar. Compounds were tested in 10-dose IC50 mode with a 3-fold serial dilution starting at 10 micromolar. After a 20-min incubation, ATP (Sigma-Aldrich, St. Louis, Mo. 63178) and [g33P] ATP (specific activity 10 microCi/microliter) purchased at PerkinElmer (Boston, Mass., 02118 Cat #BLU 003H250UC) were added at a final total concentration of 10 mM. Reactions were carried out at room temperature for 2 hr and spotted onto P81 ionexchange cellulose chromatography paper (Reaction Biology). Filter paper was washed in 0.75% phosphoric acid to remove unincorporated ATP. The percent remaining kinase activity relative to a vehicle-containing (DMSO) kinase reaction was calculated for each kinase/inhibitor pair.
Measurement: IC50 < 0.1 nM (pChEMBL 10)
Descriptors: MW 464.45, ALogP 5.35, HBA 6, HBD 2, PSA 92.69, QED 0.41
Compound (SMILES): NCC1(F)CCN(c2cnc3c(Sc4ccccc4Cl)n[nH]c3n2)CC1
InChIKey: QEYBQQBNUXWYMH-UHFFFAOYSA-N
Target: Tyrosine-protein phosphatase non-receptor type 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06124
Assay: Inhibition Assay: SHP2 is allosterically activated through binding of bis-tyrosyl-phorphorylated peptides to its Src Homology 2 (SH2) domains. The latter activation step leads to the release of the auto-inhibitory interface of SHP2, which in turn renders the SHP2 protein tyrosine phosphatase (PTP) active and available for substrate recognition and reaction catalysis. The catalytic activity of SHP2 was monitored using the surrogate substrate DiFMUP in a prompt fluorescence assay format.The inhibition of SHP2 by compounds of the disclosure (concentrations varying from 0.003-100 μM) was monitored using an assay in which 0.25 nM of SHP2 was incubated with of 0.5 μM of peptide IRS1_pY1172(dPEG8)pY1222 (sequence: H2N-LN(pY)IDLDLV(dPEG8)LST(pY)ASINFQK-amide) After 30-60 minutes incubation at 25° C., the surrogate substrate DiFMUP (Invitrogen, cat #D6567, 100 μM final) was added to the reaction and the conversion of DiFMUP to 6,8-difluoro-7-hydroxyl-4-methyl coumarin (DiFMU) was monitored continuously for 10 minutes with excitation at 355 nm and emission at 460 nm using a microplate reader (PolarStar, BMG).
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 392.89, ALogP 3.42, HBA 6, HBD 2, PSA 83.72, QED 0.71
Compound (SMILES): Cc1csc(-c2ccc(C3CNC(=O)N3c3ccc4[nH]cnc4c3)cc2)n1
InChIKey: ARSYWUVMRURROX-UHFFFAOYSA-N
Target: Glutaminyl-peptide cyclotransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q16769
Assay: QC Inhibition Assay: An inhibition activity assay of QC inhibitors was conducted. See Huang et al., J. Biol. Chem. 2011, 286, 12439-12449. A reaction mixture containing 300 μM of Gln-βNA and 0.2 units of human PAP I was prepared. QC was first incubated with an inhibitor at 25° C. for 30 minutes and the enzyme-inhibitor mixture was then added to the reaction mixture to initiate the cyclization reaction. An IC50 value was obtained by fitting an initial reaction rate versus an inhibitor concentration using KaleidaGraph. A Ki value of the inhibitor was calculated according to an equation IC50=Ki(1+[S]/Km). See Segel, Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems, pp. 100-118. New York: John Wiley & Sons, 1993. In this equation, [S] is a substrate (i.e., Gln-βNA) concentration and Km is a Michaelis-Menten constant. The lower the Ki value, the higher the inhibitor's QC inhibition rate.
Measurement: Ki = 100 nM (pChEMBL 7)
Descriptors: MW 375.46, ALogP 4.27, HBA 4, HBD 2, PSA 73.91, QED 0.56
Compound (SMILES): CCCNC(=O)CO[C@H](c1ccccc1)[C@H](CNC)c1ccc2ccccc2c1
InChIKey: IIMALBJVIZZYSR-ILBGXUMGSA-N
Target: Sodium-dependent noradrenaline transporter (SINGLE PROTEIN), Homo sapiens
UniProt: P23975
Assay: Uptake Assay Protocol: NET: This protocol was designed to measure inhibition of uptake by the human norepinephrine transporter. The reagents were human NET (HEK293F) cells, desipramine (Sigma), nomifensine, neurotransmitter transporter uptake assay kit (Molecular Devices), freestyle 293 expression medium (Invitrogen), 10× Hank's Balanced Salt Solution (HBSS; Invitrogen), 1 M HEPES (Mediatech), Biocoat poly-D-lysine 96-well, black, clear plates (Becton, Dickinson), and 500 μL polypropylene U-bottom 96-well plates (Fisher). The Assay Buffer (AB) was 1×HBSS and 0.02 M HEPES.The HEK293F cells were transfected with the human norepinephrine transporter and frozen in 1 mL aliquots at about 1E+07 cells/mL. On the day of the experiment, the cells were removed from −80° C. or liquid nitrogen and thawed in a room temperature water bath. The cells were dilute to about 1-2E+06 with Freestyle medium. A 1 mL sample (1:2 dilution) was prepared, and the cells were counted. The cells were spun at 1100 rpm for 5 minutes, and the medium was aspirated off. The cells were resuspend in medium at 1.5E+06 cells/mL for about 60,000 cells per well. 40 μL of cells were dispensed per well in the Biocoat plates. The plates were spun at 1100 rpm for 1 minute to improve homogeneity of the cell layer and were incubated at 37° C. for a minimum of 3 hours.
Measurement: Ki = 2.4 nM (pChEMBL 8.619999885559082)
Descriptors: MW 390.53, ALogP 4.43, HBA 3, HBD 2, PSA 50.36, QED 0.54
Compound (SMILES): CCOC(=O)C1(Cn2c(=O)[nH]/c(=N\c3ccc(OCC4CC4)cc3)n(Cc3ccc(C)cc3)c2=O)CC1
InChIKey: ZDOCCHXRYOAMKV-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Human P2X3 Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 10% BSA, and 0.08% Pluronic F-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), each well was added with 40 μL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.). Measurement of fluorescence intensity by FDSS 3000 was started, and 40 μl, of DMSO solutions containing different concentrations of the test, compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1.% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 50 nM ATP solution (50 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 3 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement. The 50% inhibitory concentration (IC50) was calculated under the assumption that the specific maximum fluorescence intensity without test compound is 0% inhibition and that the specific maximum fluorescence intensity when the dilution buffer was added in place of ATP solution is 100% inhibition, to evaluate the inhibitory activity of the test compound. FDSS software (Hamamatsu Photonics K.K.) was used for calculation of the specific maximum fluorescence intensity. IC50 was calculated using Microsoft Excel (Microsoft Corporation) and Ltd.).
Measurement: IC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 504.59, ALogP 3.06, HBA 8, HBD 1, PSA 107.68, QED 0.43