lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC1C2CC(Oc3ccc(C(F)(F)F)cn3)C(C2)N1C(=O)c1c(F)cccc1-n1nccn1
InChIKey: UOPGXAOIEPNGAF-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 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 461.42, ALogP 3.89, HBA 6, HBD 0, PSA 73.14, QED 0.55
Compound (SMILES): Cc1ccc2c(-c3nc(Nc4cc(C5CC5)cc(CN5C[C@@H](C)N[C@@H](C)C5)c4O)ncc3Cl)c[nH]c2c1
InChIKey: MNPVYGOUSLAGOL-HDICACEKSA-N
Target: Receptor-type tyrosine-protein kinase FLT3 (SINGLE PROTEIN), Homo sapiens
UniProt: P36888
Assay: Evaluation of Kinase Inhibitory Activity: The inhibitory activity of some of the compounds described above against FLT3-ITD, FLT3 wild type (WT), VEGFR2 (KDR), and SYK kinase was measured. The inhibitory activity of the compounds against mutant proteins, such as FLT3 VVT and ITD, was evaluated based on the LanthaScreen technology developed by Thermo Fisher Scientific Inc. This assay is based on the binding of Alexa Fluor® 647-labeled, ATP-competitive kinase inhibitor (kinase tracer-236) to kinase, and signals of fluorescence resonance energy transfer (FRET) were measured in the presence of europium-conjugated antibody. This experiment was carried out on a 384-well plate in conditions including 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA, and 1% DMSO. After measuring the background signal in the absence of proteins such as FLT3 or FLT3-ITD and measuring the non-inhibition signal by adding only the solvent (1% DMSO), an evaluation compound was used at a set concentration (for example, 50 nM to 0.05 nM, 1:10 dilution) to calculate the kinase inhibitory activity of the evaluation compound as IC50.
Measurement: IC50 = 0.4 nM (pChEMBL 9.399999618530273)
Descriptors: MW 517.08, ALogP 6.1, HBA 6, HBD 4, PSA 89.1, QED 0.23
Compound (SMILES): OC[C@H]1O[C@@H](c2ncnn2-c2ccc(C(F)(F)F)cc2)[C@H](O)[C@@H](n2cc(-c3cc(F)c(F)c(F)c3)nn2)[C@H]1O
InChIKey: CIKNZMVBCFCBNN-OBJCFNGXSA-N
Target: Galectin-3 (SINGLE PROTEIN), Homo sapiens
UniProt: P17931
Assay: Biological Assay: The Gal-3 assays were performed in 384 white Opti plates in three replicates at room temperature with gentle shaking at 250-300 rpmFrom the original stocks, 2.525× working stock concentrations of His-tagged recombinant human Gal-3 (hGal-3) and that of B-ASF were prepared. From the working stock, 20 μl of hGal-3 (15 nM) and 20 μl B-ASF (15 nM) were added to the plates. In Negative Control, only hGal-3 was added. A concentration range of 50× working stocks were prepared for the compounds in 100% DMSO. Aliquots of 1 uL of the compounds were added to the wells and pre-incubated with 20 μl hGal-3 per well for 30 minutes Then 20 μl B-ASF wereadded and incubated for another 1 hour. To detect the signal, 5 μL (final conc. of 1.0 nM) terbium labelled Anti-His antibody was added and incubated for 30 min followed by adding 5 μL (final conc. of 20 nM) Streptavidin d2 and incubation for another 1 hour. The assay signal was detected using HTRF screen protocol (Excitation wavelength=340 nm, emission wavelength=615 nm/665 nm) on Envision 2104 Multilabel Reader. Data analysed using Toolset and Curve Master. Results are reported in the experimental section (IC50 in μM).
Measurement: IC50 = 560 nM (pChEMBL 6.25)
Descriptors: MW 556.42, ALogP 2.36, HBA 10, HBD 3, PSA 131.34, QED 0.25
Compound (SMILES): C#C[C@@]1(O)[C@@H](CO[C@H](C(=O)O)c2csc(-c3ccccc3)n2)O[C@@H](n2cnc3c(N)nc(Cl)nc32)[C@@H]1O
InChIKey: RBAYPVRBAXLAQI-MEFXRUIVSA-N
Target: 5'-nucleotidase (SINGLE PROTEIN), Homo sapiens
UniProt: P21589
Assay: Inhibition of the CD73 Enzyme In Vitro: For measurements of soluble CD73 enzyme activity, recombinant CD73 was obtained from R&D Systems, Cat. No. 5795-EN-010. Serial dilutions of test compounds were incubated with recombinant CD73 and AMP in reaction buffer (25 mM Tris HCl pH7.5, 5 mM MgCl2, 50 mM NaCl, 0.25 mM DTT, 0.005% Triton X-100). The final reaction volume was 25 μL and the final concentrations of recombinant CD73 and AMP were 0.05 nM and 50 μM, respectively. Reactions were allowed to proceed for 1 hour at 37° C. before the addition of 100 μL Malachite Green (Cell Signaling Technology, Cat. No. 12776). After 5 minutes at room temperature, absorbance at 630 nm was determined on a microplate spectrophotometer. The concentration of inorganic phosphate was determined using a phosphate standard curve.
Measurement: IC50 = 263 nM (pChEMBL 6.579999923706055)
Descriptors: MW 542.96, ALogP 1.65, HBA 12, HBD 4, PSA 178.73, QED 0.2
Compound (SMILES): CC(C)(F)N1CCC(n2cc(-c3ccc4[nH]nc(C(=O)Nc5ccc(OC(F)F)cc5)c4c3)cn2)CC1
InChIKey: WAXDUCPCAWNAIN-UHFFFAOYSA-N
Target: Dual specificity tyrosine-phosphorylation-regulated kinase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: Q13627
Assay: Kinase Assay: DYRK1A: Each compound was dissolved in DMSO as a 10 mM stock and used to prepare compound source plates. Serial dilution (1:3, 11-point dose-response curves from 10 M to 0.00016 M) and compound transfer was performed using the ECHO 550 (Labcyte, Sunnyvale, Calif.) into 1536-well black-walled round bottom plates (Corning).The DYRK1A kinase assay was run using the Ser/Thr 18 peptide Z-lyte assay kit according to manufacturer's instructions (Life Technologiesa Division of Thermo-Fisher). This is a non-radioactive assay using fluorescence resonance energy transfer (FRET) between coumarin and fluorescein to detect kinase activity which is represented as a ratio of coumarin emission/fluorescein emission.Briefly, recombinant DYRK1A kinase, ATP and Ser/Thr peptide 18 were prepared in 1× Kinase buffer to final concentrations of 0.19 μg/mL, 30 μM, and 4 μM respectively. The mixture was allowed to incubate with the representative compounds for one hour at room temperature. All reactions were performed in duplicate. Unphosphorylated (0% Control) and phosphorylated (100% control) forms of Ser/Thr 18 served as control reactions. Additionally, an 11-point dose-response curve of Staurosporine (1 uM top) was run to serve as a positive compound control.After incubation, Development Reagent A was diluted in Development Buffer then added to the reaction and allowed to further incubate for one hour at room temperature. The plate was read at Ex 400 Em 455 to detect the coumarin signal and Ex 400 Em 520 to measure the signal (EnVision Multilabel Plate Reader, PerkinElmer).
Measurement: EC50 = 9940 nM (pChEMBL 5)
Descriptors: MW 512.54, ALogP 5.62, HBA 6, HBD 2, PSA 88.07, QED 0.31
Compound (SMILES): Nc1ncnc2c1c(-c1ccc(Oc3c(F)c(F)cc(F)c3F)cc1F)nn2[C@@H]1CCN(C(=O)CCCl)C1
InChIKey: WWVKQKZDYDOYRS-LLVKDONJSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: HTRF Assay: The enzyme reaction mixture of BTK wild type standard HTRF assay contained 1 nM BTK wild type, 1 μM biotin-TK1 peptide, and 30 μM ATP in a buffer. The enzyme reaction were carried out at room temperature for 60 minutes. 5 μl of 0.2 M EDTA were added to quench the reaction and then the inhibitors (5 μl) were added at final concentrations of 2 nM antibody and 62.5 nM XL665. The plates were incubated at room temperature for 60 minutes and then read in the Envision plate reader. The readouts were transformed into inhibition rate % by the equation of (Min Ratio)/(Max-Min)*100%. Hence the 1050 data of test compounds were generated by using four parameters curve fitting.
Measurement: IC50 = 148 nM (pChEMBL 6.829999923706055)
Descriptors: MW 552.89, ALogP 4.97, HBA 7, HBD 1, PSA 99.16, QED 0.21
Compound (SMILES): Cc1nc2c(F)cc(-c3nc(Nc4ccc5c(n4)CCN(C(=O)[C@@H](C)N(C)C)C5)ncc3F)cc2n1C(C)C
InChIKey: SSIOYGMEKCPFAV-MRXNPFEDSA-N
Target: Cyclin-dependent kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P11802
Assay: LANCE Ultra ULight Assay: 1. Take 10 mM stock solution of the test compound, in 96-well compound plate, DMSO was used to dilute the compound to an initial concentration of 100x, then this concentration was used as the first concentration, 3-fold diluted to make 10 serial concentrations; 1 μL each serial dilution was then added to 19 μL 1x reaction buffer to prepare 5x compound for use; 2 μL 5x compound was transferred from 96-well plate to 384-well plate; compound-free control well was added with 2 μL the following liquid: 1x reaction buffer with the addition of 1 μL DMSO; 2 μL 250 mM EDTA was added to the Min control well.2.1x reaction buffer was used to formulate the kinase, substrate and ATP into a 2.5xenzyme/substrate mixture and 2.5xATP solution respectively. In the experiment, the final concentration of CDK4/CycD3 kinase is 0.76 ng/μL, the final concentration of ATP is 8 μM; the final concentration of CDK6/CycD3 kinase is 0.5 ng/μL, the final concentration of ATP is 50M; the final concentration of CDK2/CycA2 kinase is 0.86 ng/μL, the final concentration of ATP is 1 μM; the final concentration of CDK2/CycE1 kinase is 1.016 ng/μL, the final concentration of ATP is 20M; 2.5xenzyme/substrate mixture was added to a 384-well plate, incubated at room temperature for 5 minutes; then added with 2.5xATP solution, reacted at room temperature for 30 minutes.3. LANCE Detection Buffer was used, 1xto prepare 2xLANCE Ultra Europium-anti-phospho-eIF4E-binding protein 1 (Thr37/46) for use. After the enzymatic reaction was continued for 30 minutes, 10 mM EDTA was added to 384-well plate and the mixture was reacted at room temperature for 5 minutes. Then LANCE Ultra Europium-anti-phospho-eIF4E-binding protein 1 (Thr37/46) was added, reacted at room temperature for 1 hour.4. The 384-well plate was placed in HERAEUS Multifuge X1R centrifuge, centrifuged at 2000 rpm for 2 minutes; data were measured on EnVision, 337 nM wavelength laser was used as the excitation light, test at RFU665 nM and RFU615 nM, and RFU665 nM/RFU615 nM x10000 was used as the final data for analysis.
Measurement: IC50 = 1.44 nM (pChEMBL 8.84000015258789)
Descriptors: MW 534.62, ALogP 4.63, HBA 8, HBD 1, PSA 92.07, QED 0.39
Compound (SMILES): COc1cc2c(cc1OC)[C@H]1C[C@@H](COC(=O)[C@H](N)c3ccc(C(F)(F)F)cc3)[C@H](CC(C)C)CN1CC2
InChIKey: AMJUIJNHZRYMOD-MIDQIFMJSA-N
Target: Synaptic vesicular amine transporter (SINGLE PROTEIN), Homo sapiens
UniProt: Q05940
Assay: Determining human Vmat2 Inhibitory Activity of a Compound: The human Ki's for the compounds listed in Table 11 were determined using a slightly modified procedure shown below (see data in column under the heading Ki nM). In a total volume of 0.15 mL in low-binding 96-well plates (Corning #3605), twelve concentrations of Compound 1-1 and R,R,R-DHTBZ were competed against 10 nM 3H-dihydrotetrabenezine (American Radiolabeled Chemicals, Kd 2.6 nM) on rat forebrain homogenate (100 μg membrane protein per well) or human platelet homogenate (15 μg membrane protein per well) in VMAT2 binding buffer (Dulbecco's phosphate buffered saline, 1 mM EDTA, pH 7.4). Following incubation at 25° C. for 90 minutes, bound radioligand was collected by rapid filtration onto GF/B glass fiber filters using a Unifilter-96 Harvester (PerkinElmer). Filter plates were pre-treated with 0.1% polyethylenimine and allowed to dry overnight, and following harvesting the filter plates were washed with 800 d VMAT2 binding buffer. Bound radioligand was quantified by scintillation counting using a Topcount NXT (PerkinElmer).
Measurement: Ki = 260 nM (pChEMBL 6.579999923706055)
Descriptors: MW 534.62, ALogP 5.55, HBA 6, HBD 1, PSA 74.02, QED 0.45
Compound (SMILES): CC(C)(O)[C@H](F)CN1Cc2cc(NC(=O)c3ccc[nH]c3=O)c(N3CCOCC3)cc2C1=O
InChIKey: KQUREFWMCLWQLR-LJQANCHMSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Transcreener-Fluorecescence Polarization Assay: Kinase activities were assayed using the Transcreener-Fluorecescence polarization platform (BelBrook Labs, Madison, Wis., USA) that measures amounts of the reaction product, ADP. The IRAK4 reaction conditions were optimized using an IRAK1-derived peptide (sequence H-KKARFSRFAGSSPSQSSMVAR) to provide a linear reaction rate over the course of a 90 min incubation, which resulted in 10-12% conversion of the starting ATP to ADP. Final IRAK4 assay conditions were 1.25 nM IRAK4; 125 μM ATP; 10 μM MgCl2; 125 μM peptide in reaction buffer (25 mM HEPES (pH7.4); 2 mM Dithiothreitol; 0.015% Brij-35; and 0.5% dimethyl sulfoxide. The IRAK1 activity was optimized similarly, yielding final assay conditions of 1.5 nM IRAK1; 62.5 μM ATP; 5 μM MgCl2, and 62.5 μM IRAK1 peptide in reaction buffer for 60 min.
Measurement: Ki = 8.2 nM (pChEMBL 8.09000015258789)
Descriptors: MW 458.49, ALogP 1.53, HBA 6, HBD 3, PSA 114.97, QED 0.6
Compound (SMILES): CCCCNc1ncc(C(=O)Nc2ccc(S(=O)(=O)N3CCOCC3)cc2)c(NCCCOC)n1
InChIKey: XPFLTTSBMVPAGF-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: Activity Assay: Briefly, activity assays were performed in a 384 well, polypropylene microplate in a final volume of 50 μL of 50 mM Hepes, Ph 7.4 containing 10 mM MgCl2, 1.0 mM DTT, 0.01% Triton X-100, 0.1% Bovine Serum Albumin (BSA), containing 1.0 μM fluorescent substrate and ATP at the Km for each enzyme. All reactions were terminated by addition of 20 μL of 70 mM EDTA. After a 180 min incubation, phosphorylated and unphosphorylated substrate peptides were separated in buffer supplemented with 1×CR-8 on a LabChip EZ Reader equipped with a 12-sipper chip. Data were analyzed using EZ Reader software. Assay conditions for MCE assays
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 506.63, ALogP 2.41, HBA 9, HBD 3, PSA 134.78, QED 0.35
Compound (SMILES): C=CC(=O)N(C)CCC1CCN(C(=O)N(C)[C@H](C(=O)N[C@H]2Cc3nc(cs3)-c3ccc4c(c3)c(c(-c3cccnc3[C@H](C)OC)n4CC)CC(C)(C)COC(=O)[C@@H]3CCCN(N3)C2=O)C(C)C)CC1
InChIKey: GOVKLQHDTFLFSF-HJAGXSRZSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Biochemical Assay: A variety of Ras proteins may be inhibited by compounds of the present invention (e.g., K-Ras, N-Ras, H-Ras, and mutants thereof at positions 12, 13 and 61, such as G12C, G12D, G12V, G12S, G13C, G13D, and Q61L, and others described herein). The purpose of this biochemical assay is to measure the ability of test compounds to covalently label nucleotide-loaded K-Ras isoforms. In assay buffer containing 12.5 mM HEPES pH 7.4, 75 mM NaCl, 1 mM MgCl2, 1 mM BME, 5 uM Cyclophilin A and 2 uM test compound, a 5 uM stock of GMP-PNP-loaded K-Ras (1-169) G12C is diluted 10-fold to yield a final concentration of 0.5 uM; with final sample volume being 100 uL. The sample is incubated at 25�� C. for a time period of up to 24 hours prior to quenching by the addition of 10 uL of 5% Formic Acid. Quenched samples are centrifuged at 15000 rpm for 15 minutes in a benchtop centrifuge before injecting a 10 uL aliquot onto a reverse phase C4 column and eluting into the mass spectrometer with an increasing acetonitrile gradient in the mobile phase. Analysis of raw data may be carried out using Waters MassLynx MS software, with % bound calculated from the deconvoluted protein peaks for labeled and unlabeled K-Ras.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 966.26, ALogP 7.03, HBA 12, HBD 2, PSA 171.54, QED 0.11
Compound (SMILES): CN(C)c1ccc(C2c3nnnn3CCN2Cc2ccccc2F)cc1
InChIKey: RGVZVZGJLPTNBS-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: TR-FRET Assay: In this assay, a GST-tagged human RORγ ligand binding domain (GST-hRORγ-LBD) interacts with a synthetic biotinylated TRAP220 cofactor peptide containing an LXXLL motif (amino acids 631-655 from NP_004765). The ligand-binding domain (LBD) of RORγ is expressed as fusion protein with GST in BL-21 (BL3) cells using the vector pDEST15. Cells are lysed by lysozyme-treatment and sonication, and the fusion proteins purified over glutathione sepharose (Pharmacia) according to the manufacturers instructions. The strong constitutive interaction is disrupted upon binding of functional antagonists. The strength of the interaction is monitored by TR-FRET between streptavidine APC interacting with the biotinylated peptide and Europium-labelled Anti-GST. 6 μl RORγ-LBD-GST solution (final 8.7 nM) in assay buffer (20 mM Tris-HCl, pH 6.8, 5 mM MgCl2, 60 mM KCl, 0.1% delipidated BSA, 1 mM DTT) are dispensed into black 384 well small volume plates (Greiner #784076). 6 μl TRAP220 peptide (final 400 nM), SA-APC (final 1.6 ng/μl), Eu-anti-GST (final 0.125 ng/μl) in assay buffer and 2 μl test compounds (DMSO stocks prediluted in 20 mM Tris-HCl, pH 6.8, 5 mM MgCl2, 60 mM KCl) are added and the mixture incubated 60 min at RT in the dark before measuring FRET (excitation 337 nm, emission at 615 and 665 nm).
Measurement: IC50 = 5445 nM (pChEMBL 5.260000228881836)
Descriptors: MW 352.42, ALogP 2.48, HBA 6, HBD 0, PSA 50.08, QED 0.72
Compound (SMILES): CC(C)(O)[C@H](F)CNC(=O)c1cnc(Nc2ncc(C#N)cc2F)cc1Nc1cccc(O)c1
InChIKey: PYTKVZXEXIRJQY-LJQANCHMSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 μL prepared from 15 μL additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij 35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction mixture was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 3.6 nM (pChEMBL 8.4399995803833)
Descriptors: MW 468.46, ALogP 3.52, HBA 8, HBD 5, PSA 143.19, QED 0.34
Compound (SMILES): CSc1cnc(N[C@H]2CC[C@H](Nc3ccc(-n4cnccc4=O)cn3)C2)nc1
InChIKey: ODSLDYCZVOWWHV-KBPBESRZSA-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 395.49, ALogP 2.58, HBA 9, HBD 2, PSA 97.62, QED 0.61
Compound (SMILES): Cc1cc(C(C)Nc2ncc(-c3noc(C(F)(F)F)n3)cn2)ccn1
InChIKey: OMPHAFHZEBBOGW-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: HDAC1 Assay: Human recombinant full length HDAC1 expressed in a baculovirus expression system was purchased from BPS BioSciences (San Diego, Calif., U.S.A.). The substrate used in the HDAC1 assay was 5 μM of acetyl-Gly-Ala-Lys(acetyl)-AMC.
Measurement: IC50 = 1600 nM (pChEMBL 5.800000190734863)
Descriptors: MW 350.3, ALogP 3.42, HBA 7, HBD 1, PSA 89.62, QED 0.77
Compound (SMILES): CN(CCOCCO)Cc1ccc(-c2ccc(-c3cc4nc(O[C@@H]5CO[C@H]6[C@@H]5OC[C@H]6O)[nH]c4cc3Cl)cc2)cc1
InChIKey: BZHBIBPBNWVKAG-SKKKGAJSSA-N
Target: AMP-activated protein kinase (AMPK) alpha-1/beta-1/gamma-1 (PROTEIN COMPLEX), Homo sapiens
UniProt: Q13131
Assay: In Vitro pAMPK1 Kinase Activation Assay: Compound effect on AMPK enzyme activation was determined in a cell-free format with a 12-point concentration curve. The ADP-Glo detection system was used to determine phosphorylation of a SAMS peptide substrate. Recombinant AMPK α1/β1/γ1 complex was pre-activated by phosphorylation with CAMKK2 followed by incubated with compound for 15 minutes prior to the SAMS phosphorylation reaction. Activity curves and EC50 values were fitted by interpolation to an ATP:ADP standard curve as indicated by the ADP-Glo manufacturer using Prism software.
Measurement: EC50 = 1.3 nM (pChEMBL 8.890000343322754)
Descriptors: MW 580.08, ALogP 3.9, HBA 8, HBD 3, PSA 109.3, QED 0.23
Compound (SMILES): N#Cc1cc(-c2ncnc(Nc3ccc(N4CCN(C5COC5)CC4)cc3)n2)ccc1OCCC1CNC(=O)C1
InChIKey: ZKFZDTFRAIIRBT-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: TBK1 and IKKE Biological Assays: Enzymatic activity of IKKε and TBK1 was measured using a homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay that monitors enzyme dependent phosphorylation of a biotinylated serine/threonine peptide substrate. An increase in the amount of phosphorylated peptide results in an increase in TR-FRET signal. TBK1 and IKKε were expressed and purified as full length recombinant proteins. Detection reagents for the assay were purchased from Cisbio. TBK1 and IKKε enzymes were assayed under initial rate conditions in the presence of 2×Km ATP (40-80 μM) and 1 μM peptide, hepes (pH 7), 0.1 mM orthovanadate, 0.02% NaN3, 0.01% BSA, 10 mM MgCl2, 0.01% (v/v) tritonX, 1 mM dithiothreitol, 0.5% (v/v) DMSO at the following concentrations for each enzyme: TBK1 at 2.5 nM and IKKε at 0.3 nM. After an assay reaction time of 240 minutes at 25° C., reactions were terminated with EDTA.Amount of phosphorylated peptide was determined by the addition of 125 nM streptavidin XL665 and europium cryptate labeled anti-phospho monoclonal antibody and the resulting TR-FRET signal was recorded on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 200 μs read window). Data was normalized based on a positive (1 μM Staurosporine) and negative (DMSO) controls and IC50 values calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations.
Measurement: IC50 = 95000 nM (pChEMBL 4.019999980926514)
Descriptors: MW 540.63, ALogP 2.58, HBA 10, HBD 2, PSA 128.53, QED 0.42
Compound (SMILES): Cn1nc2ccc(-c3c[nH]c4nc(N5[C@@H]6CC[C@H]5C[C@@H](N)C6)cnc34)c(Cl)c2c1C#N
InChIKey: JRHIFWQYUNDUMP-CLLJXQQHSA-N
Target: Tyrosine-protein phosphatase non-receptor type 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06124
Assay: Biochemical Assay: SHP2 activity was monitored by measuring the conversion of the surrogate substrate 6,8-difluoromethylumbelliferyl phosphate (DiFMUP) to the fluorescent product, 6,8-difluoromethylumbelliferone (DiFMU).SHP2 was pre-incubated with test compounds and the activating peptide pIRS1 (H2N-LN(pY)IDLDLV-(PEG)8-LST(pY)ASINFQK-amide) for 30 min, prior to addition of the 6,8-difluoromethylumbelliferyl phosphate (DiFMUP), (Thermo Fisher D6567). Final assay concentrations were 10 μM SHP2, 0.25 μM pIRS1 peptide, 50 μM DiFMUP, 25 mM Bis-Tris propane, pH 7.0, 150 mM NaCl, 0.05% (v/v) Tween-20, 0.5 mM TCEP and 5% (v/v) DMSO. Rates of reaction were then measured over 30 min by monitoring fluorescence on a BMG Pherastar reader at excitation 360 nm/emission 450 nm. IC50 values were calculated from the normalized dose-response plots using four parameter logistic curve fit.
Measurement: IC50 = 7.1 nM (pChEMBL 8.149999618530273)
Descriptors: MW 432.92, ALogP 3.5, HBA 7, HBD 2, PSA 112.44, QED 0.5
Compound (SMILES): Cc1cnn2ncn(Cc3nc([C@H]4[C@@H]5CN(c6ccccc6)C[C@@H]54)no3)c(=O)c12
InChIKey: IAWHXDCOTSCDSA-QLPKVWCKSA-N
Target: Transient receptor potential cation channel subfamily A member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75762
Assay: FLIPR Assay (15 minutes): The IC50 (effective concentration) of compounds on the human TRPA1 channel was determined using a FLIPR Tetra instrument. CHO cells expressing TRPA1 were plated into 384-well plates, incubated overnight at 37° C., and loaded with BD calcium indicator dye for 1 hr at 37° C. followed by 15 minutes at room temperature. The assay buffer was Hank's Balanced Salt Solution (HBSS) containing 20 mM HEPES (pH readjusted to 7.4) along with 0.02% BSA.Following dye load and plate cool down, compounds were added to the cells using FLIPR Tetra. Plates were then incubated with compounds for 10 minutes or 90 minutes at room temperature prior to adding agonist. Following this incubation, about an EC50 concentration of cinnamaldehyde (75) was added to active the channels and block of cinnamaldehyde induced calcium influx was measured.The IC50 results were fit with a standard Hill function, keeping the Hill coefficient (n) fixed to 1.5. Fixing the Hill coefficient will generally reduce variability of the IC50 determination. The IC50 results were individually examined to make sure the MIN and MAX points were set correctly prior to validation of the results.
Measurement: IC50 = 8.11 nM (pChEMBL 8.09000015258789)
Descriptors: MW 389.42, ALogP 1.48, HBA 9, HBD 0, PSA 94.35, QED 0.52
Compound (SMILES): Cc1cc(=O)c2c(OCCNS(C)(=O)=O)ncc(Cl)c2n1-c1c(Cl)cccc1Cl
InChIKey: YBFIRRLWTMWYQM-UHFFFAOYSA-N
Target: Kir3.1/Kir3.4 (PROTEIN COMPLEX), Homo sapiens
UniProt: P48544
Assay: IonWorks Quattro system: Quattro is controlled using IonWorks v2 software to perform the following steps:a. Add 3.5 ul cells plus 3.5 ul external buffer to wells of Quattro Patch Plateb. Circulate amphotericin B and internal buffer onto cellsc. Apply the following voltage protocol: Pulse 1: 15 mV for 300 milliseconds (ms), followed by Pulse 2: −120 mV for 400 ms, then Pulse 3: −15 mV for 400 ms, and finally Pulse 4: −120 mV to 40 mV over 500 ms (this is a voltage ramp).d. Measure magnitude of inward potassium current at time point between 1200-1220 ms from start of Pulse 1 (i.e. during the voltage ramp phase).e. Add 3.5 ul of diluted compounds (or DMSO) to wells and repeat steps c-d (final compound concentrations are 50 uM to 0.1 uM, for potent compounds 0.5 uM to 0.01 uM and each concentration is tested in quadruplicatei.e. in 4 separate wells).f. The difference between current magnitude pre vs. post-compound gives a measure of GIRK1/4 inhibition.7. Data analysis:IC50 values are calculated by plotting the percentage of current inhibition (normalized to the DMSO-only control) as a function of compound concentration using standard data analysis software.
Measurement: IC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 476.77, ALogP 3.58, HBA 6, HBD 1, PSA 90.29, QED 0.55
Compound (SMILES): CCOc1cc(-c2ccc(N3CCN(C(=O)C(CN)c4ccc(F)cc4)CC3)nc2)c2c(C#N)cnn2c1
InChIKey: RVLYHUHKAJONLN-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 = 39.3 nM (pChEMBL 7.409999847412109)
Descriptors: MW 513.58, ALogP 3.2, HBA 8, HBD 1, PSA 112.78, QED 0.4
Compound (SMILES): COCCn1nc(C(=O)NC[C@]2(O)CC[C@@H](S(C)(=O)=O)CC2)c(C)c1-c1ccc(CC(C)(C)C(F)(F)F)cc1OC(F)F
InChIKey: FXUAMEFLRIFASB-JKCYLYBBSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor Assay: ThermoFluor® experiments were carried out using instruments owned by Janssen Research and Development, L.L.C. through its acquisition of 3-Dimensional Pharmaceuticals, Inc. 1,8-ANS (Invitrogen) was used as a fluorescent dye. Protein and compound solutions are dispensed into black 384-well polypropylene PCR microplates (Abgene) and overlayed with silicone oil (1 μL, Fluka, type DC 200) to prevent evaporation. Bar-coded assay plates are robotically loaded onto a thermostatically controlled PCR-type thermal block and then heated at a typical ramp-rate of 1° C./min for all experiments. Fluorescence was measured by continuous illumination with UV light (Hamamatsu LC6) supplied via fiber optic and filtered through a band-pass filter (380-400 nm; >6 OD cutoff). Fluorescence emission of the entire 384-well plate was detected by measuring light intensity using a CCD camera (Sensys, Roper Scientific) filtered to detect 500±25 nm, resulting in simultaneous and independent readings of all 384 wells. Images were collected at each temperature, and the sum of the pixel intensity in a given area of the assay plate was recorded versus temperature.
Measurement: Kd = 1.9 nM (pChEMBL 8.720000267028809)
Descriptors: MW 639.68, ALogP 4.69, HBA 8, HBD 2, PSA 119.75, QED 0.32
Compound (SMILES): Cc1oc2ccc(/N=c3\[nH]c(=O)n(CC4COC(C)(C)OC4)c(=O)n3Cc3ccc(Cl)cc3)cc2c1C
InChIKey: COTYBQCSYIKFCP-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 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 525.01, ALogP 4.03, HBA 8, HBD 1, PSA 103.75, QED 0.42
Compound (SMILES): Cc1cccc(C)c1Nc1n[nH]c2nc(Nc3ccc4c(c3)CNCC4)ncc12
InChIKey: IZTDIATUHNDTOK-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Inhibitory Assay: BTK enzyme evaluation with each compound of examples of the invention was performed by using BTK enzyme inhibition diagnosis kit (Cisbio, Codolet, France). ATP (adenosine triphosphate), BTK, peptide (biotin-Aca-AAAEEIYGEI-NH2) and each compound of examples of the invention were mixed, followed by reaction for 30 minutes. Then, EDTA (ethylenediaminetetraacetic acid) was added thereto in order to terminate the reaction. At this time, the EDTA solution contained curopium-containing antibody (antiphosphoresidue antibody) and straptavidin-XL665 (SA-XL665, Cisbio). After incubation for 1 hour, fluorescence was measured. The emission values of 665 nm and 620 nm excited at 337 nm were measured with Envision reader.
Measurement: IC50 = 0.3 nM (pChEMBL 9.520000457763672)
Descriptors: MW 385.48, ALogP 4.1, HBA 6, HBD 4, PSA 90.55, QED 0.42
Compound (SMILES): C[C@@H]1C[C@@H](NC(=O)c2cc(C3CC3)on2)CCN1S(=O)(=O)CC(F)(F)F
InChIKey: WQRMRNDOWRYWNZ-KOLCDFICSA-N
Target: Histone-lysine N-methyltransferase SMYD3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H7B4
Assay: SMYD3 Biochemical Assay: The assays were all performed in a buffer consisting of 25 mM Tris-Cl pH 8.0, 1 mM TCEP, 0.005% BSG, and 0.005% Tween 20, prepared on the day of use. Compounds in 100% DMSO (1 ul) were spotted into a 384-well white opaque OptiPlate using a Bravo automated liquid handling platform outfitted with a 384-channel head (Agilent Technologies). DMSO (1 ul) was added to Columns 11, 12, 23, 24, rows A-H for the maximum signal control and 1 ul of SAH, a known product and inhibitor of SMYD3, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control. A cocktail (40 ul) containing the SMYD3 enzyme was added by Multidrop Combi (Thermo-Fisher). The compounds were allowed to incubate with SMYD3 for 30 min at room temperature, then a cocktail (10 ul) containing SAM and MEKK2 was added to initiate the reaction (final volume=51 ul). The final concentrations of the components were as follows: SMYD3 was 0.4 nM, 3H-SAM was 8 nM, MEKK2 was 12 nM, SAH in the minimum signal control wells was 1 mM, and the DMSO concentration was 2%. The assays were stopped by the addition of non-radiolabeled SAM (10 ul) to a final concentration of 100 uM, which dilutes the 3H-SAM to a level where its incorporation into MEKK2 is no longer detectable. Radiolabeled MEKK2 was detected using a scintillation proximity assay (SPA). 10 uL of a 10 mg/mL solution of SPA beads in 0.5 M citric acid was added and the plates centrifuged at 600 rpm for 1 min to precipitate the radiolabeled MEKK2 onto the SPA beads. The plates were then read in a PerkinElmer TopCount plate reader to measure the quantity of 3H-labeled MEKK2 as disintegrations per minute (dpm) or alternatively, referred to as counts per minute (cpm).% Inhibition Calculation% ⁢ ⁢ inh = 100 - ( dpm cmpd - dpm min dpm max - dpm min ) × 100Where dpm=disintegrations per minute, cmpd=signal in assay well, and min and max are the respective minimum and maximum signal controls.Four-Parameter IC50 FitY = Bottom + ( Top - Bottom ) ( 1 + ( X IC 50 ) Hill ⁢ ⁢ CoefficientWhere top and bottom are the normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit. The Hill Coefficient normally allowed to float but may also be fixed at 1 in a 3-parameter fit. Y is the % inhibition and X is the compound concentration.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 395.4, ALogP 2.03, HBA 5, HBD 1, PSA 92.51, QED 0.82
Compound (SMILES): CC(C)(OC(=O)Nc1cc(F)c2c(c1)N(S(=O)(=O)c1ccc(F)c(C(F)(F)F)c1)C[C@H](CCC(=O)O)O2)C(F)(F)F
InChIKey: NLUQNRDMQNGGFY-LBPRGKRZSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Biochemical TR-FRET Assay: To identify novel antagonists of RORgammaT, an assay was developed which employs the interaction of RORgammaT with its co-activator peptide SRC1_2. This peptide mimics the recruitment of co-activators to RORgammaT through its interaction with the LXXLL (SEQ ID NO:1) (e.g., NR box) motifs (Xie et al., J. Immunol. 175: 3800-09, 2005; Kurebayashi et al., Biochem. Biophys. Res. Commun. 315: 919-27, 2004; Jin et al., Mol. Endocrinology 24:923-29, 2010). The RORγ-Ligand Binding Domain TR-FRET Assay was run according to the following protocol.HIS-tagged RORγ-LBD protein was recombinantly expressed in Escherichia coli. The RORγ-LBD protein was purified by Ni2+-affinity resin. Purified protein was then diluted in assay buffer (50 mM Tris pH 7.0, 50 mM KCl, 1 mM EDTA, 0.1 mM DTT, 100 μg/mL bovine serum albumin, delipidated) to obtain a RORγ-LBD final concentration of 3 nM. Europium tagged anti-HIS antibody was also added to this solution (1.25 nM). Separately, SF9 cells not expressing any recombinant protein were lysed (32,000 cells per μl in 25 mM Tris, 50 mM NaCl) and the previously frozen lysate was added to the diluted RORγ-LBD solution at a ratio of 0.75 μl SF9 lysate per 15 μl of diluted RORγ-LBD.
Measurement: IC50 = 10.2 nM (pChEMBL 7.989999771118164)
Descriptors: MW 620.47, ALogP 5.69, HBA 6, HBD 2, PSA 122.24, QED 0.38
Compound (SMILES): FC(F)(F)c1cccc(CN2C[C@@H]([C@H]3CC(Br)=NO3)Cc3ccccc32)c1
InChIKey: YPTWEOXJZRWRRM-MAUKXSAKSA-N
Target: Transcriptional enhancer factor TEF-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28347
Assay: TEAD Inhibition Assay: TEAD inhibition can be assayed using Hippo Pathway TEAD ReporterMCF7 Cell Line (BPS Bioscience, Catalog #: 60618).
Measurement: EC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 439.28, ALogP 5.38, HBA 3, HBD 0, PSA 24.83, QED 0.64
Compound (SMILES): CC(C)(C)C(=O)NC12CC[C@@]3(OC1)[C@H]1Cc4ccc(O)cc4[C@@]3(CCN1CC1CC1)C2
InChIKey: SEDKKDAEQSQWMA-DALJCPOSSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Mu-Opioid Receptor Binding Assay: Radioligand dose-displacement binding assays for μ-opioid receptors used 0.3 nM [3H]-diprenorphine (Perkin Elmer, Shelton, Conn.), with 5 mg membrane protein/well in a final volume of 500 μl binding buffer (10 mM MgCl2, 1 mM EDTA, 5% DMSO, 50 mM HEPES, pH 7.4). Reactions were carried out in the absence or presence of increasing concentrations of unlabeled naloxone. All reactions were conducted in 96-deep well polypropylene plates for 2 hours at room temperature. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Perkin Elmer, Shelton, Conn.), presoaked in 0.5% polyethylenimine using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by performing three filtration washes with 500 μl of ice-cold binding buffer. Filter plates were subsequently dried at 50° C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well), and plates were counted using a Packard Top-Count for 1 min/well. The data were analyzed using the one-site competition curve fitting functions in GraphPad PRISM v. 3.0 or higher (San Diego, Calif.), or an in-house function for one-site competition curve-fitting.
Measurement: Ki = 0.056 nM (pChEMBL 10.25)
Descriptors: MW 424.59, ALogP 3.52, HBA 4, HBD 2, PSA 61.8, QED 0.78
Compound (SMILES): Cc1cccc([C@H]2CN[C@@H](C(=O)N[C@@H](C)C(=O)NCc3ccc(N)nc3C)C2)c1
InChIKey: KZTQMUPGPPSSOK-KFKAGJAMSA-N
Target: Mannan-binding lectin serine protease 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00187
Assay: Enzymatic Assay for MASP-2: The assay was run at room temperature in an assay buffer containing 20 mM Hepes, pH 7.4, 140 mM NaCl and 0.1% Tween 20. Assay parameters were adjusted such that the assay was linear with respect to time, enzyme and substrate concentrations. Under these optimized assays conditions, IC50 values were equivalent to Ki values, except in a few cases of tight binding inhibitors. Cases of tight binding or possible slow binding inhibitors were handled by the methods described in Copeland R. A. (2013) Evaluation of Enzyme Inhibitors in Drug Discovery. 2nd Ed., John Wiley and Sons, Inc., Chapters 5-7.
Measurement: Ki < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 395.51, ALogP 1.55, HBA 5, HBD 4, PSA 109.14, QED 0.59
Compound (SMILES): Nc1c(C(=O)NCCc2ccc(C3CCNC3)cc2)sc2ncccc12
InChIKey: VJECGCNVYQLLPR-UHFFFAOYSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 28 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96RU2
Assay: Biochemical Assay: USP28: The assay was performed in a final volume of 20 uL in assay buffer containing 20 mM Tris-HCl (pH 8.0, (1M Tris-HCl, pH 8.0 solution; Corning 46-031-CM)), 2 mM CaCl2 (1M Calcium Chloride solution; Sigma #21114) 2 mM BME (2-Mercaptoethanol; Sigma 63689-25ML -F), 0.01% Prionex (0.22 uM filtered, Sigma #G-0411), and 0.01% Triton X-100. Stock compound solutions were stored at â20° C. as 10 mM in DMSO. Up to 1 month prior to the assay, 2 mM test compounds were pre-dispensed into assay plates (Black, low volume; Corning #3820) and frozen at â20° C. Prestamped assay plates were allowed to come to room temperature on the day of the assay. For the screen, 100 nL of 2 mM was pre-dispensed for a final screening concentration of 10 uM (DMSO(fc)=0.5%). Enzyme (USP28, construct USP28 (USP28-5(1-1077)-TEV-6*His; LifeSensors) concentration and incubation times were optimized for the maximal signal-to -background while maintaining initial velocity conditions at a fixed substrate concentration. The final concentration of the enzyme in the assay was 400 pM. Final substrate (Ub-Rh110; Ubiquitin -Rhodamine 110, R&D Systems #U-555) concentration was 25 nM with [Ub-Rh110] Km. 10 uL of 2x enzyme was added to assay plates (pre-stamped with compound) either simultaneously with 2xUb-Rh110 or preincubated with USP28 40 minutes prior to the addition of 10 uL of 2xUb -Rh110 to compound plates. Plates were incubated stacked for 90 minutes at room temperature before fluorescence was read on the Envision (Excitation at 485 nm and Emission at 535 nm; Perkin Elmer) or on the PheraSTAR (Excitation at 485 nm and Emission at 535 nm; BMG Labtech).
Measurement: IC50 < 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 366.49, ALogP 2.93, HBA 5, HBD 3, PSA 80.04, QED 0.65
Compound (SMILES): Cc1c(NCc2ccccc2CN2CCC2)cc(F)c(S(=O)(=O)Nc2ccncn2)c1F
InChIKey: SCYAIXFDQNCSLT-UHFFFAOYSA-N
Target: Sodium channel protein type 8 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UQD0
Assay: Sodium Influx Assay (In Vitro Assay): This sodium influx assay employs the use of the cell permeable, sodium sensitive dye ANG2 to quantify sodium ion influx through sodium channels which are maintained in an open state by use of sodium channel modulators. This high throughput sodium influx assay allows for rapid profiling and characterization of sodium channel blockers.In general, Trex HEK293 cells were stably transfected with an inducible expression vector containing the full-length cDNA coding for the desired human sodium channel α-subunit and with an expression vector containing full length cDNA coding for the β1-subunit. Sodium channel expressing cell lines were induced with tetracycline (1 μg/mL) and plated on 384-well PDL-coated plates at a density of 25K-30K cells/well in culture media (DMEM, containing 10% FBS and 1% L-glutamine). After overnight incubation (37° C., 5% CO2), culture media was removed and cells were loaded with 5 uM ANG2 dye for 1-1.5 h in Buffer 1 (155 mM NMDG, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, adjusted with Tris to pH 7.4). Access dye was removed and cells were incubated with test compounds for 1 hr in buffer 1 containing sodium channel modulator(s) at room temperature. Hamamatsu FDSS μCell was used to perform a 1:1 addition of Na/K challenge buffer (140 mM NaCl, 20 mM HEPES, 1 mM CaCl2, 15 mM KCl, 1 mM MgCl2, 10 mM glucose, adjusted with Tris to pH 7.4) and simultaneously read plates at excitation wavelength of 530 nm and emission wavelength set at 558 nm. Percent inhibition of sodium ion influx was calculated for each test compound at each test concentration to determine the IC50 values.
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 459.52, ALogP 3.68, HBA 6, HBD 2, PSA 87.22, QED 0.53