lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(C)(C)Cn1cnc(-c2ccc(F)cc2)c1-c1ccc2ncc(C#N)n2n1
InChIKey: RSHMAVDNPXHMRY-UHFFFAOYSA-N
Target: TGF-beta receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P36897
Assay: Biological Assay: Assays for the compounds reported below were conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGF-βR1 T204D or HIS-TGF-βR2 WT, anti-HIS detection antibody, a labeled small molecule probe (Kd=<100 nM; koff=<0.001 s−1) and test compounds in assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij35, 4 mM DTT, and 0.05 mg/ml BSA). The reaction is incubated for 1 hour at room temperature and the HTRF signal was measured on an Envision plate reader (Ex: 340 nm; Em: 520 nm/495 nm). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay are 1 nM HIS-TGF-βR1 T204D or HIS-TGF-βR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule prode (at Kd) and 0.5% DMSO. Dose response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations.
Measurement: IC50 = 99.2 nM (pChEMBL 7)
Descriptors: MW 374.42, ALogP 4.32, HBA 6, HBD 0, PSA 71.8, QED 0.54
Compound (SMILES): COc1cc(N=S(C)(=O)C2CC2)cc2ncnc(Nc3ccc(F)cc3O[C@H]3COC[C@H]3O)c12
InChIKey: VEXWMFXNIRRCIF-WAZKHLONSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBH9
Assay: MNK2a In Vitro Kinase Assay (Assay 2): ASSAY SETUP: The inhibition of kinase activity of MNK2a was assessed using pre-activated GST-MNK2a. The white, 384-well OptiPlate F plates were purchased from PerkinElmer. The ADP-Glo Kinase Assay (including ultra pure ATP) was purchased from Promega (V9103). Activated MNK2a was obtained as described in WO2011/104340. The unlabeled eIF4E peptide (NH2-TATKSGSTTKNR-CONH2), differing from Seq. ID No. 5 of WO 2011/104340 by the C-terminal CONH2 group, was purchased from Thermo Fisher Scientific. All other materials were of highest grade commercially available. Compounds are tested in either serial dilutions or single dose concentrations. The compound stock solutions are 10 mM in 100% DMSO The serial compound dilutions are prepared in 100% DMSO followed by 1:27.3 intermediate dilution in assay buffer. The final DMSO concentration in assay will be <3%.In the 384-well plates 3 μl of test compound from the intermediate dilutionis mixed with 4 μl of the activated MNK2 enzyme (final concentration of 10 nM) and 4 μl of the peptide (final concentration of 25 μM)/ultra pure ATP (final concentration of 20 μM), all dissolved in assay buffer. This step is followed by an incubation time of 90 min, then 10 μl of ADP Glo reagent are added, followed by 40 min of incubation. Then 20 μl of kinase detection reagent are admixed. The plates are sealed and after an incubation period of 30 min, the luminescence signal is measured in an Envision reader to determine the amount of produced ADP. All incubation steps are performed at room temperature.The assay buffer consists of 20 mM HEPES, 2 mM DTT, 0.01% BSA, 20 mM MgCl2 and 0.1% Pluronic F-127.Each assay microtiter plate contains wells with vehicle controls instead of compound (1% DMSO in water) as reference for the high signal (100% CTL, high signal), and wells containing a potent MNK2 inhibitor (final 20 μM, 1% DMSO) as reference for low signal (0% CTL, low signal).The luminescent signal generated is proportional to the ADP concentration produced and is correlated with activated MNK2 activity. The analysis of the data is performed by the calculation of the percentage of ATP consumption of activated MNK2 in the presence of the test compound compared to the consumption of ATP in the presence of activated MNK2 without compound. RLU(sample)−RLU(low control))*100/(RLU(high value)−RLU(low control)) [RLU=relative luminescence units]An inhibitor of the MNK2 enzyme will give values between 100% CTL (no inhibition) and 0% CTL (complete inhibition). Values of more than 100% CTL are normally related to compound/sample specific physico-chemical properties (e.g. solubility, light absorbance, fluorescence).IC50 values based on dose response curves are calculated with the AssayExplorer software.
Measurement: IC50 = 33 nM (pChEMBL 7.480000019073486)
Descriptors: MW 488.54, ALogP 3.55, HBA 9, HBD 2, PSA 115.16, QED 0.52
Compound (SMILES): COc1ccc(OC)c(C2c3nnnn3CCN2Cc2ccccc2F)c1
InChIKey: NSRGITMVFIFXMT-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 = 24950 nM (pChEMBL 4.599999904632568)
Descriptors: MW 369.4, ALogP 2.43, HBA 7, HBD 0, PSA 65.3, QED 0.69
Compound (SMILES): CCC[C@@H](C)S(=O)(=O)Nc1cc(-c2ccc(=O)n(C)c2)nc(Oc2c(C)cccc2Cl)n1
InChIKey: ASFJJBKOTIDCQF-OAHLLOKOSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: In Vitro Enzyme Inhibition Assay-CREBBP Inhibition: Determination of the IC50 for the CREBBP inhibitors disclosed herein was performed as follows: CREBBP was cloned and expressed in E. Coli as His-tag protein and purified by Nickel affinity and gel-filtration chromatography. The protein was further characterized as a single band with the correct molecular weight by SDS-PAGE. CREBBP binding and inhibition was assessed by monitoring the interaction of biotinylated H4-tetraacetyl peptide (AnaSpec, H4K5/8/12/16(Ac), biotin-labeled) with the target using the AlphaScreen technology (Perkin Elmer). In a 384-well ProxiPlate CREBBP (50 nM final) was combined with peptide (20 nM final) in 50 mM HEPES (pH 7.3), 10 mM NaCl, 0.25 mM TCEP, 0.1% (w/v) BSA, and 0.005% (w/v) Brij-35 either in the presence of DMSO (final 0.4% DMSO) or compound dilution series in DMSO. After 20-minute incubation at room temperature, Alpha streptavidin donor beads and Nickel Chelate acceptor beads were added to a final concentration of 5 μg/mL. After two hours of equilibration, plates were read on an Envision instrument and the IC50 was calculated using a four parameter non-linear curve fit.The ability of the compounds disclosed herein to inhibit CBP activity was quantified and the respective IC50 value was determined.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 476.99, ALogP 4.53, HBA 7, HBD 1, PSA 103.18, QED 0.51
Compound (SMILES): COC(=O)[C@](C)(CO)NCc1cc(Cl)c(O[C@H]2CCc3c(-c4ccc5c(c4F)OCCO5)cccc32)cc1OCc1cncc(C#N)c1
InChIKey: OYZNJOWGTUQDNB-IVUOSGLUSA-N
Target: Programmed cell death 1 ligand 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZQ7
Assay: Enzyme-Linked Immunosorbent AssayâELISA: Plates were coated with 1 Î¼g/mL of human PD-L1 (obtained from R&D) in PBS overnight at 4Â° C. The wells were then blocked with PBS containing 2% BSA in PBS (W/V) with 0.05% TWEEN-20 for 1 hour at 37Â° C. The plates were washed 3 times with PBS/0.05% TWEEN-20 and the samples were diluted to 1:5 in dilution medium in the ELISA plates. Human PD-1 and biotin 0.3 Î¼g/mL (ACRO Biosystems) were added and incubated for 1 hour at 37Â° C. then washed 3 times with PBS/0.05% TWEEN-20. A second block was added with PBS containing 2% BSA in PBS (W/V)/0.05% TWEEN-20 for 10 min at 37Â° C. and was washed 3 times with PBS/0.05% TWEEN-20. Streptavidin-HRP was added for 1 hour at 37Â° C. then washed 3 times with PBS/0.05% TWEEN-20. TMB substrate was added and reacted for 20 min at 37Â° C. A stop solution (2 N aqueous H2SO4) was added. The absorbance was read at 450 nm using a micro-plate spectrophotometer.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 674.13, ALogP 5.84, HBA 10, HBD 2, PSA 132.16, QED 0.19
Compound (SMILES): Cc1ccc(Br)nc1NC(=O)[C@@H]1C[C@@]2(C)C[C@H]2N1C(=O)Cn1nc(C(N)=O)cc1-c1cccs1
InChIKey: XWBIJBCXIIJTRM-JCEJCQQGSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Factor D Assay: Human Factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration is incubated with test compound at various concentrations for 5 min at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBz and DTNB (Ellman's reagent) are added to final concentrations of 100 μM each. Absorbance at 405 nm (A405) is recorded at 30 second intervals for 30 min using a microplate spectrophotometer. IC50 values are calculated by nonlinear regression of complement Factor D reaction rates as a function of test compound concentration.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 543.45, ALogP 3.19, HBA 7, HBD 2, PSA 123.21, QED 0.46
Compound (SMILES): O=C(C[C@@H]1CCc2cc(C(O)(C(F)(F)F)C(F)(F)F)ccc2N1S(=O)(=O)c1ccc(F)cc1)N1CCC(N2Cc3ccccc3NC2=O)CC1
InChIKey: GTWSXKXRIJMETK-VWLOTQADSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Spa Binding Assay: The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromotography. 50 nM RORγ (A262-S507) is incubated with test compound at varying concentrations for 15 min at room temperature in PBS buffer containing 0.5% fatty acid free BSA. 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 15 min. 4 mg/mL of Ysi Copper HIS-TAG SPA Beads (Perkin Elmer) are added, and the mixture is incubated for 30 min. The reaction is read on a MicroBeta Trilux scintillation plate reader (Perkin Elmer).
Measurement: IC50 = 172 nM (pChEMBL 6.760000228881836)
Descriptors: MW 728.69, ALogP 6.08, HBA 5, HBD 2, PSA 110.26, QED 0.3
Compound (SMILES): CCc1ccnc(NC(=O)c2ccc(-c3nn([C@@H]4CCCN(C(=O)C5(C)COC5)C4)c4ccnc(N)c34)cc2)c1
InChIKey: DPGPFYMSYUQBGR-JOCHJYFZSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Btk Enzyme Activity Assay C: BTK enzymatic activity was determined with the LANCE (Lanthanide Chelate Excite) TR-FRET (Time-resolved fluorescence resonance energy transfer) assay. In this assay, the potency (IC50) of each compound was determined from an eleven point (1:3 serial dilution; final compound concentration range in assay from 1 μM to 0.017 nM) titration curve using the following outlined procedure. To each well of a black non-binding surface Corning 384-well microplate (Corning Catalog #3820), 5 nL of compound (2000 fold dilution in final assay volume of 10 μL) was dispensed, followed by the addition of 7.5 μL of 1× kinase buffer (50 mM Hepes 7.5, 10 mM MgCl2, 0.01% Brij-35, 1 mM EGTA, 0.05% BSA & 1 mM DTT) containing 5.09 pg/μL (66.67 pM) of BTK enzyme (recombinant protein from baculovirus-transfected Sf9 cells: full-length BTK, 6HIS-tag cleaved). Following a 60 minute compound & enzyme incubation, each reaction was initiated by the addition of 2.5 μL 1× kinase buffer containing 8 μM biotinylated A5 peptide (Biotin-EQEDEPEGDYFEWLE-NH2), and 100 μM ATP. The final reaction in each well of 10 μL consists of 50 pM hBTK, 2 μM biotin-A5-peptide, and 25 μM ATP. Phosphorylation reactions were allowed to proceed for 120 minutes. Reactions were immediately quenched by the addition of 20 uL of 1× quench buffer (15 mM EDTA, 25 mM Hepes 7.3, and 0.1% Triton X-100) containing detection reagents (0.626 nM of LANCE-Eu-W1024-anti-phospho Tyrosine antibody, PerkinElmer and 86.8 nM of Streptavidin-conjugated Dylight 650, Dyomics/ThermoFisher Scientific). After 60 minutes incubation with detection reagents, reaction plates were read on a PerkinElmer EnVision plate reader using standard TR-FRET protocol. Briefly, excitation of donor molecules (Eu-chelate:anti-phospho-antibody) with a laser light source at 337 nm produces energy that can be transferred to Dylight-650 acceptor molecules if this donor:acceptor pair is within close proximity. Fluorescence intensity at both 665 nm (acceptor) and 615 nm (donor) are measured and a TR-FRET ratio calculated for each well (acceptor intensity/donor intensity). IC50 values were determined by 4 parameter robust fit of TR-FRET ratio values vs. (Log10) compound concentrations.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 539.64, ALogP 4.09, HBA 8, HBD 2, PSA 128.26, QED 0.38
Compound (SMILES): Cc1ccncc1-c1cccc(S(N)(=O)=O)c1-c1nn[nH]n1
InChIKey: TYPPDRKNERPHMN-UHFFFAOYSA-N
Target: Metallo-beta-lactamase type 2 (SINGLE PROTEIN), Bacteria
UniProt: C7C422
Assay: Class B Enzyme Assay: The Class B enzyme activities were measured in the presence of the test inhibitor in a fluorescence assay against a commercially available substrate consisting of a cephalosporin core linking 7-hydroxycoumarin to fluorescein (CCF2-FA). The enzyme (NDM-1, IMP-1 or VIM-1) and the substrate were diluted in 100 mM KH2PO4 buffer (pH 7) containing 0.005% Tween-20 and 10 μM ZnSO4. In the assay, the final concentration of enzyme was 1 pM, 2 pM and 30 pM for NDM-1, IMP-1 and VIM-1, respectively, and the final concentration of CCF2-FA was 1.25 μM. The test inhibitor was dissolved in dimethylsulfoxide and diluted 1:50 in the assay, resulting in a final concentration range of 20 uM to 0.00063 uM. In a 384-well microplate, the test inhibitor was incubated with the metallo-β-lactamase enzyme and the substrate for 2 hours at 25° C. Fluorescence at 460 nm following excitation at 405 nm was measured. The IC50 value was determined from semi-logarithmic plots of enzyme inhibition versus inhibitor concentration, with a curve generated using a 4-parameter fit.
Measurement: IC50 = 5.38 nM (pChEMBL 8.270000457763672)
Descriptors: MW 316.35, ALogP 0.88, HBA 6, HBD 2, PSA 127.51, QED 0.74
Compound (SMILES): Cc1ccc(-n2cccn2)c(C(=O)N2CCC[C@@H](C)[C@H]2CNc2ccc(C#N)cn2)c1
InChIKey: RMDXIELGJALRDL-XMSQKQJNSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Cell-based Functional Assay: Measurement of [Ca2+]i using a FLIPR: CHO-OX1 or CHO-OX2 cells are seeded into black-walled clear-base 384-well plates (Costar) at a density of 20,000 cells per well in F12-K medium supplemented with 10% FBS and selection antibiotic (500 ug/ml G418 or 15 ug/ml Blasticidin) and cultured overnight. The cells are incubated with equal volume of calcium4 loading buffer (Molecular Devices, Inc.) containing 2.5 mM probenecid at 37° C. for 30 min, followed by putative OX1 or OX2 receptor antagonists (dose-range 0.1 nM-10 μM) for another 30 min.
Measurement: IC50 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 414.51, ALogP 3.8, HBA 6, HBD 1, PSA 86.84, QED 0.69
Compound (SMILES): COc1cc(Nc2nccc(Oc3ccc(NC(=O)Nc4cc(C(C)C)nn4-c4ccc(C)cc4)c4ccccc34)n2)cc(S(=O)(=O)C2CC2)c1
InChIKey: XSINXQGWUBFCGP-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 12 (SINGLE PROTEIN), Homo sapiens
UniProt: P53778
Assay: Enzyme Inhibition Assay: The inhibitory activities of compounds of the invention against p38MAPKγ (MAPK12: Invitrogen), are evaluated in a similar fashion to that described hereinabove. The enzyme (800 ng/mL, 2.5 μL) is incubated with the test compound (2.5 μL at either 4 μg/mL, 0.4 μg/mL, 0.04 μg/mL, or 0.004 μg/mL) for 2 hr at RT. The FRET peptides (8 μM, 2.5 μL), and appropriate ATP solution (2.5 μL, 400 μM) is then added to the enzymes/compound mixtures and incubated for 1 hr. Development reagent (protease, 5 μL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan® Flash, Thermo Scientific).
Measurement: IC50 = 102 nM (pChEMBL 6.989999771118164)
Descriptors: MW 703.83, ALogP 8.37, HBA 10, HBD 3, PSA 149.36, QED 0.12
Compound (SMILES): C[C@H]1[C@H](Nc2nc3ccccc3o2)CCCN1C(=O)c1ccc(F)cc1-c1ncccn1
InChIKey: NYRCHCPBPDEEDQ-HNAYVOBHSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Receptor Radioligand Binding Studies: 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). The plates were washed 3 times by aspirating 30 ml PBS through the plates. Plates were dried in 55° C. oven for 60 min, scintillation fluid was added, and the radioactivity was counted on a Topcount (Packard).
Measurement: Ki = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 431.47, ALogP 4.53, HBA 6, HBD 1, PSA 84.15, QED 0.51
Compound (SMILES): C[C@H]1CN(CC[C@H]2CC[C@](O)(CO)CC2)c2c(oc3ccc(C(F)(F)F)cc23)C(=O)N1
InChIKey: XRMOSSWFDRFEQH-DGCWBMGLSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Pim-1 Inhibitory Activity Assay: The calculation of the Pim-1 inhibitory activity of the compound was performed using the following solution according to the protocol attached to ADP-Glo Kinase Assay (cat. V9102, Promega). The purified enzyme of human Pim-1 described above was used. (i) Preparation of Solution. The kinase buffer solution (50 mmol/L HEPES (pH 7.5), 5 mmol/L MgCl2, 1 mmol/L DTT, 0.05% BSA) was prepared by dissolving HEPES (Jena Bioscience), MgCl2 (Sigma-Aldrich), DTT (Sigma-Aldrich) and BSA (Sigma-Aldrich) in purified water. The ATP solution (288 μmol/L) was prepared by dissolving 100 mmol/L ATP (Promega) in the kinase buffer solution. The enzyme/substrate solution (0.2 mmol/L Pim-1, 30 μmol/L Pim2tide) was prepared by dissolving Pim-1 (described above) and Pim2tide (custom synthetic product by GenScript USA, the same product as PIM2tide cat. 12-542 by Millipore) in the kinase buffer solution. The test compound solution (containing 12.5% DMSO) was prepared by dissolving the DMSO solution of the test compound in the kinase buffer solution. The vehicle solution (containing 12.5% DMSO) was prepared by dissolving DMSO in the kinase buffer solution. (ii) Method. To a 384-well assay plate (Corning, 4513), the test compound solution or vehicle solution (control) was added by 1 μL/well, the enzyme/substrate solution or kinase buffer solution (blank) was added by 2 μL/well, and the ATP solution was added by 2 μL/well, and they were mixed. After enzymatically reacting at room temperature for 45 minutes, ADP-Glo Reagent (Promega) was added by 5 μL/well, and they were mixed. After reacting at room temperature for 60 minutes, Kinase Detection Reagent (Promega) was added by 10 μL/well, and they were mixed. After reacting at room temperature for 30 minutes, the luminescence amount of each well was measured for 10 msec with a multi-label plate reader EnVision (PerkinElmer).
Measurement: Ki = 0.074 nM (pChEMBL 10.130000114440918)
Descriptors: MW 440.46, ALogP 3.69, HBA 5, HBD 3, PSA 85.94, QED 0.68
Compound (SMILES): COc1cnc(-c2nc(C(=O)Nc3cnn(C)c3[C@@H]3CC[C@@H](N)[C@H](F)CO3)cs2)c(C)c1
InChIKey: WVIFKINRRYYQMF-CPUCHLNUSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Biological Assay: Determination of the Pim kinase activity of a Formula I compound is possible by a number of direct and indirect detection methods. Certain exemplary compounds described herein were assayed for their Pim kinase binding activity, including isoforms Pim-1, Pim-2, andPim-3, (Example 901) and in vitro activity against tumor cells (Example 902). Certain exemplary compounds of the invention had Pim binding activity IC50 values less than about 1 micromolar (μM). Certain compounds of the invention had tumor cell-based activity EC50 values less than about 1 micromolar (μM).
Measurement: Ki = 0.043 nM (pChEMBL 10.369999885559082)
Descriptors: MW 460.54, ALogP 3.02, HBA 9, HBD 2, PSA 117.18, QED 0.6
Compound (SMILES): O=C(NO)c1cccc2c1CC1(CCN(c3cccc(C(F)(F)F)c3)C1=O)C2
InChIKey: RKBAINCYKCNTGV-UHFFFAOYSA-N
Target: Histone deacetylase 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96DB2
Assay: HDAC Inhibition Assays: This example describes in vitro inhibition properties of exemplary HDAC11 inhibitors for various HDACs. HDAC inhibition assays were performed using an electrophoretic mobility shift assay at Nanosyn, Inc. (Santa Clara, Calif.). Full length human recombinant HDAC proteins were expressed in the baculoviral system and purified by affinity chromatography. A human recombinant HDAC3 was co-expressed with nuclear receptor corepressor (Ncor2). The following peptide substrates were used: FAM-RHKK(Ac)-NH2 for HDAC3, HDAC6 and HDAC8; FITC-H3K27(Ac)-NH2 for HDAC1, HDAC2 and HDAC10; FAM-RHKK(tri-fluor-Ac)-NH2 for HDAC4, HDAC5, HDAC7, HDAC9 and HDAC11. Reactions consisting of compound, enzyme, and substrate were performed in reaction buffer (comprised of 100 mM HEPES, pH7.5, 25 mM KCl, 0.1% bovine serum albumin, 0.01% Triton X-100) at 25° C. and quenched by the addition of termination buffer (100 mM HEPES, pH7.5, 0.01% Triton X-100, 0.05% SDS). The fluorescence intensity of the electrophoretically separated de-acetylated product and substrate peptide were measured and analyzed using the LabChip® 3000 microfluidic electrophoresis instrument (Perkin Elmer/Caliper Life Sciences). The IC50 values of inhibitors were determined by fitting the %-inhibition curves with 4 parameter dose-response model using XLfit 4 software (IDBS).
Measurement: IC50 = 750 nM (pChEMBL 6.119999885559082)
Descriptors: MW 390.36, ALogP 3.35, HBA 3, HBD 2, PSA 69.64, QED 0.61
Compound (SMILES): O=S(=O)(c1nc2n(n1)[C@H](c1ccccc1)C[C@@H]2F)[C@@H]1CCC1(F)F
InChIKey: IWBCNIRIAYKNQE-SDDRHHMPSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: Biochemical Assay: The ability of the receptor interacting protein kinase (RIPK1) to catalyze the hydrolysis of adenosine-5′-triphosphate (ATP) is monitored using the Transcreener ADP (adenosine-5′-diphosphate) assay (BellBrook Labs). Purified human RIP1 kinase domain (2-375) (50 nM) derived from a baculovirus-infected insect cell expression system is incubated with test compounds for 2 hours in 50 mM Hepes buffer (pH 7.5) containing 30 mM MgCl2, 1 mM dithiothreitol, 50 uM ATP, 0.002% Brij-35, and 0.5% dimethyl sulfoxide (DMSO). Reactions are quenched by the addition of 1× Bell Brooks Stop buffer B (20 mM Hepes (ph7.5), 40 mM ethylenediaminetetraacetic acid and 0.02% Brij-35) containing an additional 12 mM EDTA and 55 ug/mL ADP2 antibody and 4 nM ADP-AlexaFluor® 633 tracer. The tracer bound to the antibody is displaced by the ADP generated during the RIP1K reaction, which causes a decrease in fluorescence polarization that is measured by laser excitation at 633 nm with a FP microplate reader M1000. Fractional activity was plotted against test article concentration. Using Genedata Screener software (Genedata; Basel, Switzerland), the data were fit to the tight-binding apparent inhibition constant (Ki app) Morrison equation [Williams, J. W. and Morrison, J. F. (1979) The kinetics of reversible tight-binding inhibition. Methods Enzymol 63: 437-67]. The following equation was used to calculate fractional activity and Ki app:Fractional ⁢ ⁢ activity = V i V o = 1 - ( [ E ] T + [ I ] T + K i app ) - ( [ E ] T + [ I ] T + K i app ) 2 - 4 ⁡ [ E ] T ⁡ [ I ] T 2 ⁡ [ E ] Twhere [E]T and [I]T are the total concentrations of active enzyme and test article, respectively.
Measurement: Ki < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 357.36, ALogP 2.85, HBA 5, HBD 0, PSA 64.85, QED 0.85
Compound (SMILES): CCc1cccc(-c2nnc(NS(=O)(=O)[C@@H](C)[C@H](O)c3ncc(C)cn3)n2-c2c(OC)cccc2OC)n1
InChIKey: IMKZIGAPROGKMW-AOMKIAJQSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: [35S]GTPγS Binding: The human APJ receptor was cloned by polymerase chain reaction and the gene encoding the receptor was subcloned in pFLAG-CMV-3 expression vector (Sigma, Saint Louis, Mo. USA) in-house at Amgen. A GTPγS binding assay was performed on membranes prepared from CHO cells stably expressing human APJ receptor. The optimum experimental conditions for the concentrations of GDP, MgCl2, and NaCl in the assay buffer were initially determined. The assay was performed in assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, and 0.1% (w/v) BSA with 200 mM NaCl, 3 M GDP] and membranes expressing human APJ receptor/well along with WGA PS beads. The reaction was initiated by addition of 0.2 nM [35S]GTPγS (Perkin Elmer Life and Analytical Sciences, Waltham USA) in the absence or presence of various ligands and incubated at RT for 90 min. Nonspecific binding was determined in the presence of 100 M GTPγS and was always less than 0.2% of total binding. All the results presented are means of several independent experiments and analyzed by non-linear regression methods using commercially available program Prism (GraphPad, San Diego, Calif.) to obtain EC50.
Measurement: EC50 = 0.39 nM (pChEMBL 9.40999984741211)
Descriptors: MW 539.62, ALogP 2.87, HBA 11, HBD 2, PSA 154.24, QED 0.31
Compound (SMILES): COC(=O)/C=C/CC[C@H](NC(=O)c1cc([N+](=O)[O-])ccc1C(=O)O)C(=O)N[C@H](C(=O)N1CC(=O)C[C@H]1C(=O)N[C@H](C(=O)N[C@H](C(N)=O)C1CCCCC1)C1CCCCC1)C(C)(C)C
InChIKey: PIFOKPGDWWGCKA-SFIJLXCDSA-N
Target: Coagulation factor XIII A chain (SINGLE PROTEIN), Homo sapiens
UniProt: P00488
Assay: Isopeptidase Assay for Estimating Inhibitor Potency: FXIIIa activity has been determined using substrate A101 (Zedira GmbH, Darmstadt, Germany), which is based on the N-terminal dodecapeptide of α2-antipiasmin. FXIIIa catalyzes by its isopeptidase activity the release of dark quencher dinitrophenyl at the original substrate glutamine position resulting in fluorescence increase (based on the N-terminal 2-aminobenzoyl fluorescent dye) (Oertel K, HunfekJ A, Specker E, Reiff C, Seitz R, Pasternack R, Dodt J. A highly sensitive fluorometric assay for determination of human coagulation factor XIII in plasma. Anal Biochem 2007; 367:152-8.)Briefly, 12 μL recombinant cFXIII-A2 (T027, Zedira GmbH, Darmstadt, Germany) or FXIII-A2B2 derived from human plasma (T007) (25 μg/mL) and 3 μL human α-thrombin (0.5 U/mL, T056, Zedira) were mixed with 270 μL assay buffer (50 mM Tris-HCl, 10 mM CaCl2), 150 mM NaCl, 5.56 mM glycine methyl ester, 5 mM DTT, pH 7.5) containing 55 μM A101 substrate. The mixture was incubated for 20 min at room temperature to activate FXIII. 15 μL of inhibitor solution (serial dilution from 1.25 μM to 1.25 nM) dissolved in DMSO/assay buffer were added, mixed and the kinetic measurement started after 3 min. Fluorescence emission was monitored at 418 nm (λex=313 nm) and 37° C. for 30 min using a CLARIOstar fluorescence micro plate reader (BMG Labtech, Ortenberg, Germany). For measurements without inhibitor, 15 μL of assay buffer/2% (v/v) DMSO were added. All measurements were performed in triplicate. The respective IC50 values were calculated by non-linear regression using the MARS software package (BMG Labtech).
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 881.98, ALogP 2.22, HBA 12, HBD 6, PSA 303.61, QED 0.05
Compound (SMILES): CS(=O)(=O)c1cc(Cl)ccc1Nc1cc(NC(=O)[C@H]2C[C@H]2F)nc2[nH]c(C(F)F)nc12
InChIKey: JRGSBABLZWCYRD-DTWKUNHWSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Binding Assay: Binding constants for compounds of the present invention against the JH2 domain were determined by the following protocol for a KINOMEscan® assay (DiscoveRx). A fusion protein of a partial length construct of human TYK2 (JH2domain-pseudokinase) (amino acids G556 to D888 based on reference sequence NP_003322.3) and the DNA binding domain of NFkB was expressed in transiently transfected HEK293 cells. From these HEK 293 cells, extracts were prepared in M-PER extraction buffer (Pierce) in the presence of Protease Inhibitor Cocktail Complete (Roche) and Phosphatase Inhibitor Cocktail Set II (Merck) per manufacturers' instructions. The TYK2 (JH2domain-pseudokinase) fusion protein was labeled with a chimeric double-stranded DNA tag containing the NFkB binding site (5′-GGGAATTCCC-3′) fused to an amplicon for qPCR readout, which was added directly to the expression extract (the final concentration of DNA-tag in the binding reaction is 0.1 nM).
Measurement: Kd = 0.75 nM (pChEMBL 9.119999885559082)
Descriptors: MW 473.86, ALogP 3.99, HBA 6, HBD 3, PSA 116.84, QED 0.5
Compound (SMILES): CN(C)c1nccc(Sc2cnc(N3CCC4(CCC[C@H]4N)CC3)n3cnnc23)c1Cl
InChIKey: AKKLKWAYGDVKIP-MRXNPFEDSA-N
Target: Tyrosine-protein phosphatase non-receptor type 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06124
Assay: SHP2 Enzyme Activity Inhibition Assay: Compound powders were dissolved in DMSO to prepare mother liquor.During the experiment, the stock solutions of the compounds were subjected to 3-fold gradient dilution with DMSO, and 10 different test concentrations of each compound were set. 1 μL of each concentration of the compounds was transferred into the wells of detection plates (Corning, Costar 3915), setting two duplicates for each concentration. The protein used was the activated protein SHP2E76A with amino acid mutation at position 76 of the protein, and the substrate used was DiFMUP (Invitrogen, E12020). SHP2E76A protein and substrate were diluted with buffer (0.1 M NaAc (pH 7.2), 0.02% Tween 20, 0.1% BSA, 1 mM EDTA, 5 mM DTT) to a concentration of 1.2 nM and 20 M, respectively. 50 L of enzyme solution was added to the detection wells, followed by 50 L of substrate. On the Spectra max i3 (Molecular Devices) instrument, the fluorescence signal was recorded (Ex 358 nm/Em 455 nm) every minute, from which the product accumulation rate was calculated to characterize the enzyme activity. GraphPad Prism 5 was used for nonlinear regression analysis, and the curve of enzyme activity versus compound concentration was fitted by the equation Y=Bottom+(Top-Bottom)/(1+10{circumflex over ( )}((Log IC50−X)*HillSlope)) to calculate the IC50 value of each compound.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 459.02, ALogP 3.49, HBA 9, HBD 1, PSA 88.47, QED 0.64
Compound (SMILES): COCCS(=O)(=O)N1[C@@H]2CCC[C@H]1C[C@H](Nc1nc(Nc3cc(C)[nH]n3)cc3ncccc13)C2
InChIKey: KWWSXWUYXMTDST-NNZMDNLPSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Biochemical JAK Assay: A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 and Tyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH 7.5, 0.01% Brij-35, 10 mM MgCl2, and 1 mM EGTA). Recombinant GST-tagged JAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained from Life Technologies.Serially diluted compounds were pre-incubated with each of the four JAK enzymes and the substrate in white 384-well microplates (Corning) at ambient temperature for lh. ATP was subsequently added to initiate the kinase reactions in 10 μL total volume, with 1% DMSO. The final enzyme concentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and 0.25 nM respectively; the corresponding Km ATP concentrations used are 25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200 nM for all four assays. Kinase reactions were allowed to proceed for 1 hour at ambient temperature before a 10 μL preparation of EDTA (10 mM final concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (Life Technologies, 2 nM final concentration) in TR-FRET dilution buffer (Life Technologies) was added. The plates were allowed to incubate at ambient temperature for lh before being read on the EnVision reader (Perkin Elmer). Emission ratio signals (520 nm/495 nm) were recorded and utilized to calculate the percent inhibition values based on DMSO and background controls.For dose-response analysis, percent inhibition data were plotted vs. compound concentrations, and IC50 values were determined from a 4-parameter robust fit model with the Prism software (GraphPad Software). Results were expressed as pIC50 (negative logarithm of IC50) and subsequently converted to pKi (negative logarithm of dissociation constant, Ki) using the Cheng-Prusoff equation.Test compounds having a higher pKi value in each of the four JAK assays show greater inhibition of JAK activity. Compounds of the invention tested in this assay typically exhibited pKi values between about 7 and about 10.3
Measurement: Ki < 0.1 nM (pChEMBL 10)
Descriptors: MW 485.61, ALogP 3.18, HBA 8, HBD 3, PSA 125.13, QED 0.44
Compound (SMILES): CN1CCN(c2cccc3c2CCN(C(=O)c2cncc(-c4cccc(Cl)c4)c2)C3C(=O)Nc2ccc(C(=O)O)cc2)CC1
InChIKey: HPPADHZZFREPQU-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Enzyme Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 158 nM (pChEMBL 6.800000190734863)
Descriptors: MW 610.11, ALogP 5.23, HBA 6, HBD 2, PSA 106.08, QED 0.31
Compound (SMILES): C=CC(=O)N1CCC(n2c(-c3ccccc3)nc3cnc(N)nc32)C1
InChIKey: UZNYBTOHYFPEAB-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Inhibition assay for BTK: To determine IC50 of a compound against a kinase, a series of concentrations of the compound were tested for the inhibition. IC50 was calculated by plotting the concentration of compound vs the percentage of inhibition in treated wells using GraphPad Prism 5.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 334.38, ALogP 2.03, HBA 6, HBD 1, PSA 89.93, QED 0.74
Compound (SMILES): N#CN1CCC(F)(C(=O)Nc2cc(-c3ccccc3)on2)C1
InChIKey: KASDLCBIWMCJCT-UHFFFAOYSA-N
Target: OTU domain-containing protein 7B (SINGLE PROTEIN), Homo sapiens
UniProt: Q6GQQ9
Assay: Biochemical Assay: Dilution plates were prepared at 21 times the final concentration (2100 μM for a final concentration of 100 μM) in 50% DMSO in a 96-well polypropylene V-bottom plate (Greiner #651201). A typical 8-point dilution series to be 100, 30, 10, 3, 1, 0.3, 0.1, 0.03 M final. Reactions were performed in duplicate in black 384 well plates (small volume, Greiner 784076) in a final reaction volume of 21 μl. Either 1 μl of 50% DMSO or diluted compound was added to the plate. Cezanne 1 was diluted in reaction buffer (40 mM Tris, pH 7.5, 0.005% Tween 20, 0.5 mg/ml BSA, 5 mM-beta-mercaptoethanol) to the equivalent of 0.005 μl/well and 10 μl of diluted Cezanne 1 was added to the compound. Enzyme and compound were incubated for 30 min at room temp. Reactions were initiated by the addition of 50 nM of TAMRA labelled peptide linked to ubiquitin via an iso-peptide bond as fluorescence polarisation substrate. Reactions were read immediately after addition of substrate and following a 2 hr incubation at room temperature. Readings were performed on a Pherastar Plus (BMG Labtech). λ Excitation 540 nm; λ Emission 590 nm.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 300.29, ALogP 2.18, HBA 5, HBD 1, PSA 82.16, QED 0.88
Compound (SMILES): Cc1ccc(N2CCN(c3nccc(COC(=O)NC(=N)N)c3F)CC2)cc1
InChIKey: GXYQLQSVXMGXTR-UHFFFAOYSA-N
Target: Amine oxidase [copper-containing] 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16853
Assay: TBD: TBD
Measurement: IC50 = 11000 nM (pChEMBL 4.960000038146973)
Descriptors: MW 386.43, ALogP 1.98, HBA 6, HBD 3, PSA 107.57, QED 0.55
Compound (SMILES): O=C(O)[C@@H]1C[C@H](OCC#Cc2ccccc2)CCN1C(=O)C(c1ccccc1)c1ccccc1
InChIKey: AHXPRBIMEKRMDN-FTJBHMTQSA-N
Target: Type-2 angiotensin II receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P50052
Assay: Receptor Binding Assay: 120 μL membrane (5 mg protein/well) was incubated with 15 μL of [125I]-CGP42112A and 15 μL of compound at RT for 1.5 hrs.The binding reaction was stopped by rapid filtration through Unifilter GF/C plates (presoaked in 0.3% (v:v) BSA).Plate was washed three times with ice cold wash buffer.The filtration plates were dried at 37° C. overnight.50 μL of scintillation cocktail was added to each well.Radioactivity was determined using MicroBetaTriluxmicroplate scintillation counter.
Measurement: IC50 = 228 nM (pChEMBL 6.639999866485596)
Descriptors: MW 453.54, ALogP 4.33, HBA 3, HBD 1, PSA 66.84, QED 0.56
Compound (SMILES): CC(=O)NCCSc1nonc1/C(=N/O)NC1CCc2ccc(Br)cc21
InChIKey: NPRFKJCVYQHSRX-UHFFFAOYSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: Enzyme Assay: HIS-tagged IDO1 protein was recombinantly expressed in Escherichia coli using ZYP5052 autoinduction media supplemented with 500 μM delta aminolevulinic acid for 48 hours at 16 degrees Celsius. IDO1 protein was purified using Ni2+-affinity resin and size exclusion chromatography. Purified protein was then diluted in assay buffer (50 mM Tris pH 7.0, 1% glycerol, 20 μM methylene blue, 0.05% Tween-20, 20 mM sodium ascorbate, 100 units/mL catalase to obtain a final IDO1 concentration of 40 nM. IDO1 solution (30 μM) or buffer alone (30 μM) were dispensed to wells of the assay plate using a BioRAPTR liquid dispenser (Beckman Coulter). Assay plates containing compound and IDO1 enzyme were incubated at room temperature for 30 minutes. Afterwards, 10 μL of 400 μM tryptophan in assay buffer were added to each well of the assay plate using a BioRAPTR liquid dispenser. Plates were incubated at room temperature for 60 minutes and reactions were quenched by addition of 10 μL of 0.5 M methyl isonipecotate in dimethyl sulfoxide. Plates were sealed and incubated at 37° C. for 4 hours or 50° C. for 2 h. The plates are allowed to cool and then centrifuged for 1 minute at 1000×g. The resulting fluoresence was measured in an Envision plate reader (Perkin Elmer) with a 400/25 nm excitation filter and an 510/20 nm emission filter.
Measurement: IC50 = 1960 nM (pChEMBL 5.710000038146973)
Descriptors: MW 440.32, ALogP 2.47, HBA 7, HBD 3, PSA 112.64, QED 0.16
Compound (SMILES): CC(C)(C)c1nc(N2CCN(CCCCNC(=O)N3CCc4c([nH]c5ccccc45)C3)CC2)cc(C(F)(F)F)n1
InChIKey: JWWLBNFBBLUIHS-UHFFFAOYSA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Radioligand Binding Assay: Stably transfected HEK cells expressing the human D2-long and the D3 dopamine receptor were developed using the pIRESneo2 bicistronic expression vector (Clontech, Palo Alto, Calif.). Levels of expression of human D2 and D3 dopamine receptors in HEK cells were 57, 941±14,686 fmol/mg protein and 4202±1516 fmol/mg protein, respectively. Competition curves were performed using 125I-IABN. Membrane homogenates (50 μL) were suspended in 50 mM Tris-HCl/150 mM NaCl/10 mM EDTA buffer, pH 7.5 and incubated with 50 μL of 125I-IABN at 37° C. for 60 min. Nonspecific binding was defined using 4 μM (+)-butaclamol. The radioligand concentration used was approximately equal to the Kd value, and the concentration of the competitive inhibitor ranged over five orders of magnitude. Binding was terminated by the addition of cold wash buffer (10 mM Tris-HCl/150 mM NaCl, pH 7.4) and filtration over a glass-fiber filter (Schleicher and Schuell No. 32). Filters were washed with 10 mL of cold buffer, and the radioactivity was quantitated. A Packard Cobra gamma counter was used for 125I-labeled radioligands (efficiency=75%). The protein concentration was determined using a BCA reagent (Pierce) with bovine serum albumin as the protein standard.
Measurement: Ki = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 557.67, ALogP 4.94, HBA 5, HBD 2, PSA 80.39, QED 0.42
Compound (SMILES): Cc1cc(Cc2ccc(C(=O)NCC(=O)N3[C@H](C4CCCCC4)CC[C@@H]3C(C)(C)C#N)cc2C)c(=O)[nH]n1
InChIKey: DGHRYDZXERNVHH-IZZNHLLZSA-N
Target: Calcitonin gene-related peptide type 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q16602
Assay: Determination of Binding Affinity for CGRP Receptor by Radioligand Binding Assay in SK-N-MC Cell Membranes: The binding affinity assay of compounds for human CGRP receptor was carried out by inhibition of radiolabeled ligand [125I]-CGRP binding in human neuroblastoma cell line SK-N-MC cell membranes.Cell membranes prepared from SK-N-MC cells expressing CGRP receptors endogenously (Muff et al., Ann N Y Acad Sci. 1992; 657: 106-116) were used for radioligand binding assay. Radioligand binding assay was performed using 96-well microplate in a total volume of 200 μL in each well. A 2.5 μL of serial dilution of compound dissolved in dimethylsulfoxide (DMSO) was mixed with SK-N-MC cell membranes (40 μg membrane protein per well) and [125I]-CGRP (PerkinElmer NEX354, final concentration of 150 pM) in an assay buffer consisting of 50 mM Tris-HCl, 5 mM MgCl2 and 0.1% bovine serum albumin (pH 7.4). The assay plate was incubated with shaking on a plate shaker at room temperature for 90 minutes. The incubation was terminated by filtration through a GF/C glass fiber filter plate (Merck Millipore) pre-soaked with 0.3% polyethyleneimine (PEI). Filters were washed 4 times with 300 μL of ice-cold assay buffer. After drying the filter plate, 100 μL of scintillation fluid (PerkinElmer, MicroScint-20) was added to each well and the radioactivity was counted using a TopCount NXT (PerkinElmer). Non-specific binding was determined in the presence of 1.25 μM unlabelled human α-CGRP (Bachem). The radioactivity was converted to the percent of specific binding using the equation below.% ⁢ ⁢ of ⁢ ⁢ specific ⁢ ⁢ binding = ( Y - Y ⁢ ⁢ min ) ( Y ⁢ ⁢ max - Y ⁢ ⁢ min ) × 100 [ Number ⁢ ⁢ 1 ]In the formulae, Y is observed radioactivity, Ymax is total bound activity, and Ymin is non-specific bound activity.From these data, the concentration of compound required for 50% inhibition of radioligand binding (IC50) was determined using Prism (GraphPad Inc.). The IC50 value is then converted to the equilibrium dissociation constant (Ki) using the Cheng-Prusoff equation below (Cheng & Prusoff (1973) Biochem. Pharmacol. 22, 3099-3108).Ki = IC 50 1 + [ L ] Kd [ Number ⁢ ⁢ 2 ]In the formulae, [L] is the concentration of radioligand, Kd is the apparent dissociation constant of the radioligand for the receptor as determined by saturation binding assay with [125I]-CGRP.
Measurement: Ki = 0.11 nM (pChEMBL 9.960000038146973)
Descriptors: MW 517.67, ALogP 4.2, HBA 5, HBD 2, PSA 118.95, QED 0.57
Compound (SMILES): CC(=O)N1CCN(c2cccc(-c3cc(-c4cn(C)nc4C(F)(F)F)c4c(N)ncnn34)c2)C(=O)C1(C)C
InChIKey: FWZBTMGBGHAOKP-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: ADP-Glo Format PI3K Assay: The ADP-Glo format PI3K assays were performed in Proxiplate 384-well plates (Perkin Elmer #6008280). The final assay volume was 2 μl prepared from 1 μl additions of enzyme/PIP2:PS lipid (Invitrogen #PV5100) mixture and 1 μl ATP (provided in kit, Promega #V9101) and test compounds in assay buffer (50 mM HEPES pH 7.5, 3 mM MgCl2, 100 mM NaCl, 0.5 mM EGTA, 2 mM DTT, 0.03% CHAPS). The reaction was initiated by the combination of enzyme/lipid, ATP, and test compounds. The reaction mixture was incubated at room temperature for 30 minutes (PI3K Alpha, Beta, Gamma) or 3 hours for PI3K Delta. ADP-Glo (2 μl), followed by Kinase Detection reagent (4 μl), were added to reactions following the initial incubation and allowed to incubate 40 minutes at room temperature. The reaction mixture was analyzed on the TOPCOUNT (Perkin Elmer). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of enzyme in the assays are PI3K Alpha [0.5 nM], PI3K Beta [2 nM], PI3K Gamma [20 nM], PI3K Delta [0.5 nM]. ATP final concentrations are as follows: for Alpha [10 μM], for Beta [12.5 μM], for Gamma [6.5 μM], for Delta [100 μM]. Lipid final concentration was the same for all enzymes, [25 μM]. Dose response curves were generated to determine the concentration required to inhibit 50% of activity. Compounds were dissolved at 0.12 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. The IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 526.52, ALogP 3.37, HBA 8, HBD 1, PSA 114.65, QED 0.44