lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): NCCCCC(NC(=O)C1CCCC1)C(=O)COc1ccccc1F
InChIKey: CJMLKFUQJAPXLR-UHFFFAOYSA-N
Target: Procathepsin L (SINGLE PROTEIN), Homo sapiens
UniProt: P07711
Assay: Inhibition of Lysine Gingipain: The capacities of compounds of the present invention to inhibit the activity of lysine gingipain were measured in a fluorogenic assay similar to those described by Barret (Biochemical Journal. 1980, 187(3), 909). The specific assay conditions were as follows. Buffer: pH=7.5, 100 mM Tris-HCl, 75 mM NaCl, 2.5 mM CaCl2), 10 mM cysteine, 1% DMSO after all additions. Protein: 0.1 nM Kgp, isolated from culture of Porphyromonas gingivalis, as described by Pike et al. (J. Biol. Chem. 1994, 269(1), 406), and Potempa and Nguyen (Current Protocols in Protein Science. 2007, 21.20.1-21.20.27). Fluorogenic substrate: 10 μM Z-His-Glu-Lys-MCA. Time=90 minutes. Temperature=37° C. Each compound: 10 concentrations, starting at either 100 μM or 100 nM, with lower concentrations generated by serial 3-fold dilutions. By testing a range of concentrations for each compound, the concentration required to inhibit the activity of lysine gingipain by 50% (the IC50) was determined. Under the described assay conditions, the signal-to-noise ratio was excellent, and the Z factor was greater than 0.6. Compounds in Table 1 were tested, as well as the compounds set forth in Table 2 below.The capacities of compounds of the present invention to inhibit the activity of cathepsins B, H, K, L, and S were measured in similar assays. Boc-Leu-Arg-Arg-AMC (20 μM) in sodium acetate buffer (50 mM, pH 5.5) containing DTT (1 mM) and EDTA (2 mM) was used for the Cathepsin B assay. L-Arg-AMC (20 μM) in sodium acetate buffer (50 mM, pH 5.5) containing DTT (1 mM) and EDTA (2 mM) was used for the Cathepsin H assay. Z-Phe-Arg-AMC (10 μM) in HEPES buffer (50 mM, pH 7.4) containing DTT (2.5 mM) and EDTA (1 mM) was used for the Cathepsin K assay. Z-Phe-Arg-AMC (20 μM) in sodium acetate buffer (50 mM, pH 5.5) containing DTT (1 mM) and EDTA (2 mM) was used for the Cathepsin L assay. Z-Leu-Arg-AMC (10 μM) in sodium acetate buffer (25 mM, pH 4.5) containing DTT (2.5 mM) and NaCl (50 mM) was used for the Cathepsin S assay.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 350.43, ALogP 2.58, HBA 4, HBD 2, PSA 81.42, QED 0.64
Compound (SMILES): Cc1cnc(-c2cc(OC3CCN(CC(F)(F)F)CC3)cc(C(=O)N[C@H](C)c3cnc(C(F)(F)F)nc3)c2)s1
InChIKey: HEBACSHOFHMYBR-OAHLLOKOSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Biological Assay: A fluorescent imaging plate reader (FILEX/FLIPR station; Molecular Devices) was used to monitor intracellular calcium levels using the calcium-chelating dye Rim-4 (Molecular Probes). The excitation and emission wavelengths used to monitor fluorescence were 470-495 nm and 515-575 nm, respectively. Cells expressing purinergic receptors P2X3 (human) or P2X2/3 (human) were plated at a density of 15,000 cells/well in collagen-coated 384-well plates approximately 20 hours before beginning the assay. On the day of the assay, 20 μl of loading buffer (Hank's balanced salt solution, 20 mM HEPES, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, 5 mM probenecid, 10 mM D-glucose monohydrate, 2 μM Fluo-4, and 5 units/mL, hexokinase, pH=7.4) was added and cells dye-loaded for 90 min at 37° C. The dye supernatant was removed and replaced with 45 μl probenecid buffer (Hank's balanced salt solution, 20 mM HEPES, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, 5 mM probenecid, 10 mM D-glucose monohydrate, pH=7.4). The test compound was added in a volume of 5 μl and allowed to incubate for 30 min at 37° C.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 573.56, ALogP 5.82, HBA 7, HBD 1, PSA 80.24, QED 0.36
Compound (SMILES): CC(C)N1CCN(c2cc(C(=O)Nc3cc4cc(-c5cnn(C)c5)ccc4cn3)ccn2)CC1
InChIKey: LRGIQHCIXRKTHV-UHFFFAOYSA-N
Target: Proto-oncogene Wnt-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P04628
Assay: The screening assay for Wnt activity: The screening assay for Wnt activity is described as follows. Reporter cell lines can be generated by stably transducing cancer cell lines (e.g., colon cancer) or primary cells (e.g., IEC-6 intestinal cells) with a lentiviral construct that includes a Wnt-responsive promoter driving expression of the firefly luciferase gene.SW480 colon carcinoma cells were transduced with a lentiviral vector expressing luciferase with a human Sp5 promoter consisting of a sequence of eight TCF/LEF binding sites. SW480 cells stably expressing the Sp5-Luc reporter gene and a hygromycin resistance gene were selected by treatment with 150 μg/mL of hygromycin for 7 days. These stably transduced SW480 cells were expanded in cell culture and used for all further screening activities. Each compound was dissolved in DMSO as a 10 mM stock and used to prepare compound source plates. Serial dilution (1:3, 10-point dose-response curves starting from 10 μM) and compound transfer was performed using the ECHO 550 (Labcyte, Sunnyvale, Calif.) into 384-well white solid bottom assay plates (Greiner Bio-One) with appropriate DMSO backfill for a final DMSO concentration of 0.1%. For Sp5-Luc reporter gene assays, the cells were plated at 4,000 cells/well in 384-well plates with a DMEM medium containing 1% fetal bovine serum, and 1% Penicillin-Streptomycin and incubated for 36 to 48 hours at 37° C. and 5% CO2. Following incubation, 15 μl of BriteLite Plus luminescence reagent (Perkin Elmer) was added to each well of the 384-well assay plates. The plates were placed on an orbital shaker for 2 min and then luminescence was quantified using the Envision (Perkin Elmer) plate reader. Readings were normalized to DMSO only treated cells, and normalized activities were utilized for EC50 calculations using the dose-response log (inhibitor) vs. responsevariable slope (four parameters) nonlinear regression feature available in GraphPad Prism 5.0 (or Dotmatics).
Measurement: EC50 = 116 nM (pChEMBL 6.940000057220459)
Descriptors: MW 455.57, ALogP 3.81, HBA 7, HBD 1, PSA 79.18, QED 0.49
Compound (SMILES): Cn1c(C(O)c2cc(-c3ncnc4cc(N5CCOCC5)ccc34)c(F)cc2Cl)cccc1=O
InChIKey: WYNSFINJUTXESJ-UHFFFAOYSA-N
Target: DNA-dependent protein kinase catalytic subunit (SINGLE PROTEIN), Homo sapiens
UniProt: P78527
Assay: Biochemical Assay: The kinase assay was carried out in streptavidin-coated 348-well microtitre flashplates. To this end, 1.5 ug of DNA-PK/protein complex and 100 ng of biotinylated substrate, such as, for example, PESQEAFADLWKK-biotin-NH2 (biotin-DNA-PK peptide), were incubated for 90 min at room temperature in a total volume of 36.5 ul (34.25 mM HEPES/KOH; 7.85 mM Tris HCl; 68.5 mM KCl; 5 uM ATP; 6.85 mM MgCl2; 0.5 mM EDTA; 0.14 mM EGTA; 0.69 mM DTT; pH 7.4) with 500 ng of DNA from calf thymus, 0.1 uCi of 33P-ATP and 1.8% of DMSO per well with and without the test compound. The reaction was stopped using 50 ul/well of 200 mM EDTA. After incubation for a further 30 min at room temperature, the liquid was removed. Each well was washed three times with 100 ul of 0.9% saline solution. A nonspecific reaction (blank value) was determined using 10 uM of an innate kinase inhibitor. The radioactivity measurement was carried out using a TopCount. IC50 values were calculated in RS1.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 480.93, ALogP 3.71, HBA 7, HBD 1, PSA 80.48, QED 0.48
Compound (SMILES): CCCCNc1ncc(C(=O)NCc2ccc(-n3ccnc3-c3ccccc3)cc2)c(N[C@H]2CC[C@H](O)CC2)n1
InChIKey: WHTNOTFWPYUVNF-YOCNBXQISA-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 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 539.68, ALogP 5.19, HBA 8, HBD 4, PSA 116.99, QED 0.19
Compound (SMILES): Cc1cc(-c2[nH]c3ccc(C4CCN(C(=O)CN(C)C)CC4)cc3c2C(C)C)cn2ncnc12
InChIKey: NZMVKBPHIXDNBP-UHFFFAOYSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
Assay: TLR7/8/9 Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 7.4 nM (pChEMBL 8.130000114440918)
Descriptors: MW 458.61, ALogP 4.58, HBA 5, HBD 1, PSA 69.53, QED 0.48
Compound (SMILES): C[C@H](NC(=O)c1c(N)nn2cccnc12)c1cc2cccc(C#Cc3cocn3)c2c(=O)n1-c1ccccc1
InChIKey: YLGVWMNBLDMDKA-SFHVURJKSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: PI3-Kinase HTRF Assay: A PI3-Kinase HTRF® assay kit (cat No. 33-016) purchased from Millipore Corporation was used to screen compounds provided herein. This assay used specific, high affinity binding of the GRP1 pleckstrin homology (PH) domain to PIP3, the product of a Class 1A or 1B PI3 Kinase acting on its physiological substrate PIP2. During the detection phase of the assay, a complex was generated between the GST-tagged PH domain and biotinylated short chain PIP3. The biotinylated PIP3 and the GST-tagged PH domain recruited fluorophores (Streptavidin-Allophycocyanin and Europium-labeled anti-GST respectively) to form the fluorescence resonance energy transfer (FRET) architecture, generating a stable time-resolved FRET signal. The FRET complex was disrupted in a competitive manner by non-biotinylated PIP3, a product formed in the PI3 Kinase assay.PI3 Kinase α, β, γ or δ activity was assayed using the PI3 Kinase HTRF® assay kit (catalogue No. 33-016) purchased from Millipore Corporation. Purified recombinant PI3Kα (catalogue No. 14-602-K), PI3Kβ (catalogue No. 14-603-K), PI3Kγ (catalogue No. 14-558-K), and PI3Kδ (catalogue No. 14-604-K) were obtained from Millipore Corporation. Purified recombinant PI3K enzyme was used to catalyze the phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP2 at 10 μM) to phosphatidylinositol 3,4,5-trisphosphate (PIP3) in the presence of 10 μM ATP. The assay was carried out in 384-well format and detected using a Perkin Elmer EnVision Xcite Multilabel Reader. Emission ratios were converted into percent inhibitions and imported into GraphPad Prism software. The concentration necessary to achieve inhibition of enzyme activity by 50% (IC50) was calculated using concentrations ranging from 20 μM to 0.1 nM (12-point curve). IC50 values were determined using a nonlinear regression model available in GraphPad Prism 5.
Measurement: IC50 = 2000 nM (pChEMBL 5.699999809265137)
Descriptors: MW 515.53, ALogP 3.49, HBA 9, HBD 2, PSA 133.34, QED 0.34
Compound (SMILES): CCCCO/N=C(\C)[C@@H]1CCc2cc([C@H]3CC[C@](N)(COP(=O)(O)O)C3)ccc2C1
InChIKey: VAOYAPAGOCRXFS-MHQSGURASA-N
Target: Sphingosine-1-phosphate lyase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O95470
Assay: S1P1 Binding Assay: Membranes were prepared from CHO cells expressing human S1P1. Cells pellets (1×109 cells/pellet) were suspended in buffer containing 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.5, 50 mM NaCl, 2 mM EDTA (Ethylenediaminetetraacetic acid) and Protease Inhibitor cocktail (Roche), and disrupted on ice using the Polytron homogenizer. The homogenate was centrifuged at 20,000 rpm (48,000 g) and the supernatant was discarded. The membrane pellets were resuspended in buffer containing 50 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM MgCl2, 2 mM EDTA and stored in aliquots at −80° C. after protein concentration determination. Membranes (2 μg/well) and 0.03 nM final concentration of 33P-S1P ligand (1 mCi/ml, Perkin elmer or American Radiolabeled Chemicals) diluted in assay buffer (50 mM HEPES, pH7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% fatty acid free BSA (bovine serum albumin), 1 mM NaF) were added to the compound plates (384 Falcon v-bottom plate (0.5 μl/well in a 11 point, 3-fold dilution). Binding was performed for 45 minutes at room temperature, terminated by collecting the membranes onto 384-well Millipore FB filter plates, and radioactivity was measured by TOPCOUNT®. The competition data of the test compounds over a range of concentrations was plotted as percentage inhibition of radioligand specific binding. The IC50 is defined as the concentration of competing ligand needed to reduce specific binding by 50%.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 438.51, ALogP 4.06, HBA 5, HBD 3, PSA 114.37, QED 0.23
Compound (SMILES): CC#CC(=O)N1CC[C@]2(C1)C[C@H](n1nc(-c3cnn(Cc4ccc(F)cc4)c3)c(C(N)=O)c1N)C2
InChIKey: ZYQIUDPJBOUHCC-FEHRVWHQSA-N
Target: Tyrosine-protein kinase TXK (SINGLE PROTEIN), Homo sapiens
UniProt: P42681
Assay: Lanthscreen Eu Kinase Binding Assay : Lanthscreen Eu Kinase Binding assay for BMX is performed as described above for BTK except that 1 nM human recombinant full length TEC (His-tagged) kinase and 1 nM Alexa Fluor647-labeled Kinase Tracer #178 were used instead.Representative compounds of the present invention are assessed for inhibition of BTK, BMX, and TXK measuring phosphorylation of a substrate (Z′-LYTE assay, Life Technologies) and TEC measuring displacement of a tracer (Lanthscreen® Eu Kinase Binding assay, Life Technologies).
Measurement: IC50 = 92 nM (pChEMBL 7.039999961853027)
Descriptors: MW 475.53, ALogP 2.19, HBA 7, HBD 2, PSA 125.06, QED 0.55
Compound (SMILES): O=C(c1cnc2ccc(-c3cnc(Cl)c(NS(=O)(=O)c4ccccc4F)c3)cn12)N1CCOCC1
InChIKey: BUVRUPZGOSXRSC-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42338
Assay: Inhibition Assay of PI3K: An exemplary system is PI 3-Kinase (human) HTRF Assay from Upstate. The assay can be carried out according to the procedures suggested by the manufacturer. Briefly, the assay is a time resolved FRET assay that indirectly measures PIP3 product formed by the activity of a PI3-K. The kinase reaction is performed in a microtitre plate (e.g., a 384 well microtitre plate). The total reaction volume is approximately 20 ul per well. In the first step, each well receives 2 ul of test compound in 20% dimethylsulphoxide resulting in a 2% DMSO final concentration. Next, approximately 14.5 ul of a kinase/PIP2 mixture (diluted in 1× reaction buffer) is added per well for a final concentration of 0.25-0.3 ug/ml kinase and 10 μM PIP2. The plate is sealed and incubated for 15 minutes at room temperature. To start the reaction, 3.5 ul of ATP (diluted in 1× reaction buffer) is added per well for a final concentration of 10 μM ATP. The plate is sealed and incubated for 1 hour at room temperature. The reaction is stopped by adding 5 ul of Stop Solution per well and then 5 ul of Detection Mix is added per well. The plate is sealed, incubated for 1 hour at room temperature, and then read on an appropriate plate reader.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 515.95, ALogP 3.46, HBA 7, HBD 1, PSA 105.9, QED 0.41
Compound (SMILES): CC[C@@H]1CN(c2nc(=O)n(C)c3ccc(C#N)nc23)[C@@H](C)CN1C(c1ccc(OC(F)(F)F)cc1)c1ccccn1
InChIKey: XSORAHGOFUGSSD-ZSFYKYPJSA-N
Target: Diacylglycerol kinase zeta (SINGLE PROTEIN), Homo sapiens
UniProt: Q13574
Assay: In Vitro DGK Inhibition Assay: The reactions were carried out in 50 mM MOPS pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 μM CaCl2, and 1 mM DTT (assay buffer). The reactions using a detergent/lipid micelle substrate also contained 50 mM octyl B-D-glucopyranoside. The lipid substrate concentrations were 11 mM PS and 1 mM DAG for the detergent/lipid micelle reactions. The lipid substrate concentrations were 2 mM PS, 0.25 mM DAG, and 2.75 mM PC for the extruded liposome reactions (5 mM total lipid). The reactions were carried out in 150 μM ATP. The enzyme concentrations for the DGKα and DGKζ were 5 nM. The compound inhibition studies were carried out as follows: 25 nL (ADPGLO assay) or 50 nL (LIPGLO assay) droplets of each test compound (top concentration 10 mM with 11 point, 3-fold dilution series for each compound) solubilized in DMSO were transferred to wells of a white 1536 well plate (Corning 3725). A 5 mL enzyme/lipid substrate solution at 2× final reaction concentration was prepared by combining 2.5 mL 4× enzyme solution (20 nM DGKα or DGKζ (prepared as described below) in assay buffer) and 2.5 mL of either 4× liposome or 4× detergent/lipid micelle solution (compositions described below) and incubated at room temperature for 10 minutes. Next, 1 μL 2× enzyme/lipid substrate solution was added to wells containing the test compound and reactions were initiated with the addition of 1 μL 300 uM ATP. The reactions were allowed to proceed for 2 hr (ADPGLO assay) or 1 hr (LIPGLO assay), after which 2 μL Glo Reagent (Promega V9101) was added and incubated for 40 minutes. Next, 4 μL Kinase Detection Reagent was added and incubated for 30 minutes. Luminescence was recorded using an EnVision microplate reader. The percent inhibition was calculated from the ATP conversion generated by no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition.
Measurement: IC50 = 290 nM (pChEMBL 6.539999961853027)
Descriptors: MW 563.58, ALogP 4.57, HBA 9, HBD 0, PSA 100.17, QED 0.34
Compound (SMILES): CC1c2nnc(C3CC4(C3)CN(c3ncccn3)C4)n2-c2ccc(Cl)cc2CN1C(=O)OC(CF)CF
InChIKey: KXHBRROPICKYDV-UHFFFAOYSA-N
Target: Vasopressin V1a receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P37288
Assay: Vasopressin V1A Receptor Antagonist Assay: The purpose of this assay was to determine the inhibitory effect of synthesized compounds on the Vasopressin V1a receptor. The assay as performed in Chinese Hamster Ovary (CHO) cells expressing the human Arginine Vasopressin Receptor 1a (AVPR1a). Arginine Vasopressin (AVP) evokes an increase in intracellular calcium in CHO-AVPR1a cells which is measured in a fluorescence assay on the FLIPRTETRA using calcium sensitive dyes. Test compounds were assessed for their ability to affect the magnitude of the response to AVP, with antagonists showing a concentration-dependent reduction in the AVP-mediated fluorescence. Compounds were tested in duplicate in a 10-point, 1:3 dilution series starting at a nominal concentration of 3 μM in the assay.CHO-AVPR1a cells were maintained in routine culture in T175 Flasks at 37° C., 5% CO2. The growth medium consists of Ham's F12 media supplemented with 10% v/v fetal bovine serum, 1× non-essential amino acids, and 0.4 mg/ml Geneticin G418.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 529.98, ALogP 4.42, HBA 8, HBD 0, PSA 89.27, QED 0.48
Compound (SMILES): COc1nccnc1C(O)c1cc(-c2ncnc3cc(N4CCOCC4)ccc23)c(F)cc1Cl
InChIKey: YNJIBQSLMOPTQF-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Patch Clamp Assay: The patch-clamp measurement was carried out at room temperature in whole-cell configuration on human embryonic kidney cells (HEK293) which have been transfected in a stable manner with the hERG gene.The whole-cell configurations were carried out using an automated patch clamp device (Patchliner, Nanion Technologies, Munich). This is a glass chip-based system with which automated whole-cell measurements on up to 8 cells simultaneously are possible. The glass chip has a hole of defined size to which the cell is transferred into the Gigaseal by application of a reduced pressure and brought into the whole-cell configuration. Buffer, cell suspension and test substances were added to microchannels of the chip using a Teflon-coated pipette. The cells were clamped to a holding potential of -80 mV. For measurement of substance-promoted inhibition of the Kv11.1 channel, the following voltage protocol was applied at 10-second intervals: 51 ms/-80 mV, 500 ms/+40 mV, 500 ms/-40 mV, 200 ms/-80 mV. The leakage current is subtracted by means of the P4 method. The cells were resuspended in extracellular buffer (EC) and applied to the chip. After the cell had been collected, the seal was improved by addition of a seal enhancer buffer. As soon as the whole-cell configuration had been reached, the seal enhancer buffer was washed out and replaced by extracellular buffer. The measurement started in EC for 1.5 min. DMSO (vehicle control, 0.1% of DMSO) was then applied, and the control current was recorded for 3 min. The test substance was subsequently added twice in the same concentration, and the potassium current was measured for 3.5 min in each case.
Measurement: Ki > 25000 nM (pChEMBL 4.6020002365112305)
Descriptors: MW 481.92, ALogP 3.81, HBA 8, HBD 1, PSA 93.49, QED 0.46
Compound (SMILES): CC(=O)N1CCC(c2cc(-c3ccc(NC(=O)c4cccn(-c5ccccc5)c4=O)cc3)c3c(N)ncnn23)CC1
InChIKey: ZRLFZFXBBUSCFN-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor TYRO3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06418
Assay: Enzymatic Assay: Tyro3, and Mer:The kinase assay buffer contained 50 mM HEPES, pH7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% NP-40 and 2 mM DTT. 0.1 ul test compounds dissolved in DMSO were transferred from compound plates to white 384-well assay plates (Greiner LUMITRAC plates). The final concentration of DMSO was 1.25%. Enzyme solutions of 5.1 nM phosphor-Axl, or 0.0625 nM c-Mer (Carna Biosciences, 08-108), or 0.366 nM Tyro3 (Life Technologies, PR7480A) were prepared in assay buffer. A 1 mM stock solution of peptide substrate Biotin-EQEDEPEGDYFEWLE-amide SEQ ID NO: 1 (Quality Controlled Biochemicals, MA) dissolved in DMSO was diluted to 1 uM in assay buffer containing 2000 uM ATP. 4 ul enzyme solution (or assay buffer for the enzyme blank) was added to the appropriate wells in each plate, and then 4 ul/well substrate solution was added to initiate the reaction. The plate was protected from light and incubated at room temperature for 60 min. The reaction was stopped by adding 4 ul detection solution containing 50 mM Tris-HCl, pH7.8, 150 mM NaCl, 0.05% BSA, 45 mM EDTA, 180 nM SA-APC (Perkin Elmer, CR130-100) and 3 nM Eu-W1024 anti-phosphotyrosine PY20 (Perkin Elmer, AD0067). The plate was incubated for 1 h at room temperature, and HTRF (homogenous time resolved fluorescence) signal was measured on a PHERAstar FS plate reader (BMG labtech).
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 547.62, ALogP 4.11, HBA 8, HBD 2, PSA 127.62, QED 0.34
Compound (SMILES): C=CC(=O)Nc1cccc(Nc2nc(Nc3cccc(S(N)(=O)=O)c3)ncc2C)c1
InChIKey: GBBOTMNQSLLOPI-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Omnia Assay: Briefly, 10x stocks of EGFR-WT (PV3872) from Invitrogen and EGFR-T790M/L858R (40350) from BPS Bioscience, San Diego, Calif., 1.13×ATP (AS001A) and appropriate Tyr-Sox conjugated peptide substrates (KCZ1001) were prepared in 1× kinase reaction buffer consisting of 20 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 5 mM β-glycerophosphate, 5% glycerol (10× stock, KB002A) and 0.2 mM DTT (DS001A). 5 μL of each enzyme were pre-incubated in a Corning (#3574) 384-well, white, non-binding surface microtiter plate (Corning, N.Y.) for 30 min. at 27° C. with a 0.5 μL volume of 50% DMSO and serially diluted compounds prepared in 50% DMSO. Kinase reactions were started with the addition of 45 μL of the ATP/Tyr-Sox peptide substrate mix and monitored every 30-90 seconds for 60 minutes at λex360/λem485 in a Synergy4 plate reader from BioTek (Winooski, Vt.). At the conclusion of each assay, progress curves from each well were examined for linear reaction kinetics and fit statistics (R2, 95% confidence interval, absolute sum of squares). Initial velocity (0 minutes to 30 minutes) from each reaction was determined from the slope of a plot of relative fluorescence units vs time (minutes) and then plotted against inhibitor concentration to estimate IC50 from log[Inhibitor] vs Response, Variable Slope model in GraphPad Prism from GraphPad Software (San Diego, Calif.).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 424.49, ALogP 3.04, HBA 7, HBD 4, PSA 139.1, QED 0.43
Compound (SMILES): CC(C)(C)S(=O)(=O)Nc1cccc(-n2ccc3cnc(Nc4ccc(C5CCNCC5)cc4)nc32)c1
InChIKey: IZLQRTZZHBLSKO-UHFFFAOYSA-N
Target: Bromodomain testis-specific protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q58F21
Assay: AlphaLisa Assay: 1. Add 20 μl of the diluted compound to each well of the 384-well opti plate2. Tap the plate gently3. Add 10 μl of the enzyme to each well of the opti plate4. Cover with plate sealer5. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.6. Incubate the plate at room temperature for 10 minutes7. Add 10 μl of the substrate8. Cover with plate sealer9. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.10. Cover with plate sealer and incubate at room temperature for 1 hour11. After incubation, transfer 10 μl of reaction mix to fresh wells of a 384-well plate.12. Add 10 μl acceptor bead from working stock.13. Cover with plate sealer14. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.15. Cover with plate sealer and incubate at room temperature for 30 minutes16. Add 10 μl donor bead from working stock (in dark).17. Cover with plate sealer18. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.19. Cover with plate sealer and incubate at room temperature for 15-30 minutes20. Read the plate in pherastar (alpha screen protocol)
Measurement: IC50 = 322 nM (pChEMBL 6.489999771118164)
Descriptors: MW 504.66, ALogP 5.17, HBA 7, HBD 3, PSA 100.94, QED 0.33
Compound (SMILES): O=C(c1c(F)ccc(NS(=O)(=O)c2ccc(C(F)(F)F)cc2)c1F)c1c[nH]c2ncnc(OCC3CC3)c12
InChIKey: SFHPGRLOFYABKA-UHFFFAOYSA-N
Target: Cytochrome P450 2D6 (SINGLE PROTEIN), Homo sapiens
UniProt: P10635
Assay: Inhibition Assay: The inhibition of CYP enzyme activity (IC50) for each of CYP1A2, CYP2C19, CYP2C9, CYP2D6, CYP3A4(BFC) and CYP3A4(BQ) is determined for compounds, where inhibition of metabolism of a known substrate leads to a decrease in the fluorescence of the metabolized product. The fluorescence of the product is monitored as a function of compound concentration. Compounds are dissolved in dimethyl sulfoxide to a concentration of 100 mM. These are diluted 1 μL into 82 μL of acetonitrile. An 11 μL aliquot of this solution is then added to 204 μL of cofactor mix (1.3% NADPH Regeneration system Solution A, 1.04% NADPH Regeneration system Solution B from BD Biosciences, 5% acetonitrile and 0.05% dimethyl sulfoxide). These are then serially diluted 1:1 (160 μL to 160 μL co-factor mix) for a total of 10 points. A 10 μL aliquot of this final mixture is dispensed into 384 well assay plates and incubated for 10 minutes at 37° C. Enzyme and substrate mix (10 μL; 0.5 pmol CYP1A2/5 μM CEC; 1.0 pmol CYP2C9/75 μM MFC; 0.5 pmol CYP2C19/25 μM CEC; 1.5 pmol CYP2D6/1.5 μM AMMC; 1.0 pmol CYP3A4/50 μM BFC; or 1.0 pmol CYP3A4/40 μM BQ) is added to these assay plates. Assay plates are incubated at 37° C. (CYP1A2-15 min; CYP2C9-45 min; CYP2C19, 2D6 and 3A4-30 min) and read in a Tecan Safire 2 plate reader (CYP1A2, 2C19 and 3A4 409 ex/460 em; CYP2C9 and 2D6 409 ex/530 em).
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 552.48, ALogP 5.08, HBA 6, HBD 2, PSA 114.04, QED 0.23
Compound (SMILES): CCCCCC[C@@H]1Cc2ccc([C@H]3CC[C@](N)(COP(=O)(O)O)C3)cc2CO1
InChIKey: MJKUOBXNYRXRCS-CEWLAPEOSA-N
Target: Sphingosine-1-phosphate lyase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O95470
Assay: Receptor [35S] GTPgammaS Binding Assays: (S1P1 GTPgammaS/S1P3 GTPgammaS): Compounds were loaded in a 384 Falcon v-bottom plate (0.5 l/well in a 11 point, 3-fold dilution). Membranes prepared from S1P1/CHO cells or EDG3-Gal5-bla HEK293T cells (EDG3 equivalent S1P3) were added to the compound plate (40 l/well, final protein 3 μg/well) with MULTIDROP®. [35S]GTP (1250 Ci/mmol, Perkin Elmer) was diluted in assay buffer: 20 mM HEPES, pH7.5, 10 mM MgCl2, 150 mM NaCl, 1 mM EGTA (ethylene glycol tetraacetic acid), 1 mM DTT (Dithiothreitol), 10 μM GDP, 0.1% fatty acid free BSA, and 10 μg/ml Saponin to 0.4 nM. 40 μl of the [35S] GTP solution was added to the compound plate with a final concentration of 0.2 nM. The reaction was kept at room temperature for 45 min. At the end of incubation, all the mixtures in the compound plate were transferred to Millipore 384-well FB filter plates via the VELOCITY11® Vprep liquid handler. The filter plate was washed with water 4 times by using the manifold Embla plate washer and dried at 60° C. for 45 min. MicroScint 20 scintillation fluid (30 μl) was added to each well for counting on the Packard TOPCOUNT®. EC50 is defined as the agonist concentration that corresponds to 50% of the Ymax (maximal response) obtained for each individual compound tested. The EC50 for Example 689 was determined to be 5.7 nM in the assay utilizing membranes prepared from S1P1/CHO cells. The EC50 for Example 689 was determined to be >2000 nM in the assay utilizing membranes prepared from EDG3-Gal5-bla HEK293T cells.A smaller value for GTPγS S1P1 EC50 value indicated greater activity for the compound in the GTPγS S1P1 binding assay. A larger value for the GTPγS S1P3 EC50 value indicated less activity in the GTPγS S1P3 binding assay. Example 689, which is the phosphate ester of Example 672, possessed activity as an agonist of S1P1 and is selective over S1P3. Example 697, which is the phosphate ester of Example 681, possessed activity as an agonist of S1P1 and is selective over S1P3. Thus the compounds of the present invention may be used in treating, preventing, or curing various S1P1 receptor-related conditions while reducing or minimizing the side effects due to S1P3 activity. The selectivity of the compounds of the present invention indicate their potential use in treating, preventing, or curing autoimmune and inflammatory diseases such as multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, lupus, psoriasis, or vascular diseases, while reducing or minimizing possible side effects due to S1P3 activity. Other potential uses of the compounds of the present invention include minimizing or reducing rejection of transplanted organs, while reducing or minimizing side effects due to S1P3 activity.
Measurement: EC50 < 10 nM (pChEMBL 8)
Descriptors: MW 411.48, ALogP 4.17, HBA 4, HBD 3, PSA 102.01, QED 0.42
Compound (SMILES): Cc1cc(-c2[nH]c3ccc(C4CCC(N5CC6(COC6)C5)CC4)nc3c2C(C)C)cn2ncnc12
InChIKey: RZANCIPEENVUSF-UHFFFAOYSA-N
Target: Toll-like receptor 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR96
Assay: TLR7/8/9 Inhibition Reporter Assays: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 880 nM (pChEMBL 6.059999942779541)
Descriptors: MW 470.62, ALogP 5.06, HBA 6, HBD 1, PSA 71.34, QED 0.45
Compound (SMILES): N#CCC1(n2ncc(-c3cc(-c4cccc(N)n4)cc4ncccc34)n2)CC(C#N)C1
InChIKey: QTLYTEJFTGGRRA-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Caliper Enzyme Assay at 1mM ATP: Compounds were added to a 384-well plate. Reaction mixtures contained 10 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% BSA, 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20% to 30% phosphorylation. The assays were stopped with 15 μL of 180 mM HEPES, pH 7.4, 20 mM EDTA, and 0.2% Coating Reagent 3. The assay plates were placed on a Caliper Life Science LC3000 instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 404 nM (pChEMBL 6.389999866485596)
Descriptors: MW 406.45, ALogP 3.68, HBA 8, HBD 1, PSA 130.09, QED 0.55
Compound (SMILES): CNCC(Cc1cccs1)c1ccc2ccccc2c1
InChIKey: APFLBUWSTCFNIE-UHFFFAOYSA-N
Target: Cytochrome P450 2D6 (SINGLE PROTEIN), Homo sapiens
UniProt: P10635
Assay: Inhibition Assay: Cytochrome P450 3A4 and 2D6:Recombinant enzymes, 3A4 and 2D6, generated using the ABL yeast expression system were used. For CYP3A4, the enzyme amount was 2 pmol per well, and the substrate (7-benzyloxy-4-(trifluoromethyl) coumarin; BFC) was used at 15 μM. The assay mixture also included K2HPO4 (pH 7.4; conc. 0.1 M) and NADPH (conc. 1 mM). The incubation time was 30 minutes, and the reference inhibitor was A-naphthoflavone. For CYP2D6, the enzyme amount was 2 pmol per well, and the substrate (7-methoxy-4-(aminomethyl)-coumarin; MAMC) was used at 20 μM. The assay mixture also included K2HPO4 (pH 7.4; conc. 0.1 M) and NADPH (conc. 0.06 mM). The incubation time was 35 minutes, and the reference inhibitor was quinidine.Briefly, 0.6 μL of serially diluted compound was added to 75 μL water. 10 μL of diluted compound in water was then added to each assay plate. 20 μL of a K2HPO4, enzyme, and substrate mixture was added. After 10 minutes of room temperature pre-incubation, 10 μL of NADPH was added, and the plates were placed at 37° C. for 30 or 35 minutes depending on the enzyme being tested. After the incubation was complete, 20 μL of 0.1% Tris/ACN was added to the plate. The plate was read on a FluorStar fluorescence plate reader.
Measurement: IC50 = 960 nM (pChEMBL 6.019999980926514)
Descriptors: MW 281.42, ALogP 4.45, HBA 2, HBD 1, PSA 12.03, QED 0.73
Compound (SMILES): Cc1cnc(-c2cc(OC[C@@]3(C)CCOC3)cc(C(=O)N[C@H](C)c3cnc(C(F)(F)F)nc3)c2)s1
InChIKey: FUZHKJYNGBDBHI-CMJOXMDJSA-N
Target: Cytochrome P450 2D6 (SINGLE PROTEIN), Homo sapiens
UniProt: P10635
Assay: CYP Inhibition and Pre-Incubation CYP Inhibition Assays: Use of in vitro assays to evaluate the inhibition potential of new drug candidates towards CYP-mediated metabolism has been shown to be effective as part of a strategy to minimise the chances of drug interactions with co-administered drugs. The inhibitory potency of the test compound towards 5 human cytochrome P450 isoforms (CYP1A2, 2C8, 2C9, 2D6, and 3A4) was determined. More preferred examples of the present invention have CYP inhibition IC50≥10 μM.For CYP3A4 time dependent inhibitory potential was also tested by applying a 30 min pre-incubation time of the test compound in metabolically active incubation system. If a time-dependent inhibition of CYP3A4 is observed, this is a hint of an irreversible mechanism-based inhibition of the CYP3A4 activity by the test compound. More preferred examples of the present invention have pre-incubation CYP inhibition IC50≥20 μM.Method Description CYP Inhibition AssayHuman liver microsomes (pooled, >30 male and female donors) were incubated with individual CYP isoform-selective standard probes (phenacetin for CYP1A2, amodiquine for CYP2C8, diclofenac for CYP2C9, dextromethorphan for CYP2D6 and midazolam for CYP3A4) in the absence and presence of increasing concentrations of the test compound in order to compare the extent of formation of the respective metabolite. In addition, a set of incubation in the absence of test compound was used as a negative control. Furthermore, the inhibitory potency of standard inhibitors was included as positive controls (fluvoxamine for CYP1A2, montelukast for CYP2C8, sulfaphenazole for CYP2C9, fluoxetine for CYP2D6, ketoconazole for CYP3A4 and mibefradil for CYP3A4-preincubation). Incubation conditions (protein and probe substrate concentration, incubation time) were optimised with regard to linearity and metabolite turnover. Incubation medium consisted of 50 mM potassium phosphate buffer (pH 7.4) containing 1 mM EDTA, NADPH regenerating system (1 mM NADP, 5 mM glucose 6-phosphate, glucose 6-phosphate dehydrogenase (1.5 U/mL). Sequential dilutions and incubations were performed on a Genesis Workstation (Tecan, Crailsheim, FRG) in 96-well plates at 37° C. A final incubation volume of 200 μL was used. Reactions were stopped by addition of 100 μL acetonitrile containing the respective internal standard. Precipitated proteins were removed by centrifugation of the well plate, supernatants were combined and analyses were performed by LC-MS/MS. The LC-MS/MS quantification of the metabolites paracetamol (CYP1A2), desethylamodiaquine (CYP2C8), 4-hydroxydiclofenac (CYP2C9), dextrorphan (CYP2D6), and 1-hydroxyidazolam (CYP3A4) was performed with a PE SCIEX API 3000 LC/MS/MS system (Applied Biosystems, MDS Sciex, Concord, Ontario, Canada).
Measurement: IC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 506.55, ALogP 5.22, HBA 7, HBD 1, PSA 86.23, QED 0.47
Compound (SMILES): Cc1cc(-c2[nH]c3ccc(C4CCN(C(=O)CN(C)C)CC4)cc3c2C(C)C)cn2ncnc12
InChIKey: NZMVKBPHIXDNBP-UHFFFAOYSA-N
Target: Toll-like receptor 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR96
Assay: TLR7/8/9 Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 3749 nM (pChEMBL 5.429999828338623)
Descriptors: MW 458.61, ALogP 4.58, HBA 5, HBD 1, PSA 69.53, QED 0.48
Compound (SMILES): C[C@H](NC(=O)c1c(N)nn2cccnc12)c1cc2cccc(C#Cc3cocn3)c2c(=O)n1-c1ccccc1
InChIKey: YLGVWMNBLDMDKA-SFHVURJKSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: PI3-Kinase HTRF Assay: A PI3-Kinase HTRF® assay kit (cat No. 33-016) purchased from Millipore Corporation was used to screen compounds provided herein. This assay used specific, high affinity binding of the GRP1 pleckstrin homology (PH) domain to PIP3, the product of a Class 1A or 1B PI3 Kinase acting on its physiological substrate PIP2. During the detection phase of the assay, a complex was generated between the GST-tagged PH domain and biotinylated short chain PIP3. The biotinylated PIP3 and the GST-tagged PH domain recruited fluorophores (Streptavidin-Allophycocyanin and Europium-labeled anti-GST respectively) to form the fluorescence resonance energy transfer (FRET) architecture, generating a stable time-resolved FRET signal. The FRET complex was disrupted in a competitive manner by non-biotinylated PIP3, a product formed in the PI3 Kinase assay.PI3 Kinase α, β, γ or δ activity was assayed using the PI3 Kinase HTRF® assay kit (catalogue No. 33-016) purchased from Millipore Corporation. Purified recombinant PI3Kα (catalogue No. 14-602-K), PI3Kβ (catalogue No. 14-603-K), PI3Kγ (catalogue No. 14-558-K), and PI3Kδ (catalogue No. 14-604-K) were obtained from Millipore Corporation. Purified recombinant PI3K enzyme was used to catalyze the phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP2 at 10 μM) to phosphatidylinositol 3,4,5-trisphosphate (PIP3) in the presence of 10 μM ATP. The assay was carried out in 384-well format and detected using a Perkin Elmer EnVision Xcite Multilabel Reader. Emission ratios were converted into percent inhibitions and imported into GraphPad Prism software. The concentration necessary to achieve inhibition of enzyme activity by 50% (IC50) was calculated using concentrations ranging from 20 μM to 0.1 nM (12-point curve). IC50 values were determined using a nonlinear regression model available in GraphPad Prism 5.
Measurement: IC50 = 3000 nM (pChEMBL 5.519999980926514)
Descriptors: MW 515.53, ALogP 3.49, HBA 9, HBD 2, PSA 133.34, QED 0.34
Compound (SMILES): CCCCO/N=C(\C)[C@@H]1CCc2cc([C@H]3CC[C@](N)(COP(=O)(O)O)C3)ccc2C1
InChIKey: VAOYAPAGOCRXFS-MHQSGURASA-N
Target: Sphingosine-1-phosphate lyase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O95470
Assay: Receptor [35S] GTPgammaS Binding Assays: (S1P1 GTPgammaS/S1P3 GTPgammaS): Compounds were loaded in a 384 Falcon v-bottom plate (0.5 l/well in a 11 point, 3-fold dilution). Membranes prepared from S1P1/CHO cells or EDG3-Gal5-bla HEK293T cells (EDG3 equivalent S1P3) were added to the compound plate (40 l/well, final protein 3 μg/well) with MULTIDROP®. [35S]GTP (1250 Ci/mmol, Perkin Elmer) was diluted in assay buffer: 20 mM HEPES, pH7.5, 10 mM MgCl2, 150 mM NaCl, 1 mM EGTA (ethylene glycol tetraacetic acid), 1 mM DTT (Dithiothreitol), 10 μM GDP, 0.1% fatty acid free BSA, and 10 μg/ml Saponin to 0.4 nM. 40 μl of the [35S] GTP solution was added to the compound plate with a final concentration of 0.2 nM. The reaction was kept at room temperature for 45 min. At the end of incubation, all the mixtures in the compound plate were transferred to Millipore 384-well FB filter plates via the VELOCITY11® Vprep liquid handler. The filter plate was washed with water 4 times by using the manifold Embla plate washer and dried at 60° C. for 45 min. MicroScint 20 scintillation fluid (30 μl) was added to each well for counting on the Packard TOPCOUNT®. EC50 is defined as the agonist concentration that corresponds to 50% of the Ymax (maximal response) obtained for each individual compound tested. The EC50 for Example 689 was determined to be 5.7 nM in the assay utilizing membranes prepared from S1P1/CHO cells. The EC50 for Example 689 was determined to be >2000 nM in the assay utilizing membranes prepared from EDG3-Gal5-bla HEK293T cells.A smaller value for GTPγS S1P1 EC50 value indicated greater activity for the compound in the GTPγS S1P1 binding assay. A larger value for the GTPγS S1P3 EC50 value indicated less activity in the GTPγS S1P3 binding assay. Example 689, which is the phosphate ester of Example 672, possessed activity as an agonist of S1P1 and is selective over S1P3. Example 697, which is the phosphate ester of Example 681, possessed activity as an agonist of S1P1 and is selective over S1P3. Thus the compounds of the present invention may be used in treating, preventing, or curing various S1P1 receptor-related conditions while reducing or minimizing the side effects due to S1P3 activity. The selectivity of the compounds of the present invention indicate their potential use in treating, preventing, or curing autoimmune and inflammatory diseases such as multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, lupus, psoriasis, or vascular diseases, while reducing or minimizing possible side effects due to S1P3 activity. Other potential uses of the compounds of the present invention include minimizing or reducing rejection of transplanted organs, while reducing or minimizing side effects due to S1P3 activity.
Measurement: EC50 < 10 nM (pChEMBL 8)
Descriptors: MW 438.51, ALogP 4.06, HBA 5, HBD 3, PSA 114.37, QED 0.23
Compound (SMILES): CCCCc1ccc(-c2nc3ccc(C(=O)NO)cc3[nH]2)cc1
InChIKey: NCLUKPQSAOTOJT-UHFFFAOYSA-N
Target: Histone deacetylase 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BY41
Assay: In vitro enzymatic HDACs assays: All Examples were tested in HDAC1, 2, 3, 6 and 8 in vitro enzymatic assays. The assay principle is well known (Hauser et al. 2009, Bradner et al. 2010) and all necessary reagents like enzymes, substrates, developer and reference compounds are commercially available (see e.g. BPS Biosciences http://www.bpsbioscience.com/). A series of dilutions of the compounds were prepared with a top concentration of 200 μM (2 μM for HDAC6). The enzymatic reactions were conducted in a mixture containing assay buffer, bovine serum albumin, HDAC substrate, and a test compound. After enzymatic reaction, developer was added and after an additional incubation time, fluorescence intensity was measured at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. A selection of the Examples were tested in a full HDAC panel containing HDAC1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 in vitro enzymatic assays. The tests were performed as above except that a series of dilutions of the compounds were prepared with a top concentration of 10 μM for all HDACs.
Measurement: IC50 = 190 nM (pChEMBL 6.71999979019165)
Descriptors: MW 309.37, ALogP 3.69, HBA 3, HBD 3, PSA 78.01, QED 0.5