lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1cc(OCCCc2c(C)n(CCC(=O)O)c3c(-c4cccnc4C)cccc23)cc(C)c1Cl
InChIKey: MIDQCWNBPAOOSE-UHFFFAOYSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07820
Assay: Fluorescence Polarization Anisotropy Competition Assay: Compound affinity was measured using a fluorescence polarization anisotropy competition assay. Anisotropy measurements were carried out in 384-well, black, flat-bottom plates (Greiner Bio-one, Monroe, N.C., USA). The assay was run using a fluorescein isothiocyanate-labeled BH3 peptide derived from Bak (FITC-AHx-GQVGRQLAIIGDDINR-NH2) that was purchased from GenScript (Piscataway, N.J.) at >95% purity and used without further purification. 10 nM FITC-Bak peptide and 14 nM recombinant Mcl-1 (residues 172-327) were added to assay buffer (3 mM dithiothreitol, 50 mM NaCl, 20 mM Tris, pH 7.5). For selectivity assays, 40 nM Bcl-2 (residues 1-207A96T,G110R, Δ35-91, replaced with Bcl-xL35-50) or 4 nM Bcl-xL (residues 1-209, loop 45-86 deleted) were incubated with 10 nM FITC-Bak in assay buffer.Compounds are diluted in DMSO in a 10-point, 3-fold serial dilution scheme. 2.5 uL compound is added to 47.5 uL of assay buffer containing FITC-Bak and protein, for a final DMSO concentration of 5% and a top concentration of 20 uM. A FITC-Bak peptide alone (100% inhibition) and peptide plus protein (0% inhibition) control is included on each assay plate. The plate was mixed and incubated for 90 minutes at room temperature. Anisotropy is measured at excitation wavelength 480 nm and emission wavelength 535 nm using an EnVision Multi-label plate reader (PerkinElmer, Wellesley, Mass., USA). Fluorescence anisotropy is plotted against compound concentration to generate an IC50 (inhibitor concentration at which 50% of bound peptide is displaced) by fitting the data to a 4-parameter logistic model using XLFit software (Guildford, Surrey, UK). IC50 is converted to a binding dissociation constant (Ki value)
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 491.03, ALogP 7.08, HBA 4, HBD 1, PSA 64.35, QED 0.25
Compound (SMILES): Cc1coc(-c2ccccc2-c2ccc3ncc(C(N)=O)n3c2)n1
InChIKey: BBGRPDUUCWDMCV-UHFFFAOYSA-N
Target: TGF-beta receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P36897
Assay: Inhibitory Activity Toward ALK5 Enzyme: 1. Preparation of kinase buffer: 40 mM Tris (pH 7.5), 20 mM MgCl2, 0.10% BSA, 1 mM DTT.2. Preparation of the compound: the final concentration of the compound was 10 μM, and the compound was prepared to a 100-fold concentration, i.e, 1 mM. 100 μL compound of the 100-fold concentration was added to the second well of a 384-well plate, and 60 μL of 100% DMSO was added to other wells. 30 μL compound in the second well was transferred to the third well, and the compound was 3-fold diluted in sequence to give 10 different concentrations. 50 nL compound was transferred to a reaction plate by echo.3. Kinase reaction: the kinase was added to 1× kinase buffer to prepare 2× kinase solution. The final concentration of kinase solution was 25 nM of ALK5. Peptide TGFbR1 (purchased from Signal Chem, catalog number T36-58) and ATP were added to 1× kinase buffer to prepare 2× substrate solution. The final concentration of substrate solution was 0.1 mg/mL of polypeptide TGFbR1 and 7 μM of ATP. 2.5 μL of 2× kinase solution was added to a 384-well reaction plate (containing 50 nL compound dissolved in 100% DMSO), 1× kinase buffer was added to the negative control well. The reaction plated was incubated at room temperature for 10 minutes. 2.5 μL of 2× substrate solution was added to the 384-well reaction plate. The 384-well plate was covered with a lid and incubate at 30° C. for 1 hour. ADP-Glo reagent (purchased from Promege, catalog number v9102) was equilibrated to room temperature. 5 μL of ADP-Glo reagent was transferred to the reaction well of the 384-well plate to terminate the reaction.4. Determination of reaction results: 10 μL of kinase detection reagent was added to each reaction well, the 384-well plate was shaken for 1 minute, and allowed to stand for 30 minutes at room temperature. The luminescence value of the sample was read by Synegy.5. Curve Fitting: the data of the luminescence reading was copied from the Synegy program.
Measurement: IC50 = 74 nM (pChEMBL 7.130000114440918)
Descriptors: MW 318.34, ALogP 3.06, HBA 5, HBD 1, PSA 86.42, QED 0.63
Compound (SMILES): Cc1cccc(OCC(C)C)c1-n1nc2c(c1-c1ccc(NC(N)=O)cc1)CN(c1ncc(C(F)(F)F)cc1F)CC2
InChIKey: DITYNAZJTIEYGH-UHFFFAOYSA-N
Target: C5a anaphylatoxin chemotactic receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21730
Assay: chemotaxis assay: Optionally, compounds may be further assayed for their ability to inhibit chemotaxis in cells. Chemotaxis assays are performed using 5 μm pore polycarbonate, polyvinylpyrrolidone-coated filters in 96-well chemotaxis chambers (Neuroprobe; Gaithersburg, Md.) using chemotaxis buffer (Hank's balanced salt solution (HBSS) and 1% FBS). C5aR ligands (i.e., C5a, R&D Systems; Minneapolis, Minn.) are use to evaluate compound mediated inhibition of C5aR mediated migration. Other chemokines (i.e., SDF-1α; R&D Systems; Minneapolis, Minn.) are used as specificity controls. The lower chamber is loaded with 29 μl of chemokine (i.e., 0.03 nM C5a) and varying amounts of compound; the top chamber contains 100,000 U937 or neutrophil cells in 20 μl. The chambers are incubated 1.5 hours at 37° C., and the number of cells in the lower chamber quantified either by direct cell counts in five high powered fields per well or by the CyQuant assay (Molecular Probes), a fluorescent dye method that measures nucleic acid content and microscopic observation.
Measurement: IC50 = 27.5 nM (pChEMBL 7.559999942779541)
Descriptors: MW 582.6, ALogP 6.49, HBA 6, HBD 2, PSA 98.3, QED 0.24
Compound (SMILES): O=c1c2cccnc2nc2n1-c1ccccc1C2=NO
InChIKey: OAYVXFMPXXTMHN-UHFFFAOYSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: Indoleamine-2,3-Dioxygenase 1 Inhibitory Activity: Experimental method: IDO-1 can oxidatively cleave the indole ring of tryptophan to form N-formylkynurenine. Referring to the method in the literature (Eddy W. Yue, et al. J. Med. Chem. 2009, 52, 7364-7367), 20 nM IDO-1, 2 mM D-tryptophan, 20 mM ascorbic acid, 3.5M methyl blue and 0.2 mg/mL antioxidant enzyme were sequentially added to 50 mM potassium phosphate buffer at room temperature. Due to the formation of N-formylkynurenine, the reaction rate was recorded by the increasing absorbance of the solution at 321 nm. IC50 values were calculated using Prism GraphPad software.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 264.24, ALogP 1.32, HBA 6, HBD 1, PSA 80.37, QED 0.38
Compound (SMILES): OCC(O)c1nc2c(OCC3CCCCC3)c(Cl)c(Cl)cc2[nH]1
InChIKey: QYFVUTPUECSKEB-UHFFFAOYSA-N
Target: GTP-binding protein Rheb (SINGLE PROTEIN), Homo sapiens
UniProt: Q15382
Assay: In Vitro Kinase Assay: For the Rheb-dependent in vitro kinase assay, HA-GST-Rheb was first loaded with GTP-γ-S (Millipore #20-176) by incubating 600 ng/μL Rheb with 10 mM EDTA and 0.4 mM GTP-γ-S at 30° C. for 10 minutes. MgCl2 was added to a final concentration of 20 mM to stop the reaction. This mixture was used at 20× in the kinase assay.Loaded HA-GST-Rheb was then incubated with compound (final DMSO concentration 1%) for 30 minutes at room temperature. mTORC1, GFP-4E-BP1 substrate (Life Technologies #PV4759), and kinase assay buffer (HEPES pH 7.4, KCl, MgCl2) were added to the Rheb-compound mixture and incubated at room temperature for 20 minutes. ATP was then added, and the kinase reaction proceeded at 30° C. for 45 min before being stopped by the addition of SDS-PAGE loading buffer for Westerns or 10 mM EDTA for the LanthaScreen readout. During the kinase reaction, the final concentration of reagents is as follows: Rheb30 ng/μL, compoundvariable, GTPγS0.02 mM, EDTA0.5 mM, MgCl21 mM, mTORC1fixed volume of prep (molarity unknown), GFP-4E-BP1 substrate15 ng/μL, ATP0.5 mM. For LanthaScreen analysis (Thermo Fisher #PV4757), the manufacturer's instructions were followed and conditions were optimized for the amount of Rheb and mTORC1. The final concentration of reagents was as follows: 100 nM loaded HA-GST-Rheb, compound (variable), 0.5 mM EDTA, 10 mM MgCl2, 25 mM HEPES pH 7.4, 50 mM KCl, mTORC1 (fixed volume0.1 μL in 10 μL total volume), 0.4 μM GFP-4E-BP1 substrate, 0.5 mM ATP. Optimization is summarized in FIG. 1. Results were read on an EnVision reader (PerkinElmer) using 495ex/520em filters (Life Technologies, #PV00315).
Measurement: IC50 = 55000 nM (pChEMBL 4.260000228881836)
Descriptors: MW 359.25, ALogP 3.85, HBA 4, HBD 3, PSA 78.37, QED 0.76
Compound (SMILES): Cc1c(Oc2cccc(CN)c2F)ccc2c1B(O)OC2
InChIKey: XEZNMDMVOXPEQL-UHFFFAOYSA-N
Target: RAC-alpha serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P31749
Assay: Competition Binding Assay: Phosphorylation of activity of ROCK1 and ROCK2 and those of other kinases, AKT1, GRK2, PKA, PKCa and RSK1 were performed by Reaction Biology (Malvern, Pa.) (Ma et al., 2008) The ROCK1 enzyme tested was GST[1-535] ROCK1 (Genbank, NP 005397), ROCK2 was GST[5-554] (Genbank, 3327051). For the kinase assay buffers contained 20 mM Hepes, 10 mM MgCl2, 2 mM DTT, 0.02 mg/mL BSA, 0.1% DMSO, 1 mM EGTA, 0.02% Brij-35, 10 uM ATP, [γP33]-ATP and for ROCK enzymes, 20 uM long S6-peptide (32 amino acids length). Appropriate substrates and activation was applied for the other kinases tested. Each enzyme was preincubated with test compound for 15 min., and the reaction started by addition of ATP. Samples were incubated at room temperature for 120 min, and applied to P81 ion exchange paper and washed extensively; the radioactive content of the filters were determined.
Measurement: IC50 > 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 287.1
Compound (SMILES): COc1ccc(C(CNc2nn(C)c(=O)c3ccccc23)N(C)C)cc1
InChIKey: XHOSXDNEUKMRJB-UHFFFAOYSA-N
Target: Histone acetyltransferase KAT2B (SINGLE PROTEIN), Homo sapiens
UniProt: Q92831
Assay: PCAF AlphaLisa Binding Assay: His/Flag epitope tagged PCAF719-832 bromodomain was cloned, expressed and purified to homogeneity in-house. PCAF bromodomain binding and inhibition of the compounds disclosed herein was assessed by monitoring the engagement of biotinylated small molecule ligand (known to bind to the PCAF bromodomain) with the target using the AlphaLisa technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate PCAF bromodomain (225 nM final) was combined with the biotinylated small molecule ligand (6 nM final) in 50 mM HEPES (pH 7.5), 75 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.2% DMSO) or compound dilution series in DMSO. After 15 minute incubation at room temperature AlphaLisa streptavidin acceptor beads and AlphaLisa anti-histidine donor beads were added to a final concentration of 12.5 μg/mL each. After 90 minutes of equilibration plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 64 nM (pChEMBL 7.190000057220459)
Descriptors: MW 352.44, ALogP 2.66, HBA 6, HBD 1, PSA 59.39, QED 0.74
Compound (SMILES): Cc1onc(-c2ccc(Cl)nc2)c1COc1ccc(C(=O)NC2CC3(COC3)C2)nn1
InChIKey: DPSSGGDIOTWPAP-UHFFFAOYSA-N
Target: Gamma-aminobutyric acid receptor subunit alpha-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14867
Assay: Electrophysiology: Electrophysiological experiments were performed on days 3 to 5 after the micro-injection of mRNA. During the experiment the oocytes were constantly superfused by a solution containing (in mM) NaCl 90, KCl 1, HEPES 5, MgCl2 1, CaCl2 1 (pH 7.4). Oocytes were impaled by two glass microelectrodes (resistance: 0.4 MΩ) which were filled with a solution containing KCl 1M+K-acetate 1.5 M and voltage-clamped to −80 mV. The recordings were performed at room temperature using the Roboocyte two-electrode voltage clamp system (Multichannelsystem). After an initial equilibration period of 1.5 min GABA was added for 1.5 min at a concentration evoking approximately 20% of a maximal current response (EC20). After another rest interval of 2.5 min GABA was again added evoking a response of similar amplitude and shape. 0.5 min after the onset of this second GABA application the test compound, at a concentration corresponding to approximatively 30 fold its Ki, was added while GABA was still present. Current traces were recorded at a digitization rate of 10 Hz during and shortly before and after the GABA application.
Measurement: Ki = 11.2 nM (pChEMBL 7.949999809265137)
Descriptors: MW 441.88, ALogP 2.98, HBA 8, HBD 1, PSA 112.26, QED 0.58
Compound (SMILES): N=C(N)c1ccc(CNC(=O)c2ccc3c(c2)[C@@H]2O[C@H]3c3ccc(C4CC4)cc32)cc1
InChIKey: ALJWHNMEZKCUCS-BJKOFHAPSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Plasma Kallikrein Inhibition Assay: The chromogenic substrate D-Pro-Phe-Arg-pNa, 2HCl (BIOPHEN CS-31(02) from Hyphen BioMed, Neuville-Sur-oise, France) was dissolved in 5 mL deionized water and stored at 4° C. Concentration was determined in the spectrophotometer at 342 nm using an extinction coefficient of 8270. All other chemicals were of analytical grade.Human plasma kallikrein was purchased from Enzyme Research Labs (South Bend, Ind., USA, batch HPKa 2830). A stock solution of 7 μM in 50% glycerol was stored at −20° C.Enzyme reactions were conducted in assay buffer comprised of 20 mM HEPES at pH 7.4, 150 mM NaCl, 0.1% PEG-8000 and 0.01% Triton X-100.Both enzyme and substrate were diluted in assay buffer.The compound solutions as well as the enzyme and the substrate solutions were transferred to 96-well plates (Clear, UV-Star, Flat-bottom, Half-Area plates; cat. No. 675801 Greiner Bio-one, purchased from VWR International, Arlington Heights, Ill., USA) using a Rainin LTS 96-channel pipettor (Rainin, Columbus, Ohio, USA). Plate measurements were conducted using a SPECTROStar Nano reader (BMG Labtech, San Francisco, Calif., USA). The SPECTROStar Nano is a spectrophotometer and absorbance was measured at 405 nm. We used discrete wavelength, precise, kinetic reads of 15 cycles with a 60 sec cycle time.Determination of IC50 valuesFor the determination of IC50 values, the assays were performed at room temperature in 96-well plates with a total assay volume of 85 μL per well. The test compound was dissolved in 100% DMSO. The compounds were serially diluted in DMSO in a 7 point dose response. For the assays, 66.5 μL of protease solution (protease in assay buffer) was added per well followed by the addition of 8.5 μL of compound in 100% DMSO. The final assay concentration of the human plasma kallikrein was 250 μM. After 30 min. incubation at room temperature on an orbital shaker, the reactions were started by the addition of 10 μL substrate solution (in assay buffer, final assay concentration was 600 uM). After the addition of the substrate solution the final DMSO concentration was 10%. The plate was placed again on the shaker for 5 sec, spun at 2000 rpm for 5 sec and read on the spectrophotometer. The effect of the compound on the enzymatic activity was obtained from the linear part of the progress curves and determined after 15 minutes.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 409.49, ALogP 4.3, HBA 3, HBD 3, PSA 88.2, QED 0.43
Compound (SMILES): N#CN1CCC(F)(C(=O)N2CCc3cc(-c4ccccc4)ccc32)C1
InChIKey: XLOPQKCBUYZPOB-UHFFFAOYSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 30 (SINGLE PROTEIN), Homo sapiens
UniProt: Q70CQ3
Assay: Biochemical Assay: Dilution plates were prepared at 21 times the final concentration (2100 M 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. USP30 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 0.05 l/well and 10 μl of diluted USP30 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 335.38, ALogP 3.14, HBA 3, HBD 0, PSA 47.34, QED 0.79
Compound (SMILES): O=C(O)c1ccncc1NCC1CCCc2cc(Cl)ccc21
InChIKey: WQNPSJUCFWEQLQ-UHFFFAOYSA-N
Target: Lysine-specific demethylase 4C (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H3R0
Assay: In Vitro Enzyme Inhibition Assay: JMJD2C: The ability of test compounds to inhibit the activity of JMJD2C was determined in 384-well plate format under the following reaction conditions: 0.3 nM JMJD2C, 300 nM H3K9me3-biotin labeled peptide (Anaspec cat #64360), 2 μM alpha-ketoglutaric acid in assay buffer of 50 mM HEPES, pH7.3, 0.005% Brij35, 0.5 mM TCEP, 0.2 mg/ml BSA, 50 μM sodium L-ascorbate, and 2 μM ammonium iron(II) sulfate. Reaction product was determined quantitatively by TR-FRET after the addition of detection reagent Phycolink Streptavidin-allophycocyanin (Prozyme) and Europium-anti-di-methylated histone H3 lysine 9 (H3K9me2) antibody (PerkinElmer) in the presence of 5 mM EDTA in LANCE detection buffer (PerkinElmer) at a final concentration of 50 nM and 1 nM, respectively.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 316.79, ALogP 3.97, HBA 3, HBD 2, PSA 62.22, QED 0.9
Compound (SMILES): C#Cc1cnc(NCc2ccc(-c3ccc4ncnc(N)c4c3)s2)c(C(=O)NCc2ccc(F)c(F)c2)c1
InChIKey: HUELUZAHWQJKHC-UHFFFAOYSA-N
Target: 3-phosphoinositide-dependent protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O15530
Assay: PDK1 kinase assay: PDK1 (amino acids 51-359) and AKT2 (amino acids 140-467 fused to PIFtide, amino acids EEQEMFRDFDYIADW) were expressed as N-terminally tagged GST fusion proteins in insect cells and purified to homogeneity. Enzyme activity was determined in a coupled PDK1/AKT/FAM-crosstide assay and phosphorylation of FAM-crosstide was determined by standard IMAP protocol (Molecular Devices). For inhibition studies, compounds were titrated 4-fold in DMSO and diluted 40-fold into assay buffer (10 mM Tris HCl pH7.2; 10 mM MgCl2; 0.01% Triton X-100; 1 mM DTT) containing PDK1, AKT2, and FAM-crosstide (final concentrations: 0.75 nM PDK1, 10 nM unphosphorylated AKT2, and 100 nM crosstide substrate). The kinase reaction was initiated by adding ATP to a final concentration of 40 μM of PDK1 and incubated at 25° C. for 30 min. To detect assay product, the kinase reaction was combined with Progressive Binding Solution (1:600 Progressive Binding Reagent, 50% Buffer A, 50% Buffer B, Molecular Devices) in a 1:3 ratio. The mixture was incubated for 2 hours at 25° C. and the plate was scanned on an Analyst AD with excitation at 485 nm and emission at 530 nm. The fluorescence polarization value P is defined by the equation below. The value mP is generated by multiplying the P value for each reaction well by a factor of 1000. The value ΔmP for each well is the mP value for that well minus the mP value for the average negative control. P=(Fpar−Fperp)/(Fpar+Fperp)  Eq.:Where par is fluorescence intensity parallel to the excitation plane; and perp is fluorescence intensity perpendicular to the excitation plane. ΔmP values were plotted as a function of compound concentration and the data were analyzed with a 4-parameter fit using GraphPad Prism software.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 526.57, ALogP 5.14, HBA 7, HBD 3, PSA 105.82, QED 0.25
Compound (SMILES): CC(C)[C@@H]1CN(c2cccc(S(C)(=O)=O)c2)CCN1c1ccc(C(F)(F)F)c(C2CC2)n1
InChIKey: NSWRBHFZGBXSGU-FQEVSTJZSA-N
Target: Oxysterols receptor LXR-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q13133
Assay: LXR alpha/beta Radioligand Binding Assay: Compounds of the invention were assessed in a competition binding assay where different concentrations of compounds were incubated with the LXR ligand binding domain (LBD) in the presence of radiolabeled LXR ligand [3H]TO901317. The amount of the LXR-LBD that complexed with [3H]T0901317 was measured by scintillation proximity assay (SPA) employing non-specific binding of LXR-LBD to poly-lysine coated Yttrium silicate beads. Partially purified LXR α or β LBD protein (15-45 nM) was incubated at rt for 30 min with 15 nM [3H]TO901317 (25-40 Ci/mmol) and different concentrations of test compounds in 80 μL of phosphate buffered saline (PBS) buffer containing 2.5% DMSO, 1% glycerol, 2 mM EDTA, 2 mM CHAPS and 5 mM DTT in 96-well plates. Poly-lysine SPA beads (50 μg) were added to each well and the total volume was adjusted to 120 μL. The plates were shaken on an orbital shaker for 20 min and then allowed to settle for 10 more min at rt before a brief centrifugation at 2,000 rpm for 1 min. The SPA signal was measured on a MicroBeta® liquid scintillation counter (Perkin Elmer, Waltham, Mass.), and the results were used for calculating IC50 values based on the total binding (DMSO control) and non-specific binding (5 μM of unlabeled TO901317) controls. The Ki values were calculated according to equation 1, where [RL] is the final concentration of [3H]TO901317 in the assay, and the Kd values of 20 nM and 10 nM of TO901317 for LBDs of LXRα and LXRβ, respectively, were determined by direct titration of the radioligand with these proteins.
Measurement: Ki = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 467.56, ALogP 4.73, HBA 5, HBD 0, PSA 53.51, QED 0.64
Compound (SMILES): COC(=O)N1c2ccc3c(nc(Cc4ccccc4)n3CCN3CCC4(COC4)C3)c2CC[C@@H]1C
InChIKey: ILDCZWFWZDQRDV-FQEVSTJZSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: HTRF Biochemical Assay for CBP and BRD4 Activity: The assay was performed in a final volume of 6 μL in assay buffer containing 50 mM Hepes (pH 7.5, (0.5M Hepes, pH 7.5 solution; Teknova H1575)), 0.5 mM GSH, 0.01% BGG (0.22 μM filtered, Sigma, G7516-25G), 0.005% BSA (0.22 μM filtered, EMD Millipore Corporation, 126575) and 0.01% Triton X-100 (Sigma, T9284-10L). Nanoliter quantities of 10-point, 3-fold serial dilution in DMSO were pre-dispensed into 1536 assay plates (Corning, #3724BC) for a final test concentration of 33 μM to 1.7 nM, top to lowest dose, respectively. 3 of 2× Protein and 3 μL of 2× Peptide Ligand were added to assay plates (pre-stamped with compound). Plates were incubated for varying times at room temperature prior to measuring the signal. TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) was measured on the PHERAstar (BMG, equipped with HTRF optic module [337/520/490]) or on the Envision (PerkinElmer, equipped with the TRF Laser unit, TRF dual mirror D400/D505 and emission filters M520 and M495). Data were reported as percent inhibition compared with control wells based on the following equation: % inh=1−((TR-FRET ratio−AveLow)/(AveHigh−AveLow)) where TR-FRET ratio=(Fluorescence at 520 nm/Fluorescence at 490 nm)*10000), AveLow=average TR-FRET ratio of no enzyme control (n=32), and AveHigh=average TR-FRET ratio of DMSO control (n=32). IC50 values were determined by curve fitting of the standard 4 parameter logistic fitting algorithm included in the Activity Base software package: IDBS XE Designer Model 205. Data is fitted using the Levenburg Marquardt algorithm. For all assay formats data were reported as percent inhibition compared with control wells based on the following equation: % inh=100*((FLU−AveLow)/(AveHigh−AveLow)) where FLU=measured Fluorescence, AveLow=average Fluorescence of no enzyme control (n=32), and AveHigh=average Fluorescence of DMSO control (n=32). IC50 values were determined by curve fitting of the standard 4 parameter logistic fitting algorithm included in the Activity Base software package: IDBS XE Designer Model 205. Data is fitted using the Levenburg Marquardt algorithm.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 474.61, ALogP 4.26, HBA 6, HBD 0, PSA 59.83, QED 0.55
Compound (SMILES): C[C@@H](COCC(=O)N[C@H]1CCN(c2ncc(C(F)(F)F)cn2)C[C@@H]1O)Nc1cn[nH]c(=O)c1C(F)(F)F
InChIKey: IQPJDFUIVMISPG-JKOKRWQUSA-N
Target: Protein mono-ADP-ribosyltransferase TIPARP (SINGLE PROTEIN), Homo sapiens
UniProt: Q7Z3E1
Assay: Enzymatic Assay: Displacement of Probe A, a biotinylated probe binding to the TIPARP active site, was measured using a time-resolved fluorescence energy transfer (TR-FRET) assay. 20 nL of a dose response curve of each test compound was spotted in black 384-well polystyrene proxiplates (Perkin Elmer) using a Mosquito (TTP Labtech). Reactions were performed in a 8 μl volume by adding 6 μL of TIPARP and Probe A in assay buffer (20 mM HEPES pH=8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002% Tween20), incubating with test compound at 25° C. for 30 min, then adding 2 μL of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin (Perkin Elmer). The final concentrations of TIPARP and Probe A were 6 nM and 2 nM, respectively. The final concentration of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin were 4 nM and 0.25 nM, respectively. Reactions were incubated at 25° C. for an additional 30 min, then read on an Envision platereader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 320 nm and emission of 615 nm and 665 nM with a 90 is delay. The ratio of the 665/615 nm emission were calculated for each well to determine the amount of complex of TIPARP and Probe Ain each well. Control wells containing a negative control of 0.25% DMSO vehicle or a positive control of 100 μM 5-(5-(piperidin-4-yloxy)isoindolin-2-yl)-4-(trifluoromethyl)pyridazin-3(2H)-one were used to calculate the % inhibition.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 539.44, ALogP 1.17, HBA 9, HBD 4, PSA 145.36, QED 0.36
Compound (SMILES): Cc1ccc(Cn2c(=O)n(CCCC(=O)O)c(=O)[nH]/c2=N\c2ccc(Oc3ccc(F)cn3)cc2)cc1
InChIKey: SLZIAROFBLAYBQ-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 cell transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 384-well PDL-coated microtiter plate at a concentration of 3000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, and 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.5% BSA, and 0.04% Pluronic P-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), and each well was added with 20 μL of the washing buffer. The plate was placed in High-Throughput Screening System FLIPR 384 (Molecular Device Co.). Measurement of fluorescence intensity by FLIPR 884 was started, and 20 μL of DMSO solutions containing different concentrations of the test compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 150 nM ATP solution (25 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 4 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement.
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 505.51, ALogP 3.12, HBA 8, HBD 2, PSA 131.57, QED 0.36
Compound (SMILES): CCc1cccnc1Oc1ccc(-c2c(C)c(=O)[nH]c(=O)n2C)cc1
InChIKey: HHUBSIFPIYFJMK-UHFFFAOYSA-N
Target: D(1A) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P21728
Assay: Receptor Binding Assay: D1 binding assays were performed using over-expressing LTK human cell lines. To determine basic assay parameters, ligand concentrations were determined from saturation binding studies where the Kd for [3H]-SCH23390 was found to be 1.3 nM. From tissue concentration curve studies, the optimal amount of tissue was determined to be 1.75 mg/mL per 96 well plate using 0.5 nM of [3H]-SCH23390. These ligand and tissue concentrations were used in time course studies to determine linearity and equilibrium conditions for binding. Binding was at equilibrium with the specified amount of tissue in 30 minutes at 37° C. From these parameters, Ki values were determined by homogenizing the specified amount of tissue for each species in 50 mM Tris (pH 7.4 at 4° C.) containing 2.0 mM MgCl2 using a Polytron and spun in a centrifuge at 40,000×g for 10 minutes. The pellet was resuspended in assay buffer [50 mM Tris (pH 7.4@ RT) containing 4 mM MgSO4 and 0.5 mM EDTA]. Incubations were initiated by the addition of 200 μL of tissue to 96-well plates containing test drugs (2.5 μL) and 0.5 nM [3H]-SCH23390 (50 μL) in a final volume of 250 μL. Non-specific binding was determined by radioligand binding in the presence of a saturating concentration of (+)-Butaclamol (10 μM), a D1 antagonist. After a 30 minute incubation period at 37° C., assay samples were rapidly filtered through Unifilter-96 GF/B PEI-coated filter plates and rinsed with 50 mM Tris buffer (pH 7.4 at 4° C.).
Measurement: Ki = 52.7 nM (pChEMBL 7.28000020980835)
Descriptors: MW 337.38, ALogP 2.8, HBA 5, HBD 1, PSA 76.98, QED 0.79
Compound (SMILES): CC(C)Oc1cccc(S(=O)(=O)c2ccc(NC(=O)NCc3cccnc3)cc2)c1
InChIKey: OFPLTPSUOUTPCC-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Biochemical Inhibition Assay: The NAMPT enzymatic reactions were carried out in Buffer A (50 mM Hepes pH 7.5, 50 mM NaCl, 5 mM MgCl2, and 1 mM THP) in 96-well V-bottom plates. The compound titrations were performed in a separate dilution plate by serially diluting the compounds in DMSO to make a 100× stock. Buffer A (89 μL) containing 33 nM of NAMPT protein was added to 1 μL of 100× compound plate containing controls (e.g. DMSO or blank). The compound and enzyme mix was incubated for 15 minutes at room temperature, then 10 μL of 10× substrate and co-factors in Buffer A were added to the test well to make a final concentration of 1 μM NAM, 100 μM 5-Phospho-D-ribose 1-diphosphate (PRPP), and 2.5 mM Adenosine 5′-triphosphate (ATP). The reaction was allowed to proceed for 30 minutes at room temperature, then was quenched with the addition of 11 μL of a solution of formic acid and L-Cystathionine to make a final concentration of 1% formic acid and 10 μM L-Cystathionine. Background and signal strength was determined by addition (or non-addition) of a serial dilution of NMN to a pre-quenched enzyme and cofactor mix.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 425.51, ALogP 4.02, HBA 5, HBD 2, PSA 97.39, QED 0.6
Compound (SMILES): Cc1c([C@@H](C)Nc2nnc(C)c3cnc(O[C@H]4CCN(C)C4)cc23)cccc1C(F)(F)F
InChIKey: HSPHZCFOXUBJSA-ZBFHGGJFSA-N
Target: Son of sevenless homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07889
Assay: Inhibition of Labeled Tracer Ligand: Table 22: This Example illustrates that exemplary compounds of the present invention bind to SOS1 and prevent a labeled tracer ligand from occupying the S0S1 binding site. The ability of a compound of Formula (I) to bind to SOS1 was measured using a HTRF displacement assay. A recombinant human SOS1 polypeptide (corresponding to amino acids 560-1049, expressed in E. Coli with N-terminal His-TEV-AviTag-SOS1 (MW=59.4 kDa) and lanthanide labeled streptavidin (CisBio) was incubated with an exemplary compound of Formula (I) (in a DMSO stock solution) in buffer (25 mM HEPES pH 7.5, 25 mM NaCl, 1 mM DTT, 0.01% Brij 35, 0.02% BSA, 0.1% DMSO). After a 10-15 minute incubation at room temperature, a solution comprised of a custom-made Cy5 labelled tracer and MAb Anti-6HIS Tb cryptate Gold (Cisbio 61HI2TLA) in buffer was added to the solution containing the SOS1 polypeptide and exemplary compound of Formula (I). After a 1-hour incubation at room temperature, the HTRF signal was measured using Clairostar plate reader (BMG Labtech) according to the manufacturer's instructions. Excitation filter EX-TR was used, and emission 1 was detected at 650-610 nm and emission 2 detected at 620-610 nm. The HTRF ratio was calculated using the formula: [emission 1/emission 2]*10000.
Measurement: Ki = 8.76 nM (pChEMBL 8.0600004196167)
Descriptors: MW 445.49, ALogP 4.92, HBA 6, HBD 1, PSA 63.17, QED 0.6
Compound (SMILES): O=C(OC(C(F)(F)F)C(F)(F)F)N1CCC2(CCN(Cc3cc(CN4CCCCC4)cc(C(F)(F)F)c3)C2)CC1
InChIKey: WUYIVANPBNYKBM-UHFFFAOYSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: In Vitro Competitive Activity-Based Protein Profiling Assay: Human: Proteomes (human prefrontal cortex or cell membrane fractions) (50 μL, 1.0-2.0 mg/mL total protein concentration) were preincubated with varying concentrations of inhibitors at 37° C. After 30 min, FP-Rh or JW912 (1.0 μL, 50 μM in DMSO) was added and the mixture was incubated for another 30 min at room temperature. Reactions were quenched with SDS loading buffer (15 μL-4X) and run on SDS-PAGE. Following gel imaging, serine hydrolase activity was determined by measuring fluorescent intensity of gel bands corresponding to MAGL using ImageJ 1.49 k software.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 589.54, ALogP 6.61, HBA 4, HBD 0, PSA 36.02, QED 0.36
Compound (SMILES): Cn1cnc2ncn(Cc3nn(CCc4ccc(-c5ccccc5)cc4)c(=O)o3)c(=O)c21
InChIKey: SCDFLVQWVCBTRY-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily A member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75762
Assay: FLIPR Assay: The IC50 (effective concentration) of compounds on the human and rat TRPA1 channels were determined using a FLIPR Tetra instrument. CHO cells expressing TRPA1 were plated into 384-well plates, incubated overnight at 37° C., and loaded with BD calcium indicator dye for 1 hr at 37° C. followed by 15 minutes at room temperature. The assay buffer was Hank's Balanced Salt Solution (HBSS) containing 20 mM HEPES (pH readjusted to 7.4) along with 0.02% BSA.Following dye load and plate cool down, compounds were added to the cells using FLIPR Tetra. Plates were then incubated with compounds for 20 minutes at room temperature prior to adding agonist. Following this incubation, about an EC50 concentration of cinnamaldehyde (75 uM for human TRPA1 and 45 uM for rat TRPA1) was added to active the channels and block of cinnamaldehyde induced calcium influx was measured.The IC50 results were fit with a standard Hill function, keeping the Hill coefficient (n) fixed to 1.5. Fixing the Hill coefficient will generally reduce variability of the IC50 determination. The IC50 results were individually examined to make sure the MIN and MAX points were set correctly prior to validation of the results.
Measurement: IC50 = 251 nM (pChEMBL 6.599999904632568)
Descriptors: MW 428.45, ALogP 2.24, HBA 9, HBD 0, PSA 100.74, QED 0.41
Compound (SMILES): Cc1c(-c2ccc(Br)s2)nc2ccc(Br)cc2c1C(=O)NC12CCC(C(=O)O)(CC1)CC2
InChIKey: YQSKYWQZSPETJF-UHFFFAOYSA-N
Target: Prostaglandin F2-alpha receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P43088
Assay: In Vitro Test of Inhibition of Human FP Receptor Activity : For the characterization of test substances in respect of FP antagonism, PGF2α-induced calcium flux in FP-expressing CHEM1 cells (Millipore, HTS093C) was used.3000 cells in 25 μl of full medium [DMEM F12, 10% FCS, 1.35 mM sodium pyruvate, 20 mM HEPES, 4 mM GlutaMAX, 2% sodium bicarbonate, 1% Pen/Strep, 1% 100× non-essential amino acids] are sown per well of a 384 multititer plate (from Greiner, TC plate, black with clear base) and incubated at 37° C./5% CO2 for 24 hours. Prior to the measurement, the medium is replaced by 30 μl of Fluo-8 AM loading buffer [calcium-free Tyrode (130 mM NaCl, 5 mM KCl, 20 mM HEPES, 1 mM MgCl2, 4.8 mM NaHCO3, pH 7.4), 2 mM CaCl2, 1× SmartBlock (from CANDOR Bioscience GmbH), 4.5 mM Probenecid, 5 μM Fluo-8 AM, 0.016% Pluronic, 0.04% Brilliant black] and incubated at 37° C./5% CO2 for 30 minutes. The test substance is prepared in DMSO in various concentrations as a dose-response curve (starting concentration 10 mM, dilution factor 3.16) and prediluted 1:50 with calcium-free Tyrode/2 mM CaCl2. 10 μl of the prediluted substance solution are added to the Fluo-8-laden cells and incubated at 37° C./5% CO2 for 10 minutes. The FP receptor is activated by adding 20 μl of 3 nM (final concentration) PGF2α in calcium-free Tyrode/2 mM CaCl2/0.04% Brilliant black, and the calcium flux is determined by measuring the fluorescence at ex. 470 nm/em. 525 nm in a fluorescence measuring instrument (FLIPR Tetra, Molecular Devices) for 120 seconds.
Measurement: IC50 = 383 nM (pChEMBL 6.420000076293945)
Descriptors: MW 578.33, ALogP 6.7, HBA 4, HBD 2, PSA 79.29, QED 0.36
Compound (SMILES): Cc1ccc(-n2cccn2)c(C(=O)N2CCC[C@@]2(C)c2nc3c(C)c(Cl)ccc3[nH]2)c1
InChIKey: AWYCCGHIHBHRDF-DEOSSOPVSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Inhibition Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 μg/ml G418, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2.Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 433.94, ALogP 5.17, HBA 4, HBD 1, PSA 66.81, QED 0.48
Compound (SMILES): N#CN=C(NCc1ccc(S(=O)(=O)c2ccc(F)c(F)c2)cc1)Nc1ccncc1
InChIKey: YANBKPJDDVRAAF-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Biochemical Inhibition Assay: The NAMPT enzymatic reactions were carried out in Buffer A (50 mM Hepes pH 7.5, 50 mM NaCl, 5 mM MgCl2, and 1 mM THP) in 96-well V-bottom plates. The compound titrations were performed in a separate dilution plate by serially diluting the compounds in DMSO to make a 100× stock. Buffer A (89 μL) containing 33 nM of NAMPT protein was added to 1 μL of 100× compound plate containing controls (e.g. DMSO or blank). The compound and enzyme mix was incubated for 15 minutes at room temperature, then 10 μL of 10× substrate and co-factors in Buffer A were added to the test well to make a final concentration of 1 μM NAM, 100 μM 5-Phospho-D-ribose 1-diphosphate (PRPP), and 2.5 mM Adenosine 5′-triphosphate (ATP). The reaction was allowed to proceed for 30 minutes at room temperature, then was quenched with the addition of 11 μL of a solution of formic acid and L-Cystathionine to make a final concentration of 1% formic acid and 10 μM L-Cystathionine. Background and signal strength was determined by addition (or non-addition) of a serial dilution of NMN to a pre-quenched enzyme and cofactor mix.
Measurement: IC50 = 0.8 nM (pChEMBL 9.100000381469727)
Descriptors: MW 427.44, ALogP 3.23, HBA 5, HBD 2, PSA 107.24, QED 0.28
Compound (SMILES): Cc1cc(S(=O)(=O)Nc2cccc(F)n2)ncc1N(C)C1CCN(Cc2ccccc2F)CC1
InChIKey: WGDILJNGFXFIAY-UHFFFAOYSA-N
Target: Sodium channel protein type 8 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UQD0
Assay: Sodium Influx Assay (In Vitro Assay): This sodium influx assay employs the use of the cell permeable, sodium sensitive dye ANG2 to quantify sodium ion influx through sodium channels which are maintained in an open state by use of sodium channel modulators. This high throughput sodium influx assay allows for rapid profiling and characterization of sodium channel blockers.In general, Trex HEK293 cells were stably transfected with an inducible expression vector containing the full-length cDNA coding for the desired human sodium channel α-subunit and with an expression vector containing full length cDNA coding for the β1-subunit. Sodium channel expressing cell lines were induced with tetracycline (1 μg/mL) and plated on 384-well PDL-coated plates at a density of 25K-30K cells/well in culture media (DMEM, containing 10% FBS and 1% L-glutamine). After overnight incubation (37° C., 5% CO2), culture media was removed and cells were loaded with 5 uM ANG2 dye for 1-1.5 h in Buffer 1 (155 mM NMDG, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, adjusted with Tris to pH 7.4). Access dye was removed and cells were incubated with test compounds for 1 hr in buffer 1 containing sodium channel modulator(s) at room temperature. Hamamatsu FDSS μCell was used to perform a 1:1 addition of Na/K challenge buffer (140 mM NaCl, 20 mM HEPES, 1 mM CaCl2, 15 mM KCl, 1 mM MgCl2, 10 mM glucose, adjusted with Tris to pH 7.4) and simultaneously read plates at excitation wavelength of 530 nm and emission wavelength set at 558 nm. Percent inhibition of sodium ion influx was calculated for each test compound at each test concentration to determine the IC50 values.
Measurement: IC50 = 72 nM (pChEMBL 7.139999866485596)
Descriptors: MW 487.58, ALogP 3.96, HBA 6, HBD 1, PSA 78.43, QED 0.51
Compound (SMILES): CC(C)(C)COc1nc(N2CCC(F)(F)C2)c2ncn(Cc3ccccc3Cl)c2n1
InChIKey: CZKXKEBHIDYZEV-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1x HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min. Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 uM to 0.13 nM cAMP. EC50 values were determined using Activity Base analysis (ID Business Solution, Limited). The EC50 values for a wide range of cannabinoid agonists generated from this assay were in agreement with the values published in the scientific literature. The compounds of the invention are CB2 receptor agonists with EC50 below 1 uM and selectivity versus CB1 in the corresponding assay of at least 10 fold. Particular compound of the invention are CB2 receptor agonists with EC50 below 0.05 uM and selectivity versus CB1 in the corresponding assay of at least 500 fold.
Measurement: EC50 = 2.7 nM (pChEMBL 8.569999694824219)
Descriptors: MW 435.91, ALogP 4.8, HBA 6, HBD 0, PSA 56.07, QED 0.58
Compound (SMILES): Nc1nc2cc(-c3ccc4ncc(C(=O)N5CC6CCC(C5)O6)n4c3)ccc2o1
InChIKey: UMPHKJHUXLTCMQ-UHFFFAOYSA-N
Target: Phosphatidylinositol 3-kinase regulatory subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O00459
Assay: PI 3-Kinase HTRF Assay: 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 1x reaction buffer) is added per well for a final concentration of 0.25-0.3 ug/ml kinase and 10 uM PIP2. The plate is sealed and incubated for 15 minutes at room temperature. To start the reaction, 3.5 ul of ATP (diluted in 1x reaction buffer) is added per well for a final concentration of 10 uM 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 < 10000 nM (pChEMBL 5)
Descriptors: MW 389.42, ALogP 2.73, HBA 7, HBD 1, PSA 98.89, QED 0.57
Compound (SMILES): O=C(Nc1ccccc1)Nc1cc(S(=O)(=O)Nc2ccccc2)ccc1-c1ccsc1
InChIKey: PLYBNKFKYCYFFB-UHFFFAOYSA-N
Target: Isocitrate dehydrogenase [NADP], mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P48735
Assay: IDH2 Enzymatic Assay: Compounds were assayed for IDH2 R140Q inhibitory activity through a cofactor depletion assay. Compounds were preincubated with enzyme, then the reaction was started by the addition of NADPH and α-KG, and allowed to proceed for 60 minutes under conditions previously demonstrated to be linear with respect for time for consumption of both cofactor and substrate. The reaction was terminated by the addition of a second enzyme, diaphorase, and a corresponding substrate, resazurin. Diaphorase reduces resazurin to the highly fluorescent resorufin with the concomitant oxidation of NADPH to NADP, both halting the IDH2 reaction by depleting the available cofactor pool and facilitating quantitation of the amount of cofactor remaining after a specific time period through quantitative production of an easily detected fluorophore.Specifically, into each of 12 wells of a 384-well plate, 1 μl of compound dilution series was placed, followed by the addition of 40 μl of buffer (50 mM potassium phosphate, pH 7.5; 150 mM NaCl; 10 mM MgCl2, 10% glycerol, 0.05% bovine serum albumin, 2 mM beta-mercaptoethanol) containing 1.25 μg/ml IDH2 R140Q. The compound was then incubated for one hour at room temperature with the enzyme; before starting the IDH2 reaction with the addition of 10 μl of substrate mix containing 50 μM NADPH and 6.3 mM α-KG in the buffer described above. After a further one hour of incubation at room temperature, the reaction was halted and the remaining NADPH measured through conversion of resazurin to resorufin by the addition of 25 μl Stop Mix (36 μg/ml diaphorase enzyme and 60 μM resazurin; in buffer). After one minute of incubation the plate was read on a plate reader at Ex544/Em590.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 449.56, ALogP 5.86, HBA 4, HBD 3, PSA 87.3, QED 0.34