lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1cccc(-c2cc(=O)oc3c(C)c4occ(C)c4cc23)c1
InChIKey: LZIPJYWQYOTMJO-UHFFFAOYSA-N
Target: Potassium voltage-gated channel subfamily A member 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22001
Assay: Patch-Clamp Assay for Inhibitory Activity on Kv1.3: For a description of a Kv1.3 patch-clamp assay, see Grissmer et al., Mol. Pharmacol. 1994, 45, 1227; whole cell patch-clamp recording was performed with n≥2 individual experiments at each compound concentration using different cells. Three or more different concentrations were determined per dose response curve.
Measurement: IC50 = 2250 nM (pChEMBL 5.650000095367432)
Descriptors: MW 320.34, ALogP 4.83, HBA 4, HBD 0, PSA 52.58, QED 0.5
Compound (SMILES): CNc1nc(NC2C3CCC2CN(c2cnnc(C)c2)C3)nc2c1C(C)(C)CN2C1CC(F)(F)C1
InChIKey: PMYFUWNKBDXODX-UHFFFAOYSA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Homo sapiens
UniProt: P05067
Assay: Cellular gamma-Secretase Assay: Human neuroglioma H4 cells overexpressing human APP695 with the Swedish double mutation (K595N/M596L) were plated at 30,000 cells/well/100 μl in 96-well plates in IMDM media containing 10% FCS, 0.2 mg/l Hygromycin B and incubated at 37° C., 5% CO2.3-4 hr post plating, compounds are a diluted in media and 50 μl is added as 1.5-fold concentrate to achieve the final concentration. Compound incubation is performed for 24 hr. Final doses typically range from 4 μM down to 0.0013 μM in half-log steps resulting in a eight point dose response curve. Appropriate controls using vehicle only and reference compound were applied to this assay. The final concentration of Me2SO was 0.4%.After incubation at 37° C., 5% CO2, the supernatant was subjected to quantification of secreted Aβ42 by the means of an AlphaLisa assay kit (Human Amyloid beta 1-42 Kit: Cat # AL203C, Perkin Elmer). 20 μl of the cell culture supernatant was transferred to an assay plate. Then 10 μl of a mixture of the AlphaLisa coupled capture antibody and the biotinylated detection antibody was added and incubated for 3 hours at room temperature while softly shaking the assay plate. After a further addition of 20 μl of the Donor beads the assay plate was incubated for 30 min at room temperature and constant shaking without exposure to direct light. The assay plate was then read on a Paradigm AlphaLisa Reader using the build-in program with excitation at 680 nm and emission at 570 nm.
Measurement: EC50 = 36 nM (pChEMBL 7.440000057220459)
Descriptors: MW 484.6, ALogP 3.84, HBA 8, HBD 2, PSA 82.1, QED 0.66
Compound (SMILES): Cn1nncc1COc1ccccc1-c1[nH]ncc1Oc1ccc(C#N)cc1
InChIKey: DGPVHBMKBOLTHR-UHFFFAOYSA-N
Target: Cytochrome P450 11B1, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P15538
Assay: Inhibition Assay: G-402 cells can be obtained from ATCC (CRL-1440) and were originally derived from a renal leiomyoblastoma.The expression plasmids contains the ORF for either human/cyno CYP11B1 or CYP11B2 under the control of a suitable promoter (CMV-promoter) and a suitable resistance marker (neomycin). Using standard techniques the expression plasmid is transfected into G-402 cells and these cells are then selected for expressing the given resistance markers. Individual cell-clones are then selected and assessed for displaying the desired enzymatic activity using 11-Deoxycorticosterone (Cyp11B2) or 11-Deoxycortisol (Cyp11B1) as a substrate.G-402 cells expressing CYP11 constructs were established as described above and maintained in McCoy's 5a Medium Modified, ATCC Catalog No. 30-2007 containing 10% FCS and 400 μg/ml G418 (Geneticin) at 37° C. under an atmosphere of 5% CO2/95% air. Cellular enzyme assays were performed in DMEM/F12 medium containing 2.5% charcoal treated FCS and appropriate concentration of substrate (0.3-10 uM 11-Deoxycorticosterone, 11-Deoxycortisol or Corticosterone). For assaying enzymatic activity, cells were plated onto 96 well plates and incubated for 16 h. An aliquot of the supernatant is then transferred and analyzed for the concentration of the expected product (Aldosterone for CYP11B2; Cortisol for CYP11B1). The concentrations of these steroids can be determined using HTRF assays from CisBio analyzing either Aldosterone or Cortisol.
Measurement: EC50 = 0.171 nM (pChEMBL 9.770000457763672)
Descriptors: MW 372.39, ALogP 3.45, HBA 7, HBD 1, PSA 101.64, QED 0.56
Compound (SMILES): CN(C)C/C=C/C(=O)N1CCOc2c1ccc1ncnc(Nc3ccc(Sc4ccc(F)cc4)cc3)c21
InChIKey: BZEDJVGMLBRRFR-ONEGZZNKSA-N
Target: Receptor tyrosine-protein kinase erbB-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P04626
Assay: Inhibition of EGFR and HER2 Kinase Activity Assay: The assay for the inhibition of EGFR and HER2 kinase activity by small molecular compounds was carried out using the method as follows:1) Dilution of the compoundsIn a 96-well plate a, the compounds were diluted with DMSO using a 3-fold gradient dilution to form 11 concentrations, the 12th concentration is pure DMSO (as a positive control); and in a new 96-well plate b the above solutions were diluted 25 times with ultrapure water (DMSO concentration is 4%).2) Transferring the compounds to 384-well plateThe compound solutions diluted with ultrapure water in the 96-well plate b above was transferred to the corresponding wells of a 384-well plate in duplicate.3) Addition of 4×kinase solution: 2.5 μl of the above 4× kinase solution was taken using multichannel pipette and added to the corresponding reaction wells of the 384-well plate, mixed well and pre-reacted at room temperature for 5 minutes.4) Addition of 2×substrate/ATP mixed solution: 5 μl of the above 2×substrate/ATP mixed solution was taken using multichannel pipette and added to the corresponding reaction wells of the 384-well plate.5) Negative control: negative control wells were set in the 384-well plate, and 2.5 μl 4×substrate, 2.5 μl 4×enzyme solution, 2.5 μl 1×Kinase Assay Buffer and 2.5 μl ultrapure water containing 4% DMSO were added to each well.6) Mixed by centrifugation and kept at room temperature for 2 hours in the dark.7) Termination of the enzymatic reaction:5 μl of the above 4× stop solution was pipetted to the corresponding wells of the 384-well plate, centrifuged and mixed, and reacted at room temperature for 5 minutes.8) Development reaction:5 μl of the above 4× detection solution was pipetted into the corresponding wells of the 384-well plate, centrifuged and mixed, and reacted at room temperature for 1 hour.9) The 384-well plate was placed into a microplate reader and the signal was detected using the corresponding program.10) IC50 analysis:Well reading value=10000*EU665 value/EU615 valueInhibition rate=(reading value of positive control well−reading value of experimental well)/(reading value of positive control well−reading value of negative control well)*100%Corresponding IC50s can be calculated by entering the drug concentrations and the corresponding inhibition rates into GraphPad Prism 5.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 515.61, ALogP 5.51, HBA 7, HBD 1, PSA 70.59, QED 0.33
Compound (SMILES): CC(C)Nc1c(-c2cccnc2)nnc2cc(-c3ccc(S(C)(=O)=O)cc3)ccc12
InChIKey: OTJBPWKTWVDPPF-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: In Vitro Kinase Activities (LRRK2 TR-FRET Peptide Assay): Compounds as described herein (compounds of Formula I, e.g., compounds of the above Examples) are tested for their in vitro kinase activities using various LRRK2 (including LRRK2 G2019S mutant) assays. For example, assays were performed in a total volume of 20 μL using the same kinase reaction buffer (50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM EGTA, 2 mM DTT and 0.01% Tween-20) for wild-type or G2019S mutant LRRK2. Serially diluted compounds (1% DMSO as co-solvent) were pre-incubated with recombinant GST-LRRK2 (wild-type, or G2019S mutant, Invitrogen) for 15 minutes at room temperature in 384-well Corning black plates. Mixtures of ATP and biotin-LRRKtide substrate (biotin-RLGRDKYKTLRQIRQ) (SEQ ID NO: 1) were added to the wells at a final concentration of 100 μM ATP and 100 nM substrate, with final kinase concentration of 10 nM. The kinase reactions were carried out at room temperature for 60 minutes, then the reaction was stopped with the addition of 10 μL/well of stop/detection buffer (25 mM HEPES pH 7.5, 66 mM EDTA, 0.8 M KF and 0.1% BSA) that contains streptavidin-XL665 (12.5 nM) and europium-conjugated phospho-specific antibody (2nM). The plates were read 1 hour later and the time-resolved fluorescence (665 nm to 615 nm ratio) measured using an Envision reader. The inhibition of each well was calculated using the control and background readings for that plate. IC50 values were determined from dose-response curves using eight concentrations from the serial dilution of the test compounds.Many compounds of the Examples are demonstrated to be inhibitors of LRRK2, with most of the compounds tested typically measuring an IC50 below 1 μM for both LRRK2 and G2019S mutant LRRK2 kinase activity.
Measurement: IC50 = 13000 nM (pChEMBL 4.889999866485596)
Descriptors: MW 418.52, ALogP 4.58, HBA 6, HBD 1, PSA 84.84, QED 0.51
Compound (SMILES): Cn1ncc2ccc(-c3cc(COC(C)(C)C(=O)O)nn3Cc3ccccc3Cl)cc21
InChIKey: IRFKTOCKUJVLSN-UHFFFAOYSA-N
Target: Monocarboxylate transporter 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O15427
Assay: MCT4-Mediated Lactate Transport in MDA-MB-231 Breast Cancer Cells: MDA-MB-231 breast cancer cells may be used to measure MCT4 activity in cells with high native levels of MCT4 and low levels of MCT1 and are known to those with skill in the art. MCT4 activity may be assessed by monitoring the intracellular pH change that accompanies lactate/proton symport, using the pH-sensitive fluorescent dye 2′,7′-bis-(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), in a manner similar to that previously reported for MCT1 and MCT4. The following is an exemplary procedure for assaying MCT4 activity of the compounds of Formula (I).
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 438.92, ALogP 4.52, HBA 6, HBD 1, PSA 82.17, QED 0.46
Compound (SMILES): Cc1c(N2C(=O)C(C)(C)N(CCCOc3ccc(-c4ccncc4)nc3)C2=S)ccc(C#N)c1Cl
InChIKey: BSDSTBGCLFMDAI-UHFFFAOYSA-N
Target: Androgen receptor (SINGLE PROTEIN), Mus musculus
UniProt: P19091
Assay: Androgen Receptor Assay: Androgen binding is measured using the hydroxylapatite (HAP) assay. In brief, the radioactive steroid [3H]R1881 solubilized in ethanol is diluted with buffer B (10 mM Tris-HCl, 1.5 mM EDTA disodium salt, 10 mM α-monothioglycerol, pH 7.4). Aliquots of the cell or prostate cytosol preparation (0.1 ml) are then incubated with 5 nM [3H]R1881 (0.1 ml, ¿100 000 cpm) in the presence or absence of the indicated concentrations of unlabeled compounds (0.1 ml, prepared in buffer B containing 30% ethanol) for 16-18 h at 0-4° C. Triamcinolone acetonide (TAC; 100 nM) is added to mask progesterone receptors. Unbound steroids are separated by incubation for 40 min at 0-4° C. with 0.3 ml HAP prepared in buffer P (50 mM Tris-HCl, 10 mM KH2PO4, pH 7.4). After incubation with HAP and 10 min of centrifugation at 1000×g, the pellet is washed 3 times with 1 ml of buffer P. Thereafter, the radioactivity is extracted from the pellet by incubation at room temperature for 60 min with 1 ml of ethanol. After centrifugation, the supernatant is decanted into a scintillation vial and the pellet is extracted again with ethanol. After the addition of scintillation liquid, the radioactivity is measured in a liquid scintillation counter.
Measurement: IC50 = 118 nM (pChEMBL 6.929999828338623)
Descriptors: MW 506.03, ALogP 5.16, HBA 6, HBD 0, PSA 82.35, QED 0.32
Compound (SMILES): O=C(O)c1nn[nH]c1Oc1cccc(C#Cc2ncccn2)c1
InChIKey: KBFPBZZFOMQMLY-UHFFFAOYSA-N
Target: 2-Hydroxyacid oxidase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UJM8
Assay: In-Vitro Human Glycolate Oxidase (hGOX) Assay: The in-vitro glycolate oxidase assay was performed using recombinant full-length human hydroxyacid oxidase 1 (HAO1), the equivalents of hGOX. The enzyme was obtained from AbCam (Catalog #ab113144) and was purified using conventional chromatography to >95% purity. Purified HAO1 was dissolved in assay buffer consisting of 10 mM NaCl, 110 mM KCl, 2 mM MgCl2, 50 mM HEPES (pH 7.4), and 0.01% Triton X-100. The assay used Corning 3575 384-well flat bottom, low flange, non-binding surface, black polystyrene plates.Test compounds in DMSO were preincubated at different concentrations with purified recombinant human GO (6 nM) for 10 min, followed by the addition of glycolate substrate (85 μM) to start the reaction. The plates were incubated for 10 min at room temperature, at which point Amplex red reagent (50 μM) was added.The fluorescence intensity signal was measured on a VariosKan LUX instrument using an excitation of 560 nm and an emission of 590 nm. The IC50 values were calculated using Graphpad Prism.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 307.27, ALogP 1.48, HBA 6, HBD 2, PSA 113.88, QED 0.7
Compound (SMILES): Cn1cncc1C(=O)N[C@H](CC1CCCCC1)C(=O)N1C[C@@H](n2nncc2C(C)(C)O)C[C@H]1C(=O)NC(CCCCNC(=O)OCc1ccccc1)C(=O)C(N)=O
InChIKey: PVXJINWBNOAFNM-LBIXZHBDSA-N
Target: Serine protease HTRA1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92743
Assay: Full Length HTRA1 Assay: Serial dilutions (1/3) from 1000 μM down to 0.051 μM of test compounds were prepared in dimethyl sulfoxide (DMSO). Then 2 μL of solution from each dilution were added to 100 μL of 4 nM full-length human His-HTRA1 in assay buffer (50 mM Tris, pH 7.5, 200 mM NaCl and 0.25% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate or CHAPS) in white non-binding 96-well plates. The assay solutions were mixed for 5 seconds on a shaker plate and incubated for 10 minutes at room temperature. Mca-H2OPT (Mca-Ile-Arg-Arg-Val-Ser-Tyr-Ser-Phe-Lys(Dnp)-Lys-OH trifluoroacetate salt) (Mca=7-methoxycoumarin-4-acetic acid; Dnp=dinitrophenyl) (5 μM) in 100 μL of assay buffer was added to the assay solutions. The reaction mixture was shaken for 5 seconds on a shaker plate and cleavage of Mca-H2OPT was monitored by spectrofluorometry (SpectraMax M3 by Molecular Devices, CA) for 10 minutes (Exλ=330 nm; Emλ=420 nm). Percent inhibition was calculated by fitting values to a standard mathematical model for determining the dose response curve.
Measurement: IC50 = 130 nM (pChEMBL 6.889999866485596)
Descriptors: MW 790.92, ALogP 1.79, HBA 13, HBD 5, PSA 245.76, QED 0.09
Compound (SMILES): CCS(=O)(=O)c1ccc([C@H](CO)NC(=O)c2cnc3c(c2)CN(C[C@H]2CC[C@H](C(F)(F)F)CO2)[C@H]3C(C)C)cc1
InChIKey: QVDGHCSMKMJNNS-VAJMTYMLSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Binding Assay: Compounds of the present invention were tested for ability to bind to ROR in a cell-free competition assay with commercially available radio-ligand (RL), 25-hydroxy [26,27-3H]-cholesterol (PerkinElmer, Cat. # NET674250UC), for a ligand binding site on a recombinant RORFigure US09796710-20171024-P00001Ligand Binding Domain (LBD) protein expressed as a 6×His-Glutathione-S-Transferase (GST) fusion. The assay was performed in 96-well SPA plates (PerkinElmer, Cat. #1450-401) in 50 mM HEPES buffer, pH 7.4, containing 150 mM NaCl, 5 mM MgCl2, 10% (v/v) glycerol, 2 mM CHAPS, 0.5 mM β-octylglucopyranoside and 5 mM DTT. Tested compounds were dissolved in DMSO, and semi-log (3.162×) serial dilutions of the compounds were prepared in the same solvent. Two μL of the DMSO solutions were mixed with 28 μL of 8.6 nM 25-hydroxy [26,27-3H]-cholesterol and 50 μL of 24 nM RORFigure US09796710-20171024-P00001LBD. The plate was shaken at 700 rpm for 20 min and incubated for 10 min at rt, after which 40 μL of poly-Lys YSi SPA beads (PerkinElmer, Cat. # RPNQ0010) were added to achieve 50 μg of the beads per well. The plate was incubated on an orbital shaker for 20 min and then for 10 min without agitation at rt. SPA signal for tritium beta radiation was registered on PerkinElmer Microbeta plate reader. Percent inhibition values were calculated based on the high signal obtained with DMSO control and the low signal observed with 10 μM standard RORFigure US09796710-20171024-P00001 inverse agonist T0901317 (SigmaAldrich, Cat. # T2320).
Measurement: Ki < 100 nM (pChEMBL 7)
Descriptors: MW 583.67, ALogP 4.21, HBA 7, HBD 2, PSA 108.83, QED 0.46
Compound (SMILES): COc1ccc(C(=O)N2CCN(CCOc3ccc(OC)c(OC)c3)CC2)cc1
InChIKey: WICIEVRBFPSOFF-UHFFFAOYSA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: D3 Radioligand Binding Assay: Compounds were tested for their ability to compete with the orthosteric radioligand [3H]methylspiperone for binding to the D3 DAR using stable HEK cell lines expressing the D3 DAR (Codex Biosciences, Gaithersburg, Md., Catalogue no. CB-80300-206) as described in the detailed protocol below presented in Table 5. Cells were cultured in Dulbecco's modified Eagle's Medium (Corning, catalogue no. 10-013) containing 10% FBS, 1,000 units/mL Penicillin, 1,000 mg/mL Streptomycin, 100 mM sodium pyruvate, 1 μg/mL Gentamicin, and 250 mg/mL G418. All cells were maintained at 37° C. in 500 CO2 and 90% humidity. For radioligand binding assays cells were removed mechanically using calcium-free Earle's balanced salt solution (EBSS). Intact cells were collected by centrifugation and then lysed with 5 mM Tris-HCl and 5 mM MgCl2 at pH 7.4. Homogenates were centrifuged at 30,000×g for 30 minutes. The membranes were re-suspended in EBSS (US Biological, catalogue no. E0249-05) pH 7.4 to a concentration of 16 μg/mL. For competition binding studies, membrane preparations were incubated for 90 minutes at room temperature with various concentrations of compound and a single concentration of [3H]methylspiperone (Perkin Elmer, NET856) in a reaction volume of 250 μL. Non-specific binding was determined in the presence of 4 μM (+)-butaclamol (Sigma-Aldrich, catalogue no. D033). Bound ligand was separated from unbound by filtration through GF/C filters using a PerkinElmer cell harvester and quantified on a Top-count (PerkinElmer). Ki values were determined using Cheng-Prusoff equation from observed IC50 values and ligand Kd values from separate saturation experiments.
Measurement: EC50 = 3500 nM (pChEMBL 5.460000038146973)
Descriptors: MW 400.48, ALogP 2.55, HBA 6, HBD 0, PSA 60.47, QED 0.68
Compound (SMILES): CCC(CC)C(=O)C1CCCN(CC(=O)N2CCC(c3ccc4[nH]c(-c5cn6ncnc6c(C)c5C)c(C(C)C)c4c3)CC2)C1
InChIKey: FVQYFDKFXQTYKG-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
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 = 3.9 nM (pChEMBL 8.40999984741211)
Descriptors: MW 610.85, ALogP 7.04, HBA 6, HBD 1, PSA 86.6, QED 0.22
Compound (SMILES): NCC1CCN(c2ccc3nc(-c4ccc(OCc5ccccc5)cc4)[nH]c3n2)CC1
InChIKey: RESDVFNQYRWDTH-UHFFFAOYSA-N
Target: Protein arginine N-methyltransferase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: O14744
Assay: Biochemical Assay : The 6 μL assay reactions are run in Greiner brand black 384-well low volume plates. All reactions contained assay buffer (phosphate buffered saline, 0.01% (v/v) Tween-20, 0.01% (w/v) albumin from chicken egg white, 1 mM dithiothreitol, 200 nM peptide substrate, 1 μM S-Adenosyl methionine, and 15 ng/reaction enzyme, with the enzyme being omitted from negative control reactions. Compounds were added in a volume of 100 nL from dilution series prepared in DMSO, positive and negative control reactions receiving the same volume DMSO without compound. The plates were sealed with adhesive seals and incubated for 4 hours at 37° C. Reaction progress was measured using the Transcreener EPIGEN methyltransferase assay (BellBrook Labs, Madison, Wis.) as recommended by the manufacturer. To each reaction 2 μL detection mix were added, containing coupling enzymes, fluorescence polarisation tracer, and AMP antibody. Plates were incubated for 90 minutes before being read on a PerkinElmer EnVision plate reader in fluorescence polarisation mode.
Measurement: IC50 = 426 nM (pChEMBL 6.369999885559082)
Descriptors: MW 413.53, ALogP 4.38, HBA 5, HBD 2, PSA 80.06, QED 0.49
Compound (SMILES): COC(=O)C#Cc1ccc2c(C(C)=O)cn(CC(=O)N3C[C@H](F)C[C@H]3C(=O)NCc3cccc(Cl)c3F)c2c1
InChIKey: RJAIMPYGLSQPAV-DVECYGJZSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Inhibition Assay: Human factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration is incubated with test compound at various concentrations for 5 minutes at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBzl and DTNB (Ellman's reagent) are added to final concentrations of 100 μM each. The increase in color is recorded at OD405 nm in a microplate in kinetic mode over 30 minutes with 30 second time points in a spectrofluorimeter. IC50 values are calculated by non-linear regression from the percentage of inhibition of complement factor D activity as a function of test compound concentration.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 555.97, ALogP 3.42, HBA 6, HBD 1, PSA 97.71, QED 0.29
Compound (SMILES): CC(=O)N1CCN(c2ccc(NC(=O)Cc3ccc(-c4ccnc(C)c4)c(F)c3)nc2)CC1
InChIKey: AWWPXGCYXOZMPB-UHFFFAOYSA-N
Target: Protein Wnt-3a (SINGLE PROTEIN), Homo sapiens
UniProt: P56704
Assay: Wnt-Luc Reporter Assay: Human embryonic kidney 293 cells (obtained from American Type Culture Collection, ATCC, Manassas, Va.) are cultured in DMEM medium (Gibco/Invitrogen, Carlsbad, Calif.) supplemented with 10% FBS (Gibco/Invitrogen, Carlsbad, Calif.), 50 unit/mL penicillin and 50 μg/mL of streptomycin (Gibco/Invitrogen, Carlsbad, Calif.) at 37° C. with 5% CO2 in air atmosphere. 293 cells in a 10 cm dish are co-transfected with 8 μg of STF-reporter plasmid containing a luciferase gene driven by Wnt-responsive elements and 2 μg of pcDNA3.1-Neo (Gibco/Invitrogen, Carlsbad, Calif.) with 30 μL of FuGENE6 (Roche Diagnostics, Indianapolis, Ind.) following the manufacturer's protocol. Stable cell lines (293 Wnt-Luc) were selected with 400 μg/mL of G418 (Gibco/Invitrogen, Carlsbad, Calif.). The 293 Wnt-Luc cells and L-cell Wnt3a cells (obtained from American Type Culture Collection, ATCC, Manassas, Va.) are trypsinized and co-cultured into a 384-well plate with DMEM medium supplemented with 2% FBS, and treated with different concentrations of a compound of the invention. After 24 hours, the firefly luciferase activities are assayed with the Bright-Glo Luciferase Assay System (Promega, Madison, Wis.). The IC50 is measured when the effect of the compound reduces the luminescence signal by 50%.
Measurement: IC50 = 0.1 nM (pChEMBL 10)
Descriptors: MW 447.51, ALogP 3.44, HBA 5, HBD 1, PSA 78.43, QED 0.65
Compound (SMILES): CNC(=O)[C@H](CCCCNC(=N)N)NC(=O)c1csc([C@@H]2C[C@H](O)CN2C(=O)c2cn3cc(Cl)ccc3n2)n1
InChIKey: GFAFFEOPNBYDEZ-MPGHIAIKSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Inhibitory Activity Assay: Plasma Kallikrein: The example compounds were evaluated for inhibition of the human activated kallikrein enzyme in two formats of an assay employing a fluorogenic peptide substrate. In one assay format, the concentrations of reagents were as follows: 20 mM Tris pH 7.5, 1 mM EDTA, 150 mM sodium chloride, 0.1% PEG-400, 0.1% Triton X-100, 500 pM activated kallikrein enzyme, 300 uM Pro-Phe-Arg-7-amido-4-methylcoumarin substrate. Prior to reaction initiation with substrate, enzyme and inhibitors were preincubated for 30 min at room temperature. After initiation with substrate, reactions were incubated for 10 min at room temperature and fluorescence emission at 460 nm from 380 nm excitation measured with a microplate reader. In another assay format, the concentrations of reagents were as follows: 20 mM Tris pH 7.5, 1 mM EDTA, 150 mM sodium chloride, 0.1% PEG-400, 0.1% Triton X-100, 5 pM activated kallikrein enzyme, 300 uM Pro-Phe-Arg-7-amido-4-methylcoumarin substrate. Prior to reaction initiation with substrate, enzyme and inhibitors were preincubated for 30 min at room temperature. After initiation with substrate, reactions were incubated for 18 hr at room temperature and fluorescence emission at 460 nm from 380 nm excitation measured with a microplate reader.
Measurement: EC50 = 3000 nM (pChEMBL 5.519999980926514)
Descriptors: MW 576.08, ALogP 0.89, HBA 9, HBD 6, PSA 190.83, QED 0.12
Compound (SMILES): CC(C(=O)Nc1ccc(C(F)(F)F)cc1)[C@H]1[C@@H]2C[C@@H](n3cnc4cc(F)c(F)cc43)C[C@@H]21
InChIKey: MQXKLAHEVORCAH-XJQPTBGDSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: IDO1 Cell-Based Assay: HeLa cells were obtained from the American Type Culture Collection (ATCC) and maintained in DMEM media containing 10% FBS. Cells (7,000/well) were seeded onto a 384 well plate in 50 μL of media and incubated at 37° C., 5% CO2 overnight. Cell media was aspirated, fresh media containing 10 ng/mL IFNgamma was added, and cells were incubated in absence or presence of various concentrations of test compound (final 0.5% DMSO) for 24 hours. Aliquots of the cell conditioned media were removed from the cell plate, and mixed with an equal volume of 200 mM ZnSO4 to precipitate media containing proteins. Two volumes of acetonitrile were added, mixed, and samples were then centrifuged at 2250 G for 20 minutes at 4° C. Aliquots of the supernatant were diluted 1:10 in 0.1% formic acid containing 3 μM of deuterated Tryptophan as an internal standard.Samples were analyzed via RFMS to quantify N-Formyl Kynurenine (AUC) and L-tryptophan (AUC). A C18 cartridge was used with mobile phases of 0.1% Formic Acid and 80% ACN/0.1% Formic Acid under isocratic conditions. Dose-response curves were analyzed using IC50 regression curve fitting (GeneData Screener). Curves were plotted as percent of control and normalized by high controls without inhibitor (100%), and low controls (0%) containing 1 μM of epacadostat, a potent cell-permeable IDO1 inhibitor. Cell viability was also assessed using the Cell Titer Glo Kit (Promega) following manufacturer recommendation.
Measurement: IC50 = 3.36 nM (pChEMBL 8.470000267028809)
Descriptors: MW 449.42, ALogP 5.81, HBA 3, HBD 1, PSA 46.92, QED 0.51
Compound (SMILES): Cn1cc(-c2cccc(S(=O)(=O)NC3CC3)c2)c2ccccc2c1=O
InChIKey: FHDKVHHTOPMSKT-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: In Vitro Enzyme Inhibition Assay: Determination of the IC50 for the heterocyclic derivative BRD4 inhibitors disclosed herein was performed as follows. His-tagged BRD4 was cloned, expressed and purified to homogeneity. Filipakopoulos et al., Nature 468:1067 (2010). BRD4 binding and inhibition was assessed by monitoring the interaction of biotinylated H4-tetraacetyl peptide (AnaSpec, H4K5/8/12/16(Ac), biotin-labeled) with the target using the AlphaScreen technology (Life Technologies). In a 384-well ProxiPlate BRD4(BD1) (2 nM final) was combined with peptide (15 nM final) in 50 mM HEPES (pH 7.3), 10 mM NaCl, 0.25 mM TCEP, 0.1% (w/v) BSA, and 0.005% (w/v) Brij-35 either in the presence of DMSO (final 0.4% DMSO) or compound dilution series in DMSO. After 20 min incubation at room temp, Alpha streptavidin donor beads and Nickel Chelate acceptor beads were added to a final concentration of 5 μg/mL. After 2 hr of equilibration, plates were read on an Envision instrument and the IC50 was calculated using a four parameter non-linear curve fit. Chemistry Example 1 (2-methyl-4-phenylisoquinolin-1-one) had an IC50 of 2.782 μM in this assay format.
Measurement: IC50 = 2750 nM (pChEMBL 5.559999942779541)
Descriptors: MW 354.43, ALogP 2.65, HBA 4, HBD 1, PSA 68.17, QED 0.78
Compound (SMILES): COc1cc(-n2ccc(N(C(=O)c3cccs3)C3CCN(C)CC3)n2)ccn1
InChIKey: STJHPTBGVAJNNB-UHFFFAOYSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Human mu-Opioid Receptor Radioligand Assay: To investigate binding properties of test compounds to human μ-opioid receptor, transfected CHO-K1 cell membranes and [3H]-DAMGO (Perkin Elmer, ES-542-C), as the radioligand, were used. The assay was carried out with 20 μg of membrane suspension, 1 nM of [3H]-DAMGO in either absence or presence of either buffer or 10 μM Naloxone for total and non-specific binding, respectively. Binding buffer contained Tris-HCl 50 mM, MgCl2 5 mM at pH 7.4. Plates were incubated at 27° C. for 60 minutes. After the incubation period, the reaction mix was then transferred to MultiScreen HTS, FC plates (Millipore), filtered and plates were washed 3 times with ice-cold 10 mM Tris-HCL (pH 7.4). Filters were dried and counted at approximately 40% efficiency in a MicroBeta scintillation counter (Perkin-Elmer) using EcoScint liquid scintillation cocktail.
Measurement: Ki = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 397.5, ALogP 3.08, HBA 7, HBD 0, PSA 63.49, QED 0.66
Compound (SMILES): Cc1cc2c(C(=O)n3nc4c(c3N)CCN(C(=O)OC(C)(C)C)C4)cccc2[nH]1
InChIKey: IHLKLCIDIHLQJB-UHFFFAOYSA-N
Target: Coagulation factor XII (SINGLE PROTEIN), Homo sapiens
UniProt: P00748
Assay: Factor XIIa (FXIIa) Inhibitory Activity: In a 96-well clear bottom plate, 80 μl of assay buffer was added to each well. Assay buffer consists of 0.5× Hank's Balanced Salt Solution (Invitrogen), buffered with 25 mM HEPES pH 7.4 (Invitrogen) and 0.5× Tris-buffered saline with Tween-20 0.05% (Santa Cruz Biotechnology). Test compounds were first dissolved in DMSO (Sigma) and then 4 μl were added to test wells containing assay buffer. Serial dilutions using an automated multi-channel pipette were used to generate a concentration range of approximately 1-100 μM. Human FXIIa (Enzyme Research Labs) was diluted in assay buffer to a final concentration of 12.5 nM. 80 μl of this enzyme solution was added to the assay wells. The enzyme/compound mixtures were incubated for 10 minutes at room temperature. Chromogenic substrate (Pefachrome XIIa; Enzyme Research Labs) was added to assay wells at a final concentration of 400 μM. The assay plate was spun for 1 minute at 1500 g and then read at 37° C. in a SpectraMax M2 plate reader (λ=405 nm). Activity was quantified as the rate of change in absorbance, which corresponds to the rate of substrate cleavage. IC50 values were determined as the concentration of inhibitor that produced 50% of the rate of change of control wells without any inhibitor. For compounds with activity <1 μM, the assay was repeated with a lower concentration range, typically from 10-1000 nM.
Measurement: IC50 = 70 nM (pChEMBL 7.159999847412109)
Descriptors: MW 395.46, ALogP 3.24, HBA 6, HBD 2, PSA 106.24, QED 0.66
Compound (SMILES): Cc1cccc(-c2cc(C(=O)Nc3nc(C)cs3)c3ncnn3c2)c1
InChIKey: BBOFIYOZOCEGFX-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 5 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P31424
Assay: Radioligand Binding Assay: The following examples of the in vitro effects of the disclosed compounds are prophetic. An example of an in vitro assay method for assessing the receptor ligand binding activity of the disclosed compounds is given below. Competition binding studies can be performed with the allosteric antagonist [.sup.3H]methoxyPEPy to determine if the disclosed compounds interact with the well-characterized allosteric binding site for the mGluR5 NAM MPEP.The allosteric antagonist MPEP analog [.sup.3H]methoxyPEPy is used to evaluate the ability of test compounds to interact with the MPEP site on mGluR5 (Cosford et al., Bioorg. Med. Chem. Lett. 2003, 13, 351). Membranes are prepared from rat mGluR5 HEK293 cells (Rodriguez et al., Mol. Pharmacol. 2005, 68, 1793). Compounds are diluted in assay buffer (50 mM Tris/0.9% NaCl, pH 7.4) to a 5.times. stock and 100 .mu.L test compound is added to each well of a 96 deep-well assay plate. 300 .mu.L aliquots of membranes diluted in assay buffer (40 .mu.g/well) are added to each well. 100 .mu.L [.sup.3H]methoxyPEPy (2 nM final concentration) is added and the reaction is incubated at room temperature for 1 hour with shaking. After the incubation period, the membrane-bound ligand is separated from free ligand by filtration through glass-fiber 96 well filter plates (Unifilter-96, GF/B, PerkinElmer Life and Analytical Sciences, Boston, Mass.). The contents of each well are transferred simultaneously to the filter plate and washed 3-4 times with assay buffer using a cell harvester (Brandel Cell Harvester, Brandel Inc., Gaithersburg, Md.). 40 .mu.L scintillation fluid is added to each well and the membrane-bound radioactivity determined by scintillation counting (TopCount, PerkinElmer Life and Analytical Sciences). Non-specific binding is estimated using 5 .mu.M MPEP. Concentration response curves were generated using a four parameter logistical equation in GraphPad Prism (GraphPad Software, Inc., La Jolla, Calif.).
Measurement: IC50 = 4770 nM (pChEMBL 5.320000171661377)
Descriptors: MW 349.42, ALogP 3.72, HBA 6, HBD 1, PSA 72.18, QED 0.61
Compound (SMILES): CN1CCC(Oc2cccc(C(=O)Nc3cc4cc(-c5cnn(C)c5)ccc4cn3)c2)CC1
InChIKey: LCINFBTUIYMEJT-UHFFFAOYSA-N
Target: Dual specificity tyrosine-phosphorylation-regulated kinase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: Q13627
Assay: Kinase Assay: The DYRKIA kinase assay was run using the Ser/Thr 18 peptide Z-lyte assay kit according to manufacturer's instructions (Life Technologies-a Division of Thermo-Fisher). This is a non-radioactive assay using fluorescence resonance energy transfer (FRET) between coumarin and fluorescein to detect kinase activity which is represented as a ratio of coumarin emission/fluorescein emission. Briefly, recombinant DYRKIA kinase, ATP and Ser/Thr peptide 18 were prepared in 1× Kinase buffer to final concentrations of 0.19 μg/mL, 30 μM, and 4 μM respectively. The mixture was allowed to incubate with the representative compounds for one hour at room temperature. All reactions were performed in duplicate. Unphosphorylated ("0% Control") and phosphorylated ("100% control") forms of Ser/Thr 18 served as control reactions. Additionally, an 11-point dose-response curve of Staurosporine (1 uM top) was run to serve as a positive compound control. After incubation, Development Reagent A was diluted in Development Buffer then added to the reaction and allowed to further incubate for one hour at room temperature. The plate was read at Ex 400 Em 455 to detect the coumarin signal and Ex 400 Em 520 to measure the signal (EnVision Multilabel Plate Reader, PerkinElmer).
Measurement: EC50 = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 441.54, ALogP 4.36, HBA 6, HBD 1, PSA 72.28, QED 0.5
Compound (SMILES): O=C(Nc1ccc(Cl)cc1F)N[C@@H]1CCN(c2ccc(-c3ccccc3CO)c(F)c2F)C1=O
InChIKey: UWBBPTCIYAQRSD-LJQANCHMSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: FPR2 and FPR1 Cyclic Adenosine Monophosphate (cAMP) Assays: A mixture of forskolin (5 μM final for FPR2 or 10 μM final for FPR1) and IBMX (200 μM final) were added to 384-well Proxiplates (Perkin-Elmer) pre-dotted with test compounds in DMSO (1% final) at final concentrations in the range of 0.020 nM to 100 μM. Chinese Hamster Ovary cells (CHO) overexpressing human FPR1 or human FPR2 receptors were cultured in F-12 (Ham's) medium supplemented with 10% qualified FBS, 250 μg/ml zeocin and 300 μg/ml hygromycin (Life Technologies). Reactions were initiated by adding 2,000 human FPR2 cells per well or 4,000 human FPR1 cells per well in Dulbecco's PBS (with calcium and magnesium) (Life Technologies) supplemented with 0.1% BSA (Perkin-Elmer). The reaction mixtures were incubated for 30 min at room temperature. The level of intracellular cAMP was determined using the HTRF HiRange cAMP assay reagent kit (Cisbio) according to manufacturer's instruction. Solutions of cryptate conjugated anti-cAMP and d2 fluorophore-labelled cAMP were made in a supplied lysis buffer separately. Upon completion of the reaction, the cells were lysed with equal volume of the d2-cAMP solution and anti-cAMP solution. After a 1-h room temperature incubation, time-resolved fluorescence intensity was measured using the Envision (Perkin-Elmer) at 400 nm excitation and dual emission at 590 nm and 665 nm. A calibration curve was constructed with an external cAMP standard at concentrations ranging from 1 μM to 0.1 pM by plotting the fluorescent intensity ratio from 665 nm emission to the intensity from the 590 nm emission against cAMP concentrations. The potency and activity of a compound to inhibit cAMP production was then determined by fitting to a 4-parametric logistic equation from a plot of cAMP level versus compound concentrations.
Measurement: EC50 = 4.2 nM (pChEMBL 8.380000114440918)
Descriptors: MW 489.88, ALogP 4.84, HBA 3, HBD 3, PSA 81.67, QED 0.48
Compound (SMILES): COc1cc(-n2ccc3c(=O)n(CC4(O)CCN(C(=O)[C@@H]5CCN(C(=O)OC(C)(C)C)C[C@H]5c5ccccc5)CC4)cnc32)ccc1F
InChIKey: APUIOPMAQKKUKI-IAPPQJPRSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q93009
Assay: Evaluation of the Inhibition of USP7 by the Fluorescence Intensity (FLINT) Readings: USP7 activity was measured using Rhodamine-110 c-terminal labelled Ubiquitin as a substrate (Viva Biosciences). Incubation with USP7 results in the release of Rhodamine-110 leading to an increase in fluorescence which can be used in the continuous measurement of USP7 activity.The USP7 reactions were performed in a 50 μL volume, in 384 well black solid low binding plates (Corning #3575). The reaction buffer consisted of 100 mM Bicine pH 8.0, 0.01% TritonX100, 1 mM TCEP, and 10% DMSO.0.25 nM His-His-USP7 (aa208-560, [C315A]) was incubated with compound (final concentration 10% DMSO) for 60 minutes at 30° C. The reaction was then initiated by the addition of 500 nM Ubiquitin-Rhodamine-110 substrate and the plate read every 3 minutes for 21 minutes to measure the release of Rhodamine-110. Fluorescence Intensity (FLINT) readings were measured using a Biomek Neo plate reader (Ex.485 nm, Em.535 nm).The inhibition of increasing doses of compound was expressed as a percentage reduction in kinetic rate compared to the kinetic rates established between DMSO only and total inhibition controls (no USP7). The inhibitory concentrations that gave a 50% reduction in kinetic rate (IC50) were determined, from 11-point dose response curves, in XL-Fit using a 4-Parameter Logistic Model (Sigmoidal Dose-Response Model).
Measurement: IC50 = 70.2 nM (pChEMBL 7.150000095367432)
Descriptors: MW 659.76, ALogP 4.73, HBA 9, HBD 1, PSA 119.13, QED 0.32
Compound (SMILES): CNC(=O)C1(NC(=O)C(=O)c2c(C)c(C(=O)Nc3ccc(F)c(Cl)c3)n3c2C[C@@H](O)C3)CC(F)(F)C1
InChIKey: OUQDBMFYGKYAOQ-GFCCVEGCSA-N
Target: Hepatic sodium/bile acid cotransporter (SINGLE PROTEIN), Homo sapiens
UniProt: Q14973
Assay: HBV DNA Quantification Assay: A HepG2 cell line overexpressing the HBV virus attachment receptor sodium-taurocholate cotransporting polypeptide (NTCP) was grown to confluency in DMEM growth medium, Dulbecco's Modified Eagle Medium without sodium pyruvate (Life Technologies, Rockville, Md.) supplemented with 10% FBS (Thermo Scientific, Waltham, Md.), 1% penicillin/streptomycin (Life Technologies, Rockville, Md.) and 2 mM L-glutamine (Life Technologies, Rockville, Md.) in T175 flasks. Cells were infected with HBV AD38 viral particles (Texcell, Frederick, USA) at 4000 genome equivalents per cell. After allowing viral infection to take place for 4 days, the infected cells were harvested from the flasks by trypsinization, washed twice with OptiMEM (Life Technologies, Rockville, Md.) and re-suspended in DMEM containing 2% FBS and 1% DMSO at a density of 0.25E6 cells/ml. Infected cells were seeded on 384 well collagen coated plates (Greiner, Austria) at a density of 20,000 cells/well containing serially diluted compounds of the present disclosure or DMSO (0.5%) in a final volume of 80 μl. The assay plates were incubated for a period of 5 days and the antiviral activity of the test compounds were assayed by detecting the presence of HBV DNA in the culture supernatant using the QuantiGene 2.0 nucleic acid quantification kit (Affymetrix, Santa Clara, Calif.).The culture supernatant was harvested and treated with lysis buffer containing Proteinase K (Affymetrix, Santa Clara, Calif.). The supernatant was incubated with HBV viral DNA specific probes (Affymetrix, Santa Clara, Calif.) for 30 minutes at 55° C. This was followed by addition of 0.2M NaOH for 30 minutes at room temperature to denature the DNA, followed by addition of Neutralization buffer (Affymetrix, Santa Clara, Calif.). The resulting lysed and neutralized supernatant was then added to QuantiGene 2.0 384 well plates coated with capture oligonucleotides and incubated overnight at 55° C. The HBV specific probe set consists of Capture Extender oligonucleotides (CE's) and blocking probes. Following the overnight incubation, the wells were incubated for one hour sequentially with a Pre-Amplifier, Amplifier and Labeled probes conjugated to alkaline phosphatase with a wash step between incubations. After the final wash step, the alkaline phosphatase substrate (Luminol APS5) was added and the resulting luminescence signal was read in an EnVision Multilabel Plate Reader (PerkinElmer, Santa Clara, Calif.). The EC50 values were calculated from the fit of the doseresponse curves to a four-parameter equation. All EC50 values represent geometric mean values of a minimum of four determinations.
Measurement: EC50 = 456 nM (pChEMBL 6.340000152587891)
Descriptors: MW 526.9, ALogP 1.97, HBA 6, HBD 4, PSA 129.53, QED 0.34
Compound (SMILES): C=CC(=O)N1CCC(Oc2cnc3[nH]cc(-c4ccc(F)nc4)c3n2)CC1
InChIKey: QMIKVVTUYYIJLW-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK Caliper Enzyme Assay: Test article was solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. An 11-point half log dilution series was created in DMSO with a top concentration of 600 μM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 resulting in a top final assay compound concentration of 10 μM and a 2% DMSO concentration.Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 4 μM ATP and 1 μM peptide substrate. The JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition 1 nM JAK3 enzyme and were incubated at room temperature 75 minutes for JAK3. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 367.38, ALogP 2.71, HBA 5, HBD 1, PSA 84, QED 0.57
Compound (SMILES): C[C@@H]1COCCN1c1cc(-c2cnn(C(F)F)c2)c2ccnc(-c3ccn[nH]3)c2n1
InChIKey: TZWCUFIKVHLRMN-GFCCVEGCSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Time-Resolved Fluorescence Energy Transfer (TR-FRET Tracer A): To determine of binding activity of the test compounds, full-length human ATR protein was expressed and purified together with ATRIP as described above. Furthermore, a fluorescently labelled compound (tracer A) was used as a tracer molecule. Detection of the binding event of the tracer was achieved by time-resolved fluorescence energy transfer (TR-FRET). We used an anti-GST-Terbium antibody (CisBio) that binds to the GST-tag at the N-terminus of ATR-kinase. Excitation of Terbium with 337 nm light results in emission of fluorescent light with 545 nm. In case a tetrameric complex has formed (antiGST-Tb+GST-ATR+Strp2-ATRIP+tracer), part of the energy will be transferred from the Terbium to the fluorophore that itself emits light of 570 nm. Displacement of the fluorescent tracer by a test compound leads to a reduction of the TR-FRET-signal.For the assay 50 nl of a 100-fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384well microtiter plate (MTP, Greiner Bio-One, Frickenhausen, Germany). To prepare the ATR-working solution, ATR/ATRIP stock solution was diluted in assay buffer [50 mM HEPES (pH 7.0), 10 mM MgCl2, 1 mM DTT, 0.01% (w/v) Igepal, 0.01% (w/v) BSA] to 4.2 nM protein concentration (concentration may vary from lot to lot of protein preparation). AntiGST-Tb antibody was diluted to 4.2 nM. The ATR-working solution was incubated for 30 min at 22° C. prior to dispensing to pre-form the complex of antiGST-Tb+GST-ATR+ATRIP. Then, 3 μl of the ATR-working solution were added to the test compound and the mixture was incubated for 10 min at 22° C. to allow pre-binding of the test compounds to ATR/ATRIP. Then, 2 μl of a 100 nM solution of either tracer A or B in assay buffer were added to the ATR-working solution. The resulting mixture was incubated for 30 min at 22° C. The measurement of the TR-FRET signal was performed in a standard HTRF-compatible MTP reader instrument (e.g. BMG Pherastar) by recording the fluorescence emissions at 545 nm and 570 nm after excitation at 337-350 nm. The ratio between emission at 570 nm divided by emission at 545 nm was calculated to give the well ratio. The experimental data (well ratios) were normalised by the following way: positive control contained ATR-working solution plus either tracer A or B solution (=0% inhibition), the negative control contained all components except GST-ATR/ATRIP (=100% inhibition).
Measurement: IC50 = 56.2 nM (pChEMBL 7.25)
Descriptors: MW 411.42, ALogP 3.5, HBA 7, HBD 1, PSA 84.75, QED 0.55
Compound (SMILES): O=C(N[C@H](Cc1ccc(-c2ccccc2)cc1)CC(O)C(=O)O)c1cc(C(=O)O)n(Cc2c(F)ccc(Cl)c2F)n1
InChIKey: LXXNNOZWYPHZSD-OEPVSBQMSA-N
Target: Neprilysin (SINGLE PROTEIN), Homo sapiens
UniProt: P08473
Assay: Enzyme Inhibition Assay: The assays were performed in 384-well white opaque plates at 37° C. using the fluorogenic peptide substrates at a concentration of 10 μM in Assay Buffer (NEP: 50 mM HEPES, pH 7.5, 100 mM NaCl, 0.01% polyethylene glycol sorbitan monolaurate (Tween-20), 10 μM ZnSO4; ACE: 50 mM HEPES, pH 7.5, 100 mM NaCl, 0.01% Tween-20, 1 μM ZnSO4). The respective enzymes were used at concentrations that resulted in quantitative proteolysis of 1 μM of substrate after 20 minutes at 37° C.Test compounds were assayed over the range of concentrations from 10 μM to pM. Test compounds were added to the enzymes and incubated for 30 minute at 37° C. prior to initiating the reaction by the addition of substrate. Reactions were terminated after 20 minutes of incubation at 37° C. by the addition of glacial acetic acid to a final concentration of 3.6% (v/v).
Measurement: Ki = 3.55 nM (pChEMBL 8.449999809265137)
Descriptors: MW 583.98, ALogP 4.4, HBA 6, HBD 4, PSA 141.75, QED 0.19