lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCN(C)[C@]1(c2ccccc2)CC[C@]2(CC1)CN(CC(=O)NC)C(=O)N2CC1CCC1
InChIKey: BTCZGQFDZOTIST-PLQXJYEYSA-N
Target: Nociceptin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41146
Assay: Human Nociceptin/Orphanin FQ Peptide (hNOP) Receptor Binding Assay: The hNOP receptor binding assay was performed as homogeneous SPA-assay (scintillation proximity assay) using the assay buffer 50 mM TRIS-HCl, 10 mM MgCl2, 1 mM EDTA (pH 7.4). The final assay volume (250 μl/well) included 0.5 nM of [leucyl-3H]nociceptin as ligand (PerkinElmer Life Sciences, Inc. Boston, Mass., USA), and either test compound in dilution series or 1 μM unlabelled nociceptin for determination of unspecific binding. The test compound was diluted with 25% DMSO in H2O to yield a final 0.5% DMSO concentration, which also served as a respective vehicle control. The assay was started by adding wheat germ agglutinin coated SPA beads (GE Healthcare UK Ltd., Buckinghamshire, UK) which had been preloaded with hMOP receptor membranes (PerkinElmer Life Sciences, Inc. Boston, Mass., USA). After incubation for 60 minutes at RT and centrifugation for 20 minutes at 500 rpm the signal rate was measured by means of a 1450 Microbeta Trilux ß-counter (PerkinElmer Life Sciences/Wallac, Turku, Finland). Half-maximal inhibitory concentration (IC50) values reflecting 50% displacement of [3H]nociceptin-specific receptor binding were calculated by nonlinear regression analysis and Ki values were calculated by using the Cheng-Prusoff equation, (Cheng and Prusoff, 1973).
Measurement: Ki = 36.5 nM (pChEMBL 7.440000057220459)
Descriptors: MW 426.61, ALogP 3.43, HBA 3, HBD 1, PSA 55.89, QED 0.73
Compound (SMILES): O=C1CCC(N2Cc3c(NC(=O)CCNC=C4C(=O)CC(c5ccccc5)CC4=O)cccc3C2=O)C(=O)N1
InChIKey: PSQFZFIGEYQUJL-HKWRFOASSA-N
Target: Microtubule-associated protein 1 light chain 3 beta (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZQ8
Assay: Assay Targeting LC3B: By constructing a prokaryotic expression system, the LC3B protein was successfully expressed and purified, and a preliminary screening and verification platform was established using fluorescence polarization experiments to determine the activity of purchased and synthesized small compound libraries. The recombinant protein GST-LC3B (final concentration 180 nM) (SEQ ID NO: 1) and N-terminal FITC-labeled peptide (SEQ ID NO: 2, final concentration 18 nM) were placed in the FP buffer (50 mM HEPES pH 7.5, 0.1 mg/ml BSA and 1 mM DTT), to which a compound serially diluted with the FP buffer was added. Then the mixture was incubated at 25° C. in the dark. The fluorescence polarization value (PerkinElmer Envision, emission wavelength 480 nm; absorption wavelength 535 nm) was monitored, and the IC50 value was calculated using the GraphPad Prism 6.0 program.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 528.57, ALogP 1.97, HBA 7, HBD 3, PSA 141.75, QED 0.22
Compound (SMILES): C=CC(=O)Nc1cccc(Nc2nc(Nc3ccc(OCCOC)cc3)ncc2C(C)=O)c1
InChIKey: VVMGMLRVJMNXAO-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ITK/TSK (SINGLE PROTEIN), Homo sapiens
UniProt: Q08881
Assay: Omnia Assay: ITK: This example describes continuous-read kinase assays to measure inherent potency of compound against active forms of ITK enzymes as described in Example 251 above except that the modified ITK-optimized reagent conditions are:[ITK]=10 nM, [ATP]=25 [Y6-Sox]=10 μM (ATP KMapp=33 μM).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 447.5, ALogP 4.32, HBA 8, HBD 3, PSA 114.47, QED 0.23
Compound (SMILES): Nc1ncnc2c1c(-c1ccc(NC(=O)Nc3cc(C4(C(F)(F)F)CCC4)no3)c(F)c1)cn2C1CC1
InChIKey: ITRBLYRMVKDPOC-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase Nek7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TDX7
Assay: Enzymatic Assay: Casein substrate (from bovine milk, hydrolyzed and partially dephosphorylated mixture of α, β and a caseins, obtained from Sigma Aldrich, catalogue #C4765, diluted in distilled water to a final concentration of 1 mg/mL) and full-length recombinant human NEK7 (expressed by baculovirus in Sf9 insect cells using a N-terminal GST tag, obtained from SignalChem, catalogue #N09-10G, 0.1 μg/μL) were mixed in assay buffer (20 mM Hepes pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO). Compounds of interest (serial 3-fold dilution in DMSO from 10 μM to 0.5 nM) or vehicle (1% DMSO) were dispensed into the kinase reaction mixture by Acoustic technology (Echo550; nanoliter range). After incubation at room temperature for 20 minutes, the kinase reaction was initiated by addition of [33P]-ATP (specific activity 10 ρCi/μl) and the mixture was incubated at room temperature for 2 hours. The reaction was then stopped by spotting the reaction mixture on strips of phosphocellulose P81 paper. Following washing, the radioactivity of the P81 paper was measured and kinase activity data were expressed as the percent remaining kinase activity in test samples compared to vehicle reactions.
Measurement: IC50 = 225 nM (pChEMBL 6.650000095367432)
Descriptors: MW 515.47, ALogP 5.77, HBA 7, HBD 3, PSA 123.89, QED 0.29
Compound (SMILES): CC(C)(C)CNc1c(C#N)cnc2c(C#N)cc(N[C@H](C3=CN(C4(C(N)=O)CC4)NN3)c3cccc4cnccc34)cc12
InChIKey: UJMZJODOKIFHDW-LJAQVGFWSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: High Throughput Biochemical Assay: EGFR activity was measured using KinEASE (Cisbio), a time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay. In this assay, EGFR-catalyzes the phosphorylation of a universal Tyrosine kinase peptide substrate labeled with XL665. Europium conjugated phosphor-tyrosine specific antibody binds the resulting phosphorylated peptide. Formation of phosphorylated peptide is quantified by TR-FRET with Europium as the donor and XL665 the acceptor. The assay was performed in two main steps. The first step is the kinase reaction step and the second step is the detection step with TR-FRET reagents. In brief, test compounds 1:3 serially diluted in DMSO were delivered into Corning white, low volume, non-binding 384 well plates using the Echo 550 acoustic liquid dispenser (Labcyte®). EGFR enzyme (Human EGFR, cytoplasmic domain [669-1210] from Carna Biosciences Cat. No. 08-115) and substrates TK substrate-biotin (included in Cisbio HTRF KinEASE-TK kit Cat. No. 62TK0PEJ) were dispensed into assay plates using a Multi-Flo (Bio-Tek Instruments). The standard 10 μL reaction mixture contained 6 μM ATP (1×Km) or 12 μM ATP (2×Km), 1 μM biotinylated peptide, 0.3 nM EGFR (for 1×Km ATP) or 0.1 nM EGFR (for 2×Km ATP) in reaction buffer (10 mM MOPs, pH 7.0, 1.5% Glycerol, 0.5 mg/ml BSA, 10 mM Mg-Acetate, 1 mM DTT, 0.025% NP-40). After 60 min of incubation at room temperature, 10 μL of Stop and Detect Solution (1:400 Europium Cryptate labeled anti-phosphorylated peptide antibody solution and 125 nM strepavidin-XL665 Tracer in a 50 mM Hepes pH 7.0 detection buffer containing sufficient EDTA) was added. The plate was then further incubated for over 60 minutes at room temperature and read using an Envision 2103 Multilabeled reader (PerkinElmer) with excitation/emission/FRET emission at 340 nm/615 nm/665 nm, respectively.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 572.68, ALogP 4.32, HBA 10, HBD 5, PSA 167.81, QED 0.21
Compound (SMILES): CN(C)CCCC[C@@H](NC1CCC2(CC1)CCN(Cc1ccccc1)C2)C(=O)N1CCN(C2=NC(C)(C)CO2)C[C@H]1C(=O)NCc1cccs1
InChIKey: LKCOGCAGIMSLQK-SCVMGEOMSA-N
Target: Coagulation factor XII (SINGLE PROTEIN), Homo sapiens
UniProt: P00748
Assay: Factor XIIa Assay: The effectiveness of a compound of the present invention as an inhibitor of Coagulation Factor XIIa can be determined using a relevant purified serine protease, and an appropriate synthetic substrate. The rate of hydrolysis of the chromogenic or fluorogenic substrate by the relevant serine protease was measured both in the absence and presence of compounds of the present invention. Assays were conducted at room temperature or at 37° C. Hydrolysis of the substrate resulted in release of amino trifluoromethylcoumarin (AFC), which was monitored spectrofluorometrically by measuring the increase in emission at 510 nm with excitation at 405 nm. A decrease in the rate of fluorescence change in the presence of inhibitor is indicative of enzyme inhibition. Such methods are known to one skilled in the art. The results of this assay are expressed as the IC50, the inhibitor concentration causing a 50% decrease in Factor XIIa protease activity.Factor XIIa activity determinations were made in 50 mM HEPES buffer containing 150 mM NaCl, 5 mM CaCl2, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific) at pH 7.4. Determinations were made using purified human Factor XIIa at a final concentration of 500 pM (Sekisui Diagnostics) and the synthetic substrate, n-Acetyl-Lys-Pro-Arg-AFC, TFA salt (Sigma #C6608) at a concentration of 100 μM.Activity assays were performed by diluting a stock solution of substrate at least tenfold to a final concentration ≤0.1 Km into a solution containing enzyme or enzyme equilibrated with inhibitor. Times required to achieve equilibration between enzyme and inhibitor were determined in control experiments. Initial velocities of product formation in the absence (Vo) or presence of inhibitor (Vi) were measured. IC50 was determined as the concentration of I yielding Vi=Vo/2.
Measurement: IC50 = 9.2 nM (pChEMBL 8.039999961853027)
Descriptors: MW 706.01, ALogP 4.57, HBA 9, HBD 2, PSA 92.75, QED 0.3
Compound (SMILES): CC(=O)NC[C@H]1CN(S(=O)(=O)c2cc(Cl)cc(C#N)c2)c2cc(NC(=O)OC(C)(C)C(F)(F)F)ccc2O1
InChIKey: NFWKEJHFVUHSKE-KRWDZBQOSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Biochemical TR-FRET assay: The compounds of the invention inhibit RORgammaT activity. Activation of RORgammaT activity can be measured using, e.g., a biochemical TR-FRET assay. In such an assay, interaction of cofactor-derived peptides with human RORgammaT-Ligand Binding Domain (LBD) can be measured. The TR-FRET technique is a sensitive biochemical proximity assay that will give information concerning the interaction of a ligand with the LBD, in the presence of cofactor-derived peptides (Zhou et al., Methods 25:54-61, 2001).To identify novel antagonists of RORgammaT, an assay was developed which employs the interaction of RORgammaT with its co-activator peptide SRC1_2. This peptide mimics the recruitment of co-activators to RORgammaT through its interaction with the LXXLL (SEQ ID NO:1) (e.g., NR box) motifs (Xie et al., J. Immunol. 175: 3800-09, 2005; Kurebayashi et al., Biochem. Biophys. Res. Commun. 315: 919-27, 2004; Jin et al., Mol. Endocrinology 24:923-29, 2010). The RORγ-Ligand Binding Domain TR-FRET Assay was run according to the following protocol.HIS-tagged RORγ-LBD protein was recombinantly expressed in Escherichia coli. The RORγ-LBD protein was purified by Ni2+-affinity resin. Purified protein was then diluted in assay buffer (50 mM Tris pH 7.0, 50 mM KCl, 1 mM EDTA, 0.1 mM DTT, 100 μg/mL bovine serum albumin, delipidated) to obtain a RORγ-LBD final concentration of 3 nM. Europium tagged anti-HIS antibody was also added to this solution (1.25 nM). Separately, SF9 cells not expressing any recombinant protein were lysed (32,000 cells per μl in 25 mM Tris, 50 mM NaCl) and the previously frozen lysate was added to the diluted RORγ-LBD solution at a ratio of 0.75 μl SF9 lysate per 15 μl of diluted RORγ-LBD.
Measurement: IC50 = 43.6 nM (pChEMBL 7.360000133514404)
Descriptors: MW 574.97, ALogP 4.19, HBA 7, HBD 2, PSA 137.83, QED 0.53
Compound (SMILES): CNC(=O)c1ccc(CN2Cc3ccc4nccn4c3C2=O)cc1
InChIKey: KVZJZNYOXKRWHY-UHFFFAOYSA-N
Target: Muscarinic acetylcholine receptor M1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11229
Assay: Cell-Based Functional Assay of Muscarinic Acetylcholine Receptor: For high throughput measurement of agonist-evoked increases in intracellular calcium, CHO-K1 cells stably expressing muscarinic receptors were plated in growth medium lacking G418 and hygromycin at 15,000 cells/20 μL/well in Greiner 384-well black-walled, tissue culture (TC)-treated, clear-bottom plates (VWR). Cells were incubated overnight at 37° C. and 5% CO2. The next day, cells were washed using an ELX 405 (BioTek) with four washes (80 μL) of assay buffer then aspirated to 20 μL. Next, 20 μL of 16 μM Fluo-4/acetoxymethyl ester (Invitrogen, Carlsbad, Calif.) prepared as a 2.3 mM stock in DMSO and mixed in a 1:1 ratio with 10% (w/v) Pluronic F-127 and diluted in assay buffer was added to the wells and the cell plates were incubated for 50 min at 37° C. and 5% CO2. Dye was removed by washing with the ELX 405 (four 80 μL washes of assay buffer) then aspirated to 20 μL. Compound master plates were formatted in an 11 point CRC format (1:3 dilutions) in 100% DMSO with a starting concentration of 10 mM using the BRAVO liquid handler (Agilent). Test compound CRCs were then transferred to daughter plates (240 nL) using the Echo acoustic plate reformatter (Labcyte, Sunnyvale, Calif.) and then diluted into assay buffer (40 μL) to a 2× stock using a Thermo Fisher Combi (Thermo Fisher Scientific, Waltham, Mass.).Calcium flux was measured using the Functional Drug Screening System (FDSS) 6000 (Hamamatsu Corporation, Tokyo, Japan) as an increase in the fluorescent static ratio. Compounds were applied to cells (20 μL, 2×) using the automated system of the FDSS 6000 at 4 s into the 300 s protocol and the data were collected at 1 Hz. At 144 s into the 300s protocol, 10 μL of an EC20 concentration of the muscarinic receptor agonist acetylcholine was added (5×), followed by the addition of 12 μL an EC8O concentration of acetylcholine at the 230 s time point (5×). Agonist activity was analyzed as a concentration-dependent increase in calcium mobilization upon compound addition. Positive allosteric modulator activity was analyzed as a concentration-dependent increase in the EC20 acetylcholine response. Antagonist activity was analyzed as a concentration-dependent decrease in the EC80 acetylcholine response. Concentration-response curves were generated using a four-parameter logistical equation in XLfit curve fitting software (IDBS, Bridgewater, N.J.) for Excel (Microsoft, Redmond, Wash.) or Prism (GraphPad Software, Inc., San Diego, Calif.).
Measurement: EC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 320.35, ALogP 1.85, HBA 4, HBD 1, PSA 66.71, QED 0.8
Compound (SMILES): C=Cc1nc(C(=O)N[C@@H](CCC(=O)O)C(=O)O)ccc1NC(=O)Cc1c(N)nc(N)[nH]c1=O
InChIKey: UTFFVIDGGZNLGQ-LBPRGKRZSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: Inhibition of MTHFD2: To determine the IC50 value of a compound, an 11-concentration dose-response curve with 3-fold difference in concentration between assay points was generated by using an acoustic dispenser (Echo 550 Liquid handler, Labcyte). Each assay point was run in duplicate and the assay was performed in a white 384-well ProxiPlate Plus (6008280, PerkinElmer). DMSO was used as negative control. The serial dilution in DMSO, from compound DMSO stock solution, was created by dispensing from a 384-well low dead volume microplate (LP-0200, Labcyte) and a 384-well polypropylene microplate 2.0 (PP-0200, Labcyte). A total of 2.5 μL MTHFD2 was preincubated with compound or DMSO for 10 min. The enzymatic reaction was initiated by adding 2.5 μL folitixorin (F680350, Toronto Research Chemicals). For background control, 5 μL buffer was added to the well. Final concentrations of the components in the assay were 3.4 nmol/L MTHFD2, 5 μmol/L folitixorin and 250 μmol/L NAD+. The final concentrations of all reagents in a total assay volume of 5 μL per well were 50 mmol/L Tris-HCl at pH 8.0, 100 mmol/L NaCl, 5 mmol/L MgCl2, 25 mmol/L Na3PO4 at pH 8.0, 0.005% (v/v) Tween-20, and 2 mmol/L 2-mercaptoethanol. After 15 min reaction time, 5 μL NAD(P)H-Glo detection reagent (G9061 or G9062, Promega) was dispensed in all wells and the plate was incubated for 60 min. Luminescence was measured on a plate reader (Envision, PerkinElmer or Sense, Hidex). The light signal produced is proportional to the amount of NAD(P)H in the sample.
Measurement: IC50 = 298 nM (pChEMBL 6.53000020980835)
Descriptors: MW 459.42, ALogP -0.8, HBA 9, HBD 7, PSA 243.48, QED 0.23
Compound (SMILES): COCCC(C(=O)Nc1ccn2ncc(C(=O)N(C)C)c2c1)n1cc(OC)c(-c2cc(Cl)ccc2C#N)cc1=O
InChIKey: PAYFVGMTFBHCRS-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Measurement of FXIa Inhibition: The factor XIa inhibition of the substances according to the invention is determined using a biochemical test system which utilizes the reaction of a peptidic factor XIa substrate to determine the enzymatic activity of human factor XIa. Here, factor XIa cleaves from the peptic factor XIa substrate the C-terminal aminomethylcoumarin (AMC), the fluorescence of which is measured. The determinations are carried out in microtitre plates. Test substances are dissolved in dimethyl sulphoxide and serially diluted in dimethyl sulphoxide (3000 μM to 0.0078 μM; resulting final concentrations in the test: 50 μM to 0.00013 μM). 1 μl of the diluted substance solutions is placed into each of the wells of white microtitre plates from Greiner (384 wells). 20 μl of assay buffer (50 mM of Tris/HCl pH 7.4; 100 mM of sodium chloride; 5 mM of calcium chloride; 0.1% of bovine serum albumin) and 20 μl of factor XIa from Kordia (0.45 nM in assay buffer) are then added successively. After 15 min of incubation, the enzyme reaction is started by addition of 20 μl of the factor XIa substrate Boc-Glu(OBzl)-Ala-Arg-AMC dissolved in assay buffer (10 μM in assay buffer) from Bachem, the mixture is incubated at room temperature (22° C.) for 30 min and fluorescence is then measured (excitation: 360 nm, emission: 460 nm). The measured emissions of the test batches with test substance are compared to those of control batches without test substance (only dimethyl sulphoxide instead of test substance in dimethyl sulphoxide), and IC50 values are calculated from the concentration/activity relationships.
Measurement: IC50 = 93 nM (pChEMBL 7.03000020980835)
Descriptors: MW 563.01, ALogP 3.61, HBA 9, HBD 1, PSA 130.96, QED 0.33
Compound (SMILES): CC(O)CN1CCC[C@@H](COc2cc(-c3ccc(C#N)cc3)c(-c3cnc4c(c3)OCCO4)n3cncc23)C1
InChIKey: JZBGHCMTMZHQAX-LWMIZPGFSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: Demethylase Biochemical Assay: LANCE LSD1/KDM1A demethylase assay¿10 uL of 1 nM LSD-1 enzyme (ENZO BML-SE544-0050) in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 25 mM NaCl, 5 mM DTT) were preincubated for 1 hour at 25° C. with 0.8 uL compound/DMSO dotted in black 384 well polystyrene plates. Reactions were started by addition of 10 uL of assay buffer containing 0.4 uM Biotin-labeled Histone H3 peptide substrate: ART-K(Me1)-QTARKSTGGKAPRKQLA-GGK(Biotin) SEQ ID NO:1 (AnaSpec 64355) and incubated for 1 hour at 25° C. Reactions were stopped by addition of 10 uL 1x LANCE Detection Buffer (PerkinElmer CR97-100) supplemented with 1.5 nM Eu-anti-unmodified H3K4 Antibody (PerkinElmer TRF0404), and 225 nM LANCE Ultra Streptavidin (PerkinElmer TRF102) along with 0.9 mM Tranylcypromine-HCl (Millipore 616431). After stopping the reactions plates were incubated for 30 minutes and read on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 = 75 nM (pChEMBL 7.119999885559082)
Descriptors: MW 525.61, ALogP 4.18, HBA 9, HBD 1, PSA 105.14, QED 0.38
Compound (SMILES): CC(C)(Sc1ccc2cncc(-c3ccc(C#N)c4c3OCCO4)c2c1)C(=O)O
InChIKey: VYOZCONAIFVTSM-UHFFFAOYSA-N
Target: Solute carrier family 22 member 12 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96S37
Assay: hURAT1 Inhibition Assay: Human embryonic kidney cells (HEK293) was incubated in DMEM tissue culture medium, at 37° C., under 5% CO2 and 95% air atmosphere. TransIT-293 transfection agent (MIRUS BIO, Cat. No. MIR2706) and model URAT1 were used to construct transfected HEK293 cells. Transfected HEK293/hURAT1 cells were used to the test for 14C-uric acid transport activity. HEK293/hURAT1 cells were seeded in a 96-well plate (BD, Cat. No. 356461) fully coating with poly-D-lysine at a density of 6×104 cells per well. Cells were incubated at 37° C. for at least 12 hrs in the calorstat, and then washed with pre-heated washing buffer (125 mM sodium gluconate, 10 mM HEPES pH=7.4) at an amount of 200 μL per well to wash out the culture medium. The uric acid [8-14C] (ARC, Cat. No. ARC0513-250UCI) containing or not containing the compound was added to 50 μL HBSS buffer which was free of chloric ion each well (HBSS buffer: 125 mM sodium gluconate, 4.8 mM potassium gluconate, 1.3 mM calcium gluconate, 1.2 mM potassium dihydrogen phosphate, 1.2 mM magnesium sulfate, 5.6 mM glucose, 25 mM HEPES pH=7.4) to make the specific concentration of the uric acid 1 μCi per well. The incubating solution was removed after 10 mins incubation, followed by adding 100 μL cold washing buffer, after washing with this buffer for 3 times, the buffer was completely removed from the well. 50 μL Lysis buffer (0.1 mM NaOH) was added to each well, and transferred to a 96-well plate (PERKIN ELMER, Cat. No. 6005040) containing scintillation fluid after 5 mins, and counted by MicroBeta Trilux (PerkinElmer) to give IC50 value eventually.
Measurement: IC50 = 62 nM (pChEMBL 7.210000038146973)
Descriptors: MW 406.46, ALogP 4.5, HBA 6, HBD 1, PSA 92.44, QED 0.64
Compound (SMILES): C=CC(=O)N[C@@H]1CCCC[C@@H]1Nc1ncc2c(n1)N(Cc1nccs1)C(=O)N(c1c(Cl)c(OC)cc(OC)c1Cl)C2
InChIKey: NOZRPKCJSLOLRB-SJORKVTESA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: Omnia Assay: Omnia Assay Protocol for Potency Assessment Against FGFR 4 Enzyme. a) [FGFR4-WT]=10 nM, [ATP]=300 uM, [Y10-Sox]=10 uM (ATP KMapp 300 uM): A 10x stock solution of FGFR4-WT (PR4380C), (from Invitrogen, Carlsbad, Calif.) corresponding to method a in Table 7, was prepared as described below. Alternatively, a 10x stock solution of FGFR4-WT (F01-11G), (SignalChem, Richmond, BC) corresponding to method b in Table 7, was prepared as described below. A solution of 1.4xATP (AS001A) and 5x Tyr-Sox conjugated peptide substrate (KNZ3101) were prepared in 1x kinase reaction buffer consisting of 20 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 5 mM β-glycerophosphate, 5% glycerol (10x stock, KB002A) and 0.2 mM DTT (DS001A). 5 uL of FGFR4 was pipetted into a Corning (#3574) 384-well, white, non-binding surface microtiter plate (Corning, N.Y.), containing a 0.5 uL volume of 100% DMSO. The serially diluted compounds were prepared on a Tecan EVO100. A second addition of 10 ul of Tyr-Sox FGFR4 substrate was added to each well and the kinase reactions were started with the addition of 35 uL of 1.4xATP. The reactions were monitored every 71 seconds for 240 minutes at λex360/λem485 in a Synergy plate reader from BioTek (Winooski, Vt.). At the conclusion of each assay, progress curves from each well were examined for linear reaction kinetics and fit statistics (R2, 95% confidence interval, absolute sum of squares). Initial velocity (0 minutes to ~60 minutes) from each reaction was determined from the slope of a plot of relative fluorescence units vs time (seconds) and then plotted against inhibitor concentration to estimate IC50 from log [Inhibitor] vs Response (Variable Slope model in GraphPad Prism from GraphPad Software (San Diego, Calif.)).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 618.55, ALogP 5.43, HBA 9, HBD 2, PSA 121.81, QED 0.3
Compound (SMILES): Cn1cnc(S(=O)(=O)N2C[C@@H](NCc3ccc(Cl)cc3Cl)[C@H](c3cccnc3)C2)c1
InChIKey: OCLXSWVVPCJPBW-PKOBYXMFSA-N
Target: Alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P26572
Assay: Radioligand Binding Assay: Radioligand binding assays using recombinant CHO cell membranes expressing human GlyT1: Radioligand binding to human GlyT1c transporter-expressing membranes was determined as described in Mezler et al., Molecular Pharmacology 74:1705-1715, 2008.
Measurement: Ki < 10000 nM (pChEMBL 5)
Descriptors: MW 466.39, ALogP 3.07, HBA 6, HBD 1, PSA 80.12, QED 0.6
Compound (SMILES): CCCCn1ncnc1-c1ccc(N2CCN(C(=O)Nc3c(Cl)cccc3Cl)CC2)cc1
InChIKey: HMROCTSHOZTYJG-UHFFFAOYSA-N
Target: Diacylglycerol O-acyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75907
Assay: FlashPlate™ Assay: DGAT activity in membrane preparations was assayed in 50 mM Tris-HCl (pH 7.4), 150 mM MgCl2, 1 mM EDTA and 0.2% BSA, containing 50 μM DAG, 32 μg/ml PC/PS and 8.4 μM [3H]-oleoylCoA (at a specific activity of 30 nCi/well) in a final volume of 50 μl in 384-well format using the red shifted Basic Image FlashPlate™ (Perkin Elmer Cat.No. SMP400). In detail, 10 μl enzyme mix and 10 μl substrate mix were added to 30 μl of assay buffer, optionally in the presence of 1 μl DMSO (blank and controls) or 1 μl of the compound to be tested. This reaction mixture was incubated for 120 minutes at 37° C. and the enzymatic reaction stopped by adding 20 μl of the stop mix. The plates were sealed and the vesicles allowed to settle overnight at room temperature. Plates were centrifuged for 5 minutes at 1500 rpm and measured in Leadseeker.
Measurement: IC50 = 407 nM (pChEMBL 6.389999866485596)
Descriptors: MW 473.41, ALogP 5.41, HBA 5, HBD 1, PSA 66.29, QED 0.52
Compound (SMILES): CCS(=O)(=O)c1ccc(C#N)cc1Nn1cnc2ccc(C(F)(F)F)cc2c1=O
InChIKey: AVTQRUYWTOODAS-UHFFFAOYSA-N
Target: Epithelial discoidin domain-containing receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q08345
Assay: LanthaScreen Assay: DDR1 binding activity was measured using the LanthaScreen (Registered trademark) Eu Kinase Binding Assay (manufactured by Life Technologies Corporation). The test compound and the Alexa Fluor® 647-labeled Kinase Tracer 178 (manufactured by Life Technologies Corporation) were added to a mixture of DDR1 and the LanthaScreen (Registered trademark) Eu-anti-GST antibody. After one-hour reaction at room temperature, the fluorescence resonance energy transfer was measured. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate relative to the test compound-free control.
Measurement: IC50 = 1800 nM (pChEMBL 5.75)
Descriptors: MW 422.39, ALogP 2.96, HBA 7, HBD 1, PSA 104.85, QED 0.69
Compound (SMILES): CS(=O)(=O)N1CC=C(c2ccc(-c3cnc4ncc(C5(c6ccc7ncccc7c6)CC5)n4n3)cc2)CC1
InChIKey: PQNUNRXWPXKMFN-UHFFFAOYSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: In Vitro c-Met Kinase Enzyme Assay: Compounds were screened in vitro for their ability to inhibit c-Met kinase activity. The IC50 values of compounds for the inhibition of c-Met kinase were determined as described in the literature with some modifications (Wang, X. et al, Mol. Cancer Ther. 2003, 2(11):1085-1092; Calic, M. et al., Croatica Chemical ACTA. 2005, 78(3):367-374). Briefly, histidine-tagged c-Met catalytic domain fusion protein (Invitrogen, #PV3143) was used for the assay. IC50 measurements were based on the degree of phosphorylation of poly Glu-Tyr (Sigma-Aldrich, #P0275) that was coated (0.01 mg/per well) on 96-well microplates (R&D systems, #DY990). The reaction was carried out in a 50 μL solution containing 50 mM HEPES (pH 7.5), 10 mM MnCl2, 10 mM MgCl2, 0.5 mM DTT, 100 μM Na3VO4, 5 μM ATP (Cell Signaling Technology, #9804) and serial dilutions of individual compounds. The reaction lasted for 25 minutes at 30° C. After the reaction was completed, the contents of the plates was discarded. Plates were then washed with TBS-T (250 μL/well, 5×) and then blocked with TBS-T containing 1% BSA for 2 hours. The contents of the plates was discarded, and 100 μL (per well) of peroxidase-labeled anti-phospho-tyrosine antibody (Sigma, #A5964) diluted (1:60,000) in 1% BSA containing TBS-T were then added and incubated for 1 hour. Plates were washed with TBS-T (250 mL/well, 5×) and followed by the color reaction using 100 μL (1:1 mixture) of H2O2 and tetramethylbenzidine (R&D Systems, #DY999). The reaction was stopped in minutes with 100 μL of 2 N H2SO4. The optical density was measured immediately using a microplate reader at 450 nm with wavelength correction at 540 nm. IC50 values were calculated with the GraphPad Prism software. The linear range (i.e., the time period over which the rate remained equivalent to the initial rate) was determined for the kinase and IC50 determinations were performed within this range.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 522.63, ALogP 4.47, HBA 7, HBD 0, PSA 93.35, QED 0.34
Compound (SMILES): FC(F)Oc1cc(-c2cnc3cnn(Cc4cccnc4)c3c2)ccc1Cl
InChIKey: UXGSWTWBSLZTEI-UHFFFAOYSA-N
Target: Glutamate NMDA receptor; GRIN1/GRIN2B (PROTEIN COMPLEX), Homo sapiens
UniProt: Q05586
Assay: Effects of Test Articles on Cloned Human NR1/GuN2B Ion Channels Expressed in Mammalian Cells: NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty-four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed, and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 137 mM NaCl, 4 mM KCl, 2 mM CaCl2, 0.5 mM MgCl2 (standard assay) or 1.5 mM MgCl2 (HTS assay), 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (137 mM NaCl (standard assay) or 150 mM (HTS assay), 4 mM KCl (standard assay) or 3 mM (HTS assay), 2 mM CaCl2), 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to EC40 (standard assay) or EC40 (HTS assay) is used to maximize the assay's signal window and ability to detect NMDA receptor antagonists and negative allosteric modulators. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 117 nM (pChEMBL 6.929999828338623)
Descriptors: MW 386.79, ALogP 4.8, HBA 5, HBD 0, PSA 52.83, QED 0.49
Compound (SMILES): C[C@H](NC(=O)C1CN(c2ncnn3cc(-c4ccc(OC(C)(C)C)cc4)cc23)C1)c1ccc(Cl)cc1
InChIKey: UTXNUWNBWCKTRD-SFHVURJKSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Kit Wild Type Assay: In each well of a 384-well plate, 0.2 ng/ul final (2 nM) of wild type Kit (Carna Bioscience 08-156) was incubated in a total of 12.5 ul of buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 10 mM MgCl2, 1 mM DTT) with 1 uM Srctide (5-FAM-GEEPLYWSFPAKKK-NH2) and 400 uM ATP at 25 C for 90 minutes in the presence or absence of a dosed concentration series of compound (1% DMSO final concentration). The reaction was stopped by the addition of 70 ul of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 35 mM EDTA and 0.2% of Coating Reagent 3 (Caliper Lifesciences)). The plate was then read on a Caliper EZReader 2 (protocol settings: −1.9 psi, upstream voltage −700, downstream voltage −3000, post sample sip 35s).
Measurement: IC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 504.03, ALogP 5.54, HBA 6, HBD 1, PSA 71.76, QED 0.37
Compound (SMILES): CC(C)C1CCC(CC(O)c2c(C3CC3)sc3cncn23)CC1
InChIKey: FGIBOPVUFJDYQF-UHFFFAOYSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: Enzymatic Assay: IDO1: Recombinant full-length human IDO1 with a N-terminal hexahistidine tag expressed in E. coli and purified to homogeneity is incubated at a final concentration of 2 nM in assay buffer consisting of 37.5 mM phosphate buffer at pH6.5 supplemented with 10 mM sodium L-ascorbate, 0.45 μM methylene blue, 50 U/ml catalase, 0.01% BSA, and 0.01% Tween 20 (protocol modified from Seegers et al, JBS 2014). Example compounds are serially diluted in DMSO, further diluted in phosphate buffer, and added to the enzyme at final concentrations ranging from 10 μM to 0.5 nM. The final DMSO concentration is 0.6%. Following a pre-incubation of 30 minutes at RT, the reaction is started by the addition of L-tryptophan at a final concentration of 5 μM in assay buffer. After 30 minutes of incubation at RT, 3 μL of the reaction mixture are transferred to a 384 deep well plate containing 25 μL of deionized water. 100 μl of 200 nM L-Tryptophan-(indole-d5) in cold 100% methanol are added followed by a 10 minutes centrifugation at 4,000 rpm at 4° C. An additional 75 μL of deionized water are then added and followed by a 10 minutes centrifugation at 4,000 rpm at 4° C. The product of the reaction N′-Formylkynurenine (NFK) is quantified by LCMS and normalized to the L-Tryptophan-(indole-d5) signal. Samples with 0.6% DMSO (0% effect) and a TDO/IDO inhibitor (100% effect) are used as control samples to set the parameters for the non-linear regression necessary for the determination of the half-maximal inhibitory concentration (IC50) for each compound. For each compound concentration the percentage of activity compared to 0% and 100% effect is calculated as average ±STDEV (each concentration measured in duplicate). IC50 values and curves are generated with XLfit software (IDBS) using Dose-Response One Site model 203 (four parameter logistic curve model). When compounds are measured multiple times, mean values are given.
Measurement: IC50 = 511 nM (pChEMBL 6.289999961853027)
Descriptors: MW 332.51, ALogP 5.16, HBA 4, HBD 1, PSA 37.53, QED 0.82
Compound (SMILES): CN(C)S(=O)(=O)c1ccc(N2C(=O)c3ccccc3[C@@H](C(=O)O)[C@@H]2c2ccc3c(c2)OCCO3)cc1
InChIKey: JPNGYQYVZDXJBG-RPWUZVMVSA-N
Target: Stimulator of interferon genes protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q86WV6
Assay: H-cGAMP Filtration Binding Assay (HAQ STING): The ability of compounds to bind STING is quantified by their ability to compete with tritiated cGAMP ligand for human STING receptor membrane using a radioactive filter-binding assay. The binding assay employs STING receptor obtained from Trichoplusia ni (T. ni; Expression Systems, cat #94-002F, Expression Systems, Davis, Calif., USA) cell membranes overexpressing full-length HAQ STING prepared in-house and tritiated cGAMP ligand also purified in-house.
Measurement: IC50 = 12250 nM (pChEMBL 4.909999847412109)
Descriptors: MW 508.55, ALogP 3.28, HBA 6, HBD 1, PSA 113.45, QED 0.56
Compound (SMILES): O=c1n(Cc2ccccc2)nnn1-c1ccc(-c2cn[nH]c2)cc1
InChIKey: MKKCGQSCZYGFDF-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL, assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50; i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 = 27.5 nM (pChEMBL 7.559999942779541)
Descriptors: MW 318.34, ALogP 1.87, HBA 6, HBD 1, PSA 81.39, QED 0.62
Compound (SMILES): CNC1=C(C(=N)Nc2cc(C)cc(-c3cnn(C)c3)c2)CN(C(C)=O)CC1
InChIKey: VPENCLLOCSRRGB-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: TR-FRET Binding Assay: His/Flag epitope tagged CBP was cloned, expressed, and purified to homogeneity. CBP binding and inhibition was assessed by monitoring the engagement of a biotinylated small molecule compound with the target using the TR-FRET assay technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate CBP (4 nM final) was combined with biotin-ligand (60 nM final) in 50 mM HEPES (pH 7.5), 50 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 10 minutes incubation at room temperature, a mixture Eu-W1024 Anti-6×His antibody (6×His disclosed as SEO ID NO: 3) (Perkin Elmer AD0110) and SureLight Allophycocyanin-Streptavidin (APC-SA, Perkin Elmer CR130-100) were added to a final concentrations of 0.2 nMolar antibody and 50 nMolar APC-SA, respectively. After twenty minutes of equilibration, the plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 366.47, ALogP 2.51, HBA 5, HBD 3, PSA 86.04, QED 0.57
Compound (SMILES): CN1C(=O)C(NC(=O)c2cc(Oc3ccc(F)cc3)ccn2)COc2ccc(C#CC(C)(C)O)cc21
InChIKey: XJKQMZYDHLEEJF-UHFFFAOYSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: biochemical assay using the ADP-Glo technology: ADP-Glo (Promega, Madison, Wis., USA) reagents were thawed at ambient temperature. Kinase Detection Reagent was prepared by mixing kinase detection buffer with the lyophilized kinase detection substrate.A 500 ml stock volume of 5× Reaction Kinase Buffer was made by mixing 1000 μl of 1M MgCl2, 500 μl of 1M Tris-HCL pH7.4, 0.5 mg/ml (25 mg) of BSA, and 3475 μl of distilled H2O. A 3 ml 2× working stock volume of Reaction Kinase Buffer was made containing a final concentration of 100 μM DTT and 4 mM MnCl2.Components of RIPK1 enzyme (Rigel Pharmaceuticals, South San Francisco, Calif., USA) were thawed on ice. Diluted RIPK1 was prepared in 1× Kinase Reaction Buffer (diluted from 2× buffer) to 31 ng/well. A 166 μM working stock ATP assay solution was prepared in 1× Kinase Reaction Buffer (diluted from 2× buffer).Compounds were serially diluted in DMSO from 250 uM in 4-fold dilutions then diluted 1:5 in 2× Reaction Buffer in a 96 well plate. 1.0 μl of diluted compound was added to a 384 well plate in duplicate. 2 μl of diluted Active RIPK1 was added to 384 well plate (do not add to column 1) add 2× rxn buffer to column 1. AKT (Anaspec, Fremont, Calif., USA) at 150 nM was combined with ATP working stock at equal volume and 2 ul/well were added to the 384 well plate. The final reaction volume was 5.0ul.The plate was quickly centrifuged and the reaction was incubated at 30° C. for 30 minutes. Adding 5 μl of ADP-Glo terminated the reaction. The plate was quickly centrifuged and the reaction was incubated at room temperature for 40 minutes. Kinase Detection Reagent was then added and incubated at room temperature for 30 minutes. The relative light unit (RLU) of kinase reaction was determined by luminescent (Luminescence 0.1 s) using a Wallac Victor2 Luminometer (PerkinElmer, Waltham, Mass., USA).
Measurement: IC50 = 39.2 nM (pChEMBL 7.409999847412109)
Descriptors: MW 489.5, ALogP 3.29, HBA 6, HBD 2, PSA 100.99, QED 0.55
Compound (SMILES): CC(=O)c1nn(CC(=O)N2[C@H](C(=O)Nc3nc(Br)ccc3C)C[C@@]3(C)C[C@@H]23)c2c(C(F)CF)cc(-c3cnc(C)nc3)cc12
InChIKey: CWYQYHQFLBAEDC-UOMJZUGYSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Factor D Assay: Human Factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration is incubated with test compound at various concentrations for 5 min at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBz and DTNB (Ellman's reagent) are added to final concentrations of 100 μM each. Absorbance at 405 nm (A405) is recorded at 30 second intervals for 30 min using a microplate spectrophotometer. IC50 values are calculated by nonlinear regression of complement Factor D reaction rates as a function of test compound concentration.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 666.53, ALogP 5.47, HBA 8, HBD 1, PSA 122.97, QED 0.2
Compound (SMILES): COCCOc1cc(N2C[C@@H](C)n3c(c(CCCOc4cc(C)c(Cl)c(C)c4)c4ccc(Cl)c(-c5c(C)ncnc5C)c43)C2=O)c2c(c1)cc(C(=O)O)n2Cc1cccc(C)n1
InChIKey: IAKVQAXWTBBVMM-GDLZYMKVSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07820
Assay: TR-FRET Binding Assay: Measurements were carried out in OptiPlate-384 White Opaque 384-well plates (Perkin Elmer. Shelton, Conn.). 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, 300 nM FITC-Bak peptide, 1 nM recombinant Maltose Binding Protein (MBP) tagged Mcl-1 (residues 172-327) and 1 nM terbium conjugated-MBP antibody were added to assay buffer (4.5 mM Monobasic potassium phosphate, 15.5 mM Dibasic potassium phosphate, 1 mM Sodium EDTA, 50 mM NaCl, 1 mM DTT, 0.05% Pluronic F-68, pH 7.5). Compounds are diluted in DMSO in a 16-point, semilog serial dilution scheme. For the BAK FITC TR-FRET assay 50 nL compound is added to 20 μL of assay buffer containing Bak-FITC. MBP tagged Mcl-1 protein and antibody, for a final DMSO concentration of 0.25% and a top concentration of 10 μM. 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 3 hours at room temperature. TR-FRET signal (Delta F) is measured on a Biotek Cytation 3 multimode plate reader equipped with a filter cube containing an Ex 340/30 Em 620/10 nm filter and an Ex 340/30 Em 520 filter.
Measurement: Ki < 0.2 nM (pChEMBL 9.699000358581543)
Descriptors: MW 875.85, ALogP 10.3, HBA 10, HBD 1, PSA 133.83, QED 0.11
Compound (SMILES): C[C@@H](Cn1c(=O)[nH]/c(=N\c2ccc(Oc3ccc(CF)cn3)cc2)n(Cc2ccc(Cl)cc2)c1=O)C(=O)O
InChIKey: CFBOJAPDHUFSMY-INIZCTEOSA-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 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 539.95, ALogP 3.65, HBA 8, HBD 2, PSA 131.57, QED 0.33
Compound (SMILES): COc1cc(C#N)cc(C)c1C(=O)Nc1ccc2[nH]nc(-c3cnco3)c2c1
InChIKey: HIBFZUCZFNEBCH-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LanthaScreen Kinase Activity Assay: The assay was carried out under the following protocol conditions: 1 mM compound in DMSO was serially diluted 1:3, 11 points in DMSO with a Biomek FX and 0.1 μL of the diluted compound was subsequently stamped into the assay plate (384-well format Lumitrac 200, Greiner, 781075) with an Echo Labcyte such that the final compound concentration in the assay was 10 μM to 169 μM. Subsequently, 5 μL of 2× kinase solution (2.9 nM final concentration) was added to the assay plate in assay buffer composed of 50 mM Tris pH 8.5 (Sigma, T6791), 5 mM MgCl2 (Fluka, 63020), 1 mM EGTA (Sigma, E3889), 0.01% BRIJ-35 (Sigma, P1254) and 2 mM DTT. The reaction was started by addition of 2×ATP/LRRKtide solution in assay buffer such that the final concentration was 400 nM LRRKtide and 25 μM ATP. After 60 min incubation at room temperature, the reaction was stopped by addition of 10 μL of 2× stop solution containing a final concentration of 2 nM anti-pERM antibody and 10 mM EDTA. After a 30 min incubation at RT, the TR-FRET signal was measured on a Wallac 2104 EnVision multilabel reader at an excitation wavelength of 340 nm and reading emission at 520 nm and 495 nm. The ratio of the 520 nm and 495 nm emission was used to analyze the data.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 373.37, ALogP 3.66, HBA 6, HBD 2, PSA 116.83, QED 0.56
Compound (SMILES): CC(C)C(=O)NCc1ccc(C(F)(F)F)c(-c2nc(-c3ccc(OCC4CC4)cn3)cc(=O)[nH]2)c1
InChIKey: RUZMUZZAKJBWJT-UHFFFAOYSA-N
Target: Prostaglandin E synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O14684
Assay: Inhibitory Activity Assay: Microsomes were prepared from COS-1 cells transiently transfected with a plasmid containing human mPGES-1 cDNA, and used as mPGES-1 enzyme. The mPGES-1 enzyme was diluted with a sodium phosphate buffer (pH 7.2) containing reduced glutathione (2.5 mM) and EDTA (1 mM), DMSO or a DMSO solution of a test compound (final concentration of DMSO was 1%) was added to the enzyme, and the mixture was preincubated at 4° C. for 15 minutes. Then, PGH2 as the substrate was added at a final concentration of 1 μM to start the enzymatic reaction, and after incubation at 4° C. for 4 minutes, a solution of ferric chloride (25 mM) and citric acid (50 mM) was added to terminate the enzymatic reaction. Generated PGE2 was measured by using Prostaglandin E2 Express EIA Kit (Cayman Chemical).
Measurement: IC50 = 37 nM (pChEMBL 7.429999828338623)
Descriptors: MW 486.49, ALogP 4.58, HBA 5, HBD 2, PSA 96.97, QED 0.49