lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COC(=O)Nc1ccc2c(c1)NC(=O)CCCC[C@H](c1ccc(-c3cc(Cl)ccc3-n3cccn3)c[n+]1[O-])c1nc-2c(C)[nH]1
InChIKey: KHTVBYNJIBMOOX-XMMPIXPASA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Factor XIa Assay: The effectiveness of a compound of the present invention as an inhibitor of Coagulation Factor XIa 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 rt 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 inhibitory constant, Ki.Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 150 mM NaCl, 5 mM CaCl2, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 40 pM (Sekisui Diagnostics) and he synthetic substrate, Z-Gly-Pro-Arg-AFC, TFA salt (Sigma #C0980) 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. Assuming competitive inhibition, and that unity is negligible compared Km/[S], [I]/e, and [I]/e (where [S], [I], and e respectively represent the total concentrations, of substrate, inhibitor and enzyme), the equilibrium constant (Ki) for dissociation of the inhibitor from the enzyme can be obtained from the dependence of Vo/Vi on [I] shown in the following equation.
Measurement: Ki = 0.35 nM (pChEMBL 9.460000038146973)
Descriptors: MW 612.09, ALogP 6.35, HBA 7, HBD 3, PSA 140.87, QED 0.16
Compound (SMILES): CCc1nc2c(-c3nnc[nH]3)cc(-c3ccnc(N)c3F)cc2n1-c1ccnc2cccc(F)c12
InChIKey: MCESJOXLJSITBN-UHFFFAOYSA-N
Target: Phosphatidylinositol 3-kinase regulatory subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O00459
Assay: TR-FRET Assay: Class I PI3K isoforms were expressed and purified as heterodimeric recombinant proteins. All assay reagents and buffers for the TR-FRET assay were purchased from Millipore. PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2× Km ATP (75-500 μM), 2 μM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, and 1% (v/v) DMSO at the following concentrations for each isoform: PI3Kα, PI3Kβ, and PI3Kδ between 25 and 50 pM, and PI3Kγ at 2 nM. The compounds were added to the assay solution and incubated for 30 minutes at 25° C. The reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIPS, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 500 μs read window).
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 468.47, ALogP 4.84, HBA 7, HBD 2, PSA 111.19, QED 0.38
Compound (SMILES): COc1cc(Cn2cccn2)cc2onc(NS(=O)(=O)c3ccc(C)c(C)c3)c12
InChIKey: ZYQASBPUHOTNAM-UHFFFAOYSA-N
Target: Histone acetyltransferase KAT6A (SINGLE PROTEIN), Homo sapiens
UniProt: Q92794
Assay: Kat6A Assay 1: A. Compound preparation    1. Prepare 10 mM stock solutions in 100% DMSO from solid material    2. Serial dilute 10 mM, 1 mM or 0.1 mM compound stocks 3-fold in 100% DMSO for 11-point dose responseB. Reagent preparation    1. Prepare 1× assay buffer containing 10 mM Tris HCL pH 8.0, 2.5 mM NaCl, 0.5 mM EDTA, 0.005% BSG and 0.02% Tween-20    2. Dilute Histone peptide (CPC Scientific) and AcCoA (Sigma) together in assay buffer to 2×.    3. Dilute KAT enzyme in assay buffer to 2×.C. Enzyme reaction    1. Final reaction conditions for each KAT assay in a 20 ul assay reaction volume:  ii. KAT6A 15 nM, 1 uM AcCoA, 2 uM H3 1-21 peptide, 45-minute reaction 2. Add 0.5 ul of diluted compound to the assay plate (384-well V-bottom polypropylene plates) or 0.5 ul of DMSO for control wells.    3. Add 10 ul of 2× Histone peptide/2×AcCoA mix to the assay plate.    4. Add 10 ul of 2× enzyme to the assay plate.    5. Stop the reaction after the indicated time with the addition of 2 ul of 5% formic acid    6. Each reaction was analyzed using self-assembled monolayer desorption/ionization time-of-flight mass spectrometry (Mrksich, Milan (2008) Mass Spectrometry of Self-Assembled Monolayers: A New Tool for Molecular Surface Science ACS Nano 2008 2 (1), 7-18; SAMDI Tech, Inc. (Chicago, Ill.)).    7. Area under the curve (AUC) for both substrate and product peaks was determined for KAT5 at M.W. 2561 [Substrate+H]+ and 2603 [Product+H]+ with a +/−1 Da tolerance, respectively    8. Area under the curve (AUC) for both substrate and product peaks was determined for KAT6a, KAT6B, KAT7 and KAT8 at M.W. 2723 [Substrate+H]+ and 2765 [Product+H]+ with a +/−1 Da tolerance, respectively. 9. Percent conversion to product was calculated by: AUCProduct/(AUCsubstrate+AUCProduct).D. Data analysis    1. IC50 values were determined by fitting the % conversion at each inhibitor concentration to the 4-parameter IC50 equation using Pfizer proprietary curve fitting software.    2. Ki values were determined by fitting the % conversion at each inhibitor concentration to the Morrison equation for tightbinding competitive inhibitors using Pfizer proprietary curve fitting software.
Measurement: Ki = 2.86 nM (pChEMBL 8.539999961853027)
Descriptors: MW 412.47, ALogP 3.5, HBA 7, HBD 1, PSA 99.25, QED 0.52
Compound (SMILES): [C-]#[N+]c1ccc(NC(=O)N2CCn3nc(-c4ccc(C5CC5)c(Cl)c4)c(C(N)=O)c3C2)cc1
InChIKey: MPAAKFNKJAWGBI-UHFFFAOYSA-N
Target: Casein kinase I isoform epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: P49674
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours and terminated by adding 45 μl of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP®3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the unphosphorylated substrate and phosphorylated product. Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 μM ATP, 1.5 μM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 460.93, ALogP 4.78, HBA 4, HBD 2, PSA 97.61, QED 0.55
Compound (SMILES): CNCC(=O)N1CCC(c2ccc3[nH]c(-c4ccc5nc(C)nn5c4)c(CC(F)F)c3c2)CC1
InChIKey: FWSGXMHWPVVBKO-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 = 810 nM (pChEMBL 6.090000152587891)
Descriptors: MW 466.54, ALogP 3.92, HBA 5, HBD 2, PSA 78.32, QED 0.45
Compound (SMILES): Cc1nc2cnc(-c3cccc(CN(C)CC(F)F)c3)c(C#N)c2c(=O)n1CC#Cc1ccc(Cl)cc1-c1ccnc2c(C(=O)O)csc12
InChIKey: IIMHFLSMCZUGPZ-UHFFFAOYSA-N
Target: Eukaryotic translation initiation factor 4E-binding protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13541
Assay: Fluorescence Polarization Assay: eIF4E protein was pre-incubated with EDA-m7GDP-ATTO-550 in FPB at 2xconcentrations for 5 minutes prior to the addition of compounds. Compounds (100x) were prepared using 3-fold serial dilutions in 100% DMSO, and subsequently diluted 1:50 in FPB to produce 2x stocks. 50 ul of 2x eIF4E/EDA-m7GDP-ATTO-550 were transferred to the wells of a 96-well half-area black flat bottom polystyrene plate. 50 ul of 2x test compound were added, and binding reactions were allowed to equilibrate for 30 minutes at room temperature while protected from light. The fluorescent polarization signal was detected using a Victor 2 multi-label counter (Perkin Elmer) and the concentration necessary to achieve inhibition of eIF4E/EDA-m7GDP-ATTO-550 binding by 50% (IC50) was calculated using data from an 8-point compound dilution series.
Measurement: IC50 = 13.4 nM (pChEMBL 7.869999885559082)
Descriptors: MW 695.15, ALogP 7.02, HBA 9, HBD 1, PSA 125, QED 0.17
Compound (SMILES): Cc1cccc2cc(CNC(=O)c3nccnc3N)c(N(Cc3ccccn3)Cc3ccccn3)nc12
InChIKey: PORKKXQXTPNGRD-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Luciferase-Coupled Chemiluminescence Assay: Kinase activity is measured as the percent of ATP consumed following the kinase reaction using luciferase-luciferin-coupled chemiluminescence. Reactions were conducted in 384 or 1536-well white medium binding microtiter plates (Greiner). Kinase reactions were initiated by combining test compounds, kinase, and ATP in a 20 μL volume (6 μL volume for 1536-well plate). The reaction mixture was incubated at ambient temperature for 2 h. Following the kinase reaction, a 20 μL (or 3 μL for 1536-well plate) aliquot of KinaseGlo (Promega) was added and the chemiluminescence signal measured using an EnVision plate reader (Perkin Elmer). Total ATP consumption was limited to 25-60% and the IC50 values correlate well with those determined by radiometric assays.
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 490.57, ALogP 3.84, HBA 8, HBD 2, PSA 122.81, QED 0.34
Compound (SMILES): N[C@H]1CC[C@H](NC(=O)c2ccc(O)nn2)CC1
InChIKey: FJOXAUHIOLWNOW-ZKCHVHJHSA-N
Target: Histone-lysine N-methyltransferase SMYD3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H7B4
Assay: SMYD3 Biochemical Assay: The assays were all performed in a buffer consisting of 25 mM Tris-Cl pH 8.0, 1 mM TCEP, 0.005% BSG, and 0.005% Tween 20, prepared on the day of use. Compounds in 100% DMSO (1 ul) were spotted into a 384-well white opaque OptiPlate using a Bravo automated liquid handling platform outfitted with a 384-channel head (Agilent Technologies). DMSO (1 ul) was added to Columns 11, 12, 23, 24, rows A-H for the maximum signal control and 1 ul of SAH, a known product and inhibitor of SMYD3, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control. A cocktail (40 ul) containing the SMYD3 enzyme was added by Multidrop Combi (Thermo-Fisher). The compounds were allowed to incubate with SMYD3 for 30 min at room temperature, then a cocktail (10 ul) containing SAM and MEKK2 was added to initiate the reaction (final volume=51 ul). The final concentrations of the components were as follows: SMYD3 was 0.4 nM, 3H-SAM was 8 nM, MEKK2 was 12 nM, SAH in the minimum signal control wells was 1 mM, and the DMSO concentration was 2%. The assays were stopped by the addition of non-radiolabeled SAM (10 ul) to a final concentration of 100 uM, which dilutes the 3H-SAM to a level where its incorporation into MEKK2 is no longer detectable. Radiolabeled MEKK2 was detected using a scintillation proximity assay (SPA). 10 uL of a 10 mg/mL solution of SPA beads in 0.5 M citric acid was added and the plates centrifuged at 600 rpm for 1 min to precipitate the radiolabeled MEKK2 onto the SPA beads. The plates were then read in a PerkinElmer TopCount plate reader to measure the quantity of 3H-labeled MEKK2 as disintegrations per minute (dpm) or alternatively, referred to as counts per minute (cpm).
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 236.28, ALogP 0.18, HBA 5, HBD 3, PSA 101.13, QED 0.68
Compound (SMILES): CCCOc1ccc(S(=O)(=O)N2CCN(CCO[N+](=O)[O-])CC2)cc1-c1nc2c(CCC)cn(CC)c2c(=O)[nH]1
InChIKey: DPONWRLZGJDHRX-UHFFFAOYSA-N
Target: cGMP-specific 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O76074
Assay: Scintillation Proximity Assay (SPA): The inhibition of recombinant human (rh) PDE5A by test compounds is measured in a radiometric assay based on Scintillation Proximity Assay (SPA) technology. The substrate [3H]cGMP/cGMP is hydrolysed to [3H] 5′ GMP/5′ GMP contingent on the activity of rhPDE5A. The ensuing [3H] 5′ GMP/5′ GMP but not [3H] cGMP/cGMP binds to SPA yttrium silicate beads in the presence of Zn++ stimulating the scintillant within the bead to emit light that is detected by a ß-counter. The assay is performed in a 96 well format.The assay is done in 20 mM Tris HCl pH 7.4, 5 mM MgCl2, 0. μM cGMP/[3H] cGMP (about 60000 dpm/well) substrate with rhPDE5A1 (GST tagged, SIGMA E9034) added to an amount not exceeding 20% cGMP hydrolysis within 20 min in Tris 20 mM pH 7.4 supplemented with 0.01% bovine serum albumin (BSA) in the presence of test compounds or vehicle (0.1% DMSO). The final assay volume amounts to 100 μl and the reaction is run for 20 min at 37° C.The hydrolysis of [3H] cGMP/cGMP by rhPDE5A is terminated by adding SPA beads at 50 l/well (Perkin Elmer, RPNQ0024), pre-diluted in water as per manufacturer's instructions and supplemented with 3-isobutyl-1-methylxanthine (1 mM). Beads are allowed to sediment for at least 30 min before measurement in a Wallac Microbeta 2 (Perkin Elmer).
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 576.68, ALogP 2.67, HBA 10, HBD 1, PSA 152.9, QED 0.24
Compound (SMILES): COc1ccc(Cn2c(=O)n3ncnc3c3cc(CN4CCOCC4)sc32)c(F)c1
InChIKey: NLPSBXRSPRVYQZ-UHFFFAOYSA-N
Target: Dual specificity calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1B (SINGLE PROTEIN), Homo sapiens
UniProt: Q01064
Assay: IMAP TR-FRET assay: The IMAP TR-FRET PDE assay was optimized for concentration of enzyme, Calmodulin, cAMP or cGMP substrate, DMSO tolerance, and incubation time. Into each well of a solid white 1536 well plate (Corning) was dispensed 250 pg full-length recombinant NH-terminal GST tagged human PDE1b enzyme (BPS Bioscience Cat #60011, San Diego, Calif.) in 2.5 μL IMAP BSA reaction buffer (Molecular Devices, Sunnyvale, Calif.) containing 10 U/mL Calmodulin and 2.5 mM CaCl2 (Sigma Aldrich.) After a brief centrifugation, 30 nL compound was added by transfer from 1 mM stock in DMSO using a Kalypsys 1536 Pintool. Plates were incubated for 5 minutes at room temperature before dispensing 1.5 μL of 533 nM 5-carboxy fluorescein (FAM)-labeled cAMP (Molecular Devices, Sunnyvale, Calif.) for a final concentration of 200 nM. After a brief centrifugation, the plates were incubated for 30 minutes at room temperature. The assay was terminated by adding 5 μL IMAP binding reagent/Tb complex (Molecular Devices, Sunnyvale, Calif.) to each well. Plates were incubated 1 hour at room temperature and read on a Viewlux multimode plate reader (Perkin Elmer). The instrument was set to excite using the DUG11 filter and measure using 490/10 nm and 520/10 nm filters.
Measurement: EC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 429.48, ALogP 2.13, HBA 9, HBD 0, PSA 73.89, QED 0.48
Compound (SMILES): CC(=O)c1nn(CC(=O)N2C[C@H](F)C[C@H]2C(=O)Nc2cccc(Br)n2)c2ccc(-c3ccc4nc(C)nn4c3)cc12
InChIKey: ZBMOINZOPNVADI-KNQAVFIVSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Human Factor D Assay: Human Factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration was 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. Absorbance at 405 nm (A405) was recorded at 30 second intervals for 30 minutes 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 619.45, ALogP 3.99, HBA 9, HBD 1, PSA 127.38, QED 0.23
Compound (SMILES): O=C(CCc1c[nH]c2ccccc12)Nc1nnc(-c2ccccn2)[nH]1
InChIKey: LROGMTUTBSSKQG-UHFFFAOYSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Inhibition Assay: Buffer-I:    50 mM Tris, pH 7.5    150 mM NaCl (optional)    1 mM MgCl2    1 mM DTT.    KRas G12D mutant protein was expressed as a His-tagged protein. Purified His-KRas G12D protein was diluted in buffer-I to a final concentration of 3-10 μg/ml.    200 μl of the diluted His-KRas G12D protein was added to a nickel coated 96 well plate and incubated overnight at 4° C.    The next day, wells were washed 3× in 200 μl of Buffer-I.    Then 200 μl of Buffer-I were added to each well in the presence of 1% DMSO.    Tested compounds were added to the protein-coated wells at a concentration of 20 μM, and incubated for 3 hours at room temperature. While performing IC50 measurements a serial dilution of all tested concentrations was prepared.Then 22 μl of Cy3-GTP or Cy5-GTP was added to each well. The labeled GTP was incubated for 45 min. at room temperature.    Following GTP incubation, wells were washed 3× in Buffer-I, and 200 μl of Buffer-I were added to each well.    Following washes, the amount of bound labeled-GTP was measured with an Eppendorf AF2200 plate reader.
Measurement: IC50 < 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 332.37, ALogP 2.92, HBA 4, HBD 3, PSA 99.35, QED 0.52
Compound (SMILES): C=Cc1nn(Cc2cc3ccccc3nc2-c2ccccc2F)c2ncnc(N)c12
InChIKey: JHLWDJHNNZQHPC-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Inhibition Assay: Class I PI3-Ks can be either purchased (p110α/p85α, p110β/p85α, p110δ/p85α from Upstate, and p110γ from Sigma) or expressed as previously described (Knight et al., 2004). IC50 values are measured using either a standard TLC assay for lipid kinase activity (described below) or a high-throughput membrane capture assay. Kinase reactions are performed by preparing a reaction mixture containing kinase, inhibitor (2% DMSO final concentration), buffer (25 mM HEPES, pH 7.4, 10 mM MgCl2), and freshly sonicated phosphatidylinositol (100 μg/ml). Reactions are initiated by the addition of ATP containing 10 μCi of γ-32P-ATP to a final concentration 10 or 100 μM and allowed to proceed for 5 minutes at room temperature. For TLC analysis, reactions are then terminated by the addition of 105 l 1N HCl followed by 160 μl CHCl3:MeOH (1:1). The biphasic mixture is vortexed, briefly centrifuged, and the organic phase is transferred to a new tube using a gel loading pipette tip precoated with CHCl3. This extract is spotted on TLC plates and developed for 3-4 hours in a 65:35 solution of n-propanol:1M acetic acid. The TLC plates are then dried, exposed to a phosphorimager screen (Storm, Amersham), and quantitated. For each compound, kinase activity is measured at 10-12 inhibitor concentrations representing two-fold dilutions from the highest concentration tested (typically, 200 μM). For compounds showing significant activity, IC50 determinations are repeated two to four times, and the reported value is the average of these independent measurements.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 396.43, ALogP 4.45, HBA 6, HBD 1, PSA 82.51, QED 0.49
Compound (SMILES): CC(C)NCCNc1nn(C)c(=O)c2ccccc12
InChIKey: CKTMJAJTVLXEEZ-UHFFFAOYSA-N
Target: Histone acetyltransferase KAT2B (SINGLE PROTEIN), Homo sapiens
UniProt: Q92831
Assay: PCAF AlphaLisa Binding Assay: His/Flag epitope tagged PCAF719-832 bromodomain was cloned, expressed and purified to homogeneity in-house. PCAF bromodomain binding and inhibition of the compounds disclosed herein was assessed by monitoring the engagement of biotinylated small molecule ligand (known to bind to the PCAF bromodomain) with the target using the AlphaLisa technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate PCAF bromodomain (225 nM final) was combined with the biotinylated small molecule ligand (6 nM final) in 50 mM HEPES (pH 7.5), 75 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.2% DMSO) or compound dilution series in DMSO. After 15 minute incubation at room temperature AlphaLisa streptavidin acceptor beads and AlphaLisa anti-histidine donor beads were added to a final concentration of 12.5 μg/mL each. After 90 minutes of equilibration plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 9400 nM (pChEMBL 5.03000020980835)
Descriptors: MW 260.34, ALogP 1.34, HBA 5, HBD 2, PSA 58.95, QED 0.8
Compound (SMILES): Cc1cnn(Cc2ccc(Cn3cc(C(=O)NCc4c(F)ccc(Cl)c4F)c(C(F)(F)F)n3)cc2)c1
InChIKey: HNSAKACOONQHKG-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Inhibitory Activity In Vitro Assay : Plasma kallikrein inhibitory activity in vitro was determined using standard published methods. Human plasma kallikrein (Protogen) was incubated at 25° C. with the fluorogenic substrate H-DPro-Phe-Arg-AFC and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in optical absorbance at 410 nm and the IC50 value for the test compound was determined.
Measurement: IC50 = 373 nM (pChEMBL 6.429999828338623)
Descriptors: MW 523.89, ALogP 5.37, HBA 5, HBD 1, PSA 64.74, QED 0.26
Compound (SMILES): O[C@@H]1[C@H](O)CS[C@H]1c1cnc2c(NCc3ccccc3I)nccn12
InChIKey: MRTZNBAMYSIEHG-KFWWJZLASA-N
Target: Adenosine receptor A3 (SINGLE PROTEIN), Homo sapiens
UniProt: P0DMS8
Assay: Assay for Binding Affinity for Adenosine Receptor A3: HEK-293 cell membrane homogenates (32 μg protein), in which A3 adenosine receptors were expressed, are incubated for 120 min at 22° C. with 0.15 nM [1251]AB-MECA in the absence or presence of the test compound in a buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 1 mM EDTA and 2 UI/ml ADA. Nonspecific binding is determined in the presence of 1 μM IB-MECA. Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard). The results are expressed as a percent inhibition of the control radioligand specific binding. The standard reference compound is IB-MECA, which is tested in each experiment at several concentrations to obtain a competition curve from which its IC50 is calculated.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 468.32, ALogP 2.46, HBA 7, HBD 3, PSA 82.68, QED 0.51
Compound (SMILES): CCNS(=O)(=O)c1cc(-c2sc(Nc3cccc(N4CCNCC4=O)n3)nc2C)cc2c1C(=O)N([C@@H](C)C1CC1)C2
InChIKey: PZHYYDQKVVSTEG-KRWDZBQOSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Enzymatic Activity Assay : The activity of recombinant human PI3Kγ ((aa144-1102)-6His) and PI3Kα, β, δ (6-His(p110-p85α)) was determined by measuring the ADP level after phosphorylation of DiC8-PIP2 using a commercially available ADP-Glo kit from Promega. The assay was carried out in white low volume 384 well plates in a final volume of 14 μl at R.T. The assay conditions contained the following: 50 mM Tris buffer pH 7.4, 2.1 mM DTT, 3 mM MgCl2, 0.05% CHAPS, 20 μM ATP, 80 μM DiC8-PIP2 and 1.2 nM PI3Kα, β, γ or 0.6 nM PI3Kδ. Potential inhibitors were made up in DMSO and then diluted in the assay to give a final concentration of not exceeding 1% (v/v) DMSO. A 10-point half-log dilution series of the inhibitors (highest concentration typically 0.1 μM for δ or γ and 33 μM for a or 1) was tested and the pIC50 determined using a 4-parameter logistic equation in a non-linear curve fitting routine. Routinely, inhibitors were pre-incubated with 3 μl of enzyme for 15 min prior to the addition of 2 μl substrate mixture for a further 60 min enzyme reaction. The phosphorylation was stopped with the addition of 3 μl ADP-Glo reagent (stop solution) followed by a 40 min incubation. Prior to detection 6 μl of ADP-Glo Kinase Detection Reagent was added and the plates were read in a micro plate reader using a Luminescence filter. All additions were followed by a short centrifugation step.
Measurement: IC50 = 0.8 nM (pChEMBL 9.100000381469727)
Descriptors: MW 595.75, ALogP 3.25, HBA 9, HBD 3, PSA 136.63, QED 0.34
Compound (SMILES): COc1cn([C@@H](CC2CCC2)C(=O)Nc2ccc(C(=O)O)cc2)c(=O)cc1-c1cc(Cl)ccc1C(C)=O
InChIKey: QYKMNJJKVQYERA-DEOSSOPVSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Biological Assay: Test Example 1: Biological Activity of the Compounds of the Present Invention onThe Inhibition of Factor XIa Detected by Absorption Photometry1. Experimental MaterialsEnzyme: Coagulation Factor XIa protease (Abcam, Art. No ab62411)Substrate: Coagulation Factor XIa specific substrate (HYPHEN1310 med, Art. No. Biophen cs-21(66))Buffer: 100 mM tris-HCl, 200 mM NaCl, 0.02% Tween20, pH 7.42. Experimental Procedure20 mM of test compound dissolved in 100% DMSO was diluted to 200, 20, 2, 0.2, 0.02, 0.002 μM with 100% DMSO; 1 μl of the compound was added to each well in a 384-well plate, blank and control wells were replaced with DMSO. The plate was centrifuged to remove the compound to the bottom. 10 μl (2.5 μg/ml) of FXIa enzyme solution was added to each well, and 10 μl of buffer was added to the blank well. The plate was centrifuged to remove the enzyme solution to the bottom.Finally, 10 μl of 2 mM substrate were added to each well, and the plate was centrifuged to remove the substrate solution to the bottom.The plate was incubated for 10 minutes at 37° C.; and then the absorbance was measured at 405 nm. The absorbance was curve-fitted by graphpad
Measurement: IC50 = 35 nM (pChEMBL 7.460000038146973)
Descriptors: MW 522.99, ALogP 5.45, HBA 6, HBD 2, PSA 114.7, QED 0.36
Compound (SMILES): CO[C@H](C(=O)Nc1nnc(N[C@@H]2CCN(c3cccnn3)C2)s1)c1cccc(N2CC(F)C2)c1
InChIKey: ZDCKWVSAJLNKEB-APWZRJJASA-N
Target: Glutaminase kidney isoform, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: O94925
Assay: GLS Enzyme Potency Assay: A Glutamate Oxidase/AmplexRed coupled assay was used to measure the ability of compounds to bind to and inhibit the activity of GLS1 in vitro. 6His tagged GLS protein (amino acids 63-669) expressed in E. Coli was purified and stored at −80° C. in aliquots. GLS1 was diluted to 2× working concentration and incubated at room temperature to allow the tetrameric/dimeric forms to reach steady state. Assay measurements were performed in buffer comprising 50 mM TRIS pH 7.8, 100 mM NaPO4, pH 7.8, 0.001% v/v Tween20. Purified recombinant GLS1 protein was diluted in assay buffer to 12 nM and pre-incubated at room temperature for 30 minutes. Test compounds were prepared by dilution in 100% DMSO to give the correct dose range for 12 point concentration response and an appropriate volume (2.5-60 nl) dispensed into 384 well micro assay plates (Greiner product code 784900) using a Labcyte Echo 555 acoustic dispenser. DMSO concentration was maintained at 2% by back filling with DMSO solution. 3 μL of diluted GLS1 protein (12 nM) was then dispensed into each well using a BioRaptr automated dispenser (Beckman-Coulter) and incubated for 15 minutes at room temperature. 3 μL of 100 mM glutamine diluted in assay buffer was then added and the reaction incubated at room temperature for 60 minutes. The reaction was then stopped by addition of 45 μM 6-(2-bromoethynyl)-2,3-dimethyl-quinazolin-4-one, 75 μM Amplex Red, 0.375 units/mL Horseradish Peroxidase, 0.12 units/mL Glutamate Oxidase in 100 mM TRIS pH7.5. After 30 minutes at room temp in the dark, plates were read on a Perkin Elmer EnVision using 535/590 nm optic filters and raw data analysed using Genedata to generate IC50 values. An artefact version of the assay where the 6His tagged GLS protein and glutamine were replaced with assay buffer was also used to rule out non specific effects on the assay components.
Measurement: IC50 = 20.6 nM (pChEMBL 7.690000057220459)
Descriptors: MW 484.56, ALogP 2.5, HBA 10, HBD 2, PSA 108.4, QED 0.5
Compound (SMILES): C[C@H]1C[C@H](NC(=O)c2cc(C3CC3)on2)CCN1C(=O)[C@H]1CC[C@H](N)CC1
InChIKey: NIAICEIVBITTPZ-QCEMKRCNSA-N
Target: Histone-lysine N-methyltransferase SMYD3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H7B4
Assay: SMYD3 Biochemical Assay: The assays were all performed in a buffer consisting of 25 mM Tris-Cl pH 8.0, 1 mM TCEP, 0.005% BSG, and 0.005% Tween 20, prepared on the day of use. Compounds in 100% DMSO (1 ul) were spotted into a 384-well white opaque OptiPlate using a Bravo automated liquid handling platform outfitted with a 384-channel head (Agilent Technologies). DMSO (1 ul) was added to Columns 11, 12, 23, 24, rows A-H for the maximum signal control and 1 ul of SAH, a known product and inhibitor of SMYD3, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control. A cocktail (40 ul) containing the SMYD3 enzyme was added by Multidrop Combi (Thermo-Fisher). The compounds were allowed to incubate with SMYD3 for 30 min at room temperature, then a cocktail (10 ul) containing SAM and MEKK2 was added to initiate the reaction (final volume=51 ul). The final concentrations of the components were as follows: SMYD3 was 0.4 nM, 3H-SAM was 8 nM, MEKK2 was 12 nM, SAH in the minimum signal control wells was 1 mM, and the DMSO concentration was 2%. The assays were stopped by the addition of non-radiolabeled SAM (10 ul) to a final concentration of 100 uM, which dilutes the 3H-SAM to a level where its incorporation into MEKK2 is no longer detectable. Radiolabeled MEKK2 was detected using a scintillation proximity assay (SPA). 10 uL of a 10 mg/mL solution of SPA beads in 0.5 M citric acid was added and the plates centrifuged at 600 rpm for 1 min to precipitate the radiolabeled MEKK2 onto the SPA beads. The plates were then read in a PerkinElmer TopCount plate reader to measure the quantity of 3H-labeled MEKK2 as disintegrations per minute (dpm) or alternatively, referred to as counts per minute (cpm).% Inhibition Calculation% ⁢ ⁢ inh = 100 - ( dpm cmpd - dpm min dpm max - dpm min ) × 100Where dpm=disintegrations per minute, cmpd=signal in assay well, and min and max are the respective minimum and maximum signal controls.Four-Parameter IC50 FitY = Bottom + ( Top - Bottom ) ( 1 + ( X IC 50 ) Hill ⁢ ⁢ CoefficientWhere top and bottom are the normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit. The Hill Coefficient normally allowed to float but may also be fixed at 1 in a 3-parameter fit. Y is the % inhibition and X is the compound concentration.
Measurement: IC50 = 1407 nM (pChEMBL 5.849999904632568)
Descriptors: MW 374.49, ALogP 2.18, HBA 5, HBD 2, PSA 101.46, QED 0.84
Compound (SMILES): NC(=O)c1c(-c2cccc(F)c2)nn2c1CN(C(=O)Nc1ccc(Cl)cc1)CC2
InChIKey: BDQDISRBEPOJFU-UHFFFAOYSA-N
Target: Casein kinase I isoform epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: P49674
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours and terminated by adding 45 μl of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP®3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the unphosphorylated substrate and phosphorylated product. Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 μM ATP, 1.5 μM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 413.84, ALogP 3.49, HBA 4, HBD 2, PSA 93.25, QED 0.69
Compound (SMILES): Cc1nc2c(F)cc(-c3nc(Nc4ccc5c(n4)CCN(CCN)C5)ncc3F)cc2n1C(C)(C)C
InChIKey: IWSDTEGBQVXXON-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P11802
Assay: LANCE Ultra ULight Assay: 1. Take 10 mM stock solution of the test compound, in 96-well compound plate, DMSO was used to dilute the compound to an initial concentration of 100x, then this concentration was used as the first concentration, 3-fold diluted to make 10 serial concentrations; 1 μL each serial dilution was then added to 19 μL 1x reaction buffer to prepare 5x compound for use; 2 μL 5x compound was transferred from 96-well plate to 384-well plate; compound-free control well was added with 2 μL the following liquid: 1x reaction buffer with the addition of 1 μL DMSO; 2 μL 250 mM EDTA was added to the Min control well.2.1x reaction buffer was used to formulate the kinase, substrate and ATP into a 2.5xenzyme/substrate mixture and 2.5xATP solution respectively. In the experiment, the final concentration of CDK4/CycD3 kinase is 0.76 ng/μL, the final concentration of ATP is 8 μM; the final concentration of CDK6/CycD3 kinase is 0.5 ng/μL, the final concentration of ATP is 50M; the final concentration of CDK2/CycA2 kinase is 0.86 ng/μL, the final concentration of ATP is 1 μM; the final concentration of CDK2/CycE1 kinase is 1.016 ng/μL, the final concentration of ATP is 20M; 2.5xenzyme/substrate mixture was added to a 384-well plate, incubated at room temperature for 5 minutes; then added with 2.5xATP solution, reacted at room temperature for 30 minutes.3. LANCE Detection Buffer was used, 1xto prepare 2xLANCE Ultra Europium-anti-phospho-eIF4E-binding protein 1 (Thr37/46) for use. After the enzymatic reaction was continued for 30 minutes, 10 mM EDTA was added to 384-well plate and the mixture was reacted at room temperature for 5 minutes. Then LANCE Ultra Europium-anti-phospho-eIF4E-binding protein 1 (Thr37/46) was added, reacted at room temperature for 1 hour.4. The 384-well plate was placed in HERAEUS Multifuge X1R centrifuge, centrifuged at 2000 rpm for 2 minutes; data were measured on EnVision, 337 nM wavelength laser was used as the excitation light, test at RFU665 nM and RFU615 nM, and RFU665 nM/RFU615 nM x10000 was used as the final data for analysis.
Measurement: IC50 = 1.14 nM (pChEMBL 8.9399995803833)
Descriptors: MW 492.58, ALogP 4.29, HBA 8, HBD 2, PSA 97.78, QED 0.43
Compound (SMILES): Cn1cc(C(F)(F)F)nc1-c1ccc(Cn2ncc3cnc(-c4c(OC5CCOC5)ncnc4C4CC4)nc32)cc1
InChIKey: SZYBOXMRKRFJRH-UHFFFAOYSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O94782
Assay: Inhibitor Assay: Deubiquitinase activity was measured using ubiquitin-rhodamine 110 as a substrate. Cleavage of the amide bond between rhodamine and the c-terminal glycine of ubiquitin yields an increase in fluorescence signal. The assay was conducted in 20 ul total volume of assay buffer (50 mM Tris-HCl, pH 7.8, 0.5 mM EDTA, 0.01% Bovine Serum Albumin, 1 mM DTT, 0.01% Tween-20), and 0.05 nM USP1/UAF1 enzyme. Reaction was initiated by addition of 150 nM Ubiquitin-rhodamine (Boston Biochem) substrate.Compounds, dissolved in DMSO were tested in dose response format, beginning at 10 uM.Compounds depicted below were added to enzyme/assay buffer mix and incubated 10 min. Substrate mix was added, and reaction mix was read in kinetic mode for 30 min at Ex480/Em540 and IC50 response curves were plotted.
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 562.56, ALogP 4.8, HBA 10, HBD 0, PSA 105.66, QED 0.28
Compound (SMILES): Cc1nc2cnc(N(C)C3CCN(CCOC(F)(F)F)CC3)c(C#N)c2c(=O)n1CC#Cc1ccc(Cl)cc1-c1ccnc2c(C(=O)O)csc12
InChIKey: SZNDNRJWMRVASV-UHFFFAOYSA-N
Target: Eukaryotic translation initiation factor 4E-binding protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13541
Assay: Fluorescence Polarization Assay: eIF4E protein was pre-incubated with EDA-m7GDP-ATTO-550 in FPB at 2xconcentrations for 5 minutes prior to the addition of compounds. Compounds (100x) were prepared using 3-fold serial dilutions in 100% DMSO, and subsequently diluted 1:50 in FPB to produce 2x stocks. 50 ul of 2x eIF4E/EDA-m7GDP-ATTO-550 were transferred to the wells of a 96-well half-area black flat bottom polystyrene plate. 50 ul of 2x test compound were added, and binding reactions were allowed to equilibrate for 30 minutes at room temperature while protected from light. The fluorescent polarization signal was detected using a Victor 2 multi-label counter (Perkin Elmer) and the concentration necessary to achieve inhibition of eIF4E/EDA-m7GDP-ATTO-550 binding by 50% (IC50) was calculated using data from an 8-point compound dilution series.
Measurement: IC50 = 17.1 nM (pChEMBL 7.769999980926514)
Descriptors: MW 736.18, ALogP 6.09, HBA 11, HBD 1, PSA 137.47, QED 0.19
Compound (SMILES): CN1CCN(C2CCN(c3cnc4c(-c5ccnc6sccc56)cnn4c3)CC2)CC1
InChIKey: JUJWPKOCUGYIMQ-UHFFFAOYSA-N
Target: Activin receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q04771
Assay: Luciferase Reporter Gene Assay for ALK2 and TGFbeta Activity: In this reporter gene cell based ALK2 and TGFβ assay, the C2Cl2 cell line was employed for the measurement of ALK2 activity, using a BRE-Luc SMAD1/5/8 reporter and BMP6 as the agonist. The HEK293T cell line was employed to measure TGFβ activity, using a SBE-Luc SMAD2/3 reporter and TGFβ as the agonist. Luciferase reporter assays were read using Promega Steady-Glo Luciferase Assay System.Cells were plated in 96 well white clear bottom assay plates at 10 k cells/well for C2Cl2-BRE, 15 k cells/well for HEK293-SBE in DMEM containing 2% FBS 1% P/S. Cells were given a minimum of 4 hours in incubator at 37° C./5% CO2 to adhere prior to further treatment. Compounds were diluted in DMSO to a 10-point dilution curve and added to the plate to reach the following final concentrations: 10000, 3000, 1000, 300, 100, and 30 nM in the HEK293-SBE assay and 1000, 300, 100, 30, 10, 3, 1, 0.3 nM in the C2Cl2-BRE assay. Negative and positive control wells received 2 μL DMSO as vehicle treatment. Plates were returned to incubator for 45 minutes and then BMP6 and TGFB were added to a final concentration of 50 ng/mL and 5 ng/mL, respectively. Plates were returned to the incubator and left overnight. After a minimum of 18 hours post BMP6/TGFβ addition, the plates were read using Promega Steady-Glo Luciferase Assay System. A 1:1 mixture of prepared steady-glo and phenol free DMEM was prepared, and 50 μL/well was added to the assay plates whose media had been flicked off. Plates were given 10 minutes post steady-glo addition before luminescence was read on a Spectramax M5e microplate reader. Negative control wells were averaged and subtracted from all other wells on the plate. Inhibition was calculated as the percent of signal loss compared to the averaged positive control wells.
Measurement: IC50 = 244 nM (pChEMBL 6.610000133514404)
Descriptors: MW 433.59, ALogP 3.22, HBA 8, HBD 0, PSA 52.8, QED 0.49
Compound (SMILES): COCc1cc(-c2[nH]c3ccc(C4CCC(NC5COC5)CC4)nc3c2C(C)C)cn2ncnc12
InChIKey: TZBGAQOLKMVVJT-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assays: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 3.9 nM (pChEMBL 8.40999984741211)
Descriptors: MW 474.61, ALogP 4.56, HBA 7, HBD 2, PSA 89.36, QED 0.41
Compound (SMILES): Cc1cc(C)nc(CNC(=O)c2cc(CNC3=NCCN3)cc(-c3ccc(F)nc3C)n2)n1
InChIKey: YNTXURRQDYMXJZ-UHFFFAOYSA-N
Target: WD repeat-containing protein 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P61964
Assay: TR-FRET Binding Assay: A Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay that measures the displacement of the 10mer-Thr-FAM probe in response to compound treatment was performed for compounds wherein the IC50 from FPA assay using 10mer-Thr-FAM was below the lower assay IC50 limit 1 nM. Excess 10mer-Thr-FAM probe was utilized with His-tagged WDR5 in conjunction with a commercial anti-His antibody containing a Terbium label. The LanthaScreen Elite Tb-anti-HIS Antibody from ThermoFisher Scientific was used for this purpose. This Tb-anti-HIS has an excitation/emission of 340 nm and 490 nm, respectively. The 10mer-Thr-FAM probe when bound to WDR5 will undergo a FRET interaction with the Tb-anti-HIS and emit at 520 nm. The ratio of the 520 and 495 signals are then utilized to generate a dose-response curve to calculate an IC50 value. By virtue of FRET there is little to no background fluorescence interference from 10mer-Thr-FAM probe allowing an excess of the probe to be used permitting an increase in the lower limit of the calculated Ki when testing against highly potent inhibitors with Ki<<1 nM. WDR5-His Tag (423, residues 24-334) is expressed and purified in our lab in sufficient quantities for screening. 10mer-Thr-FAM peptide is used anywhere from 15 to 150 nM depending on the window of sensitivity required. WDR5-His tag protein is used at 2 nM. A source plate is prepared using an Echo Liquid Handler, which distributes the compounds to the assay plate (white, flat-bottom; OptiPlate) in a 10-point, 3-fold dilution schemes with a top concentration of either 5 or 20 μM depending on the anticipated potency of the compounds, in a final volume of 20 μL. A final target (WDR5)/Tb-Ab concentration of 2 nM/1 nM is dispensed from appropriate stock solutions, respectively. The final DMSO concentration in each well of the assay plate is 1% or lower. As before the plate is covered, shielded from light, and incubated for 60 minutes at room temperature with rocking. Anisotropy is then measured on a Biotek Cytation 3 at excitation wavelength of 340 nm, and emission wavelengths of 495 nm and 520 nm. Working buffer conditions (pH 7.0) are similar to that in FPA above. TR-FRET signal is plotted and IC50 and Ki values are calculated in the same manner as the fluorescence polarization anisotropy based competition assays.
Measurement: Ki = 0.99 nM (pChEMBL 9)
Descriptors: MW 448.51, ALogP 1.98, HBA 8, HBD 3, PSA 117.08, QED 0.49
Compound (SMILES): CN[C@@H](C)C(=O)N[C@H]1CCc2ccccc2N(Cc2ccc(-c3ccccc3)cc2)C1=O
InChIKey: YSTHVSKWCXWUDG-CYFREDJKSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay: The peptide AVPIAQKSEK-(ε-biotin)-OH (SEQ ID NO: 1) 1:2 TFA (Peptide A) was identified as a substrate for the TR-FRET assay by screening the 6× Histidine-tagged (SEQ ID NO: 2) BIR2 domain and BIR3 domain of XIAP against a set of 29 peptides synthesized based on sequences reported by Sweeny et al. (Biochemistry, 2006, 45, 14740 14748). The peptides were labeled with the fluorescent tags FITC or TAMRA and Kd values were determined by fluorescence polarization assay. The sequence AVPIAQKSEK (SEQ ID NO: 3) was identified as optimal for using in an assay. The peptide sequence was derivatized with biotin to provide AVPIAQKSEK-(ε-biotin)-OH (SEQ ID NO: 1) 1:2 TFA as the substrate for the TR-FRET assay.The XIAP protein sequence was obtained from the SWISS-PROT protein sequence database and the BIR2 and BIR3 domains were derived from that. The sequence of the BIR2 domain used for the TR-FRET assay is MRHHHHHHRDHFALDRPSETHADYLLRTGQVVDISDTIYPRNPAMYSEEARLKSFQNW PDYAHLTPRELASAGLYYTGIGDQVQCFACGGKLKNWEPGDRAWSEHRRHFPNCFFVL GRNLNIRSE (SEQ ID NO: 4).The sequence of the BIR3 domain used for the TR-FRET assay is MRHHHHHHRSDAVSSDRNFPNSTNLPRNPSMADYEARIFTFGTWIYSVNKEQLARAGF YALGEGDKVKCFHCGGGLTDWKPSEDPWEQHAKWYPGCKYLLEQKGQEYINNIHLTH SLEECLVRTT (SEQ ID NO: 5).Ten nanomolar of 6× Histidine-tagged (SEQ ID NO: 2) BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(ε-biotin)-OH (SEQ ID NO: 1) 1:2 TFA, in the presence of 50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/niL bovine serum albumin (BSA). Following a 45 min. incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations.
Measurement: IC50 = 12571 nM (pChEMBL 4.900000095367432)
Descriptors: MW 427.55, ALogP 3.93, HBA 3, HBD 2, PSA 61.44, QED 0.63