lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1cc(OC(F)F)cnc1C(=O)Nc1ccc(F)c([C@@]2(CF)CS(=O)(=O)C3(CC3)C(N)=N2)c1
InChIKey: LBGDDVHLKDWVLV-HXUWFJFHSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vivo Inhibition of Beta-Secretase: Several animal models, including mouse, rat, dog, and monkey, may be used to screen for inhibition of beta-secretase activity in vivo following administration of a test compound sample. Animals used in this invention can be wild type, transgenic, or gene knockout animals. For example, the Tg2576 mouse model, prepared and conducted as described in Hsiao et al., 1996, Science 274, 99-102, and other non-transgenic or gene knockout animals are useful to analyze in vivo inhibition of Amyloid beta peptide (Abeta) production in the presence of inhibitory test compounds. Generally, 2 to 18 month old Tg2576 mice, gene knockout mice or non-transgenic animals are administered test compounds formulated in vehicles, such as cyclodextran, phosphate buffers, hydroxypropyl methylcellulose or other suitable vehicles. One to twenty-four hours following the administration of compound, animals are sacrificed, and brains as well as cerebrospinal fluid (CSF) and plasma are removed for analysis of A-beta levels and drug or test compound concentrations (Dovey et al., 2001, Journal of Neurochemistry, 76, 173-181) Beginning at time 0, animals are administered by oral gavage, or other means of delivery such as intravenous injection, an inhibitory test compound of up to 100 mg/kg in a standard, conventional formulation, such as 2% hydroxypropyl methylcellulose, 1% Tween80. A separate group of animals receive 2% hydroxypropyl methylcellulose, 1% Tween80 alone, containing no test compound, and serve as a vehicle-control group. At the end of the test period, animals are sacrificed and brain tissues, plasma or cerebrospinal fluid are collected. Brains are either homogenized in 10 volumes (w/v) of 0.2% diethylamine (DEA) in 50 mM NaCl (Best et al., 2005, Journal of Pharmacology and Experimental Therapeutics, 313, 902-908), or in 10 volumes of 0.5% TritonX-100 in Tris-buffered saline (pH at about 7.6). Homogenates are centrifuged at 355,000 g, 4° C. for 30 minutes. CSF or brain supernatants are then analyzed for the presence of A-beta peptide by specific sandwich ELISA assays based on ECL (Electrochemiluminescence) technology.
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 500.47, ALogP 2.87, HBA 7, HBD 2, PSA 123.74, QED 0.59
Compound (SMILES): CC(C)n1cnc2cc(-c3ccc4c(c3)N([C@H]3C[C@@H](N5CCCCC5)C3)C(=O)C43CCN(C(=O)C4(C)CCO4)CC3)nc(Nc3ccncc3F)c21
InChIKey: LIAQPVNQEIUKKK-ZUNFDWEASA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: Enzymatic Activity Assay: Recombinant HPK1 kinase domain produced via baculovirus infection of insect cells was obtained from Proteros (Proteros Biostructures #PR-0322) and was pre-activated in the presence of 2 mM ATP (Sigma-Aldrich, cat #GE27-2056-01) and 2 mM magnesium chloride for 16 hours at 4° C. The protein reaction mixture was then loaded to a desalting column (Thermo Fisher Scientific, Cat #89889) to remove excess ATP. HPK1 was eluted with buffer containing 20 mM Tris (2-Amino-2-(hydroxymethyl)propane-1,3-diol) pH 8.0, 150 mM NaCl, 2 mM dithiothreitol and 5% glycerol, and was frozen at −80° C. for later use. HPK1 dual phosphorylation was confirmed by mass spectrometry.Ten nanoliters of test compounds dissolved in DMSO at various concentrations were dispensed into a 384-well ProxiPlate (PerkinElmer #6008289). Five microliters of a solution of recombinant HPK1 diluted in HPK1 kinase assay buffer (50 mM BES [N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid], pH 7.0; 10 mM magnesium chloride; 0.01% Triton X-100; 1 mM dithiothreitol; 0.01% bovine serum albumin; 0.1 mM sodium orthovanadate) was added to the compound-containing plate and was incubated for 15 minutes at 25° C. Five microliters of a mixture of ATP (Sigma-Aldrich #A6559) and peptide substrate STK S1 (Cisbio #61ST1BLC) diluted in HPK1 kinase assay buffer was then added to start the reaction. Final concentrations were 0.15 nM for HPK1, 10 μM for ATP, and 1 μM for the STK S1 peptide substrate. The reaction mixture was incubated at 25° C. for 3 hours and was stopped with the addition of 10 μl of an EDTA (Ethylenediaminetetraacetic acid)-containing detection buffer (Cisbio #62SDBRDF) supplemented with Europium cryptate-labeled anti-phospho-serine/threonine antibodies (Cisbio #62ST1PEJ) and XL665-labeled streptavidin (Cisbio #610SAXLG). The mixture was incubated for 16 hours at room temperature and peptide phosphorylation was measured by time-resolved fluorescence energy transfer (665 nm/620 nm) on an Envision plate reader (PerkinElmer).
Measurement: IC50 = 0.2 nM (pChEMBL 9.699999809265137)
Descriptors: MW 706.87, ALogP 6.36, HBA 9, HBD 1, PSA 108.72, QED 0.24
Compound (SMILES): CCOC(=O)CC(C)(C)NC(=O)c1cc(NC(=O)Cc2cc(Cl)ccc2O)ccn1
InChIKey: LKMWRLLCKDOHIG-UHFFFAOYSA-N
Target: Anoctamin-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5XXA6
Assay: Automated Whole-Cell Patch Clamp Assay to Detect TMEM16A Activity in Recombinant Cells: Compound activity was quantified by measuring the increase in current upon compound addition and expressing this as a percentage increase of baseline TMEM16A current level. Percentage increases in current were determined for each concentration and the data plotted as a function of concentration using either the Qpatch software or Graphpad Prism v6.05 providing the concentration which gave 50% of its maximal effect (EC50) and maximum efficacy (percentage of baseline increase). The method of calculating the results is illustrated in FIG. 1, which shows an example trace from the Qpatch TMEM16A assay. In FIG. 1, IBL equals baseline current, I[#1] equals the peak current during test compound concentration 1 incubation period and so on.
Measurement: EC50 = 350 nM (pChEMBL 6.460000038146973)
Descriptors: MW 433.89, ALogP 3.08, HBA 6, HBD 3, PSA 117.62, QED 0.55
Compound (SMILES): CCOc1cc([C@@H](CS(C)(=O)=O)N2C(=O)Cc3cc(Br)cnc32)ccc1OC
InChIKey: QVHRUPUCZDHOLL-OAHLLOKOSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4B (SINGLE PROTEIN), Homo sapiens
UniProt: Q07343
Assay: Inhibitory Effect on the Enzyme In Vitro Assay: The enzyme activity was indicated by measuring the AMP/GMP expression and tracing AMP/GMP antibody binding based on fluorescence polarization in the biological assay.Reagents:Experimental Buffer Solution:10 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 0.01% Brij 35, 1 mM DTT and 1% DMSO.Enzyme:Recombinant human PDE4B (Gen accession number: NM_002600; amino acid 305 end) was expressed by baculovirus in Sf9 insect cells using an N-terminal GST tag. MW=78 kDa.Enzyme Substrate:1 μM cAMPDetection:Transcreener® AMP2/GMP2 antibody and AMP2/GMP2 AlexaFluor633 tracerOperation Procedures:i) Recombinant human PDE4B and the enzyme substrate (1 μM cAMP) were dissolved in the newly-prepared buffer solution, respectively.ii) The PDE4B buffer solution defined as above was transferred into the reaction wells.iii) The compound which was dissolved in 100% DMSO was added to the reaction wells of the PDE4B buffer solution by ultrasonic oscillator (echo 550; nanoliter range) and incubated at room temperature for 10 minutes.iv) The enzymatic buffer solution was then added to the reaction wells defined as above to initiate the reaction.v) The reaction was incubated at room temperature for 1 hour.vi) The reaction was terminated by adding detecting mixture (Transcreener AMP2/GMP2 antibody and AMP2/GMP2 AlexaFluor633 tracer) and incubated for 90 minutes with slowly mixing. The measurement range of fluorescence polarization is Ex/Em=620/688.
Measurement: IC50 = 47.8 nM (pChEMBL 7.320000171661377)
Descriptors: MW 469.36, ALogP 2.93, HBA 6, HBD 0, PSA 85.8, QED 0.62
Compound (SMILES): CCN1CCC(n2cnc3ccc(-c4ccc5oc(N)nc5c4)cc3c2=O)CC1
InChIKey: VIZJSLWWEGRGEI-UHFFFAOYSA-N
Target: Phosphatidylinositol 3-kinase regulatory subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O00459
Assay: PI 3-Kinase HTRF Assay: Briefly, the assay is a time resolved FRET assay that indirectly measures PIP3 product formed by the activity of a PI3-K. The kinase reaction is performed in a microtitre plate (e.g., a 384 well microtitre plate). The total reaction volume is approximately 20 ul per well. In the first step, each well receives 2 ul of test compound in 20% dimethylsulphoxide resulting in a 2% DMSO final concentration. Next, approximately 14.5 ul of a kinase/PIP2 mixture (diluted in 1x reaction buffer) is added per well for a final concentration of 0.25-0.3 ug/ml kinase and 10 uM PIP2. The plate is sealed and incubated for 15 minutes at room temperature. To start the reaction, 3.5 ul of ATP (diluted in 1x reaction buffer) is added per well for a final concentration of 10 uM ATP. The plate is sealed and incubated for 1 hour at room temperature. The reaction is stopped by adding 5 ul of Stop Solution per well and then 5 ul of Detection Mix is added per well. The plate is sealed, incubated for 1 hour at room temperature, and then read on an appropriate plate reader.
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 389.46, ALogP 3.44, HBA 7, HBD 1, PSA 90.18, QED 0.58
Compound (SMILES): Cc1nnn(Cc2cc(Cl)ccc2/C=C/C(=O)N2CCC(c3ccc(F)cc3)CC2)n1
InChIKey: RRFJXXBNOWFKOF-WEVVVXLNSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: ATX Assay: Reagents LPC (oleoyl (18:1)) was purchased from Avanti Polar Lipids (Alabaster, Ala.) and solubilized in methanol to 20 mM. Amplex Red was obtained from Invitrogen Life Technologies (Paisley, UK) and dissolved in DMSO to 10 mM. Choline oxidase and horseradish peroxidase (HRP) were obtained from Sigma Aldrich (Dorset, UK) and dissolved in HBSS to 20 U/ml and 200 U/ml respectively. All reagents were stored at −20° C. in single use aliquots. All experimental measurements were performed in assay buffer made up immediately prior to use (HBSS, 0.01% BSA essentially fatty acid free).Protein Recombinant human ATX was prepared at Novartis (Basel, CH) in a human embryonic kidney (HEK) cell preparation, and stored in single use aliquots of 26 mg/ml (26 μM) stocks stored at −80° C.Method All experimental measurements were performed in black 384 well polystyrene (low volume, round bottom, Corning (3676)) plates. PerkinElmer EnVision (Fluorescence Intensity/Absorbance Monochromator) or Tecan Infinite 200 PRO series plate reader was used to detect change in fluorescent intensity.Assessing ATX inhibition ATX activity was determined by measurement of released choline in reactions containing ATX (10 nM), choline oxidase (0.1 U/ml), HRP (100 U/ml), amplex red (50 μM) and LPC 18:1 (10 μM). Compounds of the invention were prepared as 10 point serial dilutions from 1 μM in duplicate and pre-incubated with ATX at 37° C. for 20 minutes prior to the addition of remaining reagents. The liberated choline was measured from changes in fluorescence intensity (λex 530 nm, λem 590 nm) of the product resurofin at 37° C. every 2 minutes over a 40-minute period. ATX activity was measured as a slope of the linear portion of the progress curve, typically between 14 to 24 minutes.Data analysis Slope data was exported to Graphpad prism (Graphpad software, San Diego, Calif.)
Measurement: IC50 = 31 nM (pChEMBL 7.510000228881836)
Descriptors: MW 439.92, ALogP 4.24, HBA 5, HBD 0, PSA 63.91, QED 0.56
Compound (SMILES): Cc1nc(/C=C/[C@@H]2[C@@H]3[C@@H](C)OC(=O)[C@]3(C(N)=O)CC(F)(F)[C@H]2C)ccc1-c1ccccc1C#N
InChIKey: BZWUQBGOWKHPIR-YYAHKPHASA-N
Target: Serine/threonine-protein kinase MARK2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q7KZI7
Assay: PAR-1 FLIPR Assay: This assay measures the potency of the inventive compounds as PAR-1 receptor antagonists.Frozen HEK 293 Cells were plated in 384-well PDL coated plates at 12000 cells/well in 50 uL of DMEM media containing 10% FBS, pen/strep/L-Glutamine and non-essential amino acids, incubated overnight at 37° C./5% CO2. Media was then removed from the cells, incubated with 33 ul of Calcium-5 dye in assay buffer (Hank's buffer containing 20 mM HEPES, 0.04% Chaps and 2.5 mM Probenecid) for 60 minutes at 37° C. 2 uL of varying concentrations of compound in 40% DMSO in assay buffer (final DMSO concentration is 2.3%) were then added to the cells and incubated at 25° C. for 30 minutes. The plates were added to the FLIPR Tetra, the device added 5 uL of PAR-1 selective receptor-activating peptide (sequence Ala-parafluoroPhe-Arg-Cha-Cit-Try-NH2, prepared in water) at a concentration equal to the effective concentration that achieved 80% activation of signaling on the day of the experiment. The range of peptide was from 1.5-3 μM. The final volume was 40 uL/well, with 2% DMSO. The FLIPR was read at an excitation wavelength of 480 nm and an emission wavelength of 535 nm, and performed 60 scans over a 1-2 min reading time. The data were analyzed by taking the peak signal over a portion of the range of the 60 scans and dividing this signal by the minimum signal for that same range. The data were expressed as percent inhibition of the maximum divided by the minimum signal achieved at 80% activation produced by the PAR-1 activating peptide on the test day. The compounds of Examples 1-21 were tested in the assay described above and the data collected for these compounds is provided.
Measurement: IC50 = 6.92 nM (pChEMBL 8.15999984741211)
Descriptors: MW 465.5, ALogP 4.27, HBA 5, HBD 1, PSA 106.07, QED 0.54
Compound (SMILES): CC(=O)c1cn(CC(=O)N2C[C@H](F)C[C@H]2C(=O)Nc2cccc(Br)n2)c2ccc(Oc3ncc(Br)cn3)cc12
InChIKey: ZEFNCYYEQJMOSB-IERDGZPVSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Enzyme Assay: Human factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration is incubated with test compound at various concentrations for 5 minutes at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBzl and DTNB (Ellman's reagent) are added to final concentrations of 100 M each. The increase in color is recorded at OD405 nm in a microplate in kinetic mode over 30 minutes with 30 second time points in a spectrofluorimeter. IC50 values are calculated by non-linear regression from the percentage of inhibition of complement factor D activity as a function of test compound concentration.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 660.3, ALogP 4.92, HBA 8, HBD 1, PSA 119.31, QED 0.22
Compound (SMILES): Cc1cc(-c2[nH]c3ccc(C4CCN(C(=O)CN5CCS(=O)(=O)CC5)CC4)cc3c2C(C)C)cn2ncnc12
InChIKey: YPZKPYVQRNZLBA-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 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 548.71, ALogP 3.75, HBA 7, HBD 1, PSA 103.67, QED 0.41
Compound (SMILES): C[C@@H](O)c1nc2cnc3ccsc3c2n1[C@H]1CC[C@H](CNC(=O)N(C)C)CC1
InChIKey: VZUPYIHIECIFLX-MGPQQGTHSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: In Vitro JAK Kinase Assay: Compounds herein were tested for inhibitory activity of JAK targets according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (a.a. 837-1142), JAK2 (a.a. 828-1132) and JAK3 (a.a. 781-1124) were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 μL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hour and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a PHERA star plate reader (BMG, Cary, N.C.).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 401.54, ALogP 3.7, HBA 6, HBD 2, PSA 83.28, QED 0.7
Compound (SMILES): Cn1cc(-c2cnc(Cl)nc2NC2CCCCC2)cn1
InChIKey: WGMATGBLIXKVEL-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor TYRO3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06418
Assay: Verification on Inhibitory Activities of TAM Receptor Inhibiting Compounds on Tyro 3, Axl, and Mer: For evaluation of inhibitory activities of the compounds according to the present invention on Tyro 3, Axl, and Mer, the following test was carried out.The specific test method followed the method provided by Cisbio. The compounds of the examples were prepared at various concentrations, followed by addition of Tyro 3, Axl, or Mer and substrate peptides, and then ATP was added to initiate a reaction. After 1 hour, the reaction was stopped by addition of a solution containing EDTA. Thereafter, the amount of phosphorylated peptides was measured. Here, the phosphorylated peptides were treated with an europium (Eu)-labeled antibody recognizing phosphorylated peptides, and then after 1 hour, excited by irradiation of the light of a wavelength of 320 or 340 nm using an Envision Reader. The amount of light emitted at 665 nm was measured to investigate the degrees of inhibition of TAM receptors. The TAM receptor inhibitory activities (IC50 values) of the respective compounds were analyzed using the GraphPad Prism program, and tabulated in Tables 1 to 3 below. LDC1267 (CAS no. 1361030-48-9, Calbiochem), which is a TAM receptor inhibitor compound, was used as a positive control.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 291.79, ALogP 3.28, HBA 5, HBD 1, PSA 55.63, QED 0.88
Compound (SMILES): COc1c(-c2ccccc2CO)ccc(N2CC[C@@H](NC(=O)Nc3ccc(C(F)(F)F)cc3F)C2=O)c1F
InChIKey: MFUYQDNZMUXBRY-HXUWFJFHSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: FPR2 and FPR1 Cyclic Adenosine Monophosphate (cAMP) Assays: A mixture of forskolin (5 μM final for FPR2 or 10 μM final for FPR1) and IBMX (200 μM final) were added to 384-well Proxiplates (Perkin-Elmer) pre-dotted with test compounds in DMSO (1% final) at final concentrations in the range of 0.020 nM to 100 μM. Chinese Hamster Ovary cells (CHO) overexpressing human FPR1 or human FPR2 receptors were cultured in F-12 (Ham's) medium supplemented with 10% qualified FBS, 250 μg/ml zeocin and 300 μg/ml hygromycin (Life Technologies). Reactions were initiated by adding 2,000 human FPR2 cells per well or 4,000 human FPR1 cells per well in Dulbecco's PBS (with calcium and magnesium) (Life Technologies) supplemented with 0.1% BSA (Perkin-Elmer). The reaction mixtures were incubated for 30 min at room temperature. The level of intracellular cAMP was determined using the HTRF HiRange cAMP assay reagent kit (Cisbio) according to manufacturer's instruction. Solutions of cryptate conjugated anti-cAMP and d2 fluorophore-labelled cAMP were made in a supplied lysis buffer separately. Upon completion of the reaction, the cells were lysed with equal volume of the d2-cAMP solution and anti-cAMP solution. After a 1-h room temperature incubation, time-resolved fluorescence intensity was measured using the Envision (Perkin-Elmer) at 400 nm excitation and dual emission at 590 nm and 665 nm. A calibration curve was constructed with an external cAMP standard at concentrations ranging from 1 μM to 0.1 pM by plotting the fluorescent intensity ratio from 665 nm emission to the intensity from the 590 nm emission against cAMP concentrations. The potency and activity of a compound to inhibit cAMP production was then determined by fitting to a 4-parametric logistic equation from a plot of cAMP level versus compound concentrations.
Measurement: EC50 = 8.6 nM (pChEMBL 8.069999694824219)
Descriptors: MW 535.47, ALogP 5.08, HBA 4, HBD 3, PSA 90.9, QED 0.38
Compound (SMILES): COC(=O)Nc1ccc(-c2cnc(C3CC(O)c4cc(-c5cc(Cl)ccc5-n5cnnn5)c[n+]([O-])c43)[nH]2)cc1
InChIKey: SUAUGAFXXNWOJP-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Factor XIa Assay: 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 the synthetic substrate, Z-Gly-Pro-Arg-AFC, TFA salt (Sigma #C0980) at a concentration of 10004.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.V o /V i=1+[I]/Ki.
Measurement: IC50 = 1.61 nM (pChEMBL 8.789999961853027)
Descriptors: MW 544.96, ALogP 3.75, HBA 9, HBD 3, PSA 157.78, QED 0.22
Compound (SMILES): Fc1cc(F)cc(Cn2ncc3ncc(-c4ccc(F)c(OC(F)F)c4)cc32)c1
InChIKey: FSMFXOSHKCOXSB-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 = 536 nM (pChEMBL 6.269999980926514)
Descriptors: MW 405.33, ALogP 5.17, HBA 4, HBD 0, PSA 39.94, QED 0.43
Compound (SMILES): Cc1ccc(-c2cc3c(N4CCN(C(=O)N[C@@H](C)c5cccc(OC(F)F)c5)C(C)(C)C4)ncnc3[nH]2)cn1
InChIKey: QDZKHKMBRMKOGU-SFHVURJKSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Cell-Based Assays of c-Kit Mutant Kinase Activity: The c-Kit mutant D816V inhibitors were assessed using an engineered BaF3-FL KIT D816V or BaF3-FL KIT V560G/D816V cell line. The BaF3-FL KIT D816V cell lines were created by introduction of KIT mutant (D816V) full length constructs that render the cells dependent on the introduced kinase for growth. Inhibitors of c-Kit mutant D816V kinase reduce or eliminate the mediated c-kit mutant D816V kinase activation, resulting in reduced cell proliferation of the BaF3-FL Kit mutant D816V cells. This inhibition is measured by the effect of compound concentration on cell growth to assess IC50 values. BaF3-FL KIT D816V cells were seeded at 1×104 cells per well of a 96 well cell culture plate in 50 μl of cell culture medium of RPMI Medium 1× (Invitrogen #11875-093) supplemented with 10% FBS (Invitrogen #10438), 1% Non Essential Amino Acids (Invitrogen #11140), 1% Penicillin Streptomycin (Invitrogen #15140), 1% L-Glutamine (Invitrogen #25030-081). Compounds were dissolved in DMSO at a concentration of 5 mM and were serially diluted 1:3 for a total of eight points and added to the cells to a final maximum concentration of 10 μM in 100 μl cell culture medium (final concentration 0.2% DMSO). Cells were also treated with Dasatinib as a positive control. The cells were incubated at 37° C., 5% CO2 for three days. ATPlite Buffer (Perkin Elmer #6016739) and substrate were equilibrated to room temperature, and enzyme/substrate Recombinant Firefly Luciferase/D-Luciferin was reconstituted. The cell plates were equilibrated to room temperature for 30 minutes, then lysed by addition of 25 uL per well of the ATPlite Reagent. The plate was mixed for 5 minutes on a plate shaker to lyse the cells. The plates were read on a Tecan Safire using Luminescence protocol modified to read 0.1s per well. The luminescence reading assesses the ATP content, which correlates directly with cell number such that the reading as a function of compound concentration is used to determine the IC50 value.
Measurement: Ki = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 535.6, ALogP 5.3, HBA 6, HBD 2, PSA 99.27, QED 0.35
Compound (SMILES): NC(=O)Cc1ccc(Nc2cc(-c3ccc(Cl)cc3)nc3c2CCC3)cc1
InChIKey: GHPDKLGALZTENR-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4B (SINGLE PROTEIN), Homo sapiens
UniProt: Q07343
Assay: Kinetic Assay: Assays are performed in 96-well plates in a total volume of 200 μl/well. Compounds are dissolved in dimethylsulfoxide (DMSO) and added to plates in a volume of 10 μl followed by addition of 165 μl of assay mix. Plates are pre-incubated at 25° C. for 15 min and the reactions are initiated by the addition of 25 μl of cAMP followed by thorough mixing. Reaction rates are measured by monitoring the decrease in fluorescence using excitation at 355 nm and emission at 460 nm for a period of 10 min in a fluorescence plate reader. Initial rates (slopes) are determined from linear portions of the progress curves. Final concentrations of assay components are as follows: 50 mM Tris, pH 8, 10 mM MgCl2, 50 mM KCl, 2% DMSO, 5 mM tris(2-carboxyethyl)phosphine (TCEP), 0.4 mM phosphenolpyruvate (PEP), 0.01 mM NADH, 0.04 mM adenosine triphosphate (ATP), 0.004 mM cAMP, 7.5 units myokinase from yeast, 1.6 units pyruvate kinase, 2 units lactate dehydrogenase, and either 0.5 nM human PDE4D7 or 10 nM human PDE4B1. All data are percent normalized relative to controls and are presented as percent inhibition. An inhibitory concentration 50% (IC50) value is calculated by fitting of a sigmoidal dose response curve. Human PDE4D7 contained a mutation of serine 54 to aspartic acid to mimic activation by cAMP-dependent protein kinase A (PKA). Human PDE4B1 contained a corresponding mutation of serine 133 to aspartic acid to also mimic PKA activation.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 377.88, ALogP 4.66, HBA 3, HBD 2, PSA 68.01, QED 0.68
Compound (SMILES): Cc1cnc([C@H]2c3c(nc4sc(C)nn34)CCN2C(=O)COc2ccc(C3(N(C)C)CC3)nc2Cl)c(F)c1
InChIKey: BLJTVBMOCAQULE-QHCPKHFHSA-N
Target: Tryptophan 5-hydroxylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P17752
Assay: Biochemical In Vitro Assay: To generate the enzyme, full length human TPH1 is cloned into the plasmid pET20b(+) (Novagen) and expressed in E. coli. The bacterial cells are ruptured by sonication on ice and the lysate is cleared by centrifugation. The resulting protein in the pellet is re-extracted and TPH1 is purified from the obtained lysate by affinity chromatography using a pterin cosubstrate analog immobilized to the resin of the column. The protein is further purified by size exclusion chromatography to remove protein aggregates. The activity of TPH1 is determined by using a fluorescence assay. The enzyme activity assay is carried out at 15° C. with atmosphere oxygen for the duration of 60 minutes in a volume of 64 μl. The reaction is carried out in a 0.1 M Tris-HCl buffer, adjusted to pH 7.6, containing 1 mM DTT, 0.2 mg/mL catalase, 100 μM (±)-6-methyl-5,6,7,8-tetrahydropterine dihydrochloride, 40 μM L-tryptophan, and 40-80 nM of TPH1. The reaction is started by bringing together L-tryptophan with all the other reaction substituents and stopped by quenching with perchloric acid (HClO4). The amount of 5-hydroxy-L-tryptophan produced during the enzymatic reaction is determined by fluorescence readout. Fluorescence, as determined at 540 nm when excited at 300 nm wavelength, increases proportionally to the 5-hydroxy-L-tryptophan formed. Compounds are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into the assay plate. Fluorescence is measured for each well and the fluorescence at 540 nm wavelength is compared to the fluorescence of the vehicle in place of compound. Inhibitory activities of example compounds with respect to the TPH1 protein are determined by calculating the IC50 value (the concentration of compound needed to inhibit 50% of the enzyme activity).
Measurement: IC50 = 217 nM (pChEMBL 6.659999847412109)
Descriptors: MW 556.07, ALogP 4.09, HBA 9, HBD 0, PSA 88.75, QED 0.33
Compound (SMILES): CC(C)(C)OC(=O)N1CCC2(CCN(S(=O)(=O)c3ccc(CNC(=O)c4ccc5nccn5c4)cc3)CC2)C1
InChIKey: NVPQMAXYBYTENX-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Enzymatic Reaction Assay: The NAMPT enzymatic reactions were carried out in Buffer A (50 mM Hepes pH 7.5, 50 mM NaCl, 5 mM MgCl2, and 1 mM THP) in 96-well V-bottom plates. The compound titrations were performed in a separate dilution plate by serially diluting the compounds in DMSO to make a 100× stock. Buffer A (89 μL) containing 33 nM of NAMPT protein was added to 1 μL of 100× compound plate containing controls (e.g. DMSO or blank). The compound and enzyme mixture was incubated for 15 min at rt, then 10 μL of 10× substrate and co-factors in Buffer A were added to the test well to make a final concentration of 1 μM NAM, 100 μM 5-Phospho-D-ribose 1-diphosphate (PRPP), and 2.5 mM Adenosine 5′-triphosphate (ATP). The reaction was allowed to proceed for 30 min at rt, then was quenched with the addition of 11 μL of a solution of formic acid and L-Cystathionine to make a final concentration of 1% formic acid and 10 μM L-Cystathionine. Background and signal strength was determined by addition (or non-addition) of a serial dilution of NMN to a pre-quenched enzyme and cofactor mix.
Measurement: IC50 = 29.5 nM (pChEMBL 7.53000020980835)
Descriptors: MW 553.69, ALogP 3.68, HBA 7, HBD 1, PSA 113.32, QED 0.52
Compound (SMILES): CO[C@H]1CC[C@H](C(=O)N2Cc3cccnc3Nc3ccc(N4CCOCC(C)C4)cc32)CC1
InChIKey: WOSAABPYLTXUNF-NCRIQYHFSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Enzymatic Assay: Each test compound (10 mM stock in DMSO) is diluted in DMSO to make a 10-point, 3-fold dilution series. 125 nL of each dilution or DMSO alone is dispensed to a 384-well Greiner Lumitrac 200 assay plate using an Echo® Liquid Handler. To each well of the plate is added 20 uL of enzyme in assay buffer or assay buffer alone. Assay buffer consists of 50 mM sodium phosphate, pH 7.0, 50 mM magnesium chloride, 50 mM sodium chloride, and 0.01% (w/v) bovine serum albumin. When present, the R132H mutant IDH1 enzyme is at a working concentration of 1.875 nM (final concentration in assay of 1.5 nM). The assay plate is allowed to incubate for 30 minutes at room temperature and 5 uL of 5× substrate mixture (2.5 uM nicotinamide adenine dinucleotide phosphate, 100 uM adenosine diphosphate, 7.5 mM glyceraldehyde-3-phosphate, 7.5 ug/mL of spinach glyceraldehyde-3-phosphate dehydrogenase, 25 nM phosphoglycerate kinase, and 5 mM alpha-ketoglutarate in assay buffer) is added to all wells. The reaction plate is incubated for 60 minutes followed by addition of 25 uL of Promega Kinase-GLO reagent to all wells and 10-minute incubation.Luminescence is measured using a PerkinElmer Envision plate reader. The percent activity of each dilution is determined as the ratio of background corrected signal to the background corrected signal of wells receiving only DMSO. IC50 values are determined by fitting percent activity data to a four-parameter logistic dose response equation.
Measurement: IC50 = 310 nM (pChEMBL 6.510000228881836)
Descriptors: MW 450.58, ALogP 4.35, HBA 6, HBD 1, PSA 66.93, QED 0.75
Compound (SMILES): CCc1cc2c(cc1N1CCN(CC(O)CCCC(=O)c3cc4ccccc4[nH]3)CC1)OCO2
InChIKey: PDNOIHGCVLVCCX-UHFFFAOYSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Radioligand Binding Assay: Binding at dopamine D2-like receptors was determined using previously described methods. Membranes were prepared from HEK293 cells expressing human D2R, D3R or D4R, grown in a 50:50 mix of DMEM and Ham's F12 culture media, supplemented with 20 mM HEPES, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 1× antibiotic/antimycotic, 10% heat-inactivated fetal bovine serum, and 200 μg/mL hygromycin (Life Technologies, Grand Island, N.Y.) and kept in an incubator at 37° C. and 5% CO2. Upon reaching 80-90% confluence, cells were harvested using pre-mixed Earle's Balanced Salt Solution (EBSS) with 5 μM EDTA (Life Technologies) and centrifuged at 3000 rpm for 10 min at 21° C. The supernatant was removed and the pellet was resuspended in 10 mL hypotonic lysis buffer (5 mM MgCl2.6H2O, 5 mM Tris, pH 7.4 at 4° C.) and centrifuged at 20,000 rpm for 30 min at 4° C. The pellet was then resuspended in fresh EBSS buffer made from 8.7 g/L Earle's Balanced Salts without phenol red (US Biological, Salem, Mass.), 2.2 g/L sodium bicarbonate, pH to 7.4. A Bradford protein assay (Bio-Rad, Hercules, Calif.) was used to determine the protein concentration and membranes were diluted to 500 μg/mL and stored in a −80° C. freezer for later use.
Measurement: Ki = 537 nM (pChEMBL 6.269999980926514)
Descriptors: MW 463.58, ALogP 4, HBA 6, HBD 2, PSA 78.03, QED 0.47
Compound (SMILES): Cc1cncc([C@@H](CC(=O)NC[C@H](Cc2ccc(O)cc2)N(C)C)C2(C(F)(F)F)CC2)c1
InChIKey: AINLWQRUUPNNOG-PZJWPPBQSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: MOR cAMP Agonist and Antagonist Assays: CHO Tag-lite human mOR stable cell line from Cisbio (NCBI accession number: NM_000914.3, Bedford, Mass.) were seeded and grown to approximately 80% confluence in Ham F-12 with 10% FBS, 50 U/ml penicillin, 50 μg/ml streptomycin, 2 mM Hepes, and 1 mg/ml geneticin (Invitrogen, Carlsbad, Calif.). Cells were then harvested using accutase (Corning. Coming, N.Y.), centrifuged at 1300 RPM for 5 min, and plated at 5000 cells per 5 μL per well in a 5× dilution of stimulation buffer consisting of the HTRF cAMP Gi kit, water and IBMX at 0.5 mM (Cisbio, Bedford, Mass.) in white HTRF low volume 384 well plates (Cisbio, Bedford, Mass.). Plates were then incubated at 37° C. in 5% CO2 for 10 minutes. For the agonist assay, forskolin was added to a final concentration of 4 μM. For the antagonist assay, forskolin (4 μM) and DAMGO at EC90 final concentration were added. Test compounds were dissolved in DMSO and water, and then serially diluted to working concentrations such that the concentration of DMSO was less than 0.1%. Diluted test compounds were added at 2.5 μL per well, and plates were incubated at 37° C., and 5% CO2 for 15 minutes, and then at room temperature for 15 minutes. Next 5 μl/well of cAMP Eu-cryptate and 5 μl/well of anti-cAMP-d2 (both diluted 1:20 in lysis buffer) were added, and plates were incubated at room temperature for 1 hour. Following incubation, plates were read in a Synergy Neo2 multi-mode reader (Biotek, Winooski, Vt.). Plate reader settings were set to time resolved fluorescence with excitation at 330 nm and emissions of 620 nm and 665 nm. Emission fluorescence was normalized (665/620 nm signal×1000). For the agonist assay, data was normalized using the maximal DAMGO response. Measurements were performed in triplicate and the dose response was fit using nonlinear regression.
Measurement: IC50 > 3000 nM (pChEMBL 5.5229997634887695)
Descriptors: MW 449.52, ALogP 4.2, HBA 4, HBD 2, PSA 65.46, QED 0.6
Compound (SMILES): CCOc1cc([C@@H](CS(C)(=O)=O)n2c(=O)n(C3CCC3)c3cc(-c4ccccc4F)cnc32)ccc1OC
InChIKey: UVROUISFBXUXKR-XMMPIXPASA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4B (SINGLE PROTEIN), Homo sapiens
UniProt: Q07343
Assay: Inhibitory Effect on the Enzyme In Vitro Assay: The enzyme activity was indicated by measuring the AMP/GMP expression and tracing AMP/GMP antibody binding based on fluorescence polarization in the biological assay.Reagents:Experimental Buffer Solution:10 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 0.01% Brij 35, 1 mM DTT and 1% DMSO.Enzyme:Recombinant human PDE4B (Gen accession number: NM_002600; amino acid 305 end) was expressed by baculovirus in Sf9 insect cells using an N-terminal GST tag. MW=78 kDa.Enzyme Substrate:1 μM cAMPDetection:Transcreener® AMP2/GMP2 antibody and AMP2/GMP2 AlexaFluor633 tracerOperation Procedures:i) Recombinant human PDE4B and the enzyme substrate (1 μM cAMP) were dissolved in the newly-prepared buffer solution, respectively.ii) The PDE4B buffer solution defined as above was transferred into the reaction wells.iii) The compound which was dissolved in 100% DMSO was added to the reaction wells of the PDE4B buffer solution by ultrasonic oscillator (echo 550; nanoliter range) and incubated at room temperature for 10 minutes.iv) The enzymatic buffer solution was then added to the reaction wells defined as above to initiate the reaction.v) The reaction was incubated at room temperature for 1 hour.vi) The reaction was terminated by adding detecting mixture (Transcreener AMP2/GMP2 antibody and AMP2/GMP2 AlexaFluor633 tracer) and incubated for 90 minutes with slowly mixing. The measurement range of fluorescence polarization is Ex/Em=620/688.
Measurement: IC50 = 3.5 nM (pChEMBL 8.460000038146973)
Descriptors: MW 539.63, ALogP 4.77, HBA 8, HBD 0, PSA 92.42, QED 0.3
Compound (SMILES): CCc1ccc2ncnc(N[C@H]3CC[C@H](N4CCOCC4)CC3)c2c1
InChIKey: VIQWBGZRWHMIHD-QAQDUYKDSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Kinase Assay: The kinase activity of IRAK4 is determined by its ability to catalyze the phosphorylation of a fluorescent polypeptide substrate. The extent of phosphorylation is measured using the IMAP technology (Molecular Devices) where the phosphorylated fluorescent substrate binds to the large M(III)-based nanoparticles which reduces the rotational speed of the substrate and thus increases its fluorescent polarization (FP).20 μL reaction mixture contains 10 mM TriHCl, pH 7.2, 0.5 nM GST tagged IRAK4 (SignalChem), 100 nM fluorescent peptide substrate (RP7030, Molecular Devices), 100 μM ATP, 1 mM DDT, 1 mM MgCl2, and 0.01% Tween 20. The reaction is initiated by the addition of ATP. After incubation for 30 minutes at 25° C., 60 μL of Progressive IMAP Reagent (Molecular Devices) is added to stop the reaction. Change in RP7030's FP is determined by a FP reader (Analyst HT, LJL BioSystems).
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 340.47, ALogP 3.25, HBA 5, HBD 1, PSA 50.28, QED 0.93
Compound (SMILES): Cc1cc2nc(N[C@@H](C)c3ccccn3)nc(C(=O)NCCO)c2s1
InChIKey: VJPJAFJFCWYXDT-NSHDSACASA-N
Target: Adenosine receptor A2b (SINGLE PROTEIN), Homo sapiens
UniProt: P29275
Assay: Activity Assay: Compounds (Adenosine Receptor modulators) and activity assay.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 357.44, ALogP 2.29, HBA 7, HBD 3, PSA 100.03, QED 0.63
Compound (SMILES): CC(NC(=O)CN1Cc2ccc(-c3nc(NC4CCOCC4)ncc3Cl)cc2C1=O)c1ccc(N2CCOCC2)cc1
InChIKey: JKEORDJJLJLGKD-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: ERK2 In Vitro Inhibition Assay: Activity of ERK2 enzyme (Life Technologies) was determined using a time-resolved fluorescence format measuring the phosphorylation of a truncated version of Activating transcription factor 2 labelled with green fluorescent protein (ATF2-GFP) (Life Technologies). Assay reactions containing 50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Triton X-100, 1 mM DTT, 2.5% DMSO, 0.4 mM ATF2-GFP, 20 μM ATP and 0.25 nM ERK2 were set up in the presence of compound and allowed to proceed for 30 min at room temperature. Reactions were then stopped using TR-FRET dilution buffer (Life Technologies), 25 mM EDTA and 2 nM Tb-Anti-pATF2 (Thr71) (Life Technologies). After a further incubation period of at least 30 minutes, fluorescence was read on a Pherastar reader (Lanthascreen optic module; excitation 340 nm, emission 520 nm (channel A), 495 nm (channel B)). The ratio between A and B counts was used to calculate signal. IC50 values were calculated using a sigmoidal dose response equation (Prism GraphPad software, La Jolla, Calif., USA).
Measurement: IC50 = 230 nM (pChEMBL 6.639999866485596)
Descriptors: MW 591.11, ALogP 4.06, HBA 8, HBD 2, PSA 108.92, QED 0.4
Compound (SMILES): COc1cccc(OC)c1-n1c(NS(=O)(=O)[C@@H](C)[C@@H](O)c2cccc3nccn23)nnc1-c1ccc(C)o1
InChIKey: JPUVJOPSEHLSDK-QMHKHESXSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: GTPγS Binding: The human APJ receptor was cloned by polymerase chain reaction and the gene encoding the receptor was subcloned in pFLAG-CMV-3 expression vector (Sigma, Saint Louis, Mo. USA) in-house at Amgen. A GTPγS binding assay was performed on membranes prepared from CHO cells stably expressing human APJ receptor. The optimum experimental conditions for the concentrations of GDP, MgCl2, and NaCl in the assay buffer were initially determined. The assay was performed in assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, and 0.1% (w/v) BSA with 200 mM NaCl, 3 μM GDP] and membranes expressing human APJ receptor/well along with WGA PS beads. The reaction was initiated by addition of 0.2 nM [35S]GTPγS in the absence or presence of various ligands and incubated at RT for 90 min. Nonspecific binding was determined in the presence of 100 μM GTPγS and was always less than 0.2% of total binding.
Measurement: EC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 538.59, ALogP 3.36, HBA 11, HBD 2, PSA 146.01, QED 0.29
Compound (SMILES): Oc1ccc(-n2cc(-c3ccc4cccc(Oc5ccccc5)c4n3)nn2)cc1
InChIKey: XYGGNNAJVAWHLG-UHFFFAOYSA-N
Target: Macrophage migration inhibitory factor (SINGLE PROTEIN), Homo sapiens
UniProt: P14174
Assay: Inhibition of Tautomerase Activity of Human MIF: Inhibition of the tautomerase activity of MIF was measured using 4-hydroxyphenyl pyruvic acid (HPP) as substrate, largely following previously reported protocols (Taylor et al., Biochemistry, 1999, 38, 7444-7452). HPP was dissolved in 0.5 M acetate buffer, pH 6.0 to a final concentration of 10 mM and incubated overnight at room temperature to allow equilibration of the keto and enol forms. MIF (6 μL) was premixed in 500 mM boric acid, pH 6.2 (142 μL) and transferred to a transparent U bottom 96-well plate to a final concentration of 50 nM MIF. At this concentration high signal-to-noise and linearity were observed after analysis of progress curves for enol production at different protein concentrations. Inhibitors were dissolved in DMSO to 10 mM and an initial screen was performed. For compounds that showed ca. 25% or greater inhibition at 10 μM, an inhibition constant, was measured.Compounds were placed into wells (2 μL) at 6 different concentrations and incubated for 30 minutes until the assay was started by addition of HPP (504) at two concentrations (1.0 and 2.5 mM). The negative control was MIF incubated with DMSO vehicle, which in all assays was 1% and did not influence tautomerase activity. MIF activity was monitored at 305 nm for formation of the borate-enol complex using an Infinite F500 plate reader (TECAN, Morrisville, N.C.) for 175 seconds. Calculation of initial velocities and the nonlinear regression analyses for the enzyme kinetics were repeated three times with the program Prism6 (GraphPad, La Jolla, Calif.).Data obtained for the Example compounds, obtained using the methods described in Example B, are provided in Table 3. Results are also included for a reference compound ISO-1 (Chang, K. F.; Al-Abed, Y. Bioorg. Med. Chem. Lett. 2006, 16, 3376-3379; Balachandran, S. et al. Bioorg. Med. Chem. Lett. 2009, 19, 4773-4776). X-ray crystal structures were obtained for complexes of 3a, 3b, and 3v (Examples 1, 2, and 21) with human MIF as described below in Example C, which confirm the binding of these inhibitors in the tautomerase active site.
Measurement: Ki = 2950 nM (pChEMBL 5.53000020980835)
Descriptors: MW 380.41, ALogP 4.98, HBA 6, HBD 1, PSA 73.06, QED 0.48