lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1cc([C@H]2CC[C@@H]([C@@H](C)C(=O)Nc3ccc(S(F)(F)(F)(F)F)cc3)CC2)ccn1
InChIKey: MOTXANKDGGZDRL-ZACQAIPSSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: IDO Cellular Activity Inhibition Assay: The experimental principle is summarized as follows: IDO expression is low in Hela cells under no induction, but a certain concentration of IFN-γ can induces Hela cells to express IDO which catalyzes the conversion of tryptophan to N-formyl kynurenine, which in turn is hydrolyzed by trichloroacetic acid to give kynurenine. Kynurenine then reacts with the Ehrlich reagent to give a color enabling detection of the IDO activity. The absorbance at 490 nm (OD490) is directly proportional to the IDO activity.The test compound was dissolved in DMSO (Sigma, Cat. No. D5879) and diluted to 5 mM, then serially diluted 3-fold with DMSO to a minimum concentration of 2.29 μM, and each concentration point was further diluted 50-fold with FBS-free DMEM medium (ThermoFisher, Cat. No. 11995073). If a compound's IC50 value was very low, the initial concentration of the compound was lowered.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 448.5, ALogP 7.6, HBA 2, HBD 1, PSA 41.99, QED 0.48
Compound (SMILES): OC[C@@H]1CCCN1c1nc(Nc2cn(-c3cccc(OC(F)(F)F)c3)cn2)c2cccn2n1
InChIKey: SAPKNCVHUFFNAW-HNNXBMFYSA-N
Target: Activin receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q04771
Assay: Biochemical assay: Biochemical assays to measure the inhibitory effects of the compounds were performed by ThermoFisher Scientific (Life Technologies). ALK2 inhibition was tested using LanthaScreen Eu Kinase Binding Assay screening protocol.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 459.43, ALogP 3.52, HBA 9, HBD 2, PSA 92.74, QED 0.46
Compound (SMILES): OC[C@H]1C[C@@H](Nc2nc(Nc3ccccc3O)ncc2-c2nc3ccccc3s2)[C@H](O)[C@@H]1O
InChIKey: VCSFJFINUIIOIE-KRVNLHHGSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Kinase Activity Assay: Procedure: A 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 DTT, 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).Analytical LCMS Conditions:  Condition A: Phenomenex Luna C18 (4.6×250 mm), 95:5 to 5:95 water:MeCN (0.05% TFA), over 10 min, hold 6 min.  Condition B: Agilent SBC (3.0×50 mm), solvent A: H20-0.1% TFA;  solvent B: ACN-0.1% TFA; GRADIENT TABLE: 0 min:10% B, 0.3 min: 10% B, 1.5 min: 95% B, 2.70 min: 95% B, 2.76 min: 10% B, stop time 3.60 min.
Measurement: IC50 = 118 nM (pChEMBL 6.929999828338623)
Descriptors: MW 465.54, ALogP 2.72, HBA 10, HBD 6, PSA 143.65, QED 0.24
Compound (SMILES): CCC(=O)C(=O)N1CC=C(c2c[nH]c3ncc(NC(=O)OCc4ccccc4Cl)cc23)CC1
InChIKey: OEZNXVLFKHVUEQ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: TR-FRET (Time Resolved FRET) Assay: This BTK competition assay measures compound potency (IC50) for the inactivated state of Bruton's Tyrosine Kinase using FRET (Förster/Fluoresence Resonance Energy Transfer) technology. The BTK-Eu complex was incubated on ice one hour prior to use at a starting concentration of 50 nM BTK-Bioease: 10 nM Eu-streptavidin (Perkin-Elmer Catalog #AD0062). The assay buffer consisted of 20 mM HEPES (pH 7.15), 0.1 mM DTT, 10 mM MgCl2, 0.5 mg/ml BSA with 3% Kinase Stabilizer (Fremont Biosolutions, Catalog #STB-K02). After 1 h, the reaction mixture from above was diluted 10 fold in assay buffer to make 5 nM BTK: 1 nM Eu-Streptavidin complex (donor fluorophore). 18 μl of a mixture of 0.11 nM BTK-Eu and 0.11 nM Kinase Tracer 178 (Invitrogen, Catalog #PV5593,) with BTK-Eu alone as no negative control, was then dispensed into 384-well flat bottom plates (Greiner, 784076). Compounds to be tested in assay were prepared as 10× concentrations and serial dilution in half-log increments was performed in DMSO so as to generate 10 point curves. To initiate the FRET reaction, compounds prepared as 10× stock in DMSO was added to the plates and the plates were incubated 18-24 h at 14° C.
Measurement: IC50 = 404 nM (pChEMBL 6.389999866485596)
Descriptors: MW 466.93, ALogP 4.56, HBA 5, HBD 2, PSA 104.39, QED 0.52
Compound (SMILES): CS(=O)(=O)NC(=O)c1ccc(COc2cccc(-c3cnc(OCC4CCC4)c(CO)c3)c2)c(F)c1
InChIKey: DFIRUWKGTAOFFC-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: EFS-FRET Assay: This screen is used to determine the effects of compounds on human Nav1.7 channels, utilizing electrical field stimulation (EFS) system in 96-well plate format on FDSS (Hamamatsu Photonics) platform. The changes of membrane potential are monitored with FRET dye pair, DiSBAC2(3) and PTS 18.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 514.58, ALogP 3.83, HBA 7, HBD 2, PSA 114.82, QED 0.42
Compound (SMILES): CN1CCN(C(=O)C(CN[C@@H]2C[C@H]2c2ccc(F)cc2)NC(=O)c2ccc(-c3ncccn3)cc2)CC1
InChIKey: BZZBOVNHNXFCIF-ZXABPTRBSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: In Vitro Assay: Briefly, bulk histones, histone peptides or nucleosomes are incubated with purified human recombinant KDM1A, in the histone demethylase activity (HDM) assay buffer 1 (50 mM Tris pH 8.5, 50 mM KCl, 5 mM MgCl, 0.5% BSA, and 5% glycerol) from 30 minutes to 4 hours at 37° C. A typical reaction is conducted in 100 microliters in which either 20 micrograms of purified bulk histones or 3 micrograms of modified histone peptides are used as substrates. Different amounts of KDM1A ranging from 1-20 micrograms are used in the reaction along with, as necessary, other co-factors such as FAD or CoREST, depending on the chosen substrate. The reaction mixture is analyzed by SDS-PAGE and Western blotting using histone methyl-specific antibodies or by formaldehyde formation assay to examine the removal and conversion of the methyl group to formaldehyde, or by mass spectrometry in the case of peptide substrates to identify the demethylated histone peptide.
Measurement: IC50 = 13000 nM (pChEMBL 4.889999866485596)
Descriptors: MW 502.59, ALogP 2.3, HBA 6, HBD 2, PSA 90.46, QED 0.49
Compound (SMILES): CNc1cc(-c2cnc3n(CC(=O)N4CC5(COC5)C4)cccc2-3)nc2c(C(=O)N[C@@H]3CC[C@H]3OC)cnn12
InChIKey: QGDDVCXNDIJVEK-TZIWHRDSSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: TYK2 JH2 Domain Binding Assay: The binding reaction was assembled by combining 16 μl of DNA-tagged kinase extract, 3.8 μl liganded affinity beads, and 0.18 μl test compound (PBS/0.05% Tween 20/10 mM DTT/0.1% BSA/2 μg/ml sonicated salmon sperm DNA)]. Extracts were used directly in binding assays without any enzyme purification steps at a ≥10,000-fold overall stock dilution (final DNA-tagged enzyme concentration <0.1 nM). Extracts were loaded with DNA-tag and diluted into the binding reaction in a two step process. First extracts were diluted 1:100 in 1× binding buffer (PBS/0.05% Tween 20/10 mM DTT/0.1% BSA/2 μg/ml sonicated salmon sperm DNA) containing 10 nM DNA-tag. This dilution was allowed to equilibrate at room temperature for 15 minutes and then subsequently diluted 1:100 in 1× binding buffer. Test compounds were prepared as 111× stocks in 100% DMSO. Kds were determined using an 11-point 3-fold compound dilution series with three DMSO control points. All compounds for Kd measurements are distributed by acoustic transfer (non-contact dispensing) in 100% DMSO
Measurement: Kd < 0.2 nM (pChEMBL 9.699000358581543)
Descriptors: MW 530.59, ALogP 1.51, HBA 10, HBD 2, PSA 127.91, QED 0.37
Compound (SMILES): COc1cc(CNC(=O)[C@@H](CC2CCCCC2)NC(=N)NC(=O)Cc2ccc(S(N)(=O)=O)cc2)ccc1F
InChIKey: OLERNBCSIUBSMF-JOCHJYFZSA-N
Target: Cathepsin D (SINGLE PROTEIN), Homo sapiens
UniProt: P07339
Assay: In-Vitro Fluorescence Assay: In order to identify modulators of cathepsin D activity, a continuous enzymatic test was carried out with a synthetic peptide which carries a fluorescent group (MCA=(7-methoxycoumarin-4-yl)acetyl) which is quenched by energy transfer from a Dpn (2,4 dinitrophenyl) group on the same molecule, in Greiner 384-well nb microtitre plates. Cleavage of the peptidic substrate by cathepsin D causes an increase in the fluorescence intensity. In order to determine the efficacy of substances, the time-dependent increase in the fluorescence intensity in the presence of the substance was compared with the time-dependent increase in fluorescence in the absence of substances. The reference substance used was pepstatin A (Sigma-Aldrich). The substrate used was MCA-GKPILFFRLK(Dnp)d-RNH2 (Enzo Life Sciences, Lörrach). The enzyme employed was cathepsin D isolated from the human liver (Sigma-Aldrich) in a final concentration of 1.4 nM. The test was carried out in 100 mM sodium acetate buffer, 1.25% (v/v) of DMSO, 0.25% (w/v) of Chaps, pH 5.5. 2 μl of each substance solution with serially diluted substance concentration were added to in each case 4 μl of cathepsin D solution and incubated at room temperature for 10 min. The reaction was started by addition of 2 μl of substrate solution (final concentration 5 μM). After carrying out a starting-point fluorescence measurement (excitation wavelength 340 nm/emission wavelength 450 nm) using an Envision multilabel reader (Perkin Elmer), the reaction was incubated at room temperature for 60 min. The amount of peptide fragment cleaved off during the reaction time was subsequently measured by determination of the increase in the fluorescence intensity at 450 nm (excitation wavelength 340 nm).
Measurement: IC50 = 110 nM (pChEMBL 6.960000038146973)
Descriptors: MW 547.65, ALogP 2.32, HBA 6, HBD 5, PSA 163.47, QED 0.23
Compound (SMILES): CN(C)C(=O)CC[S+]([O-])c1ccc(Cl)cc1NS(=O)(=O)c1ccc(F)cc1F
InChIKey: CTTSSPAIVNPICC-UHFFFAOYSA-N
Target: C-C chemokine receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P41597
Assay: Biological Assay: HEK-Gqi5 cells stably expressing CCR2 were cultured in (DMEM high glucose, 10% FBS, 1% PSA, 400 μg/ml geneticin and 50 μg/ml hygromycin. Appropriate positive control chemokines (MCP-1, MIP1A or RANTES) was used as the positive control agonist for screening compound-induced calcium activity assayed on the FLIPRTetra. The drug plates were prepared in 384-well microplates using the EP3 and the MuItiPROBE robotic liquid handling systems.
Measurement: IC50 = 1477 nM (pChEMBL 5.829999923706055)
Descriptors: MW 450.92, ALogP 3, HBA 4, HBD 1, PSA 89.54, QED 0.66
Compound (SMILES): Cc1ccc(Cl)cc1-c1cc2nccc(N[C@@H]3C[C@H](COS(N)(=O)=O)[C@@H](O)[C@H]3O)n2n1
InChIKey: KZMBNBXLSBWCTA-OWCDTVLYSA-N
Target: Ubiquitin-conjugating enzyme E2 E1 (SINGLE PROTEIN), Homo sapiens
UniProt: P51965
Assay: HTRF Assay : The UAE enzymatic reaction totals 50 μL and contains 50 mmol HEPES (pH 7.5), 0.05% BSA, 2.5 mM MgCl2, 0.1 uM ATP, 8 nM GST-Ubc-2, 35 nM flag-ubiquitin, 1 nM recombinant human UAE or mouse UAE. Compounds for this hUAE 1050 assay are tested at 10 point 3-fold dilution. The top concentration for this assay is 1 μM. Each compound is ordered in duplicate on the same plate. The enzymatic reaction mixture is incubated for 90 minutes at room temperature (24 degrees C.) in a 384 well plate prior to termination with a stop solution (0.1 M HEPES/0.05% Tween20, 20 mmol EDTA, 410 mM KF, 0.5 nM Eu Cryptate anti-FLAG M2-K antibody (Cis-bio International), 8 ug/mL Anti-GST XL-APC (Prozyme)). After incubation for 120 minutes, quantification of the FRET is performed on the Pherostar (BMG).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 467.94, ALogP 1.1, HBA 9, HBD 4, PSA 152.07, QED 0.42
Compound (SMILES): Cc1ccccc1-c1cc2nc(Nc3cccc(CN4CCN(C)CC4)c3)ncc2[nH]1
InChIKey: SPVDVGUFWOPGCZ-UHFFFAOYSA-N
Target: Receptor-interacting serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43353
Assay: RIPK2 Inhibition Assay: RIPK2 activity was measured using an indirect ELISA detection system. HisRIPK2 (0.6 nM) was incubated in the presence of 6 μM ATP (Sigma cat # A7699), 20 mM Hepes, pH 7.5, 1 mM EGTA, 2.5 mM MgCl2, 2.5 mM MnCl2 and 0.01% Triton X-100 in a 96 well microtitre plate pre-coated with amino terminal 6 histidine, sumo tagged TTK (amino acid residues 1-275). The reaction was allowed to proceed for 30 minutes, followed by 5 washes of the plate with Wash Buffer (phosphate buffered saline supplemented with 0.2% Tween 20), and incubation for 30 minutes with a 1:3000 dilution of primary antibody (Cell Signaling cat #9381). The plate was washed 5 times with Wash Buffer, incubated for 30 minutes in the presence of secondary antibody coupled to horse radish peroxidase (BioRad cat #1721019, 1:3000 concentration), washed an additional 5 times with Wash Buffer, and incubated in the presence of TMB substrate (Sigma cat # T0440). The colorimetric reaction was allowed to continue for 5 minutes, followed by addition of stop solution (0.5 N H2SO4), and quantified by detection at 450 nm with either a monoChromatic or filter based plate reader (Molecular Devices M5 or Beckman DTX880, respectively).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 412.54, ALogP 4.42, HBA 5, HBD 2, PSA 60.08, QED 0.51
Compound (SMILES): CC(C)c1ccccc1-c1ncc2c(n1)N(Cc1ccc(C(=O)CCc3ccc(F)cc3)cc1)C(=O)C2
InChIKey: FBSIIUJCTOYIRM-UHFFFAOYSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O94782
Assay: HTS Assay: The HTS assay was performed in a final volume of 20 μL in assay buffer containing 20 mM Tris-HCl (pH 8.0, (1M Tris-HCl, pH 8.0 solution; Corning 46-031-CM)), 2 mM CaCl2) (1M Calcium Chloride solution; Sigma #21114) 1 mM GSH (L-Glutathione reduced; Sigma #G4251), 0.01% Prionex (0.22 μM filtered, Sigma #G-0411), and 0.01% Triton X-100. Stock compound solutions were stored at −20° C. as 10 mM in DMSO. Up to 1 month prior to the assay, 2 mM test compounds were pre-dispensed into assay plates (Black, low volume; Corning #3820) and frozen at −20° C. Prestamped assay plates were allowed to come to room temperature on the day of the assay. For the screen, 100 nL of 2 mM was pre-dispensed for a final screening concentration of 10 μM (DMSO(fc)=0.5%). The final concentration of the enzyme (USP1, construct USP1 (I-785, GG670, 671AA)/UAF1 (I-677)-Flag; Viva) in the assay was 100 μM. Final substrate (Ub-Rh110; Ubiquitin-Rhodamine 110, R&D Systems #U-555) concentration was 25 nM with [Ub-Rh110]<<Km. 10 μL of 2× enzyme was added to assay plates (pre-stamped with compound) either simultaneously with 2×Ub-Rh110 or preincubated with USP1 40 minutes prior to the addition of 10 μL of 2×Ub-Rh110 to compound plates. Plates were incubated stacked for 45 minutes at room temperature before fluorescence was read on the Envision (Excitation at 485 nm and Emission at 535 nm; Perkin Elmer) or on the PheraSTAR (Excitation at 485 nm and Emission at 535 nm; BMG Labtech).
Measurement: IC50 = 125 nM (pChEMBL 6.900000095367432)
Descriptors: MW 493.58, ALogP 6.31, HBA 4, HBD 0, PSA 63.16, QED 0.27
Compound (SMILES): COc1ccc(-n2ccc3c(N4C[C@@H](N)C[C@H]4CO)c(NC(=O)c4ccnc(-c5c(F)cccc5OC)n4)ccc32)c(F)c1
InChIKey: HYKAFUSRADBHNZ-OALUTQOASA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: Kinase Binding Assay: A stock solution of 1 mM test compound was prepared in DMSO. The compound plate was prepared by 3-fold and 11-point serial dilutions. 0.1 μL of the compound in DMSO was transferred from the compound plate to the white 384 well polystyrene plates. The assay buffer contained 50 mM HEPES, pH 7.5, 0.01% Tween-20, 5 mM MgCl2, 0.01% BSA, and 5 mM DTT. 5 μL of 4 nM active HPK1 (SignalChem M23-11G) prepared in the buffer was added to the plate. The enzyme concentration given was based on the given stock concentration reported by the vender. 5 μl of 18 nM tracer 222 (ThermoFisher PV6121) and 4 nM LanthaScreen Eu-Anti GST antibody (ThermoFisher PV5595) were added. After one hour incubation at 25° C., the plates were read on a PHERAstar FS plate reader (BMG Labtech). Ki values were determined.
Measurement: Ki < 100 nM (pChEMBL 7)
Descriptors: MW 600.63, ALogP 4.53, HBA 9, HBD 3, PSA 127.76, QED 0.24
Compound (SMILES): CCS(=O)(=O)Nc1cc(-c2cn(C)c(=O)c3ccccc23)c2oc(C3CC3)cc2c1
InChIKey: MGBSZFGNAZOLFQ-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: In Vitro Enzyme Inhibition Assay: Determination of the IC50 for the heterocyclic derivative BRD4 inhibitors disclosed herein was performed as follows. His-tagged BREM was cloned, expressed and purified to homogeneity. Filipakopoulos et al., 468 Nature 1067 (2010). BRD4 binding and inhibition was assessed by monitoring the interaction of biotinylated H4-tetraacetyl peptide (AnaSpec, H4K5/8/12/16(Ac), biotinlabeled) with the target using the AlphaScreen technology (Life Technologies). In a 384-well ProxiPlate BRD4(BD1) (2 nM final) was combined with peptide (15 nM final) in 50 mM HEPES (pH 7.3), 10 mM NaCl, 0.25 mM TCEP, 0.1% (w/v) BSA, and 0.005% (w/v) Brij-35 either in the presence of DMSO (final 0.4% DMSO) or compound dilution series in DMSO. After 20 min incubation at RT, Alpha streptavidin donor beads and Nickel Chelate acceptor beads were added to a final concentration of 5 μg/mL. After 2 hr of equilibration, plates were read on an Envision instrument and the IC50 was calculated using a four parameter non-linear curve fit.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 422.51, ALogP 4.59, HBA 5, HBD 1, PSA 81.31, QED 0.51
Compound (SMILES): CC(=O)Nc1ccc(-c2cc(C(=O)N3CCOCC3)c3cccc(Cl)c3n2)cc1
InChIKey: KYCCPGCRESXZCJ-UHFFFAOYSA-N
Target: MAPK/MAK/MRK overlapping kinase (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UQ07
Assay: Inhibitory Activity Assay: Table 2-3: Step 1. Add 50 μl anti-DIAPH1 (1:160 dilution in 0.1M NaHCO3 pH 9.6)/well. Incubate overnight at 4° C.    Day 2    Step 2. Use the plate washer to aspirate anti-DIAPH11 and wash plates 4× with PBS 100 μl per well per wash.    Step 3. Add 180 μl 3% BSA in 1×PBS. Incubate for 1.5 hrs at room temperature Step 4. Use the plate washer to aspirate the blocking solution and wash 5× with PBS 300 μl per well.    Step 5. Add 50 μl of DIAPH1 containing lysate (85 μg) and incubate at RT for 3 hours.    Step 6. Aspirate the lysate and wash the plate 5×(100 μl) on the plate washer.    Step 7. Add 25 μl PBS to the wells.    Step 8. Add 0.5 μL compound per well.    Step 9. Add 24.5 μL GFP RAGE tail (125 nM) into each well for 2 hrs at room temperature.    Step 10. Aspirate and wash 5× with PBS (100 μL) on the plate washer.    Step 11. Add 100 μl PBS in each well.    Step 12. Detection: Read on fluorescence plate reader excitation 435 nm and 485 nm emission
Measurement: Kd = 43000 nM (pChEMBL 4.369999885559082)
Descriptors: MW 409.87, ALogP 3.99, HBA 4, HBD 1, PSA 71.53, QED 0.71
Compound (SMILES): COC(=O)NC[C@H]1CC[C@H](n2c(CC#N)nc3cnc4ccsc4c32)CC1
InChIKey: BHBPTSXNZNDCEX-JOCQHMNTSA-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 383.48, ALogP 3.8, HBA 7, HBD 1, PSA 92.83, QED 0.74
Compound (SMILES): CCCN(CCC)C(=O)C1=Cc2ccc(C3=NCc4cc(OC)ccc4N3)cc2N=C(N)C1
InChIKey: JOYINLZPAYPUNM-UHFFFAOYSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
Assay: Pharmacological Test: For TLR8 and TLR7 activity testing, HEK-Blue human TLR8 or TLR7 cells (Invivogen, San Diego, Calif., USA) are used, respectively. These cells are designed for studying the stimulation of human TLR8 or TLR7 by monitoring the activation of NF-κB. A SEAP (secreted embryonic alkaline phosphatase) reporter gene is placed under the control of the IFN-b minimal promoter fused to five NF-κB and AP-1-binding sites. Therefore the reporter expression is regulated by the NF-κB promoter upon stimulation of human TLR8 or TLR7 for 20 hours. The cell culture supernatant SEAP reporter activity was determined using Quanti Blue kit (Invivogen, San Diego, Ca, USA) at a wavelength of 640 nm, a detection medium that turns purple/blue in the presence of alkaline phosphatase. EC50 values were determined using Activity Base analysis (ID Business Solution, Limited).
Measurement: EC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 445.57, ALogP 4.49, HBA 6, HBD 2, PSA 92.31, QED 0.66
Compound (SMILES): CC(C)S(=O)(=O)C(C(=O)NCC(=O)NC1CC1)c1nc2cc(C(F)(F)F)c(-c3ccccc3)cc2s1
InChIKey: LNQLFPOISYBOJO-UHFFFAOYSA-N
Target: Endothelial lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5X9
Assay: Biological Activity: Endothelial lipase (EL) and hepatic lipase (HL) activities were measured using a fluorescent substrate, A10070, (Invitrogen, CA) doped into an artificial vesicle containing DMPG (Avanti Polar Lipids) as the excipient. Vesicles were prepared by combining 571 μL of 29 mM DMPG in a 1:1 mixture of MeOH and CHCl3 with 2000 μL of 1 mM A10070 in a 1:1 mixture of MeOH and CHCl3. The mixture was dried under nitrogen in multiple vials then resuspended in 20 mL total volume of 50 mM HEPES pH 8.0 buffer containing 50 mM NaCl and 0.2 mM EDTA. The sample was allowed to sit at room temperature for 15 min and then was sonicated 3×4 mins on ice with a Branson Sonicator using duty cycle 1. This preparation provides vesicles with a mole fraction of 0.11 for the FRET substrate.The enzymatic assay was measured using 384-well white Optiplates. Each well contained 20 μL of assay buffer (50 mM HEPES pH 8.0, 50 mM NaCl and 1 mM CaCl2) and 0.25 μL of a DMSO solution containing a compound of interest. EL or HL (10 μL) was added and allowed to incubate with the compound for 30 min at 37° C. The source of EL was conditioned media obtained from HT-1080 cells that were transformed using RAGE technology (Athersys) to overexpress endogenous EL, and HL was partially purified from conditioned media obtained from COS cells overexpressing HL. The reaction was started by the addition of 10 μL of a 1:10 dilution of vesicles. The final total reaction volume was 20.25 μL. The reaction rates were measured on a Gemini plate reader with an excitation wavelength of 490 nm and an emission wavelength of 530 nm. Readings were taken over a period of 60 minutes, and the slope between 300 and 900 secs of the readout was used to calculate the rate of the reaction.
Measurement: IC50 = 26 nM (pChEMBL 7.579999923706055)
Descriptors: MW 539.6, ALogP 4.24, HBA 6, HBD 2, PSA 105.23, QED 0.45
Compound (SMILES): COc1cc(NCc2cc3cc(Cl)ccc3[nH]c2=O)ccc1F
InChIKey: VKHICLNCSJFWDX-UHFFFAOYSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: IDH1-R132H and IDH1-R132C Enzymatic Assay: Assays were performed in a 384-well black plate. An aliquot of 250 nL of compound was incubated with 10 μL of 30 nM IDH1-R132H or 10 nM IDH1-R132C recombinant protein in assay buffer (50 mM Tris pH=7.5, 150 mM NaCl, 5 mM MgCl2, 0.1% (w/v) Bovine Serum Albumin, and 0.01% Triton X-100) in each well at 25° C. for 15 minutes. After the plate was centrifuged briefly, an aliquot of 10 μL of 2 mM α-ketoglutarate and 20 μM NADPH solution prepared in assay buffer was then added to each well and the reaction was maintained at 25° C. for 45 minutes. An aliquot of 10 μL of diaphorase solution (0.15 U/mL diaphorase and 30 μM Resazurin in assay buffer) was added to each well. The plate was maintained at 25° C. for 15 minutes and then read on a plate reader with excitation and emission wavelengths at 535 nm and 590 nm, respectively. The IC50 of a given compound was calculated by fitting the dose response curve of inhibition of NADPH consumption at a given concentration with the four parameter logistic equation.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 332.76, ALogP 3.94, HBA 3, HBD 2, PSA 54.12, QED 0.76
Compound (SMILES): CN(C1CN(C(=O)C2CC2)C1)[C@H]1CCCC[C@@H]1Nc1cc(F)c(S(=O)(=O)Nc2nccs2)cc1Cl
InChIKey: JLMORZJUHJQWDX-ICSRJNTNSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Electrophysiological Assay (EP) (In Vitro Assay): Patch voltage clamp electrophysiology allows for the direct measurement and quantification of block of voltage-gated sodium channels (NaV's), and allows the determination of the time- and voltage-dependence of block which has been interpreted as differential binding to the resting, open, and inactivated states of the sodium channel (Hille, B., Journal of General Physiology (1977), 69: 497-515).The following voltage clamp electrophysiology studies were performed on representative compounds using cells heterologously expressing Nav1.7 or Nav1.5 channels. cDNAs for Nav1.7 (NM_002977) and Nav1.5 (AC137587) were stably expressed in Chinese Hamstr Ovary (CHO) cells and CHL (Chinese Hamster Lung) cells respectively. Sodium currents were measured in the whole-cell configuration using Syncropatch 384PE (NanIon Technologies, Germany). 1NPC®-384 chips with custom medium resistance and single hole mode are used. Internal solution consists of (in mM): 110 CsCl, 10 CsCl, 20 EGTA, and 10 Hepes (pH adjusted to 7.2); and external solution contains (in mM): 60 NMDG, 80 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 2 D-Glucose monohydrate, 10 Hepes (pH adjusted to 7.4 with NaOH).After system flushing, testing compounds are dissolved in external solution containing 0.1% Pluronic F-127. The chip is moved into the measuring head and the instrument primes the chip with external and internal solutions. 10l cells are added to the chip from a cell hotel, and a negative pressure of −50 mBar is applied to form a seal. Following treatment with seal enhancer solution and wash-off with external solution, negative pressure of −250 mbar is applied for 1 second to achieve the whole-cell configuration, followed by three washing steps in external solution. 20 μl of compounds is added to 40 μl in each well (1:3 dilution of compounds), and after mixing, 20 μl is removed so the volume is retained at 40 μl. After approximately 13 minutes recordings, 20 μl/well of 2 uM TTX, or 333 uM Tetracaine (for Nav1.5) is added to achieve full block.For voltage protocol, an holding potential of −50 mV is applied during the whole experiment. A depolarizing step is applied to −10 mV for 10 ms, followed by a hyperpolarization step to −150 mV for 20 ms to allow channel recovery from inactivation. A second depolarizing step is applied from −150 mV to −10 mV for 10 ms, where currents were measured to derive blocking effects of compounds. Inhibition is determined based on 7.5 min of compound incubation.
Measurement: IC50 = 12.3 nM (pChEMBL 7.909999847412109)
Descriptors: MW 542.1, ALogP 4.01, HBA 7, HBD 2, PSA 94.64, QED 0.52
Compound (SMILES): C[C@@H](O)c1nc2cnc3ccsc3c2n1[C@H]1CCN(CCC#N)C1
InChIKey: NPVDXJYNFLUOPI-NEPJUHHUSA-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 < 1000 nM (pChEMBL 6)
Descriptors: MW 341.44, ALogP 2.86, HBA 7, HBD 1, PSA 77.97, QED 0.79
Compound (SMILES): O=C(NS(=O)(=O)c1ccc(NCC2CCOCC2)c([N+](=O)[O-])c1)c1ccc(C2CCC(N3CCC[C@@H]3c3ccccc3C3CC3)CC2)cc1Oc1cnc2[nH]ccc2c1
InChIKey: AMJYVMQRIKFTRG-ACGXXCRRSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Bcl-2/Bcl-X Fluorescence Polarization Assay: Compounds disclosed herein were tested for blocking of Bcl-2/Bcl-xl protein with its ligand in an assay based on fluorescence polarization methodology. Recombinant human 2.7 nM Bcl-2/1.3 nM Bcl-xl protein was pre-incubated with a serial dilution of compounds disclosed herein (maximum concentration is 1 μM for Bcl-2 assay, and 10 μM or 1 μM for Bcl-xl assay, 3-fold serially diluted, 10 points) at room temperature for 0.5 hour in an assay buffer containing 20 ml potassium phosphate buffer, pH 7.5, 50 mM NaCl, 1 mM EDTA, 0.05% Tween-20, 0.01% BSA. Then the FITC labeled Bak peptide Ac-GQVGRQLAIIGDK (FITC)INR-amide (1 nM for Bcl-2, 0.82 nM for Bcl-xl) was added to plate and further incubated at room temperature for 0.5 h. The FP signals (485 nm-520 nm-520 nm) were read on BMG PHERAstar FS or BMG PHERAstar FSX instalment. The inhibition percentage of Bcl-2/Bcl-xl interaction with its ligand in presence of increasing concentrations of compounds was calculated based on the FP signals. The IC50 for each compound was derived from fitting the data to the four-parameter logistic equation by Graphpad Prism software.
Measurement: IC50 = 862 nM (pChEMBL 6.059999942779541)
Descriptors: MW 819, ALogP 8.96, HBA 10, HBD 3, PSA 168.79, QED 0.08
Compound (SMILES): CN(C)C(=O)c1nc2c(N3CCOCC3)nc(-c3cnn(C)c3)nc2[nH]1
InChIKey: SVWFYRUHHFYPCC-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: In Vitro Enzyme PI3K Assay: The Kinase-Glo luminescent kinase assay kit (from Promega) was used to measure kinase activity. In this assay, amount of ATP remaining in solution following a kinase reaction is measured. The effect of compounds on PI3Kδ inhibition was carried out by adding 2.29 μg/ml of recombinant PI3K δ enzyme (Proteros, Germany) to reaction mixture containing assay buffer (50 mM HEPES, pH 7.4, 50 mM NaCl, 0.05% CHAPS) supplemented with 10 mM MgCl2, 5 mM DTT, 60 μM Phosphatidyl inositol bisphosphate (PIP2) and 10 μM ATP in the absence or presence of different concentrations of the compounds in a final volume of 15 μl/well, in a 384 well plate. The reaction mixture was incubated for 2 hours at room temperature. At the end of incubation period the equal volume of Kinase-Glo plus (Promega, V3772), was added per well and the luminescence was measured after incubating for 10 minutes at room temperature in dark. Results were calculated by measuring the luminescence units of test samples to blanks containing no enzyme.In certain embodiments, the compounds showed IC50 of less than 1000 nM, in another embodiment, the IC50 values range from about 100 nM to 500 nM, and in yet another embodiment, it is even less than 30 nM for PI3Kδ as shown in Table 3 and 4.The compounds of the present invention were tested for their selectivity for PI3Kδ over PI3Kα, PI3Kβ and PI3Kγ following the above assay using specific recombinant enzymes (Proteros, Germany) for each kinase. Assay condition of kinase assay (PI3Kα, PI3Kβ, PI3Kγ and PI3Kδ) was as follows. Enzyme: 2.29 μg/mL; ATP: 10 μM; PIP2 substrate: 60 μM and Reaction Time: 2 hours.
Measurement: IC50 = 76 nM (pChEMBL 7.119999885559082)
Descriptors: MW 356.39, ALogP 0.29, HBA 8, HBD 1, PSA 105.06, QED 0.72
Compound (SMILES): CC(C)(O)Cn1ncc2cc(-c3ccc(C(=O)N4CC[C@H](N)C4)cc3-c3ccc(C#N)c(F)c3)c(F)cc21
InChIKey: UBFJIQCXBYUHEJ-NRFANRHFSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: Inhibitory Activity Assay: The conditions for measuring inhibitory activity of compounds against LSD1 activity were determined with reference to a document available from the website of PerkinElmer (U-TRF #38) and a patent of GlaxoSmithKline (WO2012135113).To measure the inhibitory activity, first, the compound of the present invention was serially diluted in dimethylsulfoxide (DMSO). Sequentially, the solution of the compound of the present invention in DMSO (final concentration of DMSO: 5%) and human LSD1 protein (Abcam, ab80379) were added to a reaction buffer (25 mM Tris-HCl (pH 7.5), 50 mM KCl, 2 mM CHAPS, 1 mM DTT, 0.02% BSA). The mixture was preincubated at 25° C. for 30 minutes. Thereafter, a H3K4 (Me1)-biotin-labeled peptide (Anaspec #64355) (final concentration: 200 nM) was added thereto and reacted for 60 minutes. Tranylcypromine (final concentration: 3 mM) was then added thereto to terminate the reaction. Thereafter, a detection solution containing an Eu-labeled anti-H3K4 antibody (PerkinElmer, TRF0404) and Streptavidin Alexa Fluor 647 (Thermo Fisher Scientific, 521374) was added thereto, and the mixture was allowed to stand at room temperature for 1 hour. Finally, the intensity of fluorescence under the excitation light with a wavelength of 337 nm was measured with a PHERAstar FS (BMG Labtech) at two wavelengths: 620 nm and 665 nm. The demethylation level was calculated from the ratio of the fluorescence intensity at the two wavelengths, and the compound concentration at which demethylation was inhibited by 50% was defined as IC50 (nM).
Measurement: IC50 = 0.35 nM (pChEMBL 9.460000038146973)
Descriptors: MW 515.56, ALogP 4.46, HBA 6, HBD 2, PSA 108.17, QED 0.41
Compound (SMILES): CC(=O)N1CCC([C@H](C)c2c[nH]nc2-c2ccc3c(c2F)CCC(=O)N3C)CC1
InChIKey: HUVLJWOWKSCYTO-ZDUSSCGKSA-N
Target: Cytochrome P450 11B2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P19099
Assay: Inhibition Assay: The expression plasmids contains the ORF for either human/cyno CYP11B1 or CYP11B2 under the control of a suitable promoter (CMV-promoter) and a suitable resistance marker (neomycin). Using standard techniques the expression plasmid is transfected into G-402 cells and these cells are then selected for expressing the given resistance markers. Individual cell-clones are then selected and assessed for displaying the desired enzymatic activity using 11-Deoxycorticosterone (Cyp11B2) or 11-Deoxycortisol (Cyp11B1) as a substrate.G-402 cells expressing CYP11 constructs were established as described above and maintained in McCoy's 5a Medium Modified, ATCC Catalog No. 30-2007 containing 10% FCS and 400 μg/ml G418 (Geneticin) at 37° C. under an atmosphere of 5% CO2/95% air. Cellular enzyme assays were performed in DMEM/F12 medium containing 2.5% charcoal treated FCS and appropriate concentration of substrate (0.3-10 uM 11-Deoxycorticosterone, 11-Deoxycortisol or Corticosterone). For assaying enzymatic activity, cells were plated onto 96 well plates and incubated for 16 h. An aliquot of the supernatant is then transferred and analyzed for the concentration of the expected product (Aldosterone for CYP11B2; Cortisol for CYP11B1). The concentrations of these steroids can be determined using HTRF assays from CisBio analyzing either Aldosterone or Cortisol.
Measurement: EC50 = 0.064 nM (pChEMBL 10.1899995803833)
Descriptors: MW 398.48, ALogP 3.49, HBA 3, HBD 1, PSA 69.3, QED 0.86
Compound (SMILES): O=C1CC(=O)N(c2ccc(-n3ccnc3CCc3cccc(F)c3)cc2)c2ccc3ccccc3c2N1
InChIKey: ZHJSFESDIPFXMB-UHFFFAOYSA-N
Target: P2X purinoceptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99571
Assay: Inhibition Activity Assay: The 1321N1 cells stably expressing human P2X4 receptor were seeded on a 96-well plate, cultured under the conditions of 37° C. and 5% CO2 for 24 hours, and then used for intracellular calcium measurement. Fura-2 AM, which is a calcium fluorescent indicator, was used for the intracellular calcium measurement. Fura-2 AM dissolved in an assay buffer was added to the cells, the cells were left standing at room temperature for 45 minutes so that Fura-2 AM was incorporated into the cells, and then the plate was used for the fluorescence measurement. The treatment of the cells with each test substance was performed 15 minutes before the addition of ATP, and inflow of calcium into the cells as a response induced by the addition of ATP was measured over time by using a microplate reader. The ratio of fluorescence values obtained with excitation lights of 340 nm and 380 nm was used as an index of the change of intracellular calcium level, and the inhibitory activity of the test substance was calculated on the basis of the comparison with the value obtained in the absence of the test substance (control).
Measurement: IC50 = 51 nM (pChEMBL 7.289999961853027)
Descriptors: MW 490.54, ALogP 5.96, HBA 4, HBD 1, PSA 67.23, QED 0.31
Compound (SMILES): Cn1nnn(-c2cc(Nc3ncc(F)c(N[C@@H]4C[C@@H]5CCCN5C(C)(C)C4)n3)c(F)cc2OCCN2CCOCC2)c1=O
InChIKey: XEHHETQZLHRHFY-MOPGFXCFSA-N
Target: Interleukin-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P60568
Assay: IL-2 ELISA Assay: Primary T cell stimulation and IL2 ELISA: Human primary T cells (100,000 cells per well) were pre-incubated with or without test compound in Yssel's medium for 1 hour at 37° C. Cells were then stimulated by transferring them to round-bottom 96-well plates pre-coated with 1 μm/ml αCD3 and 5 μg/ml αCD28. For counter assay, cells were instead stimulated by adding 8× stock solutions of PMA and ionomycin in Yssels (for final concentrations of 0.5 ng/ml PMA and 0.1 uM ionomycin, both from Calbiochem). Cells were incubated at 37° C. for 24 hours before 100 μL supernatants were harvested for quantification of IL-2 by ELISA using Human IL-2 Duoset ELISA Kit from R and D Systems, Cat. # DY202E.
Measurement: EC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 600.68, ALogP 2.31, HBA 13, HBD 2, PSA 127.49, QED 0.38
Compound (SMILES): C[C@@H]1[C@H](C(N)=O)N(C2CC2)CCN1c1ccnc(-c2cnc3ccc(Cl)cn23)n1
InChIKey: SOSKUVNIGZGTPM-KZULUSFZSA-N
Target: eIF-2-alpha kinase GCN2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P2K8
Assay: Enzyme Inhibition Assay: An assay buffer containing GCN2 kinase and (RS)7 was prepared. 4.7 μL of this stock solution was placed per well of a black, low volume, 384-well microtitre plate (e.g. catalogue number 3676, Corning Inc., NY). To this was added 0.65 μM of DMSO containing serial dilutions of the test compound (typical final concentrations of test compound were 0 to 8 μM). The plate was incubated for 10 minutes at 25° C. prior to the addition of 4.7 μL of ATP stock buffer to initiate the enzyme reaction. The reaction was allowed to proceed for 1 hour at 25° C., prior to the addition of 10 μL detection buffer (consisting of appropriate concentrations of ADP2 antibody and ADP Alexa633 tracer in 1× stop and detect buffer as supplied by BellBrook Labs). The reaction was left to incubate for 1 hour at 25° C., prior to measuring the fluorescence polarisation signal (mP) in each well using a PHERAstar FS reader (BMG Labtech, Germany).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 411.9, ALogP 1.97, HBA 7, HBD 1, PSA 92.65, QED 0.71
Compound (SMILES): C=C(CN1CCCC(n2nc(-c3ccc(Oc4c(F)c(F)cc(F)c4F)cc3F)c3c(N)ncnc32)C1)C(=O)NC
InChIKey: XHMMHWWVPNKGPP-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: HTRF Assay: The enzyme reaction mixture of BTK wild type standard HTRF assay contained 1 nM BTK wild type, 1 μM biotin-TK1 peptide, and 30 μM ATP in a buffer. The enzyme reaction were carried out at room temperature for 60 minutes. 5 μl of 0.2 M EDTA were added to quench the reaction and then the inhibitors (5 μl) were added at final concentrations of 2 nM antibody and 62.5 nM XL665. The plates were incubated at room temperature for 60 minutes and then read in the Envision plate reader. The readouts were transformed into inhibition rate % by the equation of (Min Ratio)/(Max-Min)*100%. Hence the 1050 data of test compounds were generated by using four parameters curve fitting.
Measurement: IC50 = 52 nM (pChEMBL 7.28000020980835)
Descriptors: MW 573.53, ALogP 4.5, HBA 8, HBD 2, PSA 111.19, QED 0.19
Compound (SMILES): Cc1nc(-c2ccc(C(F)(F)F)cc2)sc1C(=O)NCCC1CCN(Cc2ccccc2)CC1
InChIKey: RAYKHLOTECAMTE-UHFFFAOYSA-N
Target: Muscarinic acetylcholine receptor M4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08173
Assay: Binding Assay: Binding affinity (Ki) of compounds was measured by inhibition of radioligand binding to membranes from CHO cells expressing human M1, M2, M3, M4 and M5 receptors. Membranes were prepared by nitrogen cavitation and differential centrifugation as previously described (Hoare et al., Mol. Pharmacol. 2003 March; 63(3): 751-65). The radioligand employed was tritiated N-methylscopolamine, used at a concentration of 1.5 nM. A dose-response of twelve concentrations of compound was used, ranging from 10 Î¼M to 32 Î¼M. The assay buffer was 50 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 1 mM ethylenediaminetetraacetic acid, pH-adjusted to pH 7.4.
Measurement: Ki = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 487.59, ALogP 6.17, HBA 4, HBD 1, PSA 45.23, QED 0.43
Compound (SMILES): Nc1nc2c(-c3ccc(S(=O)(=O)NC[C@]4(N)CCNC4)c(S(N)(=O)=O)c3-c3nn[nH]n3)cccc2[nH]1
InChIKey: FMJPXUXLVCEEHT-IBGZPJMESA-N
Target: Metallo-beta-lactamase type 2 (SINGLE PROTEIN), Bacteria
UniProt: C7C422
Assay: BIOLOGICAL ASSAYS: The Class B enzyme activities were measured in the presence of the test inhibitor in a fluorescence assay against a commercially available substrate consisting of a cephalosporin core linking 7-hydroxycoumarin to fluorescein (CCF2-FA). The enzyme (NDM-1, IMP-1 or VIM-1; for a review, see: Meine, M.-R.; Llarrull, L. I.; Vila, A. J. Antibiotics, 2014, 3, 285-316) and the substrate were diluted in 100 mM KH2PO4 buffer (pH 7) containing 0.005% Tween-20 and 10 μM ZnSO4. In the assay, the final concentration of enzyme was 1 pM, 2 pM and 30 pM for NDM-1, IMP-1 and VIM-1, respectively, and the final concentration of CCF2-FA was 1.25 μM. The test inhibitor was dissolved in dimethylsulfoxide and diluted 1:50 in the assay, resulting in a final concentration range of 20 μM to 0.00063 μM. In a 384-well microplate, the test inhibitor was incubated with the metallo-β-lactamase enzyme and the substrate for 2 hours at 25° C. Fluorescence at 460 nm following excitation at 405 nm was measured. The IC50 value was determined from semi-logarithmic plots of enzyme inhibition versus inhibitor concentration, with a curve generated using a 4-parameter fit.
Measurement: IC50 = 0.034 nM (pChEMBL 10.470000267028809)
Descriptors: MW 533.6, ALogP -1.39, HBA 11, HBD 7, PSA 253.54, QED 0.15
Compound (SMILES): N#Cc1cccc(-c2ccc3ncc(-c4cc(F)cc(F)c4)c(N4CC(C5COCCN5)C4)c3c2)c1O
InChIKey: OAFYIJMNLCKYFV-UHFFFAOYSA-N
Target: Somatostatin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P30874
Assay: cAMP Assay : Four days prior to the assay, 5,000 Chinese hamster ovary cells (CHO-K1, ATCC #CCL-61) stably expressing the human SSTR2 are plated in each well of a 96-well tissue culture-treated plate in Ham's F12 growth media (ThermoFisher #10-080-CM) supplemented with 10% donor bovine serum (Gemini Bio-Products #100-506), 100 U/mL penicillin; 100 ug/mL streptomycin; 2 mM L-glutamine (Gemini Bio-Products #400-110) and 0.2 mg/mL hygromycin B (GoldBio #31282-04-9). The cells are cultured at 37° C., 5% CO2 and 95% humidity. On the day of the assay, the media is aspirated and the cells are treated with 50 μL of 1.6 μM NKH477 (Sigma #N3290) plus various dilutions of compounds of the invention in assay buffer [1× Hank's Balanced Salt Solution (ThermoFisher #SH3058802), 0.5 mM HEPES pH 7.4, 0.1% bovine serum albumin, 0.2 mM 3-Isobutyl-1-methylxanthine (IBMX, VWR #200002-790)]. The cells are incubated for 20 minutes at 37° C. (the final concentration of the compounds of the invention are typically 0-10,000 nM). The cells are treated with 50 μL of lysis buffer (HRTF cAMP kit, Cisbio). The lysate is transferred to 384-well plates and cAMP detection and visualization antibodies are added and incubated for 1-24 hours at room temperature. The time-resolved fluorescent signal is read with a Tecan M1000Pro multiplate reader. The intracellular cAMP concentrations are calculated by regression to a standard curve and are plotted vs. the concentration of the compounds of the invention and the EC50 of the compounds are calculated using standard methods. All data manipulations are in GraphPad Prism v6.
Measurement: EC50 < 10 nM (pChEMBL 8)
Descriptors: MW 498.53, ALogP 4.85, HBA 6, HBD 2, PSA 81.41, QED 0.42
Compound (SMILES): O=C(O)[C@H]1c2cc(Br)ccc2C(=O)N(c2ccc3c(c2)CCC3)[C@@H]1c1ccc2c(c1)OCCO2
InChIKey: RIHATFNEIDOMAI-LOSJGSFVSA-N
Target: Stimulator of interferon genes protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q86WV6
Assay: H-cGAMP Filtration Binding Assay (HAQ STING): The ability of compounds to bind STING is quantified by their ability to compete with tritiated cGAMP ligand for human STING receptor membrane using a radioactive filter-binding assay. The binding assay employs STING receptor obtained from Trichoplusia ni (T. ni; Expression Systems, cat #94-002F, Expression Systems, Davis, Calif., USA) cell membranes overexpressing full-length HAQ STING prepared in-house and tritiated cGAMP ligand also purified in-house.
Measurement: IC50 = 10880 nM (pChEMBL 4.960000038146973)
Descriptors: MW 520.38, ALogP 5.28, HBA 4, HBD 1, PSA 76.07, QED 0.51
Compound (SMILES): CN1CCCN(Cc2ccc(C3COc4ccccc4O3)cc2)CC1
InChIKey: GIAPCXVSIVXPAC-UHFFFAOYSA-N
Target: Leukotriene A-4 hydrolase (SINGLE PROTEIN), Homo sapiens
UniProt: P09960
Assay: Enzyme Assay: The compounds of the invention are assessed for the ability to interact with human LTA4 hydrolase in an enzymatic assay that measures the ability of the enzyme to cleave the peptide bond of arginyl-aminomethylcoumarin (Arg-AMC). LTA4H Enzyme (1 nM final), Arg-AMC substrate (50 μM final), and compound are combined in a reaction buffer (50 mM Tris-HCl (pH 7.5), 100 mM KCl, 0.5% bovine serum albumin) at room temperature for 1 h. The formation of product is assessed by measuring the fluorescence of aminomethylcoumarin product (excitation wavelength 380 nm/emission wavelength 460 nm).
Measurement: IC50 = 1.77 nM (pChEMBL 8.75)
Descriptors: MW 338.45, ALogP 3.34, HBA 4, HBD 0, PSA 24.94, QED 0.86