lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1cc(NC(=S)NCCCn2cncc2C)cc(OC)c1
InChIKey: KZGHEEODIBACHZ-UHFFFAOYSA-N
Target: Glutaminyl-peptide cyclotransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q16769
Assay: Spectrophotometric Assay: This novel assay was used to determine the kinetic parameters for most of the QC substrates. QC activity was analyzed spectrophotometrically using a continuous method, that was derived by adapting a previous discontinuous assay (Bateman, R. C. J. 1989 J Neurosci Methods 30, 23-28) utilizing glutamate dehydrogenase as auxiliary enzyme. Samples consisted of the respective QC substrate, 0.3 mM NADH, 14 mM α-Ketoglutaric acid and 30 U/ml glutamate dehydrogenase in a final volume of 250 μl. Reactions were started by addition of QC and persued by monitoring of the decrease in absorbance at 340 nm for 8-15 min.The initial velocities were evaluated and the enzymatic activity was determined from a standard curve of ammonia under assay conditions. All samples were measured at 30° C., using either the SPECTRAFluor Plus or the Sunrise (both from TECAN) reader for microplates. Kinetic data was evaluated using GraFit software.
Measurement: Ki = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 334.45, ALogP 2.59, HBA 5, HBD 2, PSA 60.34, QED 0.6
Compound (SMILES): CCOc1ccc(C)c(Nc2nc(=O)c(F)cn2Cc2cc(F)c(F)c(F)c2)c1
InChIKey: VBPVQCUEKZKQLW-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Evaluation of a Human P2X7 Receptor Inhibitory Activity: Stably expressing cell line (1321N1 cell transfected with the human P2X7 receptor gene (GenBank accession number NM_002562.5 including T606C and G952A SNP)) was used. The cells were seeded in a 384-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (10% fetal bovine serum, 25 mM HEPES, 1% penicillin and streptomycin in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. After replacing with 20 μL of the HBSS buffer (20 mM HEPES, 55.6 mM D-glucose, 1×HBSS(−), pH7.4-7.5), 15 μL of 17.3 μM Yo-Pro solution in the HBSS buffer was added. The plate was placed in high-throughput cellular screening system FLIPR TETRA (Molecullar Devices, LLC.) and 15 μL of 130 μM BzATP solution in the HBSS buffer was added. Measurement of fluorescence intensity by FLIPR TETRA was started. After eight minutes, 15 μL of DMSO solutions containing different concentrations of the compound of the present invention as prepared by dilution with the HBSS buffer were dispensed to each well through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 20 minutes. The maximum fluorescence intensity without the compound of the present invention is calculated as 0% inhibition and the maximum fluorescence intensity when the reference compound was added is calculated as 100% inhibition. Changing values of fluorescence intensity by the compound of the present invention were calculated by difference between maximum and minimum fluorescence intensity for 20 minutes. IC50 was calculated using logistic approximation.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 407.37, ALogP 4.3, HBA 5, HBD 1, PSA 56.15, QED 0.49
Compound (SMILES): N#Cc1cccc(N2CC3(CC(c4nnc5n4-c4ccc(Cl)cc4CN(C4CC4)C5)C3)C2)n1
InChIKey: URNZSWQDKUKVIS-UHFFFAOYSA-N
Target: Vasopressin V1a receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P37288
Assay: Vasopressin V1A Receptor Antagonist Assay: The purpose of this assay was to determine the inhibitory effect of synthesized compounds on the Vasopressin V1a receptor. The assay as performed in Chinese Hamster Ovary (CHO) cells expressing the human Arginine Vasopressin Receptor 1a (AVPR1a). Arginine Vasopressin (AVP) evokes an increase in intracellular calcium in CHO-AVPR1a cells which is measured in a fluorescence assay on the FLIPRTETRA using calcium sensitive dyes. Test compounds were assessed for their ability to affect the magnitude of the response to AVP, with antagonists showing a concentration-dependent reduction in the AVP-mediated fluorescence. Compounds were tested in duplicate in a 10-point, 1:3 dilution series starting at a nominal concentration of 3 μM in the assay.CHO-AVPR1a cells were maintained in routine culture in T175 Flasks at 37° C., 5% CO2. The growth medium consists of Ham's F12 media supplemented with 10% v/v fetal bovine serum, 1× non-essential amino acids, and 0.4 mg/ml Geneticin G418.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 457.97, ALogP 4.05, HBA 7, HBD 0, PSA 73.87, QED 0.59
Compound (SMILES): Cc1nc(C(=O)Nc2cc(-c3cnc(N4CCOCC4)nc3)c(F)cc2N2CCN(C)[C@@H](C)C2)cs1
InChIKey: YXOSTINZUJCQDD-INIZCTEOSA-N
Target: WD repeat-containing protein 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P61964
Assay: Surface Plasmon Resonance (SPR) Assay: Exemplary compounds of the application were dissolved in 100% DMSO at 10 mM, assayed fresh, and then stored at −20° C. for repeat studies and other experiments. Full length WDR5 with an N-terminal His tag and C-terminal AviTag (Avidity Inc.) was expressed in E. coli with coexpression of BirA to biotin labelled protein in vivo. Purification of the protein was performed using Ni-NTA. The purified WDR5 protein has a molecular weight of 41976 Da.SPR studies were performed using a Biacore T200 instrument (GE Health Sciences Inc.). Biotinylated WDR5 protein (approximately 3000 RU) was stably captured to streptavidin coupled SA chips according to the manufacture's protocol (GE Health Sciences Inc.). The running buffer used was HBS-EP (20 mM Hepes pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P-20) plus 5% DMSO with a flow rate of 40 μl/min. For SPR analysis, 5 different concentrations of each exemplary compound of the application were sprayed into 96 or 384 well plates using an HP D300 digital dispenser. The concentration ranged from about 195 nM to about 12 nM in a two-fold series. Concentration ranges were adjusted higher or lower for weaker or more potent compounds respectively when necessary. For the KD determinations, single cycle kinetic analysis was performed with an on time of 60 seconds, and an off time of 300 or 600 seconds. Curve fitting and KD calculations were performed with the Biacore T200 Evaluation software (GE Health Sciences Inc).
Measurement: Kd = 113 nM (pChEMBL 6.949999809265137)
Descriptors: MW 511.63, ALogP 3.28, HBA 9, HBD 1, PSA 86.72, QED 0.56
Compound (SMILES): CNCC[C@H](Oc1ccc(CN2CCCn3nc(C)cc3C2=O)c(F)c1)c1ccccc1
InChIKey: TWOXHEPRFOVUEU-DEOSSOPVSA-N
Target: Voltage-dependent calcium channel subunit alpha-2/delta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P54289
Assay: Binding Assay : Human α2δ-1 enriched membranes (2.5 μg) were incubated with 15 nM of radiolabeled [3H]-Gabapentin in assay buffer containing Hepes-KOH 10 mM, pH 7.4.NSB (non specific binding) was measured by adding 10 μM pregabalin. The binding of the test compound was measured in five different concentrations. After 60 min incubation at 27° C., binding reaction was terminated by filtering through Multiscreen GF/C (Millipore) presoaked in 0.5% polyethyleneimine in Vacuum Manifold Station, followed by 3 washes with ice-cold filtration buffer containing 50 mM Tris-HCl, pH 7.4.Filter plates were dried at 60° C. for 1 hour and 30 μl of scintillation cocktail were added to each well before radioactivity reading.Readings were performed in a Trilux 1450 Microbeta radioactive counter (Perkin Elmer).
Measurement: Ki = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 436.53, ALogP 4.11, HBA 5, HBD 1, PSA 59.39, QED 0.58
Compound (SMILES): Cc1csc(-n2ncc3ccc(-c4cccc(C(C)N)n4)cc32)n1
InChIKey: XHDJATQVNIEAKB-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Biochemical Assay: The biochemical assay is in a AlphaScreen format. The kinase reaction is based on the IRAK-4 phosphorylation of a biotin labeled peptide. The phosphopeptide is incubated with anti-phosphothreonine antibody as well as streptavidin- and protein A-coated beads. Binding of the protein-A coated beads to the antibody and the streptavidin beads to the peptide, leads to an energy transfer from one bead to the other, ultimately producing a luminescent/fluorescent signal.Generally, the kinase reaction is carried out at 1 nM IRAK4, 1.6 μM peptide, 1 mM ATP in reaction buffer 50 mM Hepes, 60 mM NaCl, 5 mM MgCl2, 0.25 mM MnCl2, 2 mM DTT, 0.01% BSA, 0.01% Tween-20) for 3.5 h at RT.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 335.44, ALogP 3.87, HBA 6, HBD 1, PSA 69.62, QED 0.62
Compound (SMILES): N=C(N)NCC1CCC(c2ccc(C(F)(F)F)c(S(N)(=O)=O)c2-c2nnn[nH]2)CC1
InChIKey: CEVNTCRDWVUNQT-UHFFFAOYSA-N
Target: Metallo-beta-lactamase type 2 (SINGLE PROTEIN), Bacteria
UniProt: C7C422
Assay: Class B Enzyme Assay: 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) 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 uM to 0.00063 uM. 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 = 0.52 nM (pChEMBL 9.279999732971191)
Descriptors: MW 446.46, ALogP 1.29, HBA 6, HBD 5, PSA 176.52, QED 0.34
Compound (SMILES): CC(=O)OC[C@@H]1CCC[C@H]1C(C#N)(c1ccccc1)C1CCN(CC2CN(c3ccc(S(=O)(=O)C4CC4)cc3)C2)CC1
InChIKey: MPKSENOWQPDREO-KMSQYKAGSA-N
Target: Menin (SINGLE PROTEIN), Homo sapiens
UniProt: O00255
Assay: Fluorescence Polarization (FP) Competitive Binding Assay: A fluorescence polarization (FP) competitive binding assay was used to determine the binding affinities of representative menin inhibitors. A FAM labeled fluorescent probe was designed and synthesized based on a MLL1 peptide (FAM-MM2). Equilibrium dissociation constant (Kd) value of FAM-MM2 to menin protein was determined from protein saturation experiments by monitoring the total fluorescence polarization of mixtures composed with the fluorescent probe at a fixed concentration and the protein with increasing concentrations up to full saturation. Serial dilutions of the protein were mixed with FAM-MM2 to a final volume of 200 μl in the assay buffer (PBS with 0.02% Bovine γ-Globulin and 4% DMSO. 0.01% Triton X-100 was added right before assays). Final FAM-MM2 concentration was 2 nM. Plates were incubated at room temperature for 30 minutes with gentle shaking to assure equilibrium. FP values in millipolarization units (mP) were measured using the Infinite M-1000 plate reader (Tecan U.S., Research Triangle Park, N.C.) in Microfluor 1 96-well, black, v-bottom plates (Thermo Scientific, Waltham, Mash.) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Kd value of FAM-MM2, which was calculated by fitting the sigmoidal dose-dependent FP increases as a function of protein concentrations using Graphpad Prism 6.0 software (Graphpad Software, San Diego, Calif.), is determined as 1.4 nM.
Measurement: IC50 = 6900 nM (pChEMBL 5.159999847412109)
Descriptors: MW 589.8, ALogP 5.21, HBA 7, HBD 0, PSA 90.71, QED 0.35
Compound (SMILES): Cc1n[nH]c2ncnc(-c3ccc(NS(=O)(=O)c4ccc(Cl)cc4)cc3)c12
InChIKey: AWXNUNSIUAEQEB-UHFFFAOYSA-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 399.86, ALogP 3.78, HBA 5, HBD 2, PSA 100.63, QED 0.54
Compound (SMILES): CC(c1ccc2c(c1)OCC2)N1CCC(NC(=O)Cc2ccc(C(F)(F)F)cc2)CC1
InChIKey: DXZGYHGEPZHMRB-UHFFFAOYSA-N
Target: Protein O-GlcNAcase (SINGLE PROTEIN), Homo sapiens
UniProt: O60502
Assay: Human O-GlcNAcase Enzyme Inhibition Assay: 5 μl of the appropriate concentration of a solution of inhibitor in McIlvaine's Buffer (pH 6.5) in 2% DMSO (for a dose response curve calculation) is added into each well of a 384-well plate (Greiner, 781900). Then, 20 nM of His-Tagged hOGA and 10 μM of FL-GlcNAc (Fluorescein mono-beta-D-(2-deoxy-2-N-acetyl) glucopyranoside; Marker Gene Technologies Inc, M1485) were added to the 384-well plate for a final volume of 20 μl. After incubation for 60 min at room temperature, the reaction was terminated by the addition of 10 μL of stop buffer (200 mM glycine, pH 10.75). The level of fluorescence (λexc 485 nm; (λemm 520 nm) was read on a PHERAstar machine. The amount of fluorescence measured was plotted against the concentration of inhibitor to produce a sigmoidal dose response curve to calculate an IC50. All individual data was corrected by subtraction of the background (Thiamet 3 uM=100% inhibition) whilst 0.5% DMSO was considered as the control value (no inhibition).
Measurement: IC50 = 125 nM (pChEMBL 6.900000095367432)
Descriptors: MW 432.49, ALogP 4.52, HBA 3, HBD 1, PSA 41.57, QED 0.76
Compound (SMILES): CCOC(=O)c1ccc2c(n1)c(-c1cccc(S(=O)(=O)N(C)C)c1)c(C)n2C/C(F)=C/CN
InChIKey: GPMKXMJMTMCLLA-BOPFTXTBSA-N
Target: Lysyl oxidase homolog 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y4K0
Assay: Inhibition Assay: LOX and LOXL2: Lysyl oxidase (LOX) is an extracellular copper dependent enzyme which oxidizes peptidyl lysine and hydroxylysine residues in collagen and lysine residues in elastin to produce peptidyl alpha-aminoadipic-delta-semialdehydes. This catalytic reaction can be irreversibly inhibited by β-aminopropionitrile (BAPN) that binds to the active site of LOX (Tang S. S., Trackman P. C. and Kagan H. M., Reaction of aortic lysyl oxidase with beta-aminoproprionitrile. J Biol Chem 1983; 258: 4331-4338). There are five LOX family members; these are LOX, LOXL1, LOXL2, LOXL3 and LOXL4. LOX and LOXL family members can be acquired as recombinant active proteins from commercial sources, or extracted from animal tissues like bovine aorta, tendons, pig skin; or prepared from cell cultures. The inhibitory effects of the compounds of the present invention were tested against the given LOX-LOXL preparation using a high-throughput coupled colorimetric method (Holt A. and Palcic M., A peroxidase-coupled continuous absorbance plate-reader assay for flavin monoamine oxidases, copper-containing amine oxidases and related enzymes. Nat. Protoc. 2006; 1: 2498-2505). The assay was developed using either 384 or 96 well format. Briefly, in a standard 384 well plate assay 25 μL of a dilution of any of the isoenzymes and orthologues in 1.2 M urea, 50 mM sodium borate buffer (pH 8.2) were added into each well in the presence of 1 μM mofegiline and 0.5 mM pargyline (to inhibit SSAO and MAO-B and MAO-A, respectively). Test compounds were dissolved in DMSO and tested in a Concentration Response Curve (CRC) with 11 data points, typically in the micromolar or nanomolar range after incubation with the enzyme for 30 min at 37° C. Twenty five L of a reaction mixture containing twice the KM concentration of putrescine (Sigma Aldrich, e.g. 20 mM for LOX, or 10 mM for LOXL2 and LOXL3), 120 μM Amplex Red (Sigma Aldrich) and 1.5 U/mL horseradish peroxidase (Sigma Aldrich) prepared in 1.2 M urea, 50 mM sodium borate buffer (pH 8.2) were then added to the corresponding wells. The above volumes were doubled in the case of 96 wells plate.
Measurement: IC50 < 300 nM (pChEMBL 6.5229997634887695)
Descriptors: MW 474.56, ALogP 3.25, HBA 7, HBD 1, PSA 107.52, QED 0.5
Compound (SMILES): CC(=O)N1CCc2c(c(-c3cccc4cc(N5CCC[C@H]5C)ncc34)nn2C2CCOCC2)C1
InChIKey: FWVXFXNRDFOXFV-GOSISDBHSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: TR-FRET Binding Assay: His/Flag epitope tagged CBP was cloned, expressed, and purified to homogeneity. CBP binding and inhibition was assessed by monitoring the engagement of a biotinylated small molecule compound with the target using the TR-FRET assay technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate CBP (4 nM final) was combined with biotin-ligand (60 nM final) in 50 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.2% DMSO) or compound dilution series in DMSO. After 10 minutes incubation at room temperature, a mixture Eu-W1024 Anti-6xHis antibody (6xHis is disclosed as SEQ ID NO: 3) (Perkin Elmer ADO 110) and SureLight Allophycocyanin-Streptavidin (APC-SA, Perkin Elmer CR130-100) were added to a final concentrations of 0.2 nMolar antibody and 50 nMolar APC-SA, respectively. After twenty minutes of equilibration, the plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 2.99 nM (pChEMBL 8.520000457763672)
Descriptors: MW 459.59, ALogP 4.34, HBA 6, HBD 0, PSA 63.49, QED 0.58
Compound (SMILES): O=C(NCc1ccc(S(=O)(=O)N2C[C@@H]3C[C@H]2CO3)cc1)c1ccc2nccn2c1
InChIKey: IWWQGZDFHQYGHV-IRXDYDNUSA-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 = 37.4 nM (pChEMBL 7.429999828338623)
Descriptors: MW 412.47, ALogP 1.43, HBA 6, HBD 1, PSA 93.01, QED 0.68
Compound (SMILES): C[C@@H]1OCC2(CCN(c3nc(N)c(-c4ccccc4F)c(=O)n3C)CC2)[C@@H]1N
InChIKey: ZLPWXAWWEPLGCE-BLLLJJGKSA-N
Target: Tyrosine-protein phosphatase non-receptor type 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06124
Assay: SHP2 Allosteric Inhibition Assay: SHP2 is allosterically activated through binding of bis-tyrosyl-phosphorylated peptides to its Src Homology 2 (SH2) domains. The latter activation step leads to the release of the auto-inhibitory interface of SHP2, which in turn renders the SHP2 protein tyrosine phosphatase (PTP) active and available for substrate recognition and reaction catalysis. The catalytic activity of SHP2 was monitored using the surrogate substrate DiFMUP in a prompt fluorescence assay format.More specifically, the phosphatase reactions were performed at room temperature in 384-well black polystyrene plate, flat bottom, low flange, non-binding surface (Corning, Cat#3575) using a final reaction volume of 25 μL and the following assay buffer conditions: 60 mM HEPES, pH 7.2, 75 mM NaCl, 75 mM KCl, 1 mM EDTA, 0.05% P-20, 5 mM DTT.The inhibition of SHP2 by compounds of the invention (concentrations varying from 0.003-100 μM) was monitored using an assay in which 0.5 nM of SHP2 was incubated with of 0.5 μM of peptide IRS1_pY1172(dPEG8)pY1222 (sequence: H2N-LN(pY)IDLDLV(dPEG8)LST(pY)ASINFQK-amide) (SEQ ID NO:1). After 30-60 minutes incubation at 25° C., the surrogate substrate DiFMUP (Invitrogen, cat# D6567) was added to the reaction and incubated at 25° C. for 30 minutes. The reaction was then carefully diluted by the addition of 5 μL of a 160 μM solution of bpV(Phen) (Enzo Life Sciences cat# ALX-270-204). The fluorescence signal was monitored using a microplate reader (Envision, Perki-Elmer) using excitation and emission wavelengths of 340 nm and 450 nm, respectively. The inhibitor dose response curves were analyzed using normalized IC50 regression curve fitting with control based normalization.
Measurement: IC50 = 46 nM (pChEMBL 7.340000152587891)
Descriptors: MW 387.46, ALogP 1.5, HBA 7, HBD 2, PSA 99.4, QED 0.81
Compound (SMILES): COc1ccc(-c2cc(NCCCN3CCCCC3)c3cc(Cl)ccc3n2)cc1
InChIKey: XPPLTLMIURGMAM-UHFFFAOYSA-N
Target: Low molecular weight phosphotyrosine protein phosphatase (SINGLE PROTEIN), Homo sapiens
UniProt: P24666
Assay: LMPTP Primary Screening Protocol: A listing of materials is provided:    Item, source, catalog no.    LMPTP-A Enzyme Stock Solution (4.22 mg/ml or 206.8 μM), SBMRI Protein Facility,    N/A    OMFP, Sigma, M2629-100 MG    Bis-Tris pH 6.0, Fisher Sci, BP301-100    Triton-X 100, Sigma, T9284    DTT, Sigma, D9779    Mol. Grade Water, Mediatech, Inc., 46-000-CM    1536-well black High base opaque bottom plate, Nexus Biosystems, 00019120Final assay conditions are:    0.625 nM LMPTP-A Enzyme    40 μM OMFP    50 mM Bis-Tris pH 6.0    1 mM DTT    0.01% Triton-X 100    20 μM test compound    3% DMSO (2% from substrate and 1% from compounds)    6 μL reaction volume    50 minutes incubation at room tempAssay Procedure    1. Prepare Reagents as described in section F. Recipe.    2. Using LabCyte Echo, transfer 60 nL from 2 mM test compound source plate into assay plate Col. 5-48 (final concentration of test compounds is 20 μM). 60 nL of DMSO should be transferred to col. 1-4 for positive and negative control wells.    3. Spin plates at 1000 rpm for 1 minute in centrifuge.    4. Using the Beckman Coulter Bioraptr, add 3 μL/well of control buffer to columns 1 and 2.    5. Using the Bioraptr, add 3 μL/well of enzyme solution to col. 3-48.    6. Using the Bioraptr, add 3 μL/well of substrate solution to col. 1-48.    7. Spin plates at 1000 rpm for 1 minute in centrifuge.    8. Incubate plates in the dark at room temperature for 50 minutes.    9. Read plates on PerkinElmer Viewlux using a FI protocol.
Measurement: IC50 > 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 409.96, ALogP 5.85, HBA 4, HBD 1, PSA 37.39, QED 0.5
Compound (SMILES): Cc1ccc(S(=O)(=O)N[C@H]2CC[C@H](O)CC2)cc1-c1cnc2c(N)nc(-c3ccncc3)cn12
InChIKey: VSWURRZGQXHZQO-IYARVYRRSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Scintillation Proximity Assay: PI3K-γ:[γ-33P]ATP (10 mCi/mL) and Wheat Germ Agglutinin (WGA) YSi SPA Scintillation Beads was purchased from Perkin-Elmer (Waltham, Mass.). Lipid kinase substrate, D-myo-Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)D (+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho linked (PIP2), CAS 204858-53-7, was purchased from Echelon Biosciences (Salt Lake City, Utah). PI3Kγ (p110γ) Recombinant Human Protein was purchased from Life technology (Grand Island, N.Y.). ATP, MgCl2, DTT, EDTA, MOPS and CHAPS were purchased from Sigma-Aldrich (St. Louis, Mo.).The kinase reaction was conducted in polystyrene 384-well Greiner Bio-one white plate from Thermo Fisher Scientific in a final volume of 25 μL. Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 2%. The PI3Kγ assay was carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 μM PIP2, 2 μM ATP, 0.5 μCi [γ-33P] ATP, 13 nM PI3Kγ. Reactions were incubated for 120 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 163 mM potassium phosphate pH 7.8, 20% glycerol, 25 mM EDTA. The final concentration of SPA beads is 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1500 rpm for 10 min, the radioactivity of the product was determined by scintillation counting on Topcount (Perkin-Elmer).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 478.58, ALogP 2.93, HBA 8, HBD 3, PSA 135.5, QED 0.4
Compound (SMILES): O=C(Nc1ccc(F)cc1)N1C[C@H]2C[C@@H](c3ccccc3)C[C@H]2C1
InChIKey: LMMBDHPRCGAAOQ-OSYLJGHBSA-N
Target: Glutamate receptor ionotropic, NMDA 2B (SINGLE PROTEIN), Homo sapiens
UniProt: Q13224
Assay: FDSS Assay: To each well of the plate, 10 μL test compound, control (MK801) or HHnoCa buffer was added to a final concentration of 10 μM with a final concentration of DMSO of 0.1%. Following 10 minutes pre-incubation in the dark, plates are loaded onto the Hamamatsu FDSS 6000. After collecting baseline fluorescence images, 3 μM glutamate, 3 glycine, and 1 mM Ca2+ in HHnoCa buffer is added to each well, and Ca2+ is recorded for 3 minutes. Data were processed by computing ratio of fluorescence at the end of data collection to baseline fluorescence to assess degree of Ca′ influx inhibition relative to that observed in MK801.
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 324.4, ALogP 4.48, HBA 1, HBD 1, PSA 32.34, QED 0.87
Compound (SMILES): Cc1ccc(F)c([C@@H](C)[C@H](NS(=O)(=O)c2cc([N+](=O)[O-])c(Cl)cc2C(N)=O)c2n[nH]c(=O)o2)c1C
InChIKey: PWUAALLQLKFFBL-QGHHPUGFSA-N
Target: Ribonucleotide reductase (PROTEIN COMPLEX), Homo sapiens
UniProt: P23921
Assay: Human RNR Inhibition Effect: The inhibitory activity against the ribonucleotide reduction reaction (hereinafter referred to as RNR reaction) of the test compound was determined by measuring the formation of deoxycytidine diphosphate (hereinafter referred to as dCDP) from cytidine diphosphate (hereinafter referred to as CDP) by the following method.Human M1 subunit and human M2 subunit (mutant lacking amino terminal 59 amino acids), which are fused a histidine tag at the amino terminus, are overexpressed in Escherichia coli and are solubilized after collection, and histidine tagged human M1 and M2 proteins were purified on a nickel chelate column.For measuring the inhibitory activity of the test compound against the RNR reaction, the method described in the document [CANCER RESEARCH 64, 1-6, 2004] was referred to.
Measurement: IC50 = 130 nM (pChEMBL 6.889999866485596)
Descriptors: MW 527.92, ALogP 2.6, HBA 8, HBD 3, PSA 191.29, QED 0.29
Compound (SMILES): N#CC[C@@H](C1CC1)n1nc(Nc2ccc(S(=O)(=O)N3CCOC4CCC[C@H]43)cc2)c2c(=O)[nH]ccc21
InChIKey: WSKPORIUCCXCOR-RJKSWSIASA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Jak Biochemical HTRF Assay Protocol: The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer #PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at room temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading. After normalization to untreated controls, the percent inhibition of the HTRF signal at each compound concentration was calculated.
Measurement: IC50 = 620 nM (pChEMBL 6.210000038146973)
Descriptors: MW 508.6, ALogP 3.27, HBA 8, HBD 2, PSA 133.11, QED 0.5
Compound (SMILES): CC(C)(C)C#C[C@]1(O)CC[C@H]2[C@@H]3CCC4=CC(=O)CCC4=C3[C@@H](c3ccc(C(C)(CO)CO)cc3)C[C@@]21C
InChIKey: HNELNYDQAFJHIR-WMQBTLKUSA-N
Target: Glucocorticoid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04150
Assay: In Vitro GR Luciferase Reporter Assay: Cell Line: CHO-K1-GR-MMTV-Luc reporter cellsCulture Media: DMEM (with phenol red)+10% FBSAssay Media: DMEM (without phenol red)+10% CSSCulture CHO-K1-GR-MMTV-Luc reporter cells in 15 cm plates in Culture Media at conditions less than 90% confluence.Prepare 200×DMSO 1:5 serial dilutions of control and test compounds in 96-well non-sterile V bottom plate in DMSO, 8 serial dilutions for each compound.Prepare 5× Assay Media diluted compound serial dilutions in 96-well non-sterile V bottom plate: Add 97.5 uL/well of Assay Media into 96-well then add 2.5 ul of 200× concentration of compounds and mix well.Seed cells for Antagonist Assay: 1.5×106 CHO-K1-GR-MMTV-Luc reporter cells were seeded in a Corning 3707 flat clear bottom 384-well white TC plate in 20 ul of Assay Media containing 12.5 nM Dexamethasone (final concentration=10 nM).Add compounds: 5 ul of assay media diluted compounds were added to appropriate wells and followed a quick spin (1000 rpm, 10 sec) to bring media and cells to the bottom of plate. The plates were covered with SealMate film to avoid evaporation and placed in 37° C. incubator for approximately 18-24 hours.Read plates: Equilibrate appropriate amount of Promega OneGlo luciferase reagent to room temperature. Remove the plates from incubator and add 25 uL of OneGlo reagent/well by multiple channel pipette and read the plates with Tecan F500 luminometer within 3 minutes.The ability of the compounds disclosed herein to inhibit GR activity was quantified and the respective IC50 value was determined.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 516.72, ALogP 5.61, HBA 4, HBD 3, PSA 77.76, QED 0.45
Compound (SMILES): CN1C(=O)[C@@H](NC(=O)c2cc(Oc3ccccc3)ccn2)COc2ccc(C#CCO)cc21
InChIKey: NYMYXIUPODJJLO-NRFANRHFSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: biochemical assay using the ADP-Glo technology: ADP-Glo (Promega, Madison, Wis., USA) reagents were thawed at ambient temperature. Kinase Detection Reagent was prepared by mixing kinase detection buffer with the lyophilized kinase detection substrate.A 500 ml stock volume of 5× Reaction Kinase Buffer was made by mixing 1000 μl of 1M MgCl2, 500 μl of 1M Tris-HCL pH7.4, 0.5 mg/ml (25 mg) of BSA, and 3475 μl of distilled H2O. A 3 ml 2× working stock volume of Reaction Kinase Buffer was made containing a final concentration of 100 μM DTT and 4 mM MnCl2.Components of RIPK1 enzyme (Rigel Pharmaceuticals, South San Francisco, Calif., USA) were thawed on ice. Diluted RIPK1 was prepared in 1× Kinase Reaction Buffer (diluted from 2× buffer) to 31 ng/well. A 166 μM working stock ATP assay solution was prepared in 1× Kinase Reaction Buffer (diluted from 2× buffer).Compounds were serially diluted in DMSO from 250 uM in 4-fold dilutions then diluted 1:5 in 2× Reaction Buffer in a 96 well plate. 1.0 μl of diluted compound was added to a 384 well plate in duplicate. 2 μl of diluted Active RIPK1 was added to 384 well plate (do not add to column 1) add 2× rxn buffer to column 1. AKT (Anaspec, Fremont, Calif., USA) at 150 nM was combined with ATP working stock at equal volume and 2 ul/well were added to the 384 well plate. The final reaction volume was 5.0ul.The plate was quickly centrifuged and the reaction was incubated at 30° C. for 30 minutes. Adding 5 μl of ADP-Glo terminated the reaction. The plate was quickly centrifuged and the reaction was incubated at room temperature for 40 minutes. Kinase Detection Reagent was then added and incubated at room temperature for 30 minutes. The relative light unit (RLU) of kinase reaction was determined by luminescent (Luminescence 0.1 s) using a Wallac Victor2 Luminometer (PerkinElmer, Waltham, Mass., USA).
Measurement: IC50 = 27.5 nM (pChEMBL 7.559999942779541)
Descriptors: MW 443.46, ALogP 2.37, HBA 6, HBD 2, PSA 100.99, QED 0.6
Compound (SMILES): CNC(=O)c1cn(CC(F)CCc2nnc(NC(=O)Cc3ncc(Cl)cc3F)s2)nn1
InChIKey: KYULIKJFQGNHPS-UHFFFAOYSA-N
Target: Glutaminase kidney isoform, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: O94925
Assay: Enzymatic Activity Assay: The inhibition of purified recombinant human GAC by varying concentrations of inhibitors is assessed via a dual-coupled enzymatic assay. The glutamate produced by the glutaminase reaction is used by glutamate oxidase to produce α-ketoglutarate, ammonia, and hydrogen peroxide, with this hydrogen peroxide subsequently being used by horseradish peroxidase to produce resorufin in the presence of Amplex UltraRed. The assay buffer consisted of 50 mM Hepes (pH 7.4), 0.25 mM EDTA and 0.1 mM Triton X-100. GAC was incubated with potassium phosphate (10 minutes at room temperature) prior to incubation with inhibitor (10 minutes at room temperature). The final reaction conditions were as follows: 2 nM GAC, 50 mM potassium phosphate, 100 mU/mL glutamate oxidase (Sigma), 1 mM glutamine (Sigma), 100 mU/mL horseradish peroxidase (Sigma), 75 μM Amplex UltraRed (Life Technologies), and 1% (v/v) DMSO. The production of resorufin was monitored on a Perkin Elmer Envision plate reader (excitation 530 nm, emission 590 nm) either in a kinetics or endpoint mode (at 20 minutes). IC50 values were calculated using a four-parameter logistic curve fit.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 470.89, ALogP 1.83, HBA 9, HBD 2, PSA 127.58, QED 0.49
Compound (SMILES): Cc1cccc(NC(=O)Nc2ccc(-c3nn(CCCCCCCC(=O)NO)c4ncnc(N)c34)cc2)c1
InChIKey: PVZJLPGZMRIHCQ-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Enzyme-Linked Immunosorbent Assay (ELISA): Inhibition of Compound IIa, IIb, IIc and IId on the Activity of Catalyzing Substrate Phosphorylation of KDR (VEGFR2) Protein Tyrosine Kinase at the Molecular Level.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 516.61, ALogP 4.87, HBA 8, HBD 5, PSA 160.08, QED 0.11
Compound (SMILES): Cc1cc(S(=O)(=O)Nc2ccc(F)cn2)c(F)cc1N(C)[C@H]1CCN(Cc2ccccc2)C1
InChIKey: VZCNXUWHCLMKQD-FQEVSTJZSA-N
Target: Sodium channel protein type 8 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UQD0
Assay: Sodium Influx Assay (In Vitro Assay): This sodium influx assay employs the use of the cell permeable, sodium sensitive dye ANG2 to quantify sodium ion influx through sodium channels which are maintained in an open state by use of sodium channel modulators. This high throughput sodium influx assay allows for rapid profiling and characterization of sodium channel blockers.In general, Trex HEK293 cells were stably transfected with an inducible expression vector containing the full-length cDNA coding for the desired human sodium channel α-subunit and with an expression vector containing full length cDNA coding for the β1-subunit. Sodium channel expressing cell lines were induced with tetracycline (1 μg/mL) and plated on 384-well PDL-coated plates at a density of 25K-30K cells/well in culture media (DMEM, containing 10% FBS and 1% L-glutamine). After overnight incubation (37° C., 5% CO2), culture media was removed and cells were loaded with 5 uM ANG2 dye for 1-1.5 h in Buffer 1 (155 mM NMDG, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, adjusted with Tris to pH 7.4). Access dye was removed and cells were incubated with test compounds for 1 hr in buffer 1 containing sodium channel modulator(s) at room temperature. Hamamatsu FDSS ρCell was used to perform a 1:1 addition of Na/K challenge buffer (140 mM NaCl, 20 mM HEPES, 1 mM CaCl2, 15 mM KCl, 1 mM MgCl2, 10 mM glucose, adjusted with Tris to pH 7.4) and simultaneously read plates at excitation wavelength of 530 nm and emission wavelength set at 558 nm. Percent inhibition of sodium ion influx was calculated for each test compound at each test concentration to determine the IC50 values.
Measurement: IC50 = 4564 nM (pChEMBL 5.340000152587891)
Descriptors: MW 472.56, ALogP 4.18, HBA 5, HBD 1, PSA 65.54, QED 0.56
Compound (SMILES): Cc1nc([C@@]2(N)CCCN(c3cnc(-c4ccc(F)cc4)cc3Cn3cnc4c(N)ncnc43)C2)ccc1F
InChIKey: JGIQVKOGKUGKGV-MUUNZHRXSA-N
Target: Histone-lysine N-methyltransferase NSD2 (SINGLE PROTEIN), Homo sapiens
UniProt: O96028
Assay: LC-MS/MS-Based NSD2 Enzymatic Assay: This assay employed LC-MS/MS technology to monitor the SAH production from the NSD2 enzymatic reaction. The enzymatic reaction was performed in white proxiplate plus 384-well microplate (Perkin Elmer). The reaction mixture (10 μL) were composed of 8 nM NSD2 [1-1365], 1 M SAM (USB, 10601) and 400 nM nucleosome (purified from mouse liver) in reaction buffer (20 mM Tris-HCl, pH at 8.0, 0.01% Tween 20, 10 mM MgCl2, 50 mM NaCl, 1 mM DTT, 1 mM TCEP (pH7.5)). After 90 min incubation at room temperature, 3 μL quenching solution containing 2.5% TFA with 320 nM d4-SAH was added to stop the reaction.For the inhibition assay, compound solutions were transferred into the wells by Mosquito (TTP LabTech). The inhibition assays were carried out by preincubating various concentrations of the inhibitor with 5 uL reaction mixture containing 16 nM NSD2 [1-1365] and 2 uM SAM (USB, 10601) in reaction buffer. After 20 min preincubation, 5 uL solution containing 800 nM nucleosome (purified from mouse liver) in reaction buffer was added to initiate the reaction. The reaction was stopped by 3 μL quenching solution containing 2.5% TFA with 320 nM d4-SAH after 90 min.The SAH production from the enzymatic assays were monitored by LC-MS/MS on an API 4000 triple quadrupole mass spec with Turbolon Spray (Applied Biosystem) coupled with Prominenece UFLC(Shimazu). Liquid chromatography was performed on a Chromolith FastGradient HPLC column (RP-18e, 25-2 mm, from Merck) at a flow rate of 0.8 ml/min. The column was connected directly to the turbo ion electrospray operating in the positive-ion mode. Mobile phase A is 0.1% FA and 2% methanol in water and mobile phase B is 0.1% FA in ACN. Injection volume was 3 μl and the autosampler was kept at 4 degree. SAH and d4-SAH were simultaneously monitored. Data were acquired and processed by Analyst software.To quantify the formed SAH, d4-SAH was added as internal standard (IS). A series of SAH (Sigma, A9384) solution at varying concentration (1-500 nM in reaction buffer) was mixed with quenching solution mentioned above. Then LC-MS/MS was carried out on API 4000 LC/MS/MS system to detect both SAH and d4-SAH. The plot of SAH peak area/IS peak area vs SAH concentration was used to generate the normalization factor of SAH. The SAH production from real enzymatic reaction was derived from the standard curve of SAH. The down-limit of our system for the detection of SAH is around 1-2 nM, and the linear range can reach up to 500 nM.
Measurement: IC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 527.58, ALogP 3.95, HBA 9, HBD 2, PSA 124.66, QED 0.35
Compound (SMILES): CO[C@H]1CC[C@H](C(=O)N2Cc3cccnc3Nc3ccc(N4CCn5nncc5C4)cc32)CC1
InChIKey: ZOUZQCIXJJIUCY-CYWCHRQTSA-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 = 689 nM (pChEMBL 6.159999847412109)
Descriptors: MW 459.55, ALogP 3.49, HBA 8, HBD 1, PSA 88.41, QED 0.64
Compound (SMILES): COc1ncnc(OC)c1/N=C(/NNC(=O)c1ccc(C)o1)NS(=O)(=O)[C@@H](C)[C@H](C)c1ncc(Cl)cn1
InChIKey: UBERSJYMQFQBGW-STQMWFEESA-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 = 0.06 nM (pChEMBL 10.220000267028809)
Descriptors: MW 553, ALogP 1.87, HBA 11, HBD 3, PSA 182.82, QED 0.21
Compound (SMILES): COc1cccc(F)c1-c1nccc(C(=O)Nc2cc(F)c(Br)cc2N2C[C@@H]3C[C@H]2CN3)n1
InChIKey: PCYDUWDMJRORMQ-STQMWFEESA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: HPK1 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 516.35, ALogP 4, HBA 6, HBD 2, PSA 79.38, QED 0.53
Compound (SMILES): CCN1CCc2cc(-c3cc(C(=O)N(C)c4ccc(O)cc4)c4n3CCCC4)c(C(=O)N3Cc4ccccc4C[C@H]3C)cc2C1
InChIKey: MFRUQWVRZLIMDY-RUZDIDTESA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Fluorescence Polarisation Assay: The fluorescence polarisation tests were carried out on microplates (384 wells). The Bcl-2 protein, labelled (histag-Bcl-2 such that Bcl-2 corresponds to the UniProtKB® primary accession number: P10415), at a final concentration of 2.50×10−8 M, is mixed with a fluorescent peptide (Fluorescein-REIGAQLRRMADDLNAQY), at a final concentration of 1.00×10−8 M in a buffer solution (Hepes 10 mM, NaCl 150 mM, Tween20 0.05%, pH 7.4), in the presence or in the absence of increasing concentrations of test compounds. After incubation for 2 hours, the fluorescence polarisation is measured.
Measurement: IC50 = 65 nM (pChEMBL 7.190000057220459)
Descriptors: MW 602.78, ALogP 6.44, HBA 5, HBD 1, PSA 69.02, QED 0.29
Compound (SMILES): CS[C@H]1O[C@@H](c2ccc(Cl)c(Cc3ccc(OCCCNC(=O)C(C)(C)CO)cc3)c2)[C@H](O)[C@@H](O)[C@@H]1O
InChIKey: AVFNRLDOQNSMBQ-WJGLBBAVSA-N
Target: Sodium/glucose cotransporter 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P13866
Assay: In Vitro Human SGLT1 Inhibition Assay: Human sodium/glucose co-transporter type 1 (SGLT1; accession number NP_000334; GI: 4507031) was cloned into pIRESpuro2 vector for mammalian expression (construct: HA-SGLT1-pIRESpuro2). HEK293 cells were transfected with the human HA-SGLT1-pIRESpuro2 vector and the bulk stable cell line was selected in presence of 0.5 μg/mL of puromycin. Human HA-SGLT1 cells were maintained in DMEM media containing 10% FBS, 1% GPS and 0.5 μg/mL of puromycin. The HEK293 cells expressing the human HA-SGLT1 were seeded in 384 well plates (30,000 cells/well) in DMEM media containing 10% FBS, 1% GPS and 0.5 μg/mL of puromycin, then incubated overnight at 37 C, 5% CO2. Cells were then washed with uptake buffer (140 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 5 mM Tris, 1 mg/mL bovine serum albumin (BSA), pH 7.3). Twenty microliters of uptake buffer with or without testing compounds were added to the cells. Then, 20 microliters of uptake buffer containing 14C-AMG (100 nCi) were also added to cells. The cell plates were incubated at 37° C., 5% CO2 for 1-2 hours. After washing the cells with uptake buffer, scintillation fluid was added (40 microliters/well) and 14C-AMG uptake was measured by counting radioactivity using a scintillation coulter (TopCoulter NXT; Packard Instruments).
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 554.11, ALogP 2.68, HBA 8, HBD 5, PSA 128.48, QED 0.27