lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1cc(C)c(-n2ccn3nc(-c4cccnc4)cc23)cc1NC(=O)c1cccc(OC(F)F)c1
InChIKey: HUHDWROALSMQIL-UHFFFAOYSA-N
Target: Angiopoietin-1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q02763
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: For the assay, 50 nl of a 100-fold concentrated solution of the respective test substance in DMSO were pipetted into a black low-volume 384-well microtitre plate (Greiner Bio-One, Frickenhausen, Germany) 2 μl of a solution of the Tie2 fusion protein in assay buffer [50 mM HEPES/HCl pH 7, mM MgCl2, 2.5 mM MnCl2, 1 mM dithiothreitol, 0.01% (v/v) Nonidet P-40, 0.1% (w/v) bovine serum albumin (BSA), 1× Complete EDTA-free protease inhibitor mixture (Roche)] were added, and the mixture was incubated for 15 min to allow pre-binding of the substance to the enzyme prior to the kinase reaction. The kinase reaction was then started by adding 3 μl of a solution of adenosine triphosphate (ATP, 1.67 mM→final concentration in 5 μl assay volume=1 mM) and substrate (1.67 μM→final concentration in 5 μl assay volume=1 μM) in assay buffer, and the resulting mixture was incubated for a reaction time of 60 min at 22° C. The concentration of the Tie2 fusion protein was adapted to the respective activity of the enzyme and adjusted such that the assay was carried out in the linear range. Typical concentrations were in the range of 30 ng/ml. The reaction was stopped by addition of 5 μl of a solution of TR-FRET detection reagents [200 nM streptavidin-XL665 and 2 nM PT66-Eu chelate, a europium chelate-labelled anti-phosphotyrosine antibody (Perkin-Elmer); alternatively, it is also possible to use PT66-Tb cryptate (Cisbio Bioassays, Codolet, France)] in aqueous EDTA solution [90 mM EDTA, 0.28% (w/v) bovine serum albumin (BSA) in 50 mM HEPES pH 7.5]. The resulting mixture was incubated at 22° C. for 1 h to allow formation of the complex of the biotinylated phosphorylated substrate and the detection reagents. The amount of phosphorylated substrate was then determined by measuring the resonance energy transfer from PT66-Eu chelate to streptavidin-XL665. To this end, the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm were measured in a TR-FRET measuring instrument (for example Viewlux, Perkin-Elmer).
Measurement: IC50 = 43 nM (pChEMBL 7.369999885559082)
Descriptors: MW 473.48, ALogP 5.66, HBA 6, HBD 1, PSA 73.45, QED 0.35
Compound (SMILES): COC1CCC2(CC1)Oc1ccc(Br)cc1C21N=C(N)N(C)C1=O
InChIKey: XFTXOVBUZBRHQH-UHFFFAOYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE Assay: For each compound being tested, the BACE activity was monitored in a fluorescence quenching assay (FRET) using the ectodomain of BACE (aa 1-454) fused to a myc-his tag and secreted from HEK293/BACEect. cells into OptiMEM (Invitrogen) as enzyme source and a substrate peptide derived from the APP-Swedish mutation which possesses a Cy3-fluorophore at the N-terminus and a Cy5Q-quencher at the C-terminus (Cy3-SEVNLDAEFK-Cy5Q-NH2; Amersham). The substrate was dissolved at 1 mg/mL in DMSO.The assay was performed in the presence of 5 μl OptiMEM (supernatant collected over 24 hours and cleared from cellular debris by centrifugation) containing the ectodomain of BACE, 25 μl water containing the desired concentration of test compound and 1% DMSO, 1 μM substrate peptide, 20 mM NaOAc, pH 4.4 and 0.04% Triton-X100 in a total assay volume of 50 μl in a 384 well plate. In general, 25 μl of compound dilution were given to the plate followed by the addition of 10 μl of BACE containing OptiMEM diluted 1:2 in water with 0.2% Triton X-100. The reaction was started with the addition of 15 μl substrate in NaOAc buffer. The reaction was incubated at 30° C. in a fluorimeter and the cleavage of the substrate was recorded as kinetic for 60 min. at ex: 530 nm, em: 590 nm. Blank wells containing either no inhibitor or no enzyme were included on each plate.The intensity of fluorescence was regressed against time in order to derive velocities of reaction in all 384 wells. These velocities were used for calculating percent inhibition using an uninhibited control containing 1% DMSO and a fully inhibited control incubations performed in the absence of enzyme. IC50 values were calculated by fitting percent inhibition vs. inhibitor concentration using standard software like GraphPadPrism.
Measurement: IC50 = 31.6 nM (pChEMBL 7.5)
Descriptors: MW 394.27, ALogP 2.15, HBA 5, HBD 1, PSA 77.15, QED 0.79
Compound (SMILES): N#Cc1nc(C(=O)NCC2(C(=O)O)CCOCC2)c(O)c2ccc(Oc3ccccc3)cc12
InChIKey: SEEAXIMDRVUIOI-UHFFFAOYSA-N
Target: Prolyl hydroxylase EGLN2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96KS0
Assay: HIF-PH Assay: Ketoglutaric acid α-[1-14C]-sodium salt, alpha-ketoglutaric acid sodium salt, and HPLC purified peptide may be obtained from commercial sources, e.g., Perkin-Elmer (Wellesley Mass.), Sigma-Aldrich, and SynPep Corp. (Dublin Calif.), respectively. Peptides for use in the assay may be fragments of HIFα as described above or as disclosed in International Publication WO 2005/118836, incorporated by reference herein. HIF-pH, e.g., PHD1 (EGLN2) or PHD2 (EGLN1), can be expressed in, e.g., insect Hi5 cells, and partially purified, e.g., through a SP ion exchange chromatography column. Enzyme activity is determined by capturing 14CO2 using an assay described by Kivirikko and Myllyla (1982, Methods Enzymol. 82:245-304). Assay reactions contain 50 mM HEPES (pH 7.4), 100 μM α-ketoglutaric acid sodium salt, 0.30 mCi/mL ketoglutaric acid α-[1-14C]-sodium salt, 40 μM FeSO4, 1 mM ascorbate, 1541.8 units/mL Catalase, with or without 50 μM peptide substrate and various concentrations of compound of the invention. Reactions are initiated by addition of HIF-PH enzyme.
Measurement: IC50 = 1890 nM (pChEMBL 5.71999979019165)
Descriptors: MW 447.45, ALogP 3.22, HBA 7, HBD 3, PSA 141.77, QED 0.52
Compound (SMILES): O=C(O)c1ccc(S(=O)(=O)N(Cc2ccc(F)c(C(F)(F)F)c2)c2cc3ccccc3s2)cc1
InChIKey: ARMZBCKQLKBOAK-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Functional Assay: The functional activity of compounds of the formula (I) was determined by measuring changes in intracellular calcium concentration using a Ca2+-sensitive fluorescent dye. The changes in fluorescent signal were monitored by a fluorescence plate reader, either a FLIPR (Molecular Devices) or FDSS (Hamamatsu). Increases in intracellular Ca2+ concentration were readily detected upon activation with icilin.At 24 hrs prior to assay, HEK293 cells stably expressing canine TRPM8 were seeded in culture medium in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences, NJ, USA) and grown overnight in 5% CO2 at 37° C. On assay day, growth media was removed and cells were loaded with Calcium 3 Dye (Molecular Devices) for 35 min at 37° C., under 5% CO2 and then for 25 min at room temperature and atmosphere. Subsequently, cells were tested for agonist-induced increases in intracellular Ca2+ levels using FLIPR or FDSS. Cells were challenged with a compound of the Formula (I) (at varying concentrations) and intracellular Ca2+ was measured for 5 min prior to the addition of icilin to all wells to achieve a final concentration that produces approximately an 80% maximal response. EC50 or IC50 values for compounds of the present invention were determined from eight-point dose-response studies.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 509.5, ALogP 6.15, HBA 4, HBD 1, PSA 74.68, QED 0.32
Compound (SMILES): CC#CCn1c(N2CCCNCC2)nc2c1c(=O)n(CCCN1CCC(C(=O)OC(C)C)CC1)c(=O)n2C
InChIKey: OFJTZKPUEJEECX-UHFFFAOYSA-N
Target: Dipeptidyl peptidase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P27487
Assay: Inhibition of DPP4 (unknown origin) using Gly-Pro-p-nitroanilide as substrate incubated for 1 hr
Measurement: IC50 = 2600 nM (pChEMBL 5.579999923706055)
Descriptors: MW 527.67, ALogP 0.77, HBA 11, HBD 1, PSA 106.63, QED 0.4
Compound (SMILES): CCCOc1cc(F)ccc1-c1ccc(/N=N/c2cc(S(=O)(=O)[O-])c3ccccc3c2N)cn1.[Na+]
InChIKey: PPDANCVCDAFBAM-IRWWKPKRSA-M
Target: Transitional endoplasmic reticulum ATPase (SINGLE PROTEIN), Mus musculus
UniProt: Q01853
Assay: Inhibition Assay: Mouse VCP cDNA was added with a DNA sequence corresponding to histidine tag at the amino-terminal, subcloned into a baculovirus vector pVL1392 (BD Bioscience), and expressed in Sf-9 insect cells. The protein was purified with a nickel column (GE Healthcare). The concentration of the protein was adjusted to 0.25-0.5 ug/ml and the protein was stored in a solution containing 50 mM TrisCl pH 8.0, 5 mM EDTA, 10% glycerol, and 15 mM DTT. The ATPase activity was determined as follows. 500 ng of the purified VCP was mixed with 100 uM [gamma-32P]ATP (18.5 GBq/mmol) and the test substance in 20 uL of ATPase buffer (20 mM HEPES (pH7.4), 50 mM KCl, 5 mM MgCl2, 15 mM DTT), and incubated at 37° C. for 10 minutes.The enzyme reaction was stopped with addition of 200 uL of an ice-cold solution containing 7% TCA and 1 mM K2HPO4. 50ul of a solution containing 3.75% ammonium molybdate and 0.02M tungstate silicic/3 NH2SO4 was added, followed by 300 uL of n-butylacetic acid.
Measurement: IC50 = 1390 nM (pChEMBL 5.860000133514404)
Descriptors: MW 480.52, ALogP 6.07, HBA 7, HBD 2, PSA 127.23, QED 0.19
Compound (SMILES): CC1(C)CCC(CN2CCN(c3ccc(C(=O)NS(=O)(=O)c4ccc(NCC5CN(C(CF)CF)CCO5)c([N+](=O)[O-])c4)c(Oc4cnc5[nH]ccc5c4)c3)CC2)=C(c2ccc(Cl)cc2)C1
InChIKey: QYKHQDXEIXGHDM-UHFFFAOYSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Representative compounds were serially diluted in dimethyl sulfoxide (DMSO) starting at 50 uM (2x starting concentration; 10% DMSO) and 10 uL were transferred into a 384-well plate. Then 10 uL of a protein/probe/antibody mix was added to each well at final concentrations listed in TABLE 1. Protein:GST-Bcl-2, Probe: F-Bak Peptide Probe Acetyl-GQVGRQLAIIGDK(6-FAM)INR-amide(SEQ ID NO: 1), Protein(nM): 1, Probe (nM): 100, Antibody: Tb-anit-GST, Antibody (nm): 1.  The samples are then mixed on a shaker for 1 minute and incubated for an additional 3 hours at room temperature. For each assay, the probe/antibody and protein/probe/antibody were included on each assay plate as negative and positive controls, respectively. Fluorescence was measured on the ENVISION plate reader (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak peptide) and 495/510 nm (Tb-labeled anti-Histidine antibody) emission filters.
Measurement: Ki < 0.01 nM (pChEMBL 11)
Descriptors: MW 947.51, ALogP 8.24, HBA 12, HBD 3, PSA 175.27, QED 0.06
Compound (SMILES): Oc1ccc(-c2ccc3c(/C=C/c4ccccc4)n[nH]c3c2)cc1O
InChIKey: RSQWXQATLHCXOO-UXBLZVDNSA-N
Target: Serine/threonine-protein kinase Chk1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14757
Assay: Inhibition of full length human C-terminal 6His-tagged CHK1 expressed in baculovirus/insect expression system assessed as reduction in NADH oxidation using Syntide2 as substrate by pyruvate kinase/lactate dehydrogenase coupled assay
Measurement: Ki = 18 nM (pChEMBL 7.75)
Descriptors: MW 328.37, ALogP 4.81, HBA 3, HBD 3, PSA 69.14, QED 0.47
Compound (SMILES): CSc1c(Cl)nc(-c2ccc(NC(=O)Nc3ccc(N4CCN(C)CC4)nc3)cc2)nc1N1CCOCC1
InChIKey: XUUSUWLTFZPBOO-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: mTOR Kinase Assay: The activity of the compound against mTOR was examined by measuring the incorporation of 33P from [γ-33P]-ATP into 4EBP1. His-tagged, recombinant human 4EBP1 was expressed in E. coli, purified by nickel-nitrilotriacetic acid (Ni-NTA) resins, and stored at −80 °C. Phosphorylation of 4EBP1 by mTOR was assayed in the presence or absence of the compound and performed in a final volume of 25 μl reaction buffer containing 300 ng 4EBP1, 50 ng recombinant mTOR (Invitrogen), 50 mM HEPES (pH 7.5), 1 mM EGTA, 0.01% Polysorbate 20, 10 mM MnCl2, 2.5 mM DTT, 10 μM ATP, and 0.5 μCi [Î³-33P]-ATP (PerkinElmir) for 30 min at 30 °C. The reactions were terminated by adding 3% phosphoric acid. The 33P labeled 4EBP1 was transferred onto UniFilter-96 GF/B plate (PerkinElmer) and quantified by Top Count Microplate Scintillation Counter (PerkinElmer). For primary screening of kinase activity inhibition, each test compound was evaluated at 10 M in duplicate.
Measurement: IC50 = 80.7 nM (pChEMBL 7.090000152587891)
Descriptors: MW 555.11, ALogP 4.15, HBA 9, HBD 2, PSA 98.75, QED 0.34
Compound (SMILES): NC[C@@H]1CCCN1C(=O)Cn1cc(NC(=O)c2cnn3cccnc23)c(-c2cc(Cl)ccc2OC(F)F)n1
InChIKey: VYWOXOFKOATHII-HNNXBMFYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 5.35 nM (pChEMBL 8.270000457763672)
Descriptors: MW 544.95, ALogP 3.05, HBA 9, HBD 2, PSA 132.67, QED 0.35
Compound (SMILES): C[C@]12CC[C@H]3[C@@H](CC[C@H]4CC(=O)NCC[C@@]43C)C1=CC=C2c1cccnc1
InChIKey: ZIBMECBYPXQGHF-CYELZPNWSA-N
Target: Steroid 17-alpha-hydroxylase/17,20 lyase (SINGLE PROTEIN), Homo sapiens
UniProt: P05093
Assay: Inhibition of CYP17 in human testicular microsomes using 17alpha-hydroxypregnenolone as substrate assessed as decrease in DHEA levels after 90 mins by ELISA
Measurement: IC50 <= 11 nM (pChEMBL 7.959000110626221)
Descriptors: MW 362.52, ALogP 4.76, HBA 2, HBD 1, PSA 41.99, QED 0.78
Compound (SMILES): Cc1nc(C)c(-c2csc(Nc3ccc(C(=O)N4C5CCC4CC(=O)C5)cn3)n2)s1
InChIKey: SYGVRVVMHWDVPF-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBA0
Assay: Antagonist activity at human TRPV4 assessed as inhibition of 4alpha-PDD-induced activation
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 439.57, ALogP 4.36, HBA 8, HBD 1, PSA 88.08, QED 0.65
Compound (SMILES): COc1ccc(C2(OCCCCCn3cnc(CC(=O)O)c3)c3ccccc3-c3ccccc32)cc1
InChIKey: KVYCIKIBCWFWQX-UHFFFAOYSA-N
Target: GABA transporter 3 (SINGLE PROTEIN), Mus musculus
UniProt: P31649
Assay: Inhibition of mouse GAT3 expressed in HEK293 cells assessed as decrease in [3H]GABA uptake preincubated for 25 mins followed by [3H]GABA addition measured after 4 mins by liquid scintillation counter method
Measurement: IC50 = 97723.72 nM (pChEMBL 4.010000228881836)
Descriptors: MW 482.58, ALogP 5.68, HBA 5, HBD 1, PSA 73.58, QED 0.28
Compound (SMILES): Cc1ccc(-c2cc(NC(=O)c3cnn4cc(Cl)cnc34)n(-c3ccc(C)cn3)n2)cc1
InChIKey: STMQZVWBIKOEII-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Kinase Assay: The kinase activity of IRAK-4 is determined by its ability to catalyze the phosphorylation of a fluorescent polypeptide substrate. The extent of phosphorylation is measured using the IMAP technology (Molecular Devices) where the phosphorylated fluorescent substrate binds to the large M(III)-based nanoparticles which reduces the rotational speed of the substrate and thus increases its fluorescent polarization (FP). 20 μl reaction mixture contains 10 mM TriHCl, pH 7.2, 0.5 nM GST tagged IRAK-4 (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, LH, BioSystems).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 443.9, ALogP 4.5, HBA 7, HBD 1, PSA 90, QED 0.44
Compound (SMILES): N#Cc1cccc(-c2c[nH]c3ncnc(N4CCCC(F)(F)C4)c23)c1
InChIKey: VIRVQRWTXFSQOS-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Lantha Screen Technology Assay: LRRK2 kinase activity was measured using Lantha Screen technology from Invitrogen. GST-tagged truncated LRRK2 from Invitrogen (Cat # PV4874) was incubated with a fluorescein-labeled peptide substrate based upon ezrin/radixin/moesin (ERM), also known as LRRKtide (Invitrogen cat #PR8976A), in the presence of a dose response of compound. Upon completion, the assay was stopped and detected with a terbium labeled anti-phospho-ERM antibody (Invitrogen, cat #PR8975A). The assay was carried out under the following protocol: 3 uL of a working solution of substrate (233 nM LRRKtide, 117 uM ATP) prepared in assay buffer (50 mM HEEPES, pH 7.5, 3 mM MgCl2, with 2 mM DTT and 0.01% Brij35 added fresh) was added to a low volume Greiner 384-well plate. The compound dose response was prepared by diluting compound to a top concentration of 3.16 mM in 100% DMSO and serial diluted by half-log in DMSO 11 times. Aliquots (3.5 uL) of the 100% DMSO dose response were mixed with 46.5 uL water.
Measurement: IC50 = 79 nM (pChEMBL 7.099999904632568)
Descriptors: MW 339.35, ALogP 3.73, HBA 4, HBD 1, PSA 68.6, QED 0.77
Compound (SMILES): C[C@@]1(C(=O)N[C@@H](Cc2ccc(NC(=O)CCCCNC(=N)N)cc2)C(=O)O)CCCN1S(=O)(=O)c1ccccc1
InChIKey: VWSGVRISQFKGKE-CUNXSJBXSA-N
Target: Integrin alpha-V/beta-1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P06756
Assay: Inhibition of integrin alphaVbeta1 (unknown origin)-mediated CHO cell adhesion to fibronectin preincubated for 15 to 30 mins followed by 60 min incubation for adhesion by crystal violet staining based cell adhesion assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 572.69, ALogP 1.63, HBA 6, HBD 6, PSA 194.78, QED 0.12
Compound (SMILES): CNC(=O)c1cccc(Nc2ncnc(N3CC(COc4cccc(F)c4)C3)n2)c1
InChIKey: FLWVCTJYBUKLTB-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Cell-Based Assay: Compounds were tested on human Nav1.7 (hNav1.7) expressed stably in 293 cells, which have no endogenous sodium channels. Two forms of whole-cell patch-clamp assays were used. In both, peak current through Nav1.7 was evoked by a depolarizing voltage pulse, and currents were monitored as a function of time in response to exposure to several different concentrations of drug. Automated electrophysiology uses the PatchXpress 7000A system. Up to sixteen cells at a time were voltage-clamped in parallel, and the steady-state voltage dependence of inactivation was determined experimentally for each cell. For each cell, fractional inactivation was set at 20%, and peak current through Nav1.7 as a function of time was measured in response to increasing concentrations of test compound. Manual electrophysiology refers to conventional whole-cell patch-clamping (Hamill et al., 1981). After formation of the whole-cell clamp, cells were lifted off the bottom of the dish with the patch pipette and positioned directly in front of an array of glass microperfusion tubes each with internal diameter about 250 microns, with individual compound concentrations flowing from each tube. Current as a function of time in response to increasing drug concentrations was measured with Nav1.7 fully in the resting state (holding voltage set to −140 mV) and on the same cells with the holding voltage set to produce about 20% inactivation. Compounds were not applied for a prespecified time, but for as long as required to achieve a stable level of block.
Measurement: IC50 = 11250 nM (pChEMBL 4.949999809265137)
Descriptors: MW 408.44, ALogP 2.63, HBA 7, HBD 2, PSA 92.27, QED 0.62
Compound (SMILES): COCc1nc2c(C(=O)Nc3cccc(Cl)c3C)cc(NC(=O)c3ncccc3F)cc2[nH]1
InChIKey: VOVIUXFKWUCSDA-UHFFFAOYSA-N
Target: Prostaglandin E synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O14684
Assay: Inhibition Assay: mPGES-1 microsome fractions were prepared from CHO-K1 cells transiently transfected with plasmid encoding the human mPGES-1cDNA. Microsomes were diluted with potassium phosphate buffer containing reduced glutathione (pH7.4), and DMSO containing test compound or DMSO alone was added (such that DMSO final concentration would be 1% in each) and incubated at 4° C. for 20 minutes. Then, the enzymatic reactions were initiated by the addition of PGH2 substrate (final concentration 1 μM) and incubated at 4° C. for 60 seconds. The reaction was terminated by the addition of a citrate solution (final citrate concentration 50 mM) containing ferric chloride (final concentration 1 mg/mL). PGE2 production in the enzyme reaction aliquot was measured using HTRF kit (Cisbio International, catalogue #62P2APEC).
Measurement: IC50 = 56.2 nM (pChEMBL 7.25)
Descriptors: MW 467.89, ALogP 4.71, HBA 5, HBD 3, PSA 109, QED 0.38
Compound (SMILES): O=C(NCc1nnc[nH]1)c1cc(NC(=O)c2cc(-c3ccc(F)cn3)c(Cl)cc2Cl)n(-c2ccccc2)n1
InChIKey: AUAQWOGBEXNTTA-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Cell Based Assay: TRKA co-expressed with p75: The assays are based upon DiscoveRx's proprietary Enzyme Fragment Complementation (EFC) technology. In the case of the TRK cell lines, the enzyme acceptor (EA) protein is fused to a SH2 protein and the TRK receptor of interest has been tagged with a Prolink tag. Cryopreserved PathHunter cells are used from either in-house produced batches or bulk batches bought directly from DiscoveRx. Cryopreserved cells are resuscitated, spun 1000 rpm for 4 min to remove freezing media, and resuspended in MEM+0.5% horse serum (both Invitrogen) to 5e^5 cells/ml. The cells are then plated using a Multidrop into Greiner white tissue culture treated plates at 20 μl/well and incubated for 24 h at 37 °C., 5% CO2, high humidity. On the day of the assay, the cell plates are allowed to cool to room temperature for 30 min prior to the assay. 4 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using the compound of Example 135, WO2005/116035 at a top concentration of 150 μM is also prepared on each test plate. High percentage effect (HPE) is defined by 150 μM of the compound of Example 135, WO2005/116035 and 0% effect (ZPE) is defined by 100% DMSO. Plates containing 1 μl of serially diluted compound, standard and HPE/ZPE are diluted 1/66 in assay buffer (PBS minus Ca2+, minus Mg2+ with 0.05% pluronic F127) using a Wellmate. Using a Platemate Plus, 5 μl of 1/66 diluted test compounds is then transferred to the cell plate and allowed to reach equilibrium by incubating for 30 min at room temperature before addition of agonist stimulus: 10 μl/well of 2 nM (0.571 nM FAC) of the cognate neurotrophin (Peprotech) diluted in agonist buffer (HBSS with 0.25% BSA). Final assay concentration of the test compounds is 8.66 μM, (the compound of Example 135, WO2005/116035 FAC is 0.325 μM). The plates are left at room temperature for a further 2 hours before addition of 10 μl of the DiscoveRx PathHunter detection reagent (made up by adding 1 part Galacton Star, 5 parts Emerald II and 19 parts Cell Assay Buffer as per the manufacturer's instructions). After reagent addition, plates are covered and incubated at room temperature for 60 minutes. Luminescence signal is read using an Envision.
Measurement: IC50 = 49 nM (pChEMBL 7.309999942779541)
Descriptors: MW 551.37, ALogP 4.68, HBA 7, HBD 3, PSA 130.48, QED 0.27
Compound (SMILES): O=S(=O)(c1ccccn1)N(Cc1ccc(F)c(F)c1)c1cc2ccccc2s1
InChIKey: SZKFEZSCJNFMGW-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Functional Assay: The functional activity of compounds of the formula (I) was determined by measuring changes in intracellular calcium concentration using a Ca2+-sensitive fluorescent dye. The changes in fluorescent signal were monitored by a fluorescence plate reader, either a FLIPR (Molecular Devices) or FDSS (Hamamatsu). Increases in intracellular Ca2+ concentration were readily detected upon activation with icilin.At 24 hrs prior to assay, HEK293 cells stably expressing canine TRPM8 were seeded in culture medium in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences, NJ, USA) and grown overnight in 5% CO2 at 37° C. On assay day, growth media was removed and cells were loaded with Calcium 3 Dye (Molecular Devices) for 35 min at 37° C., under 5% CO2 and then for 25 min at room temperature and atmosphere. Subsequently, cells were tested for agonist-induced increases in intracellular Ca2+ levels using FLIPR or FDSS. Cells were challenged with a compound of the Formula (I) (at varying concentrations) and intracellular Ca2+ was measured for 5 min prior to the addition of icilin to all wells to achieve a final concentration that produces approximately an 80% maximal response. EC50 or IC50 values for compounds of the present invention were determined from eight-point dose-response studies.
Measurement: IC50 = 47 nM (pChEMBL 7.329999923706055)
Descriptors: MW 416.47, ALogP 4.97, HBA 4, HBD 0, PSA 50.27, QED 0.46
Compound (SMILES): CCCOc1cccc(C(=O)Nc2ccc(C)c(-n3ccn4nc(-c5cccnc5)cc34)c2)c1
InChIKey: ULTCSUZBVTTYBR-UHFFFAOYSA-N
Target: Angiopoietin-1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q02763
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: For the assay, 50 nl of a 100-fold concentrated solution of the respective test substance in DMSO were pipetted into a black low-volume 384-well microtitre plate (Greiner Bio-One, Frickenhausen, Germany) 2 μl of a solution of the Tie2 fusion protein in assay buffer [50 mM HEPES/HCl pH 7, mM MgCl2, 2.5 mM MnCl2, 1 mM dithiothreitol, 0.01% (v/v) Nonidet P-40, 0.1% (w/v) bovine serum albumin (BSA), 1× Complete EDTA-free protease inhibitor mixture (Roche)] were added, and the mixture was incubated for 15 min to allow pre-binding of the substance to the enzyme prior to the kinase reaction. The kinase reaction was then started by adding 3 μl of a solution of adenosine triphosphate (ATP, 1.67 mM→final concentration in 5 μl assay volume=1 mM) and substrate (1.67 μM→final concentration in 5 μl assay volume=1 μM) in assay buffer, and the resulting mixture was incubated for a reaction time of 60 min at 22° C. The concentration of the Tie2 fusion protein was adapted to the respective activity of the enzyme and adjusted such that the assay was carried out in the linear range. Typical concentrations were in the range of 30 ng/ml. The reaction was stopped by addition of 5 μl of a solution of TR-FRET detection reagents [200 nM streptavidin-XL665 and 2 nM PT66-Eu chelate, a europium chelate-labelled anti-phosphotyrosine antibody (Perkin-Elmer); alternatively, it is also possible to use PT66-Tb cryptate (Cisbio Bioassays, Codolet, France)] in aqueous EDTA solution [90 mM EDTA, 0.28% (w/v) bovine serum albumin (BSA) in 50 mM HEPES pH 7.5]. The resulting mixture was incubated at 22° C. for 1 h to allow formation of the complex of the biotinylated phosphorylated substrate and the detection reagents. The amount of phosphorylated substrate was then determined by measuring the resonance energy transfer from PT66-Eu chelate to streptavidin-XL665. To this end, the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm were measured in a TR-FRET measuring instrument (for example Viewlux, Perkin-Elmer).
Measurement: IC50 = 260 nM (pChEMBL 6.579999923706055)
Descriptors: MW 451.53, ALogP 5.54, HBA 6, HBD 1, PSA 73.45, QED 0.35
Compound (SMILES): O=C1C(N2CCC(c3ccc(O)cc3)C(F)(F)C2)CCN1Cc1ccc(F)cc1
InChIKey: XECUJMAUWGIGPW-UHFFFAOYSA-N
Target: Glutamate receptor ionotropic, NMDA 2B (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q00960
Assay: Radioligand Binding Assay: To perform the competition binding assay, thawed membrane homogenate was added to each well of a 96-well plate (20 ug/well). The experimental compounds were serially diluted in 100% DMSO and added to each row of the assay plate to achieve desired compound concentrations, keeping the DMSO concentration in the assay plate at 1.33% of the final reaction volume. Next, 3H Ro 25-6981 (4 nM) was added to the assay plate. After incubation for 1 hr at room temperature, the membrane bound radioligand was harvested on to GF/B filter plates (treated with 0.5% PEI for 1 hr at room temperature). The filter plates were dried at 50° C. for 20 mins, incubated with microscint 20 for 10 minutes and finally, the counts were read on TopCount (Perkin Elmer). Non-specific binding was determined using MK-0657 (the preparation of this compound is described as example 1 in WO 2004 108705 (40 uM).
Measurement: Ki = 530 nM (pChEMBL 6.28000020980835)
Descriptors: MW 404.43, ALogP 3.76, HBA 3, HBD 1, PSA 43.78, QED 0.84
Compound (SMILES): Cc1ccc(-c2cc(N3CC(c4nc5ccccc5n4C)C3)nc(C)n2)c(F)n1
InChIKey: RUWRDHSXYISSAK-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Fluorescence Polarization Assay: The fluorescence polarization assay for cyclic nucleotide phosphodiesterases was performed using an IMAP® FP kit supplied by Molecular Devices, Sunnyvale, Calif. (product # R8139). IMAP® technology has been applied previously to phosphodiesterase assays (Huang, W., et al., J. Biomol Screen, 2002, 7: 215). Assays were performed at room temperature in 384-well microtiter plates with an incubation volume of 20.2 μL. Solutions of test compounds were prepared in DMSO and serially diluted with DMSO to yield 8 μL of each of 10 solutions differing by 3-fold in concentration, at 32 serial dilutions per plate. 100% inhibition is determined using a known PDE10 inhibitor, which can be any compound that is present at 5,000 times its Ki value in the assay described as follows, such as papaverine (see Siuciak, et al. Neuropharmacology (2006) 51:386-396; Becker, et al. Behav Brain Res (2008) 186(2):155-60; Threlfell, et al., J Pharmacol Exp Ther (2009) 328(3):785-795), 2-{4-[pyridin-4-yl-1-(2,2,2-trifluoroethyl)-1H-pyrazol-3-yl]phenoxymethyl}quinoline succinic acid or 2-[4-(1-methyl-4-pyridin-4-yl-1H-pyrazol-3-yl)-phenoxymethyl]quinoline succinic acid (see Schmidt, et al. J Pharmacol Exp Ther (2008) 325:681-690; Threlfell, et al., J Pharmacol Exp Ther (2009) 328(3): 785-795). 0% of inhibition is determined by using DMSO (1% final concentrations). A Labcyte Echo 555 (Labcyte, Sunnyvale, Calif.) is used to dispense 200 mL from each well of the titration plate to the 384 well assay plate. A solution of enzyme (1/7000 dilution from aliquots; sufficient to produce 20% substrate conversion) and a separate solution of FAM-labeled cAMP PDE from Molecular Devices (product # R7506), at a final concentration of 50 nM are made in the assay buffer (10 mM Tris HCl, pH 7.2, 10 mM MgCl2, 0.05% NaN3 0.01% Tween-20, and 1 mM DTT). The enzyme is added to the assay plates by the addition of 10 μL of enzyme solution to each well, shaken to mix and incubated at room temperature for 60 minutes. The substrate is then added to the assay plates by the addition of 10 μÎ¼L of substrate solution to each well, shaken to mix, and incubated at room temperature for 60 minutes. A binding solution is then made from the kit components, comprised of 80% Solution A, 20% Solution B and binding reagent at a volume of 1/600 the total binding solution. The enzymatic reaction is stopped by addition of 60 lit of the binding solution to each well of the assay plates and the plates are sealed and shaken for 10 seconds. The plate was incubated at room temperature for at least one hour prior to determining the fluorescence polarization (FP).
Measurement: Ki = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 388.45, ALogP 3.79, HBA 6, HBD 0, PSA 59.73, QED 0.5
Compound (SMILES): Cc1ncccc1NC(=O)c1ccc2c(c1)CC[C@@H]1[C@@H]2[C@@H](O)C[C@@]2(C)[C@H]1CC[C@@]2(O)C#Cc1ccccc1
InChIKey: KOCQWYVXBLIRDI-KJOSVMAWSA-N
Target: Glucocorticoid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04150
Assay: Antagonist activity at GR in human HepG2 cells assessed as inhibition of protein mediated-transcriptional activity by MMTV-promoter driven luciferase reporter gene assay
Measurement: IC50 = 558 nM (pChEMBL 6.25)
Descriptors: MW 506.65, ALogP 5.25, HBA 4, HBD 3, PSA 82.45, QED 0.41
Compound (SMILES): CN1CC=C(c2cc(C(F)(F)F)ccc2N2CCOc3cc(S(=O)(=O)Nc4nccs4)ccc32)CC1
InChIKey: VAWHXLPBDJNIOI-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: In Vitro Assay: HEK 293 cells stably transfected with either Nav 1.7 or with Nav 1.5 were recorded in population patch-clamp mode with the IonWorks® Quattro automated electrophysiology system in accordance with the manufacturer's specifications (Molecular Devices, LLC, Sunnyvale, Calif.). Sodium channel currents were measured in response to a train of depolarizations that induced successively greater inactivation. Cells were held at −110 mV for three seconds (Nav 1.7) or half a second (Nav 1.5) from a holding voltage of −15 mV, then put through a series of 26 pulses of 150 msec duration to −20 mV at a frequency of 5 Hz. Cells were then left unclamped for a period of 3 to 8 minutes while a single concentration of test compound was added. Cells were then reclamped and put through the same voltage protocol. Current at the end of the 26th pulse to −20 mV was subtracted from the peak current evoked by the 26th pulse to −20 mV to correct for leak current.
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 536.6, ALogP 5.21, HBA 7, HBD 1, PSA 74.77, QED 0.48
Compound (SMILES): Cc1ccn2nc([C@H](C)Nc3ncnc(N)c3-c3ccc4cc[nH]c4c3)n(-c3ccccc3)c(=O)c12
InChIKey: RSVIKXZONRLYDM-KRWDZBQOSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: HTRF Assay : PI-3 Kinase HTRF kit (ref. #33-037) and the different PI3K recombinant isoforms (ref. #14-602, ref. #14-603, ref. #14-604, ref. #15-558 for Alpha, Beta, Delta and Gamma respectively) were purchased at Millipore (expressed in insect cells). ATP was purchased at Sigma Aldrich (ref. #A7699).The compounds were pre-incubated with the enzyme for 30 min before starting of the catalytic reaction. [PIP2] was used at its Km. [ATP] was used at 15 μM for all isoforms for technical reasons (Km values varied between 10 and 20 μM depending on the isoform). Time of assay and [Enzyme] were optimized to work in the linear range. Stop and Detection mixtures were used as specified in the Millipore PI-3 Kinase kit.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 476.54, ALogP 4.49, HBA 8, HBD 3, PSA 118.92, QED 0.34
Compound (SMILES): COc1ccc(C(C)C)cc1-c1cc(F)cc(C(CC(=O)O)c2nnc(C)o2)c1
InChIKey: PLRPHMKTNAMQCE-UHFFFAOYSA-N
Target: Lysosomal protective protein (SINGLE PROTEIN), Homo sapiens
UniProt: P10619
Assay: Inhibition Assay: Recombinant human cathepsin A (residues 29-480, with a C-terminal 10-His tag; R&D Systems, #1049-SE) was proteolytically activated with recombinant human cathepsin L (R&D Systems, #952-CY). Briefly, cathepsin A was incubated at 10 ug/ml with cathepsin L at 1 ug/ml in activation buffer (25 mM 2-(morpholin-4-yl)-ethanesulfonic acid (MES), pH 6.0, containing 5 mM dithiothreitol (DTT)) for 15 min at 37° C. Cathepsin L activity was then stopped by the addition of the cysteine protease inhibitor E-64 (N-(trans-epoxysuccinyl)-L-leucine-4-guanidinobutylamide; Sigma-Aldrich, # E3132; dissolved in activation buffer/DMSO) to a final concentration of 10 uM.The activated cathepsin A was diluted in assay buffer (25 mM MES, pH 5.5, containing 5 mM DTT) and mixed with the test compound (dissolved in assay buffer containing (v/v) 3% DMSO) or, in the control experiments, with the vehicle in a multiple assay plate. After incubation for 15 min at room temperature.
Measurement: IC50 = 44 nM (pChEMBL 7.360000133514404)
Descriptors: MW 398.43, ALogP 4.92, HBA 5, HBD 1, PSA 85.45, QED 0.61
Compound (SMILES): COCCN1CCC(n2cc(Nc3ncc4c(n3)-c3c(nn(C)c3-c3ccc(-c5cnn(C)c5)cc3)CC4)cn2)CC1
InChIKey: DEUXRKZJLFSYAO-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: DELFIA Assay: The kinase activity is measured by DELFIA assay (dissociation-enhanced lanthanide fluorescence immunoassay, Perkin Elmer). The cytoplasmic kinase domain of human IGF-1R (amino acids 964-1370) is expressed as a fusion protein with a glutathione-S-transferase tag (IGF-1R-GST) in High Five.TM. Cells (Invitrogen). Enzyme activity is measured in the presence of substances and a control substance. Poly-glutamate-tyrosine peptide (pEY, Sigma Aldrich) and biotinylated pEY (bio-pEY) are used as reaction substrates.10 uL of substance in 25% DMSO are mixed with 30 uL of IGF-1R-GST solution (67 mM HEPES pH 7.4, 15 ug/mL pEY, 1.7 ug/mL bio-pEY, 13.3 mM MgCl2, 3.3 mM dithiothreitol, 0.0033% Brij 35, 2 ng IGF-1R-GST) in 96-well plates. The reactions are started with 10 uL of a 750 uM ATP solution. After 40 min at RT the reactions are stopped with 50 .mu.L of stop solution (250 mM EDTA, 20 mM HEPES pH 7.4). 90 uL from each reaction are transferred.
Measurement: IC50 = 0.16 nM (pChEMBL 9.800000190734863)
Descriptors: MW 564.7, ALogP 4.27, HBA 11, HBD 1, PSA 103.74, QED 0.3
Compound (SMILES): CCOc1cc(=O)n(C)cc1-c1cc(NC(=O)Cc2ccc(C)nc2)ccc1Oc1ccc(F)cc1F
InChIKey: ZOVKYDHVNAWHPB-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Assay method C: Compound dilution series were prepared in DMSO via an approximately 3-fold serial dilution from one of the following: 0.047 mM to 0.78 nM or 5-fold serial dilution from Assay method A. Compound dilutions were added directly into white, low-volume assay plates (Perkin Elmer Proxiplate 384 Plus# 6008280) using a Labcyte Echo in conjunction with Labcyte Access and Thermo Multidrop CombinL robotics. Compounds were then suspended in eight microliters (μL) of assay buffer (20 mM Sodium Phosphate, pH 6.0, 50 mM NaCl, 1 mM Ethylenediaminetetraacetic acid disodium salt dihydrate, 0.01% Triton X-100, 1 mM DL-Dithiothreitol) containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and Alexa-647-conjugated probe. The final concentration of 1X assay mixture for assay methods A, B and C contains 2% DMSO, 8 nM His-tagged bromodomain, 1 nM Europium-conjugated anti-His-tag antibody and 100 nM or 30 nM probe (for BDI or BDII, respectively) and compound concentration in the range of: 49.02 μM-15.63 nM for method A, 9.19 μM 150 pM for method B, and 0.92 μM 15 pM for method C. After a one-hour equilibration at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 212 nM (pChEMBL 6.670000076293945)
Descriptors: MW 505.52, ALogP 5.41, HBA 6, HBD 1, PSA 82.45, QED 0.35
Compound (SMILES): CN(C)c1ccc(Cl)c(C(=O)Nc2ccc3c(c2)-c2c(c(C(N)=O)nn2-c2ccc(S(C)(=O)=O)cc2)CC3)c1
InChIKey: NIWROZCHDBLSOJ-UHFFFAOYSA-N
Target: Inhibitor of nuclear factor kappa-B kinase subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O14920
Assay: Inhibition of human recombinant C-terminal FLAG-epitope-tagged IKK2 expressed in baculovirus using biotinylated IkappaBalpha peptide as substrate assessed as decrease in [gamma-33P]-ATP incorporation in substrate after 30 mins by scintillation counting method
Measurement: IC50 <= 1000 nM (pChEMBL 6)
Descriptors: MW 564.07, ALogP 4.11, HBA 7, HBD 2, PSA 127.39, QED 0.36
Compound (SMILES): CC1CCN(c2ncnc3[nH]cc(-c4cccc(C#N)c4)c23)C1
InChIKey: OKUUUFUWBALHGR-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Lantha Screen Technology Assay: LRRK2 kinase activity was measured using Lantha Screen technology from Invitrogen. GST-tagged truncated LRRK2 from Invitrogen (Cat # PV4874) was incubated with a fluorescein-labeled peptide substrate based upon ezrin/radixin/moesin (ERM), also known as LRRKtide (Invitrogen cat #PR8976A), in the presence of a dose response of compound. Upon completion, the assay was stopped and detected with a terbium labeled anti-phospho-ERM antibody (Invitrogen, cat #PR8975A). The assay was carried out under the following protocol: 3 uL of a working solution of substrate (233 nM LRRKtide, 117 uM ATP) prepared in assay buffer (50 mM HEEPES, pH 7.5, 3 mM MgCl2, with 2 mM DTT and 0.01% Brij35 added fresh) was added to a low volume Greiner 384-well plate. The compound dose response was prepared by diluting compound to a top concentration of 3.16 mM in 100% DMSO and serial diluted by half-log in DMSO 11 times. Aliquots (3.5 uL) of the 100% DMSO dose response were mixed with 46.5 uL water.
Measurement: IC50 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 303.37, ALogP 3.34, HBA 4, HBD 1, PSA 68.6, QED 0.79
Compound (SMILES): CCOc1ccc2c(c1OC)CCN(Cc1ccc([C@H](C)NC(C)=O)cc1)C2C
InChIKey: OCNZUVWMFVQZPS-BHWOMJMDSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Spectrophotometric 384 Well Assay: Assay reactions are then carried out in 384-well plates, with hACC2 in an appropriate dilution and at final assay concentrations (f.c.) of 100 mM Tris (pH 7.5), 10 mM trisodium citrate, 25 mM KHCO3, 10 mM MgCl2, 0.5 mg/ml BSA, 3.75 mM reduced L-glutathione, 15 U/ml lactate dehydrogenase, 0.5 mM phosphoenolpyruvate, 15 U/ml pyruvate kinase, compounds at different concentrations at final DMSO concentrations of 1%.The enzymatic reaction is then started by addition of a mixture of NADH, acetyl Coenzyme A (both 2000 f.c.) and ATP (500 uM f.c.). The decrease of the optical density (slope S) is then determined at 25° C. at a wavelength of 340 nm over 15 minutes in a spectrophotometric reader.Each assay microtiter plate contains wells with vehicle instead of compound as controls for the non-inhibited enzyme (100% CTL; HIGH) and wells without acetyl-CoA as controls for non-specific NADH degradation (0% CTL; LOW).
Measurement: IC50 = 449 nM (pChEMBL 6.349999904632568)
Descriptors: MW 396.53, ALogP 4.41, HBA 4, HBD 1, PSA 50.8, QED 0.75
Compound (SMILES): C/C(=C\c1ccccc1)C(=O)O
InChIKey: XNCRUNXWPDJHGV-BQYQJAHWSA-N
Target: Aldo-keto reductase family 1 member C3 (SINGLE PROTEIN), Homo sapiens
UniProt: P42330
Assay: Discontinuous Radiometric Assay: Compounds may be evaluated as selective reversible inhibitors of AKR1C3 by screening them against homogeneous recombinant AKR1C1-AKR1C4 expressed in E. coli. In each case, a discontinuous radiometric assay may be used to monitor the inhibition of progesterone reduction (20-ketosteroid reduction) catalyzed by AKR1C1, the inhibition of Δ4-AD reduction (17-ketosteroid reduction) catalyzed by AKR1C3, and the inhibition of 5α-DHT reduction (3-ketosteroid reduction) catalyzed by AKR1C2 and AKR1C4 (by measuring the formation of 20α-hydroxyprogesterone, testosterone or 3α-androstanediol by radiochromatography). Secondary screens of the compounds of interest include: (a) a full-screen against all nine human recombinant AKR enzymes to ensure there are no-intended off-target effects (in this context AKR1B10 (retinal reductase; SEQ ID NO:5) has been shown to be potently inhibited by N-phenylanthranilates) (Endo et al., 2010, Biol. Pharm. Bull. 33:886-90); (b) a screen against COX-1 and COX-2 to reaffirm that compounds do not act as NSAIDs; and (c) an expanded screen against other nuclear receptors (especially other steroid hormone receptors).
Measurement: IC50 = 6400 nM (pChEMBL 5.190000057220459)
Descriptors: MW 162.19, ALogP 2.17, HBA 1, HBD 1, PSA 37.3, QED 0.68
Compound (SMILES): COc1cc(-c2cccc(F)c2)c(Cl)cc1-n1c(=O)ccc2cc(S(=O)(=O)Nc3ccon3)ccc21
InChIKey: IJFPYAKPQLJNJY-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Nav1.7 In Vitro PX Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to -10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes.
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 525.95, ALogP 5.25, HBA 7, HBD 1, PSA 103.43, QED 0.32
Compound (SMILES): COc1cc(CN(Cc2ccc(/C=C/C(=O)O)cc2)C(C)c2ccc3c(c2)CCO3)ccc1OCCN1C(=O)CCC1=O
InChIKey: MOUKXPFYSUILDP-RIYZIHGNSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: Displacement of [125I]-CXCL10 from human recombinant CXCR3 transfected in Flp-In-CHO cell membranes after 60 mins by gamma counting method
Measurement: IC50 = 138 nM (pChEMBL 6.860000133514404)
Descriptors: MW 584.67, ALogP 5.02, HBA 7, HBD 1, PSA 105.61, QED 0.22
Compound (SMILES): N#Cc1cccc2c(O[C@H]3CC[C@H](NC(=O)c4cc(F)cc(F)c4)CC3)ccnc12
InChIKey: ZAPKMVNTBKOLHE-WGSAOQKQSA-N
Target: Androgen receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P10275
Assay: Transactivation Assay: For determining the androgen receptor-dependent transcription, a cellular assay system was used, consisting of PC-3 cells (Kaighn et al., Invest. Urol. 17: 16-23, 1979), which express the human androgen receptor stably and recombinantly (full length, wild-type form, see Swiss-Prot Acc. No. P10275, Entry Version 159, Sequence Version 2). In addition, these PC3 cells contain a stably integrated reporter gene plasmid, which is based on the commercially available plasmid pGL4.14 (#E6691, Promega Corporation, Madison, Wis., USA) and contains the luciferase gene from the American firefly (Photinus pyralis) under the control of the MMTV promoter (Cato et al., EMBO J. 6: 363-368, 1987). These cells were propagated in routine cell culture at 37° C. and 5% CO2 in a medium containing 90% RPMI 1640 (Invitrogen GmbH, Darmstadt, Germany), 100 U penicillin, 100 μg/ml streptomycin (Invitrogen), 4 mM L-glutamine (Invitrogen), 10% fetal calf serum (FCS Serum Gold, PAA Laboratories GmbH, Cölbe, Germany), 600 μg/ml Geneticin (G418-sulphate, Invitrogen) and 10 μg/ml puromycin (Sigma Aldrich GmbH, Germany). For carrying out the transactivation assays, approx. 1000 cells per well were plated out in a 384-well cell culture plate in a medium that contained activated charcoal-treated calf serum (FCS Serum Gold, PAA Laboratories) at a concentration of 5% (v/v). The test substances were added in a concentration series from 5.12×10^−12 to 1×10^−5 M in the presence of 1×10^−1° R1881 (methyltrienolone). The test plates were incubated overnight at 37° C. and 5% CO2. After 16 hours, 15 μl of Steady Glo Lysis and Detection reagent (E2550, Promega Corporation, Madison, Wis., USA) was added per well and the luminescence was read in a Topcount Luminometer (PerkinElmer, Waltham, Mass., USA) for 4 seconds per well. The luminescence values obtained were normalized, wherein 100% corresponded to the effect of the unstimulated control (without R1881), and 0% corresponded to the effect of the stimulated control (R1881 plus DMSO instead of test substance).
Measurement: IC50 = 39 nM (pChEMBL 7.409999847412109)
Descriptors: MW 407.42, ALogP 4.5, HBA 4, HBD 1, PSA 75.01, QED 0.69
Compound (SMILES): O=C(c1ccc(Cl)cc1[N+](=O)[O-])N1CCN(c2nc(Cl)ncc2Br)CC1
InChIKey: HFDZNLJXCHTHEK-UHFFFAOYSA-N
Target: Bcr/Abl fusion protein (CHIMERIC PROTEIN), Homo sapiens
UniProt: P11274
Assay: Inhibition of BRC/ABL1 (unknown origin) after 40 mins by ADP-Glo assay
Measurement: IC50 = 2580 nM (pChEMBL 5.590000152587891)
Descriptors: MW 461.1, ALogP 3.42, HBA 6, HBD 0, PSA 92.47, QED 0.4