lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1nc2cccc(-c3cc4c([nH]3)CCNC4=O)c2nc1N(C)C(C)C
InChIKey: RWNZWAMJEWYARJ-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Homogeneous Time Resolved Fluorescence Assay (HTRF): The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 384-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction. Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 22-point dosing curve having a high dose of 1 μM. A reference compound was included on each assay plate [Costar 3658] in order to validate that plate; on one plate of every assay run, two additional reference compounds were included. The Reaction Buffer consisted of 45 mM Hepes, pH 7.0, 15 mM NaCl, and 1 mM MgCl. The quench/detection buffer consisted of 50 mM Tris, 100 mM NaCl, 0.05% BSA, 0.1% Tween and 3 mM EDTA. Biotinylated BAD peptide (Biopeptide), 10 mM ATP (Sigma), Labeled p-BAD (S112) mAb (Cell Signalling and Perkin Elmer) [with 0.05% BSA and 2 mM DTT added] streptavidin:Allophycocyanin [Perkin Elmer]. Final concentrations either Pim-1 enzyme [5 pM], or Pim-2 enzyme [0.5 pM], DMSO [1%], BLC BAD (S112) [0.5 μM], ATP [1.5 μM], streptavidin:Allophycocyanin [0.002 mg/mL] and biotinylated-BAD (S112) mAb [100 pM]. Initial incubations were carried out at RT (22° C.) for 30 min for both Pim-1 and for Pim-2. Pim enzyme is added to compound in buffer, and plates are incubated of 30 min. Biotinylated BAD and ATP are added and plates are incubated for 1 h. A mixture of labeled p-BAD (S112) mAb and quench/detection buffer are added and incubated for 2 h. Fluorescence was measured by an HTRF® Envision microplate reader.
Measurement: IC50 = 4.5 nM (pChEMBL 8.350000381469727)
Descriptors: MW 349.44, ALogP 3.06, HBA 4, HBD 2, PSA 73.91, QED 0.76
Compound (SMILES): CC1CCN(C(=O)C(CCc2ccccc2Cl)NS(=O)(=O)c2cc(Cl)c(N)c(Cl)c2)CC1
InChIKey: UQZIJONPFBRSEK-UHFFFAOYSA-N
Target: C-C chemokine receptor type 10 (SINGLE PROTEIN), Homo sapiens
UniProt: P46092
Assay: Antagonist activity at human CCR10 expressed in CHOK1 cells coexpressing aequorin/Galphaq assessed as inhibition of human CCL27-dependent calcium flux in presence of coelenterazine H by chemiluminescence assay
Measurement: IC50 = 125.89 nM (pChEMBL 6.900000095367432)
Descriptors: MW 518.89, ALogP 4.77, HBA 4, HBD 2, PSA 92.5, QED 0.52
Compound (SMILES): CCNC(=O)N1C(=O)N(c2cncc(Cl)c2)C2=C(C(=O)CC2)C1c1ccc(C#N)cc1S(C)(=O)=O
InChIKey: HEWYFKIASYMIAF-UHFFFAOYSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Human Neutrophil Elastase Assay: The following buffers were used: Compound buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5; Assay buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5, containing 0.01% BSA.Assay conditions: Test compounds were prediluted in DMSO and subsequently in compound buffer (5% DMSO final). 5 μL of these compound dilutions were mixed with 10 μl Neutrophil elastase (9 ng/ml in assay buffer) in a black 384 well OptiPlate (Perkin Elmer, Cat No.: 6007270) and incubated for 15 min at room temperature. Subsequently 10 μL substrate solution in assay buffer were added (250 μM final concentration) and the plates were incubated for 60 min at room temperature. After inactivation of the enzyme, fluorescence intensities were measured at 380 nm excitation and 460 nm emission wavelengths.
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 513.96, ALogP 3.34, HBA 7, HBD 1, PSA 140.54, QED 0.66
Compound (SMILES): CC(C)(C)C[C@H]1N[C@@H](C(=O)NC2CCC(OC(=O)[C@H](Cc3ccccc3)NC(=O)OC(C)(C)C)CC2)[C@H](c2cccc(Cl)c2F)[C@@]12C(=O)Nc1cc(Cl)c(F)cc12
InChIKey: SJZZHUDRDGTEMW-KYGSIEARSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: Fluorescence-polarization Binding Assay: The binding affinity of the MDM2 inhibitors was determined using an optimized, sensitive and quantitative fluorescence polarization-based (FP-based) binding assay using a recombinant human His-tagged MDM2 protein (residues 1-118) and a fluorescently tagged p53-based peptide. The design of the fluorescence probe was based upon a previously reported high-affinity p53-based peptidomimetic compound (5-FAM-βAla-βAla-Phe-Met-Aib-pTyr-(6-Cl-LTrp)-Glu-Ac3c-Leu-Asn-NH2 (SEQ ID NO: 1)) (García-Echeverría et al., J. Med. Chem. 43: 3205-3208 (2000)). This tagged peptide is called PMDM6-F. MDM2 protein was serially double diluted in a Dynex 96-well, black, round-bottom plate, and the PMDM6-F peptide was added at 1 nM concentration. The assay was performed in the buffer: 100 mM potassium phosphate, pH 7.5; 100 μg/mL bovine gamma globulin; 0.02% sodium azide, 0.01% Triton X-100) and the polarization values were measured after 3 h of incubation using an ULTRA READER (Tecan U.S. Inc).
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 827.8, ALogP 8.13, HBA 7, HBD 4, PSA 134.86, QED 0.16
Compound (SMILES): CN(C(=O)c1cncn1C)c1cccc(OCC(O)CN2CCc3ccccc3C2)c1
InChIKey: YARDYKVGUJOKTK-UHFFFAOYSA-N
Target: PRMT5/MEP50 complex (PROTEIN COMPLEX), Homo sapiens
UniProt: Q9BQA1
Assay: Inhibition of recombinant human fetal brain FLAG-tagged PRMT5/full length human testis 6His-tagged MEP50 expressed in baculovirus infected insect Sf9 cells using human histone H4 (1 to 15 residues) as substrate preincubated for 30 mins followed by [3H]-SAM addition
Measurement: IC50 = 1001 nM (pChEMBL 6)
Descriptors: MW 420.51, ALogP 2.49, HBA 6, HBD 1, PSA 70.83, QED 0.64
Compound (SMILES): CC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@H](C(=O)N[C@@H](CC(=O)O)C(=O)NCC(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(N)=O)C(C)C)C(C)C
InChIKey: VBYQFMLDNMLJTD-URURHIFTSA-N
Target: Tissue factor pathway inhibitor (SINGLE PROTEIN), Homo sapiens
UniProt: P10646
Assay: Binding affinity to TFPI (unknown origin) incubated for 1.5 hrs using tracer peptide JBT0131 by screening ELISA
Measurement: IC50 = 32 nM (pChEMBL 7.5)
Descriptors: MW 2415.79
Compound (SMILES): Cc1cc2ccc(C#N)cc2c(C)c1Oc1ccccc1OCCn1ccc(=O)[nH]c1=O
InChIKey: CHUQPDPBGYQCDO-UHFFFAOYSA-N
Target: Human immunodeficiency virus type 1 reverse transcriptase (SINGLE PROTEIN), Human immunodeficiency virus 1
UniProt: Q72547
Assay: Inhibition of HIV1 reverse transcriptase Y181C mutant infected in human MT2 cells assessed as protection against viral infection by MTT assay
Measurement: EC50 > 100 nM (pChEMBL 7)
Descriptors: MW 427.46, ALogP 4.05, HBA 6, HBD 1, PSA 97.11, QED 0.5
Compound (SMILES): CN(CC1CC1)c1cc(O)c(=O)[nH]n1
InChIKey: VBRWTUPXJUSBQW-UHFFFAOYSA-N
Target: D-amino-acid oxidase (SINGLE PROTEIN), Homo sapiens
UniProt: P14920
Assay: In Vitro DAAO Enzyme Assay: Human DAAO enzyme was supplied by the Takeda Pharmaceutical Company (Osaka) and each batch was tested and used at concentrations giving comparable levels of activity. The Km of D-Serine was measured for each enzyme batch to maintain consistency; this Km was used in subsequent assays.On the day of the assay compounds were serially diluted in DMSO before being diluted 1:20 with assay buffer (20 mM Tris ph 7.4). A 5 ul portion of assay buffer was added to the wells of a 384 clear base black-walled plate (Corning), 5 ul of diluted compound was then added via automated plate to plate transfer using the Bravo liquid handler (Agilent technologies) followed by 5 ul of human DAAO enzyme and then 5 ul D-Serine 50 mM was added to all but the negative control wells (final concentration of 10 mM). Finally 5 ul Amplex red reagent (Invitrogen) was added to all wells as per manufacturer's protocol. The plate was incubated for 60 minutes in the dark at 25° C., and the fluorescence in each well was measured in the Envision plate reader.
Measurement: IC50 = 220 nM (pChEMBL 6.659999847412109)
Descriptors: MW 195.22, ALogP 0.32, HBA 4, HBD 2, PSA 69.22, QED 0.73
Compound (SMILES): c1ccc2sc(CC3CCCN(c4ncnc5cc(N6CCOCC6)ccc45)C3)cc2c1
InChIKey: RAUFNYDOHVPKJZ-UHFFFAOYSA-N
Target: DNA-dependent protein kinase catalytic subunit (SINGLE PROTEIN), Homo sapiens
UniProt: P78527
Assay: Biochemical Assay: The kinase assay was carried out in streptavidin-coated 348-well microtitre FlashPlates®. To this end, 1.5 μg of the DNA-PK/protein complex and 100 ng of biotinylated substrate, such as, for example, PESQEAFADLWKK biotin-NH2 (biotin-DNA-PK peptide), in a total volume of 36.5 μl (34.25 mM HEPES/KOH, 7.85 mM Tris-HCl, 68.5 mM KCl, 5 μM ATP, 6.85 mM MgCl2, 0.5 mM EDTA, 0.14 mM EGTA, 0.69 mM DTT, pH 7.4), were incubated at room temperature for 90 min with 500 ng of DNA from calf thymus, 0.1 μCi of 33P-ATP and 1.8% of DMSO per well with or without the test compound. The reaction was stopped using 50 μl/well of 200 mM EDTA. After incubation for a further 30 min at room temperature, the liquid was removed. Each well was washed three times with 100 μl of 0.9% sodium chloride solution. A non-specific reaction (blank value) was determined using 10 μM of a proprietary kinase inhibitor.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 444.6, ALogP 5.14, HBA 6, HBD 0, PSA 41.49, QED 0.44
Compound (SMILES): CCC(C)(C)NCCn1cc(-c2ccc(F)c(C(F)(F)F)c2)nc1C1CCN(c2ncnc(N)c2C#N)CC1
InChIKey: PFBFGTPOSIOVBG-UHFFFAOYSA-N
Target: Ribosomal protein S6 kinase beta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23443
Assay: P70S6K Enzyme Assay: P70S6K inhibitor compounds are diluted and plated in 96 well plates. A reaction mixture including the following components is then added to the compound plate to initiate the enzyme reaction; P70S6K (3 nM, T412E mutant, Millipore) is mixed with 24 uM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 uM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction is incubated for 90 min at 25.degree. C., before the addition of 10 mM EDTA to stop the reaction. The proportion of substrate and product (phosphorylated) peptide is analysed on a Caliper Life Sciences Lab Chip 3000, using a pressure of -1.4 psi, and upstream and downstream voltages of -3000 and -700 respectively. Product peaks are resolved before substrate peaks on the resulting chromatograms.
Measurement: IC50 = 1.7 nM (pChEMBL 8.770000457763672)
Descriptors: MW 544.6, ALogP 5.11, HBA 8, HBD 2, PSA 108.68, QED 0.38
Compound (SMILES): O=C(Nc1ccc(-c2n[nH]c(=O)c3c2CCCC3)cc1)N1Cc2ccccc2C1
InChIKey: LUJABRZNRKRZRI-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: Inhibition of ROCK2 (unknown origin) using S6-peptide as substrate in presence of [33P]-ATP by HTRF assay
Measurement: Ki = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 386.46, ALogP 3.86, HBA 3, HBD 2, PSA 78.09, QED 0.7
Compound (SMILES): CCS(=O)(=O)Nc1cc(-c2ccc(F)cc2F)cc(-n2cnc3cc(-n4cccn4)ccc32)c1
InChIKey: BZZAURTUOCLCSA-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: TR-FRET Assay: Compounds were screened in the TR-FRET assay with FGFR1 kinase. 5 ng of FGFR1 [Upstate, USA] kinase was used for assay. The compound was incubated with the kinase for 30 minutes at RT. After the incubation, substrate mix [40 nM Ultra light poly GT (Perkin Elmer, USA) and 13 μM ATP (Sigma)] was added. The above reaction was stopped by the addition of 40 mM EDTA after the 30 mM kinase reaction. The Eu-labelled antiphospho-tyrosine antibody [Perkin Elmer, USA] was added at 0.5 nM and the fluorescence emission at 615 nm/665 nm [excitation at 340 nm] was measured. The compounds were initially screened at 100 nM and 1 μM concentrations. The compounds with >50% inhibition at 100 nM of FGFR1 were taken for the full dose response studies. The final DMSO concentration in the assay was 1%.
Measurement: IC50 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 479.51, ALogP 4.92, HBA 6, HBD 1, PSA 81.81, QED 0.37
Compound (SMILES): COc1ccc(COC(=O)C2=C(Cc3cccs3)NC(=O)NC2c2ccccc2)cc1
InChIKey: UWEYUFPUWZXACY-UHFFFAOYSA-N
Target: Sodium/iodide cotransporter (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q63008
Assay: Inhibition of sodium iodide symporter in rat FRTL5 cells assessed as inhibition of iodide uptake using NaI after 1 hr by arsenic/cerium sandell-Kolthoff method
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 434.52, ALogP 4.35, HBA 5, HBD 2, PSA 76.66, QED 0.54
Compound (SMILES): Cc1ccc(-c2noc(C3CC(=CCCO)C3)n2)cc1NC(=O)c1cnc2ccccn12
InChIKey: BWCMRRLLHHZFMK-BNCCVWRVSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Cell Proliferation Assay: The compounds was tested for inhibition of SCF dependent proliferation using human Mo7e cells which endogenously express c-kit in a 384 well format. Three-fold serially diluted test compounds (Cmax=10 mM) were evaluated for their antiproliferative activity of Mo7e cells stimulated with human recombinant SCF. After 48 hours of incubation at 37° C., cell viability was measured by adding 25 uL of CellTiter Glo (Promega) to the cells and the luminescence was measured by a CLIPR CCD camera (Molecular Devices).
Measurement: IC50 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 429.48, ALogP 4.13, HBA 7, HBD 2, PSA 105.55, QED 0.45
Compound (SMILES): CCC(CC)(c1ccc(CCC(=O)NO)c(C)c1)c1ccc(OC[C@H](O)C(C)(C)C)c(C)c1
InChIKey: RWZNNVOXLNXTEQ-VWLOTQADSA-N
Target: Histone deacetylase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O15379
Assay: Fluorogenic Inhibition Assay: Boc-Lys(Ac)-7-amino-4-methylcoumarin (Boc-Lys(Ac)-AMC) was used as substrate for the HDAC assays. Substrate solution was prepared as follow: Boc(Lys-Ac)-AMC was dissolved in DMSO and diluted with HDAC buffer (15 mM Tris-HCl [pH 8.1], 250 Î¼M EDTA, 250 mM NaCl, 10% glycerol) to give 1 mM solutions containing 1.7% DMSO. Trypsin was used to stop the reaction, releasing free AMC. The trypsin solution was prepared as follow: trypsin was dissolved in HDAC buffer to give a concentration of 10 mg/mL. Release of AMC was monitored by measuring the fluorescence at 460 nm (lex=390 nm) with a microplate reader (SpectraMax Gemini) at 37° C. The AMC signals were recorded against a blank with buffer, substrate and trypsin but without the enzyme. All experiments were carried out at least in triplicate. For HDAC inhibition assays, inhibitor diluted in 50 μL of HDAC buffer was mixed with 10 μL of diluted enzyme solution in HDAC buffer at room temperature. The HDAC reaction was started by adding 40 μL of substrate solution in HDAC buffer followed by 30 min of incubation with stirring at 37° C. The reaction was stopped by adding 100 μL, of trypsin solution. After a 10 min incubation with stirring at 37° C., the release of AMC was monitored by measuring the fluorescence.
Measurement: IC50 = 45100 nM (pChEMBL 4.349999904632568)
Descriptors: MW 455.64, ALogP 5.63, HBA 4, HBD 3, PSA 78.79, QED 0.32
Compound (SMILES): CN(C)[C@H]1Cc2c(Cl)cc(Cl)cc2[C@@H]1Oc1ccc(S(=O)(=O)NCCOCCOCCNC(=O)NCCCCNC(=O)NCCOCCOCCNS(=O)(=O)c2ccc(O[C@H]3c4cc(Cl)cc(Cl)c4C[C@@H]3N(C)C)cc2)cc1
InChIKey: MAZANAXBGWRPGC-SIAPTGDMSA-N
Target: Sodium/hydrogen exchanger 3 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P26433
Assay: Inhibition of rat NHE3-mediated sodium-dependant hydrogen ion antiport in Opossum kidney cells preincubated in NH4Cl-HEPES buffer containing BCECF-AM dye followed by washout with ammonium free HEPES and subsequent addition of compound in presence of NHE1 antagonist EIPA by fluorescence assay
Measurement: IC50 = 6.31 nM (pChEMBL 8.199999809265137)
Descriptors: MW 1205.12
Compound (SMILES): O=C(Nc1ccc(CC2CCCN2)cc1)c1ccc(Cl)cc1
InChIKey: GOMDUACECPUMGB-UHFFFAOYSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q923Y8
Assay: Radioligand Binding Assay: HEK-293 cells stably expressing mouse TAAR1 were maintained at 37° C. and 5% CO2 in DMEM high glucose medium, containing fetal calf serum (10%, heat inactivated for 30 min at 56° C.), penicillin/streptomycin (1%), and 375 ug/ml geneticin (Gibco). Cells were released from culture flasks using trypsin/EDTA, harvested, washed twice with ice-cold PBS (without Ca2+ and Mg2+), pelleted at 1,000 rpm for 5 min at 4° C., frozen and stored at -80° C. Frozen pellets were suspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 10 mM EDTA and homogenized with a Polytron (PT 6000, Kinematica) at 14,000 rpm for 20 s. The homogenate was centrifuged at 48,000Ã¿g for 30 min at 4° C. Subsequently, the supernatant was removed and discarded, and the pellet resuspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 0.1 mM EDTA using the Polytron (20 s at 14,000 rpm). This procedure was repeated and the final pellet resuspended in HEPES-NaOH containing 0.1 mM EDTA and homogenized using the Polytron. Typically, aliquots of 2 ml membrane portions were stored at -80° C. With each new membrane batch the dissociation constant (Kd) was determined via a saturation curve. The TAAR1 radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine (described in WO 2008/098857) was used at a concentration equal to the calculated Kd value, that was usually around 2.3 nM, resulting in the binding of approximately 0.2% of the radioligand and a specific binding representing approximately 85% of the total binding. Nonspecific binding was defined as the amount of 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine bound in the presence of 10 uM unlabeled ligand. All compounds were tested at a broad range of concentrations (10 pM to 10 uM) in duplicates. The test compounds (20 ul/well) were transferred into a 96 deep well plate (TreffLab), and 180 ul of HEPES-NaOH (20 mM, pH 7.4) containing MgCl2 (10 mM) and CaCl2 (2 mM) (binding buffer), 300 ul of the radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine at a concentration of 3.3xKd in nM and 500 ul of the membranes (resuspended at 50 ug protein per ml) added. The 96 deep well plates were incubated for 1 hr at 4° C. Incubations were terminated by rapid filtration through Unifilter-96 plates (Packard Instrument Company) and glass filters GF/C (Perkin Elmer) presoaked for 1 hr in polyethylenimine (0.3%) and washed 3 times with 1 ml of cold binding buffer. After addition of 45 ul of Microscint 40 (PerkinElmer) the Unifilter-96 plate was sealed and after 1 hr the ratioactivity counted using a TopCount Microplate Scintillation Counter (Packard Instrument Company).
Measurement: Ki = 2.7 nM (pChEMBL 8.569999694824219)
Descriptors: MW 314.82, ALogP 3.89, HBA 2, HBD 2, PSA 41.13, QED 0.9
Compound (SMILES): Nc1ncc(C2CCOCC2)nc1-c1ccc(C(=O)NC(c2ccccc2)C(F)(F)F)c(F)c1
InChIKey: ZQDSDJGAAHMLAJ-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Kinase Assay (New): Compound potency against activated ERK2 is determined using a kinase assay that measures ERK2-catalyzed phosphorylation of biotinylated ERKtide peptide substrate ([Biotin]-AHA-K-R-E-L-V-E-P-L-T-P-S-G-E-A-P-N-Q-A-L-L-R-[NH2], the peptide sequence derived from EGF receptor: SEQ ID NO:1). The assay is carried out in 50 mM HEPES [pH 7.5], 5 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 0.05% BSA using 0.25 nM ERK2, 200 nM ERKtide peptide and 35 uM ATP (all concentrations are final in the reaction) in a total volume of 10.25 uL. A 16-point, half-log dilution series of compounds at 41x final concentration is used for generating IC50 curves. Compound dilution series are prepared in 100% DMSO. ERK2 is preincubated with compounds for 30 minutes at ambient temperature. Reaction is initiated by addition of a substrate cocktail of ERKtide peptide and ATP and is allowed to proceed for 2-3 hours at ambient temperature. Reaction is terminated by addition of 10 uL of a 2x stop buffer consisting of 100 mM Tris-Cl [pH 7.5], 25 mM EDTA, 0.01% Tween 20, 10 ug/mL of AlphaScreen Protein A Acceptor Beads, 10 ug/mL of Streptavidin Donor Beads (PerkinElmer, Waltham, Mass.), and 1.4 ug/mL phospho-EGF Receptor (Thr669) antibody (Cat #3056, Cell Signaling Technology, Danvers, Mass.). Terminated reactions are read, after overnight incubation in the dark, on an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, Mass.), with excitation and emission wavelengths set to 680 nm and 570 nm, respectively. IC50 values are determined using a four-parameter fit.
Measurement: IC50 = 27 nM (pChEMBL 7.570000171661377)
Descriptors: MW 474.46, ALogP 4.79, HBA 5, HBD 2, PSA 90.13, QED 0.52
Compound (SMILES): CC1=C(C(=O)OCCN(C)C)C(c2ccccc2)c2c(nc3c(c2N)CC(F)(F)CC3)N1
InChIKey: RNQQNUBOXVKVNM-UHFFFAOYSA-N
Target: Acetylcholinesterase (SINGLE PROTEIN), Homo sapiens
UniProt: P22303
Assay: Inhibition Assay: Procedure: 48 Ml 1xReaction Buffer was initially added to each compound test well in the 96-well black plate arranged, followed by addition of 2 Ml 100xcompound solution; the test for each concentration was performed in duplicate. To the positive control well, 2 Ml DMSO+48 Ml 1xReaction Buffer were added; while 100 Ml 20 Mm H2O2 working solution was directly added to the positive validation well. To the negative control well, 2 Ml DMSO+98 Ml 1xReaction Buffer were added, and each test was performed in duplicate. 50 Ml 4xAchE application solution was added to each of the compound test wells and the positive control wells. 100 Ml 2xworking solution was added to each of all the wells and mixed until homogenous. The enzymatic reaction was started with the volume of the overall reaction system reaching 200 Ml, which resulted in the final concentrations of the compound solution of 10 umol/L, 2 umol/L, 0.4 umol/L, 0.08 umol/L, 0.016 umol/L, 0.0032 umol/L and 0.064 umol/L.
Measurement: IC50 = 136 nM (pChEMBL 6.869999885559082)
Descriptors: MW 442.51, ALogP 3.72, HBA 6, HBD 2, PSA 80.48, QED 0.69
Compound (SMILES): Cn1cc(-c2cc(Oc3ccc(NC(=O)N4CCN(C5CCOCC5)C4=O)nc3)ccn2)cn1
InChIKey: GBBAOXPAINQYPS-UHFFFAOYSA-N
Target: Macrophage colony-stimulating factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P07333
Assay: Enzymatic Assay: Activity of unphosphorylated c-FMS kinase (uFMS, Seq. ID no. 1) was determined by following the production of ADP from the FMS kinase reaction with ATP and poly E4Y as substrates through coupling with the pyruvate kinase/lactate dehydrogenase system (e.g., Schindler et al. Science (2000) 289: 1938-1942). In this assay, the oxidation of NADH (thus the decrease at A340 nm) was continuously monitored spectrophometrically. The reaction mixture (100 .mu.L) contained FMS (purchased from Millipore) (10 nM), polyE4Y (1 mg/mL), MgCl.sub.2 (10 mM), pyruvate kinase (4 units), lactate dehydrogenase (0.7 units), phosphoenol pyruvate (1 mM), NADH (0.28 mM) and ATP (500 .mu.M) in 90 mM Tris buffer containing 0.2% octyl-glucoside and 1% DMSO, pH 7.5. The inhibition reaction was started by mixing serial diluted test compound with the above reaction mixture. The absorption at 340 nm was monitored continuously for 4 hours at 30.degree. C. on Synergy 2 plate reader.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 463.5, ALogP 3.12, HBA 8, HBD 1, PSA 114.71, QED 0.62
Compound (SMILES): C[C@H]1CCCN1C1CC=C(c2ccc(NC(=O)OCc3ccccc3)c(F)c2)CC1
InChIKey: PGEBLGFMKXZZNY-HXBUSHRASA-N
Target: Histamine H3 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5N1
Assay: Radioligand Binding Assay: Rhesus H3 radioligand binding assay was performed using rhesus H3 receptor membranes (prepared as described above), [3H]-Methylhistamine (Perkin Elmer) and WGA SPA beads (wheat germ agglutinin scintillation proximity assay) beads (Amersham). The assay was performed in 96-well Opti-Plates (Packard). Each reaction contained 50 μl rhesus H3 membranes (20-30 μg total protein), 50 μl WGA SPA beads (0.1 μg) and 50 μl of 83 Ci/mmol [3H]-Methylhistamine (final concentration 2 nM) and 50 μl of tested compound. The compounds of this invention and/or vehicle were diluted with binding buffer from 10 mM DMSO stocks. Assay plates were sealed with TopSeal (Perkin Elmer) and mixed on shaker (25° C., 1 hour). Assay plates were read on TopCount scintillation counter (Packard).
Measurement: Ki = 6.88 nM (pChEMBL 8.15999984741211)
Descriptors: MW 408.52, ALogP 5.99, HBA 3, HBD 1, PSA 41.57, QED 0.66
Compound (SMILES): Brc1cnc(NC2CCc3ccccc32)nc1Nc1cc(C2CC2)[nH]n1
InChIKey: IQJAOJXGJBJWID-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human IGF1R kinase domain (M954 to C1367 residues) un-phosphorylated apo-enzyme form expressed in baculovirus based expression system using poly-EY peptide incubated for 2 hrs by luciferase-luciferin coupled chemiluminescent assay
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 411.31, ALogP 4.68, HBA 5, HBD 3, PSA 78.52, QED 0.57
Compound (SMILES): CN(C)C(=O)/C(C#N)=C/c1ccc(Nc2cnc3[nH]cc(C(=O)C(C)(C)C)c3n2)cc1Cl
InChIKey: NFFYSXNCFJIIDU-RIYZIHGNSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Phosphorylation Assay: Phosphorylation of the appropriate peptide substrate (fluorescently labeled Srctide) by recombinant JAK3 (catalytic domain, aa 781-1124) was measured in the presence and absence of different concentrations of test compounds. Test compound, enzyme, fluorescently labeled substrate, and cofactors (ATP and Mg2+) were combined in a well of a microtiter plate and incubated for 2 hours at 25° C. The composition of the reaction buffer was 100 mM HEPES pH 7.5, 5 mM MgCl2, 0.01% Triton-X 100, 0.1% Bovine Serum Albumin, and 1% DMSO. Enzyme concentration in the final reaction mixture was 0.5 nM while peptide substrate was at 1 uM. ATP was used at 1xKm concentration corresponding to 2 uM. Serial dilutions (3-fold steps) of compounds were prepared in DMSO. At the end of the incubation, the reaction was stopped by the addition of 20 mM EDTA. Substrate and product (i.e. phosphorylated substrate) were separated electrophoretically and both were quantified by fluorescence intensity.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 450.93, ALogP 4.58, HBA 6, HBD 2, PSA 114.77, QED 0.33
Compound (SMILES): CO[C@H]1CC[C@]2(CC1)Cc1ccc(O)cc1[C@@]21N=C(C)C(N)=N1
InChIKey: FUZQENPVBOMSKG-WBAXXEDZSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: TR-FRET Assay: The beta -secretase enzyme used in the TR-FRET is prepared as follows:The cDNA for the soluble part of the human (3-Secretase (AA 1-AA 460) was cloned using the ASP2-Fc10-1-IRES-GFP-neoK mammalian expression vector. The gene was fused to the Fc domain of IgG1 (affinity tag) and stably cloned into HEK 293 cells. Purified sBACE-Fc was stored in -80 C. in 50 mM Glycine pH 2.5, adjusted to pH 7.4 with 1 M Tris and had a purity of 40%.The enzyme (truncated form) was diluted to 6 ug/mL (stock 1.3 mg/mL) and the TruPoint BACE1 Substrate to 200 nM (stock 120 uM) in reaction buffer (NaAcetate, chaps, triton x-100, EDTA pH4.5). Enzyme and compound in dimethylsulphoxide (final DMSO concentration 5%) was mixed and pre-incubated for 10 minutes at RT. Substrate was then added and the reaction was incubated for 15 minutes at RT. The reaction was stopped with the addition of 0.35 vol Stop solution (NaAcetate, pH 9).
Measurement: IC50 = 1300 nM (pChEMBL 5.889999866485596)
Descriptors: MW 313.4, ALogP 2.51, HBA 5, HBD 2, PSA 80.2, QED 0.84
Compound (SMILES): COc1cccc(C(C)CCN2CCCCC2)c1
InChIKey: VOQBIIDKGKTMLS-UHFFFAOYSA-N
Target: Sigma non-opioid intracellular receptor 1 (SINGLE PROTEIN), Cavia porcellus
UniProt: Q60492
Assay: Displacement of [3H]-(+)-pentazocine from sigma 1 receptor in guinea pig cerebral cortex membranes after 120 mins by scintillation counting analysis
Measurement: Ki = 36 nM (pChEMBL 7.440000057220459)
Descriptors: MW 247.38, ALogP 3.67, HBA 2, HBD 0, PSA 12.47, QED 0.79
Compound (SMILES): CNC(=O)c1c(-c2ccc(F)cc2F)oc2cc(N(C)S(C)(=O)=O)c(-c3ccc4c(n3)-c3cc5c(F)cccc5n3C(CO)C4)cc12
InChIKey: LSUWDCGUCDKNAG-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: Inhibition Assay: To initiate an assay, replicon cells were plated in the presence of a dilution series of test compound in the absence of G418. Typically, the assays were performed in a 96-well plate formate for manual operation, or a 384-well plate format for automated assay. Replicon cells and compound were incubated for 96 hours. At the end of the assay, cells were washed free of media and compound, and the cells were then lysed. RNA was quantified indirectly through detection of replicon-encoded NS3/4A protein levels, through an ELISA-based assay with an antibody specific for NS3/4A.
Measurement: IC50 = 1.154 nM (pChEMBL 8.9399995803833)
Descriptors: MW 660.67, ALogP 6.05, HBA 7, HBD 2, PSA 117.67, QED 0.22
Compound (SMILES): Cc1cccc2c(=O)[nH]c(-c3cnc(N4CCN(C(=O)C5CC5)CC4)nc3)cc12
InChIKey: QUSZOYLWBHWZLQ-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase tankyrase-1 (SINGLE PROTEIN), Homo sapiens
UniProt: O95271
Assay: Chemiluminescent PARP Assay: Tankyrase activity was assayed using a 96-well format HT Universal Chemiluminescent PARP Assay Kit (Trevigen, Inc, cat. no. 4676-096-K) according to the manufacturer's instructions. In short, tankyrase/PARP activity is quantified by the incorporation of biotinylated nicotinamide adenine dinucleotide (biotin-NAD+) onto the immobilised pseudo substrate, Histone. The extent of poly(Biotin-ADP)ribosylation (PARylation) in the presence of increasing dose of inhibitor is then quantified by binding of streptavidin conjugated horse radish peroxidase (strep-HRP) followed by chemiluminescent detection. Prior to assay initiation, inhibitor stocks were prepared in aqueous DMSO (10% (v/v)) from 5 millimolar (mM) stock in 100% DMSO (Sigma Aldrich, cat. no. 265855) as 10x concentrations. For the primary assay (i.e., single dose at 1 micromolar (uM) final concentration) this corresponded to 10 uM in 10% DMSO.
Measurement: IC50 = 51 nM (pChEMBL 7.289999961853027)
Descriptors: MW 389.46, ALogP 2.35, HBA 5, HBD 1, PSA 82.19, QED 0.74
Compound (SMILES): CN(C)CCOc1ccc(/C(=C(/CCCF)c2ccccc2)c2ccc(Br)cc2)cc1.Cl
InChIKey: CWNATXNAOHYCPH-JTHROIFXSA-N
Target: Estrogen-related receptor gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P62508
Assay: Inhibition of fluorescien-conjugated coactivator PGC1a binding to GST-tagged human ERRgamma LBD after 1 hr by TR-FRET assay
Measurement: IC50 = 480 nM (pChEMBL 6.320000171661377)
Descriptors: MW 482.44, ALogP 7.1, HBA 2, HBD 0, PSA 12.47, QED 0.29
Compound (SMILES): C[C@@H]1CN([C@@H](C)CO)S(=O)(=O)c2ccc(C#Cc3cncnc3)cc2O[C@H]1CN(C)C(=O)NC1CCCCC1
InChIKey: ZJEVFKUHTXSENH-SWYRRKHMSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Inhibition of human C-terminal His8-tagged IDH1-R132H mutant expressed in Escherichia coli Rosetta assessed as inhibition of ketoreductase activity by measuring NADPH consumption preincubated for 60 mins followed by addition of alpha-KG as substrate measured after 50 mins in presence of NADPH and Mg2+ by resazurin based diaphorase-coupled fluorescence assay
Measurement: IC50 = 320 nM (pChEMBL 6.5)
Descriptors: MW 555.7, ALogP 2.62, HBA 7, HBD 2, PSA 124.96, QED 0.54
Compound (SMILES): O=C1NN=C2CCCc3[nH]c4cccc1c4c32
InChIKey: GGJVOTFHPYZESL-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: PARP-1 Enzymatic Assay: PARP-1 enzymatic assay was conducted using a method modified from HT F Homogeneous PARP Inhibition Assay Kit (Trevigen). 8.8 nM PARP-1 was pre-incubated with different concentrations of compounds in a buffer containing 100 mM Tris-HCl pH 8.0, 100 mM NaCl, 20 mM MgCl2, and 1% DMSO for 30 min at RT. The auto-PARylation reaction was initiated by addition of 500 nM NAD and 20 ng/ul activated DNA (Sigma) and incubated at RT for 40 min. The remaining NAD was detected by incubation with cycling assay solution containing 1% ethanol, 0.30 U/ml alcohol dehydrogenase, 25 uM resazurin, and 0.25 U/ml diaphorase for 50 min at RT. The concentration of NAD is proportional to the fluorescence signal at Ex 540 nm/Em 590 nm.
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 225.25, ALogP 1.95, HBA 2, HBD 2, PSA 57.25, QED 0.71
Compound (SMILES): Cc1nn(Cc2ccccc2)c(C)c1-c1ccc(N2CCc3cccc(C(=O)Nc4nc5ccccc5s4)c3C2)nc1C(=O)O
InChIKey: ZTNPPPMKYJVOKW-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07817
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The measurement of competition of compounds of Formula (I) with F-Bak for a Bcl-2 family protein (Bcl-xL) binding site using a Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) binding assay: Test compounds were serially diluted in DMSO starting at 50 M (2x starting concentration; 10% DMSO) and 10 Î¼L transferred into a 384-well plate. Then 10 Î¼L of a protein/probe/antibody mix is added to each well at final concentrations listed in Table 1. Protein: GST-Bcl-xL; 1(nM) Probe: F-Bak (GQVGRQLAIIGDK (6-FAM)INR-amide) SEQ ID NO: 1; 100(nM) Antibody: Tb-anti-GST; 1(nM). The samples are then mixed on a shaker for 1 minute then incubated for an additional 2 hours at room temperature. For each assay plate, a probe/antibody and protein/antibody/probe mixture were included as a negative and a positive control, respectively. Fluorescence was measured on the ENVISION plate reader (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak) and 495/510 nm (Tb-labeled anti-his antibody) emission filters. Dissociation constants (Ki) were determined using Wang's equation (Wang, Z.X. An 20 exact mathematical expression for describing competitive binding of two different ligands to protein molecule. FEBS Lett. 1995 360:111-114). The TR-FRET assay can be performed in the presence of varying concentrations of human serum (HS) or fetal bovine serum (FBS).
Measurement: Ki = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 614.73, ALogP 6.73, HBA 8, HBD 2, PSA 113.24, QED 0.2
Compound (SMILES): COc1ncc(-c2ccc3ncc4c(=O)[nH]c(=O)n(-c5cccc(C(F)(F)F)c5)c4c3n2)cn1
InChIKey: CCGXFNNEXOERED-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: PI3Kα Enzymatic Assay: Agents: 1-fold kinase buffer: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS, 2 mM DTT). 4-fold kinase solution: PI3KÎ± kinase was added to 1-fold kinase buffer to form 4-fold kinase solution with a final concentration of 1.65 nM. 2-fold substrate and ATP solution: the substrate PIP2 and ATP were added to 1-fold kinase buffer to form 2-fold substrate solution with final concentration of 50 μM of PIP2 and 25 μM of ATP. 4-fold test substance solutions: 100-fold test substance solutions in different gradient concentrations were formulated with 100% DMSO, and diluted by 25 folds with 1-fold kinase buffer to form 4-fold test substance solutions in different gradient concentrations. Kinase-Glo reagent kit, which was placed to warm up to room temperature. Procedure: To each well of a 384-well plate was added 2.5 μL of the 4-fold test substance solutions in gradient concentrations. To each well was added 2.5 μL of 4-fold kinase solution, and then the plate was incubated for 10 mins. Then to each well was added 5 μL of 2-fold substrate and ATP solution, and then the plate was incubated at room temperature for 1 hour. Finally, 10 μL of the detection solution was added to terminate the reaction. After 15 mins, the data Lance signal (665 nM) was read from Envision.
Measurement: IC50 = 40.7 nM (pChEMBL 7.389999866485596)
Descriptors: MW 466.38, ALogP 3.11, HBA 8, HBD 1, PSA 115.65, QED 0.41
Compound (SMILES): COc1ccccc1Oc1c(NS(=O)(=O)c2ccc(C(F)(C(F)(F)F)C(F)(F)F)cc2)nc(-c2ncccn2)nc1OCCO
InChIKey: OCRSOSTUIAIWII-UHFFFAOYSA-N
Target: Endothelin-1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P25101
Assay: Agonist activity at ETA receptor in human SKNMC cells assessed as induction of Ca2+ mobilization by fluorimetric method
Measurement: IC50 = 75.4 nM (pChEMBL 7.119999885559082)
Descriptors: MW 663.53, ALogP 5.2, HBA 10, HBD 2, PSA 145.65, QED 0.21
Compound (SMILES): COc1ccc(C2CCN(C(=O)c3ccc(C)c(-c4[nH]c(C)nc4C)c3)CC2)cc1
InChIKey: BMVGSAATCYIUNE-UHFFFAOYSA-N
Target: Fatty acid synthase (SINGLE PROTEIN), Homo sapiens
UniProt: P49327
Assay: Biochemical Assay: The FASN enzyme was isolated from SKBr3 cells. SKBr3 is a human breast cancer cell-line with high levels of FASN expression. It is estimated that FASN comprises about 25% of the cytosolic proteins in this cell line. SKBr3 cells were homogenized in a dounce homogenizer then centrifuged for 15 minutes at 4° C. to remove particulate matter. The supernatant was then analyzed for protein content, diluted to the appropriate concentration, and used to measure FASN activity. The presence of FASN was confirmed by western blot analysis. A similar method for isolation of FASN from SKBr3 cells is described in Teresa, P. et al. (Clin. Cancer Res. 2009; 15(24), 7608-7615). FASN activity of the SKBr3 cell extract was determined by measuring either NADPH oxidation or the amount of thiol-containing coenzyme A (CoA) released during the fatty acid synthase reaction. The dye CPM (7-diethylamino-3-(4'-maleimidyl-phenyl)-4-methylcoumarin) contains a thiol reactive group that increases its fluorescence emission on reaction with the sulfhydryl group of CoA.
Measurement: IC50 = 89 nM (pChEMBL 7.050000190734863)
Descriptors: MW 403.53, ALogP 5.03, HBA 3, HBD 1, PSA 58.22, QED 0.66
Compound (SMILES): CCCC[C@H](NC(C)=O)C(=O)N[C@H]1CCC(=O)NCC[C@@H](C(=O)N[C@H](Cc2c[nH]c3ccccc23)C(N)=O)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@@H](Cc2ccccc2)NC(=O)[C@H](Cc2c[nH]cn2)NC1=O
InChIKey: IJVUOKFOZUYPMK-ZUWNFKQVSA-N
Target: Melanocortin receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P32245
Assay: Competitive Inhibition Binding Assay: A competitive inhibition binding assay was performed using membrane homogenates prepared from HEK-293 cells that express recombinant hMCR-1a or hMCR-4 (in each instance where the h prefix refers to human), or alternatively membrane homogenates from B16-F10 mouse melanoma cells containing endogenous murine MCR-1. In the examples that follow, all MCR-1 and MCR-4 values are for human recombinant receptors, unless otherwise noted. Assays were performed in 96 well polypropylene round-bottom plates (VWR catalog number 12777-030). Membrane homogenates were incubated with 0.1 nM [I125]-NDP-α-MSH (Perkin Elmer) and increasing concentrations of test peptides of the present invention in buffer containing 25 mM HEPES buffer (pH 7.5) with 100 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 0.3 mM 1,10-phenanthroline, and 0.2% bovine serum albumin. After incubation for 90 minutes at 37° C., the assay mixture was filtered onto GF/B Unifilter plates (Perkin-Elmer catalog number 6005177) and washed with 3 mL of ice-cold buffer per well. Filters were air dried and 35 uL of scintillation cocktail added to each well. Plates were counted in a Microbeta counter for bound radioactivity. Non-specific binding was measured by inhibition of binding of [I125]-NDP-α-MSH in the presence of 1 uM NDP-α-MSH. Maximal specific binding (100%) was defined as the difference in radioactivity (cpm) bound to cell membranes in the absence and presence of 1 uM NDP-α-MSH. Each assay was conducted in duplicate and the actual mean values are described, with results less than 0% reported as 0%. Ki values for peptides of the present invention were determined using Graph-Pad Prism curve-fitting software.
Measurement: Ki = 33 nM (pChEMBL 7.480000019073486)
Descriptors: MW 1010.17
Compound (SMILES): CCNC(=O)c1ccc(-n2cnc3c(NCC4CCOCC4)cc(Oc4ccc(OC)c(SC)c4)cc32)cc1C
InChIKey: AQGVXNLXYIAESJ-UHFFFAOYSA-N
Target: Dual specificity protein kinase TTK (SINGLE PROTEIN), Homo sapiens
UniProt: P33981
Assay: Kinase Assay: For the assay 50 nL of a 100-fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 μl of a solution of Mps-1 in assay buffer [0.1 mM sodium-ortho-vanadate, 10 mM MgCl2, 2 mM DTT, 25 mM Hepes pH 7.7, 0.05% BSA, 0.001% Pluronic F-127] were added and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compounds to Mps-1 before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 μl of a solution of 16.7 adenosine-tri-phosphate (ATP, 16.7 μM=>final conc. in the 5 μl assay volume is 10 μM) and peptide substrate (1.67 μM=>final conc. in the 5 μl assay volume is 1 μM) in assay buffer and the resulting mixture was incubated for a reaction time of 60 min at 22° C. The concentration of Mps-1 in the assay was adjusted to the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical enzyme concentrations were in the range of about 1 nM (final conc. in the 5 μl assay volume). The reaction was stopped by the addition of 3 μl of a solution of HTRF detection reagents (100 mM Hepes pH 7.4, 0.1% BSA, 40 mM EDTA, 140 nM Streptavidin-XLent [#61GSTXLB, Fa. Cis Biointernational, Marcoule, France], 1.5 nM anti-phospho(Ser/Thr)-Europium-antibody [#AD0180, Perkin Elmer LAS, Rodgau-Jügesheinn, Germany]. The resulting mixture was incubated 1 h at 22° C. to allow the binding of the phosphorylated peptide to the anti-phospho(Ser/Thr)-Europium-antibody. Subsequently the amount of phosphorylated substrate was evaluated by measurement of the resonance energy transfer from the Europium-labelled anti-phospho(Ser/Thr) antibody to the Streptavidin-XLent. Therefore, the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm was measured in a Viewlux TR-FRET reader (PerkinElmer LAS, Rodgau-Jügesheinn, Germany).
Measurement: IC50 = 54 nM (pChEMBL 7.269999980926514)
Descriptors: MW 560.72, ALogP 6.44, HBA 8, HBD 2, PSA 86.64, QED 0.22
Compound (SMILES): O=C(Nc1cccc(C(F)(F)F)c1)c1ccc(C(F)F)c(-c2ccnc(N3CCOCC3)c2)c1
InChIKey: IYANNOOOCLOAND-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: In Vitro Enzyme Assay: Final assay conditions were 4 pM cRaf, 3 uM ATP, 10 nM biotin tagged MEK1 kinase dead protein substrate. Reactions were performed in Greiner384 well plates, catalog #784075. Reaction buffer was 50 mM Tris, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 0.01% Tween-20, 0.05% BSA and 1 mM DTT. 5 ul of 2×cRaf plus MEK1 were dispensed into plates containing 0.25 ul of compound in 100% DMSO and incubated at room temperature for 30 minutes. Reactions were started by addition of 5 ul of 2×ATP. Reactions were run for two hours and terminated by addition of 10 ul of 2× antibody and Alphascreen bead mixture in Stop Buffer. Stop Buffer was 50 mM Tris, pH 7.5, 0.005% Tween-20, 25 mM EDTA.
Measurement: IC50 = 5.035 nM (pChEMBL 8.300000190734863)
Descriptors: MW 477.43, ALogP 5.79, HBA 4, HBD 1, PSA 54.46, QED 0.47
Compound (SMILES): CCn1ccc(Nc2ncc3c(n2)-c2c(nn(C)c2Cc2cccc(-c4cnn(CC(C)C)c4)c2)CC3)n1
InChIKey: WIGMQNAICQDORZ-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 = 0.35 nM (pChEMBL 9.460000038146973)
Descriptors: MW 507.65, ALogP 5.05, HBA 9, HBD 1, PSA 91.27, QED 0.32
Compound (SMILES): O=C(Nc1cccc(CN2CCC(C(=O)NC3CCCCC3)CC2)c1)c1c(F)cccc1C(F)(F)F
InChIKey: JJVIVDPXYRNMHF-UHFFFAOYSA-N
Target: Atypical chemokine receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P25106
Assay: In Vitro Assay: CHO-K1 CXCR7 b-arrestin cells are detached from culture dishes with a cell dissociation buffer (Invitrogen, #13151-014) and collected in growing medium (F12 HAMS 90% (v/v)/FCS 10% (v/v), Penicilin/streptomycin 1% (v/v)). 5000 cells per well (in 20 μl) are seeded in a 384 well plate (white-walled, clear bottom; BD Falcon #353274). The plate is incubated at 37° C./5% CO2 for 24 hours. Medium is then replaced by 20 μl OPTIMEM (Invitrogen #31985) for 3 to 4 hours. Test compounds are dissolved at 10 mM in DMSO and serially diluted in DMSO to 200× of the final concentration for dose response curves. Compounds are then diluted 1:33.3 in HBSS1×. 5 μl/well of HBSS1×/20 mM HEPES/0.2% BSA are added to the assay plate followed by addition of 5 μl/well of diluted compounds. CXCL12 (Peprotech #300-28A) may be used as a reference agonist. The plate is incubated for 90 minutes at 37° C. 12 μl of detection reagent (Path Hunter Detection Kit, DiscoveRx, #93-0001) is transferred to the assay plate and to the plate is incubated for 1 hour at room temperature. Luminescent signal is read in a microplate reader (FLUOstar Optima, bmg).
Measurement: EC50 = 425 nM (pChEMBL 6.369999885559082)
Descriptors: MW 505.56, ALogP 5.76, HBA 3, HBD 2, PSA 61.44, QED 0.49
Compound (SMILES): O=C(Nc1nc2ccccc2s1)c1cccc2c1CN(c1ccc(-c3cnn(Cc4ccccc4OCCN4CCOCC4)c3)c(C(=O)O)n1)CC2
InChIKey: HTSMAYIADJQPMN-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07817
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The measurement of competition of compounds of Formula (I) with F-Bak for a Bcl-2 family protein (Bcl-xL) binding site using a Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) binding assay: Test compounds were serially diluted in DMSO starting at 50 M (2x starting concentration; 10% DMSO) and 10 Î¼L transferred into a 384-well plate. Then 10 Î¼L of a protein/probe/antibody mix is added to each well at final concentrations listed in Table 1. Protein: GST-Bcl-xL; 1(nM) Probe: F-Bak (GQVGRQLAIIGDK (6-FAM)INR-amide) SEQ ID NO: 1; 100(nM) Antibody: Tb-anti-GST; 1(nM). The samples are then mixed on a shaker for 1 minute then incubated for an additional 2 hours at room temperature. For each assay plate, a probe/antibody and protein/antibody/probe mixture were included as a negative and a positive control, respectively. Fluorescence was measured on the ENVISION plate reader (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak) and 495/510 nm (Tb-labeled anti-his antibody) emission filters. Dissociation constants (Ki) were determined using Wang's equation (Wang, Z.X. An 20 exact mathematical expression for describing competitive binding of two different ligands to protein molecule. FEBS Lett. 1995 360:111-114). The TR-FRET assay can be performed in the presence of varying concentrations of human serum (HS) or fetal bovine serum (FBS).
Measurement: Ki = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 715.84, ALogP 5.83, HBA 11, HBD 2, PSA 134.94, QED 0.17