lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(C)(C)Nc1c(-c2cccc(C#N)c2)nc2ccc(F)cn12
InChIKey: VPEVJZHGWCCMBL-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily A member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75762
Assay: Dose Response Assay: Harvested cells with 0.025% trypsin/EDTA. Resuspended cells in growth media without selection antibiotics. Measured cell density and diluted to 2.4×10^5 cells/ml in media containing 1 μg/ml Doxycycline Plate 25 μl/well into 384 well black/clear tissue culture-treated plates. Incubated overnight at 37° C. Growth media was removed from the cell plates (20 μl) and 20 μl of the Replacement Buffer was added followed by addition of 25 μl of diluted dye. All three steps were performed using a Plate Washer BioTek 407. The plates were then incubated for 30' at RT. After incubation, both the cell and compound plates were brought to the FLIPR and 20 μl of the diluted compounds/antagonist/bk were transferred to the cell plates by the FLIPR. Plates were then incubated for 30' at room temperature. After 30' incubation, plates were returned to the FLIPR and 20 ul of 4.5× Cinnamaldehyde was added to the cell plates. During the compound addition as well as agonist addition, fluorescence readings were taken simultaneously from all 384 wells of the cell plate every 1.5 seconds. Five readings were taken to establish a stable baseline, then 20 μl of sample was rapidly (30 μl/sec) and simultaneously added to each well of the cell plate. The fluorescence was continuously monitored before, during and after sample/agonist addition for a total elapsed time of 100 seconds (compound addition) and 120 seconds (agonist addition).
Measurement: IC50 = 20420 nM (pChEMBL 4.690000057220459)
Descriptors: MW 308.36, ALogP 4.22, HBA 4, HBD 1, PSA 53.12, QED 0.77
Compound (SMILES): N=C(N)c1ccc(CC2NCCn3c2nc2cc(C4(C(=O)N5CCCCC5)CCCC4)ccc23)cc1
InChIKey: SKHSFQDFSNIGLL-UHFFFAOYSA-N
Target: Prothrombin (SINGLE PROTEIN), Homo sapiens
UniProt: P00734
Assay: Inhibition of human thrombin assessed as reduction in release of free nitroaniline using tosyl-glycyl-prolyl-arginine-4-nitranilide acetate as substrate preincubated for 10 mins followed by substrate addition by spectrophotometer
Measurement: IC50 = 1028 nM (pChEMBL 5.989999771118164)
Descriptors: MW 484.65, ALogP 4.03, HBA 5, HBD 3, PSA 100.03, QED 0.38
Compound (SMILES): CC(C)Oc1ccc(-c2nc(-c3cccc4c3CC[C@@H]4NC(=O)N3CCC(O)CC3)no2)cc1C#N
InChIKey: KIALPTLWECVGBJ-QHCPKHFHSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: Agonist activity at human S1P1 expressed in CRE-bla CHOK1 cells assessed as inhibition of forskolin-induced cAMP formation preincubated for 4 hrs followed by FRET-based beta-lactamase fluorescent substrate addition measured after 2 hrs
Measurement: EC50 = 0.05 nM (pChEMBL 10.300000190734863)
Descriptors: MW 487.56, ALogP 4.22, HBA 7, HBD 2, PSA 124.51, QED 0.55
Compound (SMILES): O=C1NC2(CCCC2)C(=O)N[C@@H](Cc2ccccc2)C(=O)NC/C=C\c2ccccc2OCCN[C@H]1Cc1cccc2ccccc12
InChIKey: GSIGZEKAABXHPZ-MNQNQIJJSA-N
Target: Growth hormone secretagogue receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92847
Assay: Displacement of [125I]-Ghrelin from human GSHR1a transfected in HEK293 cell membranes after 30 mins by microplate scintillation and luminescence counting method
Measurement: Ki = 1000 nM (pChEMBL 6)
Descriptors: MW 630.79, ALogP 4.72, HBA 5, HBD 4, PSA 108.56, QED 0.26
Compound (SMILES): CCn1c2ccccc2c2cc(C(=O)C(=O)Nc3ccccc3)ccc21
InChIKey: HPQXNTAWOZZCNF-UHFFFAOYSA-N
Target: Pancreatic triacylglycerol lipase (SINGLE PROTEIN), Sus scrofa
UniProt: P00591
Assay: Inhibition of porcine pancreatic lipase assessed as reduction in 4-nitrophenol formation from 4-nitrophenyl butyrate substrate preincubated for 30 mins followed by substrate addition measured after 5 mins by spectrophotometer
Measurement: IC50 = 24220 nM (pChEMBL 4.619999885559082)
Descriptors: MW 342.4, ALogP 4.64, HBA 3, HBD 1, PSA 51.1, QED 0.43
Compound (SMILES): N#Cc1cc(-c2ccnc(Nc3ccc(NC(=O)CN4CC[C@H](F)C4)nc3)n2)cnc1N1CC[C@H](F)C1
InChIKey: DZIXGUZDRHLHQY-OALUTQOASA-N
Target: Inhibitor of nuclear factor kappa-B kinase subunit epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: Q14164
Assay: Time-Resolved Fluorescence-based Lanthascreen Assay: Kinase inhibition assays (10 μL) were performed at 20° C. in 384-well plate format. Compound IC50 values were determined at the apparent Km for ATP (20 μM) based on a radiometric assay (Invitrogen) using 8 or 10 point curves in duplicate. The final reaction conditions contained 400 nM fluorescein-IkBα substrate (DRHDSGLDSMKDE), 20 μM ATP, 2 nM or 8 nM IKKε or TBK1 kinase respectively, and 3% DMSO in kinase assay buffer consisting of 50 mM HEPES (pH 7.5), 10 mM MgCl, 1 mM EGTA, 0.01% Brij-35.Compound dilutions were prepared from 10 mM DMSO stocks by dilution into DMSO. Compound dilution series were further diluted in kinase assay buffer to give a 12% DMSO stock, the final concentration in the assay being 3% DMSO.The kinase phosphorylation assay was initiated by the addition of the kinase and the reaction was allowed to proceed for 1 h or 2.5 h for IKKε and TBK-1 kinase respectively.
Measurement: IC50 < 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 505.53, ALogP 3.08, HBA 9, HBD 2, PSA 122.96, QED 0.5
Compound (SMILES): COc1cc(C(=O)NCCCN(C)C)ccc1Nc1ncc2c(n1)-c1c(nn(C)c1Cc1cccc(-c3cnn(C)c3)c1)CC2
InChIKey: DANRHRCSZZGXLP-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 = 0.08 nM (pChEMBL 10.100000381469727)
Descriptors: MW 605.75, ALogP 4.4, HBA 10, HBD 2, PSA 115.02, QED 0.21
Compound (SMILES): COc1cc(OC)cc(C(=O)Nc2ccc(C)c(-n3ccn4nc(-c5cccnc5)cc34)c2)c1
InChIKey: ORZYTZBSPPUODW-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 = 770 nM (pChEMBL 6.110000133514404)
Descriptors: MW 453.5, ALogP 4.76, HBA 7, HBD 1, PSA 82.68, QED 0.4
Compound (SMILES): CCOC(=O)Cc1csc([C@H](Cc2ccc(NS(=O)(=O)O)cc2)NC(=O)OC(C)(C)C)n1
InChIKey: NKDAZIQHANNNIG-INIZCTEOSA-N
Target: Receptor-type tyrosine-protein phosphatase beta (SINGLE PROTEIN), Homo sapiens
UniProt: P23467
Assay: Inhibition of recombinant HPTPbeta (unknown origin) preincubated for 10 mins followed by substrate addition measured after 15 mins by DiFMUP probe-based fluorescence assay
Measurement: IC50 = 167 nM (pChEMBL 6.78000020980835)
Descriptors: MW 485.58, ALogP 3.27, HBA 8, HBD 3, PSA 143.92, QED 0.36
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(S(=O)(=O)C3CC3)CC2)C1
InChIKey: YBLNSIVEOFFIIM-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Inhibition of human N-terminal His-tagged JAK1 catalytic domain (837 to 1142 residues) expressed in baculovirus infected insect cells assessed as decrease in phosphorylation of biotinylated peptide substrate after 1 hr in presence of 1 mM ATP by HTRF method
Measurement: IC50 <= 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 466.57, ALogP 1.7, HBA 8, HBD 1, PSA 123.8, QED 0.59
Compound (SMILES): CCOC(=O)c1[nH]c2cc(Cl)ccc2c1C(C(=O)NC(C)(C)C)N(C=O)Cc1c(F)cc(F)cc1F
InChIKey: KKADJXPMMUATEF-UHFFFAOYSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: Fluorescence Polarization (FP) Assay: Fluorescent polarization experiments were performed as follows. Briefly, the fluorescence polarization experiments were read on an Ultra Evolution 384-well plate reader (Tecan) with the 485 nm excitation and 535 nm emission filters. The fluorescence intensities parallel (Intparallel) and perpendicular (Intperpedicular) to the plane of excitation were measured in parallel perpendicular black 384-well NBS assay plates (Corning) at room temperature (20° C). The background fluorescence intensities of blank samples containing the references buffer were subtracted and steady-state fluorescence polarization was calculated using the equation: P=(Intparallel Gintperpendicular)/(Intparallel+Gintperpendicular), and the correction factor G (G=0.998 determined empirically) was introduced to eliminate differences in the transmission of vertically and horizontally polarized light. All fluorescence polarization values were expressed in millipolarization units (mP).
Measurement: Ki = 6.8 nM (pChEMBL 8.170000076293945)
Descriptors: MW 523.94, ALogP 5.03, HBA 4, HBD 2, PSA 91.5, QED 0.32
Compound (SMILES): O=C(OC(C(F)(F)F)C(F)(F)F)N1CCN(Cc2ccc(N3CCOCC3)cc2Cl)CC1
InChIKey: VIDXUIDVWFXJOO-UHFFFAOYSA-N
Target: Fatty-acid amide hydrolase 1 (SINGLE PROTEIN), Mus musculus
UniProt: O08914
Assay: In Vitro Competitive Activity-Based Protein Assay: Proteomes (mouse brain membrane fraction or cell lysates) (50 uL, 1.0 mg/mL total protein concentration) were preincubated with varying concentrations of inhibitors at 37 C. After 30 min, FP-Rh (1.0 uL, 50 uM in DMSO) was added and the mixture was incubated for another 30 min at 37 C. Reactions were quenched with SDS loading buffer (50 uL-4x) and run on SDS-PAGE. Following gel imaging, serine hydrolase activity was determined by measuring fluorescent intensity of gel bands corresponding to MAGL, ABHD6 and FAAH using ImageJ 1.43u software.Preparation of Mouse Brain Proteomes from Inhibitor Treated Mice. Inhibitors were administered to wild-type C57Bl/6J by oral gavage in a vehicle of polyethylene glycol. Each animal was sacrificed 4 h following administration and brain proteomes were prepared and analyzed according to previously established methods (See Niphakis, M. J., et al. (2011) ACS Chem. Neurosci. and Long, J. Z., et al. Nat. Chem. Biol. 5:37-44).
Measurement: IC50 > 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 489.84, ALogP 3.92, HBA 5, HBD 0, PSA 45.25, QED 0.6
Compound (SMILES): COC(=O)c1ccc(-c2nc3cc(Cc4cncc5cc(OC)c(OC)cc45)ccc3o2)cc1
InChIKey: WUUYTBRAERHWHG-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Diaxonhit Phosphodiesterase Assay: The phosphodiesterase assay was developed using the LANCE cAMP kit (PerkinElmer). The assay buffer contained HBSS with 5 mM HEPES, 0.1% BSA, and 1.5 mM MgCl2, pH 7.4. PDE10A (BPS Bioscience) was used at 200 pg/well (with a specific activity of 3200 pmole/min/ug with assay conditions: 10 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM MnCl2, 200 uM cAMP, 2.5 kU 5' nucleotidase, 37° C., 20 min) and PDE4D3 (BPS Bioscience) was used at 100 pg/well (with a specific activity of 32713 pmole/min/ug with assay conditions: 10 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM MnCl2, 200 uM cAMP, 2.5 kU 5' nucleotidase, 37° C., 20 min). The Biotin-cAMP tracer, supplied in 10 mmol/L Tris-HCl buffered (pH 8.0) salt solution with 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 0.1% bovine serum albumin (BSA), and 0.05% sodium azide, is used at a dilution of 1/375. The assay detection mixture contained the LANCE Eu-W8044 labeled streptavidin 1/2250 (supplied in 50 mmol/L Tris-HCl buffer).
Measurement: IC50 = 61 nM (pChEMBL 7.210000038146973)
Descriptors: MW 454.48, ALogP 5.44, HBA 7, HBD 0, PSA 83.68, QED 0.32
Compound (SMILES): CCC(C)(C)S(=O)(=O)C[C@H](C1CC1)N1C(=O)[C@@](C)(CC(=O)O)C[C@H](c2cccc(Cl)c2)[C@H]1c1ccc(Cl)cc1
InChIKey: YSOOYNBAOSHOTO-QNYAKKFESA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: Homogenous Time-Resolved Fluorescence Assay (HTRF2 Assay): The standard assay conditions for the in vitro HTRF assay consisted of a 50 ul total reaction volume in black 384-well Costar polypropylene plates in 1×PBS buffer pH 7.4, 1 mM DTT, 0.1% BSA, 2.5 nM GST-hMDM2 (aa 1-188), 5 nM biotinylated-p53 (aa 1-83), 1.8 nM SA-XLent (Cisbio; Bedford, Mass.), 0.6 nM anti-GST cryptate monoclonal antibody (Cisbio; Bedford, Mass.) and 200 mM KF. Amino acid residues 1-188 of human MDM2 were expressed as an amino-terminal glutathione-S-transferase (GST) fusion protein (GST-hMDM2) in Escherichia coli. Residues 1-83 of human p53 were expressed as an amino-terminal AviTag™-TrxA-6×His fusion protein (biotinylated p53) in E. coli. Each protein was purified from cell paste by affinity chromatography.Specifically, 10 uL of GST-hMDM2 was incubated with 10 ul of diluted compound (various concentrations, serially diluted) in 10% DMSO for 20 minutes at room temperature. 20 uL of biotinylated-p53 was added to the GST-hMDM2+compound mixture, and then incubated at room temperature for 60 min. 10 uL of detection buffer consisting of SA-XLent, anti-GST cryptate antibody and KF was added to GST-hMDM2, biotinylated-p53 and compound reaction and left at room temperature to reach equilibrium for >4 hrs. The final concentration of DMSO in the reaction was 2%. Time-resolved fluorescence readings were measured on a microplate multilabel reader. Percentage of inhibition was calculated relative to nutlin-3. All assay conditions remained the same as described above, with the exception of the following changes in reagent concentrations: 0.2 nM GST-hMDM2 (1-188), 0.5 nM biotinylated-p53 (1-83), 0.18 nM SA-XLent, and 100 mM KF.
Measurement: IC50 = 0.6 nM (pChEMBL 9.220000267028809)
Descriptors: MW 594.6, ALogP 6.91, HBA 4, HBD 1, PSA 91.75, QED 0.32
Compound (SMILES): CC[C@]1(C(=O)N2CC[C@](c3ccc(C(F)(C(F)(F)F)C(F)(F)F)cc3)(S(=O)(=O)c3ccc(F)c(C)c3)C2)CC[C@H](C(=O)O)CC1
InChIKey: AHXUPPRSSHLOPV-DOYMOWJKSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Scintillation Proximity Assay (SPA): The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 ug/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 uM to 0.28 nM for 10 min at room temperature in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% Glycerol (Sigma Cat#G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat #RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce [3H] 25-hydroxycholesterol specific binding by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 41 nM (pChEMBL 7.389999866485596)
Descriptors: MW 669.63, ALogP 7, HBA 4, HBD 1, PSA 91.75, QED 0.25
Compound (SMILES): O=C(NC1CC1)c1ccc(-c2cnc3c(NCCCCO)nc(C#CC4(O)CCCC4)cn23)cc1
InChIKey: RBPPNACCGRZVCA-UHFFFAOYSA-N
Target: Dual specificity protein kinase TTK (SINGLE PROTEIN), Homo sapiens
UniProt: P33981
Assay: Mps-1 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 ul 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 ul of a solution of 16.7 adenosine-tri-phosphate (ATP, 16.7 uM=> final conc. in the 5 ul assay volume is 10 uM) and peptide substrate (1.67 uM=> final conc. in the 5 ul assay volume is 1 uM) 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 ul assay volume). The reaction was stopped by the addition of 3 ul 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, PerkinElmer LAS, Rodgau-Jugesheim, 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-Jugesheim, Germany).
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 473.58, ALogP 3.13, HBA 7, HBD 4, PSA 111.78, QED 0.3
Compound (SMILES): C[C@@H](Nc1ncnc2[nH]c(-c3ccc(OCCCC(=O)NO)cc3)cc12)c1ccccc1
InChIKey: NQJNYUNDDOUISQ-MRXNPFEDSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of N-terminal GST-fused human EGFR (His672 to Ala1210 residues) expressed in baculovirus expression system using biotinylated PTP1B at Tyr66 as substrate preincubated for 5 mins followed by substrate addition measured after 30 mins by DELFIA
Measurement: IC50 <= 100 nM (pChEMBL 7)
Descriptors: MW 431.5, ALogP 4.46, HBA 6, HBD 4, PSA 112.16, QED 0.18
Compound (SMILES): NC(=O)c1c(-c2ccc(Oc3ccccc3)cc2)nn2c1[nH]c1ccc(N)cc12
InChIKey: VARKGQPOASCLDR-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Time-Resolved Fluorescence Resonance Energy Transfer Assay: Compounds disclosed herein were tested for inhibition of Btk kinase activity in an assay based on time-resolved fluorescence resonance energy transfer methodology. Recombinant Btk was pre-incubated with the compounds disclosed herein at room temperature for 1 hour in an assay buffer containing 50 mM Tris pH7.4, 10 mM MgCl2, 2 mM MnCl2, 0.1 mM EDTA, 1 mM DT, 20 nM SEB, 0.1% BSA, 0.005% tween-20. The reactions were initiated by the addition of ATP (at the concentration of ATP Km) and peptide substrate (Biotin-AVLESEEELYSSARQ-NH2). After incubating at room temperature for 1 h, an equal volume of stop solution containing 50 mM HEPES pH7.0, 800 mM KF, 20 mM EDTA, 0.1% BSA, Eu cryptate-conjugated p-Tyr66 antibody and streptavidin-labeled XL665 was added to stop the reaction. Plates were further incubated at room temperature for 1 hour, and then the TR-FRET signals (ex337 nm, em 620 nm/665 nm) were read on BMG PHERAstar FS instrument. The residual enzyme activity in presence of increasing concentrations of compounds was calculated based on the ratio of fluorescence at 615 nm to that at 665 nm. The IC50 for each compound was derived from fitting the data to the four-parameter logistic equation by Graphpad Prism software.
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 383.41, ALogP 3.96, HBA 5, HBD 3, PSA 111.43, QED 0.41
Compound (SMILES): CNC(=O)CCc1ccc(Nc2nc(C)cc(Nc3cc(C4CC4)n[nH]3)n2)cc1
InChIKey: PJTPBKBOLOBDTA-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 391.48, ALogP 3.55, HBA 6, HBD 4, PSA 107.62, QED 0.47
Compound (SMILES): Nc1ncnc(N2CCC(c3nc(-c4ccc(F)c(C(F)(F)F)c4)cn3CCN3CCCCC3)CC2)c1-c1ccccc1F
InChIKey: UREVFJWGXHMNOI-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 = 5.1 nM (pChEMBL 8.289999961853027)
Descriptors: MW 611.66, ALogP 6.76, HBA 7, HBD 1, PSA 76.1, QED 0.23
Compound (SMILES): NC(=S)NNc1nncc2ccccc12
InChIKey: BRPCQCNGNFEDIO-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of EGFR (unknown origin) using poly (Glu, Tyr) 4:1 as substrate after 1 hr by ELISA
Measurement: IC50 = 77 nM (pChEMBL 7.110000133514404)
Descriptors: MW 219.27, ALogP 0.79, HBA 4, HBD 3, PSA 75.86, QED 0.51
Compound (SMILES): CNC(=O)CC1(NC(=O)c2cc(O[C@@H](C)C(F)(F)F)c(C3CC3)cn2)COC1
InChIKey: ASMGXYRKSILTNL-JTQLQIEISA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1×HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min. Compounds were added to a final assay volume of 100 μl and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 μl detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.
Measurement: EC50 = 116.4 nM (pChEMBL 6.929999828338623)
Descriptors: MW 401.39, ALogP 1.92, HBA 5, HBD 2, PSA 89.55, QED 0.73
Compound (SMILES): COc1cccc(OC(C)C)c1-c1cccc(C(CC(=O)O)c2nccs2)c1
InChIKey: FZPWDIXQFMGZRR-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 = 165 nM (pChEMBL 6.78000020980835)
Descriptors: MW 397.5, ALogP 5.21, HBA 5, HBD 1, PSA 68.65, QED 0.56
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccc(F)cc3)n2)CCN(c2ccc(F)nc2)CC1
InChIKey: YZAGYRRAHHADSJ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 0.4 nM (pChEMBL 9.399999618530273)
Descriptors: MW 437.45, ALogP 3.31, HBA 7, HBD 2, PSA 112.86, QED 0.57
Compound (SMILES): CCCc1nc2ccccc2n1Cc1ccc(-c2ccc(C(=O)O)cc2)cc1
InChIKey: HENIXOFDIHJWSF-UHFFFAOYSA-N
Target: Peroxisome proliferator-activated receptor gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P37231
Assay: Transactivation at Gal4 fused PPARgamma LBD (unknown origin) expressed in African green monkey COS7 cells after 42 hrs by luciferase assay
Measurement: EC50 = 1880 nM (pChEMBL 5.730000019073486)
Descriptors: MW 370.45, ALogP 5.4, HBA 3, HBD 1, PSA 55.12, QED 0.49
Compound (SMILES): O=C(CC1CCCC1)N1CCC(Oc2ccc(NC(=O)N3Cc4ccccc4C3)cn2)CC1
InChIKey: DVVAPWQWLSLATO-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 2:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1287128.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 128 nM (pChEMBL 6.889999866485596)
Descriptors: MW 448.57, ALogP 4.58, HBA 4, HBD 1, PSA 74.77, QED 0.73
Compound (SMILES): CCOCCn1c(C2CN(c3cc(-n4nc(C)nc4C)nc(C)n3)C2)nc2ccccc21
InChIKey: YCUKEQMJIZDJQK-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 = 8.5 nM (pChEMBL 8.069999694824219)
Descriptors: MW 432.53, ALogP 2.97, HBA 9, HBD 0, PSA 86.78, QED 0.42
Compound (SMILES): C[C@@H]1CN(c2cc(-c3n[nH]c4ccc(-c5cnc(N6CCCC6)s5)cc34)ncn2)C[C@H](C)O1
InChIKey: ACXSBSVOUFZGBC-IYBDPMFKSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LanthaScreen™ Assay (5 mM ATP): a) 400 nl of a 1:2.15 serial dilution of test compound(98 μM top assay concentration) is spotted via Labcyte Echo to certain wells in a 384 well black, untreated plate. Control wells contain 400 nl of either DMSO or 400 nl of a known inhibitor in DMSO. b) 10 μl of a 2.5 nM LRRK2(G2019S mutation, GST-LRRK2(amino acids 970-2527)) enzyme solution in 1× assay buffer(50 mM Tris pH 8.5, 10 mM MgCl2, 0.01% Brij-35, 1.0 mM EGTA, 2 mM DTT, 0.05 mM NaVO4) is added to all wells. c) A 30 minute room temperature incubation is followed by addition of 10 μl of 800 nM fluorescein labeled LRRKtide peptide substrate and 10 mM ATP solution in 1× assay buffer to all wells. d) After a 35 minute room temperature incubation, 20 μl of TR-FRET Dilution Buffer(Invitrogen PV3756B) containing 4 nM Tb-labeled anti-phospho LRRKtide antibody and 20 mM EDTA is added to all wells. e) Plates are incubated at room temperature for 1 hour and read on an Envision™ multi-mode plate reader with LanthaScreen™ settings. Results are analysed using Assay Data Analyzer.
Measurement: IC50 = 7.6 nM (pChEMBL 8.119999885559082)
Descriptors: MW 461.6, ALogP 4.36, HBA 8, HBD 1, PSA 83.06, QED 0.48
Compound (SMILES): C=CC(=O)Nc1cc(C)ccc1C1CCNc2c(C(N)=O)c(-c3ccc(OCC4CC4)cc3)nn21
InChIKey: LNQORQSLRHYZLO-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Time-Resolved Fluorescence Resonance Energy Transfer Assay: Compounds disclosed herein were tested for inhibition of Btk kinase activity in an assay based on time-resolved fluorescence resonance energy transfer methodology. Recombinant Btk was pre-incubated with the compounds disclosed herein at room temperature for 1 hour in an assay buffer containing 50 mM Tris pH7.4, 10 mM MgCl2, 2 mM MnCl2, 0.1 mM EDTA, 1 mM DT, 20 nM SEB, 0.1% BSA, 0.005% tween-20. The reactions were initiated by the addition of ATP (at the concentration of ATP Km) and peptide substrate (Biotin-AVLESEEELYSSARQ-NH2). After incubating at room temperature for 1 h, an equal volume of stop solution containing 50 mM HEPES pH7.0, 800 mM KF, 20 mM EDTA, 0.1% BSA, Eu cryptate-conjugated p-Tyr66 antibody and streptavidin-labeled XL665 was added to stop the reaction. Plates were further incubated at room temperature for 1 hour, and then the TR-FRET signals (ex337 nm, em 620 nm/665 nm) were read on BMG PHERAstar FS instrument. The residual enzyme activity in presence of increasing concentrations of compounds was calculated based on the ratio of fluorescence at 615 nm to that at 665 nm. The IC50 for each compound was derived from fitting the data to the four-parameter logistic equation by Graphpad Prism software.
Measurement: IC50 = 5.6 nM (pChEMBL 8.25)
Descriptors: MW 471.56, ALogP 4.28, HBA 6, HBD 3, PSA 111.27, QED 0.42
Compound (SMILES): O=C(CN1CCN(c2ccnc3cc(Cl)ccc23)CC1)Nc1ncc([N+](=O)[O-])s1
InChIKey: PFIJUFCFDBWLKM-UHFFFAOYSA-N
Target: DNA gyrase subunit B (SINGLE PROTEIN), Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155)
UniProt: A0QNE0
Assay: Inhibition of Mycobacterium smegmatis 155 6His-tagged DNA gyrase B catalytic domain ATPase activity expressed in Escherichia coli BL21 (DE3) pLysS cells assessed as inorganic phosphate release after 120 mins in presence of ATP by malachite green dye based assay
Measurement: IC50 = 14600 nM (pChEMBL 4.840000152587891)
Descriptors: MW 432.89, ALogP 3.01, HBA 8, HBD 1, PSA 104.5, QED 0.49
Compound (SMILES): COc1ccc(C2=C(C#N)C(=O)NC(c3ccc(OCCCC(F)(F)F)cc3)(C(F)(F)F)C2)cc1Cl
InChIKey: AJLMVRIHSUKYDI-UHFFFAOYSA-N
Target: Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q10469
Assay: MGAT LCMS Assay: The MGAT enzyme reactions were performed in CORNING Falcon 96-well Polypropylene plates, in a total volume of 60 uL of 50 mM Potassium Phosphate buffer pH 7.4, containing a final concentration of 100 uM 2-oleoylglycerol, 15 uM oleoyl-Coenzyme A and 0.0013 ug/uL Human or Mouse MGAT-2 or 0.0026 ug/uL Rat recombinant MGAT-2 membranes expressed in Sf9 cells. Assay plates were run through a fully automated robotics system and shaken for 5 seconds every minute for a total 10 minutes. The reactions were then quenched with 120 uL of ice cold methanol containing 1 ug/mL 1,2-distearoyl-rac-glycerol as the internal standard. Plates were shaken for 2 minutes and spun down to remove protein precipitation. After the spin, samples were transferred to LC/MS compatible PCR plates. For LC/MS analysis, a ThermoFisher Surveyor pump, utilizing a Waters Symmetry C8, 50x2.1 mm column, was used for the chromatography of enzyme products.
Measurement: IC50 = 112 nM (pChEMBL 6.949999809265137)
Descriptors: MW 532.87, ALogP 6.32, HBA 4, HBD 1, PSA 71.35, QED 0.33
Compound (SMILES): O=C(O)CC(c1cccc(-c2ccccc2)c1)c1nccs1
InChIKey: MGSWYLXRNPBIPP-UHFFFAOYSA-N
Target: Lysosomal protective protein (SINGLE PROTEIN), Homo sapiens
UniProt: P10619
Assay: Inhibition Assay: 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, as substrate then bradykinin carrying an N-terminal® Bodipy FL (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl) label (JPT Peptide Technologies GmbH; dissolved in assay buffer) was added to the mixture. The final concentration of cathepsin A was 833 ng/ml and the final concentration of labeled bradykinin 2 μM. After incubation for 15 min at room temperature the reaction was stopped by the addition of stop buffer (130 mM 2-(4-(2-hydroxy-ethyl)-piperazin-1-yl)-ethanesulfonic acid, pH 7.4, containing (v/v) 0.013%° Triton X-100, 0.13% Coating Reagent 3 (Caliper Life Sciences), 6.5% DMSO and 20 μM ebelactone B (Sigma, #E0886)).
Measurement: IC50 = 2586 nM (pChEMBL 5.590000152587891)
Descriptors: MW 309.39, ALogP 4.42, HBA 3, HBD 1, PSA 50.19, QED 0.76
Compound (SMILES): Fc1c(Cl)cc(Nc2n[nH]c3cccnc23)cc1Cl
InChIKey: WGTVTOIDLDFGBS-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14833
Assay: Receptor Activity Assay: The disclosed compounds and compositions can be evaluated for their ability to act as a potentiator of metabotropic glutamate receptor activity, in particular mGluR4 activity, by any suitable known methodology known in the art. For example, Chinese Hamster Ovary (CHO) cells transfected with human mGluR4 or HEK cells co-transfected with rat mGluR4 and the G-protein regulated Inwardly Rectifying Potassium channel (GIRK) were plated in clear bottom assay plates for assay in a Hamamatsu FDSS Fluorometric Plate Reader. The cells were loaded with either a Ca2+-sensitive fluorescent dye or the thallium responsive dye and the plates were washed and placed into a suitable kinetic plate reader. For human mGluR4 assays, a fluorescence baseline was established for 3-5 seconds, the disclosed compounds were then added to the cells, and the response in cells was measured.
Measurement: EC50 = 410 nM (pChEMBL 6.389999866485596)
Descriptors: MW 297.12, ALogP 4.15, HBA 3, HBD 2, PSA 53.6, QED 0.7
Compound (SMILES): C[C@@H](CN)NC(=O)c1cc(NC(=O)c2cc(-c3ncccc3F)c(F)cc2Cl)n(-c2ccccc2)n1
InChIKey: FYAABJULMNOBPX-AWEZNQCLSA-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 = 185 nM (pChEMBL 6.730000019073486)
Descriptors: MW 510.93, ALogP 4.2, HBA 6, HBD 3, PSA 114.93, QED 0.34
Compound (SMILES): O=C(Nc1cn(CC(=O)N2CCC(NC3CCC3)CC2)nc1-c1cc(Cl)ccc1OC(F)F)c1cnn2cccnc12
InChIKey: YWAQBIMFQJZPHY-UHFFFAOYSA-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 = 0.91 nM (pChEMBL 9.039999961853027)
Descriptors: MW 599.04, ALogP 4.23, HBA 9, HBD 2, PSA 118.68, QED 0.3
Compound (SMILES): COc1cc(-c2cc(C)cc(C)c2)ccc1-n1c(=O)ccc2cc(S(=O)(=O)Nc3ccon3)ccc21
InChIKey: IDDFFDJUOPMNJI-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 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 501.56, ALogP 5.07, HBA 7, HBD 1, PSA 103.43, QED 0.35
Compound (SMILES): N=C(N)Nc1ccc(C(=O)Oc2ccc3nc(C(=O)Nc4ccc(CC(=O)O)cc4)ccc3c2)cc1
InChIKey: LWEOUOVULLARDN-UHFFFAOYSA-N
Target: Serine protease 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P07477
Assay: Inhibition Assay: The compound was dissolved in dimethyl sulfoxide (DMSO), and diluted to an arbitrary concentration (A). A was 100-fold diluted with a buffer (0.1 M Tris (pH 8.0), 0.15 M NaCl, 10 mM CaCl2, 0.05% Brij38) (B). The r-h trypsin was diluted with a buffer to 0.088 ug/mL, and the m-trypsin was diluted with a buffer to 1/50 (C). The dilution ratio of the m-trypsin (1/50) was set to exhibit the same activity as the 0.088 ug/mL r-h trypsin as determined by kinetic analysis. The substrate solution of a substrate for the enzyme reaction, BZiPAR, (Rhodamine Reference Substrate) was diluted with a buffer to 5 umol/L (D). B; 5 uL, C; 5 uL, and D; 10 uL were added to a 384-plate, and incubated at room temperature for 30 minutes. The fluorescent signals were detected with Ex/Em = 497/520 using Tecan Safire Fluorometer. The compound was reviewed from 2500 nM to its 3-fold value, 0.0075 nM, at 12 concentrations, and the inhibitory rate of each compound was calculated.
Measurement: IC50 = 0.29 nM (pChEMBL 9.539999961853027)
Descriptors: MW 483.48, ALogP 3.64, HBA 6, HBD 5, PSA 167.49, QED 0.11