lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC1(C)CCC(CN2CCN(c3ccc(C(=O)NS(=O)(=O)c4ccc(NC5CCN(C6COC6)CC5)c([N+](=O)[O-])c4)c(Oc4cnc5[nH]ccc5c4)c3)CC2)=C(c2ccc(Cl)cc2)C1
InChIKey: AZIUZWNRHASVGU-UHFFFAOYSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Representative compounds were serially diluted in dimethyl sulfoxide (DMSO) starting at 50 uM (2x starting concentration; 10% DMSO) and 10 uL were transferred into a 384-well plate. Then 10 uL of a protein/probe/antibody mix was added to each well at final concentrations listed in TABLE 1. Protein:GST-Bcl-2, Probe: F-Bak Peptide Probe Acetyl-GQVGRQLAIIGDK(6-FAM)INR-amide(SEQ ID NO: 1), Protein(nM): 1, Probe (nM): 100, Antibody: Tb-anit-GST, Antibody (nm): 1.  The samples are then mixed on a shaker for 1 minute and incubated for an additional 3 hours at room temperature. For each assay, the probe/antibody and protein/probe/antibody were included on each assay plate as negative and positive controls, respectively. Fluorescence was measured on the ENVISION plate reader (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak peptide) and 495/510 nm (Tb-labeled anti-Histidine antibody) emission filters.
Measurement: Ki < 0.01 nM (pChEMBL 11)
Descriptors: MW 909.51, ALogP 8.1, HBA 12, HBD 3, PSA 175.27, QED 0.08
Compound (SMILES): O=C1NC(=O)/C(=C/c2cnn3c(NC4CC4)nc(NCc4nc5c(s4)CCCCC5)nc23)N1
InChIKey: MDPDMLQEIIFQRV-ZSOIEALJSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 75 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and the resid
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 465.54, ALogP 2.22, HBA 10, HBD 4, PSA 138.23, QED 0.25
Compound (SMILES): COc1ccc(-c2ccnc(C(=O)O)n2)cc1Cl
InChIKey: NIZYZXODGRQPOJ-UHFFFAOYSA-N
Target: Kynurenine 3-monooxygenase (SINGLE PROTEIN), Homo sapiens
UniProt: O15229
Assay: Inhibition of human KMO
Measurement: IC50 = 1.2 nM (pChEMBL 8.920000076293945)
Descriptors: MW 264.67, ALogP 2.5, HBA 4, HBD 1, PSA 72.31, QED 0.92
Compound (SMILES): COc1cc2c(Nc3ccc(NC(=O)Nc4ccccc4)cc3)ncnc2cc1OCc1ccccc1
InChIKey: BUGHTZSHXDNUKL-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of N-terminal GST-tagged human VEGFR-2 cytoplasmic domain (790 to 1356 residues) expressed in baculovirus expression system preincubated for 10 mins followed by FAM-labelled peptide substrate addition by caliper mobility shift assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 491.55, ALogP 6.61, HBA 6, HBD 3, PSA 97.4, QED 0.23
Compound (SMILES): CCN(CC)C(=O)c1cc(Cl)cc(S(=O)(=O)c2ccc(NC(=O)/C=C/c3cccnc3)cc2)c1
InChIKey: AINJYQJZHQACAH-KPKJPENVSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Biochemical Inhibition Assay: The NAMPT enzymatic reactions were carried out in Buffer A (50 mM Hepes pH 7.5, 50 mM NaCl, 5 mM MgCl2, and 1 mM THP) in 96-well V-bottom plates. The compound titrations were performed in a separate dilution plate by serially diluting the compounds in DMSO to make a 100x stock. Buffer A (89 uL) containing 33 nM of NAMPT protein was added to 1 uL of 100x compound plate containing controls (e.g. DMSO or blank). The compound and enzyme mix was incubated for 15 minutes at room temperature, then 10 uL of 10x substrate and co-factors in Buffer A were added to the test well to make a final concentration of 1 uM NAM, 100 uM 5-Phospho-D-ribose 1-diphosphate (PRPP), and 2.5 mM Adenosine 5'-triphosphate (ATP). The reaction was allowed to proceed for 30 minutes at room temperature, then was quenched with the addition of 11 uL of a solution of formic acid and L-Cystathionine to make a final concentration of 1% formic acid and 10 uM L-Cystathionine.
Measurement: IC50 = 19.4 nM (pChEMBL 7.710000038146973)
Descriptors: MW 498, ALogP 4.7, HBA 5, HBD 1, PSA 96.44, QED 0.45
Compound (SMILES): O=C(NCc1ccc2c(c1)oc1ccccc12)c1ccc(-c2cnccn2)cc1
InChIKey: VYCNSPHXPXIAHF-UHFFFAOYSA-N
Target: Protein-serine O-palmitoleoyltransferase porcupine (SINGLE PROTEIN), Mus musculus
UniProt: Q9JJJ7
Assay: Inhibition of porcupine in mouse L Wnt3A cells co-cultured with HEK293 cells after 48 hrs by Super-top flash reporter gene assay
Measurement: IC50 = 701 nM (pChEMBL 6.150000095367432)
Descriptors: MW 379.42, ALogP 4.97, HBA 4, HBD 1, PSA 68.02, QED 0.48
Compound (SMILES): O=C(O)COc1ccc(Cl)cc1C1c2scnc2CCN1C(=O)OCc1cccc(F)c1
InChIKey: RFTNSYMSKCQXLQ-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Radioligand Displacement Assay: Binding assay was performed in a final assay volume of 250 μl. First, 25 μl of test compound, previously diluted in Binding-Buffer (Binding-Buffer: 50 mM Tris-Base, 100 mM NaCl, 1 mM EDTA, 0.1% BSA (protease free), 0.01% NaN3, 10 mM MnCl2, pH 7.0) was placed into each well. After addition of 75 μl Binding-Buffer, 50 μl of the radioligand 3H-PGD2 (at 2.5 nM (220.000 dpm/well) from ANAWA ART0662) was added to each well. Binding assay was started by addition of 100 μl CRTH2 membrane fragments, reaching a final concentration of 20 μg/well. For non-specific binding, PGD2 was added to the reaction mixture to 10 mM final concentration. This assay mix was incubated for 90 minutes at room temperature and then filtered through a GF/C filter 96-well plate which was pre-soaked for 3 hours in 0.5% polyethyleneimine (PEI). The filter-wells were washed three times with ice cold Binding-Buffer. Then, 40 μl of Microscint-40 (Packard) was added to each well and the re
Measurement: IC50 = 4.4 nM (pChEMBL 8.359999656677246)
Descriptors: MW 476.91, ALogP 4.68, HBA 6, HBD 1, PSA 88.96, QED 0.56
Compound (SMILES): N#Cc1ccc2c(c1)CN(C(=O)Nc1ccc(C(=O)NCC3CCCC3)cc1)C2
InChIKey: YUUNMEWAIWZTQG-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 = 2.07 nM (pChEMBL 8.680000305175781)
Descriptors: MW 388.47, ALogP 4.03, HBA 3, HBD 2, PSA 85.23, QED 0.83
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3F)ncnc2cc1OC[C@@H]1CN2CCC[C@H]2C(=O)N1C
InChIKey: RPLCLUCJTHAIOW-UGSOOPFHSA-N
Target: Ephrin type-B receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P54760
Assay: Inhibition of recombinant human N-terminal His6-tagged EphB4 kinase domain (E605 to E890 residues) expressed in baculovirus using poly-AEKY as substrate after 150 mins in presence of gamma33P-ATP by liquid scintillation spectrometry
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 520.39, ALogP 4.51, HBA 7, HBD 1, PSA 79.82, QED 0.48
Compound (SMILES): CCOc1ccc2c(c1)CN(C(=O)c1nc(N)nc3ccc(C)cc13)C2
InChIKey: RIZKVPUYNDIGMM-UHFFFAOYSA-N
Target: Heat shock protein HSP 90-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P07900
Assay: Recombinant Hsp90 [His-Tev-huHsp90-alpha (9-236)] was immobilized on a Biacore CM5 chip at 25 degrees C and a flow rate of 30 uL/min using amine coupling at pH 4.50 according to Biacore standard protocol. Hsp90 was applied at a concentration of 20 ug/mL with 50uM 17-DMAG.Data sets were processed and analyzed using the software Biacore 4000 Evaluation. Solvent corrected and double-referenced association and dissociation phase data were fitted to a simple 1:1 interaction model with mass transport limitations
Measurement: Kd = 4826.33 nM (pChEMBL 5.320000171661377)
Descriptors: MW 348.41, ALogP 3.08, HBA 5, HBD 1, PSA 81.34, QED 0.79
Compound (SMILES): CCCN(C)C(=O)c1ccc(NC(=O)N2Cc3cccnc3C2)cc1
InChIKey: NLPPZTLUNXVYJZ-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 = 2230 nM (pChEMBL 5.650000095367432)
Descriptors: MW 338.41, ALogP 3.11, HBA 3, HBD 1, PSA 65.54, QED 0.93
Compound (SMILES): O=C(O)c1cc2ccccc2nc1/C=C/c1cn2nccc(N3CCOCC3)c2n1
InChIKey: UUKXULSAWQOVJN-AATRIKPKSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q9QYJ6
Assay: In Vitro Assay: Rat recombinant PDE10a (rPDE10a) was expressed in Sf9 cells using a recombinant rPDE10a baculovirus construct. Cells were harvested after 48 h of infection and the rPDE10a protein was purified by metal chelate chromatography on Ni-sepharose 6FF. Tested compounds were dissolved and diluted in 100% DMSO to a concentration 100 fold of the final concentration in the assay. Compound dilutions (0.4 uL) were added in 384 well plates to 20 uL of incubation buffer (50 mM Tris pH 7.8, 8.3 mM MgCl2, 1.7 mM EGTA). 10 uL of rPDE10a enzyme in incubation buffer was added and the reaction was started by addition of 10 uL substrate to a final concentration of 60 nM cAMP and 0.008 uCi3H-cAMP. The reaction was incubated for 60 min at rt. After incubation, the reaction was stopped with 20 uL of 17.8 mg/mL PDE SPA beads. After sedimentation of the beads during 30 min the radioactivity was measured in a Perkin Elmer Topcount scintillation counter.
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 401.43, ALogP 2.98, HBA 7, HBD 1, PSA 92.85, QED 0.56
Compound (SMILES): O=C(Nc1ccc(S(=O)(=O)NCC(O)CO)cc1)c1cc(N(CC2CC2)C2CCCCC2)ncn1
InChIKey: DSRUTEKAQLWZOK-UHFFFAOYSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: 35S-GTPgammaS Binding: Measurements of 35S-GTPgammaS Binding: Membranes (1 to 10 ug protein) prepared as described above, were incubated in 96-well Scintiplates (PerkinElmer) with test compounds diluted in DMSO, in 180 ul of 20 mM HEPES, pH 7.4, 10 mM MgCl2, 2 ug/well Saponin, 0.2% fatty acid free BSA (Assay buffer), 140 mM NaCl and 1.7 uM GDP. The assay was initiated with the addition of 20 ul of 1.5 nM [35S]-GTPgammaS (1100 Ci/mmol; GE Healthcare) in assay buffer. After 60 min incubation at 30° C. on a shaker, plates were centrifuged for 10 min at 2000 RPM. Supernatant was discarded and membrane bound radioactivity was measured on a PerkinElmer 1450 MicroBeta counter. Triplicate samples were averaged and expressed as % response relative to S1P activation in absence of compound (n=2).
Measurement: EC50 = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 503.63, ALogP 1.91, HBA 8, HBD 4, PSA 144.75, QED 0.36
Compound (SMILES): COc1ncccc1S(=O)(=O)N(Cc1ccc(F)c(C(F)(F)F)c1)c1sc2ccccc2c1C
InChIKey: KEZCXZXEMNUNCO-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Functional Assay: The functional activity of compounds of the formula (I) was determined by measuring changes in intracellular calcium concentration using a Ca2+-sensitive fluorescent dye. The changes in fluorescent signal were monitored by a fluorescence plate reader, either a FLIPR (Molecular Devices) or FDSS (Hamamatsu). Increases in intracellular Ca2+ concentration were readily detected upon activation with icilin.At 24 hrs prior to assay, HEK293 cells stably expressing canine TRPM8 were seeded in culture medium in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences, NJ, USA) and grown overnight in 5% CO2 at 37° C. On assay day, growth media was removed and cells were loaded with Calcium 3 Dye (Molecular Devices) for 35 min at 37° C., under 5% CO2 and then for 25 min at room temperature and atmosphere. Subsequently, cells were tested for agonist-induced increases in intracellular Ca2+ levels using FLIPR or FDSS. Cells were challenged with a compound of the Formula (I) (at varying concentrations) and intracellular Ca2+ was measured for 5 min prior to the addition of icilin to all wells to achieve a final concentration that produces approximately an 80% maximal response. EC50 or IC50 values for compounds of the present invention were determined from eight-point dose-response studies.
Measurement: IC50 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 510.53, ALogP 6.17, HBA 5, HBD 0, PSA 59.5, QED 0.29
Compound (SMILES): COc1nn2cc(-c3ccc(Cl)cc3)nc2s1
InChIKey: UKCLNFBWHOFDDT-UHFFFAOYSA-N
Target: Proteinase-activated receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96RI0
Assay: Antagonist activity at PAR4 in human platelets assessed as inhibition of gamma-thrombin-induced PAC1 binding response preincubated for 20 mins followed by agonist addition for 20 mins by FITC-based flow cytometric analysis
Measurement: IC50 = 621 nM (pChEMBL 6.210000038146973)
Descriptors: MW 265.73, ALogP 3.12, HBA 5, HBD 0, PSA 39.42, QED 0.71
Compound (SMILES): CC(C)OC(=O)/C=C\n1cnc(-c2csc(Cl)c2)n1
InChIKey: ZJWVIEHPTNDCCB-ARJAWSKDSA-N
Target: Exportin-1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14980
Assay: Inhibition of Crm1 in human U2OS cells expressing GFP-fused HIV1 Rev assessed as decrease in nuclear export of GFP-fused HIV1 Rev after 1 hr by Hoechst 33258 staining-based assay
Measurement: IC50 <= 1000 nM (pChEMBL 6)
Descriptors: MW 297.77, ALogP 3.08, HBA 6, HBD 0, PSA 57.01, QED 0.64
Compound (SMILES): CC1CCCCN1C(=O)c1ccc(NC(=O)N2Cc3ccccc3C2)cc1
InChIKey: UCOGVUIJXRKQLZ-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 1: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-1251667.0. 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 = 284 nM (pChEMBL 6.550000190734863)
Descriptors: MW 363.46, ALogP 4.25, HBA 2, HBD 1, PSA 52.65, QED 0.87
Compound (SMILES): CC(C)(CNS(=O)(=O)c1ccccc1)C(=O)NC1C2CC3CC1CC(C(N)=O)(C3)C2
InChIKey: ZSDIIUALSOGCPA-UHFFFAOYSA-N
Target: 11-beta-hydroxysteroid dehydrogenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28845
Assay: HTRF Assay : The inhibiting activity of 11β-HSD1 derived from microsomal fractions was measured using the HTRF assay (62CO2PEB, Cisbio). Different concentrations of compounds were added to 96-well plates, followed by the addition of TE buffer (20 mM Tris buffer and 5 mM EDTA, pH 6.0) containing 200 μM NADPH (N1630, Sigma) and 160 nM cortisone (C2755, Sigma), The reactions were initiated by the addition of human microsomal fractions (M0317, Sigma), and were allowed to incubate for 2 h at 37° C. Europium (Eu3+) cryptate and XL665-conjugated cortisol were then added to each well and incubated for an additional 2 h at room temperature.
Measurement: IC50 = 28.6 nM (pChEMBL 7.539999961853027)
Descriptors: MW 433.57, ALogP 1.79, HBA 4, HBD 3, PSA 118.36, QED 0.61
Compound (SMILES): Cc1cccc2cc(Cn3nc(I)c4c(N)ncnc43)n(-c3ccccc3Cl)c(=O)c12
InChIKey: MLINGYDIVFWDJY-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Inhibition of recombinant human C-terminal His6-tagged p110gamma expressed in insect cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 542.77, ALogP 4.33, HBA 7, HBD 1, PSA 91.62, QED 0.34
Compound (SMILES): CC(CO)n1cc(C(=O)c2cc(NC(=O)Cc3ccc(C(F)(F)F)cc3)ccn2)c2cnccc21
InChIKey: CRJGVCIGPRPYOO-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: HTRF Assay: Isolated TRK Enzyme assays use the HTRF KinEASE-TK kit (Cisbio Cat#62TK0PEJ) with recombinant His-tagged cytoplasmic domains of each TRK receptor sourced from Invitrogen (see table below). This activity-assay measures the phosphorylation of tyrosine residues within a substrate from the HTRF kit which has been validated by Cisbio for a variety of tyrosine kinases including the TRK receptors.Assay Details: Invitrogen Amino FAC FAC Assay ReactionTarget Cat# acids enzyme ATP TimeTRKA PV3144 aa 441-4 nM 40 uM 35 min (NTRK1) 796 TRKB PV3616 aa 526-1 nM 1.4 uM 40 min (NTRK2) 838 TRKC PV3617 aa 510- 10 nM 15 uM 30 min (NTRK3) 8250.5 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using the compound of Example 135 disclosed in WO2005/116035 of 150 uM is also prepared on each test plate. High percentage effect (HPE) is defined by 150 uM.
Measurement: IC50 = 141 nM (pChEMBL 6.849999904632568)
Descriptors: MW 482.46, ALogP 4.42, HBA 6, HBD 2, PSA 97.11, QED 0.38
Compound (SMILES): CCC1CC(=O)NN=C1c1ccc2nc(-c3cccc(OCCN(CC(C)C)CC(C)C)c3)oc2c1
InChIKey: JEYUAUFCHBKBGD-UHFFFAOYSA-N
Target: Glucose-dependent insulinotropic receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TDV5
Assay: AlphaScreen cAMP Assay: MIN6 cells [Miyazaki J et al. Endocrinology. 1990 July; 127(1):126-32] were stably transfected with an expression vector for human GPR119 cDNA (Acc. No. NP-848566). Min-6/hGPR119 cells are cultured in DMEM, 10% FBS, 50 uM beta-mercaptoethanol, 0.3 mg/mL Geniticin, 2 mM GlutaMAX at 37 C. 5% CO2. For the assay, the cells are placed in Optiplates (white, 384-well, 160W-barcoded, TC, sterile with lid, Cat. No. #6007688 (Perkin Elmer); 10000 cells/well; 50 ul). The plates covered with lids are then incubated for 24 hours at 37 C./5% CO2.After the medium has been sucked out of the wells completely, 10 ul of the test compound are added, the compounds are diluted beforehand with stimulating buffer (140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 10 mM Hepes, 5 mM NaHCO3; pH 7.4. 0.5 mM IBMX and 0.1% BSA, the final DMSO concentration is 1%). After 45 minutes' incubation at RT, the cAMP concentrations are determined using the AlphaScreen cAMP Assay.
Measurement: EC50 = 248 nM (pChEMBL 6.610000133514404)
Descriptors: MW 490.65, ALogP 5.74, HBA 6, HBD 1, PSA 79.96, QED 0.37
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccc(S(=O)(=O)C(F)F)cc3)n2)CCC(N2CCC(F)(F)CC2)CC1
InChIKey: GYSAXDBJNHZIQI-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 = 0.1 nM (pChEMBL 10)
Descriptors: MW 556.59, ALogP 4, HBA 8, HBD 2, PSA 134.11, QED 0.47
Compound (SMILES): CCCCCCCCCCCCCCO[C@@H]1[C@@H](OCCCCCCCCCCCCCC)[C@@H](OC)O[C@H](COC(=O)CCC(=O)OC[C@H](NC(=O)OCC2c3ccccc3-c3ccccc32)C(=O)O)[C@H]1OC
InChIKey: RZWDXHPALWPAIZ-KYRVERLDSA-N
Target: Tumor necrosis factor (SINGLE PROTEIN), Homo sapiens
UniProt: P01375
Assay: Biological Assay: The inhibitory activity of the novel conjugates on LPS-induced TNFα production was investigated in vitro using THP-1 macrophages. THP-1 macrophages are a useful system for studying inflammatory processes and serve as a model for peripheral monocytes/macrophages and their responses to bacterial infection.
Measurement: IC50 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 1010.36
Compound (SMILES): COC(C)(C(=O)Nc1cnn(Cc2c(C)noc2C)c1)c1ccccc1
InChIKey: YUHWNDOAGXIUDW-UHFFFAOYSA-N
Target: Taste receptor type 2 member 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYW2
Assay: Fluorescence Polarization Assays: In another embodiment, Fluorescence Polarization ("FP") based assays may be used to detect and monitor ligand binding. Fluorescence polarization is a versatile laboratory technique for measuring equilibrium binding, nucleic acid hybridization, and enzymatic activity. Fluorescence polarization assays are homogeneous in that they do not require a separation step such as centrifugation, filtration, chromatography, precipitation, or electrophoresis. These assays are done in real time, directly in solution and do not require an immobilized phase. Polarization values can be measured repeatedly and after the addition of reagents since measuring the polarization is rapid and does not destroy the sample. Generally, this technique can be used to measure polarization values of fluorophores from low picomolar to micromolar levels.
Measurement: IC50 = 2100 nM (pChEMBL 5.679999828338623)
Descriptors: MW 354.41, ALogP 3.04, HBA 6, HBD 1, PSA 82.18, QED 0.74
Compound (SMILES): COc1ccccc1-n1[se]c2ccccc2c1=O
InChIKey: KKKQSDGDCNBMFN-UHFFFAOYSA-N
Target: Methionine aminopeptidase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P50579
Assay: Non-covalent/mixed type inhibition of recombinant human MetAP2 using Met-AMC as substrate preincubated for 30 mins followed by substrate addition by spectrofluorimetric method
Measurement: IC50 = 9980 nM (pChEMBL 5)
Descriptors: MW 304.21
Compound (SMILES): Nc1ncnc2c1c(-c1ccc(O)cc1)nn2Cc1nc2cccc(C#CCCCC(=O)N3CCOCC3)c2c(=O)n1Cc1ccccc1Cl
InChIKey: LZLZWYUFFAROFC-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Inhibition Assay: PI3 kinases catalyse the phosphorylation of phosphatidylinositol 4,5-biphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3) in the presence of ATP and Mg2+ ions. The PIP3 product can be detected by displacement of biotin-PIP3 from energy transfer complexes consisting of europium labelled anti-GST monoclonal antibody, a GST-tagged Pleckstrin homology (PH) domain, biotinylated PIP3 and streptavidin-allophycocyanin (APC) by the time-resolved fluorescence resonance energy transfer (TR-FRET) (HTRF® PI3K enzyme assay, Millipore). Excitation (330 nm) of europium in the complex results in an energy transfer to the APC and a fluorescent emission at 665 nm although europium itself emits at its characteristic 620 nm. The PIP3 product formed by PI3K activity displaces biotin-PIP3 from the complex and results in a loss of energy transfer (decreasing signal). The compound to be tested was added, at the desired final concentrations, to a mixture of PIP2 substrate and recombinant PI3 kinase α, δ or γ enzymes (Millipore), and the mixture incubated for 2 hr at RT. Following this incubation period, ATP (20 μM) was added to the enzyme/compound/PIP2 substrate mixture and the resulting mixture was incubated for 30 min at RT. A stopping solution containing biotinylated PIP3 and the detection mix containing the GST tagged GRP1 pleckstrin homology (PH) domain and fluorophores were then added and the mixture was incubated at RT for 15-18 hr, prior to detection in a fluorescence microplate reader (Varioskan® Flash, ThermoFisher Scientific).
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 689.18, ALogP 4.62, HBA 11, HBD 2, PSA 154.28, QED 0.17
Compound (SMILES): O=C(C1CCCC1)N1CCC(c2ccc(C(O)(C(F)(F)F)C(F)(F)F)cc2)(S(=O)(=O)c2ccc(F)cc2)C1
InChIKey: AWOZLZVEQAYRHZ-UHFFFAOYSA-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 = 114 nM (pChEMBL 6.940000057220459)
Descriptors: MW 567.52, ALogP 5.23, HBA 4, HBD 1, PSA 74.68, QED 0.4
Compound (SMILES): COCOc1ccc(CNC(=O)CCCCCN2CCN(c3ccccc3-c3ccc(OC)cc3)CC2)cc1
InChIKey: AKRZFAUTJFZOAO-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: P34969
Assay: Displacement of [3H]-5-CT from human 5-HT7b receptor expressed in HEK293 cell membranes after 1 hr by microbeta counting analysis
Measurement: Ki = 6.5 nM (pChEMBL 8.1899995803833)
Descriptors: MW 531.7, ALogP 5.34, HBA 6, HBD 1, PSA 63.27, QED 0.22
Compound (SMILES): CCn1cnc(S(=O)(=O)N2CCC(C(=O)Nc3cccc(-c4ccno4)c3)CC2)c1
InChIKey: NHRIWFBGYCNGQC-UHFFFAOYSA-N
Target: Dihydrolipoyl dehydrogenase (SINGLE PROTEIN), Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
UniProt: P9WHH9
Assay: Inhibition Assay: The assay was performed in a manner similar to that described in Bryk et al., Biochemistry (2010) 49:1616-1627 and modified for an online robotics screening system.A solution containing Lpd (100 nM) and 100 mM sodium phosphate buffer (pH 7.0) was prepared. Another solution containing the substrate lipoamide (2 mM), 100 mM sodium phosphate buffer (pH 7.0), EDTA (4 mM), and NADH (200 uM) was also prepared. The detection reagent, DTNB was dissolved in DMSO and diluted to a concentration of 375 uM with a 100 mM sodium phosphate buffer (pH 7.0).4 uL/well of Lpd solution was dispensed into 1536-well clear bottom plates. Next, 50 nL of each test compounds (1 mM dissolved in DMSO) was added into each well. After one hour of incubation, 4 uL/well of lipoamide (LPA) solution was added, so that in each well, the concentrations of Lpd, LPA, and the test compound were 50 nM, 1 mM, and 6.25 uM, respectively.
Measurement: IC50 = 2680 nM (pChEMBL 5.570000171661377)
Descriptors: MW 429.5, ALogP 2.6, HBA 7, HBD 1, PSA 110.33, QED 0.64
Compound (SMILES): COc1ccc(C(=O)c2ccc(C(=O)c3ccc(N(C)c4ccc(Cl)cc4)cn3)cc2C(=O)O)c(OC)c1
InChIKey: UQZGVUWEXXPOQI-UHFFFAOYSA-N
Target: Leukotriene C4 synthase (SINGLE PROTEIN), Homo sapiens
UniProt: Q16873
Assay: Inhibition of recombinant human LTC4 synthase expressed in Pichia pastoris using LTA4 as substrate preincubated for 10 mins followed by substrate addition measured after 5 mins by HTRF method
Measurement: IC50 = 68 nM (pChEMBL 7.170000076293945)
Descriptors: MW 530.96, ALogP 5.68, HBA 7, HBD 1, PSA 106.03, QED 0.28
Compound (SMILES): Cc1cc(CNc2c(C)ccc(OCC(=O)O)c2C)c(F)c(-c2cccc(F)c2)c1
InChIKey: DBLPFYSZBJWSQE-UHFFFAOYSA-N
Target: Prostaglandin E2 receptor EP2 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P43116
Assay: Cellular Functional Assay: Human CHO cells expressing the recombinant human prostanoid EP2 receptor were suspended in assay medium (HBSS buffer (Invitrogen) containing 20 mM HEPES (pH7.4) and 500 μM isobutyl-methylxanthine IBMX) and plated out to yield approximately 1×104 cells/well in a 96-well. Following this, cells were incubated with agonists for 30 min in the presence of the test compounds. For stimulated control measurements, separate assay wells contain 10 μM PGE2 (control specific agonist). All incubations were carried out at 37° C. in a 5% CO2 atmosphere. Following incubation, the amount of cAMP in each well was determined by HTRF method. The cells are lysed and the fluorescence acceptor (D2-labeled cAMP) and fluorescence donor (anti-cAMP antibody labeled with europium cryptate) are added.
Measurement: EC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 411.45, ALogP 5.63, HBA 3, HBD 2, PSA 58.56, QED 0.53
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CC3CCC(C2)N3C(=O)C2CCC(F)(F)CC2)C1
InChIKey: GIIDKVLLPDMGDB-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 534.62, ALogP 4.09, HBA 7, HBD 1, PSA 106.73, QED 0.53
Compound (SMILES): CC(C)(C)OC(=O)N1CC[C@H](n2cc(C(N)=O)c(Nc3ccccc3)n2)[C@@H](C#N)C1
InChIKey: MFISRUZXZNTIAN-YOEHRIQHSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Biochemical HTRF Assay: The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at ambient temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at ambient temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at ambient temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading.
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 410.48, ALogP 3.05, HBA 7, HBD 2, PSA 126.27, QED 0.8
Compound (SMILES): CCOc1ccc(C[C@@H](NC(=O)c2cnc(Cl)c(Cl)c2)C(=O)N[C@@H](Cc2ccccc2)C(=O)NCc2ccc(CN)cc2)cc1
InChIKey: XKUREAYXZXNALY-XZWHSSHBSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Inhibition Assay: Plasma kallikrein inhibitory activity in vitro was determined using standard published methods (see e.g. Johansen et al., Int. J. Tiss. Reac. 1986, 8, 185; Shori et al., Biochem. Pharmacol., 1992, 43, 1209; Stiirzebecher et al., Biol. Chem. Hoppe-Seyler, 1992, 373, 1025). Human plasma kallikrein (Protogen) was incubated at 37° C. with the fluorogenic substrate H-DPro-Phe-Arg-AFC and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in optical absorbance at 410 nm and the IC50 value for the test compound was determined.
Measurement: IC50 = 133 nM (pChEMBL 6.880000114440918)
Descriptors: MW 648.59, ALogP 4.63, HBA 6, HBD 4, PSA 135.44, QED 0.15
Compound (SMILES): CC(C)(C)OC(=O)/C=C/c1ccc([N+](=O)[O-])cc1
InChIKey: SULXLGCTOCXWEE-RMKNXTFCSA-N
Target: Protein-glutamine gamma-glutamyltransferase 2 (SINGLE PROTEIN), Cavia cutleri
UniProt: P08587
Assay: Enzyme Inhibition Assay: For enzymology studies of these compounds, recombinant guinea pig liver TGase was expressed in Escherichia coli and effectively purified (Gillet, S. M. F. G. et al J. N., Prot. Exp. & Purif. 2004, 33, 256). In addition to being easy to obtain in excellent yield and solubility, guinea pig liver TGase was chosen because it shows 80% homology with human tissue TGase (Aeschlimann, D.; Paulsson, M., Throm. Haemost. 1994, 71, 402) and may thus serve as a model for the evaluation of inhibitors of potential therapeutic utility.The IC50 values of synthetic analogues 14a-38a were determined from inhibition of the reaction of 54.4 mM of the chromogenic TGase substrate N-Cbz-Glu(Î³-p-nitrophenyl ester)Gly with 0.010 U of recombinant guinea pig liver TGase as previously reported (Leblanc, A.; Gravel, C.; Labelle, J.; Keillor, J. W. Biochemistry 2001, 40, 8335) and described in detail in the Materials section below. The mode of inhibition was determined for the representative lead compound.
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 249.27, ALogP 2.95, HBA 4, HBD 0, PSA 69.44, QED 0.36
Compound (SMILES): CC(=O)Nc1cc(-c2ccc(F)cc2)cc(-n2cnc3cc(-c4ccc(F)nc4)ccc32)c1
InChIKey: LCTCHIBAXSORHF-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 = 59 nM (pChEMBL 7.230000019073486)
Descriptors: MW 440.45, ALogP 5.99, HBA 4, HBD 1, PSA 59.81, QED 0.35
Compound (SMILES): Cn1ncc2ccc(-c3ccnc(NCC(O)CN4CCc5ccccc5C4)c3)cc21
InChIKey: CQZZLUBQEFIVIB-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 < 100 nM (pChEMBL 7)
Descriptors: MW 413.53, ALogP 3.47, HBA 6, HBD 2, PSA 66.21, QED 0.51
Compound (SMILES): C[C@H]1C[C@H]2CSC(N)=N[C@@]2(c2cc(NC(=O)c3ccc(Cl)cn3)ccc2F)CO1
InChIKey: UOHXUSCZQJMWQO-PVUDRZGPSA-N
Target: Cytochrome P450 2D6 (SINGLE PROTEIN), Homo sapiens
UniProt: P10635
Assay: CYP2D6 Inhibition Assay: CYP2D6 inhibition was obtained by measuring inhibition of 5 uM Dextromethorphan in pooled HLM (HL-MIX-102).
Measurement: IC50 < 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 434.92, ALogP 3.81, HBA 6, HBD 2, PSA 89.6, QED 0.77
Compound (SMILES): COc1ccc(C(C)(C)C)cc1-c1cccc(C(CC(=O)O)c2ncco2)c1
InChIKey: FYDPVPNHLKTGDO-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 = 103 nM (pChEMBL 6.989999771118164)
Descriptors: MW 379.46, ALogP 5.25, HBA 4, HBD 1, PSA 72.56, QED 0.63
Compound (SMILES): O=C(/C=C/c1ccc([N+](=O)[O-])cc1)c1c[nH]nn1
InChIKey: GWCZBIMTBRUMMV-ZZXKWVIFSA-N
Target: Protein-glutamine gamma-glutamyltransferase 2 (SINGLE PROTEIN), Cavia cutleri
UniProt: P08587
Assay: Enzyme Inhibition Assay: For enzymology studies of these compounds, recombinant guinea pig liver TGase was expressed in Escherichia coli and effectively purified (Gillet, S. M. F. G. et al J. N., Prot. Exp. & Purif. 2004, 33, 256). In addition to being easy to obtain in excellent yield and solubility, guinea pig liver TGase was chosen because it shows 80% homology with human tissue TGase (Aeschlimann, D.; Paulsson, M., Throm. Haemost. 1994, 71, 402) and may thus serve as a model for the evaluation of inhibitors of potential therapeutic utility.The IC50 values of synthetic analogues 14a-38a were determined from inhibition of the reaction of 54.4 mM of the chromogenic TGase substrate N-Cbz-Glu(Î³-p-nitrophenyl ester)Gly with 0.010 U of recombinant guinea pig liver TGase as previously reported (Leblanc, A.; Gravel, C.; Labelle, J.; Keillor, J. W. Biochemistry 2001, 40, 8335) and described in detail in the Materials section below. The mode of inhibition was determined for the representative lead compound.
Measurement: IC50 = 45000 nM (pChEMBL 4.349999904632568)
Descriptors: MW 244.21, ALogP 1.61, HBA 5, HBD 1, PSA 101.78, QED 0.38
Compound (SMILES): Cc1noc(C)c1Cn1cc(NS(=O)(=O)c2ccccc2F)cn1
InChIKey: HAPUNHPFCABVDD-UHFFFAOYSA-N
Target: Taste receptor type 2 member 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYW2
Assay: Fluorescence Polarization Assays: In another embodiment, Fluorescence Polarization ("FP") based assays may be used to detect and monitor ligand binding. Fluorescence polarization is a versatile laboratory technique for measuring equilibrium binding, nucleic acid hybridization, and enzymatic activity. Fluorescence polarization assays are homogeneous in that they do not require a separation step such as centrifugation, filtration, chromatography, precipitation, or electrophoresis. These assays are done in real time, directly in solution and do not require an immobilized phase. Polarization values can be measured repeatedly and after the addition of reagents since measuring the polarization is rapid and does not destroy the sample. Generally, this technique can be used to measure polarization values of fluorophores from low picomolar to micromolar levels.
Measurement: IC50 = 8510 nM (pChEMBL 5.070000171661377)
Descriptors: MW 350.38, ALogP 2.48, HBA 6, HBD 1, PSA 90.02, QED 0.76
Compound (SMILES): FC(F)(F)Oc1ccc(Nc2ncc(Br)c(Nc3cc(C4CC4)[nH]n3)n2)c(Br)c1
InChIKey: FEYKRGMWWMTATG-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 >= 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 534.13, ALogP 5.99, HBA 6, HBD 3, PSA 87.75, QED 0.36
Compound (SMILES): CCN1CC(C)(C)Oc2nc(N3CC4CC35CC5O4)nc(-c3ccc(NC(=O)Nc4ccnc(C)c4)c(F)c3)c2C1=O
InChIKey: WRJWCEAWELAFRH-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The test detects the phosphorylation of the substrate GFP-4EBP1 using a terbium-labelled antibody that is specific for the phospho-Thr46 epitope. Binding of the Tb-antibody to the phosphorylated GFP peptide allows the transfer of energy from Tb, excited at 340 nm, to GFP with an increase in the fluorescence emission of the GFP at 520 nm. The test is performed in 96-well format (Corning/Costar 96 black flat-bottomed half-wells plate, ref. 3694) in a total volume of 30 ul. To 1 ul of inhibitor in 100% DMSO are added (final concentrations) 400 nM of GFP-4EBP1 (Invitrogen PV4759), 8 uM of ATP, 200 ng/ml of enzyme (human recombinant aa mTOR 1360-2549, Invitrogen PV4753) in a buffer of HEPES 50 mM pH 7.5, EGTA 1 mM, MnCl2 10 mM, BSA 0.1 mg/ml, glycerol 0.5%, NV-10 0.1 mg/ml, DTT 2 mM. The mixture is incubated for 30 minutes at room temperature. 10 ul of solution are taken up and the reaction is quenched by adding 10 ul of Tb-antibody solution (Invitrogen PV4757)/EDTA.
Measurement: IC50 = 41 nM (pChEMBL 7.389999866485596)
Descriptors: MW 573.63, ALogP 4.38, HBA 8, HBD 2, PSA 121.81, QED 0.46
Compound (SMILES): Clc1cc(Nc2n[nH]c3cccnc23)cnc1N1CCOCC1
InChIKey: RPEKYFVVWRUAQX-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 = 2380 nM (pChEMBL 5.619999885559082)
Descriptors: MW 330.78, ALogP 2.59, HBA 6, HBD 2, PSA 78.96, QED 0.77
Compound (SMILES): COc1c(C(C)Nc2ncnc(N)c2C#N)cc(Cl)c(F)c1-c1cncc(C#N)c1
InChIKey: RYVODQGTYUBTFL-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Scintillation Proximity Assay (SPA): The kinase reaction was conducted in polystyrene 384-well matrix white plate from Thermo Fisher Scientific in a final volume of 25 uL. Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 0.5%. The PI3K assays were carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 uM PIP2, 20 uM ATP, 0.2 uCi [gamma-33P]ATP, 4 nM PI3K delta. Reactions were incubated for 210 min and terminated by the addition of 40 uL SPA beads suspended in quench buffer: 150 mM potassium phosphate pH 8.0, 20% glycerol. 25 mM EDTA, 400 uM ATP. The final concentration of SPA beads was 1.0 mg/mL.
Measurement: IC50 < 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 423.84, ALogP 3.84, HBA 8, HBD 2, PSA 133.53, QED 0.63
Compound (SMILES): CC(=O)O[C@H]1C[C@@H](Nc2nc3c(nc2N2CCN(Cc4ccc(F)cc4F)CC2)CCN(C(C)=O)C3)C1
InChIKey: JVADGRPSAFEECF-OYRHEFFESA-N
Target: G-protein coupled receptor 6 (SINGLE PROTEIN), Homo sapiens
UniProt: P46095
Assay: GPR6 In Vitro Assay (A.2): This cell based assay measures the ability of compounds to inhibit the constitutive cAMP activity of GPR6 receptor expressed in CHO-K1 cells. CHO cells were stably expressed with GPR6 receptor, whose expression is controlled by a tetracycline inducable element. The cells were cultured in medium containing F12K, 10% FBS, 1% PennStrep, 200 ug/mL Hygromycin. GPR6 receptor expression was induced for 20 hrs with 2 ug/ml doxycycline (sigma D9891) in growth media. After addition of doxycycline cells were plated at a density of 450-750 cells per well in 96-well half-volume black tissue culture plates (Costar) and placed in an incubator (37°, 5% CO2) for 20 hours prior to cAMP assays. Culture media was removed from cells and they were washed with 50 uL/well of Ringer's Buffer (MgCl2 0.047 mg/mL, NaH2PO4 0.18 mg/mL, Na2HPO4 0.1 mg/mL, KCl 0.34 mg/mL, NaHCO3 1.26 mg/mL, D-glucose 1.8 mg/mL, NaCl 7 mg/mL; pH=7.4). Compounds suspended in DMSO were diluted in Ringer's Buffer.
Measurement: IC50 = 0.2198 nM (pChEMBL 9.65999984741211)
Descriptors: MW 514.58, ALogP 2.49, HBA 8, HBD 1, PSA 90.9, QED 0.59