lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1cccc(OC)c1C1CCCC(=O)N1Cc1cccc(-c2nccs2)n1
InChIKey: HOHUOHPDNVNIFD-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: In Vitro Assay: Intracellular Calcium Measurements : In Vitro Assay: Intracellular Calcium Measurements:Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 g/ml G418, 100 U/ml penicillin, 100 g/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2. Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH:water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 179 nM (pChEMBL 6.75)
Descriptors: MW 409.51, ALogP 4.48, HBA 6, HBD 0, PSA 64.55, QED 0.6
Compound (SMILES): COc1cc(F)c(C2CNC(=O)N2c2ccc3nc[nH]c3c2)c(F)c1
InChIKey: BMVLGILWYACRJJ-UHFFFAOYSA-N
Target: Glutaminyl-peptide cyclotransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q16769
Assay: Spectrophotometic Assay: Spectrophotometic Assay: This novel assay was used to determine the kinetic parameters for most of the QC substrates. QC activity was analyzed spectrophotometrically using a continuous method, that was derived by adapting a previous discontinuous assay (Bateman, R.C.J. 1989 J Neurosci Methods 30, 23-28).
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 344.32, ALogP 3.12, HBA 3, HBD 2, PSA 70.25, QED 0.77
Compound (SMILES): O=C1NCC(c2ccccc2OCCN2CCOCC2)N1c1ccc2nc[nH]c2c1
InChIKey: XSPFROIVDYNUKL-UHFFFAOYSA-N
Target: Glutaminyl-peptide cyclotransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q16769
Assay: Spectrophotometic Assay: Spectrophotometic Assay: This novel assay was used to determine the kinetic parameters for most of the QC substrates. QC activity was analyzed spectrophotometrically using a continuous method, that was derived by adapting a previous discontinuous assay (Bateman, R.C.J. 1989 J Neurosci Methods 30, 23-28).
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 407.47, ALogP 2.54, HBA 5, HBD 2, PSA 82.72, QED 0.66
Compound (SMILES): COCc1ccc(C(C)=O)c2sc(C(=O)Nc3ccc4c(C(C)O)cccc4n3)c(C)c12
InChIKey: UTKKKENTWPNDDJ-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Activity Assay: A human acute lymphoblastic lymphoma T cell line MOLT-4 (which can be purchased under ATCC No. CRL-1582 from ATCC) was cultured in an RPMI1640 medium containing 10% fetal bovine serum to obtain 5108 MOLT-4 cells. The cells were recovered by centrifugation and suspended in 10 ml of buffer solution A (25 mM Tris-HCl, 5 mM 2-mercaptoethanol, 2 mM benzamidine, 2 mM EDTA, and 0.1 mM 4-(2-aminoethyl)benzenesulfonyl hydrochloride, pH 7.5). The cells were homogenized using a Polytron homogenizer and centrifuged (4° C., 25,000 g, 10 minutes). Then, the supernatant was further ultracentrifuged (4° C., 100,000 g, 60 minutes). The obtained supernatant was filtered through a 0.2um filter to obtain a soluble fraction.HiTrap Q HP column (5 ml2, GE Healthcare Japan Corp.) equilibrated with buffer solution A was charged with the obtained soluble fraction. Phosphodiesterase was eluted with 300 ml of buffer solution A containing a linear gradient solution of 0 to 0.8 M NaCl to recover sixty 5-ml fractions. Each fraction was tested for cAMP-metabolizing phosphodiesterase activity. In each fraction, a fraction eluted as an active peak centered around 250 mM NaCl was collected from fractions that had cAMP metabolic activity and did not lose the metabolic activity by 10uM rolipram (PDE4 selective inhibitor) and 10uM milrinone (PDE3 selective inhibitor). In order to confirm whether or not PDE10A mRNA was expressed in the MOLT-4 cells, total RNA was prepared from the MOLT-4 cells according to a standard method and analyzed by RT-PCR using PDE10A gene-specific primers (PDE10A sense primer: 5'-TGCTCCATGGTGGAAGTGGA-3' (SEQ ID NO: 1) and PDE10A antisense primer: 5'-CAACTGGAAGCATGCGGTCA-3' (SEQ ID NO: 2)). As a result, PDE10A mRNA was detected from the total RNA of the MOLT-4 cells. On the other hand, total RNA was prepared from Jurkat cells, one type of human T cell, and similarly subjected to RT-PCR. As a result, PDE10A mRNA was rarely detected. An active peak centered around 250 mM NaCl was not observed in fractions obtained by the treatment of Jurkat cells in the same way as above. Eur J Biochem. 1999, 266 (3), 1118-2 has reported that PDE10A from the rat striatum and testis is eluted as an active peak centered around 250 mM NaCl.
Measurement: IC50 = 0.53 nM (pChEMBL 9.279999732971191)
Descriptors: MW 448.54, ALogP 5.41, HBA 6, HBD 2, PSA 88.52, QED 0.39
Compound (SMILES): COC(C)c1ccc(C(C)=O)c2sc(C(=O)Nc3cc(C(C)(C)O)c4ccccc4n3)c(C)c12
InChIKey: WUFBKOARFGVHKI-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Activity Assay: A human acute lymphoblastic lymphoma T cell line MOLT-4 (which can be purchased under ATCC No. CRL-1582 from ATCC) was cultured in an RPMI1640 medium containing 10% fetal bovine serum to obtain 5108 MOLT-4 cells. The cells were recovered by centrifugation and suspended in 10 ml of buffer solution A (25 mM Tris-HCl, 5 mM 2-mercaptoethanol, 2 mM benzamidine, 2 mM EDTA, and 0.1 mM 4-(2-aminoethyl)benzenesulfonyl hydrochloride, pH 7.5). The cells were homogenized using a Polytron homogenizer and centrifuged (4° C., 25,000 g, 10 minutes). Then, the supernatant was further ultracentrifuged (4° C., 100,000 g, 60 minutes). The obtained supernatant was filtered through a 0.2um filter to obtain a soluble fraction.HiTrap Q HP column (5 ml2, GE Healthcare Japan Corp.) equilibrated with buffer solution A was charged with the obtained soluble fraction. Phosphodiesterase was eluted with 300 ml of buffer solution A containing a linear gradient solution of 0 to 0.8 M NaCl to recover sixty 5-ml fractions. Each fraction was tested for cAMP-metabolizing phosphodiesterase activity. In each fraction, a fraction eluted as an active peak centered around 250 mM NaCl was collected from fractions that had cAMP metabolic activity and did not lose the metabolic activity by 10uM rolipram (PDE4 selective inhibitor) and 10uM milrinone (PDE3 selective inhibitor). In order to confirm whether or not PDE10A mRNA was expressed in the MOLT-4 cells, total RNA was prepared from the MOLT-4 cells according to a standard method and analyzed by RT-PCR using PDE10A gene-specific primers (PDE10A sense primer: 5'-TGCTCCATGGTGGAAGTGGA-3' (SEQ ID NO: 1) and PDE10A antisense primer: 5'-CAACTGGAAGCATGCGGTCA-3' (SEQ ID NO: 2)). As a result, PDE10A mRNA was detected from the total RNA of the MOLT-4 cells. On the other hand, total RNA was prepared from Jurkat cells, one type of human T cell, and similarly subjected to RT-PCR. As a result, PDE10A mRNA was rarely detected. An active peak centered around 250 mM NaCl was not observed in fractions obtained by the treatment of Jurkat cells in the same way as above. Eur J Biochem. 1999, 266 (3), 1118-2 has reported that PDE10A from the rat striatum and testis is eluted as an active peak centered around 250 mM NaCl.
Measurement: IC50 = 9.1 nM (pChEMBL 8.039999961853027)
Descriptors: MW 476.6, ALogP 6.15, HBA 6, HBD 2, PSA 88.52, QED 0.33
Compound (SMILES): FC(F)(F)c1ccc(Nc2nc(-c3ccncc3)nc3c2CCN(c2ncccc2C(F)(F)F)CC3)cc1
InChIKey: HTUVWWLYSOYLAR-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: Functional Assay: HEK293 cells were transfected with human TRPV1 cloned in pcDNA3.1zeo(+) using the Effectene non-liposomal lipid based transfection kit (Qiagen) (hTRPV1/HEK293). hTRPV1/HEK293 cells were routinely grown as monolayers under selection in zeocin (200 μg/mL; Invitrogen) in Dulbecco's Modified Eagle Medium (DMEM, Gibco BRL) supplemented with 10% fetal bovine serum, and penicillin/streptomycin (50 units/mL) in 5% CO2 at 37° C. Cells were passaged frequently, every 3-5 days, to avoid overgrowth, depletion of essential medium components, or acidic medium exposure. Cells were passaged using a brief wash in 0.05% trypsin with 1 mM EDTA, followed by dissociation in divalent-free phosphate-buffered saline (Hyclone #SH30028.02). Dissociated cells were seeded onto poly-D-lysine coated black-walled 96-well plates (Biocoat; Becton Dickinson #354640) at about 40,000 cells per well and grown for approximately 1 day in culture medium to near confluency. The assay buffer was composed of 130 mM NaCl, 2 mM KCl, 2 mM MgCl2, 10 mM HEPES, 5 mM glucose, and either 2 mM or 20 μM CaCl2. On the day of the experiment, the culture medium was replaced with 2 mM calcium assay buffer using an automated plate washer (ELx405; Biotek, VT). The cells were incubated in 100 μL/well Fluo-3/AM (2 μM; TEFLabs #0116) with Pluronic F127 (100 μg/mL; Sigma #P2443) for 1 h at rt in the dark. After loading the cells, the dye solution was replaced with 50 μL/well of 20 μM calcium assay buffer using the ELx405 plate washer. Test compounds (50 μL/well) were added to the plate and incubated for 30 min. Intracellular Ca2+ levels were subsequently assayed using a Fluorometric Imaging Plate Reader (FLIPR™ instrument, Molecular Devices, CA) to simultaneously monitor Fluo-3 fluorescence in all wells (λexcitation=488 nm, λemission=540 nm) during challenge with agonist (capsaicin). The IC50 values were determined. Cells were challenged with 150 nM capsaicin and the fluorescence counts were captured following agonist addition at a sampling rate of 0.33 Hz. The contents of the wells were mixed 3 times (40 μL mix volume) immediately after the additions were made. Concentration dependence of block was determined by exposing each well of cells in duplicate rows of a 96 well plate to a serial dilution of test compound. The concentration series usually started at 10 Î¼M with a three-fold serial decrement in concentration. The magnitude of the capsaicin response was determined by measuring the change in fluo3 fluorescence before and 100 seconds after the addition of the agonist.
Measurement: IC50 = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 530.48, ALogP 6.32, HBA 6, HBD 1, PSA 66.83, QED 0.31
Compound (SMILES): Cc1c(-c2cccc(C(F)(F)F)c2)c(=O)c(C(=O)NCc2ccc(S(C)(=O)=NC#N)cc2)cn1-c1ccccc1
InChIKey: LKJSBLFRMZHVSS-UHFFFAOYSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Human Neutrophil Elastase Assay: The following buffers were used: Compound buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5; Assay buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5, containing 0.01% BSA.Assay conditions: Test compounds were prediluted in DMSO and subsequently in compound buffer (5% DMSO final). 5 μL of these compound dilutions were mixed with 10 μl Neutrophil elastase (9 ng/ml in assay buffer) in a black 384 well OptiPlate (Perkin Elmer, Cat No.: 6007270) and incubated for 15 min at room temperature. Subsequently 10 μL substrate solution in assay buffer were added (250 μM final concentration) and the plates were incubated for 60 min at room temperature. After inactivation of the enzyme, fluorescence intensities were measured at 380 nm excitation and 460 nm emission wavelengths.
Measurement: IC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 564.59, ALogP 5.7, HBA 6, HBD 1, PSA 104.32, QED 0.3
Compound (SMILES): O=C(O)Cc1cccc(C(=O)Nc2ccc3c(c2)OCO3)c1
InChIKey: FJGUUPQXRZGZTC-UHFFFAOYSA-N
Target: Cytosolic carboxypeptidase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5U5Z8
Assay: Inhibition of recombinant N-terminal His-tagged AGBL2 (unknown origin) assessed as tyrosine release using tyrosinated C-terminal tail of tubulin as substrate in presence of hydroxycoumarin preincubated for 30 to 60 mins followed by substrate addition by FRET assay
Measurement: Ki = 4.89 nM (pChEMBL 8.3100004196167)
Descriptors: MW 299.28, ALogP 2.29, HBA 4, HBD 2, PSA 84.86, QED 0.9
Compound (SMILES): CC(O)C(=O)Nc1cccc(-c2n[nH]c3ccc(-c4nc[nH]n4)cc23)c1
InChIKey: DROWREYOLCHQPX-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P45984
Assay: Inhibition of His6-tagged JNK2 (unknown origin) assessed as reduction in c-Jun phosphorylation using GST-c-Jun (1 to 79 residues) and [gamma32P]ATP incubated for 1 hr by scintillation counting assay
Measurement: IC50 <= 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 348.37, ALogP 2.33, HBA 5, HBD 4, PSA 119.58, QED 0.45
Compound (SMILES): O=C(Cc1ccc(-n2cnnn2)nc1)N1CCN2C[C@@H](c3cccc(-n4cnnn4)c3)OC[C@@H]2C1
InChIKey: BOYIAGVYLPPPJL-PMACEKPBSA-N
Target: ATP-sensitive inward rectifier potassium channel 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P48048
Assay: Thallium Flux Assay: The ROMK channel functional thallium flux assay is performed in 384 wells, using the FLIPR-Tetra instrument. HEK-hKir1.1 cells are seeded in Poly-D-Lysine microplates and kept in a 37° C.-10% CO2 incubator overnight. On the day of the experiment, the growth media is replaced with the FluxOR™ reagent loading buffer and incubated, protected from light, at ambient temperature (23-25° C.) for 90 min. The loading buffer is replaced with assay buffer±test compound followed by 30 min incubation at ambient temperature, where the Thallium/Potassium stimulant is added to the microplate. The fluorescence intensity of wells containing 3 μM of a standard control ROMK inhibitor of the present invention is used to define the ROMK-sensitive component of thallium flux. Fluorescence in the presence of test compounds is normalized to control values to provide % fluorescence change. IC50 values represent the concentration of compound that inhibits 50% of the ROMK thallium flux signal.
Measurement: IC50 = 340 nM (pChEMBL 6.46999979019165)
Descriptors: MW 473.5, ALogP -0.14, HBA 12, HBD 0, PSA 132.87, QED 0.39
Compound (SMILES): CNC(=O)c1c(-c2cnn(-c3ccccc3)c2)oc2cc(N(C)S(C)(=O)=O)c(-c3ccc4c(n3)-c3cc5c(F)cccc5n3CC4)cc12
InChIKey: LDLYXUSVZRCWSC-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: Inhibition Assay: To initiate an assay, replicon cells were plated in the presence of a dilution series of test compound in the absence of G418. Typically, the assays were performed in a 96-well plate formate for manual operation, or a 384-well plate format for automated assay. Replicon cells and compound were incubated for 96 hours. At the end of the assay, cells were washed free of media and compound, and the cells were then lysed. RNA was quantified indirectly through detection of replicon-encoded NS3/4A protein levels, through an ELISA-based assay with an antibody specific for NS3/4A.
Measurement: IC50 = 5479 nM (pChEMBL 5.260000228881836)
Descriptors: MW 660.73, ALogP 6.42, HBA 8, HBD 1, PSA 115.26, QED 0.22
Compound (SMILES): CN(c1ccc(S(=O)(=O)NC2CC2)cn1)[C@@H]1CCN(c2ncnc3[nH]ccc23)C1
InChIKey: RJDFEVIMBGLEBT-CQSZACIVSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzymatic Assay: JAK1/2/3 kinase assay are performed in vitro using Kit-Tyr 6 Peptide (Invitrogen, Cat. No. PV4122). TYK2 kinase assay are performed in vitro using Z′-LYTE Kinase Assay Kit-Tyr 3 Peptide (Invitrogen, Cat. No. PV3192). Recombinant human JAK1/2/3 or TYK2 catalytic domains are from Invitrogen (Cat No. PV4774/PV4210/PV3855/PV4790); All reactions (20 μL) are started by adding 2.5 μL of the testing compound in 4% DMSO solution, 5 μL of Kinase/Peptide substrate Mixture (3.2, 0.04, 0.2 or 8 μg/mL for Recombinant human JAK1/2/3 catalytic domains, 4 μM for Z-LYTE Tyr 6 peptide or Z-LYTE Tyr 3 peptide) or Phospho-Peptide solution (Invitrogen, Cat. No. PV3192, diluted with 1.33× Kinase Buffer), 2.5 μL ATP Solution (300/100/40/100 μM, JAK1/JAK2/JAK3/TYK2) or 1.33× Kinase Buffer (Invitrogen, Cat. No. PV3189, 5× diluted with distilled water). The 384-well assay plate (Corning, Cat. No. 3575) is mixed and incubated at room temperature for 1 hour. 5 μL of the Development Solution (Dilute Development Reagent A (Cat. No. PV3297) is diluted to 1/64 with Development Buffer (Cat. No. PV3127) for JAK1, JAK2 and JAK3 assay; Development Reagent A (Cat. No. PV3297) is diluted to 1/2048 with Development Buffer (Cat. No. PV3127) for TYK2 assay. The diluted Development Solution is then added to each well, mixed and incubated at room temperature for another 1 hour. The kinase reaction is then stopped by adding 5 μL of the Stop Reagent (Invitrogen, Cat. No. PV3094), and the plate is read with Wallac 1420 VICTOR3 Multilabel Counter (PerkinElmer) at 445 nm and 520 nm fluorescence. All compounds are initially tested at 8 different concentrations (1 μM down to 0.0003 μM) using a 1:3 serial dilution scheme.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 413.51, ALogP 1.51, HBA 7, HBD 2, PSA 107.11, QED 0.63
Compound (SMILES): Cc1cc(-c2nc(-c3ccc4c(c3)CCC(N)(CO)C4)no2)ccc1OC(C)C
InChIKey: OYOXQRDPFPJOHL-UHFFFAOYSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: GTPγS Binding Assay: Test method: wetting membranes by saponin solution, and preparing [35S]-GTPγS in binding solution (4×). The test compound was prepared as 4× of the final concentration which was then performed serial 3× dilution in 100% DMSO. Membranes with high-expressed S1P1 were collected in 5 ml binding buffer, and 50 μL was added to 96-well (containing 5 μg membrane protein with high-expressed S1P1). 25 μL, test compound was added in each well. The compound was incubated with the membranes for 30 min at room temperature, then 25 μL [35S]-GTPγS was added, and the experiment was initiated. The 96-well detection plate was incubated for 1 hour at room temperature, filtered by filtermat B, and washed with washing solution for three times. After the plate was dried for 1 h at 50° C., 8 ml scintillator solution was added and counted using scintillation counter.
Measurement: IC50 = 473 nM (pChEMBL 6.329999923706055)
Descriptors: MW 393.49, ALogP 3.68, HBA 6, HBD 2, PSA 94.4, QED 0.69
Compound (SMILES): COc1cc(OC)cc(N(CCNC(C)C)c2ccc3ncc(-c4cnn(C5CCN(C)CC5)c4)nc3c2)c1.Cl
InChIKey: WWJUKYMCFMIUBZ-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 47.86 nM (pChEMBL 7.320000171661377)
Descriptors: MW 529.69, ALogP 4.91, HBA 9, HBD 1, PSA 80.57, QED 0.31
Compound (SMILES): CC(=O)c1ccccc1NC(=O)c1ccc([N+](=O)[O-])cc1
InChIKey: QFTKDGUBCGPCME-UHFFFAOYSA-N
Target: Shiga toxin subunit A (SINGLE PROTEIN), Shigella dysenteriae
UniProt: Q9FBI2
Assay: Inhibition of Shigella dysenteriae type 1 Shiga toxin A subunit assessed as change in transition temperature by SYPRO orange dye based fluorescence-based thermal shift assay
Measurement: Ki = 5000 nM (pChEMBL 5.300000190734863)
Descriptors: MW 284.27, ALogP 3.05, HBA 4, HBD 1, PSA 89.31, QED 0.53
Compound (SMILES): CCC(CC)(NC(=O)c1ccc(C(F)(F)F)c(OCC2CC2)n1)C(=O)NC
InChIKey: IZRAKQFSHFVXGF-UHFFFAOYSA-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.times.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.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul 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 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM 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.
Measurement: EC50 = 5.1 nM (pChEMBL 8.289999961853027)
Descriptors: MW 387.4, ALogP 2.92, HBA 4, HBD 2, PSA 80.32, QED 0.72
Compound (SMILES): O=C(c1ccc(OCC(F)(F)F)nc1)N1CCCC(c2ccccc2-c2ccc(F)nc2)C1
InChIKey: JIGZWXGHTJQGHD-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FLIPR Assay: The assay buffer was formulated by removing 120 mL from a 1 L bottle of fresh, sterile dH2O (Mediatech, Herndon, Va.) and adding 100 mL of 10×HBSS that does not contain Ca++ or Mg++ (Gibco, Invitrogen, Grand Island, N.Y.) followed by 20 mL of 1.0 M Hepes, pH 7.3 (Fisher Scientific, BP299-100). The final buffer consisted of 20 mM Hepes, pH 7.3, 1.261 mM CaCl2, 0.493 mM MgCl2, 0.407 mM Mg(SO)4, 5.33 mM KCl, 0.441 mM KH2PO4, 137 mM NaCl, 0.336 mM Na2HPO4 and 0.556 mM D-glucose (Hanks et al., Proc. Soc. Exp. Biol. Med. 71:196 (1949)), and the simple formulation was typically the basic buffer throughout the assay (i.e., all wash and addition steps).
Measurement: IC50 = 954 nM (pChEMBL 6.019999980926514)
Descriptors: MW 459.44, ALogP 5.24, HBA 4, HBD 0, PSA 55.32, QED 0.39
Compound (SMILES): CCC(CC)(NC(=O)c1ccc(N2CCC(F)(F)C2)c(OCC2CC2)n1)C(=O)NC
InChIKey: PNKMGWWBPULYAC-UHFFFAOYSA-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.times.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.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul 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 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM 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.
Measurement: EC50 = 4.6 nM (pChEMBL 8.34000015258789)
Descriptors: MW 424.49, ALogP 2.75, HBA 5, HBD 2, PSA 83.56, QED 0.64
Compound (SMILES): COc1ccc(Nc2nc(C#N)nc(NC3CCCCC3)n2)cc1
InChIKey: HCJPYLNVLFDHCY-UHFFFAOYSA-N
Target: Cathepsin K (SINGLE PROTEIN), Homo sapiens
UniProt: P43235
Assay: Inhibition of human cathepsin K expressed in Pichia pastoris using Z-Phe-Arg-AMC as substrate after 30 mins by spectromicrofluorimetric method
Measurement: IC50 = 23 nM (pChEMBL 7.639999866485596)
Descriptors: MW 324.39, ALogP 3.24, HBA 7, HBD 2, PSA 95.75, QED 0.87
Compound (SMILES): OC[C@H]1CN(Cc2ccncc2)C[C@@H](O)[C@@H]1O
InChIKey: HEUKMGNKIWGRQK-IJLUTSLNSA-N
Target: Lysosomal acid glucosylceramidase (SINGLE PROTEIN), Homo sapiens
UniProt: P04062
Assay: Enzyme Assay: To 2.5 μl of supernatant (in 96-well plates) was added 17.5 μl reaction buffer (citrate phosphate buffer, pH 4.5, no Triton X-100), and 50 μl of 4-methyl umbelliferone (4-MU)-labeled substrate, β-glucopyranoside, or a labeled negative controls α-glucopyranoside or α-galacatopyranoside). Plates were incubated at 370 for 1 hour, followed by the addition of 70 μl stop buffer (0.4 M glycine-NaOH, pH 10.6). Activity of GCase was determined by measuring the emission at 460 nm by exciting at 355 nm using a 1 second read time per well (Victor2 multilabel counter-Wallac).
Measurement: Ki = 8020 nM (pChEMBL 5.099999904632568)
Descriptors: MW 238.29, ALogP -0.77, HBA 5, HBD 3, PSA 76.82, QED 0.64
Compound (SMILES): CCc1ccccc1-n1c(Cn2nc(-c3cn[nH]c3)c3c(N)ncnc32)cc2cccc(C)c2c1=O
InChIKey: RMFKLMYYMKWEOP-UHFFFAOYSA-N
Target: PI3-kinase p110-alpha/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P42336
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110alpha/untagged recombinant full length human p85alpha expressed in baculovirus infected Sf21 cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 476.54, ALogP 4.02, HBA 8, HBD 2, PSA 120.3, QED 0.39
Compound (SMILES): CC(O)c1nnc(-c2ccc3[nH]nc(-c4ccc(F)cc4)c3c2)[nH]1
InChIKey: GVVJMGSPVVYTNT-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P45984
Assay: Inhibition of His6-tagged JNK2 (unknown origin) assessed as reduction in c-Jun phosphorylation using GST-c-Jun (1 to 79 residues) and [gamma32P]ATP incubated for 1 hr by scintillation counting assay
Measurement: IC50 <= 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 323.33, ALogP 3.21, HBA 4, HBD 3, PSA 90.48, QED 0.54
Compound (SMILES): CC[C@@H]1CNC(=O)c2cc(-c3cccc4ccc(Nc5ccccc5)nc34)[nH]c21
InChIKey: JRPMCVBHTMZOFM-OAHLLOKOSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Homogeneous Time Resolved Fluorescence Assay (HTRF): The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction. Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 μM. A reference compound was included on each assay plate in order to validate that plate; on one plate of every assay run, two additional reference compounds were included. The final buffer conditions were as follows: 60 mM Hepes, pH 7.0, 0.05% BSA, 2 mM DTT. Incubations were carried out at RT (22° C.) for 2 h for Pim-1, 1 hour and 30 min for Pim-3, and 45 min for Pim-2. The reaction was stopped by the addition of 3 mM EDTA, and fluorescence was measured by an HTRF® Rubystar microplate reader.
Measurement: IC50 = 2.4 nM (pChEMBL 8.619999885559082)
Descriptors: MW 382.47, ALogP 5.21, HBA 3, HBD 3, PSA 69.81, QED 0.45
Compound (SMILES): OC(CNc1cccc(-c2cccc3[nH]cnc23)c1)CN1CCc2ccccc2C1
InChIKey: APHASUFVZNWOIN-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 = 101 nM (pChEMBL 7)
Descriptors: MW 398.51, ALogP 4.06, HBA 4, HBD 3, PSA 64.18, QED 0.46
Compound (SMILES): Cc1ccc(C(=O)N2CCC(c3ccc(C#N)cc3)CC2)cc1-c1[nH]c(CC#N)nc1Cl
InChIKey: XHWYZLDRAZANKZ-UHFFFAOYSA-N
Target: Fatty acid synthase (SINGLE PROTEIN), Homo sapiens
UniProt: P49327
Assay: Biochemical Assay: The FASN enzyme was isolated from SKBr3 cells. SKBr3 is a human breast cancer cell-line with high levels of FASN expression. It is estimated that FASN comprises about 25% of the cytosolic proteins in this cell line. SKBr3 cells were homogenized in a dounce homogenizer then centrifuged for 15 minutes at 4° C. to remove particulate matter. The supernatant was then analyzed for protein content, diluted to the appropriate concentration, and used to measure FASN activity. The presence of FASN was confirmed by western blot analysis. A similar method for isolation of FASN from SKBr3 cells is described in Teresa, P. et al. (Clin. Cancer Res. 2009; 15(24), 7608-7615). FASN activity of the SKBr3 cell extract was determined by measuring either NADPH oxidation or the amount of thiol-containing coenzyme A (CoA) released during the fatty acid synthase reaction. The dye CPM (7-diethylamino-3-(4'-maleimidyl-phenyl)-4-methylcoumarin) contains a thiol reactive group that increases its fluorescence emission on reaction with the sulfhydryl group of CoA.
Measurement: IC50 = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 443.94, ALogP 5, HBA 4, HBD 1, PSA 96.57, QED 0.61
Compound (SMILES): O=C(CN1C(=O)Cc2ccccc21)N1CCN(c2scnc2-c2nc3ccccc3[nH]2)CC1
InChIKey: BDANYOOWQHBQMA-UHFFFAOYSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: FLIPR Assay: The bioactivity of compounds is tested in a fluorometric imaging plate reader (FLIPR: Molecular Devices) using engineered CHO-K1 cells expressing the human CXCR3A coupled to a G protein (Galpha(16)). Cells are plated the day prior to bioassay in F12 medium supplemented with 10% FBS and G418 and hygromycin antibiotics to maintain recombinant selection. At the day of bioassay, cells are washed and dye loaded for one hour with Fluo-4-AM (Invitrogen) in Hanks Balanced Salt Solution (Invitrogen), buffered with 20 mM Hepes at pH 7.4 and sodium bicarbonate (0.015%), containing 5 mM probenecid. This buffer, but lacking the dye and containing probenecid at a concentration of 2.5 nM, is also is used for washing steps (wash buffer); or lacking both dye and probenecid but supplemented with 0.1% BSA for compound dilution steps (dilution buffer). Cells are washed free of excess dye and 60 microliter of wash buffer is added. Stock solutions of test compounds are made up at a concentration of 10 mM in DMSO, and serially diluted in dilution buffer to concentrations required for inhibition dose response curves. After a 10 minute incubation period at 37° C., 10 microliters of each compound dilution are transferred from a compound plate to the plate containing the recombinant cells in the FLIPR instrument according to the manufacturer's instructions. Following basal readings, 10 microliter CXCL10 agonist at a concentration of 20 nM (from Peprotech) is added, again using the FLIPR instrument. Changes in fluorescence are monitored before and after addition of the test compounds.
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 458.55, ALogP 2.92, HBA 6, HBD 1, PSA 85.43, QED 0.51
Compound (SMILES): C[C@@H](NC1CCCC(c2ccc(C(=O)O)cc2)C1)c1cccc2ccccc12
InChIKey: XHECHHFQBPQRSS-PPNDLDFTSA-N
Target: Cytochrome P450 2D6 (SINGLE PROTEIN), Homo sapiens
UniProt: P10635
Assay: Inhibition Assay: Incubations were conducted in 96 well microtiter plates based on a method described by BD Biosciences. To the first well in each row, a NADPH regenerating system and test compound was added. In the second well and all remaining wells, NADPH regenerating system and acetonitrile (final concentration of 2%) was added. The final assay concentration of the NADPH regenerating system was 8.2 uM NADP+, 0.41 mM glucose-6-phosphate, 0.41 mM magnesium chloride hexahydrate and 0.4 U/ml glucose-6-phosphate dehydrogenase and 0.01 mg/mL control insect cell membrane protein. The test compound solution was serially diluted 1:3 through the eighth wells.The final concentration of the test compounds were in the range 100 uM to 45.7 nM in the eight rows. Wells 9 and 10 contained no test compound (only NADPH regenerating system and enzyme/substrate mix) and wells 11 and 12 were used as controls for background fluorescence (enzyme and substrate were added after the reaction was terminated). The plate was then pre-incubated at 37° C. for 10 min, and the reaction was initiated by the addition of pre-warmed enzyme/substrate mix. The assay concentration of the enzyme/substrate mix was 100 mM potassium phosphate, pH 7.4, 1.5 pmol recombinant human P450 CYP2D6 and 1.5 uM of the fluorescent substrate 3-[2-(N,N diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcounnarin (AMMC). The assay was conducted in duplicate in a final volume of 200 uL per well. Reactions were terminated after 30 min by addition of a 4:1, acetonitrile:0.5 M Tris base solution. Quinidine was used as positive control, 0.5 uM as highest concentration. Fluorescence per well was measured using a fluorescence plate reader (excitation: 390 nm, emission: 460 nm).
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 373.5, ALogP 5.92, HBA 2, HBD 2, PSA 49.33, QED 0.58
Compound (SMILES): c1ccc(-c2ccc(-c3cnc(-c4ccccc4)[nH]3)cn2)cc1
InChIKey: LXRPAWBWBSNMJX-UHFFFAOYSA-N
Target: Hematopoietic prostaglandin D synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O60760
Assay: Inhibition of HPGDS (unknown origin) by fluorescence polarization assay
Measurement: IC50 = 31.6 nM (pChEMBL 7.5)
Descriptors: MW 297.36, ALogP 4.81, HBA 2, HBD 1, PSA 41.57, QED 0.59
Compound (SMILES): NCc1ccc(CNC(=O)[C@H](Cc2ccccc2)NC(=O)[C@@H](Cc2ccc(OCc3ccc(F)cc3)cc2)NC(=O)c2ccccc2)cc1
InChIKey: WEVQLDNHQXUOLS-PQQNNWGCSA-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 = 2491 nM (pChEMBL 5.599999904632568)
Descriptors: MW 658.77, ALogP 5.25, HBA 5, HBD 4, PSA 122.55, QED 0.12
Compound (SMILES): Cc1ccc(-n2nc(-c3ccc(N[SH](=O)=O)cc3)cc2NC(=O)c2cnn3cccnc23)cc1
InChIKey: LDTTUYVBNXADJR-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Kinase Assay: The kinase activity of IRAK-4 is determined by its ability to catalyze the phosphorylation of a fluorescent polypeptide substrate. The extent of phosphorylation is measured using the IMAP technology (Molecular Devices) where the phosphorylated fluorescent substrate binds to the large M(III)-based nanoparticles which reduces the rotational speed of the substrate and thus increases its fluorescent polarization (FP). 20 μl reaction mixture contains 10 mM TriHCl, pH 7.2, 0.5 nM GST tagged IRAK-4 (SignalChem), 100 nM fluorescent peptide substrate (RP7030, Molecular Devices), 100 μM ATP, 1 mM DTT, 1 mM MgCl2, and 0.01% Tween 20. The reaction is initiated by the addition of ATP. After incubation for 30 minutes at 25° C., 60 μl of Progressive IMAP Reagent (Molecular Devices) is added to stop the reaction. Change in RP7030's FP is determined by a FP reader (Analyst HT, LH, BioSystems).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 473.52, ALogP 3.08, HBA 8, HBD 3, PSA 123.28, QED 0.33
Compound (SMILES): CN1C(=O)CCC1c1cc2[nH]c(-c3ccccc3)cc2cc1Oc1ccc(CCC(=O)O)cc1
InChIKey: UCRAOTLBHOKJPK-UHFFFAOYSA-N
Target: Hexokinase-4 (SINGLE PROTEIN), Homo sapiens
UniProt: P35557
Assay: Glucokinase-Activating Assay: To test the exemplified compounds, the following assay was employed. Recombinant human liver glucokinase was expressed as a FLAG fusion protein in E. coli, and purified on ANTIFLAG M2 AFFINITY GEL (Sigma). The assay was carried out at 30° C. in a 96-well plate. In the plate was distributed 69 ul each of assay buffer (25 mM Hepes Buffer: pH=7.2, 2 mM MgCl2, 1 mM ATP, 0.5 mM TNAD, 1 mM dithiothreitol), to which was added 1 ul of a DMSO solution of the compound or DMSO as control. Then, 20 ul of pre-ice-cooled enzyme mixture (FLAG-GK, 20 U/ml G6PDH) was distributed thereto, to which was added 10 ul of 25 mM glucose as substrate to initiate the reaction (final glucose concentration=2.5 mM). After starting the reaction, the absorbance at 405 nm was measured every 30 seconds for 10 minutes to evaluate the compound based on the initial increase for 5 minutes. FLAG-GK was added so that the increase of absorbance after 5 minutes fell between 0.05 to 0.1 in the presence of 1% DMSO. The OD values of the respective compounds were measured in the respective concentrations, wherein the OD value of DMSO as control is regarded as 100%. From the OD values at the respective concentrations, Emax (%, 2.5 mM Glu) and EC50 (nM, 2.5 mM Glu) were calculated and used as indicators of the GK activation capability of the compounds. According to the above assay, the GK activation capability of the exemplified compounds of the present invention was determined. The following table shows the results. Where different enantiomers of the same compound were tested, two numbers are provided. Where 3 numbers are provided, for example, Ex. #30, data for the racemic form is also shown.
Measurement: EC50 = 455.9 nM (pChEMBL 6.340000152587891)
Descriptors: MW 454.53, ALogP 5.94, HBA 3, HBD 2, PSA 82.63, QED 0.36
Compound (SMILES): NS(=O)(=O)OC[C@H]1O[C@@H](n2cnc3c(NCCN4CCCC4)ncnc32)[C@H](O)[C@@H]1O
InChIKey: QHLHQTWFNWUANH-XNIJJKJLSA-N
Target: NEDD8-activating enzyme E1 catalytic subunit (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TBC4
Assay: Inhibition of recombinant human untagged NAEalpha/N-terminal His-tagged NAEbeta expressed in Bac-to-Bac expression system using FLAG-tagged NEDD8 as substrate after 90 mins in presence of GST-tagged Ubc12 by HTRF assay
Measurement: IC50 <= 100 nM (pChEMBL 7)
Descriptors: MW 443.49, ALogP -1.83, HBA 12, HBD 4, PSA 177.95, QED 0.36
Compound (SMILES): Cc1c(C(=O)Nc2cccc(C(F)(F)F)c2)cc(C2CCNCC2)n1CCCN1CCOCC1
InChIKey: UHWGANGBODUJKW-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBA0
Assay: Inhibition Assay: Test Example 1: (1) The TRPV4 inhibitory activity was measured using CHO-K1 cells stably expressing human TRPV4 (hTRPV4/CHO cells). (2) In the day before the experimental day, frozen cell were thawed and washed with the culture medium (MEM-α, 10% FBS, 2 mmol/L GlutaMax, 50 unit Penicillin, 50 μg/mL Streptomycin). Then cells were suspended in the culture medium. (3) The 384-well plates which hTRPV4/CHO cells were seeded at densities of 4000 cells/well in the culture medium and cultured in a CO2 incubator in the presence of 5% CO2 at 37° C. for overnight was used as assay plates. (4) The assay plate was washed with assay buffer (Hanks, 20 mmol/L HEPES, 2.5 mmol/L probenecid, pH7.4), and the buffer were remained at 20 μL/well. (5) 10 μl of dye loading buffer (9 μmol/L Fluo 4-AM, 0.09% Pluronic F-127/assay buffer) was added to each well, then the assay plate was incubated in a CO2 incubator in the presence of 5% CO2 at 37° C. for one hour. (final 3 μmol/L Fluo4-AM). (6) The assay plate was washed with the assay buffer, and the buffers were remained at 20 μL/well. Then the assay plate was incubated for 10 min at room temperature. (7) Diluted compound solution was dispensed to each well in the compound plate, and mixed with built-in Pipette and Mixer of the Fluorescence analysis system FLIPR TETRA (Molecular devices). (8) After incubation for 5 min, 20 μL of 4α-PDD solution were applied to each well of assay plate and mixed with the FLIPR TETRA. (final 1 Î¼mol/L 4α-PDD). (9) The fluorescent intensity was measured with FLIPR TETRA system for 10 min from the point of time addition the compound solution, at Ex 470-495 nm, Em 515-575 nm wavelength.
Measurement: Ki = 4700 nM (pChEMBL 5.329999923706055)
Descriptors: MW 478.56, ALogP 4.26, HBA 5, HBD 2, PSA 58.53, QED 0.62
Compound (SMILES): CCn1ncc2cc3nc(c21)CCCO[C@H](CO)[C@H](c1ccccc1)NC(=O)N3
InChIKey: ILQXAAGAKNRVJZ-MJGOQNOKSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Inhibition of human activated ERK2 assessed as reduction in phosphorylation activity preincubated for 15 mins followed by addition of IMAP peptide as substrate and ATP measured after 60 mins by fluorescence polarization method
Measurement: IC50 = 3.443 nM (pChEMBL 8.460000038146973)
Descriptors: MW 395.46, ALogP 2.64, HBA 6, HBD 3, PSA 101.3, QED 0.63
Compound (SMILES): CN(c1ccc(NS(=O)(=O)C2=CCCS2)cn1)[C@@H]1CCN(c2ncnc3[nH]ccc23)C1
InChIKey: JEILQWKCGZRZNP-OAHLLOKOSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzymatic Assay: JAK1/2/3 kinase assay are performed in vitro using Kit-Tyr 6 Peptide (Invitrogen, Cat. No. PV4122). TYK2 kinase assay are performed in vitro using Z'-LYTE™ Kinase Assay Kit-Tyr 3 Peptide (Invitrogen, Cat. No. PV3192). Recombinant human JAK1/2/3 or TYK2 catalytic domains are from Invitrogen (Cat No. PV4774/PV4210/PV3855/PV4790); All reactions (20 μL) are started by adding 2.5 μL of the testing compound in 4% DMSO solution, 5 μL of Kinase/Peptide substrate Mixture (3.2, 0.04, 0.2 or 8 μg/mL for Recombinant human JAK1/2/3 catalytic domains, 4 μM for Z-LYTE™ Tyr 6 peptide or Z-LYTE™ Tyr 3 peptide) or Phospho-Peptide solution (Invitrogen, Cat. No. PV3192, diluted with 1.33× Kinase Buffer), 2.5 μL ATP Solution (300/100/40/100M, JAK1/JAK2/JAK3/TYK2) or 1.33× Kinase Buffer (Invitrogen, Cat. No. PV3189, 5× diluted with distilled water). The 384-well assay plate (Corning, Cat. No. 3575) is mixed and incubated at room temperature for 1 hour. 5 μL of the Development Solution (Dilute Development Reagent A (Cat. No. PV3297) is diluted to 1/64 with Development Buffer (Cat. No. PV3127) for JAK1, JAK2 and JAK3 assay; Development Reagent A (Cat. No. PV3297) is diluted to 1/2048 with Development Buffer (Cat. No. PV3127) for TYK2 assay. The diluted Development Solution is then added to each well, mixed and incubated at room temperature for another 1 hour. The kinase reaction is then stopped by adding 5 μL of the Stop Reagent (Invitrogen, Cat. No. PV3094), and the plate is read with Wallac 1420 VICTOR3 Multilabel Counter (PerkinElmer™) at 445 nm and 520 nm fluorescence. All compounds are initially tested at 8 different concentrations (1 μM down to 0.0003 μM) using a 1:3 serial dilution scheme.
Measurement: IC50 = 0.055 nM (pChEMBL 10.260000228881836)
Descriptors: MW 457.59, ALogP 2.79, HBA 8, HBD 2, PSA 107.11, QED 0.58
Compound (SMILES): Cc1nc(C(=O)N2Cc3cc4c(cc3C[C@H]2C(=O)N[C@@H](Cc2ccc(-c3ccnc(C)c3C)cc2)C(=O)O)OC[C@H](c2ccc(OCC3CCC(C(F)(F)F)CC3)cc2)O4)c(C)o1
InChIKey: SMCXOOICFFLLPU-NNXAUVBWSA-N
Target: Glucagon-like peptide 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P43220
Assay: cAMP Functional Assay: The efficacy of GLP-1 receptor agonists was studied in a cAMP functional assay using HEK-293 Cells expressing the cloned human GLP-1 receptor. GLP-1-expressing cells (10,000-20,000 cells per 0.1 mL) were plated in 96-well plates in Dulbecco's modified eagles media (DMEM) containing 10% fetal bovine serum, 2 mg/mL G418 and penicillin-streptomycin. Following overnight incubation of cells, media was removed, and compounds (at concentrations ranging from 0.0001 to 100 μM) were added to monolayer cells in Iscove's modified dulbecco's medium (IMDM), 100 μM RO 20-1724 PDE inhibitor, 0.1% BSA, 2% DMSO in a final volume of 100 μL, and incubated for 30 min at 37° C. 95%02, 5% CO2 in a humidified incubator. Alternatively, cells were harvested and combined with compounds in suspension using the buffer and protocol described above. cAMP was quantified using a homogenous time-resolved fluorescence detection system (cAMP dynamic, CIS bio International).
Measurement: EC50 = 183 nM (pChEMBL 6.739999771118164)
Descriptors: MW 866.93, ALogP 8.61, HBA 9, HBD 2, PSA 153.32, QED 0.13
Compound (SMILES): O=C/C=C1\O[C@@H]2CC(=O)N2C1(C/C=C/c1ccccc1)C(=O)OCc1ccccc1
InChIKey: KBUJSCWFZMYQLR-QQKZXBOMSA-N
Target: Beta-lactamase (SINGLE PROTEIN), Escherichia coli K-12
UniProt: P00811
Assay: Cell Free Inhibition Assay: The inhibitory concentrations (IC50, [uM]) of the beta -lactamase inhibitors against purified TEM-1, SHV-1 and AmpC beta -lactamases are assessed by determining the concentration of inhibitor at which 50% of the nitrocefin hydrolysis is inhibited by the particular enzyme. Assays are performed with beta -lactamases expressed in the pET system (Novagen, San Diego, Calif.) without signal peptides. They contain an N-terminal hexa-Histidine tag that is used for purification on Ni-NTA (Qiagen, Hilden, Germany). The compounds are prepared as 50 mM stocks in DMSO and diluted into buffer P1 (50 mM phosphate, pH 7) to a final concentration of 10% DMSO. All further dilutions are done in P2 (P1 with 10% DMSO). Enzyme and compound dilutions are pre-incubated for 10 min at 37 C. and the reaction is started with the addition of pre-warmed (37 C.) nitrocefin at a final concentration of 50 mM. The change in absorption at 490 nm is followed at 37 C. for 10 min with 30 s intervals.
Measurement: IC50 = 5820 nM (pChEMBL 5.239999771118164)
Descriptors: MW 403.43, ALogP 3.24, HBA 5, HBD 0, PSA 72.91, QED 0.31
Compound (SMILES): CC(C)(C)NC(=O)C1CCN(Cc2cccc(NC(=O)Cc3ccccc3Cl)c2)CC1
InChIKey: RYJGOOFWXBVDKG-UHFFFAOYSA-N
Target: Atypical chemokine receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P25106
Assay: In Vitro Assay: CHO-K1 CXCR7 b-arrestin cells are detached from culture dishes with a cell dissociation buffer (Invitrogen, #13151-014) and collected in growing medium (F12 HAMS 90% (v/v)/FCS 10% (v/v), Penicilin/streptomycin 1% (v/v)). 5000 cells per well (in 20 μl) are seeded in a 384 well plate (white-walled, clear bottom; BD Falcon #353274). The plate is incubated at 37° C./5% CO2 for 24 hours. Medium is then replaced by 20 μl OPTIMEM (Invitrogen #31985) for 3 to 4 hours. Test compounds are dissolved at 10 mM in DMSO and serially diluted in DMSO to 200× of the final concentration for dose response curves. Compounds are then diluted 1:33.3 in HBSS1×. 5 μl/well of HBSS1×/20 mM HEPES/0.2% BSA are added to the assay plate followed by addition of 5 μl/well of diluted compounds. CXCL12 (Peprotech #300-28A) may be used as a reference agonist. The plate is incubated for 90 minutes at 37° C. 12 μl of detection reagent (Path Hunter Detection Kit, DiscoveRx, #93-0001) is transferred to the assay plate and to the plate is incubated for 1 hour at room temperature. Luminescent signal is read in a microplate reader (FLUOstar Optima, bmg).
Measurement: EC50 = 1 nM (pChEMBL 9)
Descriptors: MW 442, ALogP 4.65, HBA 3, HBD 2, PSA 61.44, QED 0.69