lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(C)CN(Cc1cc(Cl)c2c(c1)OCCCO2)C(=O)C(C)CNCc1ccccc1F
InChIKey: JEAKPDQKDUXKJF-UHFFFAOYSA-N
Target: Prokineticin receptor 2 (SINGLE PROTEIN), Cricetulus griseus
UniProt: G3H407
Assay: Calcium Mobilization Assay: An aequorin-based luminescent assay for calcium mobilization was used to measure mobilization of intracellular Ca2+ (Bullock et al., Mol Pharmacol 65, 582-588, 2004). Chinese hamster ovary (CHO) cells stably expressing photoprotein aequorin and recombinant PKR1 or PKR2 were tested by this method. Briefly, the cells were charged in Opti-MEM (Invitrogen) containing 8 μM of coelenterazine cp at 37° C. for 2 hours. Cells were detached by brief typsinization and maintained in Hank's Balanced Salt Solution (HBSS) plus 10 mM HEPES (pH7.5) and 0.1% BSA at about 5×105 cells/ml. Luminescence measurements were made using a Berthold luminometer.
Measurement: Ki = 8.62 nM (pChEMBL 8.0600004196167)
Descriptors: MW 462.99, ALogP 5.05, HBA 4, HBD 1, PSA 50.8, QED 0.57
Compound (SMILES): CC(C)CN(Cc1cc(Cl)c2c(c1)OCCCO2)C(=O)C(C)CNCc1cccc(O)c1
InChIKey: HCMJCMYVOXIEMD-UHFFFAOYSA-N
Target: Prokineticin receptor 1 (SINGLE PROTEIN), Cricetulus griseus
UniProt: G3HIE5
Assay: Calcium Mobilization Assay: An aequorin-based luminescent assay for calcium mobilization was used to measure mobilization of intracellular Ca2+ (Bullock et al., Mol Pharmacol 65, 582-588, 2004). Chinese hamster ovary (CHO) cells stably expressing photoprotein aequorin and recombinant PKR1 or PKR2 were tested by this method. Briefly, the cells were charged in Opti-MEM (Invitrogen) containing 8 μM of coelenterazine cp at 37° C. for 2 hours. Cells were detached by brief typsinization and maintained in Hank's Balanced Salt Solution (HBSS) plus 10 mM HEPES (pH7.5) and 0.1% BSA at about 5×105 cells/ml. Luminescence measurements were made using a Berthold luminometer.
Measurement: Ki = 23.5 nM (pChEMBL 7.630000114440918)
Descriptors: MW 461, ALogP 4.62, HBA 5, HBD 2, PSA 71.03, QED 0.57
Compound (SMILES): Cc1ccccc1CNCC(C)C(=O)N(Cc1cc(Cl)c2c(c1)OCCCO2)CC(C)C
InChIKey: DLEIOLCZRQPXMP-UHFFFAOYSA-N
Target: Prokineticin receptor 1 (SINGLE PROTEIN), Cricetulus griseus
UniProt: G3HIE5
Assay: Calcium Mobilization Assay: An aequorin-based luminescent assay for calcium mobilization was used to measure mobilization of intracellular Ca2+ (Bullock et al., Mol Pharmacol 65, 582-588, 2004). Chinese hamster ovary (CHO) cells stably expressing photoprotein aequorin and recombinant PKR1 or PKR2 were tested by this method. Briefly, the cells were charged in Opti-MEM (Invitrogen) containing 8 μM of coelenterazine cp at 37° C. for 2 hours. Cells were detached by brief typsinization and maintained in Hank's Balanced Salt Solution (HBSS) plus 10 mM HEPES (pH7.5) and 0.1% BSA at about 5×105 cells/ml. Luminescence measurements were made using a Berthold luminometer.
Measurement: Ki = 19.6 nM (pChEMBL 7.710000038146973)
Descriptors: MW 459.03, ALogP 5.22, HBA 4, HBD 1, PSA 50.8, QED 0.56
Compound (SMILES): CC(C)CN(Cc1cc(Cl)c2c(c1)OCCCO2)C(=O)C(C)CNCc1cccc(N)c1
InChIKey: JUTHYLUQHVXSQU-UHFFFAOYSA-N
Target: Prokineticin receptor 1 (SINGLE PROTEIN), Cricetulus griseus
UniProt: G3HIE5
Assay: Calcium Mobilization Assay: An aequorin-based luminescent assay for calcium mobilization was used to measure mobilization of intracellular Ca2+ (Bullock et al., Mol Pharmacol 65, 582-588, 2004). Chinese hamster ovary (CHO) cells stably expressing photoprotein aequorin and recombinant PKR1 or PKR2 were tested by this method. Briefly, the cells were charged in Opti-MEM (Invitrogen) containing 8 μM of coelenterazine cp at 37° C. for 2 hours. Cells were detached by brief typsinization and maintained in Hank's Balanced Salt Solution (HBSS) plus 10 mM HEPES (pH7.5) and 0.1% BSA at about 5×105 cells/ml. Luminescence measurements were made using a Berthold luminometer.
Measurement: Ki = 19.8 nM (pChEMBL 7.699999809265137)
Descriptors: MW 460.02, ALogP 4.49, HBA 5, HBD 2, PSA 76.82, QED 0.54
Compound (SMILES): COc1ncccc1CNCC(C)C(=O)N(Cc1cc(Cl)c2c(c1)OCCCO2)CC(C)C
InChIKey: AJVTYHYPVKOHDL-UHFFFAOYSA-N
Target: Prokineticin receptor 2 (SINGLE PROTEIN), Cricetulus griseus
UniProt: G3H407
Assay: Calcium Mobilization Assay: An aequorin-based luminescent assay for calcium mobilization was used to measure mobilization of intracellular Ca2+ (Bullock et al., Mol Pharmacol 65, 582-588, 2004). Chinese hamster ovary (CHO) cells stably expressing photoprotein aequorin and recombinant PKR1 or PKR2 were tested by this method. Briefly, the cells were charged in Opti-MEM (Invitrogen) containing 8 μM of coelenterazine cp at 37° C. for 2 hours. Cells were detached by brief typsinization and maintained in Hank's Balanced Salt Solution (HBSS) plus 10 mM HEPES (pH7.5) and 0.1% BSA at about 5×105 cells/ml. Luminescence measurements were made using a Berthold luminometer.
Measurement: Ki = 1.82 nM (pChEMBL 8.739999771118164)
Descriptors: MW 476.02, ALogP 4.32, HBA 6, HBD 1, PSA 72.92, QED 0.55
Compound (SMILES): CC(CNCc1ccccc1)C(=O)NC(CCc1ccc2c(c1)OCCCO2)C(C)C
InChIKey: YDYGYVQRRINDBI-UHFFFAOYSA-N
Target: Prokineticin receptor 1 (SINGLE PROTEIN), Cricetulus griseus
UniProt: G3HIE5
Assay: Calcium Mobilization Assay: An aequorin-based luminescent assay for calcium mobilization was used to measure mobilization of intracellular Ca2+ (Bullock et al., Mol Pharmacol 65, 582-588, 2004). Chinese hamster ovary (CHO) cells stably expressing photoprotein aequorin and recombinant PKR1 or PKR2 were tested by this method. Briefly, the cells were charged in Opti-MEM (Invitrogen) containing 8 μM of coelenterazine cp at 37° C. for 2 hours. Cells were detached by brief typsinization and maintained in Hank's Balanced Salt Solution (HBSS) plus 10 mM HEPES (pH7.5) and 0.1% BSA at about 5×105 cells/ml. Luminescence measurements were made using a Berthold luminometer.
Measurement: Ki = 573 nM (pChEMBL 6.239999771118164)
Descriptors: MW 424.59, ALogP 4.35, HBA 4, HBD 2, PSA 59.59, QED 0.6
Compound (SMILES): CC(C)CN(Cc1cc(Cl)c2c(c1)OCCO2)C(=O)C(C)CNCc1ccccc1
InChIKey: JSGXCKQZBFNOOT-UHFFFAOYSA-N
Target: Prokineticin receptor 1 (SINGLE PROTEIN), Cricetulus griseus
UniProt: G3HIE5
Assay: Calcium Mobilization Assay: An aequorin-based luminescent assay for calcium mobilization was used to measure mobilization of intracellular Ca2+ (Bullock et al., Mol Pharmacol 65, 582-588, 2004). Chinese hamster ovary (CHO) cells stably expressing photoprotein aequorin and recombinant PKR1 or PKR2 were tested by this method. Briefly, the cells were charged in Opti-MEM (Invitrogen) containing 8 μM of coelenterazine cp at 37° C. for 2 hours. Cells were detached by brief typsinization and maintained in Hank's Balanced Salt Solution (HBSS) plus 10 mM HEPES (pH7.5) and 0.1% BSA at about 5×105 cells/ml. Luminescence measurements were made using a Berthold luminometer.
Measurement: Ki = 134 nM (pChEMBL 6.869999885559082)
Descriptors: MW 430.98, ALogP 4.52, HBA 4, HBD 1, PSA 50.8, QED 0.63
Compound (SMILES): CC(C)CN(Cc1cc(Cl)c2c(c1)OCCCO2)C(=O)C1CN(Cc2ccccc2)CCO1
InChIKey: PJETZGQSLQTKAL-UHFFFAOYSA-N
Target: Prokineticin receptor 1 (SINGLE PROTEIN), Cricetulus griseus
UniProt: G3HIE5
Assay: Calcium Mobilization Assay: An aequorin-based luminescent assay for calcium mobilization was used to measure mobilization of intracellular Ca2+ (Bullock et al., Mol Pharmacol 65, 582-588, 2004). Chinese hamster ovary (CHO) cells stably expressing photoprotein aequorin and recombinant PKR1 or PKR2 were tested by this method. Briefly, the cells were charged in Opti-MEM (Invitrogen) containing 8 μM of coelenterazine cp at 37° C. for 2 hours. Cells were detached by brief typsinization and maintained in Hank's Balanced Salt Solution (HBSS) plus 10 mM HEPES (pH7.5) and 0.1% BSA at about 5×105 cells/ml. Luminescence measurements were made using a Berthold luminometer.
Measurement: Ki = 23 nM (pChEMBL 7.639999866485596)
Descriptors: MW 473.01, ALogP 4.39, HBA 5, HBD 0, PSA 51.24, QED 0.6
Compound (SMILES): CC(C)CN(Cc1cc(Cl)c2c(c1)OCCCO2)C(=O)C(C)CNCc1cccc2[nH]ccc12
InChIKey: WVARBMDYVFPBSM-UHFFFAOYSA-N
Target: Prokineticin receptor 2 (SINGLE PROTEIN), Cricetulus griseus
UniProt: G3H407
Assay: Calcium Mobilization Assay: An aequorin-based luminescent assay for calcium mobilization was used to measure mobilization of intracellular Ca2+ (Bullock et al., Mol Pharmacol 65, 582-588, 2004). Chinese hamster ovary (CHO) cells stably expressing photoprotein aequorin and recombinant PKR1 or PKR2 were tested by this method. Briefly, the cells were charged in Opti-MEM (Invitrogen) containing 8 μM of coelenterazine cp at 37° C. for 2 hours. Cells were detached by brief typsinization and maintained in Hank's Balanced Salt Solution (HBSS) plus 10 mM HEPES (pH7.5) and 0.1% BSA at about 5×105 cells/ml. Luminescence measurements were made using a Berthold luminometer.
Measurement: Ki = 87.8 nM (pChEMBL 7.059999942779541)
Descriptors: MW 484.04, ALogP 5.39, HBA 4, HBD 2, PSA 66.59, QED 0.43
Compound (SMILES): CC(C)CN(Cc1cc(Cl)c2c(c1)OCCCO2)C(=O)C(C)CNCc1cccc2cc[nH]c12
InChIKey: KAPDWKOAFWYLSJ-UHFFFAOYSA-N
Target: Prokineticin receptor 1 (SINGLE PROTEIN), Cricetulus griseus
UniProt: G3HIE5
Assay: Calcium Mobilization Assay: An aequorin-based luminescent assay for calcium mobilization was used to measure mobilization of intracellular Ca2+ (Bullock et al., Mol Pharmacol 65, 582-588, 2004). Chinese hamster ovary (CHO) cells stably expressing photoprotein aequorin and recombinant PKR1 or PKR2 were tested by this method. Briefly, the cells were charged in Opti-MEM (Invitrogen) containing 8 μM of coelenterazine cp at 37° C. for 2 hours. Cells were detached by brief typsinization and maintained in Hank's Balanced Salt Solution (HBSS) plus 10 mM HEPES (pH7.5) and 0.1% BSA at about 5×105 cells/ml. Luminescence measurements were made using a Berthold luminometer.
Measurement: Ki = 6.96 nM (pChEMBL 8.15999984741211)
Descriptors: MW 484.04, ALogP 5.39, HBA 4, HBD 2, PSA 66.59, QED 0.43
Compound (SMILES): CC(C)CN(Cc1cc(Cl)c2c(c1)OCCCO2)C(=O)C(C)CNCc1cccc2c1ccn2C
InChIKey: XHIQXRHIAJLOTA-UHFFFAOYSA-N
Target: Prokineticin receptor 2 (SINGLE PROTEIN), Cricetulus griseus
UniProt: G3H407
Assay: Calcium Mobilization Assay: An aequorin-based luminescent assay for calcium mobilization was used to measure mobilization of intracellular Ca2+ (Bullock et al., Mol Pharmacol 65, 582-588, 2004). Chinese hamster ovary (CHO) cells stably expressing photoprotein aequorin and recombinant PKR1 or PKR2 were tested by this method. Briefly, the cells were charged in Opti-MEM (Invitrogen) containing 8 μM of coelenterazine cp at 37° C. for 2 hours. Cells were detached by brief typsinization and maintained in Hank's Balanced Salt Solution (HBSS) plus 10 mM HEPES (pH7.5) and 0.1% BSA at about 5×105 cells/ml. Luminescence measurements were made using a Berthold luminometer.
Measurement: Ki = 3.96 nM (pChEMBL 8.399999618530273)
Descriptors: MW 498.07, ALogP 5.4, HBA 5, HBD 1, PSA 55.73, QED 0.43
Compound (SMILES): CCCN(Cc1cc(Cl)c2c(c1)OCCCO2)C(=O)C(C)CNCc1cccc2[nH]ccc12
InChIKey: QPQXYADDRSUJTK-UHFFFAOYSA-N
Target: Prokineticin receptor 1 (SINGLE PROTEIN), Cricetulus griseus
UniProt: G3HIE5
Assay: Calcium Mobilization Assay: An aequorin-based luminescent assay for calcium mobilization was used to measure mobilization of intracellular Ca2+ (Bullock et al., Mol Pharmacol 65, 582-588, 2004). Chinese hamster ovary (CHO) cells stably expressing photoprotein aequorin and recombinant PKR1 or PKR2 were tested by this method. Briefly, the cells were charged in Opti-MEM (Invitrogen) containing 8 μM of coelenterazine cp at 37° C. for 2 hours. Cells were detached by brief typsinization and maintained in Hank's Balanced Salt Solution (HBSS) plus 10 mM HEPES (pH7.5) and 0.1% BSA at about 5×105 cells/ml. Luminescence measurements were made using a Berthold luminometer.
Measurement: Ki = 19.7 nM (pChEMBL 7.710000038146973)
Descriptors: MW 470.01, ALogP 5.15, HBA 4, HBD 2, PSA 66.59, QED 0.45
Compound (SMILES): COc1ncc(Nc2ncc(C(C)CO)cc2-c2nc(C)nc(N)n2)cc1F
InChIKey: JCDSCTRINIXUJR-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Alphascreen Assay: The PI3K AlphaScreen assay (PerkinElmer, Waltham, Mass.) measures the activity of a panel of four phosphoinositide 3-kinases: PI3Kalpha, PI3Kbeta, PI3Kgamma, and PI3Kdelta. Each of these enzymes phosphorylates the 3'-hydroxyl group on phosphatidylinositiol (4,5)-bisphosphate (PIP2) to produce phosphatidylinositol (3,4,5)-trisphosphate (PIP3). This phosphorylation activity is measured using a GST-tagged PIP3 binding protein (Echelon Biosciences, Salt Lake City, Utah), an anti-GST-tagged Acceptor bead, and streptavidin-Donor bead. The interaction of biotinylated-PIP3 analog (IP4) and the PIP3 binding protein brings both Acceptor and Donor beads together producing, upon excitation of the Donor beads at 680 nm, a singlet oxygen species leading to the luminescent AlphaScreen signal. When PIP3 is produced via phosphorylation of PIP2 by a PI3K, PIP3 competes with biotinylated-PIP3 analog (IP4) for binding to the PIP3 binding protein.
Measurement: Ki = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 385.4, ALogP 2.21, HBA 9, HBD 3, PSA 131.96, QED 0.58
Compound (SMILES): CC(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@H]1CCCNC(=O)CC[C@@H](C(N)=O)NC(=O)[C@H](Cc2c[nH]c3ccccc23)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@@H](Cc2ccccc2)NC(=O)[C@H](CCC(N)=O)NC1=O
InChIKey: SUYDUGVDUFCDHQ-IQSCWYJSSA-N
Target: Melanocyte-stimulating hormone receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q01726
Assay: cAMP Assay: Accumulation of intracellular cAMP was examined as a measure of the ability of the peptides of the present invention to elicit a functional response in HEK-293 cells that express MC4-R. Confluent HEK-293 cells that express recombinant hMC4-R were detached from culture plates by incubation in enzyme-free cell dissociation buffer. Dispersed cells were suspended in Earle's Balanced Salt Solution containing 10 mM HEPES (pH 7.5), 1 mM MgCl2, 1 mM glutamine, 0.5% albumin and 0.3 mM 3-isobutyl-1-methyl-xanthine (IBMX), a phosphodiesterase inhibitor. The cells were plated in 96-well plates at a density of 0.5x105 cells per well and pre-incubated for 10 minutes. Cells were exposed for 15 minutes at 37° C. to peptides of the present invention dissolved in DMSO (final DMSO concentration of 1%) at a concentration range of 0.05-5000 nM in a total assay volume of 200 uL. NDP-α-MSH was used as the reference agonist. cAMP levels were determined by an HTRF cAMP cell-based assay system from Cisbio Bioassays utilizing cryptate-labeled anti-cAMP and d2-labeled cAMP, with plates read on a PerkinElmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. The maximum efficacies of the test peptides of the present invention were compared to that achieved by the reference melanocortin agonist NDP-αMSH.
Measurement: EC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 1058.22
Compound (SMILES): CC(=O)N[C@H](CCCNC(=N)N)C(=O)N[C@H]1CCCNC(=O)CC[C@@H](C(N)=O)NC(=O)[C@H](Cc2c[nH]c3ccccc23)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@@H](Cc2ccccc2)NC(=O)[C@H](CCC(N)=O)NC1=O
InChIKey: SUYDUGVDUFCDHQ-XEFNOBGUSA-N
Target: Melanocyte-stimulating hormone receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q01726
Assay: cAMP Assay: Accumulation of intracellular cAMP was examined as a measure of the ability of the peptides of the present invention to elicit a functional response in HEK-293 cells that express MC4-R. Confluent HEK-293 cells that express recombinant hMC4-R were detached from culture plates by incubation in enzyme-free cell dissociation buffer. Dispersed cells were suspended in Earle's Balanced Salt Solution containing 10 mM HEPES (pH 7.5), 1 mM MgCl2, 1 mM glutamine, 0.5% albumin and 0.3 mM 3-isobutyl-1-methyl-xanthine (IBMX), a phosphodiesterase inhibitor. The cells were plated in 96-well plates at a density of 0.5x105 cells per well and pre-incubated for 10 minutes. Cells were exposed for 15 minutes at 37° C. to peptides of the present invention dissolved in DMSO (final DMSO concentration of 1%) at a concentration range of 0.05-5000 nM in a total assay volume of 200 uL. NDP-α-MSH was used as the reference agonist. cAMP levels were determined by an HTRF cAMP cell-based assay system from Cisbio Bioassays utilizing cryptate-labeled anti-cAMP and d2-labeled cAMP, with plates read on a PerkinElmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. The maximum efficacies of the test peptides of the present invention were compared to that achieved by the reference melanocortin agonist NDP-αMSH.
Measurement: EC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 1058.22
Compound (SMILES): CC(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@H]1CCCNC(=O)CC[C@@H](C(N)=O)NC(=O)[C@H](Cc2c[nH]c3ccccc23)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@@H](Cc2ccccc2)NC(=O)[C@H](CC(N)=O)NC1=O
InChIKey: BOAIJXQJMLWMSF-VWUVXGKKSA-N
Target: Melanin-concentrating hormone receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99705
Assay: Competitive Inhibition Assay: A competitive inhibition binding assay is performed using membrane homogenates prepared from HEK-293 cells that express recombinant hMC4-R, hMC3-R, or hMC5-R, and from B-16 mouse melanoma cells (containing endogenous MC1-R). In some instances, HEK-293 cells that express recombinant hMC1-R were employed. In the examples that follow, all MC3-R, MC4-R and MC5-R values are for human recombinant receptors. MC1-R values are for B-16 mouse melanoma cells, unless the heading is hMC1-R, in which case the value is for human recombinant MC1-R. Assays were performed in 96 well GF/B Millipore multiscreen filtration plates (MAFB NOB10) pre-coated with 0.5% bovine serum albumin (Fraction V). Membrane homogenates were incubated with 0.2 nM (for hMC4-R) 0.4 nM (for MC3-R and MC5-R) or 0.1 nM (for mouse B16 MC1-R or hMC1-R) [I125]-NDP-α-MSH (Perkin Elmer) and increasing concentrations of test peptides of the present invention in buffer containing 25 mM HEPES buffer (pH 7.5) with 100 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 0.3 mM 1,10-phenanthroline, and 0.2% bovine serum albumin. After incubation for 60 minutes at 37° C., the assay mixture was filtered and the membranes washed three times with ice-cold buffer. Filters were dried and counted in a gamma counter for bound radioactivity. Non-specific binding was measured by inhibition of binding of [I125]-NDP-α-MSH in the presence of 1 μM NDP-α-MSH. Maximal specific binding (100%) was defined as the difference in radioactivity (cpm) bound to cell membranes in the absence and presence of 1 μM NDP-α-MSH. Radioactivity (cpm) obtained in the presence of test compounds was normalized with respect to 100% specific binding to determine the percent inhibition of [I125]-NDP-α-MSH binding.
Measurement: Ki = 688 nM (pChEMBL 6.159999847412109)
Descriptors: MW 1044.19
Compound (SMILES): CC(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@H]1CCCCNC(=O)C[C@@H](C(N)=O)NC(=O)[C@H](Cc2c[nH]c3ccccc23)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@@H](Cc2ccccc2)NC(=O)[C@H](CC(N)=O)NC1=O
InChIKey: GTMASPVXPKDKOK-VWUVXGKKSA-N
Target: Melanocyte-stimulating hormone receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q01726
Assay: cAMP Assay: Accumulation of intracellular cAMP was examined as a measure of the ability of the peptides of the present invention to elicit a functional response in HEK-293 cells that express MC4-R. Confluent HEK-293 cells that express recombinant hMC4-R were detached from culture plates by incubation in enzyme-free cell dissociation buffer. Dispersed cells were suspended in Earle's Balanced Salt Solution containing 10 mM HEPES (pH 7.5), 1 mM MgCl2, 1 mM glutamine, 0.5% albumin and 0.3 mM 3-isobutyl-1-methyl-xanthine (IBMX), a phosphodiesterase inhibitor. The cells were plated in 96-well plates at a density of 0.5x105 cells per well and pre-incubated for 10 minutes. Cells were exposed for 15 minutes at 37° C. to peptides of the present invention dissolved in DMSO (final DMSO concentration of 1%) at a concentration range of 0.05-5000 nM in a total assay volume of 200 uL. NDP-α-MSH was used as the reference agonist. cAMP levels were determined by an HTRF cAMP cell-based assay system from Cisbio Bioassays utilizing cryptate-labeled anti-cAMP and d2-labeled cAMP, with plates read on a PerkinElmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. The maximum efficacies of the test peptides of the present invention were compared to that achieved by the reference melanocortin agonist NDP-αMSH.
Measurement: EC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 1044.19
Compound (SMILES): CC(=O)N[C@H](CCCNC(=N)N)C(=O)N[C@H]1CCCNC(=O)CC[C@@H](C(N)=O)NC(=O)[C@H](Cc2c[nH]c3ccccc23)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@@H](Cc2ccccc2)NC(=O)[C@H](CC(N)=O)NC1=O
InChIKey: BOAIJXQJMLWMSF-GWZAZKCQSA-N
Target: Melanocortin receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P32245
Assay: Competitive Inhibition Assay: A competitive inhibition binding assay is performed using membrane homogenates prepared from HEK-293 cells that express recombinant hMC4-R, hMC3-R, or hMC5-R, and from B-16 mouse melanoma cells (containing endogenous MC1-R). In some instances, HEK-293 cells that express recombinant hMC1-R were employed. In the examples that follow, all MC3-R, MC4-R and MC5-R values are for human recombinant receptors. MC1-R values are for B-16 mouse melanoma cells, unless the heading is hMC1-R, in which case the value is for human recombinant MC1-R. Assays were performed in 96 well GF/B Millipore multiscreen filtration plates (MAFB NOB10) pre-coated with 0.5% bovine serum albumin (Fraction V). Membrane homogenates were incubated with 0.2 nM (for hMC4-R) 0.4 nM (for MC3-R and MC5-R) or 0.1 nM (for mouse B16 MC1-R or hMC1-R) [I125]-NDP-alpha -MSH (Perkin Elmer) and increasing concentrations of test peptides of the present invention in buffer containing 25 mM HEPES buffer (pH 7.5) with 100 mM NaCl.
Measurement: Ki = 28 nM (pChEMBL 7.550000190734863)
Descriptors: MW 1044.19
Compound (SMILES): CC(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@H]1CCNC(=O)CCC[C@@H](C(N)=O)NC(=O)[C@H](Cc2c[nH]c3ccccc23)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@@H](Cc2ccccc2)NC(=O)[C@H](CCC(N)=O)NC1=O
InChIKey: FSTDLRINZOKGRA-IQSCWYJSSA-N
Target: Melanocyte-stimulating hormone receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q01726
Assay: Competitive Inhibition Assay: A competitive inhibition binding assay is performed using membrane homogenates prepared from HEK-293 cells that express recombinant hMC4-R, hMC3-R, or hMC5-R, and from B-16 mouse melanoma cells (containing endogenous MC1-R). In some instances, HEK-293 cells that express recombinant hMC1-R were employed. In the examples that follow, all MC3-R, MC4-R and MC5-R values are for human recombinant receptors. MC1-R values are for B-16 mouse melanoma cells, unless the heading is hMC1-R, in which case the value is for human recombinant MC1-R. Assays were performed in 96 well GF/B Millipore multiscreen filtration plates (MAFB NOB10) pre-coated with 0.5% bovine serum albumin (Fraction V). Membrane homogenates were incubated with 0.2 nM (for hMC4-R) 0.4 nM (for MC3-R and MC5-R) or 0.1 nM (for mouse B16 MC1-R or hMC1-R) [I125]-NDP-alpha -MSH (Perkin Elmer) and increasing concentrations of test peptides of the present invention in buffer containing 25 mM HEPES buffer (pH 7.5) with 100 mM NaCl.
Measurement: Ki = 133 nM (pChEMBL 6.880000114440918)
Descriptors: MW 1058.22
Compound (SMILES): CC(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@H]1CCCC(=O)NCC[C@@H](C(N)=O)NC(=O)[C@H](Cc2c[nH]c3ccccc23)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@@H](Cc2ccccc2)NC(=O)[C@H](CC(N)=O)NC1=O
InChIKey: ZLLOVWBBACAVIV-VWUVXGKKSA-N
Target: Melanin-concentrating hormone receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99705
Assay: Competitive Inhibition Assay: A competitive inhibition binding assay is performed using membrane homogenates prepared from HEK-293 cells that express recombinant hMC4-R, hMC3-R, or hMC5-R, and from B-16 mouse melanoma cells (containing endogenous MC1-R). In some instances, HEK-293 cells that express recombinant hMC1-R were employed. In the examples that follow, all MC3-R, MC4-R and MC5-R values are for human recombinant receptors. MC1-R values are for B-16 mouse melanoma cells, unless the heading is hMC1-R, in which case the value is for human recombinant MC1-R. Assays were performed in 96 well GF/B Millipore multiscreen filtration plates (MAFB NOB10) pre-coated with 0.5% bovine serum albumin (Fraction V). Membrane homogenates were incubated with 0.2 nM (for hMC4-R) 0.4 nM (for MC3-R and MC5-R) or 0.1 nM (for mouse B16 MC1-R or hMC1-R) [I125]-NDP-α-MSH (Perkin Elmer) and increasing concentrations of test peptides of the present invention in buffer containing 25 mM HEPES buffer (pH 7.5) with 100 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 0.3 mM 1,10-phenanthroline, and 0.2% bovine serum albumin. After incubation for 60 minutes at 37° C., the assay mixture was filtered and the membranes washed three times with ice-cold buffer. Filters were dried and counted in a gamma counter for bound radioactivity. Non-specific binding was measured by inhibition of binding of [I125]-NDP-α-MSH in the presence of 1 μM NDP-α-MSH. Maximal specific binding (100%) was defined as the difference in radioactivity (cpm) bound to cell membranes in the absence and presence of 1 μM NDP-α-MSH. Radioactivity (cpm) obtained in the presence of test compounds was normalized with respect to 100% specific binding to determine the percent inhibition of [I125]-NDP-α-MSH binding.
Measurement: Ki = 295 nM (pChEMBL 6.53000020980835)
Descriptors: MW 1044.19
Compound (SMILES): CC(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@H]1CCC(=O)NCCC[C@@H](C(N)=O)NC(=O)[C@H](Cc2c[nH]c3ccccc23)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@@H](Cc2ccccc2)NC(=O)[C@H](CC(N)=O)NC1=O
InChIKey: HYNXKHHGAXLIEQ-VWUVXGKKSA-N
Target: Melanocortin receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P32245
Assay: cAMP Assay: Accumulation of intracellular cAMP was examined as a measure of the ability of the peptides of the present invention to elicit a functional response in HEK-293 cells that express MC4-R. Confluent HEK-293 cells that express recombinant hMC4-R were detached from culture plates by incubation in enzyme-free cell dissociation buffer. Dispersed cells were suspended in Earle's Balanced Salt Solution containing 10 mM HEPES (pH 7.5), 1 mM MgCl2, 1 mM glutamine, 0.5% albumin and 0.3 mM 3-isobutyl-1-methyl-xanthine (IBMX), a phosphodiesterase inhibitor. The cells were plated in 96-well plates at a density of 0.5x105 cells per well and pre-incubated for 10 minutes. Cells were exposed for 15 minutes at 37° C. to peptides of the present invention dissolved in DMSO (final DMSO concentration of 1%) at a concentration range of 0.05-5000 nM in a total assay volume of 200 uL. NDP-α-MSH was used as the reference agonist. cAMP levels were determined by an HTRF cAMP cell-based assay system from Cisbio Bioassays utilizing cryptate-labeled anti-cAMP and d2-labeled cAMP, with plates read on a PerkinElmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. The maximum efficacies of the test peptides of the present invention were compared to that achieved by the reference melanocortin agonist NDP-αMSH.
Measurement: EC50 = 0.413 nM (pChEMBL 9.380000114440918)
Descriptors: MW 1044.19
Compound (SMILES): CC(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@H]1CCCCNC(=O)C[C@@H](C(N)=O)NC(=O)[C@H](Cc2ccc3ccccc3c2)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@@H](Cc2ccccc2)NC(=O)[C@H](CC(N)=O)NC1=O
InChIKey: KOMGNGDDTPNSKC-PDBPWWFNSA-N
Target: Melanocortin receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P32245
Assay: Competitive Inhibition Assay: A competitive inhibition binding assay is performed using membrane homogenates prepared from HEK-293 cells that express recombinant hMC4-R, hMC3-R, or hMC5-R, and from B-16 mouse melanoma cells (containing endogenous MC1-R). In some instances, HEK-293 cells that express recombinant hMC1-R were employed. In the examples that follow, all MC3-R, MC4-R and MC5-R values are for human recombinant receptors. MC1-R values are for B-16 mouse melanoma cells, unless the heading is hMC1-R, in which case the value is for human recombinant MC1-R. Assays were performed in 96 well GF/B Millipore multiscreen filtration plates (MAFB NOB10) pre-coated with 0.5% bovine serum albumin (Fraction V). Membrane homogenates were incubated with 0.2 nM (for hMC4-R) 0.4 nM (for MC3-R and MC5-R) or 0.1 nM (for mouse B16 MC1-R or hMC1-R) [I125]-NDP-alpha -MSH (Perkin Elmer) and increasing concentrations of test peptides of the present invention in buffer containing 25 mM HEPES buffer (pH 7.5) with 100 mM NaCl.
Measurement: Ki = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 1055.21
Compound (SMILES): C[C@H]1CC[C@H](Cn2c(N3CCO[C@@H]4CCC[C@H]43)nc3cc(-c4noc(=O)[nH]4)nc(-c4cncc(Cl)c4)c32)CC1
InChIKey: FDBWTMMKIZGUEZ-IEGYKGNPSA-N
Target: Cytochrome P450 2D6 (SINGLE PROTEIN), Homo sapiens
UniProt: P10635
Assay: Inhibition of CYP2D6 (unknown origin)
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 550.06, ALogP 5.07, HBA 9, HBD 1, PSA 114.96, QED 0.37
Compound (SMILES): C[C@H]1CC[C@H](Cn2c(N3CCOC4CCCCC43)nc3cc(-c4noc(=O)[nH]4)nc(-c4cncc(Cl)c4)c32)CC1
InChIKey: UARAEDGQJRIWEY-YTFADCBGSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: FRET Assay: Methods: An HDM2 FRET assay was developed to assess the compounds' inhibitory activity towards binding of p53 protein. A truncated version of HDM2 with residues 17 to 125 (containing p53 binding surface, Science (1994) 265, 346-355), with N-terminal His and Thioredoxin tag was generated in pET32a expression vector and expressed in E. coli strain BL21(DE3)Rosetta. Protein was purified using Ni-affinity chromatography, followed by size exclusion chromatography using Superdex 75 26/60 column. To assess inhibition of p53 binding to HDM2, a FITC labeled 8-mer peptide (SEQ ID NO:1: Ac-Phe-Arg-Dpr-Ac6c-(6-Br)Trp-Glu-Glu-Leu-NH2; Anal Biochem. 2004 Aug. 1; 331(1):138-46) with strong affinity towards the p53 binding pocket of HDM2 was used. The HDM2 assay buffer contained 1x Phosphate Buffered Saline (Invitrogen, Cat#14190), 0.01% BSA (Jackson ImmunoResearch, Cat#001-000-162), 0.01% Tween-20.
Measurement: IC50 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 564.09, ALogP 5.46, HBA 9, HBD 1, PSA 114.96, QED 0.34
Compound (SMILES): C[C@H]1CC[C@H](Cn2c(N3CCOC4CCCCC43)nc3cc(-c4n[nH]c(=O)o4)nc(-c4cncc(Cl)c4)c32)CC1
InChIKey: NDSBBPYRNSOZKT-YTFADCBGSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: FRET Assay: Methods: An HDM2 FRET assay was developed to assess the compounds' inhibitory activity towards binding of p53 protein. A truncated version of HDM2 with residues 17 to 125 (containing p53 binding surface, Science (1994) 265, 346-355), with N-terminal His and Thioredoxin tag was generated in pET32a expression vector and expressed in E. coli strain BL21(DE3)Rosetta. Protein was purified using Ni-affinity chromatography, followed by size exclusion chromatography using Superdex 75 26/60 column. To assess inhibition of p53 binding to HDM2, a FITC labeled 8-mer peptide (SEQ ID NO:1: Ac-Phe-Arg-Dpr-Ac6c-(6-Br)Trp-Glu-Glu-Leu-NH2; Anal Biochem. 2004 Aug. 1; 331(1):138-46) with strong affinity towards the p53 binding pocket of HDM2 was used. The HDM2 assay buffer contained 1x Phosphate Buffered Saline (Invitrogen, Cat#14190), 0.01% BSA (Jackson ImmunoResearch, Cat#001-000-162), 0.01% Tween-20.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 564.09, ALogP 5.46, HBA 9, HBD 1, PSA 114.96, QED 0.34
Compound (SMILES): C[C@H]1CC[C@H](Cn2c(N3CCCC4CCCC43)nc3cc(-c4noc(=O)[nH]4)nc(-c4cncc(Cl)c4)c32)CC1
InChIKey: CJKBAYXWIIJQPF-VMXWJKLDSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: FRET Assay: Methods: An HDM2 FRET assay was developed to assess the compounds' inhibitory activity towards binding of p53 protein. A truncated version of HDM2 with residues 17 to 125 (containing p53 binding surface, Science (1994) 265, 346-355), with N-terminal His and Thioredoxin tag was generated in pET32a expression vector and expressed in E. coli strain BL21(DE3)Rosetta. Protein was purified using Ni-affinity chromatography, followed by size exclusion chromatography using Superdex 75 26/60 column. To assess inhibition of p53 binding to HDM2, a FITC labeled 8-mer peptide (SEQ ID NO:1: Ac-Phe-Arg-Dpr-Ac6c-(6-Br)Trp-Glu-Glu-Leu-NH2; Anal Biochem. 2004 Aug. 1; 331(1):138-46) with strong affinity towards the p53 binding pocket of HDM2 was used. The HDM2 assay buffer contained 1x Phosphate Buffered Saline (Invitrogen, Cat#14190), 0.01% BSA (Jackson ImmunoResearch, Cat#001-000-162), 0.01% Tween-20.
Measurement: IC50 = 63 nM (pChEMBL 7.199999809265137)
Descriptors: MW 548.09, ALogP 6.09, HBA 8, HBD 1, PSA 105.73, QED 0.32
Compound (SMILES): CCOc1cc2ncc(C#N)c(Nc3ccc(OCc4ccccn4)c(Cl)c3)c2cc1NC(=O)/C=C/[C@H]1CCCN1C
InChIKey: SADXACCFNXBCFY-IYNHSRRRSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of wild type human N-terminal GST tagged EGFR (669 to 1210 residues) expressed in Sf21 insect cells incubated for 30 mins in presence of ATP by HTRF analysis
Measurement: IC50 = 0.98 nM (pChEMBL 9.010000228881836)
Descriptors: MW 583.09, ALogP 6.47, HBA 8, HBD 2, PSA 112.4, QED 0.2
Compound (SMILES): CCOC(=O)CNC(=O)NCc1ccc(N)cc1
InChIKey: UCMPTJMHPJQEED-UHFFFAOYSA-N
Target: Peptidyl-prolyl cis-trans isomerase D (SINGLE PROTEIN), Homo sapiens
UniProt: Q08752
Assay: Enzymatic Assay: Cyclophilin PPlase activity was measured at 20 C. by using the standard chymotrypsin coupled assay (Kofron J L, Kuzmic P, Kishore V, Colon-Bonilla E, Rich D H. Determination of kinetic constants for peptidyl prolyl cis-trans isomerases by an improved spectrophotometric assay. Biochemistry. 1991 Jun. 25; 30(25):6127-34). The assay buffer (25 mM Hepes, 100 mM NaCl, pH 7.8) and CypA, B or D (1900 nM stock solution) were pre-cooled to 4 C., to which then was added 5 uL of 50 mg/ml chymotrypsin in 1 mM HCl. The reaction was initiated by adding 20 uL of 3.2 mM peptide substrate (Suc-Ala-Ala-cis-Pro-Phe-pNA) in LiCl/TFE solution with rapid inversion. After a delay from the onset of mixing, the absorbance of p-nitroaniline was followed at 390 nM until the reaction was complete (1 min). The final concentration of LiCl in the assay was 20 mM; TFE was present at a concentration of 4% (v/v). Absorbance readings were collected every 1 s by spectrophotometer.
Measurement: IC50 = 6200 nM (pChEMBL 5.210000038146973)
Descriptors: MW 251.29, ALogP 0.63, HBA 4, HBD 3, PSA 93.45, QED 0.53
Compound (SMILES): COc1ccccc1C1CCCN1C(=O)CNC(=O)NCc1ccc(N)cc1
InChIKey: VJEMAKQKNYWEFQ-UHFFFAOYSA-N
Target: Peptidyl-prolyl cis-trans isomerase D (SINGLE PROTEIN), Homo sapiens
UniProt: Q08752
Assay: Enzymatic Assay: Cyclophilin PPlase activity was measured at 20 C. by using the standard chymotrypsin coupled assay (Kofron J L, Kuzmic P, Kishore V, Colon-Bonilla E, Rich D H. Determination of kinetic constants for peptidyl prolyl cis-trans isomerases by an improved spectrophotometric assay. Biochemistry. 1991 Jun. 25; 30(25):6127-34). The assay buffer (25 mM Hepes, 100 mM NaCl, pH 7.8) and CypA, B or D (1900 nM stock solution) were pre-cooled to 4 C., to which then was added 5 uL of 50 mg/ml chymotrypsin in 1 mM HCl. The reaction was initiated by adding 20 uL of 3.2 mM peptide substrate (Suc-Ala-Ala-cis-Pro-Phe-pNA) in LiCl/TFE solution with rapid inversion. After a delay from the onset of mixing, the absorbance of p-nitroaniline was followed at 390 nM until the reaction was complete (1 min). The final concentration of LiCl in the assay was 20 mM; TFE was present at a concentration of 4% (v/v). Absorbance readings were collected every 1 s by spectrophotometer.
Measurement: IC50 = 11400 nM (pChEMBL 4.940000057220459)
Descriptors: MW 382.46, ALogP 2.44, HBA 4, HBD 3, PSA 96.69, QED 0.67
Compound (SMILES): Nc1ccc(CNC(=O)NCC(=O)N2CCCC2c2ccccc2Br)cc1
InChIKey: QASXSMHVNVTEBM-UHFFFAOYSA-N
Target: Peptidyl-prolyl cis-trans isomerase B (SINGLE PROTEIN), Homo sapiens
UniProt: P23284
Assay: Enzymatic Assay: Cyclophilin PPlase activity was measured at 20 C. by using the standard chymotrypsin coupled assay (Kofron J L, Kuzmic P, Kishore V, Colon-Bonilla E, Rich D H. Determination of kinetic constants for peptidyl prolyl cis-trans isomerases by an improved spectrophotometric assay. Biochemistry. 1991 Jun. 25; 30(25):6127-34). The assay buffer (25 mM Hepes, 100 mM NaCl, pH 7.8) and CypA, B or D (1900 nM stock solution) were pre-cooled to 4 C., to which then was added 5 uL of 50 mg/ml chymotrypsin in 1 mM HCl. The reaction was initiated by adding 20 uL of 3.2 mM peptide substrate (Suc-Ala-Ala-cis-Pro-Phe-pNA) in LiCl/TFE solution with rapid inversion. After a delay from the onset of mixing, the absorbance of p-nitroaniline was followed at 390 nM until the reaction was complete (1 min). The final concentration of LiCl in the assay was 20 mM; TFE was present at a concentration of 4% (v/v). Absorbance readings were collected every 1 s by spectrophotometer.
Measurement: IC50 = 760 nM (pChEMBL 6.119999885559082)
Descriptors: MW 431.33, ALogP 3.19, HBA 3, HBD 3, PSA 87.46, QED 0.64
Compound (SMILES): CSc1ccccc1C1CCCN1C(=O)CNC(=O)NCc1ccc(N)cc1
InChIKey: BWOIOAFNMATNEP-UHFFFAOYSA-N
Target: Peptidyl-prolyl cis-trans isomerase D (SINGLE PROTEIN), Homo sapiens
UniProt: Q08752
Assay: Enzymatic Assay: Cyclophilin PPlase activity was measured at 20 C. by using the standard chymotrypsin coupled assay (Kofron J L, Kuzmic P, Kishore V, Colon-Bonilla E, Rich D H. Determination of kinetic constants for peptidyl prolyl cis-trans isomerases by an improved spectrophotometric assay. Biochemistry. 1991 Jun. 25; 30(25):6127-34). The assay buffer (25 mM Hepes, 100 mM NaCl, pH 7.8) and CypA, B or D (1900 nM stock solution) were pre-cooled to 4 C., to which then was added 5 uL of 50 mg/ml chymotrypsin in 1 mM HCl. The reaction was initiated by adding 20 uL of 3.2 mM peptide substrate (Suc-Ala-Ala-cis-Pro-Phe-pNA) in LiCl/TFE solution with rapid inversion. After a delay from the onset of mixing, the absorbance of p-nitroaniline was followed at 390 nM until the reaction was complete (1 min). The final concentration of LiCl in the assay was 20 mM; TFE was present at a concentration of 4% (v/v). Absorbance readings were collected every 1 s by spectrophotometer.
Measurement: IC50 = 640 nM (pChEMBL 6.190000057220459)
Descriptors: MW 398.53, ALogP 3.15, HBA 4, HBD 3, PSA 87.46, QED 0.52
Compound (SMILES): CCN(OCc1ccccc1)C(=O)CNC(=O)NCc1ccc(N)cc1
InChIKey: PBTXVZDJFSIFNB-UHFFFAOYSA-N
Target: Peptidyl-prolyl cis-trans isomerase B (SINGLE PROTEIN), Homo sapiens
UniProt: P23284
Assay: Enzymatic Assay: Cyclophilin PPlase activity was measured at 20 C. by using the standard chymotrypsin coupled assay (Kofron J L, Kuzmic P, Kishore V, Colon-Bonilla E, Rich D H. Determination of kinetic constants for peptidyl prolyl cis-trans isomerases by an improved spectrophotometric assay. Biochemistry. 1991 Jun. 25; 30(25):6127-34). The assay buffer (25 mM Hepes, 100 mM NaCl, pH 7.8) and CypA, B or D (1900 nM stock solution) were pre-cooled to 4 C., to which then was added 5 uL of 50 mg/ml chymotrypsin in 1 mM HCl. The reaction was initiated by adding 20 uL of 3.2 mM peptide substrate (Suc-Ala-Ala-cis-Pro-Phe-pNA) in LiCl/TFE solution with rapid inversion. After a delay from the onset of mixing, the absorbance of p-nitroaniline was followed at 390 nM until the reaction was complete (1 min). The final concentration of LiCl in the assay was 20 mM; TFE was present at a concentration of 4% (v/v). Absorbance readings were collected every 1 s by spectrophotometer.
Measurement: IC50 = 4800 nM (pChEMBL 5.320000171661377)
Descriptors: MW 356.43, ALogP 2.05, HBA 4, HBD 3, PSA 96.69, QED 0.5
Compound (SMILES): Cc1ncccc1CNC(=O)c1ccc2c(c1)CC[C@@H]1C[C@@](O)(CCC(F)(F)F)CC[C@@]21Cc1ccccc1
InChIKey: KCAXPRFNWUOBNC-RDAHNBDFSA-N
Target: Disintegrin and metalloproteinase domain-containing protein 17 (SINGLE PROTEIN), Homo sapiens
UniProt: P78536
Assay: Fluorescence Polarization Ligand Binding Assay: The glucocorticoid receptor fluorescence polarization ligand binding (GRFP) assay is used to evaluate direct binding of testing compounds to full-length glucocorticoid (GR) protein. Reagents for this assay are purchased from Invitrogen in a test kit. A fluorescent labeled GR ligand is used as a fluorescent tracer and test compounds compete with the fluorescent tracer for GR binding. The change in polarization value in the presence of test compounds is due to binding of test compounds to GR and is used to determine IC50 and relative binding affinity of test compounds for GR.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 536.64, ALogP 6.62, HBA 3, HBD 2, PSA 62.22, QED 0.36
Compound (SMILES): Cc1nn(C2CCCCC2)c2sc(C(=O)Nc3ccc(N4CCN(C)CC4)cc3)cc12
InChIKey: ISQKDGBSKUJRSX-UHFFFAOYSA-N
Target: Phosphodiesterase 4B (SINGLE PROTEIN), Mus musculus
UniProt: Q8VBU5
Assay: Inhibition Assay: The PDE 4 (phosphodiesterase IV) inhibiting effect of the compounds of the present invention was performed by the following method, which was modified assay method described in Biochemical. Pharmacol. 48(6), 1219-1223 (1994).  (1) The active fraction of PDE 4 (phosphodiesterase IV) was obtained. That is, the livers obtained from three Balb/c mice (male, 12 weeks: obtainable from CLEA Japan, Inc.) were suspended with 30 mL of buffer solution B [20 mM of bis-tris, 5 mM of 2-mercaptoethnol, 2 mM of benzamidine, 2 mM of EDTA, 0.1 mM of 4-(2-aminoethyl)benzensulfonyl hydrochloride, 50 mM of sodium acetate; pH 6.5], then homogenized by Polytron homogenizer. The homogenate were centrifuged under 25,000xG for 10 minutes at 4 C. The supernatant was separated and thus obtained supernatant was further centrifuged under 100,000xG for 60 minutes at 4Â° C., and then filtrated with 0.2 um filter to obtain the soluble fraction.
Measurement: IC50 = 1200 nM (pChEMBL 5.920000076293945)
Descriptors: MW 437.61, ALogP 4.92, HBA 6, HBD 1, PSA 53.4, QED 0.63
Compound (SMILES): Cc1nn(C2CCCCC2)c2sc(C(=O)Nc3ccc(N4CCC(O)CC4)cc3)cc12
InChIKey: QMTADOHBGLSRDC-UHFFFAOYSA-N
Target: High affinity 3',5'-cyclic-AMP phosphodiesterase 7A (SINGLE PROTEIN), Homo sapiens
UniProt: Q13946
Assay: Inhibition Assay: The PDE 7 (phosphodiesterase VII) inhibiting effect of the compounds of the present invention was performed by the following method, which was modified assay method described in Biochemical. Pharmacol. 48(6), 1219-1223 (1994).  (1) The active fraction of PDE 7 (phosphodiesterase VII) was obtained. That is, MOLT-4 (obtainable from ATCC as ATCC No. CRL-1582), which was cell line of human acute lymphoblastic lymphoma T cells, was incubated in RPMI1640 culture medium containing 10% fetal bovine serum to obtain 5x108 MOLT-4 cells. The cells were collected by centrifugation and suspended with 10 mL of buffer solution A [25 mM of tris-HCl, 5 mM of 2-mercaptoethnol, 2 mM of benzamidine, 2 mM of EDTA, 0.1 mM of 4-(2-aminoethyl)benzensulfonyl hydrochloride; pH 7.5], then homogenized by Polytron homogenizer. The homogenate were centrifuged under 25,000xG for 10 minutes at 4 C. The supernatant was separated and thus obtained supernatant was further centrifuged under 100,000xG for 60 minutes.
Measurement: IC50 = 26 nM (pChEMBL 7.579999923706055)
Descriptors: MW 438.6, ALogP 5.12, HBA 6, HBD 2, PSA 70.39, QED 0.59
Compound (SMILES): CCn1cnc2c(-c3ccc(F)c(-c4ccc(S(=O)(=O)CC)cc4OC)c3)cnnc21
InChIKey: PTTQXDBPTFOCMT-UHFFFAOYSA-N
Target: GABA-A receptor; alpha-3/beta-3/gamma-2 (PROTEIN COMPLEX), Homo sapiens
UniProt: P28472
Assay: Displacement of [3H]-flumazenil from human GABAA alpha3beta3gamma2 receptor expressed in HEK293 cell membranes measured after 2 hrs by liquid scintillation counting method
Measurement: Ki = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 440.5, ALogP 4.12, HBA 7, HBD 0, PSA 86.97, QED 0.45
Compound (SMILES): C[C@@H](O)[C@@H]1NC(=O)[C@H](CCCCN)NC(=O)[C@@H](Cc2c[nH]c3ccccc23)NC(=O)[C@H](Cc2ccncc2)NC(=O)[C@H](Cc2ccccc2)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](NC[C@H](Cc2ccc(O)cc2)NC(=O)CSC[C@@H]2C[C@@H]3c4cccc5[nH]cc(c45)C[C@H]3N(C)C2)CCCCNC(=O)[C@H](Cc2ccccc2)NC1=O
InChIKey: PTIFFBYOEZNBTJ-BGUUXQBZSA-N
Target: Somatostatin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30872
Assay: Radioligand Binding Assay: Membranes for in vitro receptor binding assays were obtained by the following procedures. CHO-K1 cells expressing one of the somatostatin receptors were homogenized in ice-cold buffer with 10 mM Tris-HCl, 5 mM EDTA, 3 mM EGTA, 1 mM phenylmethylsuphonyl fluoride, pH 7.6, using Polytron PT10-35GT (Kinematica) at 18,000 rpm for 30 seconds and centrifuged at 500xg for 10 minutes. The supernatant containing the plasma membranes was centrifuged at 100,000xg for 30 minutes and the pellet was resuspended in buffer containing 20 mM glycine-glycine, 1 mM MgCl2, 250 mM sucrose, pH 7.2, for storage at -80 C.For the SSTR1, 2 and 5 assays, membranes and various concentrations of test compounds were incubated in 96-well plates for 60 minutes at 25 C. with 0.05 nM [125I-Tyr11]-SRIF-14 (for hSSTR1; PerkinElmer Life Science), 0.05 nM [125I-Tyr]-seglitide (for hSSTR2; PerkinElmer Life Science) or 0.05 nM [125I-Tyr]-[DPhe-cyclo(Cys-Tyr-DTrp-Lys-Val-Cys)-Thr-NH2] (for hSSTR5.
Measurement: Ki = 38.1 nM (pChEMBL 7.420000076293945)
Descriptors: MW 1604.01
Compound (SMILES): C[C@@H](O)[C@@H]1NC(=O)[C@H](CCCCN)NC(=O)[C@@H](Cc2c[nH]c3ccccc23)NC(=O)[C@H](Cc2ccccc2)NC(=O)[C@H](Cc2ccccc2)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](NC[C@H](Cc2ccc(O)cc2)NC(=O)CSC[C@@H]2C[C@@H]3c4cccc5[nH]cc(c45)C[C@H]3N(C)C2)CCCCNC(=O)[C@H](Cc2ccc(F)cc2)NC1=O
InChIKey: NKLVDXHPTVXWQC-ZGMREDCLSA-N
Target: Somatostatin receptor type 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P31391
Assay: Radioligand Binding Assay: Membranes for in vitro receptor binding assays were obtained by the following procedures. CHO-K1 cells expressing one of the somatostatin receptors were homogenized in ice-cold buffer with 10 mM Tris-HCl, 5 mM EDTA, 3 mM EGTA, 1 mM phenylmethylsuphonyl fluoride, pH 7.6, using Polytron PT10-35GT (Kinematica) at 18,000 rpm for 30 seconds and centrifuged at 500xg for 10 minutes. The supernatant containing the plasma membranes was centrifuged at 100,000xg for 30 minutes and the pellet was resuspended in buffer containing 20 mM glycine-glycine, 1 mM MgCl2, 250 mM sucrose, pH 7.2, for storage at -80 C.For the SSTR1, 2 and 5 assays, membranes and various concentrations of test compounds were incubated in 96-well plates for 60 minutes at 25 C. with 0.05 nM [125I-Tyr11]-SRIF-14 (for hSSTR1; PerkinElmer Life Science), 0.05 nM [125I-Tyr]-seglitide (for hSSTR2; PerkinElmer Life Science) or 0.05 nM [125I-Tyr]-[DPhe-cyclo(Cys-Tyr-DTrp-Lys-Val-Cys)-Thr-NH2] (for hSSTR5.
Measurement: Ki = 25.2 nM (pChEMBL 7.599999904632568)
Descriptors: MW 1621.01
Compound (SMILES): C[C@@H](O)[C@@H]1NC(=O)[C@H](CCCCN)NC(=O)[C@@H](Cc2c[nH]c3ccccc23)NC(=O)[C@H](Cc2ccccc2)NC(=O)[C@H](Cc2ccccc2)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](NCCSC[C@@H]2C[C@@H]3c4cccc5[nH]cc(c45)C[C@H]3N(C)C2)CCCCNC(=O)[C@H](Cc2ccc(F)cc2)NC1=O
InChIKey: TTWNKLQDNLHBQI-NEAURRHBSA-N
Target: Somatostatin receptor type 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P35346
Assay: Radioligand Binding Assay: Membranes for in vitro receptor binding assays were obtained by the following procedures. CHO-K1 cells expressing one of the somatostatin receptors were homogenized in ice-cold buffer with 10 mM Tris-HCl, 5 mM EDTA, 3 mM EGTA, 1 mM phenylmethylsuphonyl fluoride, pH 7.6, using Polytron PT10-35GT (Kinematica) at 18,000 rpm for 30 seconds and centrifuged at 500xg for 10 minutes. The supernatant containing the plasma membranes was centrifuged at 100,000xg for 30 minutes and the pellet was resuspended in buffer containing 20 mM glycine-glycine, 1 mM MgCl2, 250 mM sucrose, pH 7.2, for storage at -80 C.For the SSTR1, 2 and 5 assays, membranes and various concentrations of test compounds were incubated in 96-well plates for 60 minutes at 25 C. with 0.05 nM [125I-Tyr11]-SRIF-14 (for hSSTR1; PerkinElmer Life Science), 0.05 nM [125I-Tyr]-seglitide (for hSSTR2; PerkinElmer Life Science) or 0.05 nM [125I-Tyr]-[DPhe-cyclo(Cys-Tyr-DTrp-Lys-Val-Cys)-Thr-NH2] (for hSSTR5.
Measurement: Ki = 0.52 nM (pChEMBL 9.279999732971191)
Descriptors: MW 1457.83