Agonist activity at human MC1R expressed in HEK293 cells assessed as induction of intracellular cAMP accumulation measured after 3 minsAgonist activity at human MC1R expressed in HEK293 cells assessed as induction of intracellular cAMP accumulation measured after 3 mins

Agonist activity at human MC1R expressed in HEK293 cells assessed as induction of intracellular cAMP accumulation measured after 3 minsAgonist activity at human MC1R expressed in HEK293 cells assessed as induction of intracellular cAMP accumulation measured after 3 mins

Agonist activity at mouse MC1R by alphascreen cAMP assayAgonist activity at mouse MC1R by alphascreen cAMP assay

Agonist activity at mouse MC1R by alphascreen cAMP assayAgonist activity at mouse MC1R by alphascreen cAMP assay

Agonist activity at mouse MC1R by alphascreen cAMP assayAgonist activity at mouse MC1R by alphascreen cAMP assay

Agonist activity at mouse MC1R by alphascreen cAMP assayAgonist activity at mouse MC1R by alphascreen cAMP assay

Agonist activity at mouse MC1R by alphascreen cAMP assayAgonist activity at mouse MC1R by alphascreen cAMP assay

Agonist activity at mouse MC1R by alphascreen cAMP assayAgonist activity at mouse MC1R by alphascreen cAMP assay

Agonist activity at mouse MC1R by alphascreen cAMP assayAgonist activity at mouse MC1R by alphascreen cAMP assay

Agonist activity at mouse MC1R by alphascreen cAMP assayAgonist activity at mouse MC1R by alphascreen cAMP assay

Agonist activity at mouse MC1R expressed in HEK293 cells incubated for 2 hrs by AlphaScreen cAMP assayAgonist activity at mouse MC1R expressed in HEK293 cells incubated for 2 hrs by AlphaScreen cAMP assay

Agonist activity at mouse MC1R expressed in HEK293 cells incubated for 2 hrs by AlphaScreen cAMP assayAgonist activity at mouse MC1R expressed in HEK293 cells incubated for 2 hrs by AlphaScreen cAMP assay

Agonist activity at mouse MC1R expressed in HEK293 cells incubated for 2 hrs by AlphaScreen cAMP assayAgonist activity at mouse MC1R expressed in HEK293 cells incubated for 2 hrs by AlphaScreen cAMP assay

Agonist activity at mouse MC1R expressed in HEK293 cells incubated for 2 hrs by AlphaScreen cAMP assayAgonist activity at mouse MC1R expressed in HEK293 cells incubated for 2 hrs by AlphaScreen cAMP assay

Agonist activity at mouse MC1R expressed in HEK293 cells incubated for 2 hrs by AlphaScreen cAMP assayAgonist activity at mouse MC1R expressed in HEK293 cells incubated for 2 hrs by AlphaScreen cAMP assay

Agonist activity at mouse MC1R expressed in HEK293 cells incubated for 2 hrs by AlphaScreen cAMP assayAgonist activity at mouse MC1R expressed in HEK293 cells incubated for 2 hrs by AlphaScreen cAMP assay

Agonist activity at mouse MC1R expressed in HEK293 cells incubated for 2 hrs by AlphaScreen cAMP assayAgonist activity at mouse MC1R expressed in HEK293 cells incubated for 2 hrs by AlphaScreen cAMP assay

Agonist activity at mouse MC1R expressed in HEK293 cells incubated for 2 hrs by AlphaScreen cAMP assayAgonist activity at mouse MC1R expressed in HEK293 cells incubated for 2 hrs by AlphaScreen cAMP assay

Agonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP Alpha-screen assayAgonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP Alpha-screen assay

Agonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP Alpha-screen assayAgonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP Alpha-screen assay

Agonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP Alpha-screen assayAgonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP Alpha-screen assay

Agonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP Alpha-screen assayAgonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP Alpha-screen assay

Agonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP Alpha-screen assayAgonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP Alpha-screen assay

Agonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP Alpha-screen assayAgonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP Alpha-screen assay

Agonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP Alpha-screen assayAgonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP Alpha-screen assay

Agonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP Alpha-screen assayAgonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP Alpha-screen assay

Agonistic activity against human MC1RAgonistic activity against human MC1R

Agonistic activity against human MC1RAgonistic activity against human MC1R

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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.

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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as induction of cAMP level at 100 uM incubated for 2 hr by alphaScreen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as induction of cAMP level at 100 uM incubated for 2 hr by alphaScreen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as induction of cAMP level at 100 uM incubated for 2 hr by alphaScreen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as induction of cAMP level at 100 uM incubated for 2 hr by alphaScreen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as induction of cAMP level at 100 uM incubated for 2 hr by alphaScreen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as induction of cAMP level at 100 uM incubated for 2 hr by alphaScreen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as induction of cAMP level at 100 uM incubated for 2 hr by alphaScreen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as induction of cAMP level at 100 uM incubated for 2 hr by alphaScreen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as induction of cAMP level at 100 uM incubated for 2 hr by alphaScreen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as induction of cAMP level at 100 uM incubated for 2 hr by alphaScreen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as induction of cAMP level at 100 uM incubated for 2 hr by alphaScreen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as induction of cAMP level at 100 uM incubated for 2 hr by alphaScreen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as induction of cAMP level at 100 uM incubated for 2 hr by alphaScreen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as induction of cAMP level at 100 uM incubated for 2 hr by alphaScreen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as induction of cAMP level at 100 uM incubated for 2 hr by alphaScreen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as induction of cAMP level at 100 uM incubated for 2 hr by alphaScreen assay

Agonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP AlphaScreen assayAgonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP AlphaScreen assay

Agonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP AlphaScreen assayAgonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP AlphaScreen assay

Agonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP AlphaScreen assayAgonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP AlphaScreen assay

Agonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP AlphaScreen assayAgonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP AlphaScreen assay

Agonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP AlphaScreen assayAgonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP AlphaScreen assay

Agonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP AlphaScreen assayAgonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP AlphaScreen assay

Agonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP AlphaScreen assayAgonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP AlphaScreen assay

Agonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP AlphaScreen assayAgonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP AlphaScreen 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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.

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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.

Agonistic activity against mouse MC1RAgonistic activity against mouse MC1R

Agonistic activity against mouse MC1RAgonistic activity against mouse MC1R

Agonistic activity against mouse MC1RAgonistic activity against mouse MC1R

Agonistic activity against mouse MC1RAgonistic activity against mouse MC1R

Agonistic activity against mouse MC1RAgonistic activity against mouse MC1R

Agonistic activity against mouse MC1RAgonistic activity against mouse MC1R

Agonistic activity against mouse MC1RAgonistic activity against mouse MC1R

Agonistic activity against mouse MC1RAgonistic activity against mouse MC1R

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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.

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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as increase in forskolin-induced cAMP accumulation preincubated for 2 hrs followed by biotinylated cAMP addition and subsequent incubation for 2 hrs by AlphaScreen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as increase in forskolin-induced cAMP accumulation preincubated for 2 hrs followed by biotinylated cAMP addition and subsequent incubation for 2 hrs by AlphaScreen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as increase in forskolin-induced cAMP accumulation preincubated for 2 hrs followed by biotinylated cAMP addition and subsequent incubation for 2 hrs by AlphaScreen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as increase in forskolin-induced cAMP accumulation preincubated for 2 hrs followed by biotinylated cAMP addition and subsequent incubation for 2 hrs by AlphaScreen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as increase in forskolin-induced cAMP accumulation preincubated for 2 hrs followed by biotinylated cAMP addition and subsequent incubation for 2 hrs by AlphaScreen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as increase in forskolin-induced cAMP accumulation preincubated for 2 hrs followed by biotinylated cAMP addition and subsequent incubation for 2 hrs by AlphaScreen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as increase in forskolin-induced cAMP accumulation preincubated for 2 hrs followed by biotinylated cAMP addition and subsequent incubation for 2 hrs by AlphaScreen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as increase in forskolin-induced cAMP accumulation preincubated for 2 hrs followed by biotinylated cAMP addition and subsequent incubation for 2 hrs by AlphaScreen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as increase in forskolin-induced cAMP accumulation preincubated for 2 hrs followed by biotinylated cAMP addition and subsequent incubation for 2 hrs by AlphaScreen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as increase in forskolin-induced cAMP accumulation preincubated for 2 hrs followed by biotinylated cAMP addition and subsequent incubation for 2 hrs by AlphaScreen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as increase in forskolin-induced cAMP accumulation preincubated for 2 hrs followed by biotinylated cAMP addition and subsequent incubation for 2 hrs by AlphaScreen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as increase in forskolin-induced cAMP accumulation preincubated for 2 hrs followed by biotinylated cAMP addition and subsequent incubation for 2 hrs by AlphaScreen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as increase in forskolin-induced cAMP accumulation preincubated for 2 hrs followed by biotinylated cAMP addition and subsequent incubation for 2 hrs by AlphaScreen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as increase in forskolin-induced cAMP accumulation preincubated for 2 hrs followed by biotinylated cAMP addition and subsequent incubation for 2 hrs by AlphaScreen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as increase in forskolin-induced cAMP accumulation preincubated for 2 hrs followed by biotinylated cAMP addition and subsequent incubation for 2 hrs by AlphaScreen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as increase in forskolin-induced cAMP accumulation preincubated for 2 hrs followed by biotinylated cAMP addition and subsequent incubation for 2 hrs by AlphaScreen 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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.

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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.

Evaluated for agonist activity against human Melanocortin 1 receptor using hMC1-R assayEvaluated for agonist activity against human Melanocortin 1 receptor using hMC1-R assay

Evaluated for agonist activity at cloned mammalian human MSH1 receptorEvaluated for agonist activity at cloned mammalian human MSH1 receptor

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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.

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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.

Agonist activity at mouse melanocortin 1 receptor expressed in HEK293 cells after 48 hrs by cAMP based beta-galactosidase reporter gene assayAgonist activity at mouse melanocortin 1 receptor expressed in HEK293 cells after 48 hrs by cAMP based beta-galactosidase reporter gene assay

Agonist activity at mouse melanocortin 1 receptor expressed in HEK293 cells after 48 hrs by cAMP based beta-galactosidase reporter gene assayAgonist activity at mouse melanocortin 1 receptor expressed in HEK293 cells after 48 hrs by cAMP based beta-galactosidase reporter gene assay

Agonist activity at mouse melanocortin 1 receptor expressed in HEK293 cells after 48 hrs by cAMP based beta-galactosidase reporter gene assayAgonist activity at mouse melanocortin 1 receptor expressed in HEK293 cells after 48 hrs by cAMP based beta-galactosidase reporter gene assay

Agonist activity at mouse melanocortin 1 receptor expressed in HEK293 cells after 48 hrs by cAMP based beta-galactosidase reporter gene assayAgonist activity at mouse melanocortin 1 receptor expressed in HEK293 cells after 48 hrs by cAMP based beta-galactosidase reporter gene assay

Agonist activity at mouse melanocortin 1 receptor expressed in HEK293 cells after 48 hrs by cAMP based beta-galactosidase reporter gene assayAgonist activity at mouse melanocortin 1 receptor expressed in HEK293 cells after 48 hrs by cAMP based beta-galactosidase reporter gene assay

Agonist activity at mouse melanocortin 1 receptor expressed in HEK293 cells after 48 hrs by cAMP based beta-galactosidase reporter gene assayAgonist activity at mouse melanocortin 1 receptor expressed in HEK293 cells after 48 hrs by cAMP based beta-galactosidase reporter gene assay

Agonist activity at mouse melanocortin 1 receptor expressed in HEK293 cells after 48 hrs by cAMP based beta-galactosidase reporter gene assayAgonist activity at mouse melanocortin 1 receptor expressed in HEK293 cells after 48 hrs by cAMP based beta-galactosidase reporter gene assay

Agonist activity at mouse melanocortin 1 receptor expressed in HEK293 cells after 48 hrs by cAMP based beta-galactosidase reporter gene assayAgonist activity at mouse melanocortin 1 receptor expressed in HEK293 cells after 48 hrs by cAMP based beta-galactosidase reporter gene assay

Functional activity at the mouse melanocortin 1 receptorFunctional activity at the mouse melanocortin 1 receptor

Agonist activity at mouse melanocortin 1 receptor expressed in HEK293 cells by AlphaScreen cAMP assayAgonist activity at mouse melanocortin 1 receptor expressed in HEK293 cells by AlphaScreen cAMP assay

Agonist activity at mouse melanocortin 1 receptor expressed in HEK293 cells by AlphaScreen cAMP assayAgonist activity at mouse melanocortin 1 receptor expressed in HEK293 cells by AlphaScreen cAMP assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as induction of cAMP accumulation after 2 hrs by alpha screen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as induction of cAMP accumulation after 2 hrs by alpha screen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as induction of cAMP accumulation after 2 hrs by alpha screen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as induction of cAMP accumulation after 2 hrs by alpha screen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as induction of cAMP accumulation after 2 hrs by alpha screen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as induction of cAMP accumulation after 2 hrs by alpha screen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as induction of cAMP accumulation after 2 hrs by alpha screen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as induction of cAMP accumulation after 2 hrs by alpha screen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as induction of cAMP accumulation after 2 hrs by alpha screen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as induction of cAMP accumulation after 2 hrs by alpha screen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as induction of cAMP accumulation after 2 hrs by alpha screen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as induction of cAMP accumulation after 2 hrs by alpha screen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as induction of cAMP accumulation after 2 hrs by alpha screen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as induction of cAMP accumulation after 2 hrs by alpha screen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as induction of cAMP accumulation after 2 hrs by alpha screen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as induction of cAMP accumulation after 2 hrs by alpha screen assay

Agonist activity to the mouse Melanocortin-1 receptor (mMC1R)Agonist activity to the mouse Melanocortin-1 receptor (mMC1R)

Agonist activity to the mouse Melanocortin-1 receptor (mMC1R)Agonist activity to the mouse Melanocortin-1 receptor (mMC1R)

Agonist activity to the mouse Melanocortin-1 receptor (mMC1R)Agonist activity to the mouse Melanocortin-1 receptor (mMC1R)

Decrease in frog skin reflectivity (metabotropic activity).Decrease in frog skin reflectivity (metabotropic activity).

Effective concentration against mouse Melanocortin 1 receptorEffective concentration against mouse Melanocortin 1 receptor

Effective concentration against mouse Melanocortin 1 receptorEffective concentration against mouse Melanocortin 1 receptor

Agonist activity at mouse MC1R expressed in HEK293 cells by beta-galactosidase assayAgonist activity at mouse MC1R expressed in HEK293 cells by beta-galactosidase assay

Agonist activity at mouse MC1R expressed in HEK293 cells by beta-galactosidase assayAgonist activity at mouse MC1R expressed in HEK293 cells by beta-galactosidase assay

Agonist activity at mouse MC1R expressed in HEK293 cells by beta-galactosidase assayAgonist activity at mouse MC1R expressed in HEK293 cells by beta-galactosidase assay

Agonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as increase in cAMP accumulation after 2 hrs by AlphaScreen assayAgonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as increase in cAMP accumulation after 2 hrs by AlphaScreen assay

Agonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as increase in cAMP accumulation after 2 hrs by AlphaScreen assayAgonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as increase in cAMP accumulation after 2 hrs by AlphaScreen assay

Agonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as increase in cAMP accumulation after 2 hrs by AlphaScreen assayAgonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as increase in cAMP accumulation after 2 hrs by AlphaScreen assay

Agonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as increase in cAMP accumulation after 2 hrs by AlphaScreen assayAgonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as increase in cAMP accumulation after 2 hrs by AlphaScreen assay

Agonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as increase in cAMP accumulation after 2 hrs by AlphaScreen assayAgonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as increase in cAMP accumulation after 2 hrs by AlphaScreen assay

Agonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as increase in cAMP accumulation after 2 hrs by AlphaScreen assayAgonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as increase in cAMP accumulation after 2 hrs by AlphaScreen assay

Agonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as increase in cAMP accumulation after 2 hrs by AlphaScreen assayAgonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as increase in cAMP accumulation after 2 hrs by AlphaScreen assay

Agonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as increase in cAMP accumulation after 2 hrs by AlphaScreen assayAgonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as increase in cAMP accumulation after 2 hrs by AlphaScreen assay

Functional activity at the mouse melanocortin 1 receptorFunctional activity at the mouse melanocortin 1 receptor

Agonist activity at mouse MC1R expressed in HEK293 cells by beta-galactosidase assayAgonist activity at mouse MC1R expressed in HEK293 cells by beta-galactosidase assay

Agonist activity at mouse MC1R expressed in HEK293 cells by beta-galactosidase assayAgonist activity at mouse MC1R expressed in HEK293 cells by beta-galactosidase assay

Agonist activity at mouse MC1R expressed in HEK293 cells by beta-galactosidase assayAgonist activity at mouse MC1R expressed in HEK293 cells by beta-galactosidase assay

Agonist activity at mouse MC1R expressed in HEK293 cells by beta-galactosidase assayAgonist activity at mouse MC1R expressed in HEK293 cells by beta-galactosidase assay

Agonist activity at mouse MC1R expressed in HEK293 cells by beta-galactosidase assayAgonist activity at mouse MC1R expressed in HEK293 cells by beta-galactosidase assay

Agonist activity at mouse MC1R expressed in HEK293 cells by beta-galactosidase assayAgonist activity at mouse MC1R expressed in HEK293 cells by beta-galactosidase assay

Agonist activity at mouse MC1R expressed in HEK293 cells by beta-galactosidase assayAgonist activity at mouse MC1R expressed in HEK293 cells by beta-galactosidase assay

Agonist activity at mouse MC1R expressed in HEK293 cells by beta-galactosidase assayAgonist activity at mouse MC1R expressed in HEK293 cells by beta-galactosidase assay

Evaluated for agonist activity at cloned mammalian human MSH1 receptorEvaluated for agonist activity at cloned mammalian human MSH1 receptor

Evaluated for agonist activity at cloned mammalian human MSH1 receptorEvaluated for agonist activity at cloned mammalian human MSH1 receptor

Evaluated for agonist activity at cloned mammalian human MSH1 receptorEvaluated for agonist activity at cloned mammalian human MSH1 receptor

Evaluated for agonist activity at cloned mammalian human MSH1 receptorEvaluated for agonist activity at cloned mammalian human MSH1 receptor

Evaluated for agonist activity at cloned mammalian human MSH1 receptorEvaluated for agonist activity at cloned mammalian human MSH1 receptor

Evaluated for agonist activity at cloned mammalian human MSH1 receptorEvaluated for agonist activity at cloned mammalian human MSH1 receptor

Evaluated for agonist activity at cloned mammalian human MSH1 receptorEvaluated for agonist activity at cloned mammalian human MSH1 receptor

Evaluated for agonist activity at cloned mammalian human MSH1 receptorEvaluated for agonist activity at cloned mammalian human MSH1 receptor

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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.

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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.

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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.

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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.

Evaluated for agonist activity against mouse Melanocortin 1 receptor using mMC1R assayEvaluated for agonist activity against mouse Melanocortin 1 receptor using mMC1R assay

Activity at mouse MC1R expressed in HEK293 cells assessed as cAMP accumulation by CRE/beta-galactosidase reporter gene assayActivity at mouse MC1R expressed in HEK293 cells assessed as cAMP accumulation by CRE/beta-galactosidase reporter gene assay

Activity at mouse MC1R expressed in HEK293 cells assessed as cAMP accumulation by CRE/beta-galactosidase reporter gene assayActivity at mouse MC1R expressed in HEK293 cells assessed as cAMP accumulation by CRE/beta-galactosidase reporter gene assay

Activity at mouse MC1R expressed in HEK293 cells assessed as cAMP accumulation by CRE/beta-galactosidase reporter gene assayActivity at mouse MC1R expressed in HEK293 cells assessed as cAMP accumulation by CRE/beta-galactosidase reporter gene assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as cAMP accumulation incubated for 2 hrs by alphascreen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as cAMP accumulation incubated for 2 hrs by alphascreen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as cAMP accumulation incubated for 2 hrs by alphascreen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as cAMP accumulation incubated for 2 hrs by alphascreen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as cAMP accumulation incubated for 2 hrs by alphascreen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as cAMP accumulation incubated for 2 hrs by alphascreen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as cAMP accumulation incubated for 2 hrs by alphascreen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as cAMP accumulation incubated for 2 hrs by alphascreen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as cAMP accumulation incubated for 2 hrs by alphascreen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as cAMP accumulation incubated for 2 hrs by alphascreen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as cAMP accumulation incubated for 2 hrs by alphascreen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as cAMP accumulation incubated for 2 hrs by alphascreen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as cAMP accumulation incubated for 2 hrs by alphascreen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as cAMP accumulation incubated for 2 hrs by alphascreen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as cAMP accumulation incubated for 2 hrs by alphascreen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as cAMP accumulation incubated for 2 hrs by alphascreen assay

Functional activity at the mouse melanocortin 1 receptorFunctional activity at the mouse melanocortin 1 receptor

Functional activity at the mouse melanocortin 1 receptorFunctional activity at the mouse melanocortin 1 receptor

Functional activity at the mouse melanocortin 1 receptorFunctional activity at the mouse melanocortin 1 receptor

Functional activity at the mouse melanocortin 1 receptorFunctional activity at the mouse melanocortin 1 receptor

Functional activity at the mouse melanocortin 1 receptorFunctional activity at the mouse melanocortin 1 receptor

Functional activity at the mouse melanocortin 1 receptorFunctional activity at the mouse melanocortin 1 receptor

Functional activity at the mouse melanocortin 1 receptorFunctional activity at the mouse melanocortin 1 receptor

Functional activity at the mouse melanocortin 1 receptorFunctional activity at the mouse melanocortin 1 receptor

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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.

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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.

In vitro agonist potency for Mouse Melanocortin 1 receptorIn vitro agonist potency for Mouse Melanocortin 1 receptor

In vitro agonist potency for Mouse Melanocortin 1 receptorIn vitro agonist potency for Mouse Melanocortin 1 receptor

In vitro agonist potency for Mouse Melanocortin 1 receptorIn vitro agonist potency for Mouse Melanocortin 1 receptor

In vitro agonist potency for Mouse Melanocortin 1 receptorIn vitro agonist potency for Mouse Melanocortin 1 receptor

In vitro agonist potency for Mouse Melanocortin 1 receptorIn vitro agonist potency for Mouse Melanocortin 1 receptor

In vitro agonist potency for Mouse Melanocortin 1 receptorIn vitro agonist potency for Mouse Melanocortin 1 receptor

In vitro agonist potency for Mouse Melanocortin 1 receptorIn vitro agonist potency for Mouse Melanocortin 1 receptor

In vitro agonist potency for Mouse Melanocortin 1 receptorIn vitro agonist potency for Mouse Melanocortin 1 receptor

Functional activity at the mouse melanocortin 1 receptorFunctional activity at the mouse melanocortin 1 receptor

Evaluated for agonist activity against human Melanocortin 1 receptor using hMC1-R assayEvaluated for agonist activity against human Melanocortin 1 receptor using hMC1-R assay

Evaluated for agonist activity at cloned mammalian human MSH1 receptorEvaluated for agonist activity at cloned mammalian human MSH1 receptor

Functional activity at the mouse melanocortin 1 receptorFunctional activity at the mouse melanocortin 1 receptor

Agonist activity to the mouse Melanocortin-1 receptor (mMC1R)Agonist activity to the mouse Melanocortin-1 receptor (mMC1R)

Agonist activity to the mouse Melanocortin-1 receptor (mMC1R)Agonist activity to the mouse Melanocortin-1 receptor (mMC1R)

Agonist activity to the mouse Melanocortin-1 receptor (mMC1R)Agonist activity to the mouse Melanocortin-1 receptor (mMC1R)

Agonist activity to the mouse Melanocortin-1 receptor (mMC1R)Agonist activity to the mouse Melanocortin-1 receptor (mMC1R)

Agonist activity to the mouse Melanocortin-1 receptor (mMC1R)Agonist activity to the mouse Melanocortin-1 receptor (mMC1R)

Agonist activity to the mouse Melanocortin-1 receptor (mMC1R)Agonist activity to the mouse Melanocortin-1 receptor (mMC1R)

Agonist activity to the mouse Melanocortin-1 receptor (mMC1R)Agonist activity to the mouse Melanocortin-1 receptor (mMC1R)

Agonist activity to the mouse Melanocortin-1 receptor (mMC1R)Agonist activity to the mouse Melanocortin-1 receptor (mMC1R)

Evaluated for agonist activity against mouse Melanocortin 1 receptor using mMC1R assayEvaluated for agonist activity against mouse Melanocortin 1 receptor using mMC1R assay

Agonist activity at mouse MC1 receptor expressed in HEK293 cells after 2 hrs by cAMP alpha screen assayAgonist activity at mouse MC1 receptor expressed in HEK293 cells after 2 hrs by cAMP alpha screen assay

Agonist activity at mouse MC1 receptor expressed in HEK293 cells after 2 hrs by cAMP alpha screen assayAgonist activity at mouse MC1 receptor expressed in HEK293 cells after 2 hrs by cAMP alpha screen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as increase in cAMP production incubated for 2 hrs by AlphaScreen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as increase in cAMP production incubated for 2 hrs by AlphaScreen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as increase in cAMP production incubated for 2 hrs by AlphaScreen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as increase in cAMP production incubated for 2 hrs by AlphaScreen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as increase in cAMP production incubated for 2 hrs by AlphaScreen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as increase in cAMP production incubated for 2 hrs by AlphaScreen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as increase in cAMP production incubated for 2 hrs by AlphaScreen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as increase in cAMP production incubated for 2 hrs by AlphaScreen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as increase in cAMP production incubated for 2 hrs by AlphaScreen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as increase in cAMP production incubated for 2 hrs by AlphaScreen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as increase in cAMP production incubated for 2 hrs by AlphaScreen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as increase in cAMP production incubated for 2 hrs by AlphaScreen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as increase in cAMP production incubated for 2 hrs by AlphaScreen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as increase in cAMP production incubated for 2 hrs by AlphaScreen assay

Agonist activity at mouse MC1R expressed in HEK293 cells assessed as increase in cAMP production incubated for 2 hrs by AlphaScreen assayAgonist activity at mouse MC1R expressed in HEK293 cells assessed as increase in cAMP production incubated for 2 hrs by AlphaScreen assay

Agonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP AlphaScreen assayAgonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP AlphaScreen assay

Agonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP AlphaScreen assayAgonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP AlphaScreen assay

Agonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP AlphaScreen assayAgonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP AlphaScreen assay

Agonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP AlphaScreen assayAgonist activity at mouse melanocortin receptor 1 expressed in HEK293 cells assessed as stimulation of cAMP accumulation incubated for 2 hrs by cAMP AlphaScreen assay

In vitro agonist potency for Mouse Melanocortin 1 receptorIn vitro agonist potency for Mouse Melanocortin 1 receptor

Agonist activity at human MC1R expressed in HEK2936E cells assessed as cAMP production in presence of IBMX by time resolved fluorescence assayAgonist activity at human MC1R expressed in HEK2936E cells assessed as cAMP production in presence of IBMX by time resolved fluorescence assay

Agonist activity at human MC1R expressed in HEK2936E cells assessed as cAMP production in presence of IBMX by time resolved fluorescence assayAgonist activity at human MC1R expressed in HEK2936E cells assessed as cAMP production in presence of IBMX by time resolved fluorescence assay

Agonist activity at human MC1R transfected in HEK293 cellsAgonist activity at human MC1R transfected in HEK293 cells

Agonist activity at human MC1R transfected in HEK293 cellsAgonist activity at human MC1R transfected in HEK293 cells

Functional activity at the mouse melanocortin 1 receptorFunctional activity at the mouse melanocortin 1 receptor

Maximal agonist response of mouse melanocortin 1 receptor (MC1R)Maximal agonist response of mouse melanocortin 1 receptor (MC1R)

Maximal agonist response of mouse melanocortin 1 receptor (MC1R)Maximal agonist response of mouse melanocortin 1 receptor (MC1R)

Maximal agonist response of mouse melanocortin 1 receptor (MC1R)Maximal agonist response of mouse melanocortin 1 receptor (MC1R)

Maximal agonist response of mouse melanocortin 1 receptor (MC1R)Maximal agonist response of mouse melanocortin 1 receptor (MC1R)

Maximal agonist response of mouse melanocortin 1 receptor (MC1R)Maximal agonist response of mouse melanocortin 1 receptor (MC1R)

Maximal agonist response of mouse melanocortin 1 receptor (MC1R)Maximal agonist response of mouse melanocortin 1 receptor (MC1R)

Maximal agonist response of mouse melanocortin 1 receptor (MC1R)Maximal agonist response of mouse melanocortin 1 receptor (MC1R)

Maximal agonist response of mouse melanocortin 1 receptor (MC1R)Maximal agonist response of mouse melanocortin 1 receptor (MC1R)

Agonist activity at human MC1R expressed in HEK2936E cells assessed as cAMP production in presence of IBMX by time resolved fluorescence assayAgonist activity at human MC1R expressed in HEK2936E cells assessed as cAMP production in presence of IBMX by time resolved fluorescence assay

Agonist activity at human MC1R expressed in HEK2936E cells assessed as cAMP production in presence of IBMX by time resolved fluorescence assayAgonist activity at human MC1R expressed in HEK2936E cells assessed as cAMP production in presence of IBMX by time resolved fluorescence assay

Maximal agonist response of mouse melanocortin 1 receptor (MC1R)Maximal agonist response of mouse melanocortin 1 receptor (MC1R)

Agonist activity at mouse MC1 receptor expressed in HEK293 cells after 2 hrs by cAMP alpha screen assayAgonist activity at mouse MC1 receptor expressed in HEK293 cells after 2 hrs by cAMP alpha screen assay

Agonist activity at mouse MC1 receptor expressed in HEK293 cells after 2 hrs by cAMP alpha screen assayAgonist activity at mouse MC1 receptor expressed in HEK293 cells after 2 hrs by cAMP alpha screen assay

Agonist activity at mouse MC1 receptor expressed in HEK293 cells after 6 hrs by cAMP-based beta-galactosidase reporter gene assayAgonist activity at mouse MC1 receptor expressed in HEK293 cells after 6 hrs by cAMP-based beta-galactosidase reporter gene assay

Agonist activity at mouse MC1 receptor expressed in HEK293 cells after 6 hrs by cAMP-based beta-galactosidase reporter gene assayAgonist activity at mouse MC1 receptor expressed in HEK293 cells after 6 hrs by cAMP-based beta-galactosidase reporter gene assay

Agonist activity at mouse MC1 receptor expressed in HEK293 cells after 6 hrs by cAMP-based beta-galactosidase reporter gene assayAgonist activity at mouse MC1 receptor expressed in HEK293 cells after 6 hrs by cAMP-based beta-galactosidase reporter gene assay

Agonist activity at mouse MC1 receptor expressed in HEK293 cells after 6 hrs by cAMP-based beta-galactosidase reporter gene assayAgonist activity at mouse MC1 receptor expressed in HEK293 cells after 6 hrs by cAMP-based beta-galactosidase reporter gene assay

Agonist activity at mouse MC1 receptor expressed in HEK293 cells after 6 hrs by cAMP-based beta-galactosidase reporter gene assayAgonist activity at mouse MC1 receptor expressed in HEK293 cells after 6 hrs by cAMP-based beta-galactosidase reporter gene assay

Agonist activity at mouse MC1 receptor expressed in HEK293 cells after 6 hrs by cAMP-based beta-galactosidase reporter gene assayAgonist activity at mouse MC1 receptor expressed in HEK293 cells after 6 hrs by cAMP-based beta-galactosidase reporter gene assay

Agonist activity at mouse MC1 receptor expressed in HEK293 cells after 6 hrs by cAMP-based beta-galactosidase reporter gene assayAgonist activity at mouse MC1 receptor expressed in HEK293 cells after 6 hrs by cAMP-based beta-galactosidase reporter gene assay

Agonist activity at mouse MC1 receptor expressed in HEK293 cells after 6 hrs by cAMP-based beta-galactosidase reporter gene assayAgonist activity at mouse MC1 receptor expressed in HEK293 cells after 6 hrs by cAMP-based beta-galactosidase reporter gene assay

Agonistic activity against human Melanocortin 1 receptorAgonistic activity against human Melanocortin 1 receptor

Agonistic activity against human Melanocortin 1 receptorAgonistic activity against human Melanocortin 1 receptor

Full agonist activity at mouse MC1R expressed in HEK293 cells co-expressing CRE/beta-galactosidase reporter gene after 6 hrs by beta-galactosidase cAMP assayFull agonist activity at mouse MC1R expressed in HEK293 cells co-expressing CRE/beta-galactosidase reporter gene after 6 hrs by beta-galactosidase cAMP assay

Full agonist activity at mouse MC1R expressed in HEK293 cells co-expressing CRE/beta-galactosidase reporter gene after 6 hrs by beta-galactosidase cAMP assayFull agonist activity at mouse MC1R expressed in HEK293 cells co-expressing CRE/beta-galactosidase reporter gene after 6 hrs by beta-galactosidase cAMP assay

Full agonist activity at mouse MC1R expressed in HEK293 cells co-expressing CRE/beta-galactosidase reporter gene after 6 hrs by beta-galactosidase cAMP assayFull agonist activity at mouse MC1R expressed in HEK293 cells co-expressing CRE/beta-galactosidase reporter gene after 6 hrs by beta-galactosidase cAMP assay

Full agonist activity at mouse MC1R expressed in HEK293 cells co-expressing CRE/beta-galactosidase reporter gene after 6 hrs by beta-galactosidase cAMP assayFull agonist activity at mouse MC1R expressed in HEK293 cells co-expressing CRE/beta-galactosidase reporter gene after 6 hrs by beta-galactosidase cAMP assay

Full agonist activity at mouse MC1R expressed in HEK293 cells co-expressing CRE/beta-galactosidase reporter gene after 6 hrs by beta-galactosidase cAMP assayFull agonist activity at mouse MC1R expressed in HEK293 cells co-expressing CRE/beta-galactosidase reporter gene after 6 hrs by beta-galactosidase cAMP assay

Full agonist activity at mouse MC1R expressed in HEK293 cells co-expressing CRE/beta-galactosidase reporter gene after 6 hrs by beta-galactosidase cAMP assayFull agonist activity at mouse MC1R expressed in HEK293 cells co-expressing CRE/beta-galactosidase reporter gene after 6 hrs by beta-galactosidase cAMP assay

Full agonist activity at mouse MC1R expressed in HEK293 cells co-expressing CRE/beta-galactosidase reporter gene after 6 hrs by beta-galactosidase cAMP assayFull agonist activity at mouse MC1R expressed in HEK293 cells co-expressing CRE/beta-galactosidase reporter gene after 6 hrs by beta-galactosidase cAMP assay

Full agonist activity at mouse MC1R expressed in HEK293 cells co-expressing CRE/beta-galactosidase reporter gene after 6 hrs by beta-galactosidase cAMP assayFull agonist activity at mouse MC1R expressed in HEK293 cells co-expressing CRE/beta-galactosidase reporter gene after 6 hrs by beta-galactosidase cAMP 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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.

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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.

Evaluated for agonist activity against human Melanocortin 1 receptor using hMC1-R assayEvaluated for agonist activity against human Melanocortin 1 receptor using hMC1-R assay

Evaluated for agonist activity at cloned mammalian human MSH1 receptorEvaluated for agonist activity at cloned mammalian human MSH1 receptor

Agonist activity at human melanocortin receptor 1 expressed in human T-Rex-293 cells assessed as stimulation of intracellular cAMP accumulation incubated for 45 mins by LANCE cAMP assayAgonist activity at human melanocortin receptor 1 expressed in human T-Rex-293 cells assessed as stimulation of intracellular cAMP accumulation incubated for 45 mins by LANCE cAMP assay

Agonist activity at mouse MC1 receptor expressed in HEK293 cells after 2 hrs by cAMP alpha screen assayAgonist activity at mouse MC1 receptor expressed in HEK293 cells after 2 hrs by cAMP alpha screen assay

Agonist activity at mouse MC1 receptor expressed in HEK293 cells after 2 hrs by cAMP alpha screen assayAgonist activity at mouse MC1 receptor expressed in HEK293 cells after 2 hrs by cAMP alpha screen assay

Decrease in frog skin reflectivity (metabotropic activity).Decrease in frog skin reflectivity (metabotropic activity).

Agonist activity at human melanocortin receptor 1 expressed in human T-Rex-293 cells assessed as stimulation of intracellular cAMP accumulation incubated for 45 mins by LANCE cAMP assayAgonist activity at human melanocortin receptor 1 expressed in human T-Rex-293 cells assessed as stimulation of intracellular cAMP accumulation incubated for 45 mins by LANCE cAMP 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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.

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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.

Agonist activity at human MC1RAgonist activity at human MC1R

Agonist activity at human MC1RAgonist activity at human MC1R

Agonist activity at human MC1R expressed in HEK293 cells assessed as increase in cAMP production incubated for 2 hrs by AlphaScreen assayAgonist activity at human MC1R expressed in HEK293 cells assessed as increase in cAMP production incubated for 2 hrs by AlphaScreen assay

Agonist activity at human MC1R expressed in HEK293 cells assessed as increase in cAMP production incubated for 2 hrs by AlphaScreen assayAgonist activity at human MC1R expressed in HEK293 cells assessed as increase in cAMP production incubated for 2 hrs by AlphaScreen assay

Agonist activity at human MC1R expressed in HEK293 cells assessed as increase in cAMP production incubated for 2 hrs by AlphaScreen assayAgonist activity at human MC1R expressed in HEK293 cells assessed as increase in cAMP production incubated for 2 hrs by AlphaScreen assay

Agonist activity at human MC1R expressed in HEK293 cells assessed as increase in cAMP production incubated for 2 hrs by AlphaScreen assayAgonist activity at human MC1R expressed in HEK293 cells assessed as increase in cAMP production incubated for 2 hrs by AlphaScreen assay

Agonist activity at human MC1R expressed in HEK293 cells assessed as increase in cAMP production incubated for 2 hrs by AlphaScreen assayAgonist activity at human MC1R expressed in HEK293 cells assessed as increase in cAMP production incubated for 2 hrs by AlphaScreen assay

Agonist activity at human MC1R expressed in HEK293 cells assessed as increase in cAMP production incubated for 2 hrs by AlphaScreen assayAgonist activity at human MC1R expressed in HEK293 cells assessed as increase in cAMP production incubated for 2 hrs by AlphaScreen assay

Agonist activity at human MC1R expressed in HEK293 cells assessed as increase in cAMP production incubated for 2 hrs by AlphaScreen assayAgonist activity at human MC1R expressed in HEK293 cells assessed as increase in cAMP production incubated for 2 hrs by AlphaScreen assay

Agonist activity at human MC1R expressed in HEK293 cells assessed as increase in cAMP production incubated for 2 hrs by AlphaScreen assayAgonist activity at human MC1R expressed in HEK293 cells assessed as increase in cAMP production incubated for 2 hrs by AlphaScreen assay

Agonistic activity against human Melanocortin 1 receptorAgonistic activity against human Melanocortin 1 receptor

In vitro agonist potency was evaluated in HEK293 cells transfected with human melanocortin receptor (hMC1R)In vitro agonist potency was evaluated in HEK293 cells transfected with human melanocortin receptor (hMC1R)

In vitro agonist potency was evaluated in HEK293 cells transfected with human melanocortin receptor (hMC1R)In vitro agonist potency was evaluated in HEK293 cells transfected with human melanocortin receptor (hMC1R)

In vitro agonist potency was evaluated in HEK293 cells transfected with human melanocortin receptor (hMC1R)In vitro agonist potency was evaluated in HEK293 cells transfected with human melanocortin receptor (hMC1R)

In vitro agonist potency was evaluated in HEK293 cells transfected with human melanocortin receptor (hMC1R)In vitro agonist potency was evaluated in HEK293 cells transfected with human melanocortin receptor (hMC1R)

In vitro agonist potency was evaluated in HEK293 cells transfected with human melanocortin receptor (hMC1R)In vitro agonist potency was evaluated in HEK293 cells transfected with human melanocortin receptor (hMC1R)

In vitro agonist potency was evaluated in HEK293 cells transfected with human melanocortin receptor (hMC1R)In vitro agonist potency was evaluated in HEK293 cells transfected with human melanocortin receptor (hMC1R)

In vitro agonist potency was evaluated in HEK293 cells transfected with human melanocortin receptor (hMC1R)In vitro agonist potency was evaluated in HEK293 cells transfected with human melanocortin receptor (hMC1R)

In vitro agonist potency was evaluated in HEK293 cells transfected with human melanocortin receptor (hMC1R)In vitro agonist potency was evaluated in HEK293 cells transfected with human melanocortin receptor (hMC1R)

Activity at mouse MC1R expressed in HEK293 cells assessed as cAMP accumulation by CRE/beta-galactosidase reporter gene assayActivity at mouse MC1R expressed in HEK293 cells assessed as cAMP accumulation by CRE/beta-galactosidase reporter gene assay

Activity at mouse MC1R expressed in HEK293 cells assessed as cAMP accumulation by CRE/beta-galactosidase reporter gene assayActivity at mouse MC1R expressed in HEK293 cells assessed as cAMP accumulation by CRE/beta-galactosidase reporter gene 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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.

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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.

Agonist activity at human MC1R expressed in HEK2936E cells assessed as cAMP production in presence of IBMX by time resolved fluorescence assayAgonist activity at human MC1R expressed in HEK2936E cells assessed as cAMP production in presence of IBMX by time resolved fluorescence assay

Agonist activity at human MC1R expressed in HEK2936E cells assessed as cAMP production in presence of IBMX by time resolved fluorescence assayAgonist activity at human MC1R expressed in HEK2936E cells assessed as cAMP production in presence of IBMX by time resolved fluorescence assay

Agonist activity at human MC1R expressed in HEK2936E cells assessed as cAMP production in presence of IBMX by time resolved fluorescence assayAgonist activity at human MC1R expressed in HEK2936E cells assessed as cAMP production in presence of IBMX by time resolved fluorescence assay

Agonist activity at human MC1R expressed in HEK2936E cells assessed as cAMP production in presence of IBMX by time resolved fluorescence assayAgonist activity at human MC1R expressed in HEK2936E cells assessed as cAMP production in presence of IBMX by time resolved fluorescence assay

Agonist activity at human MC1R expressed in HEK2936E cells assessed as cAMP production in presence of IBMX by time resolved fluorescence assayAgonist activity at human MC1R expressed in HEK2936E cells assessed as cAMP production in presence of IBMX by time resolved fluorescence assay

Agonist activity at human melanocortin receptor 1 expressed in human T-Rex-293 cells assessed as stimulation of intracellular cAMP accumulation incubated for 45 mins by LANCE cAMP assayAgonist activity at human melanocortin receptor 1 expressed in human T-Rex-293 cells assessed as stimulation of intracellular cAMP accumulation incubated for 45 mins by LANCE cAMP assay

Agonist activity at human melanocortin receptor 1 expressed in human T-Rex-293 cells assessed as stimulation of intracellular cAMP accumulation incubated for 45 mins by LANCE cAMP assayAgonist activity at human melanocortin receptor 1 expressed in human T-Rex-293 cells assessed as stimulation of intracellular cAMP accumulation incubated for 45 mins by LANCE cAMP assay

Agonist activity at human melanocortin receptor 1 expressed in human T-Rex-293 cells assessed as stimulation of intracellular cAMP accumulation incubated for 45 mins by LANCE cAMP assayAgonist activity at human melanocortin receptor 1 expressed in human T-Rex-293 cells assessed as stimulation of intracellular cAMP accumulation incubated for 45 mins by LANCE cAMP assay

Agonist activity at human melanocortin receptor 1 expressed in human T-Rex-293 cells assessed as stimulation of intracellular cAMP accumulation incubated for 45 mins by LANCE cAMP assayAgonist activity at human melanocortin receptor 1 expressed in human T-Rex-293 cells assessed as stimulation of intracellular cAMP accumulation incubated for 45 mins by LANCE cAMP assay

Agonist activity at human melanocortin receptor 1 expressed in human T-Rex-293 cells assessed as stimulation of intracellular cAMP accumulation incubated for 45 mins by LANCE cAMP assayAgonist activity at human melanocortin receptor 1 expressed in human T-Rex-293 cells assessed as stimulation of intracellular cAMP accumulation incubated for 45 mins by LANCE cAMP 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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.

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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.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 a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 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 0.4ÿ105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells 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 Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.

Agonist activity at human MC1R expressed in HEK2936E cells assessed as cAMP production in presence of IBMX by time resolved fluorescence assayAgonist activity at human MC1R expressed in HEK2936E cells assessed as cAMP production in presence of IBMX by time resolved fluorescence assay

Agonist activity at human MC1R expressed in HEK2936E cells assessed as cAMP production in presence of IBMX by time resolved fluorescence assayAgonist activity at human MC1R expressed in HEK2936E cells assessed as cAMP production in presence of IBMX by time resolved fluorescence assay

Agonist activity at human melanocortin receptor 1 expressed in human T-Rex-293 cells assessed as stimulation of intracellular cAMP accumulation incubated for 45 mins by LANCE cAMP assayAgonist activity at human melanocortin receptor 1 expressed in human T-Rex-293 cells assessed as stimulation of intracellular cAMP accumulation incubated for 45 mins by LANCE cAMP assay