INTRODUCTION

1. INTRODUCTION • a-MSH and ACTH are two classical hormones derived from proopiomelanocortin (POMC) precursor protein. They have been studied for many years for their biological activities at the pigmentary melanocortin 1 receptor (MC1R) and at the adrenal receptor (MC2R), respectively. • Recently, three additional melanocortin receptors have been cloned and stably transfected in cell lines: the melanocortin 3 receptor (MC3R -- found primarily in the brain); the melanocortin 4 receptor (MC4R -- found primarily in the brain); and the melanocortin 5 receptor (MC5R -- found throughout the body). • The endogenous ligands for these receptors are unknown but likely candidates include POMC peptides such as a-MSH, g-MSH, b-MSH and modified versions of these peptides.

2. POSSIBLE BIOLOGICAL ACTIVITIES ASSOCIATED WITH NEW MCRs • FEEDING BEHAVIOR -- Obesity, Anorexia • ERECTILE FUNCTION • CARDIOVASCULAR FUNCTION -- Blood Pressure and Heart Rate • KIDNEY FUNCTION -- Natriuresis • THERMOREGULATION • LEARNING • OTHERS

3. GOALS OF RESEARCH To develop peptides and peptidomimetic derivatives and analogues of POMC peptides that have high affinity and selectivity for melanocortin 3, 4 and 5 receptors and that are stable to enzymatic breakdown invitro and in vivo. Both AGONISTS and ANTAGONISTS are needed.

4. METHODS • Computer assisted design based on conformational and topographical considerations. • Solid phase synthesis including cyclic (backbone - backbone, side chain - side chain, and backbone - side chain) and acyclic analogues. • Biological assays including binding affinity determination and second messenger measurements using cloned receptors, and invitro and in vivo bioassays. • Conformational analysis, X-ray crystallography, NMR, and other biophysical methods for selected analogues.

5. IMPORTANT STRUCTURES

6. FROG SKIN BIOASSAY FOR CYCLIC “SOMATOSTATIN-RELATED” ANALOGUES

7. AFFINITIES OF CYCLIC “SOMATOSTATIN-RELATED” ANALOGUES

8. ANTAGONIST ACTIVITIES OF CYCLIC LACTAM ANALOGUES

9. BINDING AFFINITIES OF CYCLIC MELANOTROPIN PEPTIDES

10. CONCLUSIONS I • Effective ligands for the MC1R based on Somatostatin have been successfully designed, synthesized and bio-assayed. • All the synthesized peptides are being tested at the MC3-5 receptors. • It was demonstrated that one class of compounds can be converted into another class via rational design based on our understanding of conformationally and topographically constrained ligands and their receptor interactions. • New applications of these compounds and of this novel concept for designing effective bioactive compounds are being applied to other areas.

11. CONCLUSIONS II • CYCLIC LACTAM ANALOGUES of MT-II substituted in positions 6, 7 and 9 with various modified, constrained aromatic residues can lead to potent antagonists. • SELECTIVITY for MC4RvsMC3R and MC5R most readily obtained. • POTENCY and SELECTIVITY of cyclic Somatostatin-derived melanotropin analogues at MCRs are highly dependent on ring conformation. • MC3, MC4 and MC5 receptors have different structure-activity relationships with melanotropins.

12. SUMMARY OF CURRENT STATUS OF SELECTIVE AGONIST AND ANTAGONIST LIGANDS FOR MC3-5Rs • MC3R • Have Both Agonists and Antagonists • Selectivity • Good vsMC5R (>300) • Modest vsMC4R (10-50) • MC4R • Have Both Agonists and Antagonists • Selectivity • Good vsMC3R (>100) • Excellent vsMC5R (>500) • MC5R • Have Agonists and Recently Discovered Antagonists • Selectivity • A Few Equil. vsMC3R • A Few Equil. vsMC4R