Insights into receptor/ligand specificity: A structural and pharmacological study of inverse agonists and their respective receptors

The range of skin colors seen across the world are a result of the skin attempting to maximize vitamin D production while minimizing UV radiation related cellular damage. Due to large-scale human migration skin colors no longer necessarily correspond to UV radiation exposure, which has lead to increases in skin cancer, cataracts and vitamin D deficiency related skeletal-muscular disorders. This imbalance has created a need for drugs that can modulate pigmentation. Pigmentation can be modulated by Melanocortin 1 Receptor (MC1R) ligands, however, many of the native and synthetic MC1R ligands bind to other melanocortin receptors (MCRs) and can alter food consumption or metabolism. We aimed to determine the major contact points within MC1R that are necessary for high affinity binding and an appropriate cellular response. This problem was studied using two native inverse agonists to the MCRs, Agouti Related Protein (AgRP) and Agouti Signalling Protein (ASIP). AgRP is known to bind MC3R and MC4R while ASIP can bind to MC1R, MC3R and MC4R. By swapping the three critical regions of ASIP and AgRP in numerous combinations we were able to determine the role of various regions that helped identify hotspots that are necessary for high affinity binding. In addition, we identified that the last six residues of ASIP, which make up the C-terminal loop are essential for inverse agonist activity.;MCRs are difficult to crystallize and are too large for conventional NMR studies. One of the few ways to obtain information regarding the binding pockets of these proteins is by studying the structure of their ligands. Structural and pharmacological data of ligands provides clues regarding the nature of the receptors binding pocket. Previous structural studies on ASIP determined there were two interconverting structures due to proline isomerization and based on pharmacological studies our lab surmised that only one structure---the cis/cis form was active at the MCRs. This assumption was based on Pro→Ala mutants. We went back and conducted NMR studies on the ASIP double mutant P103A, P105A and on the truncated ASIP also with the P103A, P105A mutations. All truncated proteins in previous studies contained the P103A, P105A mutations, due to production issues; therefore we wished to confirm that there is minimum structural deviation from ASIP P103A, P105A and the truncated ASIP. Our NMR studies indicate that the cis/cis form is likely not the active form, instead the cis/trans forms is likely the active form. In addition, we confirmed that removing the C-terminal loop of ASIP does not greatly alter the global structure.;Through our structural and pharmacological studies we identified three critical contact points for high affinity binding, confirmed that the C-terminal loop is not structurally necessary but, is essential for inverse agonist activity at MC1R and our NMR studies suggest that the pharmacologically active form of ASIP is actually the cis/trans form. This work provides critical information to appropriately model MC1R. By improving the models of MC1R, researchers can improve their designs of MC1R specific drugs and help to alleviate pigmentation related disorders.