Development of Molecular Probes for the Central Melanocortin System

Abstract

The gain or loss of weight as a therapeutic strategy is challenging, as the modulation of weight requires integrated approaches of dietary, environmental, and behavioral changes. The centrally-located melanocortin-3 and -4 receptors (MC3R and MC4R) both regulate food intake and energy homeostasis, and are associated with a variety of feeding-related behavioral phenotypes. Data suggest that the MC3R and MC4R modulate body weight through different feeding behaviors. For instance, the MC3R may promote food intake through dopamine-mediated reward circuitry, while the MC4R may promote food intake under hypocaloric conditions to maintain a caloric state of homeostasis. To understand these possible physiological roles and to test hypotheses regarding possible therapeutic interventions for disease of positive or negative energy balance, selective and potent polypharmacological probes are highly valuable. This thesis describes the generation of several valuable chemical probes for these receptors, and provides additional foundational work for the development of future molecular probes. In Chapter 3 and Chapter 4, derivatives of a truncated octapeptide macrocyclic mimetic of the endogenous melanocortin antagonist/inverse agonist agouti-related protein (AGRP) were created. This study utilized a single substitution strategy in Chapter 3 to identify a position in the octapeptide template that may be modified to increase MC4R antagonist activity. In Chapter 4, the body of knowledge surrounding the structure-activity relationship of this structure was used to employ a multi-substitution strategy. This study resulted in the discovery of the most potent and selective MC4R antagonist discovered to date, and the serendipitous discovery of a substitution pattern that allows this macrocycle to nearly recapitulate the dual antagonist pharmacology of the native hormone. An MC4R-selective antagonist ligand may only be useful in some behavioral models of motivated feeding in wildtype animals, as the dual antagonist AGRP is expressed under hypocaloric conditions. Therefore, it was sought to increase the potency of previously discovered polypharmacological melanocortin probes that possess MC3R agonist activity and MC4R antagonist activity. In Chapter 5, a combinatorial tetrapeptide library was generated based on screening results from a mixture-based positional scanning library previously reported. This resulted in the identification of a tetrapeptide with a more potent polypharmacological profile than previously reported ligands, and resulted in the identification of a 100-fold MC3R selective tetrapeptide. In these studies, the rapid iterative prototyping capabilities of peptide synthesis were employed for the creation of molecular probes. These probes do not address the basal level of MC4R activity or its physiological relevance. To identify the physiological relevance of the signaling cascade mechanisms produced by MC4R constitutive activity, probes that replicate these signaling pathways in vivo are required. The basal level of MC4R activation may be due to autostimulation by the MC4R extracellular N-terminal domain, since a synthetic hMC4R N-terminal domain construct H-hMC4R(1-26)-NH2 possess µM agonist activity at the receptor. In Chapter 6, N-terminal domain constructs possessing disease-associated polymorphisms were synthesized and characterized, and the structure of the N-terminal domain peptide was studied using nuclear magnetic resonance spectroscopy. This resulted in the discovery that two of the polymorphism-containing N-terminal peptides possessed increased potency compared to the wildtype N-terminal peptide, while the other peptides were equipotent with the wild type peptide. This may indicate that the MC4R N-terminal domain possesses a different biased signaling cascade profile than other endogenous agonists. Because these polymorphisms have been identified in obese patients, this profile difference may play an important physiological role. It was also found that the N-terminal domain peptide adopts a random coil in solution, and thus may undergo a disorder-to-order transition upon binding to the receptor. These results suggest that in future probe development, rigidifying the N-terminal domain peptide to a bioactive conformation may increase its potency.