Discovery of Nanomolar Melanocortin-3 Receptor (MC3R)-Selective Small Molecule Pyrrolidine Bis-Cyclic Guanidine Agonist Compounds Via a High-Throughput “Unbiased” Screening Campaign

Abstract

The central melanocortin-3 and melanocortin-4 receptors (MC3R, MC4R) are key regulators of body weight and energy homeostasis. Herein, the discovery and characterization of first-in-class small molecule melanocortin agonists with selectivity for the melanocortin-3 receptor over the melanocortin-4 receptor are reported. Identified via “unbiased” mixture-based high-throughput screening approaches, pharmacological evaluation of these pyrrolidine bis-cyclic guanidines resulted in nanomolar agonist activity at the melanocortin-3 receptor. The pharmacological profiles at the remaining melanocortin receptor subtypes tested indicated similar agonist potencies at both the melanocortin-1 and melanocortin-5 receptors and antagonist or micromolar agonist activities at the melanocortin-4 receptor. This group of small molecules represents a new area of chemical space for the melanocortin receptors with mixed receptor pharmacology profiles that may serve as novel lead compounds to modulate states of dysregulated energy balance.

Ligands for Melanocortin Receptors: Beyond Melanocyte-Stimulating Hormones and Adrenocorticotropin

“…To avoid undesirable side effects, targeting of the centrally located MC3R, which also plays a role in food intake and fat disposition [ 18 , 19 , 140 ], has emerged as a valuably alternative approach. As recently reported, pyrrolidine bis-cyclic guanidines have nanomolar agonist potency at the MC3R and over 10-fold selectivity for the MC3R over the MC4R through “unbiased” mixture-based high-throughput screening approaches [ 141 ]. Recently, Ericson et al (2017) [ 26 ] and Yeo et al (2021) [ 135 ] published two outstanding reviews that summarized recent small molecule ligands of MCRs at humans and rodents.…”

The Parallel Structure–Activity Relationship Screening of Three Compounds Identifies the Common Agonist Pharmacophore of Pyrrolidine Bis-Cyclic Guanidine Melanocortin-3 Receptor (MC3R) Small-Molecule Ligands

“…To identify potential MC3R-selective small molecules, 69 distinct mixture-based scaffold libraries were previously assayed at the MC3R and MC4R [ 29 ]. Using a combined metric of MC3R activity as well as MC3R over MC4R selectivity, both pyrrolidine bis-cyclic guanidine and pentaamine scaffolds were advanced to a mixture-based positional scan [ 29 ], an approach previously utilized to identify MC4R polymorphic rescue compounds [ 30 ], MC3R agonist tetrapeptides [ 24 , 31 ], and MC4R antagonist ligands [ 32 ].…”

“…To identify potential MC3R-selective small molecules, 69 distinct mixture-based scaffold libraries were previously assayed at the MC3R and MC4R [ 29 ]. Using a combined metric of MC3R activity as well as MC3R over MC4R selectivity, both pyrrolidine bis-cyclic guanidine and pentaamine scaffolds were advanced to a mixture-based positional scan [ 29 ], an approach previously utilized to identify MC4R polymorphic rescue compounds [ 30 ], MC3R agonist tetrapeptides [ 24 , 31 ], and MC4R antagonist ligands [ 32 ]. While many of the pentaamine mixtures and individual compounds were observed to be toxic, deconvolution of the pyrrolidine bis-cyclic guanidine library led to the synthesis of 37 compounds, of which 9 possessed full agonist efficacy at the MC3R with sub-micromolar potencies [ 29 ].…”

“…Using a combined metric of MC3R activity as well as MC3R over MC4R selectivity, both pyrrolidine bis-cyclic guanidine and pentaamine scaffolds were advanced to a mixture-based positional scan [ 29 ], an approach previously utilized to identify MC4R polymorphic rescue compounds [ 30 ], MC3R agonist tetrapeptides [ 24 , 31 ], and MC4R antagonist ligands [ 32 ]. While many of the pentaamine mixtures and individual compounds were observed to be toxic, deconvolution of the pyrrolidine bis-cyclic guanidine library led to the synthesis of 37 compounds, of which 9 possessed full agonist efficacy at the MC3R with sub-micromolar potencies [ 29 ]. While similar agonist efficacies and potencies were observed for these 9 compounds at the MC1R and MC5R, none were full agonists at the MC4R and all 9 possessed antagonist activity at the MC4R (pA 2 values between 5.5 and 7.0) [ 29 ].…”

Functional Mixture-Based Positional Scan Identifies a Library of Antagonist Tetrapeptide Sequences (LAtTeS) with Nanomolar Potency for the Melanocortin-4 Receptor and Equipotent with the Endogenous AGRP(86-132) Antagonist

“…An unbiased method exploring a large area of chemical space may identify new scaffolds, leading to ligands that are not readily apparent from previously known compounds or activity trends. Mixture-based positional scanning libraries are one technique that can identify novel active compounds, as reviewed. − Rescue compounds for MC4R polymorphisms that are properly expressed at the cell surface but do not respond to the endogenous melanocortin ligands, agonist peptides selective for the MC3R over MC4R, , and small molecule MC3R ligands have all been identified using this technology. These melanocortin examples all involved identifying new agonist compounds.…”