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

Abstract

The melanocortin-4 receptor (MC4R) plays an important role in appetite. Agonist ligands that stimulate the MC4R decrease appetite, while antagonist compounds increase food consumption. Herein, a functional mixture-based positional scan identified novel MC4R antagonist sequences. Mixtures comprising a library of 12,960,000 tetrapeptides were screened in the presence and absence of the NDP-MSH agonist. These results led to the synthesis of 48 individual tetrapeptides, of which 40 were screened for functional activity at the melanocortin receptors. Thirteen compounds were found to possess nanomolar antagonist potency at the MC4R, with the general tetrapeptide sequence Ac-Aromatic-Basic-Aromatic-Basic-NH2. The most notable results include the identification of tetrapeptide 48 [COR1-25, Ac-DPhe­(pI)-Arg-Nal(2′)-Arg-NH2], an equipotent MC4R antagonist to agouti-related protein [AGRP­(86-132)], more potent than miniAGRP­(87-120), and possessing 15-fold selectivity for the MC4R versus the MC3R. These tetrapeptides may serve as leads for novel appetite-inducing therapies to treat states of negative energy balance, such as cachexia and anorexia.

Discovery of a Pan-Melanocortin Receptor Antagonist [Ac-DPhe(pI)-Arg-Nal(2′)-Orn-NH2] at the MC1R, MC3R, MC4R, and MC5R that Mediates an Increased Feeding Response in Mice and a 40-Fold Selective MC1R Antagonist [Ac-DPhe(pI)-DArg-Nal(2′)-Arg-NH2]

“…Previously, an Arg or DNal(2′) was substituted at the second position, resulting in the tetrapeptides Ac-DPhe(pI)-Arg-Nal(2′)-Arg-NH 2 (same as lead 1, LTT1-20 in the present experiment) and Ac-DPhe(pI)-DNal(2′)-Nal(2′)-Arg-NH 2 . 61 The tetrapeptide possessing an Arg in the second position was a more potent mMC3R and mMC4R antagonist (pA 2 values of 7.8 and 9.0, respectively) 61 compared to the DNal(2′)-substituted compound (pA 2 values of <5.5 and 5.8), 61 indicating that a basic charge in the second position was important for mMC3R and mMC4R antagonist potency. The SAR presented herein demonstrates that amino acids possessing L-stereochemistry and a basic guanidyl group in the second position of this tetrapeptide scaffold (Arg, 1, LTT1-20 and hArg, 7, LTT1-40) result in the most potent mMC3R and mMC4R antagonist ligands.…”

“…65,66 The lead tetrapeptide 1 [Ac-DNal(2′)-Arg-Nal(2′)-Arg-NH 2 ] was previously reported to minimally stimulate the MC1R and MC4R (25% activation at 100 μM concentrations) 61 and possessed antagonist activity at the mMC3R and mMC4R (pA 2 = 7.8 and 9.0, respectively). 61 It was resynthesized in this study (1, and was found to possess similar activation at the mMC1R and mMC4R at 100 μM concentrations (35 and 25%, respectively; Table 2 and Figure 2) and antagonist activity at the mMC3R and mMC4R (pA 2 values of 7.5 and 8.9, respectively; Table 3 and Figure 2) compared to the prior report. Due to the minimal agonist response of 1 (LTT1-20) at the mMC1R and mMC5R, this ligand was assayed for antagonist activity at these two receptors for the first time herein (Figure 2).…”

“…Previously reported substitutions at the fourth position for this tetrapeptide scaffold have included a DLys (same as 19, SSM1-18 in the present study), DNal(2′), and Arg (same as 1, LTT1-20 in the present study) residues. 61 The reported pA 2 values at the mMC3R and mMC4R for Ac-DPhe(pI)-Arg-Nal(2′)-DNal(2′)-NH 2 (<5.5 and 6.7), 61 Ac-DPhe(pI)-Arg-Nal(2′)-DLys-NH 2 (6.8 and 8.0), 61 and Ac-DPhe(pI)-Arg-Nal(2′)-Arg-NH 2 (7.8 and 9.0) 61 indicated that having a basic in the fourth position increased mMC3R and mMC4R antagonist potency. This work identifies that residues at the fourth position possessing a basic amine or guanidyl side chain group result in potent mMC3R antagonists in this scaffold, while having a basic amine, guanidyl, or Cit substitution result in MC4R antagonist activity with sub-10 nM potency.…”

“…Since the "AlphaScreen" cAMP assay is a loss-of-signal assay, in which higher concentrations of compound result in lower assay signal, the data were normalized to baseline and maximal NDP-MSH signal for illustrative purposes, as previously described. 65,66 The lead tetrapeptide 1 [Ac-DNal(2′)-Arg-Nal(2′)-Arg-NH 2 ] was previously reported to minimally stimulate the MC1R and MC4R (25% activation at 100 μM concentrations) 61 and possessed antagonist activity at the mMC3R and mMC4R (pA 2 = 7.8 and 9.0, respectively). 61 It was resynthesized in this study (1, and was found to possess similar activation at the mMC1R and mMC4R at 100 μM concentrations (35 and 25%, respectively; Table 2 and Figure 2) and antagonist activity at the mMC3R and mMC4R (pA 2 values of 7.5 and 8.9, respectively; Table 3 and Figure 2) compared to the prior report.…”

Functional Characterization of Melanocortin-3 Receptor in a Hibernating Cavefish Onychostoma macrolepis

“…MC5R is expressed in various peripheral tissues and participates in regulating the function of exocrine glands [ 4 , 5 ]. MC3R and MC4R are mainly expressed in the hypothalamus and are involved in maintaining energy homeostasis [ 6 , 7 ]. The latter two have similar physiological functions; for example, the knockout or loss-of-function mutations of MC3R or MC4R can cause obesity in mice and humans [ 8 , 9 ].…”