Selective, High Affinity Peptide Antagonists of α-Melanotropin Action at Human Melanocortin Receptor 4:  Their Synthesis and Biological Evaluation in Vitro

Bednarek, Maria A.; MacNeil, Tanya; Kalyani, Rubana N.; Tang, Rui; Ploeg, Lex H.T. Van der; Weinberg, David H.

doi:10.1021/jm010165y

Cited by 93 publications

(94 citation statements)

References 27 publications

(126 reference statements)

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“…Agonist activity was measured by using CHO cells expressing the cloned melanocortin receptors as described (23). Compounds were evaluated in three independent experiments, and data were analyzed with PRISM curve-fitting software (GraphPad, San Diego).…”

Section: Methodsmentioning

confidence: 99%

A role for the melanocortin 4 receptor in sexual function

Ploeg

Martin

Howard

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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284

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By using a combination of genetic, pharmacological, and anatomical approaches, we show that the melanocortin 4 receptor (MC4R), implicated in the control of food intake and energy expenditure, also modulates erectile function and sexual behavior. Evidence supporting this notion is based on several findings: (i) a highly selective nonpeptide MC4R agonist augments erectile activity initiated by electrical stimulation of the cavernous nerve in wild-type but not Mc4r-null mice; (ii) copulatory behavior is enhanced by administration of a selective MC4R agonist and is diminished in mice lacking Mc4r; (iii) reverse transcription (RT)-PCR and non-PCR based methods demonstrate MC4R expression in rat and human penis, and rat spinal cord, hypothalamus, brainstem, pelvic ganglion (major autonomic relay center to the penis), but not in rat primary corpus smooth muscle cavernosum cells; and (iv) in situ hybridization of glans tissue from the human and rat penis reveal MC4R expression in nerve fibers and mechanoreceptors in the glans of the penis. Collectively, these data implicate the MC4R in the modulation of penile erectile function and provide evidence that MC4R-mediated proerectile responses may be activated through neuronal circuitry in spinal cord erectile centers and somatosensory afferent nerve terminals of the penis. Our results provide a basis for the existence of MC4R-controlled neuronal pathways that control sexual function.O ur understanding of the physiology and anatomy of erectile function has advanced considerably in recent years (1-4). Penile erection is a highly coordinated reflex that is subject to modulation at many levels of the neuraxis. Relaxation of smooth muscle fibers of erectile tissue and concomitant dilatation of the arterial supply in the penis produce penile erection. Activation of neurons in the sacral spinal cord triggers activity in the pelvic nerve and, subsequently, the cavernous nerve, which can lead to the release of mediators of vasorelaxation, including nitric oxide. These mediators modulate cyclic nucleotide levels resulting in Ca 2ϩ sequestration and relaxation of smooth muscle fibers of the corpora cavernosa and corpus spongiosum in the shaft of the penis to produce arterial dilatation, engorgement of the penis with blood, and tumescence. Erections can be triggered either by peripheral (tactile) or by central (visual, olfactory, auditory, or imaginative cues) activation of somatic pathways and, as such, are influenced by tonic and phasic activity in the lumbosacral spinal cord and the brain.Five melanocortin heterotrimeric GTP-binding protein (G protein)-coupled receptors have been identified as expressed in different tissues (5, 6). The functional role of each of these five melanocortin receptors is being defined. Rodent and human genetic and pharmacological evidence indicates that activation of melanocortin 4 receptor (MC4R) results in a lean phenotype, whereas inactivation of the MC4R results in obesity (7-10). Recent studies have demonstrated that MTII, a cyclic analogue of ␣-mel...

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Section: Methodsmentioning

confidence: 99%

A role for the melanocortin 4 receptor in sexual function

Ploeg

Martin

Howard

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

284

207

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“…Potential pharmaceutical benefit can be reaped from development of ligands that can discriminate between MC3 and MC4 receptors, and therefore, it is important to identify the molecular determinants of the receptor that enable MC4/MC3 receptor discrimination. A number of MC4-selective ligands have been developed, including the agonist peptide HfRWK (Bednarek et al, 1999), the antagonist peptide M10 (Bednarek et al, 2001), and the nonpeptide agonist tetrahydroisoquinoline (THIQ) (Van der Ploeg et al, 2002) (Table 1). Conversely, ␥-MSH and a modification of the peptide ␥trp9 show some selectivity for the MC3 receptor over the MC4 receptor.…”

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confidence: 99%

“…In all cases, a large array of ligands have been used to define the effect of the mutation, allowing inferences in regard to effects on specific ligands and effects on specific residues, regions, or physical properties of the ligands. (Bednarek et al, 2001), HfRWK (Bednarek et al, 1999), and ␥trp9 were synthesized by solid-phase methodology on a Beckman Coulter 990 peptide synthesizer (Fullerton, CA) using t-N-tert-butoxycarbonyl-protected amino acids, and the assembled peptide was deprotected with hydrogen fluoride. The crude peptide products were purified by preparative high-performance liquid chromatography.…”

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confidence: 99%

Molecular Determinants of Melanocortin 4 Receptor Ligand Binding and MC4/MC3 Receptor Selectivity

Nickolls

Cismowski²,

Wang³

et al. 2003

The Journal of Pharmacology and Experimental Therapeutics

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The molecular basis of ligand recognition by the melanocortin 4 receptor (MC4R) has not been fully defined. In this study, we investigated the molecular determinants of MC4R ligand binding, employing a large array of ligands, using three approaches. First, molecular modeling of the receptor was used to identify Phe284, in transmembrane (TM) 7, as a potential site of ligand interaction. Mutation of Phe284 to alanine reduced binding affinity and potency of peptides containing L-Phe by up to 71-fold but did not appreciably affect binding of linear peptides containing D-Phe, consistent with a hydrophobic interaction between the Phe7 of ␣-melanocyte-stimulating hormone and Phe284. Second, we examined the effect of a naturally occurring mutation in TM3 (I137T) that is linked to obesity. This mutation decreased affinity and potency of cyclic, rigid peptides but not more flexible peptides, consistent with an indirect effect of the mutation on the tertiary structure of the receptor. Third, we examined the residues that support ligand selectivity for the MC4R over the MC3R. Mutation of Ile125 (TM3) of the MC4R to the equivalent residue of the MC3R (phenylalanine) selectively decreased affinity and potency of MC4R-selective ligands. This effect was mirrored by the reciprocal MC3R mutation F157I. The magnitude of this effect indicates that this locus is not of major importance. However, it is considered that an isoleucine/phenylalanine mutation may affect the orientation of Asp122, which has been identified as a major determinant of ligand binding affinity. Thus, this study provides further characterization of the MC4R binding pocket.

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“…The lactam ring of this antagonist encompasses the 7-9 segment of ␣MSH with Phe 7 replaced by D-Nal(2Ј). This small fragment, D-Nal(2Ј) 7 -Arg 8 -Trp 9 , was recognized by us as the "es- sential core" required for high affinity and high selectivity of peptide antagonists at hMC-4R (11). The amide bond between the carboxyl group of succinic acid in position 6 and the ⑀-amino group of Lys in position 10 closes the 20-membered ring of this peptide.…”

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confidence: 99%