Development of potent and proteolytically stable human neuromedin U receptor agonists

Abstract

Neuromedin U (NMU) is a highly conserved endogenous peptide that is involved in a wide range of physiological processes such as regulation of feeding behavior, the stress response and nociception. The major limitation to use NMU as a therapeutic is its short half-life. Here, we describe the development of a set of novel NMU-analogs based on NMU-8, by introducing unnatural amino acids into the native sequence. This approach shows that it is possible to generate molecules with increased potency and improved plasma stability without major changes of the peptidic nature or the introduction of large conjugates. When compared to the native NMU-8 peptide, compounds 16, 18 and 20 have potent agonist activity and affinity for both NMU receptors. Selectivity towards NMUR1 was observed when the Phe residue in position 4 was modified, whereas higher potencies at NMUR2 were found when substitutions of the Pro residue in position 6 were executed. To study the effect of the modifications on the proteolytic stability of the molecules, an in vitro stability assay in human plasma at 37 °C was performed. All analyzed analogs possessed an increased resistance against enzymatic degradation in human plasma resulting in half-lifes from 4 min for NMU-8, up to more than 23 h for compound 42.

Strengthening peptide-based drug activity with novel glyconanoparticle

“…The stability of peptides can be measured in serum, plasma, or whole blood from humans or animals to assess the suitability of peptides as therapeutic drugs [ 7 , 9 – 15 ]. Many potential therapeutic peptides have shown to be partially or fully degraded in biological matrices, including antibacterial peptides [ 7 , 10 ], hormone peptides [ 9 , 15 ], neuropeptides [ 16 , 17 ], and anti-cancer peptides [ 18 ], which has ultimately limited their use as therapeutics. Enzyme degradation in the blood has led to the development of different strategies to improve peptide stability, including chemical modification, peptide self-assembly, the use of protease and peptidase inhibitors, or formulating the peptide in a drug delivery system.…”

“…Enzyme degradation in the blood has led to the development of different strategies to improve peptide stability, including chemical modification, peptide self-assembly, the use of protease and peptidase inhibitors, or formulating the peptide in a drug delivery system. Chemical modifications that can be used to prevent enzyme recognition and reduce degradation include altering the N- and C-terminals, cyclisation, polymerization, and the incorporation of non-natural amino acids [ 2 , 8 , 9 , 15 , 16 , 19 – 21 ]. However, modifications to the peptide structure carry the inherent risk that activity, receptor affinity, or receptor selectivity may be reduced or lost upon modification [ 20 ].…”

Synthesis and in Vitro Evaluation of Stabilized and Selective Neuromedin U-1 Receptor Agonists

“…Tritiated NMU-8, which was used for the binding studies, was obtained as described before (see Supporting Information for experimental details). 13 Evaluation of the affinities and agonistic activities of the NMU-analogs at the NMURs was performed in the present study on HEK293 cells transiently expressing human (h)NMUR1 and hNMUR2 as reported before. 13 Evaluation of the affinity of the novel NMU-analogs for the NMURs was performed with a competitive binding assay using [ 3 H]-NMU-8 as radioligand.…”

“…13 Evaluation of the affinities and agonistic activities of the NMU-analogs at the NMURs was performed in the present study on HEK293 cells transiently expressing human (h)NMUR1 and hNMUR2 as reported before. 13 Evaluation of the affinity of the novel NMU-analogs for the NMURs was performed with a competitive binding assay using [ 3 H]-NMU-8 as radioligand. An inositol triphosphate (IP 3 ) accumulation assay was carried out to study the functional activity of the NMU-analogs.…”

“…4 Interestingly, we successfully modified this critical region by modification of the Pro 6 residue and found that modification of this residue could lead to selectivity toward NMUR2. 13 In the current study, this critical and conserved region was further explored. In a first step, Arg 7 was replaced by the homologated β 3 homoArg (β 3 hArg) (to give compound 1, Ac-Tyr-Phe-Leu-Phe-Arg-Proβ 3 hArg-Asn-NH 2 ).…”