Discovery of Potent Hexapeptide Agonists to Human Neuromedin U Receptor 1 and Identification of Their Serum Metabolites

Abstract

Neuromedin U (NMU) and S (NMS) display various physiological activities, including an anorexigenic effect, and share a common C-terminal heptapeptide-amide sequence that is necessary to activate two NMU receptors (NMUR1 and NMUR2). On the basis of this knowledge, we recently developed hexapeptide agonists 2 and 3, which are highly selective to human NMUR1 and NMUR2, respectively. However, the agonists are still less potent than the endogenous ligand, hNMU. Therefore, we performed an additional structure−activity relationship study, which led to the identification of the more potent hexapeptide 5d that exhibits similar NMUR1-agonistic activity as compared to hNMU. Additionally, we studied the stability of synthesized agonists, including 5d, in rat serum, and identified two major biodegradation sites: Phe 2 -Arg 3 and Arg 5 -Asn 6 . The latter was more predominantly cleaved than the former. Moreover, substitution with 4-fluorophenylalanine, as in 5d, enhanced the metabolic stability at Phe 2 -Arg 3 . These results provide important information to guide the development of practical hNMU agonists.

Identification of a degrading enzyme in human serum that hydrolyzes a C‐terminal core sequence of neuromedin U

“…As previously reported, Fmoc‐amino acids/R‐COOH (0.141 mmol) were sequentially coupled to an Fmoc‐NH‐SAL Resin (100 mg, 0.047 mmol) using the DIPCI (0.141 mmol)‐HOBt (0.141 mmol) method. Coupling steps were performed for 2 h in DMF (1.0 mL) after removal of each Fmoc group with 20% piperidine‐DMF (1.5 mL, 20 min) to obtain resin‐bound peptide.…”

“…In our previous study, we focused on this common heptapeptide sequence and performed a structure–activity relationship study to develop potent and receptor subtype–selective agonists. Specifically, based on the des ‐amino derivative 2 (3‐phenylpropionyl‐Leu 1 ‐Phe 2 ‐Arg 3 ‐Pro 4 ‐Arg 5 ‐Asn 6 ‐NH 2 ), we developed a potent NMUR1 agonist 6 ( 5d in reference 7), an NMUR1‐selective agonist 7 ( 8d in reference 8), and an NMUR2‐selective agonist 10 ( 6b in reference 8) (Figure ) . In addition, we identified two biodegradation sites, the amide bonds between Phe(4‐F) 2 ‐Arg 3 and Arg 5 ‐Asn 6 , on 6 in rat and human serum .…”

“…Specifically, based on the des ‐amino derivative 2 (3‐phenylpropionyl‐Leu 1 ‐Phe 2 ‐Arg 3 ‐Pro 4 ‐Arg 5 ‐Asn 6 ‐NH 2 ), we developed a potent NMUR1 agonist 6 ( 5d in reference 7), an NMUR1‐selective agonist 7 ( 8d in reference 8), and an NMUR2‐selective agonist 10 ( 6b in reference 8) (Figure ) . In addition, we identified two biodegradation sites, the amide bonds between Phe(4‐F) 2 ‐Arg 3 and Arg 5 ‐Asn 6 , on 6 in rat and human serum . Using reversed‐phase high‐performance liquid chromatography (RP‐HPLC), a peak corresponding to the metabolite derived from the former site ( 5d‐m2 in reference 7) was clearly detected after incubation for 30 (rat serum) or 120 min (human serum).…”

“…On the other hand, the latter site was more rapidly degraded than the former, yielding the detectable metabolite 6‐m1 ( 5d‐m1 in Ref. 7, 2‐thienylacetyl‐Trp 1 ‐Phe(4‐F) 2 ‐Arg 3 ‐Pro 4 ‐Arg 5 ‐OH, Supporting Information, Figure S1) after incubation for 10 min in human serum . This preferential degradation at the Arg–Asn site was also observed in the endogenous ligand, human NMU (hNMU, 25 amino acids [aa]) .…”

“…7, 2‐thienylacetyl‐Trp 1 ‐Phe(4‐F) 2 ‐Arg 3 ‐Pro 4 ‐Arg 5 ‐OH, Supporting Information, Figure S1) after incubation for 10 min in human serum . This preferential degradation at the Arg–Asn site was also observed in the endogenous ligand, human NMU (hNMU, 25 amino acids [aa]) . Generally, peptides are readily degraded by proteases (i.e., t 1/2 <30 min) in vivo .…”