Synthesis and evaluation of novel lipidated neuromedin U analogs with increased stability and effects on food intake

Abstract

Neuromedin U (NMU) is a 25 amino acid peptide expressed and secreted in the brain and gastrointestinal tract. Data have shown that peripheral administration of human NMU decreases food intake and body weight and improves glucose tolerance in mice, suggesting that NMU receptors constitute a possible anti-diabetic and anti-obesity drug target. However, the clinical use of native NMU is hampered by a poor pharmacokinetic profile. In the current study, we report in vitro and in vivo data from a series of novel lipidated NMU analogs. In vitro plasma stability studies of native NMU were performed to investigate the proteolytic stability and cleavage sites using LC-MS. Native NMU was found to be rapidly cleaved at the C-terminus between Arg(24) and Asn(25) , followed by cleavage between Arg(16) and Gly(17) . Lipidated NMU analogs were generated using solid-phase peptide synthesis, and in vitro potency was investigated using a human embryonic kidney 293-based inositol phosphate accumulation assay. All lipidated analogs had preserved in vitro activity on both NMU receptors with potency improving as the lipidation site was moved away from the receptor-interacting C-terminal octapeptide segment. In vivo efficacy was assessed in lean mice as reduction in food intake after acute subcutaneous administration of 1, 0.3, 0.1, and 0.03 µmol/kg. These lipidated NMU analogs prolonged the anorectic effect of NMU in a dose-dependent manner. This was likely an effect of improved pharmacokinetic properties because of improved vitro plasma stability. Accordingly, the data demonstrate that lipidated NMU analogs may represent drug candidates for the treatment of obesity.

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

“…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 .…”

Half‐Life Extension of Biopharmaceuticals using Chemical Methods: Alternatives to PEGylation

“…A successful approach has been to employ NHS‐activated fatty acid constructs for acylation of available amino groups . However, this approach often requires a two‐step HPLC purification process, which decreases the overall yield of reaction …”

“…Typically, the point of lipidation will be either the N‐terminus or a Lys residue carrying an orthogonal protecting group. In Fmoc‐SPPS, this could be for example, allyloxycarbonyl (Alloc), 2‐acetyl‐5,5‐dimethyl‐1,3‐cyclohexanedionyl (Dde), or (4‐methoxyphenyl)diphenylmethyl (Mmt) . During assembly of the peptide chain, the orthogonal protecting group may be selectively removed to reveal an attachment point for anchoring of the lipid moiety.…”

“…During assembly of the peptide chain, the orthogonal protecting group may be selectively removed to reveal an attachment point for anchoring of the lipid moiety. By performing the lipidation on‐resin, purification in one step is usually sufficient to achieve a pure product …”

“…Also, Dalbøge et al. reported that lipidation of neuromedin U (NMU) with C16‐γGlu‐ at either the N‐terminus or an engineered Lys at position nine resulted in a 15‐fold prolongation of in vitro plasma stability at 37 °C …”