Adrenomedullin 2.0: Adjusting Key Levers for Metabolic Stability

Abstract

The 52 amino acid peptide hormone adrenomedullin (ADM) plays a major role in the development and regulation of the cardiovascular and lymphatic system and has therefore gained significant interest for clinical applications. Because adrenomedullin exhibits low metabolic stability, enhancement of the plasma half-life is essential for peptide-based drug design. Fluorescently labeled ADM analogues synthesized by Fmoc/t-Bu solid phase peptide synthesis were used to analyze their enzymatic degradation and specific fragmentation pattern in human blood plasma. The determination of important cleavage sites allowed the development of selectively modified peptides in a rational approach. By combination of palmitoylation, lactam-bridging, and Nα-methylation, ADM analogues protected from enzymatic cleavage in human blood were developed and revealed an explicitly elongated half-life of 5 days in comparison to the wild-type in vitro. This triple-modification did not alter the selectivity of the analogues at the AM1 receptor, highlighting their potential for therapeutic applications.

The Impact of Adrenomedullin Thr22 on Selectivity within the Calcitonin Receptor‐like Receptor/Receptor Activity‐Modifying Protein System

“…Adrenomedullin represents ap romising starting point for selectivity research, as it already possesses ad istinct intrinsic preference for the AM 1 Ra nd the AM 2 Ro ver the CGRPR. [26] Amino acids of the activation motif of a-CGRP were introduced into ADM as af irst step. [17,18,25] Because earlier studies also demonstrated that this analogue exhibits the same selectivity profile as the full-length wild-type, it was used as al ead peptide foro ur investigations.…”

“…Olefins tapled analogues of 1 were synthesized using (S)-2-(4-pentenyl)alanine staplinga tt he N-terminal (23)(24)(25)(26)(27)10)o r the C-terminal end of the helix (26)(27)(28)(29)(30)11)i no rder to study whether the position of the stabilizing modification makes a difference. Olefins tapled analogues of 1 were synthesized using (S)-2-(4-pentenyl)alanine staplinga tt he N-terminal (23)(24)(25)(26)(27)10)o r the C-terminal end of the helix (26)(27)(28)(29)(30)11)i no rder to study whether the position of the stabilizing modification makes a difference.…”

“…[26] Manual couplings of Fmoc-(S)-2-(4-pentenyl)Ala-OH (Sigma-Aldrich), Fmoc-(S)-2-(4-pentenyl)Phe-OH and Fmoc-(S)-2-(4-pentenyl)Ser(tBu)-OH (synthesized by Bayer AG according to published procedures) [22] were carried out with a5 -fold molar excess of amino acids, 1-hydroxybenzotriazole (HOBt, Sigma-Aldrich) and N,N'-diisopropylcarbodiimide (DIC, Iris Biotech) in N,N-dimethylformamide (DMF,V WR) at room temperature for 24 h. For the coupling of amino acids N-terminally adjacent to Fmoc-(S)-2-(4pentenyl)Ala-OH a1 0-fold excess of amino acid, HOBt and DIC was used. Automated peptide synthesis was performed as described before.…”

“…[26] Human embryonic kidney cells (HEK293) were cultured in am ixture of HAM'sF -12 with l-glutamine (Lonza), Dulbecco'sm odified Eagle'sm edium (DMEM, Lonza) and sterile-filtered, heat-inactivated fetal calf serum (FCS, Biochrom) (42.5:42.5:15; v/v/v). [26] Human embryonic kidney cells (HEK293) were cultured in am ixture of HAM'sF -12 with l-glutamine (Lonza), Dulbecco'sm odified Eagle'sm edium (DMEM, Lonza) and sterile-filtered, heat-inactivated fetal calf serum (FCS, Biochrom) (42.5:42.5:15; v/v/v).…”

“…[1] Thus, understanding the structurald ifferences required for preferential receptor activation is challenging. While ADM does not adopt ap articular secondary structure in pure water,N MR measurementsi nS DS micelles revealed ah elical segment (residues [22][23][24][25][26][27][28][29][30] next to the ring structure and it was suggested that receptor interaction induces as imilar structurale ffect (Figure 1B). [9] These findings were also in accordance with the "two-domain binding model" previously described as ap otential mechanism of receptor interaction for other class B GPCR.…”