β‐amino acid substitution to investigate the recognition of angiotensin II (AngII) by angiotensin converting enzyme 2 (ACE2)

Abstract

In spite of the important role of angiotensin converting enzyme 2 (ACE2) in the cardiovascular system, little is known about the substrate structural requirements of the AngII-ACE2 interaction. Here we investigate how changes in angiotensin II (AngII) structure affect binding and cleavage by ACE2. A series of C3 β-amino acid AngII analogs were generated and their secondary structure, ACE2 inhibition, and proteolytic stability assessed by circular dichroism (CD), quenched fluorescence substrate (QFS) assay, and LC-MS analysis, respectively. The β-amino acid-substituted AngII analogs showed differences in secondary structure, ACE2 binding and proteolytic stability. In particular, three different subsets of structure-activity profiles were observed corresponding to substitutions in the N-terminus, the central region and the C-terminal region of AngII. The results show that β-substitution can dramatically alter the structure of AngII and changes in structure correlated with ACE2 inhibition and/or substrate cleavage. β-amino acid substitution in the N-terminal region of AngII caused little change in structure or substrate cleavage, while substitution in the central region of AngII lead to increased β-turn structure and enhanced substrate cleavage. β-amino acid substitution in the C-terminal region significantly diminished both secondary structure and proteolytic processing by ACE2. The β-AngII analogs with enhanced or decreased proteolytic stability have potential application for therapeutic intervention in cardiovascular disease.

Structural determinants for binding to angiotensin converting enzyme 2 (ACE2) and angiotensin receptors 1 and 2

“…Notable drops in ACE2 binding were also seen for the two β-Phe Ang II analogs with binding completely abolished in DRVYIYPβF and 15% inhibition ( Table 4 ) observed for the DRVYVYPβF analog. Notable decreases in ACE2 inhibition were also observed for β-Pro and β-Phe substitutions in native Ang II in a previous study ( Clayton et al, 2011 ). Native Ang II and chimeric analogs showing high levels of ACE2 binding at 10 μM were then assessed at reduced concentration, first at 1 μM and then at 0.1 μM if measurable inhibition was observed.…”

“…Peptide sequences and the apparent binding values are listed in Table 1 . ACE2 binding was assessed indirectly by use of a QFS assay where the ability of the compound to reduce the proteolytic cleavage of a fluorogenic substrate shows relative ACE2 binding ( Clayton et al, 2011 ). Several substitutions (Val, Tyr, Ala, Gly, Phe, Trp, Pro, His, Ile, and Leu) were made in order to further probe the highly hydrophobic and cyclic specificity of the C-terminus of Ang II.…”

“…These peptides contained the most inhibitory acetylated C-terminal tetrapeptides ( Table 3 ) grafted to the native N-terminal sequence of Ang II to further enhance ACE2 binding and are listed in Table 4 . Two β-amino acid analogs of Ang II which stabilize the scissile-bond (β-Pro 7 and β-Phe 8 ) in Ang II ( Clayton et al, 2011 ) were also tested for comparative purposes ( Table 4 ).…”

“…We previously described the influence of single β-amino acid substitutions on the structure and binding of Ang II to ACE2 ( Clayton et al, 2011 ). We demonstrated that three different regions of Ang II that exert different effects on Ang II structure and binding, namely the N-terminus, the central and the C-terminal region.…”

β-Pro7Ang III is a novel highly selective angiotensin II type 2 receptor (AT2R) agonist, which acts as a vasodepressor agent via the AT2R in conscious spontaneously hypertensive rats

“…Our relatively simple strategy of incorporating β-amino acid substitutions on an Ang template [6,23] has increased AT 2 R:AT 1 R selectivity from ∼1000-fold using Ang II analogues (e.g. β-Ile 5 Ang II; [6]) to >20 000-fold with Ang III analogues such as β-Pro 7 Ang III.…”