ß-Amino acid substitution to investigate the recognition of angiotensin II (AngII) by angiotensin converting enzyme 2 (ACE2).

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 ß-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 ß-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 ß-substitution can dramatically alter the structure of AngII and changes in structure correlated with ACE2 inhibition and/or substrate cleavage. ß-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 ß-turn structure and enhanced substrate cleavage. ß-amino acid substitution in the C-terminal region significantly diminished both secondary structure and proteolytic processing by ACE2. The ß-AngII analogs with enhanced or decreased proteolytic stability have potential application for therapeutic intervention in cardiovascular disease.

Synthesis and pharmacological evaluation of dual acting antioxidant A(2A) adenosine receptor agonists.

A series of adenosine-5'-N-alkylcarboxamides and N(6)-(2,2-diphenylethyl)adenosine-5'-N-alkylcarboxamides bearing antioxidant moieties in the 2-position were synthesized from the versatile intermediate, O(6)-(benzotriazol-1-yl)-2-fluoro-2',3'-O-isopropylideneinosine-5'-N-alkylcarboxamide (1). These compounds were evaluated as A(2A) adenosine receptor (A(2A)R) agonists in a cAMP accumulation assay, and a number of potent and selective agonists were identified. Three of these compounds were evaluated further in an ischemic injury cell survival assay and a reactive oxygen species (ROS) production assay whereby 15b and 15c were shown to reduce ROS activity and cell death due to ischemia.

A single beta-amino acid substitution to angiotensin II confers AT2 receptor selectivity and vascular function.

Novel AT(2)R ligands were designed by substituting individual ß-amino acid in the sequence of the native ligand angiotensin II (Ang II). Relative ATR selectivity and functional vascular assays (in vitro AT(2)R-mediated vasorelaxation and in vivo vasodepressor action) were determined. In competition binding experiments using either AT(1)R- or AT(2)R- transfected HEK-293 cells, only ß-Asp(1)-Ang II and Ang II fully displaced [(125)I]-Ang II from AT(1)R. In contrast, ß-substitutions at each position of Ang II exhibited AT(2)R affinity, with ß-Tyr(4)-Ang II and ß-Ile(5)-Ang II exhibiting ≈ 1000-fold AT(2)R selectivity. In mouse aortic rings, ß-Tyr(4)-Ang II and ß-Ile(5)-Ang II evoked vasorelaxation that was sensitive to blockade by the AT(2)R antagonist PD123319 and the nitric oxide synthase inhibitor L-NAME. When tested with a low level of AT(1)R blockade, ß-Ile(5)-Ang II (15 pmol/kg per minute IV for 4 hours) reduced blood pressure (BP) in conscious spontaneously hypertensive rats (ß-Ile(5)-Ang II plus candesartan, -24 ± 4 mm Hg) to a greater extent than candesartan alone (-11 ± 3 mm Hg, n=7, P<0.05), an effect that was abolished by concomitant PD123319 infusion. However, in an identical experimental protocol, ß-Tyr(4)-Ang II had no influence on BP (n=10), and it was less stable than ß-Ile(5)-Ang II in plasma stability assays. Thus, this study demonstrated that a single ß-amino acid substitution resulted in a compound that demonstrated both in vitro vasorelaxation and in vivo depressor activity via AT(2)R. This approach to the design and synthesis of novel AT(2)R-selective peptidomimetics shows great potential to provide insight into AT(2)R function.