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. 2016 Mar 8;4(2):e00226.
doi: 10.1002/prp2.226. eCollection 2016 Apr.

Structure-activity relationship study of angiotensin II analogs in terms of β-arrestin-dependent signaling to aldosterone production

Thairy Reyes Valero  1 , Emmanuel Sturchler  2 , Malika Jafferjee  1 , Giuseppe Rengo  3 , Vassiliki Magafa  4 , Paul Cordopatis  4 , Patricia McDonald  2 , Walter J Koch  5 , Anastasios Lymperopoulos  1
Affiliations

Structure-activity relationship study of angiotensin II analogs in terms of β-arrestin-dependent signaling to aldosterone production

Thairy Reyes Valero et al. Pharmacol Res Perspect. .

Abstract

The known angiotensin II (AngII) physiological effect of aldosterone synthesis and secretion induction, a steroid hormone that contributes to the pathology of postmyocardial infarction (MI) heart failure (HF), is mediated by both Gq/11 proteins and β-arrestins, both of which couple to the AngII type 1 receptors (AT1Rs) of adrenocortical zona glomerulosa (AZG) cells. Over the past several years, AngII analogs with increased selectivity ("bias") toward β-arrestin-dependent signaling at the AT1R have been designed and described, starting with SII, the gold-standard β-arrestin-"biased" AngII analog. In this study, we examined the relative potencies of an extensive series of AngII peptide analogs at relative activation of G proteins versus β-arrestins by the AT1R. The major structural difference of these peptides from SII was their varied substitutions at position 5, rather than position 4 of native AngII. Three of them were found biased for β-arrestin activation and extremely potent at stimulating aldosterone secretion in AZG cells in vitro, much more potent than SII in that regard. Finally, the most potent of these three ([Sar(1), Cys(Et)(5), Leu(8)]-AngII, CORET) was further examined in post-MI rats progressing to HF and overexpressing adrenal β-arrestin1 in vivo. Consistent with the in vitro studies, CORET was found to exacerbate the post-MI hyperaldosteronism, and, consequently, cardiac function of the post-MI animals in vivo. Finally, our data suggest that increasing the size of position 5 of the AngII peptide sequence results in directly proportional increases in AT1R-dependent β-arrestin activation. These findings provide important insights for AT1R pharmacology and future AngII-targeted drug development.

Keywords: Aldosterone; angiotensin II; angiotensin II type 1 receptor; biased ligand; structure–activity relationship (SAR); β‐arrestin; post‐MI HF.

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Figures

Figure 1
Figure 1
Comparison of potencies of CRSML, CORML, and CORET at stimulating AT1R‐ β‐arrestin coupling. (A) Comparison of CORET versus AngII (DiscoveRx assay). (B) Comparison of CORET, CORSML, and CORML versus SII (DiscoveRx assay). (C) Concentration–response curves for AT1R‐β‐arrestin binding stimulation, as derived from the CellKey assay (Cys: Sar1, Cys5, Leu8‐AngII). n = 5 independent determinations for each peptide in both assays (two‐way ANOVA with Bonferroni test).
Figure 2
Figure 2
AngII analog peptides and aldosterone turnover in vitro. (A) Schematic representation of the AT1R‐elicited pathways mediated by G proteins (G prt) and β‐arrestin1 (β arr1) converging on aldosterone synthesis and secretion in adrenocortical zona glomerulosa (AZG) cells. (B) In vitro aldosterone secretion from H295R cells overexpressing β‐arrestin1 and stimulated for 4 h with 100 nmol/L CORSML, CORML, or CORET. Data are presented as % of the secretion induced by 10 μmol/L SII. *P < 0.05, versus SII, ^P < 0.05, versus. CORML or CORSML, n = 5 independent experiments/treatment. (C, D) Western blotting for StAR protein levels in the same cells treated as in (B). Representative blots of five independent experiments are shown in (C), including blots from nontransfected (NT) H295R cells for β‐arrestin1 (β arr1) to confirm its overexpression in the transfected cells, and for GAPDH (glyceraldehyde 3‐phosphate dehydrogenase) as loading control, and the StAR protein induction (as % of the SII response), as derived by densitometric quantification, is shown in (D). *P < 0.05, versus SII, ^P < 0.05, versus CORML or CORSML, n = 5 independent experiments/treatment (two‐way ANOVA & chi‐square test with Tukey's multiple comparison test).
Figure 3
Figure 3
CORET in post‐MI HF in vivo. (A) Circulating aldosterone levels in 3‐week post‐MI rats, overexpressing β‐arrestin1 in their adrenals, and treated for seven consecutive days either with saline (MI‐Saline) or with daily i.p. injections of CORET. Aldosterone levels in age‐matched, sham‐operated, healthy, and untreated (and with normal adrenal β‐arrestin1 expression levels) animals are also included for comparison. *P < 0.05, versus Sham, # P < 0.05, versus MI‐Saline, n = 5 rats/group. (B) Ejection fraction (EF %) of the same rat groups at the end of the 7‐day treatment. *P < 0.05, versus Sham, # P < 0.05, versus MI‐Saline, n = 5 rats/group. (C) Increases in cardiac contractile function in the post‐MI groups at the end of the 7‐day treatment, as inferred by the % increase in +dP/dtmax that a maximal dose of isoproterenol (Max. Iso) induces. *P < 0.05, versus. MI‐Saline, n = 5 rats/group (two‐way ANOVA & chi‐square test with Tukey's multiple comparison test).
Figure 4
Figure 4
AngII's Ile5 substitution and AT1R‐elicited β‐arrestin activation. (A) The amino acid sequences of all of the peptides studied. The substitutions differing from AngII are highlighted in bold. (B) Concentration‐response curves for stimulation of AT1R‐β‐arrestin binding, based on the DiscoveRx assay. (C) Concentration–response curves for stimulation of AT1R‐β‐arrestin binding based on the CellKey assay. See also Table 1. n = 5 independent determinations for each analog in both assays (two‐way ANOVA with Tukey's multiple comparison test).
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