Stabilization of Angiotensin-(1-7) by key substitution with a cyclic non-natural amino acid

Abstract

Angiotensin-(1-7) [Ang-(1-7)], a heptapeptide hormone of the renin-angiotensin-aldosterone system, is a promising candidate as a treatment for cancer that reflects its anti-proliferative and anti-angiogenic properties. However, the peptide's therapeutic potential is limited by the short half-life and low bioavailability resulting from rapid enzymatic metabolism by peptidases including angiotensin-converting enzyme (ACE) and dipeptidyl peptidase 3 (DPP 3). We report the facile assembly of three novel Ang-(1-7) analogues by solid-phase peptide synthesis which incorporates the cyclic non-natural δ-amino acid ACCA. The analogues containing the ACCA substitution at the site of ACE cleavage exhibit complete resistance to human ACE, while substitution at the DDP 3 cleavage site provided stability against DPP 3 hydrolysis. Furthermore, the analogues retain the anti-proliferative properties of Ang-(1-7) against the 4T1 and HT-1080 cancer cell lines. These results suggest that ACCA-substituted Ang-(1-7) analogues which show resistance against proteolytic degradation by peptidases known to hydrolyze the native heptapeptide may be novel therapeutics in the treatment of cancer.

Keywords: ACCA; Angiotensin-(1–7); Angiotensin-converting enzyme; Dipeptidyl peptidase 3; Non-natural amino acid; Peptidomimetic.

Fig. 1

Proteolytic cleavage of angiotensin-(1–7) by angiotensin-converting enzyme (ACE) and dipeptidyl peptidase 3 (DPP 3)

Fig. 2

Structures of the Ang-(1–7) analogues ACCA1 1, ACCA2 2, and ACCA3 3

Scheme 1

Synthesis of Fmoc-ACCA 5. i) Fmoc-OSu (1.5 eq.), 10% (w/v) aq. Na₂CO₃, dioxane, 0 °C—r.t., 1 h 45 min, 95%

Fig. 3

Two orientations of the X-ray crystal structure of Fmoc-ACCA 5 (CCDC 1514790)

Fig. 4

HPLC–UV profile of the ACCA peptides and following exposure to ACE. a Ang-(1–7) (A7) standard. b ACE metabolizes Ang(1–7) to Ang-(1–5) (A5). c ACCA1 (AC1) standard. d ACCA1 is not metabolized by ACE. e ACCA2 (AC2) standard. f ACCA2 is not metabolized by ACE. g ACCA3 (AC3) standard. h ACCA3 (AC3) is metabolized by ACE to ACCA3-(1–5) [AC3(1–5)]

Fig. 5

HPLC–UV profile of the ACCA peptides and following exposure to DPP 3. a Ang-(1–7) (A7) standard. b DPP 3 metabolizes Ang-(1–7) to Ang-(3–7) and ultimately to Ang-(5–7) (A5–7). c ACCA1 (AC1) standard. d ACCA1 is metabolized by DPP 3 to likely ACCA1-(3–7) [AC1(3–7)], eluting at 6.2 min. e ACCA2 (AC2) standard. f ACCA2 is likely degraded fully to ACCA2-(5–7) [AC2 (5–7)]. g ACCA3 (AC3) standard. h ACCA3 is more stable to DPP 3 hydrolysis in comparison to Ang-(1–7) and the other analogues

Fig. 6

Effect of Ang-(1–7) or each of the Ang-(1–7)-ACCA analogues on the proliferation of 4T1 murine triple negative breast cancer cells. Ang-(1–7) (100 nM) was added daily to rapidly growing cells, while each of the ACCA analogues was added once to rapidly growth cells at day 0. The total cell number per well was counted after 3–4 days. n = 4–5 in triplicate; *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 7

Effect of Ang-(1–7) or each of the Ang-(1–7)-ACCA analogues on the proliferation of HT-1080 human sarcoma cells. Ang(1–7) (100 nM) was added daily to rapidly growing cells, while each of the ACCA analogues was added once to rapidly growth cells at day 0. The total cell number per well was counted after 3–4 days. n = 4–5 in triplicate; *p < 0.05 and **p < 0.01