Design of a MCoTI-Based Cyclotide with Angiotensin (1-7)-Like Activity

Abstract

We report for the first time the design and synthesis of a novel cyclotide able to activate the unique receptor of angiotensin (1-7) (AT1-7), the MAS1 receptor. This was accomplished by grafting an AT1-7 peptide analog onto loop 6 of cyclotide MCoTI-I using isopeptide bonds to preserve the α-amino and C-terminal carboxylate groups of AT1-7, which are required for activity. The resulting cyclotide construct was able to adopt a cyclotide-like conformation and showed similar activity to that of AT1-7. This cyclotide also showed high stability in human serum thereby providing a promising lead compound for the design of a novel type of peptide-based in the treatment of cancer and myocardial infarction.

Keywords: MAS1 receptor; angiotensin; cyclotide.

Figure 1

Design of grafted cyclotide MCo-AT1-7 to activate the receptor MAS1. The upper part of the panel shows the primary and tertiary structures of the cyclotide MCoTI-I (structure is based on a homology model using the solution structure of MCoTI-II as template (PDB: 1IB9) [14]), and the primary structure of peptide angiotensin 1-7 (AT1-7). The cyclized polypeptide is stabilized by the three-disulfide bonds (shown in red). The lower part of the figure shows the strategy used to graft an AT1-7-derived peptide onto the loop 6 of cyclotide MCoTI-I. The AT1-7-derived peptide was linked to the cyclotide backbone through the side-side chains of the N- and C-terminal residues forming two isopeptide bonds. The sequence corresponding to the AT1-7-derived peptides is shown in blue. Residue X represents l-2,3-diaminopropionic acid. Molecular graphics were built with Yasara (www.yasara.org).

Figure 2

Chemical synthesis and characterization of cyclotide MCo-AT1-7. (A) Analytical HPLC traces of for the linear thioester precursor, GSH-induced cyclization/folding crude after 24 h and purified cyclotide. An asterisk indicates the desired peptide; (B) Analytical HPLC trace and ES-MS characterization of pure MCo-AT1-7. The expected average molecular weight is shown in parenthesis.

Figure 3

¹H-NMR characterization of folded cyclotide MCo-AT1-7. Chemical shifts differences of the backbone, Nα-H and Hα protons between the common sequence (residues 1 through 32) of MCoTI-I and MCo-AT1-7 (Table S1).

Figure 4

Stability of cyclotides MCo-AT1-7 and MCoTI-I, and peptide AT1-7 and reduced linear MCo-AT1-7 precursor to human serum at 37 °C. Undigested peptides were quantified by HPLC-MS/MS.

Figure 5

Biological activity of cyclotide MCo-AT1-7. MAS1 stably transfected CHO cells were tested using different concentrations of cyclotide MCo-AT1-7 and peptide AT1-7 in the absence or presence of MAS1 antagonist A779. The amount of intracellular NO was measured by fluorescence as described in the experimental section. Cyclotide MCoTI-I was used as negative control. The average of standard deviation of two experiments is shown.