An engineered cyclic peptide alleviates symptoms of inflammation in a murine model of inflammatory bowel disease

Abstract

Inflammatory bowel diseases (IBDs) are a set of complex and debilitating diseases for which there is no satisfactory treatment. Recent studies have shown that small peptides show promise for reducing inflammation in models of IBD. However, these small peptides are likely to be unstable and rapidly cleared from the circulation, and therefore, if not modified for better stability, represent non-viable drug leads. We hypothesized that improving the stability of these peptides by grafting them into a stable cyclic peptide scaffold may enhance their therapeutic potential. Using this approach, we have designed a novel cyclic peptide that comprises a small bioactive peptide from the annexin A1 protein grafted into a sunflower trypsin inhibitor cyclic scaffold. We used native chemical ligation to synthesize the grafted cyclic peptide. This engineered cyclic peptide maintained the overall fold of the naturally occurring cyclic peptide, was more effective at reducing inflammation in a mouse model of acute colitis than the bioactive peptide alone, and showed enhanced stability in human serum. Our findings suggest that the use of cyclic peptides as structural backbones offers a promising approach for the treatment of IBD and potentially other chronic inflammatory conditions.

Keywords: NF-κB (NF-KB); SFTI-1; TNBS; colitis; cyclic peptide; nuclear magnetic resonance (NMR); peptide chemical synthesis.

Figure 1.

Schematic representation of grafting into the SFTI-1 scaffold. The three-dimensional structure of annexin A1 is shown on the left of the diagram (PDB ID code 1HM6). MC-12, highlighted on the structure of annexin A1, forms a helical structure in the full-length protein. The helical region of MC-12 is schematically represented as grafted into the binding loop of SFTI-1. SFTI-1 comprises two β-strands connected by a disulfide bond. The cyclization loop is also labeled on the diagram. The figure was generated using MOLMOL (37).

Figure 2.

Sequences of the grafted peptides. The sequences of SFTI-1 (19), and the grafted peptides are shown. The MC-12 sequence is highlighted in bold. The disulfide bond linking the two cysteine residues is shown in gray, and the cyclic backbone is shown with a black line.

Figure 3.

Secondary shift analysis of peptides. The secondary shifts for SFTI-1 are shown in red, cyc-MC12 in blue, lin-MC12(n) in cyan, and lin-MC12(l2) in yellow. The secondary shifts were calculated by subtracting the random coil shifts (26) from the αH shift. The overall shifts are similar for all peptides with the exception of lin-MC12(l2), where the shifts for the cysteine residues (residues 3 and 11) differ significantly from the other peptides.

Figure 4.

Structural analysis of cyc-MC12. The three-dimensional structure of the 20 lowest energy structures of cyc-MC12 is shown on the left of the diagram, highlighting the well defined nature of the peptide, including the grafted region. The PDB ID code is 5VAV, and the BMRB (Biological Magnetic Resonance Data Bank) code is 30274. The lowest energy structure for cyc-MC12 is shown in the middle of the diagram with the side chains of the grafted residues included. The structure of SFTI-1 is shown on the right of the diagram with the side chains of the residues replaced with MC-12 shown. The figure was made using MOLMOL (37).

Figure 5.

Three-dimensional structure of Ac2–26. The three-dimensional structure of the 20 lowest energy structures was determined using NMR derived constraints. The helical region is shown with a thickened ribbon. The PDB ID code is 5VFW and the BMRB code is 30281. The figure was made using MOLMOL (37).

Figure 6.

Protective effects of cyc-MC12 against weight loss and clinical symptoms induced by TNBS colitis. Mice were treated with TNBS at day 0 and weighed daily before termination at day 3. A, percent weight loss (****, p < 0.0001). B, representative photomicrographs of colons at day 3. C, colon lengths at day 3 (**, p = 0.0079; *, p = 0.0397). D, macroscopic score of colon pathology at day 3 (**, p = 0.0079; *, p = 0.0476). All peptides were administered at a dosage of 3 mg/kg corresponding to injection solution with molar concentrations of ∼0.18 mm for the grafted peptides and 0.1 mm for Ac2–26. Data show the mean ± S.E. from a representative experiment of 3, with n = 5.

Figure 7.

Serum stability of SFTI-1-grafted peptides. The percentage of peptide remaining in the serum stability assay as assessed by RP-HPLC. The grafted peptides and SFTI-1 showed better stability than MC-12 and the longer Ac2–26 peptide. All data are represented as the mean ± S.D. and were recorded in triplicate.

Figure 8.

Overlay of Ac2–26 with annexin A1 (PDB ID code 1HM6). Structures were superimposed over residues 9–11 (purple) in Ac2–26 and residues 10–12 (orange) in annexin A1 (these residues correspond to the MC-12 sequence Gln-Ala-Trp). The labeling corresponds to the numbering in cyc-MC12. The figure was made using MOLMOL (37).