A chameleonic macrocyclic peptide with drug delivery applications

Abstract

Head-to-tail cyclized peptides are intriguing natural products with unusual properties. The PawS-Derived Peptides (PDPs) are ribosomally synthesized as part of precursors for seed storage albumins in species of the daisy family, and are post-translationally excised and cyclized during proteolytic processing. Here we report a PDP twice the typical size and with two disulfide bonds, identified from seeds of Zinnia elegans. In water, synthetic PDP-23 forms a unique dimeric structure in which two monomers containing two β-hairpins cross-clasp and enclose a hydrophobic core, creating a square prism. This dimer can be split by addition of micelles or organic solvent and in monomeric form PDP-23 adopts open or closed V-shapes, exposing different levels of hydrophobicity dependent on conditions. This chameleonic character is unusual for disulfide-rich peptides and engenders PDP-23 with potential for cell delivery and accessing novel targets. We demonstrate this by conjugating a rhodamine dye to PDP-23, creating a stable, cell-penetrating inhibitor of the P-glycoprotein drug efflux pump.

Fig. 1. LC-MS analysis confirm that Zinnia elegans seeds contain the cyclic, 28 amino acid, two disulfide bond peptide PDP-23. (A) Zinnia elegans seeds, ruler placed for scale. (B) Image of a Zinnia elegans flower. (C) Total ion chromatogram of the seed extract marked with the retention time of PDP-23, confirming its presence at the protein level. (D) Extracted ion chromatogram (XIC) for PDP-23 showing the [M + 3H]³⁺ (m/z) state of the observed mass-to-charge ratio and its isotopic peak envelope.

Fig. 2. Characterization of synthetic disulfide isomers of PDP-23. (A) Cysteine connectivity of the three synthesized isomers with corresponding sequence. (B) MALDI-TOF MS showing a well-distributed experimental isotopic peak envelope with an observed monoisotopic mass-to-charge ratio of 3109.28⁺ for isomer I, 3109.29⁺ for isomer II and 3109.33⁺ for isomer III. (C) Analytical RP-HPLC trace of synthesized isomers in reduced and alkylated and fully oxidized state. The isomers eluted in the following order: isomer III (41.78 min), isomer II (43.02 min) and isomer I (44.22 min). (D) One-dimensional (1D) ¹H NMR spectra of the three fully oxidized isomers.

Fig. 3. 3D structure of PDP-23 in monomeric and dimeric forms. (A) Superposition of the structural ensemble of the PDP-23 monomer in 80 : 20 H₂O/CD₃CN. The backbone is shown in cyan, with side chains colored according to types: disulfides in orange, hydrophobic in green, basic in blue, acidic in red, and polar in grey. Residues are labeled with numbers and one letter amino acid codes. (B) Schematic representation of PDP-23 showing the two anti-parallel β-sheets, disulfide bonds (solid lines), turns and hydrogen bonds (dashed arrows). (C) Superposition of the structural ensemble of the PDP-23 dimer in water. The same coloring is used as in panel A but the cyclic backbone is displayed in magenta. Upper and lower case lettering are used to distinguish between the two PDP-23 molecules. (D) Comparison of the hydrophobic core regions of monomeric and dimeric PDP-23. Monomeric PDP-23 is shown in cyan and dimeric PDP-23 in magenta and purple.

Fig. 4. 3D structure of PDP-23 in micelles. (A) Superposition of the structural ensemble of PDP-23 when exposed to SDS micelles. The same coloring is used as in Fig. 3, but the backbone is displayed in yellow. (B) Superposition of the structural ensemble of PDP-23 when exposed to DPC micelles. The same coloring is used as in Fig. 3 but the backbone is displayed in black.

Fig. 5. Stability of PDP-23. Non-linear regression fit of the decay of PDP-23 over time when exposed to human serum (black), simulated gastric fluid (blue) and simulated intestinal fluid (red). All data are presented as mean ± SEM, n = 3.

Fig. 6. Internalization of PDP-23 in CHO cells. PDP-23 labeled with ATTO 488 (5 μM concentration) was incubated with the cells for 2 h at 37 °C. The plasma membrane was labeled with WGA-Alexa 633 as seen in red and the nucleus labeled with DAPI as seen in blue. The labeled PDP-23 is visible in green. The magnification used was 63×.

Fig. 7. Reversal of daunorubicin resistance when dosed in combination with the PDP-23-rhodamine conjugate in P-gp-overexpressing cells. (A) Western blot of KB-3-1 and KB-V-1 cells confirming P-gp overexpression in the KB-V-1 cell line. (B) Dose–response curve for daunorubicin with KB-3-1 (purple) and KB-V-1 (green) cell lines. Cells were treated for 24 h with a range of nine concentrations of the drug. Cell populations determined via quantification of DNA with CyQuant Cell Proliferation Assay Kit and normalized to the untreated control. All data are means ± SEM, n = 4. (C) Comparison of toxicity of a single concentration of daunorubicin (1 μM) in sensitive (KB-3-1, purple) and resistant (KB-V-1, green) cells, in the presence or absence of the PDP-23-rhodamine conjugate. Verapamil (30 μM), a P-gp inhibitor, is included as a control for P-gp inhibition. Cell populations were determined via quantification of DNA with the CyQuant Cell Proliferation Assay Kit and normalized to an untreated control. All data are mean ± SEM, n = 4; significance determined by two-way ANOVA (****p < 0.0001), (***p < 0.001).

Fig. 8. Three-dimensional structures of naturally occurring cyclic peptides. (A) SFTI-1 isolated from the seeds of the common sunflower Helianthus annuus, PDB code: 1JBL. (B) θ-defensin found in the leukocytes of rhesus macaques Macaca mulatta, PDB code: 2LYF. (C) PDP-23 isolated from the seeds of Zinnia elegans. (D) Kalata B1 isolated from the leaves of Oldenlandia affinis, PDB code: 1NB1. (E) Overlay of type I′ turns from θ-defensin RTD-1 (red) and PDP-23 (cyan). Residues in the turn region are shown in stick format and are numbered according to their position in the sequence. (F) Overlay of type VIa1 turns from kalata B1 (blue) and PDP-23 (cyan). Residues in the turn region are shown in stick format and are numbered according to their position in the sequence.