Dual-targeting anti-angiogenic cyclic peptides as potential drug leads for cancer therapy

Abstract

Peptide analogues derived from bioactive hormones such as somatostatin or certain growth factors have great potential as angiogenesis inhibitors for cancer applications. In an attempt to combat emerging drug resistance many FDA-approved anti-angiogenesis therapies are co-administered with cytotoxic drugs as a combination therapy to target multiple signaling pathways of cancers. However, cancer therapies often encounter limiting factors such as high toxicities and side effects. Here, we combined two anti-angiogenic epitopes that act on different pathways of angiogenesis into a single non-toxic cyclic peptide framework, namely MCoTI-II (Momordica cochinchinensis trypsin inhibitor-II), and subsequently assessed the anti-angiogenic activity of the novel compound. We hypothesized that the combination of these two epitopes would elicit a synergistic effect by targeting different angiogenesis pathways and result in improved potency, compared to that of a single epitope. This novel approach has resulted in the development of a potent, non-toxic, stable and cyclic analogue with nanomolar potency inhibition in in vitro endothelial cell migration and in vivo chorioallantoic membrane angiogenesis assays. This is the first report to use the MCoTI-II framework to develop a 2-in-1 anti-angiogenic peptide, which has the potential to be used as a form of combination therapy for targeting a wide range of cancers.

Figure 1. An overview of the screening process for the development of dual-targeting anti-angiogenic cyclic peptides, with a focus on the MCoTI-II framework.

(A) Several potent anti-angiogenic sequences (pigment epithelium-derived factor (PEDF), somatostatin (SST) and polyR) were grafted onto SFTI-1 (PDB ID: 1JBL) and MCoTI-II (PDB ID: 1HA9) for initial in vitro bioactivity screening and NMR characterization. The PEDF sequence comprises residues Tyr 388 to Phe 394 from a human PEDF protein (Uniprot ID: P36955), two somatostatin mimetic sequences (SST-01 and SST-02) were derived from a human somatostatin receptor comprising residues Phe 109 to Thr 112, and an anti-VEGF mimetic (polyR) was derived from a phage display library. The first tryptophan in the somatostatin epitopes was designed with a d-amino acid conformation, as a previous study showed this change is vital for its β-turn formation to selectively target SST2 and SST5 receptors. (B) Anti-angiogenic epitopes that gave potent activity among these first-generation cyclic peptides were selected, and further grafted into the MCoTI-II framework. (C) The second-generation peptides then underwent similar screening as the first-generation peptides. (D) Only the best analogues, along with corresponding single-targeting counterparts, were tested in in vivo assays and further characterized by NMR. The location of epitope insertion into SFTI-1 and MCoTI-II are indicated with red arrows. Different epitope insertions into loops of the SFTI-1 and MCoTI-II frameworks are showed using different colored circles (i.e. red (represents polyR), blue (represents PEDF), pink (represents SST-01), and green (represents SST-02)).

Figure 2. Sequences of grafted peptides with corresponding epitopes and native cyclic peptide frameworks.

First-generation grafted cyclic peptides were designed with a single anti-angiogenic epitope inserted into a single loop. For the second-generation grafted cyclic peptides, two anti-angiogenic epitopes were inserted into two separate loops. Chosen anti-angiogenic epitopes are highlighted in bold. d-tryptophan is represented by ‘w’.

Figure 3. Structural analysis using NMR.

(A) Comparison of SFTI-1 αH secondary shifts. (B) Comparison of MCoTI-II αH secondary shifts. All 2D NMR spectra were recorded at 298 K. All anti-angiogenic sequences are written in red and the regions for comparing native and grafted sequence are outlined by red dotted boxes. Disulfide bond connectivity is highlighted in yellow, and bold lines are used represent the cyclic nature of the peptides. Each cysteine is labeled with a Roman numeral and each loop is represented with the letter ‘L’. The loop of insertion of an anti-angiogenic sequence is circled in red for both SFTI-1 and MCoTI-II structures. All spectra were assigned using CCPNMR and each of the amino acid spin systems were specifically assigned based on Wuthrich et al.. The αH secondary shifts were analyzed by subtracting the random coil ¹H NMR chemical shifts of Wishart et al. from the experimental αH chemical shifts. The 3D molecular structure of SFTI-1 and MCoTI-II were illustrated using MOLMOL.

Figure 4. Hemolytic assay.

Comparison of percentage hemolysis against human red blood cells for all peptides. Melittin was used as a positive control with 100% hemolysis. Drug controls (cilengitide, octreotide, and sunitinib) were also included in the assay. All data are shown as mean ± SD (n = 3).

Figure 5. Cell migration on VEGF-mediated HUVECs.

The percentage of HUVEC migration was analyzed using Prism Version 6 (GraphPad). (A–T) VEGF was used as a positive control with 100% HUVEC migration. All peptides were tested at concentrations ranging from 0.001–50 μM except for cilengitide and octreotide, which were tested in 10-fold dilutions from 10 μM. Peptides were added in the presence of 0.3 nM VEGF (bottom chamber). Data are shown as mean ± SD (n ≥ 3). Data were normalized to the mean VEGF control. P-values are represented as follows: ****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, and *p ≤ 0.05. A one-way ANOVA with Dunnett’s post-test using a multiple comparison test was used for statistical analysis.

Figure 6. Chorionallantoic membrane and stability assays.

(A) Comparison of blood vessel count of the linear and grafted peptides. Error bars indicate ± SD (n ≥ 6). The dotted line indicates ~50% inhibition of blood vessels. A one-way ANOVA with Dunnett’s post-test using a multiple comparison test was used for statistical analysis. In addition, unpaired t-test was used to test the significance of MCoAA-02 against MCo-SST-01 and MCo-PEDF. ****p ≤ 0.0001 and ***p ≤ 0.05. All peptides were compared to 0.3 nM VEGF (highlighted in grey), which is represented as 100% blood vessel growth. (B) This diagram shows the blood vessel growth of MCoAA-02 at various concentrations compared to the cyclic frameworks SFTI-1 and MCoTI-II in the CAM assay. VEGF was used as the positive control, and octreotide and cilengitide as the negative controls. All images were taken with an original magnification of x16 on an Olympus SZX12 dissecting microscope with a light box. DP capture and DP manager software packages were used during image acquisition. (C) This graph illustrates the percentage of peptide remaining over 24 h in the serum stability assay. All compounds showed better stability than the linear PEDF peptide (highlighted with red dashed lines). All data are represented as mean ± SD and were recorded in triplicate. Peptides labeled with an asterisk (*) were tested using the same method except with an additional step – dissolving the centrifuged pellet with 8 M guanidinium chloride before RP-HPLC analysis.

Figure 7. A comparison of recent work using cyclic disulfide-rich frameworks for bi-functional studies and a schematic overview of how MCoAA-02 could inhibit the angiogenic process.

(A) A comparison of dual-targeting peptides; specifically, a comparison of the findings of this study to previous work using cyclic disulfide-rich peptide frameworks originating from plant and animal sources. Previous work focused only on grafting two identical epitopes. Our work is the first utilizing the MCoTI-II cyclic framework for the insertion of two different anti-angiogenic targets. (B) Schematic diagram depicting how MCoAA-02 has the potential to act as a dual-targeting cyclic peptide by blocking indirect signaling pathways to exert a synergistic anti-angiogenic effect.