Increasing the bioactive space of peptide macrocycles by thioamide substitution

Abstract

We show that substituting a single atom, O to S (amide to thioamide), in a peptide bond results in global restriction of the conformational flexibility in peptide macrocycles with minimal perturbation of the parent conformation. The van der Waals interactions between the C[double bond, length as m-dash]S group and the surrounding atoms are the major driving force in inducing the conformational restriction, resulting in well-defined structures of these cyclic peptides with static 3-D presentation of the pharmacophores. Utilizing this property of thioamides, we report the development of a superactive antagonist of pro-angiogenic αvβ3, αvβ5 and α5β1 integrins, which are responsible for cancer cell proliferation and survival. Using simple thio-scanning and spatial screening of a non-efficacious and conformationally flexible cyclic peptide, we could achieve a more than 10⁵ fold enhancement in its efficacy in cellulo via a single O to S substitution. The developed peptide shows better efficacy in inhibiting the pro-angiogenic integrins than the drug candidate cilengitide, with a significantly enhanced serum half-life of 36 h compared to that of cilengitide (12 h). The long shelf-life, absence of non-specific toxicity and resistance to degradation of the thioamidated macrocyclic peptides in human serum suggest the promise of thioamides in markedly improving the affinity, efficacy and pharmacology of peptide macrocycles.

Fig. 1. (A) Solution conformation of cyclo(aA₄) (P) and its thio analogs (1–5). The T₁ values of the resolved C^α are indicated adjacent to the residues. The i + 1 residue in the β-turn, and the residue about which the γ-turn is centered, are highlighted. (B) The amide proton temperature coefficients (in ppb K^–1) determined from the ¹H NMR spectra acquired between 298 K and 323 K. The value for the thioamide NH is highlighted.

Fig. 2. (A) ¹H NMR spectra of cyclo(RGDfV) (6) and its thio-analogs 7–11. The γ-CH₃ of Val in both the conformers (A and B) of 6 is shown. (B) The solution structures of 7–11 deduced from 2D ¹H NMR spectroscopy in aqueous conditions. (C) ¹H NMR spectra of cyclo(RGDFV) (12) showing two conformers. (D) Solution structures of spatial screen analogs of 9 (9a–9d). The temperature coefficients of the amide protons (ppb K^–1) are shown and the asterisk denotes the site of the d-amino acid (Gly in 9b). Significant resonance overlap precluded the structure determination of 6 and 12. The hydrogen atoms are omitted for the sake of clarity.

Fig. 3. (A) The interaction of cilengitide (shown as a green ball and stick model) bound to the αvβ3 integrin in the PDB entry 1L5G. (B) The interaction of docked 9b (shown as a yellow ball and stick model) with the αvβ3 integrin. (C) The interaction of docked 7 (shown as an orange ball and stick model) with the αvβ3 integrin. (D) An overlay of the docked poses of 9b and 7 onto that of Cilen co-crystallized with the αvβ3 integrin in ; 1L5G. The green, magenta and cyan dashed lines represent hydrogen bonds, salt bridges and aromatic hydrogen bonds, respectively. The binding site of the protein is shown as a surface representation and the carbon atoms of the protein residues are shown in grey. Oxygen, nitrogen, hydrogen and sulphur atoms are shown in red, blue, white and yellow, respectively. Please see the ESI for a detailed description of the interactions between the various ligands and the αvβ3 integrin.

Fig. 4. Proliferation of (A) breast cancer cells and (B) glioblastoma cells at 48 h; n = 4 ± SEM. Note the slow dose-dependent decrease in proliferation and minimal cytotoxicity even with a 2 × 10⁴ fold increase in initial concentration. Please refer to the ESI for the entire dose-dependent study.

Fig. 5. The ex vivo metabolic stability of the macrocyclic peptides in freshly isolated human serum for 72 h; n = 6 ± SEM. Note the rapid degradation of the unmodified cyclic peptides 6 and 12. Even at 72 h, 1/4 of the thioamidated peptides 9b and 11 are intact.