Stapled Peptides-A Useful Improvement for Peptide-Based Drugs

Abstract

Peptide-based drugs, despite being relegated as niche pharmaceuticals for years, are now capturing more and more attention from the scientific community. The main problem for these kinds of pharmacological compounds was the low degree of cellular uptake, which relegates the application of peptide-drugs to extracellular targets. In recent years, many new techniques have been developed in order to bypass the intrinsic problem of this kind of pharmaceuticals. One of these features is the use of stapled peptides. Stapled peptides consist of peptide chains that bring an external brace that force the peptide structure into an α -helical one. The cross-link is obtained by the linkage of the side chains of opportune-modified amino acids posed at the right distance inside the peptide chain. In this account, we report the main stapling methodologies currently employed or under development and the synthetic pathways involved in the amino acid modifications. Moreover, we report the results of two comparative studies upon different kinds of stapled-peptides, evaluating the properties given from each typology of staple to the target peptide and discussing the best choices for the use of this feature in peptide-drug synthesis.

Keywords: cellular uptake; helicity; peptide drugs; stapled peptide; structurally constrained peptide.

Figure 1

Peptides approved and in active development by therapeutic area (2016) [6].

Figure 2

Example of methylation of the N-terminus (A) and of the $\alpha$-carbon (B).

Scheme 1

Example of peptide stapling [16].

Figure 3

Example of i, i + 4 (A), i, i + 7 (B) and double i, i + 4 (C) stapling [17].

Figure 4

Ramachandran diagram [23].

Figure 5

Pinocytosis mechanism [27].

Scheme 2

Example of Ring Closing Metathesis Stapling [28].

Scheme 3

Ring closing metathesis reaction mechanism.

Scheme 4

Olefinic amino acid synthesis by nucleophilic substitution.

Scheme 5

Nickel-catalysed olefinic amino acid synthesis. R = -H, -Me.

Scheme 6

Example of CuAAC Stapling.

Scheme 7

Copper catalysed azide-alkyne cycloaddition (CuAAC) reaction mechanism.

Scheme 8

Alkynyl-amino acid synthesis by nucleophilic substitution. X = -O, -S, -COO, -CONH, -CH${}_2$COO, -CH${}_2$CONH.

Scheme 9

Ni-catalysed alkynyl-amino acid synthesis. R = -H, -Me.

Scheme 10

Azide-amino acid synthesis by Mitsunobu reaction.

Scheme 11

Azide-amino acid synthesis by Weinreb amide.

Scheme 12

Azide-amino acid synthesis by Hoffmann rearrangement.

Scheme 13

Azide-amino acid synthesis by Ullmann coupling.

Scheme 14

Example of Lactamization Stapling [28].

Scheme 15

PyBOP/HOBt coupling mechanism.

Scheme 16

Example of Cysteine-Xylene Stapling [28].

Scheme 17

Example of Cysteine-Perfluorobenzene Stapling [28].

Scheme 18

Example of Alkyne/Alkene - Hydrothiolation Stapling [28].

Scheme 19

Alkynyl-amino acid synthesis.

Scheme 20

Example of Selenocysteine Stapling [54].

Scheme 21

Example of Tryptophan Stapling [33].

Scheme 22

Tryptophan-aldehyde coupling mechanism.

Scheme 23

Example of Indole C-H activation Stapling [56].

Figure 6

From Huisgen, R. J. Org. Chem. 1976, 41, 403–419.

Scheme 24

Example of stapling strategy with nitrones.

Scheme 25

Example of Nitrile Oxide Stapling [28].

Scheme 26

Nitrile oxide-amino acid synthesis from double bond oxidation.

Scheme 27

Nitrile oxide-amino acid synthesis by alkyne hydroboration/oxidation.

Scheme 28

Nitrile oxide-amino acid synthesis by Negishi cross-coupling.

Figure 7

Circular dichroism spectra of peptides measured in ddH${}_2$O (A) and in 30 mM SDS solution (B). Schematic comparison of the relative helical contents in ddH${}_2$O (C) and 30 mM SDS solution (D) (1 = unstapled; 2 = all-hydrocarbon-; 3 = lactam-; 4 = triazole-; 5 = vinyl sulfide-; 6 = xylene-; 7 = perfluoroaryl-)). The relative helicity was normalized with respect to the peptide with a lactam crosslink. n.d.: not determined as a result of additional absorption [28].

Figure 8

Normalized FAM intensity versus normalized retention time of FAM-labelled peptides L1b-L7b by using flow cytometry analysis in different cell lines and reversed-phase liquid chromatography. FAM intensity and retention times were normalized with respect to the linear peptide (1) (1 = unstapled; 2 = all-hydrocarbon-; 3 = lactam-; 4 = triazole-; 5 = vinyl sulfide-; 6 = xylene-; 7 = perfluoroaryl-) [28].

Figure 9

Flow cytometry analysis of stapled $\alpha$-helical peptides with different cross-links in different cell-lines. Values are normalized with respect to Tat peptide (1 = unstapled; 2 = all-hydrocarbon-; 3 = lactam-; 4 = triazole-; 5 = vinyl sulfide-; 6 = xylene-; 7 = perfluoroaryl-) [28].

Figure 10

Haemolytic activity of stapled $\alpha$-helical peptides with different types crosslinks [28].