Two‐Component Stapling of Biologically Active and Conformationally Constrained Peptides: Past, Present, and Future

Abstract

Peptides are an emerging class of therapeutics in the pharmaceutical world. Whilst small molecules have dominated the therapeutic landscape for decades, the design and application of peptide drugs is emerging among the pharmaceutical industries and academia. Although highly selective and efficacious, peptides are characterized by poor pharmacokinetic properties and amelioration of their bioavailability remains a major hurdle. Incorporation of conformational constraints within the peptide (such as peptide stapling) has been extensively used to improve the bioavailability of these molecules; consequently, it is not surprising that a plethora of stapling techniques has been developed and has had a significant impact on the development of peptide therapeutics. Among the numerous stapling techniques known, two‐component methodologies allow facile and divergent functionalization of peptides. The authors have pioneered a stapling technique that makes use of the double Cu‐catalyzed azide–alkyne cycloaddition between di‐azido peptides and functionalized di‐alkynyl staples. In recent years, the authors have created biologically active, conformationally constrained peptides functionalized with cell‐penetrating peptides, fluorescent tags, and photo cross‐linking moieties, demonstrating the wide applicability of this methodology. Herein, the impact, advantages, limitations, and future applications of this technology and other two‐component peptide stapling techniques on the development of clinically relevant peptides are highlighted.

Multicomponent Peptide Stapling as a Diversity‐Driven Tool for the Development of Inhibitors of Protein–Protein Interactions

“…[3,4,9,10] As aresult, peptide stapling is now am ature technology [11][12][13][14][15][16] that has been applied to target protein-protein interactions related to infectious diseases,cancer, neurological, endocrine,metabolic, and cardiovascular disorders,and afirst example is tested in clinical trials. [1,2,8,10,[17][18][19] As depicted in Scheme 1, besides RCM, one-component stapling techniques (that is,t he peptide as the only component) currently include the traditional construction of lactam and disulfide bridges as well as others based on oxime and thioether bond formation [3,20] or the Ugi cyclization between Lysand Asp/Glu side chains. [21,22] On the other hand, there is an increasing interest in two-component stapling approaches (Scheme 1A), in which eventually biorthogonal processes, such as the click Cu I -catalyzed alkyne-azide cycloaddition [3,10,23] and dithiol (Cys) bis-alkylation, [24] introduce as tapling linker capable to stabilize the a-helical structure,w ith the former one also enabling the functionalization of the staple moiety.T his strategy could be potentially more efficient for the rapid optimization of the linker,since parallel syntheses and biological screening can be undertaken using as ingle peptide sequence and varying the length, flexibility, and hydrophobicity of the linker.Multi-component reactions (MCRs) are excellent diversity-generating tools and have recently emerged as powerful stapling tools capable to lock specific peptide conformations and simultaneously diversify the staple moiety by variation of endo-a nd exo-cyclic moieties during the multicomponent formation of the side chain cross-linker.…”

“…[1][2][3][4][5] While the original term "stapled peptides" was employed for hydrocarbon bridged a-helical peptides synthesized by ringclosing metathesis (RCM), [1,2,8] several stapling techniques have been developed over the last decade. [3,4,9,10] As aresult, peptide stapling is now am ature technology [11][12][13][14][15][16] that has been applied to target protein-protein interactions related to infectious diseases,cancer, neurological, endocrine,metabolic, and cardiovascular disorders,and afirst example is tested in clinical trials. [1,2,8,10,[17][18][19] As depicted in Scheme 1, besides RCM, one-component stapling techniques (that is,t he peptide as the only component) currently include the traditional construction of lactam and disulfide bridges as well as others based on oxime and thioether bond formation [3,20] or the Ugi cyclization between Lysand Asp/Glu side chains.…”

Multicomponent Peptide Stapling as a Diversity‐Driven Tool for the Development of Inhibitors of Protein–Protein Interactions

“…As depicted in Scheme , besides RCM, one‐component stapling techniques (that is, the peptide as the only component) currently include the traditional construction of lactam and disulfide bridges as well as others based on oxime and thioether bond formation or the Ugi cyclization between Lys and Asp/Glu side chains . On the other hand, there is an increasing interest in two‐component stapling approaches (Scheme A), in which eventually biorthogonal processes, such as the click Cu I ‐catalyzed alkyne–azide cycloaddition and dithiol (Cys) bis‐alkylation, introduce a stapling linker capable to stabilize the α‐helical structure, with the former one also enabling the functionalization of the staple moiety. This strategy could be potentially more efficient for the rapid optimization of the linker, since parallel syntheses and biological screening can be undertaken using a single peptide sequence and varying the length, flexibility, and hydrophobicity of the linker.…”

“…An effective strategy for α‐helix stabilization comprises the tethering of residues at either i , i +4 or i , i +7 positions in the peptide chain, so that the covalent bridge expands one or two turns of the α‐helical peptide . While the original term “stapled peptides” was employed for hydrocarbon bridged α‐helical peptides synthesized by ring‐closing metathesis (RCM), several stapling techniques have been developed over the last decade . As a result, peptide stapling is now a mature technology that has been applied to target protein–protein interactions related to infectious diseases, cancer, neurological, endocrine, metabolic, and cardiovascular disorders, and a first example is tested in clinical trials …”

Constrained Peptides with Fine‐Tuned Flexibility Inhibit NF‐Y Transcription Factor Assembly

“…To compensate for the lack of such structural constraints in small helices, preorganization has been artificially achieved through backbone rigidification or macrocyclization strategies, including the formation of hydrogen‐bond surrogates and inter‐side‐chain crosslinks . The latter approach is often referred to as peptide stapling and can be implemented through a variety of crosslinking strategies, such as lactam formation, 1,3‐dipolar cycloaddition, thiol reactive ligation, and C−C bond formation . So‐called hydrocarbon stapled peptides combine two constraints: 1) backbone derivatization through amino acid α‐methylation and 2) the crosslinking of two alkene‐bearing side chains through ring‐closing metathesis .…”