Peptide Backbone Composition and Protease Susceptibility: Impact of Modification Type, Position, and Tandem Substitution

Abstract

The clinical utility of peptides is limited by their rapid degradation by endogenous proteases. Modification of the peptide backbone can generate functional analogues with enhanced proteolytic stability. Existing principles for the design of such oligomers have focused primarily on effective structural mimicry. A more robust strategy would incorporate a rational approach for engineering maximal proteolytic stability at minimal unnatural residue content. We report here the systematic comparison of the proteolytic resistance imparted by four backbone modifications commonly employed in the design of protease-stable analogues of peptides with complex folding patterns. The degree of protection is quantified as a function of modification type, position, and tandem substitution in the context of a long, unstructured host sequence and a canonical serine protease. These results promise to inform ongoing work to develop biostable mimics of increasingly complex peptides and proteins.

Protease-Resistant and Cell-Permeable Double-Stapled Peptides Targeting the Rab8a GTPase

“…In analogy to cell penetrating peptides (CPP), 24,25 cellular uptake can be improved by incorporation of positively charged amino acids. 17,26 Proteolytic stability can be increased by incorporation of non-natural building blocks including N-methylated, 27,28 β- [29][30][31] or D-amino acids 32,33 . Macrocyclization of linear peptides is a strategy that can enhance both cellular uptake and proteolytic stability by constraining the conformational freedom.…”

Development of Potent, Protease-Resistant Agonists of the Parathyroid Hormone Receptor with Broad β Residue Distribution

“…40, 41 In addition, these cyclic β residues enhance resistance to proteolysis relative to β 3 residues. 42, 43 We expected that cyclic β residues would be tolerated in the C-terminal portion of analogues of 1 , 13 because this region must adopt an α-helix-like conformation, 23 but it was necessary to test this prediction. Since the receptor-bound conformation of the N-terminal portion of 1 is unknown, the effect on agonist activity of placing cyclic β residues in this region could not be predicted.…”

Comparison of design strategies for α-helix backbone modification in a protein tertiary fold

“…The result is a “heterogeneous-backbone” oligomer consisting of a mixture of α-residues and unnatural counterparts that collectively display a native-like sequence of side chains (e.g., an α/β-peptide that blends α- and β-amino acid residues 6 ). If modifications are made carefully, such analogues can show similar folding behaviour and biological function as the prototype natural sequence but improved stability to enzymatic degradation in vitro 7 and in vivo . 8 …”