Motivated by this gap in knowledge, we sought to address a number of open questions: (1) How do the variables of backbone modification type and position relative to a cleavage site affect protease efficiency? (2) What are the molecular mechanisms by which various modified backbones exert proteolytic protection? (3) How do multiple backbone modifications work in concert to protect a peptide from hydrolysis? Gaining a deeper understanding of these issues promises new design strategies for achieving maximal proteolytic protection in heterogeneous-backbone foldamers with minimal unnatural residue content. Although each of these building blocks has been examined in isolation, no prior report has sought to compare their effectiveness at shielding a substrate from proteolytic hydrolysis. We report here a systematic examination of the proteolytic protection imparted by four of the most common modifications employed in the design of heterogeneous-backbone foldamers ( Figure 1): d-α-residues, the C α-methylated α-residue Aib, N-Me-α-residues, and β 3-residues. While this can generate adequate proteolytic stability for biological applications, a more rational substitution approach would enable the construction of oligomers where proteolytic protection is considered alongside structural issues at the outset. In most examples of heterogeneous-backbone foldamer design, α-residue replacements are made in a manner guided primarily by structural considerations (e.g., maintaining local folding pattern and key interactions for receptor binding). An advantage of heterogeneous-backbone foldamers over homogeneous-backbone counterparts is the prospect of drawing from the wellspring of natural peptides for the design of unnatural analogues. Such backbone heterogeneity takes on another dimension when many classes of unnatural building blocks are incorporated alongside α-residues in a single chain. Recent results suggest significant potential for oligomers in which ~20–30% of the α-residues in a bioactive sequence are replaced by some unnatural analogue. At the other extreme are highly unnatural oligomeric backbones with protein-like folds and functions, often termed “foldamers.” Foldamers comprised entirely of a single type of unnatural monomer are inert to proteases and can show interesting biological activities however, the design of completely unnatural sequences that effectively mimic natural peptides can be challenging. This tactic offers the advantage of employing a biological sequence as the prototype and demonstrates that strategic placement of limited unnatural backbone content can lead to dramatically altered properties. Found at one end of the spectrum of such efforts is the introduction of one or two unnatural building blocks in a natural α-peptide to improve efficacy or stability. More recent work has explored modified backbones in the context of larger oligomers, seeking to recreate complex functions of diverse bioactive peptides and proteins on protease-resistant scaffolds. Īlterations to the chemical connectivity of the l-α-peptide backbone can be a useful means to improve proteolytic stability, and targeted backbone modification in short peptides has a rich history in the field of peptidomimetics research. A significant contributor to poor peptide bioavailability is rapid degradation by endogenous proteases, which can result in very short in vivo lifetimes. The promise of peptide therapeutics is attenuated in part by poor oral bioavailability, often necessitating administration by invasive and inconvenient parenteral methods. One solution to this problem is the use of larger peptide and protein based scaffolds, which have shown the unique ability to inhibit certain PPIs where small molecules have failed. While small molecules can effectively bind to pockets that natively recognize small ligands, disrupting protein-protein interactions (PPIs) involving extended interfaces remains a substantial challenge. The involvement of proteins in human pathology is a significant research interest, and pharmaceuticals that target proteins are in high demand.
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