Serum Stable Natural Peptides Designed by mRNA Display

Abstract

Peptides constructed with the 20 natural amino acids are generally considered to have little therapeutic potential because they are unstable in the presence of proteases and peptidases. However, proteolysis cleavage can be idiosyncratic, and it is possible that natural analogues of functional sequences exist that are highly resistant to cleavage. Here, we explored this idea in the context of peptides that bind to the signaling protein Gαi1. To do this, we used a two-step in vitro selection process to simultaneously select for protease resistance while retaining function–first by degrading the starting library with protease (chymotrypsin), followed by positive selection for binding via mRNA display. Starting from a pool of functional sequences, these experiments revealed peptides with 100–400 fold increases in protease resistance compared to the parental library. Surprisingly, selection for chymotrypsin resistance also resulted in similarly improved stability in human serum (~100 fold). Mechanistically, the decreases in cleavage results from both a lower rate of cleavage (kcat) and a weaker interaction with the protease (Km). Overall, our results demonstrate that the hydrolytic stability of functional, natural peptide sequences can be improved by two orders of magnitude simply by optimizing the primary sequence.

Retrocyclins neutralize bacterial toxins by potentiating their unfolding

“…θ -defensins are likely to be more potent under in vivo conditions due to the overall higher stability and longer lifetime of cyclic peptides in serum and under condition of protease attack [47]. Therefore, initially introduced as promising anti-viral humanized synthetic peptides, analogous to those that are still active in old-world primates [6] and widely proposed as a topical microbicide [48–50], RCs should be seriously considered as first-line antidotes against a broad range of bacterial toxins.…”

Peptiligase, an Enzyme for Efficient Chemoenzymatic Peptide Synthesis and Cyclization in Water

“…[19] Macrocylic peptides are a very promising class of pharmaceuticals, mainly due to their high stability in blood serum. [20] We embarked on the cyclization (Figure 2) of linear Microcin J25, the precursor of a 21 amino acid long lasso-peptide with antibiotic properties that is naturally produced by a strain of E. coli isolated from human feces. [21] Chemical head-totail cyclization of such a long peptide is very challenging and the reaction has to be performed with extremely diluted substrate to prevent polymerization, making it difficult for scale-up.…”

Directed Evolution of Scanning Unnatural‐Protease‐Resistant (SUPR) Peptides for in Vivo Applications

“…While our prior work demonstrated that a dual selection for protease resistance and binding gave serum stable natural peptide sequences [11] , incorporation of an appropriate 21 st amino acid results in peptides with substantially higher proteolytic stabilities that retain high-affinity target binding in vitro and in vivo . Moreover, SUPR peptide design is general and does not require specific structures or even a priori knowledge of the target structure in order to work.…”