title: 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.
authors.affiliation: Department of Chemistry, University of Southern California, Los Angeles, California 90089, United States.
authors.forename: Shannon M
authors.initials: SM
authors.lastname: Howell
authors.affiliation: Department of Chemistry, University of Southern California, Los Angeles, California 90089, United States.
authors.forename: Stephen V
authors.initials: SV
authors.lastname: Fiacco
authors.affiliation: Department of Chemistry, University of Southern California, Los Angeles, California 90089, United States.
authors.forename: Terry T
authors.initials: TT
authors.lastname: Takahashi
authors.affiliation: Mork Family Department of Chemical Engineering and Materials Science, Viterbi School of Engineering, University of Southern California, Los Angeles CA, 90089-1211.
authors.forename: Farzad
authors.initials: F
authors.lastname: Jalali-Yazdi
authors.affiliation: Department of Cancer Systems Imaging, University of Texas MD Anderson Cancer Center, Houston, TX.
authors.forename: Steven W
authors.initials: SW
authors.lastname: Millward
authors.affiliation: Mork Family Department of Chemical Engineering and Materials Science, Viterbi School of Engineering, University of Southern California, Los Angeles CA, 90089-1211.
authors.forename: Biliang
authors.initials: B
authors.lastname: Hu
authors.affiliation: Mork Family Department of Chemical Engineering and Materials Science, Viterbi School of Engineering, University of Southern California, Los Angeles CA, 90089-1211.
authors.forename: Pin
authors.initials: P
authors.lastname: Wang
authors.affiliation: 1] Department of Chemistry, University of Southern California, Los Angeles, California 90089, United States [2] Mork Family Department of Chemical Engineering and Materials Science, Viterbi School of Engineering, University of Southern California, Los Angeles CA, 90089-1211.
authors.forename: Richard W
authors.initials: RW
authors.lastname: Roberts
mesh_terms: D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000737:chemistry* / Q000627:therapeutic use; D006801:Humans; D010447:Peptide Hydrolases / Q000737:chemistry; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry* / Q000627:therapeutic use; D011485:Protein Binding; D059748:Proteolysis; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*
chemical_list: D001798:Blood Proteins; D019151:Peptide Library; D010455:Peptides; D012333:RNA, Messenger; D010447:Peptide Hydrolases