Faculty, Staff and Student Publications
Publication Date
7-1-2023
Journal
Protein Science
Abstract
Proteases are involved in many physiologic processes, and dysregulated proteolysis is basis of a variety of diseases. Specific inhibition of pathogenetic proteases via monoclonal antibodies therefore holds significant therapeutic promise. Inspired by the competitive mechanism utilized by many naturally occurring and man-made protease inhibitors, we hypothesized that substrate-like peptide sequences can act as protease subsite blocking motifs if they occupy only one side of the reaction center. To test this hypothesis, a degenerate codon library representing MMP-14 substrate profiles at P1-P5' positions was constructed in the context of an anti-MMP-14 Fab by replacing its inhibitory motif in CDR-H3 with MMP-14 substrate repertoires. After selection for MMP-14 active-site binders by phage panning, results indicated that diverse substrate-like sequences conferring antibodies inhibitory potencies were enriched in the isolated clones. Optimal residues at each of P1-P5' positions were then identified, and the corresponding mutation combinations showed improved characteristics as effective inhibitors of MMP-14. Insights on efficient library designs for inhibitory peptide motifs were further discussed. Overall, this study proved the concept that substrate-derived sequences were able to behave as the inhibitory motifs in protease-specific antibodies. With accumulating data available on protease substrate profiles, we expect the approach described here can be broadly applied to facilitate the generation of antibody inhibitors targeting biomedically important proteases.
Keywords
Humans, Peptide Hydrolases, Amino Acid Sequence, Protease Inhibitors, Antibodies, Monoclonal, Peptides, Substrate Specificity
DOI
10.1002/pro.4691
PMID
37278099
PMCID
PMC10285753
PubMedCentral® Posted Date
7-1-2023
PubMedCentral® Full Text Version
Post-print
Published Open-Access
yes