Selection of High-Affinity Peptidic Serine Protease Inhibitors with Increased Binding Entropy from a Back-Flip Library of Peptide-Protease Fusions.
We have developed a new concept for designing peptidic protein modulators, by recombinantly fusing the peptidic modulator, with randomized residues, directly to the target protein via a linker and screening for internal modulation of the activity of the protein. We tested the feasibility of the concept by fusing a 10-residue-long, disulfide-bond-constrained inhibitory peptide, randomized in selected positions, to the catalytic domain of the serine protease murine urokinase-type plasminogen activator. High-affinity inhibitory peptide variants were identified as those that conferred to the fusion protease the lowest activity for substrate hydrolysis. The usefulness of the strategy was demonstrated by the selection of peptidic inhibitors of murine urokinase-type plasminogen activator with a low nanomolar affinity. The high affinity could not have been predicted by rational considerations, as the high affinity was associated with a loss of polar interactions and an increased binding entropy.
Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark.
Hans Peter
HP
Sørensen
Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark.
Peng
P
Xu
Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, 350002 Fuzhou, People's Republic of China.
Longguang
L
Jiang
Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark.
Tobias
T
Kromann-Hansen
Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark; Department of Chemistry, Faculty of Science, University of Copenhagen, 2100 Copenhagen, Denmark.
Knud J
KJ
Jensen
Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark; Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, 350002 Fuzhou, People's Republic of China.
Mingdong
M
Huang
Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark; Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, 350002 Fuzhou, People's Republic of China. Electronic address: pa@mbg.au.dk.
Peter A
PA
Andreasen
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D018360:Crystallography, X-Ray; D055808:Drug Discovery; D019277:Entropy; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D019151:Peptide Library; D010456:Peptides, Cyclic / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism
D019151:Peptide Library; D010456:Peptides, Cyclic; D011993:Recombinant Fusion Proteins; D015842:Serine Proteinase Inhibitors; C530075:mupain-1; D012697:Serine Endopeptidases; D014568:Urokinase-Type Plasminogen Activator
conformation; fusion protein; glycine; mupain-1; phage display