Design and characterization of a hybrid miniprotein that specifically inhibits porcine pancreatic elastase.
Studying protease/peptide inhibitor interactions is a useful tool for understanding molecular recognition in general and is particularly relevant for the rational design of inhibitors with therapeutic potential. An inhibitory peptide (PMTLEYR) derived from the third domain of turkey ovomucoid inhibitor and optimized for specific porcine pancreatic elastase inhibition was introduced into an inhibitor scaffold to increase the proteolytic stability of the peptide. The trypsin-specific squash inhibitor EETI II from Ecballium elaterium was chosen as the scaffold. The resulting hybrid inhibitor HEI-TOE I (hybrid inhibitor from E. elaterium and the optimized binding loop of the third domain of turkey ovomucoid inhibitor) shows a specificity and affinity to porcine pancreatic elastase similar to the free inhibitory peptide but with significantly higher proteolytic stability. Isothermal titration calorimetry revealed that elastase binding of HEI-TOE I occurs with a small unfavorable positive enthalpy contribution, a large favorable positive entropy change, and a large negative heat capacity change. In addition, the inhibitory peptide and the hybrid inhibitor HEI-TOE I protected endothelial cells against degradation following treatment with porcine pancreatic elastase.
Hilpert
Kai
K
Humboldt University of Berlin, Medical Faculty Charité, Department of Biochemistry, Monbijoustrasse 2, Germany.
Wessner
Helga
H
Schneider-Mergener
Jens
J
Welfle
Karin
K
Misselwitz
Rolf
R
Welfle
Heinz
H
Hocke
Andreas C
AC
Hippenstiel
Stefan
S
Höhne
Wolfgang
W
D000595:Amino Acid Sequence; D000818:Animals; D015195:Drug Design; D004730:Endothelium, Vascular / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D013552:Swine
D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D010196:Pancreatic Elastase