Characterizing and Optimizing Protease/Peptide Inhibitor Interactions, a New Application for Spot Synthesis

Hilpert, Kai; Hansen, Gerd; Wessner, Helga; Schneider‐Mergener, Jens; Höhne, Wolfgang

doi:10.1093/oxfordjournals.jbchem.a022833

Cited by 30 publications

(27 citation statements)

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“…It can be concluded that a complete substitutional analysis is a valuable method for optimizing peptide inhibitors, as we previously showed in a similar approach for elastase inhibition (Hilpert et al, 2000). In that case, the directly measured elastase binding to cellulose-bound peptide spots correlated satisfactorily to inhibition measurements with solubilized peptides.…”

Section: Discussionsupporting

confidence: 65%

Phage display screening for peptidic chitinase inhibitors

Petter

Scholz

Wessner

et al. 2008

J of Molecular Recognition

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A phage display library with disulfide-cyclized peptides was screened for peptides binding to chitinases from Serratia marcescens. One of those peptides was found to efficiently inhibit chitinase A and two others were inhibitors of chitinase B. Complete substitutional analysis of all three peptides using cellulose-bound peptide spot synthesis revealed key interaction positions and allowed optimization of the chitinase B inhibitory peptides towards higher affinity, with inhibitory constants in the lower nanomolar range. Inhibition by all peptides proved to be competitive and highly specific for the chitinase used to select them, as shown with a series of chitinases from different organisms.

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Section: Discussionsupporting

confidence: 65%

Phage display screening for peptidic chitinase inhibitors

Petter

Scholz

Wessner

et al. 2008

J of Molecular Recognition

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“…Immobilized arrays of peptides have been used for several applications including the identification of important residues in proteinprotein recognition, 1,2 the study of peptide-DNA interactions, 3 the enzymatic modification of peptides, 4,5 and the characterization of peptide motifs important to cell adhesion. 6,7 Many of these advances have been accomplished using multiplexed peptide arrays fabricated by SPOT synthesis.…”

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confidence: 99%

Characterization and Optimization of Peptide Arrays for the Study of Epitope−Antibody Interactions Using Surface Plasmon Resonance Imaging

2002

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The characterization of peptide arrays on gold surfaces designed for the study of peptide-antibody interactions using surface plasmon resonance (SPR) imaging is described. A two-step process was used to prepare the peptide arrays: (i) a set of parallel microchannels was used to deliver chemical reagents to covalently attach peptide probes to the surface by a thiol-disulfide exchange reaction; (ii) a second microchannel with a wraparound design was used as a small-volume flow cell (5 microL) to introduce antibody solutions to the peptide surface. As a demonstration, the interactions of the FLAG epitope tag and monoclonal anti-FLAG M2 were monitored by SPR imaging using a peptide array. This peptide-antibody pair was studied because of its importance as a means to purify fusion proteins. The surface coverage of the FLAG peptide was precisely controlled by creating the peptide arrays on mixed monolayers of alkanethiols containing an amine-terminated surface and an inert alkanethiol. The mole fraction of peptide epitopes was also controlled by reacting solutions containing FLAG peptide and the non-interacting peptide HA or cysteine. By studying variants based on the FLAG binding motif, it was possible to distinguish peptides differing by a single amino acid substitution using SPR imaging. In addition, quantitative analysis of the signal was accomplished using the peptide array to simultaneously determine the binding constants of the antibody-peptide interactions for four peptides. The binding constant, K(ads), for the FLAG peptide was measured and found to be 1.5 x 10(8) M(-1) while variants made by the substitution of alanine for residues based on the binding motif had binding constants of 2.8 x 10(7), 5.0 x 10(6), and 2.0 x 10(6) M(-1).

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“…Also, to maintain the important disulfide bridges, the sequence of the inhibitory peptide PMT-LEYR was modified in position 2 to PCTLEYR. Furthermore, to avoid problems with an adjacent positive charge, and since a substitution for methionine is possible without decreasing inhibition (2,8), the Arg-7 was changed to methionine, a conserved position in most squash inhibitors. This inhibitor with the N-terminal sequence now reading PCTLEYM (see Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Previously, we described a method for characterizing and optimizing peptide inhibitor/ protease interactions using cellulose-bound peptides (2). We demonstrated an optimization process for a 9-mer peptide originating from the third domain of the turkey ovomucoid inhibitor, OMTKY3.…”

mentioning

confidence: 99%

Design and Characterization of a Hybrid Miniprotein That Specifically Inhibits Porcine Pancreatic Elastase

Hilpert

Wessner

Schneider‐Mergener

et al. 2003

Journal of Biological Chemistry

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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 trypsinspecific 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.

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Characterizing and Optimizing Protease/Peptide Inhibitor Interactions, a New Application for Spot Synthesis

Cited by 30 publications

References 0 publications

Phage display screening for peptidic chitinase inhibitors

Phage display screening for peptidic chitinase inhibitors

Characterization and Optimization of Peptide Arrays for the Study of Epitope−Antibody Interactions Using Surface Plasmon Resonance Imaging

Design and Characterization of a Hybrid Miniprotein That Specifically Inhibits Porcine Pancreatic Elastase

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