Elastase Inhibition Assay with Peptide Substrates – An Example for the Limited Comparability of in vitro Results

Abstract

Many factors such as buffer, pH, or enzyme concentration influence the inhibitory activity of test compounds in in vitro enzyme inhibition assays. The purpose of this study was to evaluate whether the variation of the synthetic substrate has an influence on the IC₅₀ values as well. As an exemplary enzyme, we have chosen polymorphonuclear neutrophil elastase (PMNE), which is considered as a promising target in the therapy for inflammatory and tumour diseases. A well established, validated in vitro PMNE inhibition assay was performed with three different synthetic peptide substrates (S-2484, L-1335, I-1270). As inhibitors ursolic acid, unfractionated heparin (UFH), the semisynthetic glucan sulfate PS3, and sulfated polysaccharides from the red algae Delesseria sanguinea (D.s.-SP), were used. The maximum achievable PMNE inhibition by the test compounds was shown to be determined by the substrate: 60 to 70 % (L-1335 and S-2484) or 90 % (I-1270). Furthermore, the IC₅₀ values of the inhibitors turned out to strongly vary in dependence on the substrate, although the used peptide substrates are structurally very similar. The derived activities differed by up to 480 %. In addition, the potencies of the test compounds in relation to each other altered considerably. In conclusion, by the choice of the enzyme substrate, both the apparent maximum activity and the IC₅₀ of an inhibitor can be influenced. Therefore, only results from identical test systems should be compared.

Abbreviations

D.s.-SP:sulfated polysaccharides from the red alga Delesseria sanguinea

I-1270:methoxysuccinyl-alanine-alanine-proline-valine-7-amido-4-metyhlcoumarin

L-1335:methoxysuccinyl-alanine-alanine-proline-valine-p-nitroanilide

PMN:polymorphonuclear neutrophil

PMNE:polymorphonuclear neutrophil elastase

S-2484:L-pyroglutamyl-proline-valine-p-nitroanilide

UFH:unfractionated heparin

elastase - PMNE - synthetic substrate - chromogenic assay - enzyme inhibition assay - sulfated polysaccarides - ursolic acid

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