Elastase inhibition assay – the choice of the synthetic substrate determines the result

It is well known that many factors like buffer concentration, pH, or enzyme concentration influence the inhibitory activity of test compounds in in vitro enzyme inhibition assays. The aim of the present study was to investigate whether different synthetic substrates have an influence on the inhibitory data as well.

As an example, we have chosen a well established validated in vitro PMNE (polymorphonuclear neutrophil elastase)-inhibition assay. The basic assay was modified by using the following synthetic peptide-substrates: 1. S-2484 (Chromogenix), 2. MOSAAPVPNA (Bachem), 3. I-1270 (Bachem). The concentration-dependent elastase inhibition by unfractionated heparin (UFH), PS3 (a semisynthetic glucan sulfate, US7008931-B2), and a sulfated polysaccharide from the red alga Delesseria sanguinea (D.s.-SP) was determined.

	IC50[µg/ml]/relative IC50 values
	S-2484	MOS	I-1270
PS3	0.19 1.0	0.21 1.0	0.16 1.0
UFH	0.59 3.1	1.21 5.9	0.28 1.7
D.s.-SP	0.41 2.2	0.40 1.9	0.18 1.1

The inhibitory potency of each inhibitor showed to be clearly dependent on the used substrate: this concerns both its maximal inhibitory activity, the gradient of its concentration-dependent inhibition, and its IC50-value. In addition, even the ratios of the IC50-values of any inhibitors alter by change of the substrate (see table). Only the order of the inhibitors concerning their activity is independent of the substrate and always keeps constant.

Thus also activities of inhibitors given in relation to a reference compound may vary in dependence on the respective assay procedure. As a consequence, even the use of reference substances allows only a rough estimation of an inhibitor's potency.

In conclusion, in vitro enzyme assays with synthetic substrates are useful tools in pharmacological research, but one should always be aware of their manifold limitations. IC50 values are strongly dependent on the respective test system and even relative declarations are not essentially reproducible in a modified test system.