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Detection of competitive enzyme inhibition with end point progress curve data
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Centre for Research Ethics and Bioethics. (Department Of Biochemistry and Organic Chemistry. Uppsala University)ORCID iD: 0000-0003-3522-0196
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
2006 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 358, no 1, 11-19 p.Article in journal (Refereed) Published
Abstract [en]

A model for a dimensionless factor, the inhibition detection limit (IDL), which describes the limit of detection of competitive inhibition for end point assays as a function of the proportion of substrate converted into product, has been developed. For a given end point enzymatic assay, the IDL function has a maximum that is dependent on the error structure parameters (four parameters) of the assay, the value of [S]o/K(ms), and the extent of product inhibition (K(ms)/K(mp)). Accordingly, the substrate conversion level that maximized the ability to detect samples with high Ki/[I] ratios was predicted for each member of a population of simulated assays. Furthermore, we identified a consensus substrate conversion level where the probability of a near-optimal robustness and detection limit for all the members of the assay population is maximal. Unlike the optimal substrate conversion level for individual assays, this consensus substrate conversion level was dependent only on [S]o/K(m), K(ms)/K(mp), and whether the signal increases or decreases during the course of the reaction. Consensus substrate conversion levels were beyond the initial velocity range for almost all the analyzed assay populations. It was shown that the IDL factor was a more informative indicator of assay quality than the popular Z' factor.

Place, publisher, year, edition, pages
2006. Vol. 358, no 1, 11-19 p.
Keyword [en]
Limit of detection, Enzyme inhibition, High-throughput screening, Primary screening, Robustness, Z′ factor, Enzymatic assays, Reaction progress curves, Error structure
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:uu:diva-94505DOI: 10.1016/j.ab.2006.07.008ISI: 000241428000002PubMedID: 16978578OAI: oai:DiVA.org:uu-94505DiVA: diva2:168375
Available from: 2006-05-08 Created: 2006-05-08 Last updated: 2015-03-01
In thesis
1. Sensitivity, Noise and Detection of Enzyme Inhibition in Progress Curves
Open this publication in new window or tab >>Sensitivity, Noise and Detection of Enzyme Inhibition in Progress Curves
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Starting with the development of an enzymatic assay, where an enzyme in solution hydrolysed a solid-phase bound peptide, a model for the kinetics of enzyme action was introduced. This model allowed the estimation of kinetic parameters and enzyme activity for a system that has the peculiarity of not being saturable with the substrate, but with the enzyme. In a derivation of the model, it was found that the sensitivity of the signal to variations in the enzyme concentration had a transient increase along the reaction progress with a maximum at high substrate conversion levels.

The same behaviour was derived for the sensitivity in classical homogeneous enzymatic assays and experimental evidence of this was obtained. The impact of the transient increase of the sensitivity on the error structure, and on the ability of homogeneous end-point enzymatic assays to detect competitive inhibition, came into focus. First, a non-monotonous shape in the standard deviation of progress curve data was found and it was attributed to the random dispersion in the enzyme concentration operating through the transient increase in the sensitivity. Second, a model for the detection limit of the quantity Ki/[I] (the IDL-factor) as a function of the substrate conversion level was developed for homogeneous end-point enzymatic assays.

It was found that the substrate conversion level where the IDL-factor reached an optimum was beyond the initial velocity range. Moreover, at this optimal point not only the ability to detect inhibitors but also the robustness of the assays was maximized. These results may prove to be relevant in drug discovery for optimising end point homogeneous enzymatic assays that are used to find inhibitors against a target enzyme in compound libraries, which are usually big (>10000) and crowded with irrelevant compounds.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 50 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 184
Biochemistry, Progress curves, limit of detection, Z'-factor, primary screening, enzyme inhibition, error structure, sensitivity analysis, High-throughput screning, Biokemi
urn:nbn:se:uu:diva-6886 (URN)91-554-6569-2 (ISBN)
Public defence
2006-05-29, Room B41, BMC, Husarg. 3, Uppsala, 10:00
Available from: 2006-05-08 Created: 2006-05-08 Last updated: 2012-04-13Bibliographically approved

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Gutierrez Arenas, OmarDanielson, U. Helena
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