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Putting Thought to Paper: A µARCS Protease Screen

duke.groebe{at}abbott.com

Mary L. Maus

Biological Screening, Abbott Laboratories, Abbott Park, IL

Terry Pederson

Metabolic Diseases Research, Abbott Laboratories, Abbott Park, IL

Jill Clampit

Protein Purification, Abbott Laboratories, Abbott Park, IL

Stevan Djuric

Metabolic Diseases Research, Abbott Laboratories, Abbott Park, IL

James Trevillyan

Protein Purification, Abbott Laboratories, Abbott Park, IL

Chun W. Lin

Metabolic Diseases Research, Abbott Laboratories, Abbott Park, IL

David J. Burns

Usha Warrior

Biological Screening, Abbott Laboratories, Abbott Park, IL

In micro-arrayed compound screening (µARCS), an agarose gel is used as a reaction vessel that maintains humidity and compound location as well as being a handling system for reagent addition. Two or more agarose gels may be used to bring test compounds, targets, and reagents together, relying on the pore size of the gel matrix to regulate diffusion of reactants. It is in the microenvironment of the agarose matrix that all the components of an enzymatic reaction interact and result in inhibitable catalytic activity. In an effort to increase the throughput of µARCS-based screens, reduce the effort involved in manipulating agarose gels, and reduce costs, blotter paper was used rather than a second agarose gel to introduce a substrate to a gel containing a target enzyme. In this assay, the matrix of the blotter paper did not prevent the substrate from diffusing into the enzyme gel. The compound density of the µARCS format, the ease of manipulating sheets of paper for reagent addition, and a scheduled protocol for running multiple gels allowed for a throughput capacity of more than 200,000 tests per hour. A protease assay was developed and run in the µARCS format at a rate of 200,000 tests per hour using blotter paper to introduce the substrate. Picks in the primary screen were retested in the µARCS format at a density of 384 compounds per sheet. IC₅₀ values were confirmed in a 96-well plate format. The screen identified several small molecule inhibitors of the enzyme. The details of the screening format and the analysis of the hits from the screen are presented.

Key Words: µARCS • HTS • high-throughput screening

Journal of Biomolecular Screening, Vol. 8, No. 6, 668-675 (2003)
DOI: 10.1177/1087057103258587


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