Use of Activity-Based Probes to Develop High Throughput Screening Assays That Can Be Performed in Complex Cell Extracts

Department of Pathology, Stanford School of Medicine, Stanford, California, United States of America.
PLoS ONE (Impact Factor: 3.23). 08/2010; 5(8):e11985. DOI: 10.1371/journal.pone.0011985
Source: PubMed


High throughput screening (HTS) is one of the primary tools used to identify novel enzyme inhibitors. However, its applicability is generally restricted to targets that can either be expressed recombinantly or purified in large quantities.
Here, we described a method to use activity-based probes (ABPs) to identify substrates that are sufficiently selective to allow HTS in complex biological samples. Because ABPs label their target enzymes through the formation of a permanent covalent bond, we can correlate labeling of target enzymes in a complex mixture with inhibition of turnover of a substrate in that same mixture. Thus, substrate specificity can be determined and substrates with sufficiently high selectivity for HTS can be identified. In this study, we demonstrate this method by using an ABP for dipeptidyl aminopeptidases to identify (Pro-Arg)2-Rhodamine as a specific substrate for DPAP1 in Plasmodium falciparum lysates and Cathepsin C in rat liver extracts. We then used this substrate to develop highly sensitive HTS assays (Z'>0.8) that are suitable for use in screening large collections of small molecules (i.e >300,000) for inhibitors of these proteases. Finally, we demonstrate that it is possible to use broad-spectrum ABPs to identify target-specific substrates.
We believe that this approach will have value for many enzymatic systems where access to large amounts of active enzyme is problematic.

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    • "Often, a recombinant protein of interest can be difficult to express or purify (especially for P. falciparum which has an A/T-rich genome and asparagine-rich inserts in proteins ). As a result, target-based screens have also been developed using crude cell preparations such as whole-cell lysates where the specific activity of a protein of interest can be resolved from other related activities present in the sample [17]. In the case of proteases activity, assays are often substrate based, with the readout being a change in either fluorescence or absorbance of a reaction mixture, for example, monitoring the release of a fluorescent group from a peptide substrate upon its recognition and cleavage by an enzyme target. "
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