Use of activity-based probes to develop high throughput screening assays that can be performed in complex cell extracts

• Published in: PloS one 2010-08, Vol.5 (8), p.e11985-e11985
• Main Authors: Deu, Edgar, Yang, Zhimou, Wang, Flora, Klemba, Michael, Bogyo, Matthew
• Format: Article
• Language: English
• Subjects: Aminopeptidases; Animals; Biochemistry; Biochemistry/Drug Discovery; Biochemistry/Small Molecule Chemistry; Biological properties; Biological samples; Biotechnology; Cathepsin C - antagonists & inhibitors; Cathepsin C - metabolism; Cathepsins; Cell Extracts; Dipeptidyl-peptidase I; Discovery tools; Drug Evaluation, Preclinical - methods; Enzyme inhibitors; Enzymes; Erythrocytes; Genetic engineering; High-throughput screening; High-Throughput Screening Assays - methods; Humans; Infectious Diseases/Tropical and Travel-Associated Diseases; Inhibitors; Investigations; Labeling; Liver; Liver - cytology; Lysates; Malaria; Medical screening; Medicine; Molecular Probes - metabolism; Parasites; Pathology; Plasmodium falciparum; Plasmodium falciparum - cytology; Probes; Protease inhibitors; Protease Inhibitors - pharmacology; Proteins; Rats; Rhodamine; Selectivity; Small Molecule Libraries - pharmacology; Studies; Substrate inhibition; Substrate Specificity; Substrates; Target recognition
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0011985

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.