ArticlePloS one2010
Use of activity-based probes to develop high throughput screening assays that can be performed in complex cell extracts.
Article in PloS one, 2010. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 11 papers.
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Who cites it
11 citing papers in PubMed, 17 citations in OpenAlex.
- Characterization of P. falciparum dipeptidyl aminopeptidase 3 specificity identifies differences in amino acid preferences between peptide-based substrates and covalent inhibitors.The FEBS journal · 2019Article
- Identification of Plasmodium dipeptidyl aminopeptidase allosteric inhibitors by high throughput screening.PloS one · 2019Article
- Discovery of antimicrobial compounds targeting bacterial type FAD synthetases.Journal of enzyme inhibition and medicinal chemistry · 2018Article
- Article
- Ferrous iron-dependent drug delivery enables controlled and selective release of therapeutic agents in vivo.Proceedings of the National Academy of Sciences of the United States of America · 2013Article
- Target deconvolution techniques in modern phenotypic profiling.Current opinion in chemical biology · 2013Review
- New approaches for dissecting protease functions to improve probe development and drug discovery.Nature structural & molecular biology · 2012Review
- Proteases as regulators of pathogenesis: examples from the Apicomplexa.Biochimica et biophysica acta · 2012Review
- Development of SNAP-tag fluorogenic probes for wash-free fluorescence imaging.Chembiochem : a European journal of chemical biology · 2011Article
- Functional studies of Plasmodium falciparum dipeptidyl aminopeptidase I using small molecule inhibitors and active site probes.Chemistry & biology · 2010Article
- Chemical biology approaches for the study of apicomplexan parasites.Molecular and biochemical parasitologyReview
Corrections and comments
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Authors and funding
5 authors at 2 institutions in 1 country.
Funding
Abstract
backgroundHigh 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. METHODOLOGY AND PRINCIPAL
findingsHere, 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.
conclusionsWe 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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Registered trials
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