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.
Highlights
One of the most common techniques used by the pharmaceutical industry to identify novel drug leads is high throughput screening (HTS)
We demonstrate the use of activity-based probes (ABPs) to assess the selectivity of reporter substrates in crude cell extracts. We demonstrate that this approach facilitates the identification of substrates whose kinetics of turnover inhibition perfectly correlate with the kinetics of labeling of the target enzyme by the ABP
We use the probe in crude cell extracts from the human malaria parasite Plasmodium falciparum and in crude rat liver extracts to identify a highly selective substrate for dipeptidyl aminopeptidase 1 (DPAP1) and cathepsin C (Cat C). We demonstrate that this substrate can be used to develop a highly sensitive and stable assay that is suitable for use in High throughput screening (HTS) with large libraries of small molecules
Summary
One of the most common techniques used by the pharmaceutical industry to identify novel drug leads is high throughput screening (HTS). This method allows inhibition effects of large numbers of compounds to be determined in a relative short period of time. HTS has been performed using both cell-based and extract-based assays [2,3] While these types of assays avoid the need to express and purify a target enzyme, they often rely on genetically engineered reporter systems that tend to have a high rate of false positives. Its applicability is generally restricted to targets that can either be expressed recombinantly or purified in large quantities
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