The first cell-based and label-free, high-throughput assay of specific enzyme activities

Project SummaryAbstract Cell based assays that investigate the effect of small molecules on cellular activities are becoming increasingly popular screening tools for drug discoveryThe ability to assess small molecules in a physiologically relevant environment is informativeas the assays simultaneously evaluate compound permeabilitytoxicityand activityYetcell based assays are often limited to detect global changes in cellular behaviorsuch as gene expression or phenotypeThe current challenge is to develop cell based assays for specific enzyme activitiesHoweverthe most common strategies for measuring enzyme activitiessuch as radioactivityabsorbanceand fluorescence labelsoften cannot be delivered to the appropriate target within the cellular environmentThis proposal describes a method that combines cell culture and lysis on customized surface chemistries that present cell adhesion ligands along with substrates for distinct enzyme activitiesThe cells are cultured and lysed directly on the monolayers in the presence of substrates that record the activity of enzymes within the lysateThe activity is measured using a high throughputmass spectrometry based readout termed SAMDIself assembled monolayers for matrix assisted laser desorption ionizationMALDImass spectrometryMSThe techniquetermed Tandem Culture and Lysis SAMDITCALSAMDIis amenable to any adherent cell line and can measure virtually any enzyme activityTCAL SAMDI will offer drug discovery companies in a broad range of diseases a platform for cell basedhigh throughputand label free screen of specific enzyme activities!Project Narrative This proposal addresses the current challenge to develop cell based high throughput assays capable of measuring specific enzyme activitiesThe project aims to commercialize the tandem culture and lysis with self assembled monolayer desorption ionizationTCAL SAMDIassaywhich utilizes self assembled monolayers of alkanethiolates on gold engineered to present a cellular adhesion ligand along with a substrate for a specific enzyme activityThe substrate and corresponding product is analyzed by SAMDIa quantitative label free mass spectrometry based readout for biochemical reactionsWe will apply the TCAL SAMDI assay to perform a high throughput screen ofcompounds against protein tyrosine phosphatase activity in two breast cancer cell linesThe goal is to identify novel candidates for an important therapeutic target and provide proof ofconcept for the TCAL SAMDI assay as the leading assay for measuring endogenous enzyme activities in a cellular context!