Single-Cell Biochemical Assays for the Molecular Targets of Disease

Abstract

Molecularly targeted therapies are at the forefront of clinical science, and are expected to lead to personalization of medical treatments for each patient. Most such therapies are directed at inhibiting specific signal transduction enzymes or pathways, thus creating a critical need for assays capable of measuring the activities of these proteins in disease models and in patient samples. The ability to measure relevant enzyme activity in primary cell samples at baseline and/or after treatment would provide the ability to tailor patient therapy based on aberrant signal transduction, validate mechanisms of resistance in patients, and would offer an invaluable pharmacodynamic tool to assess whether resistance is associated with inadequate target inhibition. Here we report our current efforts to create the analytical and chemical tools needed to directly measure the enzymatic activities of therapeutic targets including protein kinases, lipid modifying enzymes and the proteasome. Fluorescent reagents are under development that report the activity of these various enzymes in model cells lines and primary cells. The basic design incorporates enzyme substrates that are modified to create compounds which can be loaded into cells where they are modified by the enzyme of interest. Work has included modification of peptides to confer membrane permeability and to achieve long intracellular lifetimes. Microelectrophoretic separations combined with low-level fluorescence detection enable the quantitative analysis of these compounds from single mammalian cells. This capability addresses three major issues currently faced in the biochemical analysis of clinical samples: the need for direct measurement of the enzymatic activity of target proteins; sample size requirements that are feasible for clinical implementation; and sample heterogeneity that can mask pertinent aspects related to therapeutic response.