Development of capillary electrophoresis techniques for detecting signal transduction events: Applications to drug screening.

Abstract

Many of the signaling entities in the G protein signal transduction cascade have potential as therapeutic targets, therefore development of novel and sensitive assays is of interest. Capillary electrophoresis (CE) is one approach, where an electric field separates analytes based on their charge-to-size ratios. This work describes the development of CE assays for the detection of adenylyl cyclase (AC) activity and G protein binding. A CE-UV assay was developed for detecting AC activity by monitoring the ATP to cAMP turnover. In this assay, AC activators and inhibitors modulated AC activity resulting in increased and decreased cAMP formation, respectively. The assay was further tailored for detection of G protein coupled receptor (GPCR) agonist-stimulated AC activity using membranes co-expressing the beta₂ adrenergic receptor (beta₂AR) and AC. beta₂AR agonists increased AC activity while drugs for other GPCRs did not. To improve the sensitivity of the assay, BODIPY FL ATP (BATP) was investigated as an AC substrate using purified AC. AC activity was quantified by monitoring BODIPY FL cAMP (BcAMP) product formation, with AC activators and inhibitors modulating BcAMP formation in a dose-dependent manner. BATP was then investigated as a substrate for membrane-bound AC with the goal of developing a CE-LIF drug screening assay. GPCR drugs were screened and AC activity quantified using membranes co-expressing beta₂beta₂AR and AC. beta₂AR agonists and an inverse agonist increased and decreased AC activity, respectively, while drugs for other GPCRs had no effect. The CE-LIF assay had a 1000-fold lower limit of detection than the CE-UV assay, and has potential for use in drug screening. CE assays were developed for the detection of G proteins using a fluorescent non-hydrolyzable GTP analogue, BGTPgammaS. Detection of G protein-BGTPgammaS complex was facilitated using high electric field (1000 V/cm) and low separation temperature (15 °C), thereby reducing the time the complex was under non-equilibrium conditions. The focusing step of capillary isoelectric focusing (cIEF) was then examined as an alternative separation mechanism for the detection of G protein-BGTPgammaS complexes. Two G protein-BGTPgammaS complexes were resolved using cIEF, with a 30 pM limit of detection for one of the complexes.