Fluorescence-Based Adenylyl Cyclase Assay Adaptable to High Throughput Screening

Kannan      Vadakkadathmeethal; Jennifer   M.   Cunliffe; Jennifer      Swift; Robert   T.   Kennedy; Richard   R.   Neubig; Roger   K.   Sunahara

Abstract

The second messenger cAMP has been implicated in numerous cellular processes such as glycogen metabolism, muscle contraction, learning and memory, and differentiation and development. Genetic evidence suggests that the enzyme that produces cAMP, adenylyl cyclase (AC), may be involved in pathogenesis in many of these cellular processes. In addition, these data suggest that membrane-bound ACs may be valuable targets for therapeutics to treat pathogenesis of these processes. The development of a robust real-time adenylyl cyclase assay that can be scalable to highthroughput screening could help in the development of novel therapeutics. Here we report a novel fluorescence-based cyclase assay using Bodipy FL GTPγS (BGTPγS). The fluorescence of the Bodipy™ moiety of BGTPγS was dramatically enhanced by incubation with the minimal catalytic core of wild-type-AC (wt-AC) and a mutant with decreased purine selectivity (mut-AC), in an AC activation-dependent manner. No increase in fluorescence was observed using Bodipy FL ATPγS (BATPγS) as substrate for either wt-AC or mut-AC. Using BGTPγS, forskolin, Gsα.GTPγS and the divalent cation Mn2+ potently enhanced the rate of fluorescence increase in a concentration-dependent manner. The fluorescence enhancement of the Bodipy moiety was inhibited by known inhibitors of AC such as 2deoxy,3AMP and 2,5-dideoxy- 3ATP. Furthermore, the fluorescence assay is adaptable to 96-well and 384-well multiplate format and is thus applicable to high throughput screening methodologies.

Keywords: Adenylyl cyclase, fluorescence, high-throughput screening, Bodipy-FL-GTPγS

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