Fluorescence-based adenylyl cyclase assay adaptable to high throughput screening.
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 high-throughput screening could help in the development of novel therapeutics. Here we report a novel fluorescence-based cyclase assay using Bodipy FL GTPgammaS (BGTPgammaS). The fluorescence of the Bodipy moiety of BGTPgammaS 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 ATPgammaS (BATPgammaS) as substrate for either wt-AC or mut-AC. Using BGTPgammaS, forskolin, Gsalpha.GTPgammaS and the divalent cation Mn(2+) 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 2'deoxy,3'AMP and 2',5'-dideoxy-3'ATP. Furthermore, the fluorescence assay is adaptable to 96-well and 384-well multiplate format and is thus applicable to high throughput screening methodologies.
Vadakkadathmeethal
Kannan
K
Department of Pharmacology, University of Michigan, Ann Arbor, MI 48109-0632, USA.
Cunliffe
Jennifer M
JM
Swift
Jennifer
J
Kennedy
Robert T
RT
Neubig
Richard R
RR
Sunahara
Roger K
RK
D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000032:analysis*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005453:Fluorescence; D013329:Structure-Activity Relationship; D013997:Time Factors
D000067956:Adenylyl Cyclase Inhibitors; D004791:Enzyme Inhibitors; D000262:Adenylyl Cyclases