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RESEARCH PROFILES
An improved assay for adenylyl cyclase NH2 N N O O O HO P O P O P O O– O– O– 3 Na +
Adenylyl cyclase
N
NH2
N
N N
O 3´
2´
H O
N H
O
CH3
O
O
H N
N N B F F O
O CH3
O P O– O
CH3
N N
O O
N H
H N
N N B F F O
CH3
AC catalyzes the conversion of BATP (left) to BcAMP (right).
enzyme with BODIPY ATP (BATP). CE-laser induced fluorescence (LIF) was then used to separate potential products from the reactants. In the presence of AC, a new fluorescence peak with higher electrophoretic mobility than that of BATP was detected. However, this peak was not observed when denatured AC was used. This result suggests that AC is capable of converting fluorescently labeled BATP into BODIPY cAMP (BcAMP). Further evidence was provided by the analysis of substrate peaks. Commercially available BATP has two isomers, because BODIPY can be attached to either the 2′ or 3′ O-ribosyl position of ATP. These BATP isomers appear as two closely spaced peaks on the CE-LIF electropherogram. However, only 2′-BATP can be converted to BcAMP, because a 3′oxyanion is needed for nucleophilic attack. And indeed, the researchers found that the increase in the BcAMP peak mirrored the decrease in the fluorescence peak of only one of the substrate isomers, presumably 2′-BATP. Once Kennedy and colleagues had determined that AC efficiently catalyzed the conversion of BATP to BcAMP, and that they could detect the product by CE-LIF, they examined the effects of AC inhibitors and activators on product formation. Two known AC activators, forskolin and GS-GTPS, each activated the enzyme. Nearly identical dose–response curves were obtained for the BATP substrate and the normal, nonfluorescent ATP substrate. Similarly, the AC
inhibitor MANT-GTP inhibited AC’s conversion of BATP with the same concentration dependency as that for ATP. According to Kennedy, “These results suggested to us that this fluorescent substrate [BATP] is able to interact with the enzyme in a way that is very similar to ATP, and therefore our assay could be used to screen for drugs that would inhibit or activate AC.” Kennedy emphasizes that, unlike earlier AC assays, the new method enables the high-throughput screening of combinatorial drug libraries to identify new AC activators or inhibitors. CE instruments developed for gene sequencing have the capacity to simultaneously analyze up to 384 samples for AC activity, and the process can be easily automated. Furthermore, nine isoforms of AC have been identified, raising the possibility of identifying isoform-specific activators or inhibitors to treat particular diseases. “I think the method’s best applicability is for doing a drug screen,” says Kennedy. “You don’t have to use radioactivity, and it’s just a matter of minutes to do a given assay. Not only that, but it’s very easy to scale up to a parallel system using CE array instruments.” Given the relative ease of the new method and the physiological importance of AC, Kennedy anticipates that pharmaceutical companies will be very interested in adapting the assay to screen for novel therapeutic drugs that target the enzyme. a —Laura Tomky Cassiday
A P R I L 1 , 2 0 0 6 / A N A LY T I C A L C H E M I S T R Y
2087
ROBERT KENNEDY
Aberrant activity of the enzyme adenylyl cyclase (AC) has been linked to several diseases, including addiction, heart disease, and various neurological disorders. For this reason, the enzyme is an important therapeutic target. However, common assays for AC activity are laborious, requiring a radioactive substrate and ion-exchange chromatography to isolate the radioactive product. In the March 15 issue of Analytical Chemistry (pp 1731–1738), Jennifer Cunliffe, Roger Sunahara, and Robert Kennedy of the University of Michigan report a highthroughput, nonradioactive method for assaying AC activity. AC plays a vital role in cellular signal transduction pathways. The enzyme is embedded in the plasma membrane and catalyzes the conversion of adenosine triphosphate (ATP) into cyclic adenosine monophosphate (cAMP) in response to extracellular signals. cAMP, an important “second messenger” molecule, then relays the signal to other proteins within the signaling pathway inside the cell. The new AC assay developed by Kennedy and colleagues uses a fluorescent ATP substrate in combination with CE to monitor the production of fluorescent cAMP. Researchers were initially concerned that fluorescently labeled ATP might not interact with the enzyme. Kennedy says, “ATP is a pretty small molecule, and here we’re putting a fluorophore on it. The question we had is, will fluorescently labeled ATP serve as a good substrate to test for AC enzyme activity?” This was not a trivial issue, because another fluorescently labeled ATP molecule, MANT-ATP, is known to inhibit AC. Kennedy and colleagues chose to use the fluorophore BODIPY, which was attached to ATP via a long linker to allow close association of the ATP with the enzyme’s active site. To conduct the assay, the investigators mixed the two catalytically active cytosolic domains of the purified AC
