8 January 2010. Tweaking the signaling pathway between cyclic AMP (cAMP) and proteins involved in memory has the potential to treat a variety of cognitive disorders—unfortunately, altering that pathway also causes vomiting and other intolerable side effects. In the December 27 Nature Biotechnology online, scientists from deCODE genetics in Reykjavik, Iceland, propose a workaround, describing several compounds that improve memory in animals without making them sick. The drugs partially inhibit the enzyme phosphodiesterase 4 (PDE4), which breaks down cAMP, instead of blocking it entirely like other inhibitors. If effective in people, the experimental drugs might be useful for a wide array of neurological disorders including Alzheimer’s, Huntington’s, schizophrenia, and depression.
PDE4 is part of a large family of phosphodiesterases, all involved in regulating cAMP and cGMP to modulate a variety of biological functions. In the case of memory, cAMP activates protein kinase A, which phosphorylates Creb, which activates transcription of genes involved in memory consolidation. Since PDE4 inhibitors prevent cAMP hydrolysis, they are likely candidates to improve memory. As an added bonus, cyclic AMP also promotes proteasome function, so drugs aimed at increasing cAMP levels might also help the brain battle pathogenic proteins, said Michael Shelanski of the Taub Institute at Columbia University in New York City, who was not involved with the current study.
Several PDE inhibitors exist, but they have a big drawback. In the brainstem, they induce pathways leading to emesis—nausea and vomiting. PDE inhibitors such as rolipram do improve memory in animal models, including AD mice (see ARF related news story on Gong et al., 2004), but also act as emetics, so many scientists shy away from trying them in people. “We were forced to seek something novel in terms of mechanism,” said Mark Gurney, who led the work with joint first authors Olafur Magnusson, also at deCODE, and Alex Burgin of Emerald BioStructures (formerly deCODE biostructures) on Bainbridge Island, Washington.
Burgin and colleagues approached the problem by first solving the structure for PDE4 together with various inhibitors. Their work suggests that PDE4 is a dimer with two catalytic domains plus segments that function together as a single regulatory domain. Traditional inhibitors compete with cAMP for the active sites, fully blocking enzyme activity. The researchers used the structural information to design small molecules that might inhibit the enzyme allosterically via the regulatory domain. They synthesized more than 800 candidates, and came up with 140 partial inhibitors. Several of these inhibited cAMP hydrolysis by 70 to 90 percent in an in vitro assay Magnusson developed.
The researchers selected four partial inhibitors to study in standard animal assays, comparing them to rolipram. To test the drugs’ effect on memory, they treated mice with scopolamine, which induces amnesia, plus one of the various compounds, and measured how much time the animals spent exploring different parts of a Y-maze. Mice with better memories tend not to repeatedly re-enter the same arm, since they know they were just there. They also used the novel object recognition test, exposing the animals to one item they’ve seen before and one that is new. If the mice remember the familiar item, they should spend less time examining it than the new object. In both assays, the partial PDE inhibitors improved memory just like rolipram.
Next, the scientists tested for emetic effects from their new compounds. Rodents don’t vomit, but PDE4 inhibitors have another side effect that the researchers could measure: they reduce the amount of time that an anesthetic combination of ketamine and xylanzine keeps the animals unconscious (Robichaud et al., 2002). Rolipram reduces anesthesia times, but the partial PDE4 inhibitors did not, even when used at 1,000 times the dose that improved cognition. The new inhibitors also did not cause emesis in shrews, dogs, or monkeys.
Gurney thinks that the new compounds work because they interact only weakly with the catalytic domains of PDE4, but strongly with the regulatory domain. Since there is only one regulatory domain for two active sites, it cannot block both at the same time. Therefore these drugs cannot fully prevent cAMP hydrolysis. “This mechanism of allosteric modulation puts a ceiling on the magnitude of inhibition of the enzyme,” Gurney said. “We think that because of this, we see greatly enhanced tolerability.”
Shelanski and his Taub Institute colleague Ottavio Arancio both said the new inhibitors deserved further study. Beyond neurological diseases, they suggested that if it has minimal side effects, it might even make a good “memory tonic,” Shelanski said, for elderly people who are dealing with the normal memory decline concomitant with aging.
Gurney hopes to initiate Phase 1 clinical trials, and said that if the drugs prove safe, people with early or mild Alzheimer’s would be prime candidates for a Phase 2 study.—Amber Dance.
Burgin AB, Magnusson OT, Singh J, Witte P, Staker BL, Bjornsson JM, Thorsteinsdottir M, Hrafsdottir S, Hagen T, Kiselyov AS, Stewart LJ, Gurney ME. Design of phosphodiesterase 4D (PDE4D) allosteric modulators for enhancing cognition with improved safety. Nat Biotechnol. 2009 Dec 27. Abstract