This is Part 2 of a four-part series. See also Part 1, Part 3, Part 4. Read a PDF of the entire series.
7 January 2013. Held 7-10 December 2012 in Zuers, Austria, the 8th International Winter Conference on Alzheimer’s Disease was titled “AD Drug Therapy—Hope and Reality. New Targets in Sight?” True to form, then, new targets and compounds with which to hit them took up much space on the program. Here are some examples. Researchers are drilling down on targets found in the area of receptor neuropharmacology, such as the σ-1 receptor, as well as among the defining pathogenic proteins of neurodegenerative diseases, such as the synucleins.
First, σ-1 receptors. They are strangers to published AD drug development, though pharmaceutical industry research on compounds active in the CNS comes across them frequently. First identified in the 1970s, these receptors were studied pharmacologically until Austrian researchers cloned the gene (Hanner et al., 1996). In the years after, scientists learned that many endogenous peptides, for example, pregnanolone and neuropeptide Y, interact with these receptors, and subsequent knockout and other biological studies showed they act as chaperones associated with the endoplasmic reticulum (Hayashi and Su, 2007). Unusually for chaperones, a slew of endogenous ligands that act as agonists or antagonists operate the σ-1 receptor, Tangui Maurice at INSERM in Montpellier, France, told the audience in Zuers.
The σ-1 receptor’s role in neurodegenerative diseases is poorly understood. One mutation appears to cause juvenile ALS (Al-Saif et al., 2011), but beyond that, data on its genetic contribution remain sparse (see AlzGene listing).
The receptor is ubiquitous. Besides neurons, astrocytes, oligodendrocytes, and Schwann cells, the liver, spleen, heart, kidney, intestine, and other organs express it. It is a transmembrane protein that lives in the ER, particularly at touch points with mitochondria. There, it modulates calcium and influences the composition of lipid rafts. The receptor becomes active in response to ligand binding and in conditions of ER stress. It protects mitochondria by influencing production of radical oxygen species and expression of the anti-apoptotic gene BCL2. It also sits in the plasma membrane, where it interacts with receptors ranging from TrkB, the muscarinic acetylcholine receptor, to sodium and potassium channels. “The σ-1 receptor is an important activity-dependent signaling modulator of multiple intracellular pathways in the cell,” Maurice told the audience in Zuers.
How can the σ-1 receptor influence myriad functions in cells? It does so by way of cooperating with other receptors. At least in some cases, it forms heteromers with them, said Abraham Fisher, Israel Institute for Biological Research, Ness-Ziona (e.g., see Navarro et al., 2010). This constellation is how the σ-1 receptor may work as a target for certain drugs. For example, haloperidol treats schizophrenia by acting on the dopamine D2 and σ-1 receptors; fluoxetine treats depression through a combined effect on the serotonin and σ-1 receptors, and donepezil treats AD through an effect on a cholinesterase and the σ-1 receptor.
In Zuers, both Fisher and Maurice presented their respective efforts at finding small molecules that tickle this receptor in a way that might treat AD better than current drugs do. Fisher introduced AF710B, a bicyclic heterocyclic spiro-compound that he said selectively activates both the muscarinic M1 receptor and the σ-1 receptor. In detailing its effects on a list of phenotypic parameters—it increases sAPPα secretion, decreases tau hyperphosphorylation and GSK-3β activity, decreases Bax, and increases BCL2 expression in mitochondria—Fisher emphasized that the former two effects come through the M1 receptor and the latter two through the σ-1 receptor. Unlike previous compounds Fisher developed, which were mainly M1-selective orthosteric agonists, AF710B is an allosteric M1 receptor agonist. Its heteromer-specific effects differentiate it from other M1 agonists and modulators. Fisher claimed that the new compound is exquisitely potent, acting as a cognitive enhancer in rats at 1 to 30 micrograms (not milligrams) per kilogram body weight. According to Fisher, the compound is orally available with a safety margin of more than 50,000 times the minimally active dose. “Those are orders of magnitude more potent than donepezil and other agonists acting either on the M1 or the σ-1 receptors, respectively,” Fisher said.
Fisher proposed that the compound has a unique mechanism of action, whereby it sensitizes the M1 receptor through heterodimerization with the σ-1 receptor in the membrane of the ER, adding, however, that heteromerization of these receptors has not been formally proven. “We are looking to license it for drug development,” Fisher said.
For his part, Maurice presented data on two joint M1/σ-1 agonists called Anavex1-41 and Anavex2-73, developed by the biotech company Amylgen. Maurice tested these compounds by injecting them into the brain of a mouse model that develops an Alzheimer's-like phenotype after brain injection of synthetic Aβ oligomers (e.g., Villard et al., 2009). In this model, the compounds both treated and prevented the model’s learning and memory deficits, hippocampal cell death, astrogliosis, and tau hyperphosphorylation via GSK-3β, Maurice told the audience. Chronic oral treatment of Tg2576 mice at 3 milligrams per kilogram per day is ongoing, but preliminary data suggest that compound 2-73 reduces both Aβ load and memory deficits. “Compounds that act on both the M1 and σ-1 receptors could have therapeutic potential,” Maurice said.
Several scientists quizzed about this approach praised the science, but they also cautioned that the σ-1 receptor is generally considered a challenging drug target because it is so ubiquitously expressed not just in the body, but also in several organelles within cells. Its modulation by a plethora of ligands is tied to a range of diseases on both sides of the agonist-antagonist divide. Therefore, these scientists said, drugs against it might be prone to side effects, and safety for chronic treatment in the elderly would have to be demonstrated with special care. Emphasizing the large therapeutic safety window his compound had shown thus far, Fisher countered that future toxicology studies would test the safety of targeting the modulatory action of these two receptors.
Another alternative treatment approach featured in this session focused on synucleins. α-synuclein is such a fixture in dementing and movement disorders that a drug against it might pay off broadly. “If we target α-synuclein, it might be useful not just for Parkinson’s, but also for dementia with Lewy bodies, Parkinson’s disease dementia, multiple systems atrophy, perhaps even some cases of AD,” said Eliezer Masliah of the University of San California, San Diego. Its cousin β-synuclein is a poorly understood homologue expressed in the brain that is thought to counteract α-synuclein and even Aβ42 fibril formation, much in the way Aβ40 appears to lessen Aβ42 aggregation in some circumstances.
Manfred Windisch of the CRO QPS, formerly JSW, in Graz, Austria, cued up a pair of talks on peptidomimetic drug discovery for synucleins. First, he reviewed a published study testing three active β-synuclein peptides in transgenic mice expressing the Swedish and London APP mutations, and in mice transgenic for mutant α-synuclein (Windisch et al., 2004). Both intraperitoneal and intranasal delivery of these peptides reduced pathology while improving spatial learning and memory; however, the results were so variable that they would have required large studies going forward. Moreover, the peptides were degraded within an hour. “This was not good enough,” Windisch said.
Next, the Austrian researchers created D-enantiomeric peptides, which stay intact in the body much longer than L-enantiomers, and tested these compounds in preventive and treatment studies in transgenic mice. The new peptides seemed to work like the original ones. They reduced Aβ load measured in different fractions and improved performance on memory tests. Apparently, the D-stereoisomer works both via aggregation and an anti-apoptotic pathway, Windisch said. He hopes its structure and activity can be recapitulated in a small-molecule compound; otherwise, the peptide itself might become a candidate for drug development. “I do believe there is something there, because I have seen very many amyloid-reducing drugs that do not have the pronounced behavioral improvements we see with this peptidomimetic,” Windisch said. Until recently, Windisch headed JSW, an Austrian contract research organization that measures anti-amyloid and behavioral effects of hundreds of putative therapeutics on behalf of academic, biotech, and pharma labs around the globe. In August 2012, he sold JSW to QPS, a larger CRO headquartered in Newark, Delaware.
A separate effort to move from peptides to small molecules is underway for α-synuclein. Masliah presented work done by a biotech partnership set up between the University of California, San Diego, and EVER Neuro Pharma, the Austrian purveyor of cerebrolysin, nutritional supplements for “mental agility,” and apomorphin, a dopamine agonist injection for advanced Parkinson’s. Called Neuropore Therapies, Inc., the new company tries to turn insight gleaned from peptides that block α-synuclein aggregation into small molecules that can prevent dimerization and subsequent formation of oligomers. The idea is to stabilize monomeric α-synuclein into a functional molecule that does not enter a toxic aggregation pathway, Masliah said. In Zuers, he described how scientists in his research group had found a motif in the C-terminus of α-synuclein that seemed suitable for peptide docking. They tested some peptides for aggregation blocking and then turned them into a peptidomimetic to create analogues. Many assays later, the scientists came out with an isoindole pyrimido pyrazine compound called NPT100-18A.
This molecule blocks the formation of dimers and higher species in cell-free blood, and performed to expectations on calcium flux and neurite formation readouts in neuroblastoma and primary cell culture, Masliah said. In α-synuclein transgenic mice, the compound improved motor and memory performance and other readouts in young, but not old, mice. Masliah believes the compound works by blocking the dimerization of α-synuclein somewhere near glutamine 38 of the peptide. Mutating that site blocks the ability of the compound to keep α-synuclein from aggregating. Because the pharmacokinetics of this compound are not right just yet, the scientists are currently working on finding a series of derivatives that would more readily get into the brain and reach an effective concentration there when given by mouth.—Gabrielle Strobel.
This is Part 2 of a four-part series. See also Part 1, Part 3, Part 4. Read a PDF of the entire series.