BioCentury Innovations: Why Neuro-Bio thinks it’s time to revisit the role of AChE in AD

For the full article, please click here. Neuro-Bio Ltd. has identified a peptide derived from AChE — the enzyme responsible for breaking down acetylcholine — and believes the fragment is the main pathological driver in Alzheimer’s disease, acting upstream of β-amyloid and tau in the brain centers hit earliest in the disease. The company, which was formed in 2013, is developing inhibitors of the peptide to create a disease-modifying therapy, and is seeking investors to help fund preclinical studies. Its approach departs from the strategy behind the standard of care in AD, in which AChE inhibitors are used to enhance levels of ACh in the synaptic cleft, boosting neurotransmission to compensate for the loss of cholinergic neurons that commonly occurs in the disease. CEO Susan Greenfield told BioCentury that the marketed drugs provide only modest and transient symptomatic relief, and because their goal is to eke out more transmission from the remaining cells, they don’t address the root cause of the disease. Indeed, most companies have moved on to targeting molecules thought to drive the pathology – in particular β-amyloid and tau, each of which forms toxic protein aggregates in AD brains. But Greenfield said Neuro-Bio has a fresh take on AChE that places a cleavage product of the enzyme at the center of a mechanism that drives the earliest events, including deposition of β-amyloid and tau (see “Triggering AD,” page 3). “My view is that amyloid and tau are secondary in the cascade,” said Greenfield, adding that because both markers form aggregates in many brain centers, neither can explain why degeneration begins in specific, localized regions. “One of the problems with the amyloid theory is that it is potentially ubiquitous in the brain, yet we know only certain cells are lost,” she said. “We’ve pinpointed a much more specific mechanism in brainstem and midbrain neurons that leads to the generation of amyloid and tau downstream.” At least 20 studies have shown that AD pathology arises in a collection of deep brain centers, including the areas that produce ACh, serotonin, dopamine and norepinephrine. Greenfield said two features unite the cells: they all express AChE, even the non-cholinergic cells, and they have a common developmental origin. “These cells have a different embryological provenance from the rest of the cells in the brain: they come from the basal plate, not the alar plate. Our suggestion is that gives them different properties that make them more vulnerable” to a variety of insults. One such property, she said, is that the cells retain a higher sensitivity to trophic factors in adulthood than those in higher brain centers, which makes them more likely to switch on developmental pathways as a means of repairing damage. Neuro-Bio’s strategy rests on targeting a form of AChE that is normally only expressed in appreciable quantities during development, but gets up-regulated in the brains of AD patients. Whereas the adult form of the enzyme exists mainly as tetrameric complexes, during development it is found primarily as a monomer. “If you look at Alzheimer’s brains and compare them with the developing brain, the pattern of AChE is similar, and very different in both cases from normal adult humans,” said Greenfield. Although the monomeric form has a positive effect in development, strengthening immature synapses and promoting growth of axons and dendrites, it can kick off a toxic positive-feedback loop in mature neurons, she said. “We see neurodegeneration as an inappropriate form of development.” AGE-INAPPROPRIATE ACTIVITY The company was founded by Greenfield, who is also a senior research fellow at University of Oxford’s Lincoln College, where her work focuses on mechanisms driving AD and Parkinson’s disease. The lab identified a 14-amino acid peptide cleaved from the C-terminus of AChE monomers, and showed it could be trophic or toxic to cultured cells, depending on the dose. Although the group hasn’t described how the peptide is cleaved, Greenfield believes that it shifts the balance towards the monomeric form of AChE, which adds to the pathology. “The reason the enzyme goes from a single catalytic unit to four is that it is able to oligomerize by virtue of disulfide bonds,” she said. “But if our peptide has been cleaved, then that can no longer occur, and it reverts to the monomeric form.” While increasing trophic support in adulthood “might seem like a good thing,” said Greenfield, whether it is harmful or helpful depends on the levels of the peptide, especially considering mature cells are more vulnerable to rises in intracellular calcium. “If the calcium signal is either too excessive or goes on for too long then that can turn toxic to mitochondria, compromising oxidative phosphorylation,” said Greenfield. She thinks the peptide contributes to the non-enzymatic effects of AChE monomers, including providing trophic support to developing neurites and synapses by boosting entry of calcium to neurons. Previously, the team published a study in the Journal of Neurochemistry demonstrating the peptide is a positive allosteric modulator (PAM) of the nicotinic ACh receptor CHRNA7, and causes more calcium to flow through the receptor channel after it binds ligand. PAYING IT FORWARD This year, the company reported in Neuropharmacology that the peptide exists independently of AChE in the human brain, and its levels were twice as high in midbrain tissue samples from postmortem AD patient brains as in samples from healthy controls. The peptide levels were also 24% higher in the CSF of patients, which has prompted the company to pursue it as a biomarker of the disease, Greenfield said. In PC12 cells, which resemble neurons by releasing dopamine and norepinephrine but are derived from the adrenal medulla, the peptide potentiated ACh-evoked calcium influxes. In addition, it produced AD-like phenotypes, including increased phosphorylation of GSK3, which leads to phosphorylation of tau and secretion of β-amyloid, and decreased cell viability. Moreover, increased calcium flux led to a rise in the amount of AChE released from the cells, which Greenfield said could result in a feed-forward mechanism for generating more peptide in the AD brain. The company synthesized a cyclized version of the AChE-derived peptide — dubbed NBP14 — that was more stable than the linear peptide and also bound the nicotinic receptor. NBP14 had no effect on the cells when administered alone, but was able to almost completely block the effects of the linear peptide. In addition, NBP14 suppressed the effect of β-amyloid on the cells, restoring calcium influxes and cell viability to control levels, which suggested that in patients the compound could decrease the secretion of β-amyloid triggered by the AChE-derived peptide and the direct effects of β-amyloid on cells. β-amyloid shares some sequence similarity with the AChE-derived peptide, and has likewise been shown to bind the nicotinic receptor and potentiate calcium influx and AChE release. Greenfield said Neuro-Bio has multiple patents on the program, which cover the target and the use of peptide variants for treating neurodegenerative disorders, and is seeking a development partner. The next step, she said, is to do high throughput screening for “smaller compounds, peptides and peptidomimetics that can mimic the function of NBP14” and have improved properties for in vivo use. The company also plans to develop a CSF- or blood-based biomarker of AD that measures levels of the AChE-derived peptide. “That one I’d like to retain in-house,” said Greenfield. However, Howard Fillit, founding executive director and CSO of the Alzheimer’s Drug Discovery Foundation, told BioCentury he thinks the program has “a long way to go” before it’s ready to partner. While he said the company has “an interesting and novel proposal for the role of AChE in the disease process,” he noted that all of its mechanistic data are in in vitro systems. “I’d like to see some proof of mechanism in vivo.” It’s also unclear whether NBP14, or another peptide inhibitor, would be orally bioavailable or get into the brain, he said. “They really need to find a small molecule where there’s a very clear binding and mechanism of action.” UP OR DOWN? The company’s data suggest overactivation of CHRNA7 is toxic, driving early pathological events deep in the brain. But Fillit thinks that hypothesis is hard to reconcile with current thinking about AD. He said dialing down the activity after the disease is underway could exacerbate the cognitive symptoms of AD, some of which are almost certainly caused by loss of cholinergic transmission. For that reason, he said it’s difficult to neatly fit the work into the context of the broader literature on the role of ACh in AD, which, on balance, suggests that turning up cholinergic activity is the way to go. “The tonic effects of the cholinergic transmission on multiple brain systems are very important for many neurobiological mechanisms, yet there is a clear decline in the cholinergic system with aging.” In contrast to Neuro-Bio’s hypothesis that too much cholinergic activity is harmful, he thinks the system should be driven even harder. “There is some additional evidence and interest now in the possibility that ramping up the cholinergic system could have additional benefits” beyond sparing cholinergic transmission, including slowing the rate of hippocampal atrophy, he said. He said several companies are testing CHRNA7 agonists in AD. “The interest in this receptor has almost entirely been in terms of agonism. It certainly would be true that blocking the receptor wouldn’t be good,” said Fillit. However, he noted if the company is right, its results might provide some insight into why clinical-stage CHRNA7 agonists haven’t fared well in trials. In addition, he thought the fact that Neuro-Bio’s strategy rests on negative allosteric modulation could, as opposed to direct agonist or antagonist, to some degree “open up the prospect of a novel therapeutic approach.” At least four companies have either agonists or PAMs of CHRNA7 in preclinical or clinical development for AD. Like AChE inhibitors, agonists of the receptor aim to counter the loss of cholinergic neurons in the brain by boosting transmission from the remaining cells. However, if Greenfield’s hypothesis is correct, those compounds could have the unintended consequence of accelerating degeneration mediated by the AChE-derived peptide. She said many academic groups as well as companies have targeted the nicotinic receptor over the years with little success. “What they’re neglecting is the pathological situation in real Alzheimer’s, which involves our peptide. By the time a person comes to the clinic the brain will be swamped in it.” By contrast, her company has shown that the AChE-derived peptide specifically up-regulates the β-amyloid and tau pathways to kick off degeneration in the most vulnerable brain regions, she said. “We are suggesting that our drug is backed up by a physiological story in a way that the others are not.”