Scientists ID compounds that target amyloid fibrils in Alzheimer’s, other brain diseases

UCLA chemists and molecular biologists have for the first time used a “structure-based” approach to drug design to identify compounds with the potential to delay or treat Alzheimer’s disease, and possibly Parkinson’s, Lou Gehrig’s disease and other degenerative disorders.

All of these diseases are marked by harmful, elongated, rope-like structures known as amyloid fibrils, linked protein molecules that form in the brains of patients.

Structure-based drug design, in which the physical structure of a targeted protein is used to help identify compounds that will interact with it, has already been used to generate therapeutic agents for a number of infectious and metabolic diseases.

The UCLA researchers, led by David Eisenberg, director of the UCLA–Department of Energy Institute of Genomics and Proteomics and a Howard Hughes Medical Institute investigator, report the first application of this technique in the search for molecular compounds that bind to and inhibit the activity of the amyloid-beta protein responsible for forming dangerous plaques in the brain of patients with Alzheimer’s and other degenerative diseases.

In addition to Eisenberg, who is also a professor of chemistry, biochemistry and biological chemistry and a member of UCLA’s California NanoSystems Institute, the team included lead author Lin Jiang, a UCLA postdoctoral scholar in Eisenberg’s laboratory and Howard Hughes Medical Institute researcher, and other UCLA faculty.

The research was published July 16 in eLife, a new open-access science journal backed by the Howard Hughes Medical Institute, the Max Planck Society and the Wellcome Trust.

A number of non-structure-based screening attempts have been made to identify natural and synthetic compounds that might prevent the aggregation and toxicity of amyloid fibrils. Such studies have revealed that polyphenols, naturally occurring compounds found in green tea and in the spice turmeric, can inhibit the formation of amyloid fibrils. In addition, several dyes have been found to reduce amyloid’s toxic effects, although significant side effects prevent them from being used as drugs.

Armed with a precise knowledge of the atomic structure of the amyloid-beta protein, Jiang, Eisenberg and colleagues conducted a computational screening of 18,000 compounds in search of those most likely to bind tightly and effectively to the protein.

Those compounds that showed the strongest potential for binding were then tested for their efficacy in blocking the aggregation of amyloid-beta and for their ability to protect mammalian cells grown in culture from the protein’s toxic effects, which in the past has proved very difficult. Ultimately, the researchers identified eight compounds and three compound derivatives that had a significant effect.

While these compounds did not reduce the amount of protein aggregates, they were found to reduce the protein’s toxicity and to increase the stability of amyloid fibrils — a finding that lends further evidence to the theory that smaller assemblies of amyloid-beta known as oligomers, and not the fibrils themselves, are the toxic agents responsible for Alzheimer’s symptoms.

The researchers hypothesize that by binding snugly to the protein, the compounds they identified may be preventing these smaller oligomers from breaking free of the amyloid-beta fibrils, thus keeping toxicity in check.

An estimated 5 million patients in the U.S. suffer from Alzheimer’s disease, the most common form of dementia. Alzheimer’s health care costs in have been estimated at $178 billion per year, including the value of unpaid care for patients provided by nearly 10 million family members and friends.

In addition to uncovering compounds with therapeutic potential for Alzheimer’s disease, this research presents a new approach for identifying proteins that bind to amyloid fibrils — an approach that could have broad applications for treating many diseases.

New clues illuminate Alzheimer’s roots

Scientists at Rice University and the University of Miami have figured out how synthetic molecules designed at Rice latch onto the amyloid peptide fibrils thought to be responsible for Alzheimer’s disease. Their discovery could point the way toward therapies to halt or even reverse the insidious disease.

The metallic dipyridophenazine ruthenium molecules strongly bind to pockets created when fibrils form from misfolded proteins that cells fail to destroy. When excited under a spectroscope, the molecules luminesce, which indicates the presence of the fibrils. That much was known by Rice researchers, but until now the process was a mystery.

By combining their talents in biophysics (at Rice) and computer simulation (at Miami), researchers pinpointed four such pockets along the fibril where the hydrophobic (water-averse) molecules can bind. They believe their work will help chemists design molecules to keep the fibrils from forming the plaques found in Alzheimer’s patients.

The teams led by Rice chemist Angel Martí and Miami chemist Rajeev Prabhakar reported their results in the Journal of the American Chemical Society this month.

Two years ago, Martí and Nathan Cook, a graduate student in his lab and lead author of the new paper, combined ruthenium complexes with solutions containing the spaghetti-like amyloid fibrils. The complexes don’t luminesce by themselves, but when they link to an amyloid fibril, they can be triggered by light at one wavelength to glow at another; this helps the researchers “see” the fibrils.

This ability to track amyloids was a great step forward, but left open the question of why the complexes latched onto the fibrils at all, Cook said.

“We had no way to figure it out because our experimental techniques can’t identify binding sites,” he said. “The standard (used to analyze proteins) is to crystallize your material and use X-rays to determine where everything is positioned. The problem with amyloid beta is the fibrils are not uniform, and you can’t crystallize them. All you would get is an amorphous lump.”

But a door opened when Prabhakar, a theoretical and computational chemist who specializes in amyloids, contacted Martí and suggested a collaboration. “We both knew the other was working with amyloid betas,” Martí said. “We were able to figure out how many amyloid beta monomers (molecules that can bind with each other) had to come together to form fibrils, while he modeled the interactions. When we brought all the data together, we had a perfect match.”

“Basically, we learned from the model that we need two monomers to form a binding site,” Marti said. “The cleft where the ruthenium complex binds is completely hydrophobic, the same as the complex. Neither wants to be exposed to water, so when they find each other, they don’t have a choice but to come together. It turns out that’s exactly what needs to happen to turn on the photoluminescent response of the compound.”

Martí said testing various concentrations of monomers with ruthenium complexes helped them determine that a little more than two monomers, on average, was sufficient to get the “light switch” effect. Prabhakar’s analysis found four specific locations along the aggregating monomers where the ruthenium complexes could bind: two at the ends where the monomers tend to bind to each other, and two in the middle.

“It was a complicated system to model and we tried hard, using a variety of computational techniques,” Prabhakar said. “In the end, we were amazed to find our results in perfect agreement with the experiments performed in the Martí lab.”

The researchers called the end locations “A and B,” and the middle clefts “C and D.” The hydrophobic A and B sites exist only at the edges of the fibrils, which limits their exposure to the complexes, Martí said. “But there are lots of C and D sites,” he said. “That explains why the ruthenium complexes don’t inhibit the aggregation of fibrils. It seems the system prefers to bind another monomer, rather than a ruthenium complex, at the ends.

“But now that we understand the mechanism, we can design more hydrophobic complexes that could bind strongly to the ends and prevent further elongation of the fibril,” he said.

“There’s a whole variety of ways to tweak this that could potentially disrupt a binding pocket,” Cook said.

More challenges lie beyond the new discovery, he said. New research indicates toxic oligomers may be catalyzed by the formation of amyloid fibrils. “We might be able to prevent the formation of these oligomeric species by binding ruthenium complexes to the surface, which would completely change the surface chemistry of the fibrils,” Martí said. “These are the things we are really interested in doing right now.”

From trauma to tau - Researchers tie brain injury to toxic form of protein

University of Texas Medical Branch at Galveston researchers have uncovered what may be a key molecular mechanism behind the lasting damage done by traumatic brain injury.

The discovery centers on a particular form of a protein that neuroscientists call tau, which has also been associated with Alzheimer’s disease and other neurodegenerative conditions. Under ordinary conditions, tau is essential to neuron health, but in Alzheimer’s the protein aggregates into two abnormal forms: so-called “neurofibrillary tangles,” and collections of two, three, or four or more tau units known as “oligomers.”

Neurofibrillary tangles are not believed to be harmful, but tau oligomers are toxic to nerve cells. They are also are thought to have an additional damaging property — when they come into contact with healthy tau proteins, they cause them to also clump together into oligomers, and so spread toxic tau oligomers to other parts of the brain.

Now, in experiments with laboratory rats, using novel antibodies developed at UTMB, scientists have found that traumatic brain injuries also generate tau oligomers. The destructive protein assemblages formed within four hours after injury and persisted for at least two weeks — long enough to suggest that they might contribute to lasting brain damage.

Significantly, the rats used in the experiments were normal, unlike the genetically modified animals used in most tau research. The findings are thus likely to be more relevant to human traumatic brain injuries.

“Although people have given some attention to the formation of neurofibrillary tangles after traumatic brain injury, we were the first to look at tau oligomers, because we have an antibody that allows us to separate them out and see how much of the total tau is the toxic species,” said Bridget Hawkins, lead author of a paper on the research now online in the Journal of Biological Chemistry. “We saw that it’s a substantial amount — enough to play an important role in the effects of traumatic brain injury.”

Those effects can include memory deficits, which have been recently shown by UTMB researchers to be induced by tau oligomers. Other long-term ramifications of TBI include seizures, and disruptions in the sleep-wake cycle. The UTMB scientists hypothesize that these problems could be avoided if physicians had a way to stop the process of tau oligomerization.

One possibility is a treatment based on the antibodies used to label tau oligomers in this project, which were developed as part of an effort to develop a vaccine against different neurodegenerative disorders.

“We have antibodies that can specifically target these tau oligomers without interfering with the function of healthy tau,” said UTMB associate professor Rakez Kayed, the senior author on the paper. “This is a new approach — we’re starting by targeting them in animals — but we hope to eventually humanize these antibodies for clinical trials.”

Scientists identify molecular trigger for Alzheimer’s disease

Researchers have pinpointed a catalytic trigger for the onset of Alzheimer’s disease – when the fundamental structure of a protein molecule changes to cause a chain reaction that leads to the death of neurons in the brain.

For the first time, scientists at Cambridge’s Department of Chemistry, led by Dr Tuomas Knowles, Professor Michele Vendruscolo and Professor Chris Dobson working with Professor Sara Linse and colleagues at Lund University in Sweden have been able to map in detail the pathway that generates “aberrant” forms of proteins which are at the root of neurodegenerative conditions such as Alzheimer’s.

They believe the breakthrough is a vital step closer to increased capabilities for earlier diagnosis of neurological disorders such as Alzheimer’s and Parkinson’s, and opens up possibilities for a new generation of targeted drugs, as scientists say they have uncovered the earliest stages of the development of Alzheimer’s that drugs could possibly target.

The study, published today in the Proceedings of the US National Academy of Sciences, is a milestone in the long-term research established in Cambridge by Professor Christopher Dobson and his colleagues, following the realisation by Dobson of the underlying nature of protein ‘misfolding’ and its connection with disease over 15 years ago.

The research is likely to have a central role to play in diagnostic and drug development for dementia-related diseases, which are increasingly prevalent and damaging as populations live longer.

In 2010, the Alzheimer’s Research UK showed that dementia costs the UK economy over £23 billion, more than cancer and heart disease combined. Just last week, PM David Cameron urged scientists and clinicians to work together to “improve treatments and find scientific breakthroughs” to address “one of the biggest social and healthcare challenges we face.”

The neurodegenerative process giving rise to diseases such as Alzheimer’s is triggered when the normal structures of protein molecules within cells become corrupted.

Protein molecules are made in cellular ‘assembly lines’ that join together chemical building blocks called amino acids in an order encoded in our DNA. New proteins emerge as long, thin chains that normally need to be folded into compact and intricate structures to carry out their biological function.

Under some conditions, however, proteins can ‘misfold’ and snag surrounding normal proteins, which then tangle and stick together in clumps which build to masses, frequently millions, of malfunctioning molecules that shape themselves into unwieldy protein tendrils.

The abnormal tendril structures, called ‘amyloid fibrils’, grow outwards around the location where the focal point, or ‘nucleation’ of these abnormal “species” occurs.

Amyloid fibrils can form the foundations of huge protein deposits – or plaques – long-seen in the brains of Alzheimer’s sufferers, and once believed to be the cause of the disease, before the discovery of ‘toxic oligomers’ by Dobson and others a decade or so ago.

A plaque’s size and density renders it insoluble, and consequently unable to move. Whereas the oligomers, which give rise to Alzheimer’s disease, are small enough to spread easily around the brain - killing neurons and interacting harmfully with other molecules - but how they were formed was until now a mystery.

The new work, in large part carried out by researcher Samuel Cohen, shows that once a small but critical level of malfunctioning protein ‘clumps’ have formed, a runaway chain reaction is triggered that multiplies exponentially the number of these protein composites, activating new focal points through ‘nucleation’.

It is this secondary nucleation process that forges juvenile tendrils, initially consisting of clusters that contain just a few protein molecules. Small and highly diffusible, these are the ‘toxic oligomers’ that careen dangerously around the brain cells, killing neurons and ultimately causing loss of memory and other symptoms of dementia.

“There are no disease modifying therapies for Alzheimer’s and dementia at the moment, only limited treatment for symptoms. We have to solve what happens at the molecular level before we can progress and have real impact,” said Dr Tuomas Knowles from Cambridge’s Department of Chemistry, lead author of the study and long-time collaborator of Professor Dobson and Professor Michele Vendruscolo.

“We’ve now established the pathway that shows how the toxic species that cause cell death, the oligomers, are formed. This is the key pathway to detect, target and intervene – the molecular catalyst that underlies the pathology.”

The researchers brought together kinetic experiments with a theoretical framework based on master equations, tools commonly used in other areas of chemistry and physics but had not been exploited to their full potential in the study of protein malfunction before.

The latest research follows hard on the heels of another ground breaking study, published in April of this year again in PNAS, in which the Cambridge group, in Collaboration with Colleagues in London and at MIT, worked out the first atomic structure of one of the damaging amyloid fibril protein tendrils. They say the years spent developing research techniques are really paying off now, and they are starting to solve “some of the key mysteries” of these neurodegenerative diseases.

“We are essentially using a physical and chemical methods to address a biomolecular problem, mapping out the networks of processes and dominant mechanisms to ‘recreate the crime scene’ at the molecular root of Alzheimer’s disease,” explained Knowles.

“Increasingly, using quantitative experimental tools and rigorous theoretical analysis to understand complex biological processes are leading to exciting and game-changing results. With a disease like Alzheimer’s, you have to intervene in a highly specific manner to prevent the formation of the toxic agents. Now we’ve found how the oligomers are created, we know what process we need to turn off.”