Developing new drugs against diseases like Alzheimer's can be a long and tortuous process. It can take even longer, if initial tests to find new candidate drugs aren’t quite testing for the right activity.
David Vaux and colleagues at the Dunn School of Pathology believe that may be happening with some of the test-tube assays used to identify potential drugs for diseases like Alzheimer’s and type II diabetes, in which deposits of broken and misfolded proteins build up.
Their results that highlight the problem – and a new assay they have developed to overcome it – have just been published online in FASEB journal.
Alzheimer’s disease has been known since the beginning of the 20th century to be associated with a build up of deposits in the brain called amyloid plaques. More and more has been learnt about the formation of these plaques since. Small fragments get broken off from an innocuous protein normally present in the brain. One of these fragments – beta amyloid – becomes distorted and misfolded, and begins to stick together into fibres that eventually aggregate to form the amyloid plaques.
But sorting out the connection of this many-staged build up of protein with disease progression in Alzheimer’s has been difficult. A consensus is now building, says David Vaux, that it is the very early stages when the beta amyloid fragments first start accumulating into clusters a few strong that is toxic to nerve cells. Big pharma is now targeting these early protein assemblies in the hope of finding new drugs.
The standard way new drugs are found is first to take a protein that’s implicated in the cause or progression of a disease, and throw thousands of compounds at it. The hope is that a few compounds will be found that stop the protein in its tracks. The few ‘hits’ that are generated in this way are used as the starting point for drug development.
But there may be a problem in this case, suggests David Vaux. ‘Attempts by multinational pharmaceutical companies to identify potential drugs that might inhibit the assembly of amyloid precursors into neurotoxic intermediates have relied on assays in multi-well plates. Although they have generated hits, these have not yet translated into in vivo active drug candidates.’
Multi-well plates are plastic trays with many lines of little cylindrical wells in which separate reactions can be carried out. It’s like hundreds or even thousands of tiny test tubes lined up in rows. Using multi-well plates allows tens of thousands of potential drug compounds to be tested swiftly and easily. But each well is open to the air, and this ‘air-water interface’ is key: beta amyloid tends to gather at the surfaces of solutions, where the protein fragments begin to organise and come together.
‘All the protein fragments need to come together in a particular orientation. It’s like trying to build a tower of lego bricks,’ explains David Vaux. ‘If you took a sack of bricks and shook it around, it’s very unlikely indeed that they’d come together to form that tower. But if you float the bricks on the surface of water, they are far more likely to join up.’
Of course, such surfaces, or air-water interfaces, aren’t present in the nerve cells of the brain. So the multi-well assays don’t capture the situation in the body. Potential drug compounds that appear to block the assembly of amyloid protein fragments in a multi-well plate may not go on to work in tissue cultures. Or worse, potential compounds that could lead to valuable new drugs could be lost and never tried because they don’t show an effect in this initial screen.
In cells, the proteins are known to orient and assemble mostly at the cell membranes. So David Vaux and his team set out to come up with a new assay that replicated this situation much better.
They introduced a simple Perspex cover to fit over the reaction wells in the multi-well plate, getting rid of the air-water interface at a step. They also introduce liposomes – lipid capsules that mimic cell membranes – into the mix to provide a template on which the Alzheimer’s protein fragments could assemble. The researchers have shown that beta amyloid assembles as expected in this new assay, but not because of any surface effects at the top of the well.
The team, with funding from Synaptica, is now using this new assay to screen for potential new drug compounds. They are just beginning to get tantalising hints that some compounds do act differently in their assay, which mimics the situation in cells much better.
This allows David Vaux to conclude: ‘I am sure that the new assay can identify potentially valuable hits that would be missed by conventional assays.’