Treating protein folding diseases

The importance of protein folding has been known for almost 50 years � but this became a major focus for pharmaceutical companies when scientists discovered that apparently unrelated diseases such as Alzheimer�s disease and cancer were linked by defects in the folding of proteins. Now, protein misfolding has been identified as the cause of around 20 diseases, and is thought to occur in many ways (see Protein folding diseases). In one mechanism, accumulation of insoluble and toxic misfolded protein junk seems to be a key step on the way to Alzheimer�s disease and prion disorders such as Creutzfeldt-Jacob disease (CJD). In other conditions, such as cystic fibrosis, hereditary emphysema and some cancers, misfolding leaves too little normally folded protein around to do its job. Insights into the defects that result in both these misfolding mechanisms have allowed scientists to come tantalisingly close to designing successful treatments for these diseases.

Alzheimer�s disease
As one of the most common protein folding diseases, and of increasing concern to an ageing population, Alzheimer�s disease is an obvious case for treatment. One of its hallmarks, and probably its cause rather than consequence, is accumulation of b -amyloid plaques. These are formed when amyloid precursor protein (APP) is cut by protease enzymes and releases amyloid-b peptide fragments (Fig. 1). These then misfold and aggregate into insoluble clumps of b -amyloid plaques that surround and damage the neurons (see Protein folding diseases).

Treatments that can interrupt this process are being sought, including developing protease inhibitors to stop amyloid-b fragment release. But drug developers� two main strategies are finding ways to break down plaques in the brain, or prevent amyloid-b peptide fragments from aggregating in the first place.

A toxic intermediate
The latter approach generated recent interest, following reports that �intermediate aggregates� might be more toxic than the deposits themselves. According to the theory, proposed by researchers in Florence and Cambridge and led by Christopher Dobson, cytotoxicity is "much more pronounced for the rapidly formed non-fibrillar aggregates than for the highly organised fibrillar structures", from which they concluded that mature fibrils "were essentially harmless to cells". (Nature 416, 507�511 (2002)).

As Dobson explains, mature aggregates might be less harmful than their predecessors because the reactive regions of the latter are exposed to the elements. With more mature aggregates, he says, "a lot of the sticky ends get covered up and you end up with a rather innocuous type of material". Because Dobson�s work looked at the trouble caused to cells in culture by aggregates of proteins that are not associated with Alzheimer�s disease, this could mean that it is not specific proteins that cause problems but, in fact, their aggregation. Indeed, compelling evidence indicates that this aggregation could also be involved in other disorders of protein folding, including Parkinson�s disease and systemic amyloidosis.

Preventing aggregation is therefore a primary therapeutic target, but there is more than one stage that can be targeted. Strategies so far have focused on disrupting polymerization and fibril aggregation. These include using low-molecular-weight substances such as congo red dye, the antibiotic rifampicin, peptides that bind to amyloid-b peptide and drugs that chelate metals such as copper and zinc, which themselves accelerate amyloid-b deposition. Indeed, interim results from a placebo-controlled trial of the antibiotic iodochlorhydroxyquin (Clioquinol), which chelates these metals in vitro, showed slowing of cognitive decline in the most severely affected of 32 Alzheimer's disease patients studied.

Along similar lines, clinical trials of NC-758 (Alzhemed) are underway after this small, sulphonated molecule was shown to reduce plaque deposition in a mouse model of Alzheimer's disease, probably by preventing polysaccharides from stabilizing the amyloid-b peptide.

But, it has recently been shown that aggregation could be prevented earlier on in the process. A study showed that synaptic plasticity (a crucial process in which a synapse changes its functional properties, usually as a result of repetitive use) in vivo is disrupted by amyloid-b oligomers � the form before fibrillar aggregates are made. If these data reflect what�s going on in humans, focusing on blocking amyloid-b dimer and trimer formation (and hence preventing the formation of amyloid-b oligomers) could also be an important approach.

A vaccine setback
However, as promising as such approaches look, excitement about them has been inevitably dampened by the recent failure of the Alzheimer�s vaccine AN-1792 (Nature 415, 462 (2002)). Multinational trials began in Autumn 2001 but stopped within 6 months when 17 out of 300 subjects developed signs of brain inflammation.

It�s never been entirely clear how the AN-1792 vaccine works (Fig. 2). The vaccine is a synthetic version of amyloid-b protein, which was developed for human use after it was found that transgenic mice that normally get Alzheimer�s-like neuropathology suffered no symptoms after injection of the amyloid-b protein. This presumably stimulates antibody production and both prevents development of symptoms and slows disease progression. So, why it causes the side effect of brain inflammation in humans is puzzling. David Holtzman, a neurologist at the Washington University School of Medicine believes that "there aren�t enough antibodies crossing the blood-brain barrier (BBB) to activate microglia [immune cells in the brain that engulf and remove cells]", after studies in mice showed only 0.05% of the antibodies in the blood make it into the cerebrospinal fluid.

Holtzman and colleagues have another explanation for how the vaccine works � the �sink� hypothesis. In this model, antibodies bind to b -amyloid deposits circulating in the blood, which in turn �pulls� it out of the brain. Indeed, the level of b -amyloid in the blood of vaccinated Alzheimer�s mice was 1,000-fold greater than in those that were not vaccinated, and treated mice also had fewer brain plaques after five months.

The presence of antibody�amyloid complexes in the spleen of mice given a b -amyloid nasal vaccine suggests that these might indeed be being cleared from the body. Clinical trials of this vaccine are set to begin at the end of this year, and AN-1792 variants are also on the cards.

A new treatment?
Just as the vaccine might work by drawing amyloid away from the brain, a new compound called CPHPC, which has been developed by Mark Pepys of the Royal Free and University College Medical School in London and international collaborators looks like it might do something similar (Nature 417, 254�259 (2002)).

One of the main players involved in making plaques, and which apparently prevents their breakdown, is a protein called �serum amyloid P component� (SAP). So, interfering with SAP seems an obvious target, and CPHPC fulfils this criterion in two ways.

First, CPHPC makes SAP molecules stick to each other. This helps the liver to identify them as abnormal, and clear them from the body. CPHPC also binds to other proteins that are thought to be involved in plaque formation, which reduces their ability to participate in this process. The first effect may be not dissimilar from what the vaccine does: researchers suggest that CPHPC may be too big to cross the BBB, but can instead clear SAP from the blood, drawing it away from the brain and hence preventing or reducing plaque formation.

Pepys and colleagues have promising results using CPHPC to treat 19 people with systemic amyloidosis, a condition of abnormal protein folding that is very similar to that of Alzheimer�s disease, but which affects many organs other than the brain. They report that, after taking CPHPC for nine and a half months, patients� blood levels of SAP were reduced, apparently without severe side effects.

The team is about to start using CPHPC in patients with Alzheimer�s disease and believes that it also has the potential to treat other relatively common conditions such as adult-onset diabetes, in which amyloid deposits destroy pancreatic insulin-producing cells.

Whether getting rid of SAP will be enough to stop Alzheimer�s disease is unclear. Pepys himself said: "Even if everything works and the amyloid deposits disappear, I can�t guarantee that it would arrest the progression of dementia". But he and his team also have preliminary evidence that even if brain damage happens before the deposits are fully formed, removing SAP could still be beneficial.

Prion diseases
The transmissable spongiform encephalopathies (TSEs) such as mad cow disease (bovine spongiform encephalopathy; BSE) and its human form Creutzfeld-Jakob disease (CJD) also involve disordered protein folding. These conditions seem to occur when normal human protein particles called prions misfold, setting up a cascade by which newly formed prion proteins are also prompted to misfold (Fig. 3). These prions then stick together, forming matted deposits in the brain. This can happen apparently without cause (sporadic CJD), as a result of a mutation in the prion protein gene, (familial CJD) or when abnormal prion is taken in through the diet, as in variant (v)CJD.

Although human TSEs are rare, they are horribly distressing and ultimately fatal. The disquieting thought that such devastation lurks in our beefburgers and the questions that vCJD raised about the workings of the meat production industry made them a high-profile focus for treatment research.

Treating prion diseases
The possibility that chlorpromazine and quinacrine � drugs used to treat schizophrenia and malaria, respectively � might stop prion misfolding moved from bench to bedside in 2000. Both drugs can cross the BBB, and a CJD sufferer from the United Kingdom called Rachel Forber was treated with them in an American trial pioneered by the prion pioneer and Nobel Prize winner Stanley Prusiner (http://www.nature.com/nsu/010823/010823-1.html). Despite the reported early reversal of her symptoms, Rachel died four months after starting treatment. Other patients are apparently still being treated similarly.

It has been suggested that when a misfolded prion prompts normal prions to misfold (the process that it was hoped these drugs would block), it does so with the aid of a �macromolecular helper� � the identity of which is currently unknown, and sometimes called �Protein X�.

Daniel Cox, Rajiv Singh and collaborators at the University of California at Davis, pioneers of physical modelling work in this area, believe that the key intermediary could in fact be a �multiprotein seed�. Cox says that their work strongly suggests that we�d all be dead from prion diseases if one abnormal prion causes the next one to go wrong, and others reached somewhat similar conclusions a few years ago on the basis of chemical kinetics. "There have now been experiments [from Prusiner and colleagues] pointing to the possible role of multiprotein seeds in mammalian prion diseases", he says.

Whereas drugs that bind to protein X and keep it occupied might slow down prion conversion, Cox and colleagues, notably Alex Slepoy, think that introducing a decoy could help to treat prion diseases. Stressing that this is at present a theoretical possibility, Cox explains, "If we added normal prion protein from other suitable species to a prion infection in progress, we might be able to block the infectious prions from converting normal ones".

Future directions
Clarifying the stage at which misfolding and aggregation must be disrupted is clearly vital, as well as identifying co-factors in these processes that could be therapeutic targets. But in the cases of conditions like Alzheimer�s disease and CJD, this is tricky, as the damage occurs on the inaccessible side of the BBB � in the brain. The problem will be that if �sink� methods, which draw the harmful agents out of the brain by reducing their blood levels, don�t do their job successfully, the challenge of developing drugs that cross the BBB will remain.

Drug developers will also need to decide whether they can hope to prevent, reverse or cure diseases. Cox is cautiously optimistic that techniques such as those that he envisages for TSEs "might significantly prolong the incubation times". Prolong these times longer than any of us live and you�ve essentially cured the disease. But, as Cox believes that there must be many features that are common to all amyloid diseases, discoveries made for one disease could provide an encouraging outlook for the treatment of all these diseases.

Further Reading
Thomasson, W. A. Breakthroughs in Bioscience. Unraveling the Mystery of Protein Folding