The Next Revolution: Amorphics - The Science of Protein Misfolding

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By George Adams Ph.D and Neil Cashman M.D.

Our understanding of proteins and its functions has advanced through a series of technological and scientific revolutions over the past 50 years: direct sequencing of proteins, to nucleic acid sequencing, to proteomics for rapid identification of proteins and currently to structural genomics for the three-dimensional structure of proteins.

Now we are on the threshold of another revolution, this time to catalog and characterize protein misfolding in disease. Misfolded proteins can form large aggregate 'snowballs' which accrete until they can be seen easily through a microscope. The term 'amorphics' captures our current scientific challenge, as it highlights the amorphous transition that normal proteins may undergo in disease, and underscores the extensive study required to move from simplistic notions about 'aggregates' (without a recognizable repeating structure) or 'amyloid' (organized multimers with specific structural and staining properties). Just as Watson's and Crick's elucidation of a double stranded helix crystallized our understanding of DNA, the precise identification of the mechanisms through which proteins participate in large protein multimeric complexes will provide an essential window on the biology of a number of important diseases.

What are the aggregated misfolded protein (AMP) diseases, and why should the biotech industry care? Many AMP disorders are among the most disabling, least treatable diseases of humankind. These include Alzheimer's disease (AD), Parkinson's disease (PD), Amyotrophic Lateral Sclerosis (ALS), and peripheral disorders accompanied by misfolded protein deposition, such as type II diabetes. All of these diseases present in large segments of the population, and in which better medical treatments are critically needed. All of these diseases are also typically related to aging, with the expectation of vastly increased prevalence in the next 20 years as the post-war baby boom come into the high-risk age brackets. The poster child for this group of diseases is prion disease, including bovine spongiform encephalopathy (BSE) and the human form, Creutzfeldt-Jakob disease (CJD). It is the infectious nature of these misfolded protein aggregates that has stimulated study. The Nobel Prize was awarded to Dr. Prusiner in 1997 for describing how prions grow and propagate through template-directed misfolding, in which the prion acts as a scaffold for the misfolding of normal prion protein. This is more akin to a crystallization process than microbial replication and while initially thought to be restricted to prion disease, increasingly appears to be operative in AD, PD, and ALS - for which the misfolded protein templates are Abeta, alpha-synuclein, and superoxide dismutase 1, respectively.

A protein is composed of a long chain of amino acids which must be folded into a specific three dimensional state to be functional. The rules for protein folding fall within the province of structural genomics and are not fully understood. Amorphics is focused on how normal proteins are induced to misfold. Some disorders, such as cystic fibrosis, occur as a result of a primary defect in protein folding, for example mutations in CFTR, the cystic fibrosis transmembrane conductance regulator. As a result, improperly folded proteins accumulate in the cell. In contrast, protein misfolding disorders are generally acquired diseases, usually presenting in senescence. A majority of cases are 'sporadic' and not attributable to a genetic mutation. Why then is the normal protein, which has been functional for decades, now induced to misfold and accumulate as large aggregates in non-prion diseases where an infectious event has not been identified? It is speculated that the spontaneous generation of a 'seed' triggers massive misfolding through a template-directed process, which then presents as disease. In the case of familial forms, where a mutation is present, the mutant protein is likely inherently more susceptible to misfolding. Amorphics must discern the interchange between primary structure, conformation and misfolding in order to build a mechanistic understanding of misfolding and aggregate formation.

The protein misfolding diseases usually damage and destroy cells through a toxic gain of function acquired when proteins misfold into aggregates. The AMPs are cytotoxic to the normal cells found in the areas in which they arise, causing further progression of disease. For example, in AD, Aβ-AMPs are formed in the brain and kill neurons, as is the case in diabetes where IAPP-AMPs form in the pancreas and kill islet cells.

In order to control these AMP diseases, the science of amorphics must be advanced. The study of protein misfolding is not something that lends itself to high throughput, at least not yet, although some approaches using dynamic NMR, deuterium exchange and circular dichroism are providing some low-resolution data. Diagnosis of protein misfolding diseases is still in its infancy; for example, the only definitive diagnosis of AD is made by microscopic examination of the brain after death. And in the realm of therapeutics, progress is even more dismal; symptomatic therapies are available for patients with AD, PD and ALS, but disease modifying therapies are only being explored now and none are curative.

One of the company's working in this area is Amorfix Life Sciences, a Canadian biotech company, which has developed new platforms for the diagnosis and treatment of protein misfolding diseases. New diagnostic technologies include epitope protection. The fundamental aspect of this approach is the selective chemical covalent modification of epitopes on mono - or oligomeric normal proteins, whereas epitopes buried in the interior of an aggregate are not chemically modified, and are detectable by conventional antibodies when the aggregate has been broken up by denaturing agents. This has been used to develop a blood test for vCJD, in which infectious aggregates of prion protein in the blood can be detected over a million-fold background of normal prion protein. The same platform is now being applied to aggregated Aβ to develop a blood test for AD.

The company is also developing therapeutics based on the specific exposure of protein domains when the target proteins become misfolded in disease. The molecular surface exposure of disease specific epitopes has enabled the development of prion specific antibodies, and is now informing the search for protein misfolding specific epitopes in other proteins, such as SOD1 in ALS. In both the prion protein and in SOD1 there has been a loss of tertiary structures, which causes exposure of peptides normally inaccessible to antibody binding in the native state of the protein. Thus, disease-specific epitopes might provide a means of selectively targeting misfolded proteins, while sparing the autoimmune consequences of targeting the normal protein. Disease specific antibodies for therapy of protein misfolding diseases might be the 'killer app' of immunotherapeutics. The specific recognition of sites on a protein which have lost structure is not a job for small molecules, which tend to bind tightly to a more rigidly defined target, such as a pit or cleft in the molecular surface. In the case of AMPs, the proteins are quite flexible and can easily change their three-dimensional structure. This flexibility makes its study even more difficult as they do not crystallize and so three-dimensional structures cannot be accurately resolved.

Amorphics is in its infancy and represents a frontier for the study of the molecular dynamics of normal proteins which spontaneously change shape and become pathogenic. Immunotherapy seems to hold promise in addressing misfolded-protein diseases which will only become more prevalent as the population ages. It will be interesting to see how things unfold.

Dr. Adams has been the CEO of five successful companies and a founding investor in several others. He serves on the research review committees for the Canadian Government, federal and provincial centres of excellence and is Chairman of Sernova Technologies Inc (TSXV:SVA). In 2007, Amorfix Life Sciences received the Technology Pioneer Award from the World Economic Forum and the TSXV50 award from the Toronto Stock Exchange.

Dr. Neil Cashman is a clinical neurologist and neuroscientist. He currently holds a Canada Research Chair in Neurodegeneration and Protein Misfolding Diseases at the University of British Columbia. He was scientific founder to Caprion Pharmaceuticals and Amorfix Life Sciences, two Canadian biotechnology companies dedicated to proteomics, diagnostics and therapeutics of disease. He serves as chief scientific officer to Amorfix.