A molecular mechanism for a general amyloid re-modelling activity

Brain degeneration is one of the principal reasons why our quality of life is eroded as we age. The appearance of diseases like Alzheimer's and Parkinson's is strongly linked to the formation of amyloid aggregates in the brain. Amyloids are pathogenic fibrous assemblies of otherwise normal protein molecules. The removal of these assemblies has been shown to prevent disease progression in model animals and even lead to cognitive improvements. Strong contenders for the design of disease-modifying agents are molecules that are able to modify existing amyloid fibrils as well as prevent their formation. However, our current understanding of how these molecules work is insufficient to direct a powerful knowledge-based drug discovery programme. Our proposal will address this deficiency by using state-of-the-art structural biology techniques to unravel the mechanism behind the activity of these molecules. We have chosen as our model agent the protein G3P because of its unusually efficacious effect in animal studies and its ability to tackle amyloids of many different types. The latter argument is particularly important as more than one kind of amyloid is often found in people suffering with these degenerative conditions.

Of key importance will be the definition of the atomic surfaces involved as this information will directly feed into future drug design processes. Using nuclear magnetic resonance spectroscopy, we can map changes in the surface of the molecule using a novel hydrogen atom labelling assay. To complement this work, a molecular footprint for the amyloids on the surface of the G3P will also be measured using "FPOP", which will decorate the surface of the complex with highly reactive hydroxyl radicals, readily detected by mass spectrometry. Finally, we will also produce an array of different atomic surfaces on the G3P using genetic engineering and, by a process of elimination, work out the key components of its activity. Not only is G3P able to stick to amyloids but we suspect it uses its ability to act as a "molecular packman" to then open its mouth and expose a more powerful interface responsible for the dismantling activity. We will investigate whether this is true by exploiting the available extremely detailed knowledge of how this protein uses this ability to operate in bacterial infection.

Compared with small molecule effectors, the larger size of the G3P molecule will also allow us to visualise how it interacts with the amyloid fibrils directly using the imaging technology that the University of Sheffield has recently invested in. The relative orientations of molecules within the amyloid assemblies have the potential to reveal with near atomic detail how the G3P grabs the amyloid for dismantling. We will explore what happens to these structures as they become re-modelled into less toxic molecular assemblies. Our single particle analysis techniques are ideal for observing these mixed populations.

Finally, we will investigate the key steps G3P is affecting within the molecular processes defining amyloid assembly and disassembly. We will do this by carefully measuring how this amyloid modifying agent affects the different populations of the precursor molecule, then the partially assembled forms referred to as "oligomers" and also the mature amyloidogenic protein structures as we incubate them together. By feeding the data into existing mathematical models, we will start visualising what its activity consists of: for example, does it promote the breakage of fibrils by straining the structure or does it favour dissociation of single protein molecules one at a time? By the end of the project, we will have details of the molecular events involved and be able to apply this knowledge not only to improving the design of future therapeutics but also to an improved regulation of fibril formation by novel amyloid-based biomaterials.

Protein misfolding and aggregation is responsible for some of the most prevalent aging diseases in humans. Fibrillar "amyloid" species accumulating as extracellular plaques or intracellular tangles act as sinks for the more neurotoxic soluble species and at later stages of the disease dominate the conditions. Drugs able to target only soluble species have a limited use and so we have chosen to study an amyloid binding and modifying agent, G3P. Despite the existence of many candidate drugs, our understanding of how these exert their activity at the molecular level is poor and we propose here to visualise this process directly using our state-of-the-art "Imagine" centre. We will study the molecular interfaces of G3P responsible for the activity using a combination of site-directed mutagenesis and labelling approaches including hydrogen-exchange (NMR) and Fast Photochemical Oxidation of Proteins (MS). G3P is a 25KDa two domain protein which exhibits archetypal domain movements on substrate binding. We will define the relevance of key residues involved in domain dynamics to amyloid binding activity. We will then interrogate the changes in the structure of the amyloids (Abeta(1-42) and alpha-synuclein) directly using state-of-the-art Cryo-EM and time-resolved AFM. Finally, we will investigate the kinetics and thermodynamics of the re-modelling reaction of beta-amyloid as well as exploring the impact of G3P on the assembly process. As well as standard methods, we propose to detect changes in the population of different intermediate species directly using asymmetric flow fractionation methods uniquely suited to the properties and molecular weight range of these reactions. Using current mathematical models, we will deduce the key stages of its inhibitory and remodelling activities. We expect our results to contribute to a step change in the design of crucial disease modifying drugs for degenerative diseases.

Our quality of life as we age is severely impaired by degenerative conditions which include Alzheimer's, Parkinson's and other notable diseases. The aggregation of different proteins into amyloids is responsible for this, but can also drive a number of other lesser known processes such as bacterial biofilm formation and HIV infectivity. Our proposal will address fundamental questions regarding how protein amyloid formation can be regulated, and hence make a step change in the ability of the scientific community to control this reaction and importantly, prevent toxic side reactions. Specifically, we will determine how an amyloid re-modelling agent binds to amyloids, what the molecular determinants of this apparent broad specificity are and how the re-modelling activity takes effect on the amyloid assemblies.

These studies will provide a firmer foundation upon which to
1. accelerate progress in knowledge-based anti-amyloid drug design for use in Alzheimer's disease, Parkinson's disease and many other conditions including bacterial infection and HIV
2. provide material scientists working with beta-sheet rich protein fibres with better tools to regulate their toxicity and control their formation.

In order to ensure successful knowledge transfer of the results of our study, we will engage regularly in discussions with individuals from outside of the academic sector. We will achieve this in the following ways:
- We will choose to publish in journals and attend conferences that are designed to attract a broad audience and have major readership/attendance from industry
- We will continue participation in joint academic-industrial forums including collaboration with our local SITraN Institute (Sheffield Institute for Translational Neuroscience) and our Science and Medicine gateways as well as BBSRC funded networks (such as Bioprocessing, Metals in Biology, Biocatalyst discovery, Natural Products Discovery and Bioengineering, which are supported by Merck, BASF, Unilever, Pfizer, Codexis, Syngenta and Bayer).
- We have planned regular meetings with researchers in industry, in particular with Astrazeneca (monthly), C4X Discovery (monthly), Eli Lily (annual) and GSK (six monthly)
- The work proposed will be a source of useful discussions with NeuroPhage Pharmaceuticals with whom the research originally started and from whom we will receive information regarding clinical trials
- All investigators engage regularly with members of the public through public lectures as well as café scientifique, national science week and further outreach activities
- Last but not least, we will continue our support for the University of Sheffield's local "Dementia Interest Group" who organises regular events and publications aimed at local patient groups and their families who live with dementia.

The strength of these meetings will arm us with the correct information to bridge any gaps between translation to industry and our work. This will be done by engaging with current collaborators and new contacts which we have now identified within the international research community.