Designer Chemistry to Probe Supramolecular Assembly Mechanism and Function

Self-assembly permits the construction of large complex 3D functional structures that are biologically important both in health and disease. The manner in which such structures are formed (i.e. their mechanism or pathway of assembly) is not well-understood; this is because assembly pathways involve simultaneous and dynamic formation of multiple structures some of which are relevant to the final functional structure, some which are not and even others which are functional in their own right. Therefore, a fundamental challenge is to characterise such assemblies in real-time so as to understand their assembly pathways at a structural level and to inform our ability to intervene in these processes. This challenge is even greater for biologically functional assemblies arising from a need to be able to track individual assembly intermediates in cells; this would allow scientists to monitor their location and what other components of the cellular machinery they interact with to exert their function.

To address this challenge, this work will develop an approach to allow individual species in a self-assembly pathway to be trapped and tagged for further characterisation. Using specialised photochemistry will allow us to take a snapshot of an assembling system and, in combination with a mass spectrometry technique able to separate and characterise individual species within a complex mixture, allow characterisation of assembly intermediates in residue-specific detail and real-time. In tandem, we will develop tailored synthetic chemistry to allow individual/ populations of, assembly intermediates to be further functionalised once they have been trapped and this will allow us to begin to study the role of these intermediates in a cellular environment.

We will apply our approach to an amyloid-forming peptide from the Abeta peptide which forms amyloid fibrils and plays a central role in the development and progression of Alzheimer's disease. Thus, in addition to the fundamental and generic knowledge, tools and methods that the project will develop, we will generate new understanding on a self-assembly pathway of huge medical and societal significance. Extracellular plaques of the amyloid-beta (Abeta) peptide are a signature of Alzheimer's disease, one of several neurodegenerative conditions that result in dementia. Neurodegeneration is a major global problem; according to Alzheimer's Research, dementia affects ~ 800,000 people in the UK meaning some 25 million of the UK population have a close friend or family member who suffers from it. Indeed, it has been estimated that dementia costs the UK economy £23 billion a year which is more than cancer and heart disease combined. However, it is unclear at this stage how to best target the condition because the way in which Abeta functions at a molecular level is not understood.

Beta-sheet peptide nanostructures (e.g., amyloid fibrils) are important entities e.g. extracellular plaques of the amyloid-beta (Abeta) peptide are a signature of Alzheimer's disease. Thus, beyond the fundamental tools and understanding we will develop on the supramolecular nature of assembly, the peptide we have selected as a model for this study relates to a significant challenge of immense biochemical and medical importance. The current proposal will explore the use of photo-induced covalent-crosslinking (PIC) to probe the nature of peptide assembly in a temporal manner and develop a toolkit for further functionalization of assembly intermediates to permit studies of their function. Uniquely, we propose to exploit the advantages of diaizine chemistry for PIC, combined with the unique analytical and seperative powers of MS linked to IMS to analyse the results of the cross-linking experiments. In addition to the academic beneficiaries directly involved and the wider academic community, there are several beneficiaries with whom we will interact through our "Pathways to Impact Plan" to ensure maximal impact of the research as follows:
(1) Pharmaceutical and Biopharmaceutical Companies - these stand to benefit from the research because:
(a) The fundamental tools and methodology we are developing on the use of diazirine chemistry and PIC, together with further orthogonal functionalization, married with state-of-the-art analytical mass spectrometry techniques will be applicable to a broad range of dynamic and complex biological processes - a major challenge in drug discovery and development is to understand biological processes more effectively, hence the proposed research will exemplify the use of designer chemistry for this purpose.
(b) The knowledge we obtain from the research will be directly applicable in that it will provide a better understanding of the oligomeric peptide species involved in the self-assembly of Abeta(1-40) and potentially provide starting points for better informed drug design.
(2) Wider society - It is critical that young people are (a) aware of societal challenges whose solutions will be underpinned by basic science and (b) inspired to pursue careers in scientific research, teaching and policy development. This project is ideal to address this issue and can do so through our strong desire to make the basic science accessible to the public e.g. through our proposed outreach activities and as an activity in which undergraduate and summer project students looking to gain their first experience of research can become involved.