Protein aptamers to enable structure-based drug discovery - making the undruggable druggable

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Protein aptamers (PAs eg. Affimers, nanobodies and DARPins) are molecules which can recognise and bind to proteins to help visualise and study them. They can be modified to bind specific targets and have been shown to be as effective as antibodies for this application. However PAs also offer many advantages over antibodies, including better stability, improved ability to reach a range of tissues within the body, and simpler production. There are many potential applications for PAs within the fields of drug discovery and structural biology. They could be used to capture targets onto a solid surface or associated with a fluorescent chemical probe to carry out drug binding experiments. PAs could also be attached to a target to alter its properties and make it more amenable structural determination by current methods. The primary aim of this project is to use PAs known as DARPins to aid the drug discovery process by facilitating the visualisation of small targets usually unsuitable for imaging by cryo EM. Cryo EM is a method by which biological specimens are observed using an electron microscope. Many intracellular proteins cannot currently be visualised by cryo EM due to their small size. DARPins consist of repeating modules, the number of which can be altered depending on the application to produce either small or large DARPins. They also have a rigid, helical structure when several repeating units are bound together. Therefore attaching DARPins through protein engineering to smaller protein targets could make them amenable to imaging via cryo EM by adding bulk and stability. This would offer a technological solution to biological problem through exploiting understanding of structural biology and protein-protein interactions. Following evaluation of DARPin scaffolds for imaging of known targets using cryo EM, the same method will be evaluated for imaging of novel drug targets with structures that remain unsolved. This will include targets which would be considered difficult to prosecute by other means, again due to their small size, flexibility or incompatibility with other structural methods. A cyclic peptides library will be screened against targets of interest, with the resultant high affinity cyclic peptides engineered on to the DARPin scaffold. The DARPin scaffold-target complex will then be used to determine the structure of the target using cryo EM. The overall aim is to implement this methodology of using PAs to enable cryo EM within the Newcastle University Center for Cancer drug discovery programme, in order to improve the interrogation of potential drug targets and facilitate structure based drug discovery.