Development of an improved SMALP toolkit to extract active membrane proteins

Life depends on tiny machines within our bodies made from protein. These machines have a wide range of functions from digesting our food, through to defending us from infection and allowing us to see. One of most important classes of protein machines is those that live in the membrane that surround cells. These membrane proteins allow nutrients into the cell and allow waste products to leave; they also mediate the movement of signals into and out of the cell. These functions make them exceptionally interesting to researchers, but perhaps more importantly they are also the most important targets for developing new therapeutics for plant and animal health. For example more than 50% of all drugs used today target these membrane proteins.

Unfortunately these proteins have proved to be exceptionally difficult to study and have confounded a large number of scientists over the past 50 years. In 2009 we found an entirely new way of making these proteins by using a simple polymer more commonly found in car dashboards and paint. This method "wraps" the protein in a protective polymer "belt" that keeps it stable allowing us to study it. This new method has opened up a whole range of new opportunities for scientists to study these important proteins. This has included significant interest from scientists in pharmaceutical companies who have found that the method makes it a lot easier for them to develop new drugs.

However as with any method, our method has some small but significant limitations that mean that it cannot currently be used to study all aspects of how these protein machines work. For example our method prevents these proteins from changing shape. Such changes are often important for the function of the protein. You could say that the polymer belt is not elastic enough! In this project we aim to develop a new improved "tool kit" that contains new polymers and new methods that we hope will allow these proteins to be studied. We are hoping to first work with academic scientists to ensure that the methods work with the most important families of proteins encountered in life. We will then extend our collaborations to encompass commercial scientists ensuring that progress in developing new drugs and agrochemicals continues at a rapid rate.

Understanding membrane protein function is fundamental to our understanding of biology. Their importance in our lives is illustrated by the fact that more than 50% of therapeutics and 70% of agrochemicals target membrane proteins. However biochemical and structural studies of membrane proteins remain frustratingly challenging with, for example, only 1 in every 50 protein structures being of membrane proteins.

One of the most important issues confronting membrane protein studies is the process of extraction from membranes to produce a sample that is pure, active and stable.

In 2009 we developed the first method that allowed the extraction of membrane proteins from cell membranes complete with their local lipid environment intact. The method is based on the use of a simple polymer, Styrene Maleic Acid (SMA) which are able to auto-assemble in lipid membranes to excise a 10 nm diameter disc (SMALP) which contains the membrane protein and local lipid. We showed that the method extracts a wide range of membrane proteins from membranes to produce samples with high stability and activity. The samples are amenable to a wide range of downstream studies and have been used to produce X-ray and cryo-EM structures.

The method has been adopted by a wide range of commercial and academic scientists. This widespread use has highlighted that while for most proteins the method works well, it is not completely successful for a minority of proteins, yielding samples with diminished activity.

In this project we will generate an improved "tool kit" of new polymers and methods that will be developed using proteins that have shown low activity in SMALPs and that are both academically and commercially important (e.g. GPCRs, Cytochrome P450s & ABC transporters). The generation of these methods will be accelerated using a novel HT method for SMALP optimisation and will provide a route for generating bespoke methods for other challenging membrane proteins.

This project is the direct product of discussions with academic and commercial scientists who have used the SMALP method successfully and that are keen to see it developed to address it's limitations. As a result the project is unashamedly a methods development project which is not focused on providing direct insights into specific biological mechanisms. The project will instead produce new reagents and protocols that address the needs of scientists working on membrane proteins helping them to produce direct biological insights. In addition we have ensured that the work will also address commercial opportunities which will inevitably benefit the population as a whole. In this section we will delineate these impacts.

PRIMARY IMPACT
The project will provide tools that will allow an improved knowledge of membrane protein structure and function which is of direct benefit to industry (see letters of support from Medimmune, UCB, Sanofi, Domainex, Iontas and Syngenta). Membrane proteins represent 50% of pharmaceutical and 70% of agrochemical targets making them fundamentally important to both sectors. The team's close collaboration with these industries (TD, MW and SK have all been funded by companies in these sectors) will ensure that the outputs of the project will be tailored to the requirements of industry. In addition new reagents produced in the project that have commercial potential, will be exploited through Polyscope (who both TD & BK collaborate with) the largest producer of SMA for bio-research, enabling the growth of this company (see letter of support from Polyscope).

SECONDARY IMPACT
Improvements in the SMALP method will ensure a wider range of membrane proteins will be amenable to study increasing data on the structure and function of these proteins. This will improve the efficiency of commercial discovery pipelines. The availability of a wider range of conformational subtypes of target proteins will also improve the hit rate for agent discovery reducing costs and thereby the eventual price of the compound.

TERTIARY IMPACT
The development of a new SMA toolkit for producing new commercial targets will influence productivity of both the pharmaceutical and agrochemical sectors. This in turn will positively influence the provision of food and healthcare. An improvement in both areas has obvious benefits for society. In the case of food security, future predictions are stark. To feed the growing population, farmers will need to achieve at least a 70% increase in food production by 2050. This will be a real challenge considering the megatrends of growing population, greater affluence, and increasing urbanization. Improved agrichemicals will play a big role in attaining these goals. For the pharmaceutical industry the challenge to find new targets and hence therapeutics to address disease remains central. Much of the low hanging fruit in terms of targets have been addressed. The SMALP technology allows previously challenging targets to enter the drug discovery pathway widening the opportunities for drug development.

IMPACT ON UKPLC
Given that these developments are going to be carried out by a UK research group using UK research infrastructure in collaboration with an number of UK companies it is clear that success will have significant impact on UKplc. Central to this is an increase in competitiveness of the UK agrochemicals and pharmaceuticals industry in the world market.

IMPACT ON PUBLIC SERVICE AND POLICY
The UK government has highlighted food security and health as cornerstones of it's innovation goals (reflected in RCUK strategy). Success in this project will play a small but significant role in achieving these goals.