Improving tractability of membrane proteins as targets for drug discovery

Membrane proteins have already been identified as defining the most important drug targets for this millenium. Unfortunately, major technical challenges exist in gaining the structural information required to exploit them fully in modern, structure-based drug discovery. The key milestones in this process, which are each associated with specific challenges are: Screening for promising drug target production conditions --> Production and purification of the drug target --> Structural analysis to enable structure-based drug design Typically, the design of the initial screening step includes many variables, as the critical parameters are not yet clear in defining a successful production experiment. One variable, which is routinely assessed is the host cell. Mammalian cells, insect cells and micro organisms are often explored simultaneously as these different expression systems offer different benefits whilst having different limitations. This is an expensive, inefficient strategy, which could be streamlined by using only a single host cell. This work will therefore focus on optimising the success rate of yeast as an expression platform for drug discovery. Particular benefits of microbial eukaryotes such as yeasts are fast growth rates and the achievement of high cell density on comparatively low cost media. This means that high target yields can be obtained in relatively low culture volumes, which is attractive for high-throughput screening campaigns. To develop innovation in yeast expression and subsequent purification leading to a predictable, reproducible and reliable technology platform to produce membrane protein samples, mere trial and error is not likely to be successful. A systematic approach, based on scientific rationale is essential to tackle the complexity of this problem. Therefore we will approach the challenges of producing recombinant membrane proteins as a scientific problem requiring an understanding of underlying biological bottlenecks. This is in stark contrast to approaches used by the majority of laboratories which still rely on trial-and-error without ever understanding why most attempts fail. In brief this project will focus on three scientific objectives, which are in turn informed by the three milestones in the drug discovery workflow, detailed above: 1. Develop and evaluate efficient smart-throughput screening for expression of membrane proteins in yeast. The benefit of this will be to shorten timelines to identify successful combinations of strains, genetic modifications, co-expressed proteins, target construct variations and purification tags; 2. Develop and evaluate innovative cultivation regimes for membrane protein production. These methods and materials will help to improve yields of functional membrane proteins: a milestone in the drug discovery process is overcoming thresholds of 1 mg/L culture; 3. Develop and evaluate downstream enabling technologies for structural analysis of membrane proteins. This will improve the success rate of retrieving structural information. Structural information allows rational drug design, which is often key to successful drug discovery. In summary, the overall aim of this work is to overcome some of the bottlenecks in the drug discovery pipeline in exploiting membrane proteins as drug discovery targets more fully. This new approach will finally work towards predictable, reproducible and reliable methods to produce membrane protein samples. Ultimately this will allow their structural and functional characterisation, which lies at the heart of modern drug discovery.