Crystallographic fragment screening - improvement of workflow, tools and procedures, and application for the development of enzyme and protein-protein interaction modulators

Abstract

One of the great societal challenges of today is the fight against diseases which reduce life expectancy and lead to high economic losses. Both the understanding and the addressing of these diseases need research activities at all levels. One aspect of this is the discovery and development of tool compounds and drugs. Tool compounds support disease research and the development of drugs. For about 20 years, the discovery of new compounds has been attempted by screening small organic molecules by high-throughput methods. More recently, X-ray crystallography has emerged as the most promising method to conduct such screening. Crystallographic fragment-screening (CFS) generates binding information as well as 3D-structural information of the target protein in complex with the bound fragment. This doctoral research project is focused primarily on the optimization of the crystallographic fragment screening workflow. Investigated were the requirements for more successful screening campaigns with respect to the crystal system studied, the fragment libraries, the handling of the crystalline samples, as well as the handling of the data associated with a screening campaign. The improved CFS workflow was presented as a detailed protocol and as an accompanying video to train future CFS users in a streamlined and accessible way. Together, these improvements make CFS campaigns a more high-throughput method, offering the ability to screen larger fragment libraries and allowing higher numbers of campaigns performed per year. The protein targets throughout the project were two enzymes and a spliceosomal protein-protein complex. The enzymes comprised the aspartic protease Endothiapepsin and the SARS-Cov-2 main protease. The protein-protein complex was the RNaseH-like domain of Prp8, a vital structural protein in the spliceosome, together with its nuclear shuttling factor Aar2. By performing the CFS campaigns against disease-relevant targets, the resulting fragment hits could be used directly to develop tool compounds or drugs. The first steps of optimization of fragment hits into higher affinity binders were also investigated for improvements. In summary, a plethora of novel starting points for tool compound and drug development was identified.