Summary
The mismatch repair (MMR) system safeguards genomic stability by detecting and correcting replication errors. In some cancers, an active MMR system can help cancer cells survive lethal genomic stress. MMR can prevent the cancer cell from being detected by the immune system and can be involved in cancer resistances. Hence, MMR is an attractive therapeutic target in several pathological contexts.
Small-molecule development targeting MMR system can follow different approaches for covalent and non-covalent compounds, with native MS and HDX-MS as preferred analytical approach to map non-covalent binding.
In the present study, HDX-MS was first used to understand the mechanism of action of a protein heterodimer (>230kDa) involved in the MMR system. In-depth setup allowed measuring the DNA binding site on the protein complex as well as the ejection of an ADP molecule. For further development of inhibitors of the complex, binding site mapping of a tool compound was achieved. It highlighted a binding area corresponding to the one exposed in the presence of DNA. The structural changes induced by DNA in the heterodimer therefore represent a potential druggable pocket to investigate.
HDX-MS was applied as screening tool for drug development. Its use is often limited by time-consuming manual data curation, making it unsuitable for high-throughput hit validation. To address this, a single labeling time point combined with focused data curation was implemented.
Nevertheless, as the first compound tested indicated that its effect was possibly taking place remotely from the initially expected binding site area, data curation was extended successfully to the full protein sequences. This allowed the discovery of new promising druggable areas, close to the DNA binding site within the heterodimer. Single labelling time-point coupled to these ‘two-step’ approach (focused region / full sequences analysis) increase our throughput to select compounds of interest in focused areas of protein systems that are difficult to characterize otherwise. This approach is now routinely used as a biophysical validation tool to support structure-activity relationship (SAR) studies of low-micromolar.
