Synchrotron technique illuminates small-molecule binding on p67phox
p67phox might, to some of us, sound like the name of a Star Wars robot but it is in fact a protein linked to oxidative stress in the human brain. Oxidative stress can cause harmful chain reactions that damage cells and may cause disease. Anders Bach and his team are trying to find out how to neutralize p67phox by targeting it with drug-like small-molecules. In order to get a more exact picture of where molecules bind to p67phox, Anders and his team applied for a HALOS research grant to be able to study the process with help of protein x-ray crystallography technique at MAX IV. We asked Anders a few questions about how it was experimenting at a large-scale synchrotron facility!
Anders, can you tell us about your experiment?
- We are interested in oxidative stress in the brain. We try to develop inhibitors of various proteins to see if these can be targeted with small molecules. Thereby we hope to pave the way for new treatment strategies. In the HALOS project we investigated the protein p67phox, which is part of a multi-domain protein complex called NADPH oxidase 2. This protein's main role is to produce superoxide, which causes damage in relation to several diseases. We have used fragment-based drug discovery in order to find new fragment hits towards p67phox, and we now wish to optimize these fragments to potent, biological active, and drug-like small-molecules. The specific experiments in this HALOS project were about confirming that our fragment hits actually bind p67phox and to find out exactly where on the protein they bind by using X-ray crystallography. Such information is essential for the subsequent chemical optimization phase.
What was it like to get started with the experiment?
- There is a lot of preparation to be done at home before travelling to the facility for doing the actual experiments. You should know all about your protein and compounds of interest, e.g. stability over time, DMSO tolerability, solubility etc. If unexpected things happen when you are on the site, you must be ready to quickly adjust the experimental setup to make the best use out of the valuable time you have. However, the facilities at MAX IV were very good and the assistance from the scientific staff very professional, so our data collection trips were very efficient.
What was the most challenging and rewarding part about experimenting at a large-scale facility?
- The most rewarding part is when you get to see the final data and think about what a huge setup it takes to generate them. So many costs, coordination, and time from dedicated and highly trained people have gone into it. You feel grateful to benefit from all this, and hope to pay back with some important scientific results. The most challenging part is perhaps that. You feel this is very important, so there is also a lot of pressure on you. Not only should the experiments be successful, but the cause must also be important. We want to and should do impactful things.
What type of information/data were able to collect that would not have been possible using an alternative method?
- Protein X-ray crystallography depends on large-scale synchrotron facilities. From an experimental point of view, our alternative would be computational docking, which is much less reliable; or 2D NMR experiments, which are less informative and give much less details about the fragment-protein interaction. In practice, X-ray crystallography is the only way forward when doing fragment-based drug discovery, especially when working with new targets where there is less prior knowledge to guide the hypotheses.