Poster Presentation The 48th Lorne Conference on Protein Structure and Function 2023

High-throughput techniques enable structure guided drug discovery against an inflammatory target: NLRP3 (#424)

Andrew P Thompson 1 2 3 , Tryfon Zarganis-Tzitzikas 3 , Martin Lowe 4 , Emma Murphy 3 , Frank von Delft 2 , Paul E Brennan 3
  1. Walter and Eliza Hall Institute, Parkville, VIC, Australia
  2. Centre for Medicines Discovery, University of Oxford, Oxford, Oxfordshire, United Kingdom
  3. Oxford Drug Discovery Institute, University of Oxford, Oxford, Oxfordshire, United Kingdom
  4. Exscientia, Oxford, Oxfordshire, United Kingdom

The NLRP3 inflammasome is a key regulator of the pyroptosis response in the innate immune system. It is through this highly regulated signalling cascade that macrophages respond to danger and damage associated molecules. Consequently, NLRP3 mutants are associated with inflammatory diseases. Targeting NLRP3 with small molecule inhibitors is identified as an attractive strategy for the treatment of cancer and neurodegenerative diseases such as Alzheimer’s disease. Currently, no NLRP3 inhibitors have reached the clinic, causing the hunt for alternate scaffolds to garner broad pharmaceutical investment.

 

Progress in the structural investigation of NLRP3 has been notedly challenging due to its inherent instability and membrane association, which has hampered the establishment of a structure-guided drug discovery pipeline that can consistently deliver high-resolution structural information. In recent years, several high impact structures of the various domains and binding partners of NLRP3 have revealed its biological mechanism. Among these studies, it was confirmed that the NACHT nucleotide hydrolysis domain is the target for current small molecules, however the highest published resolution of this domain is 2.8Å, limiting medicinal chemistry efforts to optimise inhibitors. In collaboration with the pharmaceutical company Exscientia, the Oxford Drug Discovery Institute aimed to improve upon this resolution using an in-house structure guided drug discovery pipeline.

 

Here, we describe the use of high-throughput cloning, expression, purification and crystallography techniques for the development of a structure-guided drug discovery pipeline against the NACHT domain of NLRP3. Using this approach, 8 high resolution structures of NLRP3 bound to lead molecules have been solved (1.9-2.5Å), greatly informing medicinal chemistry efforts. The use of formalised high-throughput processes was crucial to overcoming the technical challenges of working with an unstable protein. Furthermore, broad assessment of compounds and the N- and C-terminal boundaries for the NLRP3 NACHT domain constructs greatly influenced the crystallisation habit and the resulting resolution. The techniques and mindset employed in this program are broadly applicable to many projects where higher throughput and accuracy of structure guided small molecule development would be beneficial.