Fragment-based Screening of Cellular Proteomics for Multi-target Drug Discovery

This project falls within the EPSRC Synthetic Organic Chemistry research area.
Fragment-based ligand and drug discovery (FBLD) has been effective in generating lead compounds for various targets. Fragments are small molecules with molecular weight ranging between 120-250 Dalton, and have generally less functionality and lower binding affinity compared to normal hits. A major advantage of using fragment screening for hit identification instead of fully functionalized drug-like molecules is that much fewer compounds are needed to cover the same amount of chemical space, making the screening process much more efficient. The detection techniques, such as X-ray crystallography, used in fragment-based screening could also provide structural understanding on fragment binding.
Fragment compounds were typically used for in-vitro assays with purified proteins for single-target screening. However, it has been recently demonstrated that differential fragment-protein interactions can be observed when cells are directly incubated with fragments in the medium.
These exciting results have inspired us to apply similar principles to fragment screening using cellular proteomics as measures of efficacy. Depicting global cellular responses using gene expression signatures to better understand induced perturbations in biological pathways and key interactions caused by drug-like molecules has been gaining popularity in the field of biomedicine. In this project, we propose to apply the concept of "connectivity map" to cellular proteomics by quantitatively measuring the changes in whole-cell protein levels induced by fragment compounds.
Overall, fragment-based screening using cellular proteomics as signatures can be extremely useful in providing insights in the absence of a distinct biological target. This project aims to demonstrate the effectiveness of such an approach in two separate cases.
Our first target system was a mouse microglia cell line, BV2, which has been reported to play an active role in multiple neurodegenerative conditions including Alzheimer's disease (AD). Microglia cells are the most abundant macrophage-like cells in the central nervous system (CNS), but they exhibit diverse phenotypes based on cellular context, making target-based screening techniques relatively challenging. Proteomic analysis of microglia cells treated with different stimuli may be able to provide a global view of the cellular changes to better elucidate key proteins involved in the processes of neurodegeneration. Our next target would be the Wnt/b-catenin signaling pathway. Many of the current efforts have been focused on down-regulating b-catenin in order to reduce cancer proliferation. Nonetheless, b-catenin plays an active role in maintaining neurons and therefore also a crucial target for neurodegenerative diseases.
Once a hit has been identified, analogues of the fragment structure will be synthesized and tested and reverse-matched against proteomics signatures generated by the disease model. Structure-activity relationship (SAR) may also be inferred if enough analogues are tested. The main part of the project will be dedicated to synthesizing functionally diverse molecules from fragment hits using various synthetic organic strategies. The ultimate goal would be to successfully develop lead compounds that reverses a majority of key changes in cellular proteomics of the disease model. These compounds could then be further tested in various animal models for in vivo efficacy.
In summary, this project aims to develop cellular screening of fragment compounds using quantitative proteomics to identify multiple biological pathways and interactions that are relevant to diseases of interest, as well as finding "hit" compounds that can be further functioalized, tested and potentially developed into drug molecules or probes.