Imaging agents for genetic disease: structural, chemical and cellular dissection

Background: Lysosomal storage diseases are the most prevalent genetic disease. Diagnostics for many are sub-optimal with both false positives and negatives, those that are treatable are done so via a combination of enzyme replacement therapy, “substrate reduction” and pharmacological chaperones.

Our objective is to use modern “activity-based probes” to (a) image and analyse levels of key enzymes in cell extracts (b) use structure based design to improve the chemistry and selectivity of these compounds (c) apply these compounds in living cells, in a diagnostic and therapy context (d) expand the use of these probes into the imaging of the anti-cancer target heparanase (e) extend these compounds for CryoEM tomography coupled to fluorescent imaging in order to visualize active enzyme in the lysosome, and track enzymes as they migrate through the cell compartments. ABPs are the most cutting edge and novel of chemical biology tools, and we have good proof of principle data (see refs 1-3, below). The work is extremely timely, as the first molecular chaperones come on the market and as the need for early diagnostics becomes clearer.

Our experimental approach will be to clone and express both homologs and human enzymes involved in diverse lysosomal storage diseases, we will then use preliminary in vitro data with fluorescent epoxides and aziridines that will be developed by the student in conjunction with Hermen Overkleeft in Leiden (where the student will have the opportunity to visit).

These compounds will be analysed at the 3D level by protein crystallography and second generation, specific compounds developed. These compounds will be applied to image enzymes in patient samples, and uses to assess the efficacy of chaperone and/or inhibitor treatments. The culmination of the PhD may be to combine the imaging agents, and independently fluorescently labelled protein to couple CryoEM tomography with fluorescent imaging for observation of these enzymes in living tissues.

References
Lysosomal Storage Disease
1. Jiang et al. Detection of Active Mammalian GH31 alpha-Glucosidases in Health and Disease Using In-Class, Broad-Spectrum Activity-Based Probes. ACS Central Science 2, 351-358 (2016).
2. Jiang et al., In vitro and in vivo comparative and competitive activity-based protein profiling of GH29 alpha-L-fucosidases. Chemical Sci 6, 2782-2789 (2015)

Heparanase and Cancer
3. Wu et al Structural characterization of human heparanase reveals insights into substrate recognition. Nature Struct Mol Biology 2015, 22, 1016–1022

Shortlisting will take place as soon as possible after the closing date and successful applicants will be notified promptly. Shortlisted applicants will be invited for an interview to take place at the University of York on a date to be confirmed. Candidates will be asked to give a 10 minute presentation prior to their interview by an academic panel.

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