This talk will describe how we used clickable photoaffinity probes for (off-)target identification/validation and to measure target engagement in live cells for β-secretase BACE1 inhibitors relevant to Alzheimer’s Disease. We generated a clickable photoprobe analog of a lead Pfizer BACE1 inhibitor that exhibited ocular toxicity in rats and performed photoaffinity labeling studies in live cells. We found that the photoprobe labeled the off-target Cathepsin D (CatD) in retinal pigment epithelium (RPE) cells and used it to create the first cellular CatD target engagement assay. Many of the BACE inhibitors that we tested showed &gt;100-fold potency increases for CatD in live cells compared to purified enzyme – resulting in a significant lowering of the BACE1/CatD selectivity window and providing an explanation for the ocular toxicity phenotype. In addition, we developed a platform to image organelle-targetable HaloTag proteins with clickable HaloTag ligands in various subcellular compartments and progress toward developing trans-cyclooctene BACE probes for imaging will be described.

Clickable photoaffinity probes to evaluate the selectivity of β-secretase inhibitors in live cells

253rd National Meeting (2017)

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