Protein prenylation is the enzymatic addition of isoprenoid substrates to the C-terminus of a “CAAX” peptide sequence, were “C” represents cysteine, “A” represents an aliphatic amino acid and “X” is a variable amino acid. Design Ankyrin Repeat Proteins (DARPins) are an alternative binding scaffold that can be engineered to recognize specific targets with high affinity and selectivity. A C-terminal CVIA motif was added to the DARPins. This motif allows the DARPins to undergo protein prenylation with the enzyme protein farnesyltransferase (PFTase). PFTase naturally catalyzes the transfer of the farnesyl moiety from its natural substrate farnesyl diphosphate (FPP) to the cysteine of the “CAAX” motif. PFTase has promiscuous substrate specificity. This attribute enables PFTase to accept modifications of its natural substrate FPP. Shown here is the synthesis of a farnesyl analog functionalized with a terminal azide. This azide functionality allows for copper-free azide-alkyne cycloaddition. Utilizing this synthetic azide analog to modify the DARPins allows for the subsequent site-specific labeling with an alkyne containing fluorophore. The DARPins used in this study were engineered to selectively bind to epithelial cell adhesion molecules (EpCAM). EpCAM is a protein that is over expressed in the membrane of various cancer cells, including breast or pancreas tumors. By introducing these fluorescently modified DARPins the visualization of the EpCAM in the cell membrane of these cancer cells becomes possible. This selective targeting method could be used to selectively locate cancerous cells <i>in vivo</i> when the azide is coupled with PET imaging reagent instead of a fluorophore.

Utilizing protein prenylation to modify EpCAM targeting DARPins with an azide-containing isoprenoid analogue

255th National Meeting (2018)

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