DEVELOPMENT OF PROTEASE RESISTANT PEPTIDE REPORTERS FOR INTRACELLULAR ASSAYS

Dysregulation of various enzymatic pathways is implicated in a great deal of diseases. The ability to monitor enzyme activity within cells is becoming increasingly important, for promoting further discovery, as well as to enable early detection and patient monitoring during treatment. The use of peptide substrates for such assays is extremely advantageous, as they are the best mimics of the enzymes’ natural substrates. Furthermore, the ease of synthesizing peptides allows them to be easily modified for specialized function and detection, making them applicable to multiple types of assays[i]. While they work quite well for in vitro assays, they are incompatible with the cytosolic environment, as they are rapidly destroyed by cytosolic peptidases[ii], making them impractical to use for intracellular assays.

Herein a synthetically simple strategy for imparting resistance to cytosolic peptidases via dimerization of a peptide substrate with various linkers is presented. This method takes advantage of the predominance of aminopeptidases in the cytosol of cells[iii]. Dimerization at the N-terminus of a peptide is shown to efficiently “mask” it, thereby significantly slowing down the degradation by cytosolic peptidases, making it more amenable to use in intracellular assays.

[i] Wu, D., Sylvester, J.E., Parker, L.L., Zhou, G., Kron, S. J. Biopolymers 2010, 94, 475-486.

[ii] Yewdell, J.W., REits, E., Neefjes, J. Nat. Rev. Immumol. 2003, 3, 952-961.

[iii] Akkad, N., Schatz, M., Dengjel, J., Tenzer, S., Schild, H. Med. Microbiol. Immunol. 2012, 201, 463-473.