| Summary: | APETx2 is a peptide isolated from the sea anemone <em>Anthopleura elegantissima</em>.<em> </em>It is the most potent and selective inhibitor of acid-sensing ion channel 3 (ASIC3) and it is currently in preclinical studies as a novel analgesic for the treatment of chronic inflammatory pain. As a peptide it faces many challenges in the drug development process, including the potential lack of stability often associated with therapeutic peptides. In this study we determined the susceptibility of wild-type APETx2 to trypsin and pepsin and tested the applicability of backbone cyclisation as a strategy to improve its resistance to enzymatic degradation. Cyclisation with either a six-, seven- or eight-residue linker vastly improved the protease resistance of APETx2 but substantially decreased its potency against ASIC3. This suggests that either the <em>N</em>- or <em>C</em>-terminus of APETx2 is involved in its interaction with the channel, which we confirmed by making <em>N</em>- and <em>C</em>-terminal truncations. Truncation of either terminus, but especially the <em>N</em>-terminus, has detrimental effects on the ability of APETx2 to inhibit ASIC3. The current work indicates that cyclisation is unlikely to be a suitable strategy for stabilising APETx2, unless linkers can be engineered that do not interfere with binding to ASIC3.
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