Relation Between Scaffold Size and Membrane Bound Enzyme Caused Degradation of Two Novel Cystine Knot Microproteins

Cystine knot microproteins are promising novel scaffolds for the oral delivery of pharmacophoric sequences. In order to cross mucosal membranes, these scaffolds should be stable towards membrane bound enzymes (MBE) of the intestinal mucosa. Therefore, two novel cystine-knot microprotein scaffolds have been evaluated regarding their stability towards MBE of excised rat small intestine and the most abundant membrane bound exopeptidase aminopeptidase N. It was shown, that SE-ET-TP-020 (37 amino acids) is degraded by membrane bound enzymes of rat intestinal mucosa, whereas SE-MC-TR-020 (32 amino acids) is not degraded by these enzymes. Neither of the two evaluated scaffolds was degraded by aminopeptidase N. The results of this study further support the hypothesis that scaffolds with 37 amino acids are more susceptible towards MBE caused degradation in comparison to scaffolds with about 30 amino acids.