γ-Secretase Cleaves Bifunctional Fatty Acid-Conjugated Small Molecules with Amide Bonds in Mammalian Cells

Connecting two small molecules, such as ligands, fluorophores, or lipids, together via a linker with amide bonds is a widely used strategy to generate synthetic bifunctional molecules for various biological and biomedical applications. Such bifunctional molecules have been used in live-cell experiments under the assumption that they should be stable in cells. However, we recently found that a membrane-targeting bifunctional molecule, composed of a lipopeptide and the small-molecule ligand trimethoprim, referred to as mgcTMP, underwent amide-bond cleavage in mammalian cells. In this work, we first identified γ-secretase as the major protease degrading mgcTMP in cells. We next investigated the intracellular degradation of several different types of amide-linked bifunctional compounds and found that <i>N</i>-terminally fatty acid-conjugated small molecules are susceptible to γ-secretase-mediated amide-bond cleavage. In contrast, amide-linked bifunctional molecules composed of two small molecules, such as ligands and hydrophobic groups, which lack lipid modification, did not undergo intracellular degradation. These findings highlight a previously overlooked consideration for the development and application of lipid-based bifunctional molecules in chemical biology research.