γ‑Secretase\nCleaves Bifunctional Fatty\nAcid-Conjugated Small Molecules\nwith Amide Bonds in Mammalian Cells

Connecting\ntwo small molecules, such as ligands, fluorophores,\nor lipids, together via a linker with amide bonds is a widely used\nstrategy to generate synthetic bifunctional molecules for various\nbiological and biomedical applications. Such bifunctional molecules\nhave been used in live-cell experiments under the assumption that\nthey should be stable in cells. However, we recently found that a\nmembrane-targeting bifunctional molecule, composed of a lipopeptide\nand the small-molecule ligand trimethoprim, referred to as mgcTMP,\nunderwent amide-bond cleavage in mammalian cells. In this work, we\nfirst identified γ-secretase as the major protease degrading\nmgcTMP in cells. We next investigated the intracellular degradation\nof several different types of amide-linked bifunctional compounds\nand found that <i>N</i>-terminally fatty acid-conjugated\nsmall molecules are susceptible to γ-secretase-mediated amide-bond\ncleavage. In contrast, amide-linked bifunctional molecules composed\nof two small molecules, such as ligands and hydrophobic groups, which\nlack lipid modification, did not undergo intracellular degradation.\nThese findings highlight a previously overlooked consideration for\nthe development and application of lipid-based bifunctional molecules\nin chemical biology research.