Controlling Toxicity of Peptide-Drug Conjugates by Different Chemical Linker Structures

The side effects of chemotherapy can be overcome by linking toxic agents to tumor-targeting peptides with cleavable linkers. Herein, this concept is demonstrated by addressing the humanY(1) receptor (hY(1)R), overexpressed in breast tumors, with analogues of the hY(1)R-preferring [F-7,P-34]NPY. First, carboxytetramethylrhodamine was connected to [F-7,P-34]NPY by an amide, ester, disulfide, or enzymatic linkage. Live imaging revealed hY(1)R-mediated delivery and allowed visualization of time-dependent intracellular release. Next, the fluorophore was replaced by the toxic agent methotrexate (MTX). In addition to linkage through the amide, ester, disulfide bond, or enzymatic cleavage site, a novel disulfide/ester linker was designed and coupled to [F-7,P-34]NPY by solid-phase peptide synthesis. Internalization studies showed hY(1)R subtype selective uptake, and cell viability experiments demonstrated hY(1)R-mediated toxicity that was clearly dependent on the linkage type. Fast release profiles for fluorophore-[F-7,P-34]NPY analogues correlated with high toxicities of MTX conjugates carrying the same linker types and emphasize the relevance of new structures connecting the toxophore and the carrier.