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 human Y1 receptor (hY1 R), overexpressed in breast tumors, with analogues of the hY1 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 hY1 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 hY1 R subtype selective uptake, and cell viability experiments demonstrated hY1 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.