Rational Tuning of Intracellular Release Kinetics in PROTAC–Peptide Conjugates via Positional Modulation of Functional Peptide Domains

Abstract Proteolysis-targeting chimeras (PROTACs) can induce degradation of otherwise challenging proteins, but their large size and unfavorable physicochemical properties often limit cellular delivery. We designed PROTAC–peptide conjugates containing a nona-arginine cell-penetrating peptide (R9), a redox-sensitive disulfide linker, and an aromatic-rich peptide sequence previously described as an endosomal escape domain (EED). The EED sequence was positioned at either the N- or C-terminus to examine how peptide-domain arrangement affects intracellular fluorescence, payload release, and BRD4 degradation. At 10 and 100 nM, the C-terminal variant MZ1-R9-EED produced a greater reduction in BRD4 abundance at 24 h than MZ1-R9 and MZ1-EED-R9, although the two-concentration comparison did not permit determination of DC50, Dmax, or hook effects. FAM-based flow cytometry yielded pH-sensitive cell-associated fluorescence signals that could not be interpreted as an absolute measure of total cellular uptake. Qualitative confocal microscopy showed punctate fluorescence with apparent LysoTracker overlap but did not provide quantitative evidence of endosomal escape. Under a defined cell-free reducing condition, MZ1-R9-EED underwent faster apparent disulfide cleavage, with a half-life of 0.21 h versus 0.36–0.47 h for the other conjugates, and yielded higher apparent intracellular free MZ1 levels under identical analytical conditions. Square-wave voltammetry revealed no substantial differences in reduction potential, whereas molecular dynamics simulations suggested a possible contribution of local steric accessibility. These findings associate peptide-domain positioning with cleavage kinetics, free-payload levels, and BRD4 reduction, identifying domain arrangement as a design variable for disulfide-linked PROTAC–peptide conjugates without establishing cleavage as the rate-limiting or causal mechanism.