Rational Tuning of Intracellular Release Kinetics in PROTAC-Peptide Conjugates via Positional Modulation of Functional Peptide Domains.
5 affiliations
- 1Graduate School of Medical Life Science, Yokohama City University, 1-7-29, Yokohama 230-0045, Japan.
- 2Division of Organic Chemistry, National Institute of Health Sciences, 3-25-26 Tonomachi, Kawasaki 210-9501, Japan.
- 3Department of Chemistry and Life Science, Yokohama National University, Yokohama 240-8501, Japan.
- 4Division of Medicinal Safety Science, National Institute of Health Sciences, 3-25-26, Tonomachi, Kawasaki 210-9501, Japan.
- 5Division of Pharmaceutical Sciences, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan.
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, D max, 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.
Related
- Phosphorylation of spleen tyrosine kinase Y130 positively regulates intracellular signaling and functional responses in platelets
- Postelectroacupuncture Fecal Microbiota Transplantation (post‐EA FMT) Regulates Gastrointestinal Hormones in Functional Dyspepsia Rats Is Associated With Modulation of the Intestinal Microbiota‐Bile Acid Metabolic Pathway
- Comparative RP-HPTLC Lipophilicity Analysis of Bioactive 1,3,4-Thiadiazole–Resorcinol Conjugates
- Cell-resolved pharmacodynamic profiling reveals tissue- and cell-population-specific activity of antisense oligonucleotide chemical conjugates in vivo
- Anisomycin-induced ribosome collisions follow a dose-dependent positional rule, distinct from the UV-induced collision landscape
BibTeX
@article{miyamoto2026rational,
title = {{Rational Tuning of Intracellular Release Kinetics in PROTAC-Peptide Conjugates via Positional Modulation of Functional Peptide Domains.}},
author = {Maho Miyamoto and Atsuki Hirama and Kosuke Saito and Naoki Shida and Mahito Atobe and Hidetomo Yokoo and Yosuke Demizu},
journal = {ACS bio \& med chem Au},
year = {2026},
month = aug,
doi = {10.1021/acsbiomedchemau.6c00158},
url = {https://europepmc.org/article/MED/42921034},
note = {CC BY-NC-ND},
}
RIS
TY - JOUR
TI - Rational Tuning of Intracellular Release Kinetics in PROTAC-Peptide Conjugates via Positional Modulation of Functional Peptide Domains.
AU - Maho Miyamoto
AU - Atsuki Hirama
AU - Kosuke Saito
AU - Naoki Shida
AU - Mahito Atobe
AU - Hidetomo Yokoo
AU - Yosuke Demizu
PY - 2026
DA - 2026/08/20
JO - ACS bio & med chem Au
DO - 10.1021/acsbiomedchemau.6c00158
UR - https://europepmc.org/article/MED/42921034
AB - 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, D max, 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.
SN - 2694-2437
ER -