The selective and temporal control of protein activity in living cells provides a powerful tool to manipulate cellular function and to develop pro-protein therapeutics (PPT) for targeted therapy. In this work, we reported a facile but general chemical approach to design PPT by modulating protein activity in response to endogenous enzyme of disease cells, and its potential for targeted cancer therapy. We demonstrated that the chemical modification of a protein with quinone propionic acid (QPN), a ligand that could be reduced by tumor-cell-specific NAD­(P)H dehydrogenase [quinone] 1 (NQO1), was reversible in the presence of NQO1. Importantly, the QPN-modified cytochrome c (Cyt c-QPN) and ribonuclease A (RNase A-QPN) showed NQO1-regulated protein activity in a highly selective manner. Furthermore, the intracellular delivery of RNase A-QPN using a novel type of lipid-based nanoparticles, and subsequent protein activation by cellular NQO1, selectively inhibit cancer cell growth in vitro and effectively suppress tumor growth in vivo. We believe that our approach increases the number of potentially useful chemical tools for reversibly controlling the structure and function of protein using a disease-cell-specific enzyme, opening opportunities in the study of dynamic biological processes and developing precise protein therapeutics.

Proteolysis targeting chimera (PROTAC) technology is a chemical protein knockdown approach that degrades protein by hijacking the cellular ubiquitin-proteasome system. Its therapeutic potential, however, is difficult to be defined due to the lack of control over the cell selectivity of PROTACs, particularly if the therapeutic purpose is to be executed in a specific type of cells. Herein we report the design of Pro-PROTAC and its catalytic activation by endogenous enzyme overexpressed in cancer cells for cell-selective protein degradation. We demonstrate that the chemical modification of the binding site between PROTAC and E3 ligase with trimethyllocked quinone efficiently blocks the protein degradation capability of PROTAC. However, NAD(P)H quinone dehydrogenase 1 (NQO1), an enzyme overexpressed in cancer cells can reduce trimethyl-locked quinone to remove the chemical modification, and to activate NQO1-PROTAC for cancer cell-selective protein degradation.Further, we show that NQO1-catalyzed β-lapachone reduction can potentiate cellular oxidative stress to activate aryl boronic acid-caged ROS-PROTAC in living cells for bromodomain-containing protein 4 (BRD4) degradation with enhanced cell selectivity. Collectively, our strategy of designing Pro-PROTAC in response to endogenous species of disease cells expands the chemical biology toolbox for cell-selective protein degradation, and it would be of great interest for targeted therapeutics discovery.

The integration of reactive oxygen species (ROS)-responsive thioketal group into lipids nanoparticles enables the efficient delivery of siRNA into cells, and selectively cancer cell gene expression silencing in response to the high level of intracellular ROS in cancer cells.