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.
Recent innovations in genome editing have enabled the precise manipulation of the genetic information of mammalians, and benefitted the development of next‐generation gene therapy. Despite these advances, several barriers to the clinical translation of genome editing remain, including the intracellular delivery of genome editing machinery, and the risk of off‐target editing effect. Here, we review the recent advance of spatiotemporal delivery of CRISPR/Cas9 genome editing machinery, which is composed of programmable Cas9 nuclease and a single‐guide RNA (sgRNA) using stimuli‐responsive nanoparticles. We discuss the specific chemistries that have been used for controlled Cas9/sgRNA delivery and intracellular release in the presence of endogenous or external signals. These methodologies can leverage biological signals found locally within disease cells, or exogenous signals administrated with spatiotemporal control, through which an improved genome editing could be achieved. We also discuss the future in exploiting these approaches for fundamental biomedical applications and therapeutic genome editing.
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.
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