In Vivo activation of pro-protein therapeutics via chemically engineered enzyme cascade reaction

Abstract

Selective and temporal control over protein activity is of great importance for the advancement of the protein of interest into precise molecular medicine. Simply installing synthetic ligands to proteins for activity regulation, however, is often obscured by either nonspecificity or insufficient efficiency. This study reports a chemical approach in which enzymatic cascade reactions were designed for selective activation of pro-protein both in vitro and in vivo. Specifically, the system consisted of aromatic boronic-acid-modified nanoparticles, reactive oxygen species (ROS)-responsive pro-protein (RNase A-NBC), a small molecule drug, β-Lapachone (β-Lap), and strategically screened synthetic lipids, required for the assembly of the nanocomplexes. Once target-delivered into tumor cells, the reduction of β-Lap produces massive H2O2 in response to NAD (P)H quinone oxidoreductase 1 (NQO1), a tumor-specific enzyme, which triggers further induction by selective chemical modification of ROS-responsive cytosolic protein ribonuclease A (RNase A)-NBC, thus, switching from “inert” pro-protein to active therapeutics, that ultimately prohibit tumor cell growth. Moreover, the designed enzymatic cascade activation of the pro-protein was effective in vivo, displaying superior therapeutic efficacy to either the pro-protein alone or the β-Lap via tumor-targeted delivery and the consequent suppression of the tumor growth. As both RNase A and β-Lap have been evaluated clinically as antitumor therapeutics, our chemical multi-step cascade methodology is, therefore, a promising strategy for selective modulation of pro-protein chemistry in the living system for fundamental investigations, favorable toward potential anticancer applications.