Engineered Toxins “Zymoxins” Are Activated by the HCV NS3 Protease by Removal of an Inhibitory Protein Domain

Abstract

The synthesis of inactive enzyme precursors, also known as “zymogens,” serves as a mechanism for regulating the execution of selected catalytic activities in a desirable time and/or site. Zymogens are usually activated by proteolytic cleavage. Many viruses encode proteases that execute key proteolytic steps of the viral life cycle. Here, we describe a proof of concept for a therapeutic approach to fighting viral infections through eradication of virally infected cells exclusively, thus limiting virus production and spread. Using the hepatitis C virus (HCV) as a model, we designed two HCV NS3 protease-activated “zymogenized” chimeric toxins (which we denote “zymoxins”). In these recombinant constructs, the bacterial and plant toxins diphtheria toxin A (DTA) and Ricin A chain (RTA), respectively, were fused to rationally designed inhibitor peptides/domains via an HCV NS3 protease-cleavable linker. The above toxins were then fused to the binding and translocation domains of Pseudomonas exotoxin A in order to enable translocation into the mammalian cells cytoplasm. We show that these toxins exhibit NS3 cleavage dependent increase in enzymatic activity upon NS3 protease cleavage in vitro. Moreover, a higher level of cytotoxicity was observed when zymoxins were applied to NS3 expressing cells or to HCV infected cells, demonstrating a potential therapeutic window. The increase in toxin activity correlated with NS3 protease activity in the treated cells, thus the therapeutic window was larger in cells expressing recombinant NS3 than in HCV infected cells. This suggests that the “zymoxin” approach may be most appropriate for application to life-threatening acute infections where much higher levels of the activating protease would be expected.

Suppression of hepatitis C virus by the flavonoid quercetin is mediated by inhibition of NS3 protease activity

“…In vivo inhibition of quercetin was also analysed in T‐Rex‐293 cells stably expressing the full‐length NS3‐4A protease (N‐ terminally fused to EGFP) under the inducible tetracycline promoter (Tet‐on NS3) model system [17]. These cells were transiently transfected with a plasmid encoding NS3 cleavable substrate, pCMV/MBP‐EGFP‐full 1b NS5AB‐CBD (Fig.…”

“…To assay the inhibition of NS3 catalytic activity in cells, we established a tetracycline‐inducible NS3 expression system based on the T‐REx™‐293 cell line (Invitrogen) as described [17].…”

“…The transfected cells were induced with 10 ng/mL tetracycline 24 h after transfection, at the same time quercetin (10 μg) or solvent was added. Twenty‐four hours later, the cells were washed, lysed and subjected to immunoblotting using polyclonal mouse serum anti‐MBP and anti‐β actin for loading control, followed by goat anti‐mouse antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) as described [17]. Western blots were analysed with the Odyssey infrared imaging system (LI‐COR Biosciences, Lincoln, NE, USA).…”

Protein Engineering: A New Frontier for Biological Therapeutics

“…The zymoxins were delivered to the cytosol as a transgene by an adenoviral vector or as a fusion protein containing the binding and translocation domain of Pseudomonas exotoxin A to facilitate entry of the zymogen into the cytosol of the target cells [51, 55]. The MazF system had the most promising results, being well tolerated by healthy cells, while eradicating Hepatitus C virus infected cells [55].…”

Fusion Proteins with Toxic Activity

“…In an in vitro model, the viral NS3 protease in hepatitis C virus (HCV) infected cells cleaved the linker between diphtheria toxin and a protective defensin or the spacer connecting ricin catalytic domain with a stalk peptide [25]. The presence of these proteases can be utilized to increase the specificity of therapeutic toxins that can eliminate infected cells.…”