<!DOCTYPE html> Design

DESIGN

Overview

Team SYSU-Medicine designs the TAICHI, the Target-Activated In situ Cancer Hijack system. We aim to develop a more powerful oncolytic virus with strict regulations by combining prodrug enzyme system and riboswitch, providing a more effective and safer cancer treatment.

Oncolytic virus M1 directed prodrug enzyme system

What is virus directed prodrug enzyme system?

In Enzyme-activating prodrug therapy, an exogenous drug-activating enzyme is selectively expressed in tumor cells, which can convert the systemically administered prodrug to an active anticancer drug in tumors with high local concentration. When the delivering vector is virus, it is called virus directed enzyme prodrug therapy. In VDEPT system, the virus vector targets the prodrug-activating enzyme to tumor cells selectively and transduces the prodrug efficiently.

Enzyme

Nitroreductase
Escherichia coli Nitroreductase is an enzyme that is frequently used as a metabolic trigger for ‘directed’ prodrugs including gene-directed enzyme prodrug therapy and antibody-directed enzyme prodrug therapy . Nitroreductase can catalyse the reduction of nitro groups in a wide range of substrates to form the corresponding hydroxylamine product. The enzyme-mediated reduction of nitro groups to the corresponding hydroxylamines, which can be further metabolised to form cytotoxic DNA cross-linking agents, is the basis for the pharmaceutical application of nitroreductases as prodrug activators .

Prodrug

Actinomycin D
Actinomycin D is an important chemical drug that can exert their clinical effects via interference with DNA function by blocking DNA replication and gene transcription . It is reported that Actinomycin D can markedly enhance the growth of alphavirus by inhibited interferon production .

CB1954
CB1954 is a weak monofunctional alkylating agent, which is converted by the Escherichia coli bacterial enzyme Nitroreductase to a cytotoxic species [5-(aziridin-1-yl)-4-N-acetoxy-2-nitrobenzamide], which induces cell death by forming interstrand DNA cross-links . Cell killing by activated CB1954 is cell cycle independent.It is reported that in vitro, Nitroreductase-expressing cancer cell lines are up to 500-2000-fold more sensitive to CB1954 than parental cell lines .

Thus our TAICHI system, using Alphavirus M1-mediated Nitroreductase combined with CB1954, may offer tumor-specific cell killing, because only cells expressing Nitroreductase should be directly susceptible to CB1954 and enhance the bystander effect, where surrounding cells not expressing the enzyme are also killed by a cell-permeable metabolite.

Riboswitch

Riboswitch is an RNA-regulatory element, which can be used to control gene expression from mRNA. Riboswitch consists of a sensor domain comprised of an RNA aptamer and an actuator domain comprised of a ribozyme. By changing the RNA aptamer in the sensor domain, switches have been engineered to respond to different small molecules, such as theophylline and tetracycline. This ribozyme–aptamer switch is incorporated into the 3’ untranslated region (UTR) of a target mRNA, where ligand binding at the sensor domain leads to modulation of ribozyme cleavage and ultimately expression of the target gene.

Riboswitches can be implemented as safety switches to terminate replication of viruses, to enhance the safety. It has been shown that insertion of riboswitches into the untranslated genomic regions of viral genes in both an oncolytic adenovirus and measles virus led to a significant decrease in viral protein expression and replication upon addition of modulating ligand.

In our TAICHI system, in order to enhance the regulation of the Nitroreductase expression and the replication of M1, tandem riboswitches are inserted into the 3’ UTR of the replicon of M1. The riboswitches act to control Nitroreductase expression by regulating cleavage of the RNA in response to theophylline and the structure of the ribozyme is disrupted in the absence of theophylline, leading to lower Nitroreductase expression and replication of M1. Combining riboswitches, the safety of TAICHI system is increased.

Rational design

We are in attempt to get two synthetic drugs(figure 3 and 4). It is reported that lV-(4-nitrobenzyloxycarbonyl)-AMD(figure3) is a potential prodrug of Actinomycin D which can be activated by Nitroreductase. Besides, we design a novel prodrugs of Actinomycin D activated by carboxypeptidase G2(CPG2) (figure4). CPG2 catalyzes the scission of the amidic, urethanic or ureidic, linkage between an aromatic nucleus and L-glutamic acid. CPG2 has been expressed both intracellularly (called CPG2*) and tethered to the outer cell surface (called stCPG2(Q)3) in a range of tumor cell lines. The extracellular tethering overcomes the need for the prodrug to cross the tumor cell membrane for activation. This method of expression is expected to improve the bystander effect, whereby a prodrug is activated to an active drug that kills neighboring tumor cells not expressing the activating enzyme.

Summary

Our TAICHI system works as follows:

Oncolytic virus M1 can selectively infect tumor cells. In the absence of theophylline, engineered M1 can replicate within tumor and constantly express the Nitroreductase which can convert the prodrug into an active anticancer drug, providing an amplification effect and enhancing the bystander killing effect. Riboswitches are capable of regulating the gene expression of M1 to ensure the safety. When the theophylline is added, which allows the ribozyme to form its active conformation and initiate the cleavage of the RNA, the riboswitches act to turn off gene expression, therefore terminating replication of viruses irreversibly to ensure the safety. In all, TAICHI system utilizes different mechanisms with oncolytic viruses and chemical drugs to kill tumor cells, providing precise and efficient drug delivery and the enhancement of therapeutic efficacy.

REFERENCE

[1]Sharma K, Sengupta K, Chakrapani H, et al. Nitroreductase-activated nitric oxide (NO) prodrugs[J]. Bioorganic & Medicinal Chemistry Letters, 2013, 23(21): 5964-5967.

[2]Emptage C D, Knox R J, Danson M J, et al. Nitroreductase from Bacillus licheniformis:a stable enzyme for prodrug activation[J]. Biochemical Pharmacology, 2009, 77(1): 21-29.

[3]Wang S, Lee Y, Lai Y, et al. Spermine Attenuates the Action of the DNA Intercalator, Actinomycin D, on DNA Binding and the Inhibition of Transcription and DNA Replication[J]. PLOS ONE, 2012, 7(11).

[4]Gifford G E, Heller E. EFFECT OF ACTINOMYCIN D ON INTERFERON PRODUCTION BY 'ACTIVE' AND 'INACTIVE' CHIKUNGUNYA VIRUS IN CHICK CELLS.[J]. Nature, 1963, 200(4901): 50-51.

[5]Knox R. J., Friedlos F., Jarman M., Roberts J. J. A new cytotoxic, DNA interstrand crosslinking agent, 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide, is formed from 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) by a nitroreductase enzyme in Walker carcinoma cells. Biochem. Pharmacol., 37: 4661-4669, 1988.

[6]Green N. K., Youngs D. J., Neoptolemos J. P., Friedlos F., Knox R. J., Springer C. J., Anlezark G. M., Michael N. P., Melton R. G., Ford M. J., Young L. S., Kerr D. J., Searle P. F. Sensitization of colorectal and pancreatic cancer cell lines to the prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) by retroviral transduction and expression of the E. coli nitroreductase gene. Cancer Gene Ther., 4: 229-238, 1997.

[7]Chungfaye G, Palmer D H, Anderson D M, et al. Virus-directed, Enzyme Prodrug Therapy with Nitroimidazole Reductase: A Phase I and Pharmacokinetic Study of its Prodrug, CB1954[J]. Clinical Cancer Research, 2001, 7(9): 2662-2668.

Team:SYSU-Medicine/Design