Enzymes to die for: exploiting nucleotide metabolizing enzymes for cancer gene therapy

Abstract

Suicide gene therapy is an attractive strategy to selectively destroy cancer cells while minimizing unnecessary toxicity to normal cells. Since this idea was first introduced more than two decades ago, numerous studies have been conducted and significant developments have been made to further its application for mainstream cancer therapy. Major limitations of the suicide gene therapy strategy that have hindered its clinical application include inefficient directed delivery to cancer cells and the poor prodrug activation capacity of suicide enzymes. This review is focused on efforts that have been and are currently being pursued to improve the activity of individual suicide enzymes towards their respective prodrugs with particular attention to the application of nucleotide metabolizing enzymes in suicide cancer gene therapy. A number of protein engineering strategies have been employed and our discussion here will center on the use of mutagenesis approaches to create and evaluate nucleotide metabolizing enzymes with enhanced prodrug activation capacity and increased thermostability. Several of these studies have yielded clinically important enzyme variants that are relevant for cancer gene therapy applications because their utilization can serve to maximize cancer cell killing while minimizing the prodrug dose, thereby limiting undesirable side effects.

Fig. 1

Overview of suicide gene therapy.

Fig. 2

Prodrug activation pathways and mechanisms of action. A representative prodrug for each suicide enzyme and their corresponding mechanism(s) of action are denoted in this figure. CD, cytosine deaminase; HSVTK, Herpes Simplex Virus thymidine kinase; dCK, deoxycytidine kinase; PNP, E. coli purine nucleoside phosphorylase; Dm-dNK, Drosophila melanogaster deoxynucleoside kinase; 5FC, 5-fluorocytosine; MeP-dR, 9-(2-deoxy-β-D-arabinofuranosyl)-6-methylpurine; GCV, ganciclovir; dFdC, gemcitabine; AZT, azidothymidine. Dotted lines = enzyme inhibition.

Fig. 3

Crystal structures of suicide enzymes. Residues targeted by mutagenesis are highlighted in red and the substrate of each enzyme is highlighted in orange. (A) HSVTK (PDB: 1K12), (B) bCD (PDB: 1K70), (C) yCD (PDB: 1P60), (D) Dm-dNK (PDB: 10T3), (E) dCK (PDB: 2A30), (F) E. coli PNP (PDB: 1PK7).