Investigation of antiviral and anticancer nucleoside analog substrate recognition of drosophila melanogaster and herpes virus deoxyribonucleoside kinases

Abstract

The deoxyribonucleoside kinase of the fruit fly Drosophila melanogaster (Dm-dNK) is a multisubstrate enzyme that phosphorylates pyrimidine and purine deoxyribonucleosides as well as several anticancer and antiviral nucleoside analogs. Dm-dNK is sequence related to the human deoxycytidine kinase (dCK), deoxyguanosine kinase (dGK) and thymidine kinase 2 (M), as well as to the herpes simplex virus type-1 thymidine kinase (Hsv-1 TK). The human and viral deoxyribonucleoside kinases can phosphorylate multiple deoxyribonucleosides, whereas Dm-dNK has the ability to phosphorylate all naturally occurring deoxyribonucleosides required for DNA replication. In addition to its broad substrate specificity, Dm-dNK also exhibits higher catalytic rates for nucleoside and nucleoside analog phosphorylation compared to other nucleoside kinases.

Nucleoside kinases are being investigated for possible use as suicide genes in combined gene/chemotherapy of cancer. The most commonly studied nucleoside kinase suicide gene is the HSV-1 TK gene used in combination with the guanosine nucleoside analog ganciclovir. The suicide nucleoside kinase is ratelimiting in the pharmacological activation of the cytotoxic nucleoside analogs, and mutants of HSV-1 TK with improved biochemical properties for nucleoside analog phosphorylation are more efficient suicide genes. The broad substrate specificity of Dm-dNK and its high catalytic rate makes it an interesting candidate gene for suicide gene therapy. The possible use of Dm-dNK as a suicide gene has been evaluated and it has been shown that over-expression of Dm-dNK enhances the sensitivity of cancer cells to several cytotoxic nucleoside analogs.

Although Dm-dNK phosphorylates both purine and pyrimidine nucleosides, the enzyme has a preference for pyrimidine nucleosides. The maximal catalytic rate of purine and pyrimidine nucleoside phosphorylation is similar, but the enzyme exhibits higher affinity for pyrimidine nucleosides and nucleoside analogs. For suicide gene therapy application, purine nucleoside analogs may be preferred because these compounds appear to induce a higher bystander cell killing, i.e. killing of untransduced neighboring cells by transfer of phosphorylated nucleoside analogs via gap junctions.

The solved structures of Dm-dNK, dGK and HSV-1 TK, reveal a common folding of these enzymes and in particular the amino acid residues involved in substrate interactions are highly conserved. However, the substrate binding site also exhibits some major differences between Dm-dNK and HSV-1 TK. Based on this structural information we performed site directed mutagenesis of the residues Asn28, I1e29, Phe1 14 and G1n81 in order to understand the determinants of the substrate specificity of the enzyme and to find Dm-dNK mutants with improved kinetic properties for application in suicide gene therapy.

It has also been shown that Dm-dNK with 20 amino acid C-terminal deletion has even higher catalytic rates for deoxyribonucleosides compared to wild-type, and it was also reported that the mutagenesis of a few amino acids allows to change the substrate specificity from pyrimidines to purines. Based on these previous studies we constructed the reported mutated enzymes and designed new mutations, with and without the 20 amino acid C-terminal deletion. We measured the ability to phosphorylate ganciclovir (GCV). We have finally selected the most efficient enzymes phosphorylating GCV and expressed in an osteosarcoma TK cell line and determined the sensitivity to nucleoside analogs. The cells expressing the Met88Arg mutant enzyme showed the highest increased sensitivity to purine nucleoside analogs with 8 to 80-fold decreased IC50 compared to untransduced control cells or cells expressing the wild-type nucleoside kinase.

We have also created a Dm-dNK protein targeted to the mitochondrial matrix by fusing a mitochondrial targeting signal to the N-terminus of the protein. We showed that the mitochondrial Dm-dNK was enzymatically active and that overexpression of the enzyme in an osteosarcoma TK‾deficient cell line resulted in an increased sensitivity to some nucleoside analogs such as 1-β-D-arabinofuranosylthymine (araT), (E)-5-(2-bromovinyl)-2''-deoxyuridine (BVDU), 5-bromo-2'' eoxyuridine (5-Br-dUrd) and 5fluoro2''deoxyuridine (S-F-dUrd). Labeling studies using [3H]-dThd also showed that mitochondrial expression of Dm-dNK, compared to nuclear expression, resulted in a higher specific [3H]-dTTP activity in the total MP pool and as a result a higher rate of [3H]-dTTP incorporation into nuclear DNA.

We conducted structural studies on HSV-1 TK. In particular we performed site-directed mutagenesis on A1a167 and A1a168 of the HSV-1 TK. The mutated A1a168His and A1a167Phe enzymes turned out to have knocked-out the dThd activity while retaining full GCV phosphorylation ability.