Molecular Basis of NDT-Mediated Activation of Nucleoside-Based Prodrugs and Application in Suicide Gene Therapy.

Javier Acosta,Cristina Fillat,Jesús Fernández-Lucas,Elena Pérez,Pedro A Sánchez-Murcia

doi:10.3390/biom11010120

Abstract

Herein we report the first proof for the application of type II 2′-deoxyribosyltransferase from Lactobacillus delbrueckii (LdNDT) in suicide gene therapy for cancer treatment. To this end, we first confirm the hydrolytic ability of LdNDT over the nucleoside-based prodrugs 2′-deoxy-5-fluorouridine (dFUrd), 2′-deoxy-2-fluoroadenosine (dFAdo), and 2′-deoxy-6-methylpurine riboside (d6MetPRib). Such activity was significantly increased (up to 30-fold) in the presence of an acceptor nucleobase. To shed light on the strong nucleobase dependence for enzymatic activity, different molecular dynamics simulations were carried out. Finally, as a proof of concept, we tested the LdNDT/dFAdo system in human cervical cancer (HeLa) cells. Interestingly, LdNDT/dFAdo showed a pronounced reduction in cellular viability with inhibitory concentrations in the low micromolar range. These results open up future opportunities for the clinical implementation of nucleoside 2′-deoxyribosyltransferases (NDTs) in cancer treatment.

Highlights

According to the World Health Organization (WHO), cancer was responsible for around 9.6 million deaths in 2018
Since dFAdo and d6MetPRib cannot be cleaved in mammal cells [33], LdNDT expressing cells could cleave these prodrugs, generating corresponding cytotoxic purine bases, which can readily diffuse across cell membranes, leading to a bystander activity in surrounding cells
Transfection of human cervical cancer (HeLa) cells with pLdNDT followed by dFAdo prodrug treatment revealed significant cytotoxicity at 1 μM dFAdo. At this concentration similar cytotoxicity was achieved with pLdNDT/dFAdo and 2-FAde, highlighting the efficiency of the enzymatic activity in transforming dFAdo into 2-FAde (Figure 5). These results indicate that the pro-drug dFAdo diffuses across cell membranes of tumor cells and is effectively metabolized to the diffusible cellular toxic compound 2-FAde within 24 h in cells that express LdNDT

Summary

Introduction

According to the World Health Organization (WHO), cancer was responsible for around 9.6 million deaths in 2018. Despite the great advances in its treatment, it is expected that this number will nearly double in 2040 [1]. One of the first strategies in cancer treatment was the use of nucleoside analogues as anticancer agents. In 2013, up to 13 purine and pyrimidine antimetabolites had been approved as chemotherapeutic agents by the Food and Drug Administration (FDA) [2]. The clinical use of nucleoside analogues as therapeutic molecules is often limited due to several drawbacks, such as insufficient drug concentrations in tumors, systemic toxicity, lack of selectivity for tumor cells, and the appearance of drug-resistant tumor cells [3,4].

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Results

Conclusion