Cell-Dependent Activation of ProTide Prodrugs and Its Implications in Antiviral Studies

Abstract

The ProTide prodrug design is a powerful tool to improve cell permeability and enhance the intracellular activation of nucleotide antiviral analogues. Previous in vitro studies showed that the activation of ProTide prodrugs varied in different cell lines. In the present study, we investigated the activation profiles of two antiviral prodrugs tenofovir alafenamide (TAF) and sofosbuvir (SOF) in five cell lines commonly used in antiviral research, namely, Vero E6, Huh-7, Calu-3, A549, and Caco-2. We found that TAF and SOF were activated in a cell-dependent manner with Vero E6 being the least efficient and Huh-7 being the most efficient cell line for activating the prodrugs. We also demonstrated that TAF was activated at a significantly higher rate than SOF. We further analyzed the protein expressions of the activating enzymes carboxylesterase 1, cathepsin A, histidine triad nucleotidebinding protein 1, and the relevant drug transporters P-glycoprotein and organic anion-transporting polypeptides 1B1 and 1B3 in the cell lines using the proteomics data extracted from the literature and proteome database. The results revealed significant differences in the expression patterns of the enzymes and transporters among the cell lines, which might partially contribute to the observed celldependent activation of TAF and SOF. These findings highlight the variability of the abundance of activating enzymes and transporters between cell lines and emphasize the importance of selecting appropriate cell lines for assessing the antiviral efficacy of nucleoside/nucleotide prodrugs.

Recent Advances in Molecular Mechanisms of Nucleoside Antivirals

“…Firstly, ProTides have a reduced risk of mitochondrial toxicity compared to the parent NA [ 117 , 118 ]. Secondly, when using ProTides, preferential metabolism in target organs and tissues can be achieved [ 119 ]. The mentioned enzymes are highly active in the liver, and they are the keys to the success of McGuigan’s prodrugs for HBV and HCV treatment [ 120 , 121 ].…”