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 nucleotide-binding 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 cell-dependent 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. 

https://pubs.acs.org/doi/pdf/10.1021/acsptsci.3c00050

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

We assessed factors that determine the tissue-specific bioactivation of ProTide prodrugs by comparing the disposition and activation of remdesivir (RDV), its methylpropyl and isopropyl ester analogues (MeRDV and IsoRDV, respectively), the oral prodrug GS-621763, and the parent nucleotide GS-441524 (Nuc). RDV and MeRDV yielded more active metabolite remdesivir-triphosphate (RDV-TP) than IsoRDV, GS-621763, and Nuc in human lung cell models due to superior cell permeability and higher susceptivity to cathepsin A. Intravenous administration to mice showed that RDV and MeRDV delivered significantly more RDV-TP to the lung than other compounds. Nevertheless, all four ester prodrugs exhibited very low oral bioavailability (<2%), with Nuc being the predominant metabolite in blood. In conclusion, ProTides prodrugs, such as RDV and MeRDV, are more efficient in delivering active metabolites to the lung than Nuc, driven by high cell permeability and susceptivity to cathepsin A. Optimizing ProTides' ester structures is an effective strategy for enhancing prodrug activation in the lung. 

Differential Bioactivation Profiles of Different GS-441524 Prodrugs in Cell and Mouse Models: ProTide Prodrugs with High Cell Permeability and Susceptibility to Cathepsin A Are More Efficient in Delivering Antiviral Active Metabolites to the Lung.

The role of rapid molecular diagnostics in managing bloodstream infections: a microbiological insight

Additional file 3 of Tilorone mitigates the propagation of α-synucleinopathy in a midbrain-like organoid model

Lipid transfer proteins (LTPs) are crucial players in nonvesicular lipid trafficking. LTPs sharing a lipocalin lipid transfer domain (lipocalin-like proteins) have a wide range of biological functions, such as regulating immune responses and cell proliferation, differentiation, and death as well as participating in the pathogenesis of inflammatory, metabolic, and neurological disorders and cancer. Therefore, the development of small-molecule inhibitors targeting these LTPs is important and has potential clinical applications. Herein, we summarize the structure and function of lipocalin-like proteins, mainly including retinol-binding proteins, lipocalins, and fatty acid-binding proteins and discuss the recent advances on small-molecule inhibitors for these protein families and their applications in disease treatment. The findings of our Perspective can provide guidance for the development of inhibitors of these LTPs and highlight the challenges that might be faced during the procedures.

Recent Advances on Small-Molecule Inhibitors of Lipocalin-like Proteins.

Identification of Andrographolide as a novel FABP4 inhibitor for osteoarthritis treatment.

Cellular drug response (concentration required for obtaining 50% of a maximum cellular effect, EC50) can be predicted by the intracellular bioavailability (Fic) and biochemical activity (half-maximal inhibitory concentration, IC50) of drugs. In an ideal model, the cellular negative log of EC50 (pEC50) equals the sum of log Fic and the negative log of IC50 (pIC50). Here, we measured Fic’s of remdesivir, favipiravir, and hydroxychloroquine in various cells and calculated their anti-SARS-CoV-2 EC50’s. The predicted EC50’s are close to the observed EC50’s in vitro. When the lung concentrations of antiviral drugs are higher than the predicted EC50’s in alveolar type 2 cells, the antiviral drugs inhibit virus replication in vivo, and vice versa. Overall, our results indicate that both in vitro and in vivo antiviral activities of drugs can be predicted by their intracellular bioavailability and biochemical activity without using virus. This virus-free strategy can help medicinal chemists and pharmacologists to screen antivirals during early drug discovery, especially for researchers who are not able to work in the high-level biosafety lab.

Predicting In Vitro and In Vivo Anti-SARS-CoV-2 Activities of Antivirals by Intracellular Bioavailability and Biochemical Activity

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Identification of Andrographolide as a novel FABP4 inhibitor for osteoarthritis treatment.

Predicting In Vitro and In Vivo Anti-SARS-CoV-2 Activities of Antivirals by Intracellular Bioavailability and Biochemical Activity