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

Abstract

Cellular drug response (concentration required for obtaining 50% of a maximum cellular effect, EC 50 ) can be predicted by the intracellular bioavailability (F ic ) and biochemical activity (half-maximal inhibitory concentration, IC 50 ) of drugs. In an ideal model, the cellular negative log of EC 50 (pEC 50 ) equals the sum of log F ic and the negative log of IC 50 (pIC 50 ). Here, we measured F ic 's of remdesivir, favipiravir, and hydroxychloroquine in various cells and calculated their anti-SARS-CoV-2 EC 50 's. The predicted EC 50 's are close to the observed EC 50 's in vitro. When the lung concentrations of antiviral drugs are higher than the predicted EC 50 '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.

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 mRNA expression data of the enzymes and transporters of interest were extracted from the Human Protein Atlas (HPA) (), in which the highest single-cell mRNA expression value found in a particular cell type or cell line was adopted. The mRNA expression of enzymes and transporters in Vero E6 and Calu-3 cells was extracted from a recent study . We selected several human tissue single cells for data analysis, including three types of human lung tissue cells: alveolar epithelial type 1 (AT1) and type 2 (AT2) cells, macrophages, endothelial cells, and two types of human bronchus cells, basal respiratory cells and respiratory ciliated cells.…”

“…The mRNA expression of enzymes and transporters in Vero E6 and Calu-3 cells was extracted from a recent study. 44 We selected several human tissue single cells for data analysis, including three types of human lung tissue cells: alveolar epithelial type 1 (AT1) and type 2 (AT2) cells, macrophages, endothelial cells, and two types of human bronchus cells, basal respiratory cells and respiratory ciliated cells. Among these, AT2 and respiratory ciliated cells are the major targets infected by SARS-CoV-2.…”

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

“…We analyzed the expressions of key enzymes and transporters involved in TAF and SOF activation and disposition in the cell lines by extracting proteomics data from the literature and the Human Protein Atlas (HPA) database. , The target activating enzymes include CES1, CatA, and HINT1, − and the target transporters are the influx transporters OATP1B1 and OATP1B3 and the efflux transporter P-gp, given that OATP1B1 and OATP1B3 facilitate the cellular uptake of TAF, and both TAF and SOF are the substrates of P-gp. ,, …”

“…The lysis buffer contained 70% methanol (v/v, methanol:water = 7:3), 0.5 μM 2-chloroadenosine, 5 μM adenosine-15 N5 5′-triphosphate (internal standards), 2% formic acid, 10% PhosStop, and 50 μM DTNB as the protectors for the MP-Nuc and TP-Nuc metabolites. 21 Then, each well was washed with 0.3 mL of water, and the solution was combined with the lysate from the well. The protein concentration was determined using the Qubit protein assay kit according to the manufacturer's instructions.…”

“…0.3 mL of ice-cold lysis buffer was then added to each well, and the cells were scraped and transferred to a 2 mL tube. The lysis buffer contained 70% methanol (v/v, methanol:water = 7:3), 0.5 μM 2-chloroadenosine, 5 μM adenosine- 15 N5 5′-triphosphate (internal standards), 2% formic acid, 10% PhosStop, and 50 μM DTNB as the protectors for the MP-Nuc and TP-Nuc metabolites . Then, each well was washed with 0.3 mL of water, and the solution was combined with the lysate from the well.…”