Tissue-Specific Proteomics Analysis of Anti-COVID-19 Nucleoside and Nucleotide Prodrug-Activating Enzymes Provides Insights into the Optimization of Prodrug Design and Pharmacotherapy Strategy

Abstract

Nucleoside and nucleotide analogs are an essential class of antivirals for COVID-19 treatment. Several nucleoside/nucleotide analogs have shown promising effects against SARS-CoV-2 in vitro ; however, their in vivo efficacy is limited. Nucleoside/nucleotide analogs are often formed as ester prodrugs to improve pharmacokinetics (PK) performance. After entering cells, the prodrugs undergo several enzymatic metabolism steps to form the active metabolite triphosphate nucleoside (TP-Nuc); prodrug activation is therefore associated with the abundance and catalytic activity of the corresponding activating enzymes. Having the activation of nucleoside/nucleotide prodrugs occur at the target site of action, such as the lung, is critical for anti-SARS-CoV-2 efficacy. Herein, we conducted an absolute quantitative proteomics study to determine the expression of relevant activating enzymes in human organs related to the PK and antiviral efficacy of nucleoside/nucleotide prodrugs, including the lung, liver, intestine, and kidney. The protein levels of prodrug-activating enzymes differed significantly among the tissues. Using catalytic activity values reported previously for individual enzymes, we calculated prodrug activation profiles in these tissues. The prodrugs evaluated in this study include nine McGuigan phosphoramidate prodrugs, two cyclic monophosphate prodrugs, two l -valyl ester prodrugs, and one octanoate prodrug. Our analysis showed that most orally administered nucleoside/nucleotide prodrugs were primarily activated in the liver, suggesting that parenteral delivery routes such as inhalation and intravenous infusion could be better options when these antiviral prodrugs are used to treat COVID-19. The results also indicated that the l -valyl ester prodrug design can plausibly improve drug bioavailability and enhance effects against SARS-CoV-2 intestinal infections. This study further revealed that an octanoate prodrug could provide a long-acting antiviral effect targeting SARS-CoV-2 infections in the lung. Finally, our molecular docking analysis suggested several prodrug forms of favipiravir and GS-441524 that are likely to exhibit favorable PK features over existing prodrug forms. In sum, this study revealed the activation mechanisms of various nucleoside/nucleotide prodrugs relevant to COVID-19 treatment in different organs and shed light on the development of more effective anti-COVID-19 prodrugs.

Activation of Tenofovir Alafenamide and Sofosbuvir in the Human Lung and Its Implications in the Development of Nucleoside/Nucleotide Prodrugs for Treating SARS-CoV-2 Pulmonary Infection

“…A more recent study, however, demonstrated that human lung tissues also have sufficient CatA and CES1 to activate RDV [13]. Our recent study found that CES1 protein abundance was over 200-fold higher in the liver than in the lung, while CatA protein expression was comparable between the lung and the liver [16]. As such, we hypothesized that a greater susceptivity to CatA and lower susceptivity to CES1 might facilitate the pulmonary activation of a ProTide.…”

“…Proteomics data were analyzed using the Spectronaut Pulsar software (version 11.0; Biognosys AG, Schlieren, Switzerland) with a spectral library generated from pooled HLS9 and HlungS9 in a previous study [16] and the human proteome FASTA file downloaded from UniProtKB. The human serine hydrolases proteomics data were extracted using a FASTA file containing 238 human serine hydrolases [16]. The normalized abundance of a specific serine hydrolase was the ratio of its intensity to the intensity of biotinylated BSA (internal standard) in each sample.…”

“…The estimated contribution of CatA and CES1 to TAF and SBV hydrolysis was calculated by timing the activity of rhCatA and rhCES1 with the enzyme abundance in a specific biological system (e.g., HlungS9, HLS9, and primary human cells) [16]. It was assumed that the activity of a recombinant human enzyme was similar to the activity of its native counterpart in human tissue S9 and primary human cells.…”

“…When evaluating an enzyme's capability to metabolize its substrate drug in a specific tissue, both the abundance and catalytic activity of the enzyme should be taken into consideration. Unlike CatA, which is expressed in a broad range of tissues [16,30], CES1 is predominantly expressed in the liver [16,31]. In the context of COVID-19, human respiratory systems, including the airway and lung tissues, are the major infection sites [32], and thus are the most important target activating sites for antiviral prodrugs.…”

“…The protein expression data of CatA, CES1, and HINT1 in human respiratory (lungs and airways) and hepatic systems were retrieved from the literature. The protein expression data in HLS9 and HLungS9 were obtained from two recent studies [13,16]. The protein expression data of autopsy samples from COVID-19 patients' lung and liver tissues were extracted from a recent study by Nie et al [26].…”