Substrate Report for: Tenofovir-alafenamide

The prodrug tenofovir alafenamide (TAF) is a first-line antiviral agent for the treatment of chronic hepatitis B infection. TAF activation involves multiple steps, and the first step is an ester hydrolysis reaction catalyzed by hydrolases. This study was to determine the contributions of carboxylesterase 1 (CES1) and cathepsin A (CatA) to TAF hydrolysis in the human liver. Our in vitro incubation studies showed that both CatA and CES1 catalyzed TAF hydrolysis in a pH-dependent manner. At their physiological pH environment, the activity of CatA (pH 5.2) was approximately 1,000-fold higher than that of CES1 (pH 7.2). Given that the hepatic protein expression of CatA was approximately 200-fold lower than that of CES1, the contribution of CatA to TAF hydrolysis in the human liver was estimated to be much greater than that of CES1, which is contrary to the previous perception that CES1 is the primary hepatic enzyme hydrolyzing TAF. The findings were further supported by a TAF incubation study with the CatA inhibitor telaprevir and the CES1 inhibitor bis-(p-nitrophenyl) phosphate. Moreover, an in vitro study revealed that the CES1 variant G143E (rs71647871) is a loss-of-function variant for CES1-mediated TAF hydrolysis. In summary, our results suggest that CatA may play a more important role in the hepatic activation of TAF than CES1. Additionally, TAF activation in the liver could be affected by CES1 genetic variation, but the magnitude of impact appears to be limited due to the major contribution of CatA to hepatic TAF activation. Significance Statement Contrary to the general perception that carboxylesterase 1 (CES1) is the major enzyme responsible for tenofovir alafenamide (TAF) hydrolysis in the human liver, the present study demonstrated that cathepsin A (CatA) may play a more significant role in TAF hepatic hydrolysis. Furthermore, the CES1 variant G143E (rs71647871) was found to be a loss-of-function variant for CES1-mediated TAF hydrolysis.

ProTide technology is a powerful tool for the design of nucleoside/nucleotide analog prodrugs. ProTide prodrug design improves cell permeability and enhances intracellular activation. The hydrolysis of the ester bond of a ProTide is a determinant of the intracellular activation efficiency and final antiviral efficacy of the prodrug. The hydrolysis is dictated by the catalytic activity and abundance of activating enzymes. The antiviral agents tenofovir alafenamide (TAF) and sofosbuvir (SBV) are typical ProTides. Both TAF and SBV have also been proposed to treat patients with COVID-19. However, the mechanisms underlying the activation of the two prodrugs in the lung remain inconclusive. In the present study, we profiled the catalytic activity of serine hydrolases in human lung S9 fractions using an activity-based protein profiling assay. We evaluated the hydrolysis of TAF and SBV using human lung and liver S9 fractions and purified enzymes. The results showed that CatA and CES1 were involved in the hydrolysis of the two prodrugs in the human lung. More specifically, CatA exhibited a nearly 4-fold higher hydrolytic activity towards TAF than SBV, whereas the CES1 activity on hydrolyzing TAF was slightly lower than that for SBV. Overall, TAF had a nearly 4-fold higher hydrolysis rate in human lung S9 than SBV. We further analyzed protein expression levels of CatA and CES1 in the human lung, liver, and primary cells of the two tissues using proteomics data extracted from the literature. The relative protein abundance of CatA to CES1 was considerably higher in the human lung and primary human airway epithelial cells than in the human liver and primary human hepatocytes. The findings demonstrated that the high susceptivity of TAF to CatA-mediated hydrolysis resulted in efficient TAF hydrolysis in the human lung, suggesting that CatA could be utilized as a target activating enzyme when designing antiviral ester prodrugs for the treatment of respiratory virus infection.

Tenofovir alafenamide (TAF) is a prodrug of tenofovir (TFV) currently in clinical evaluation for treatment for HIV and hepatitis B virus (HBV) infections. Since the target tissue for HBV is the liver, the hepatic delivery and metabolism of TAF in primary human hepatocytes in vitro and in dogs in vivo were evaluated here. Incubation of primary human hepatocytes with TAF resulted in high levels of the pharmacologically active metabolite tenofovir diphosphate (TFV-DP), which persisted in the cell with a half-life of >24 h. In addition to passive permeability, studies of transfected cell lines suggest that the hepatic uptake of TAF is also facilitated by the organic anion-transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3, respectively). In order to inhibit HBV reverse transcriptase, TAF must be converted to the pharmacologically active form, TFV-DP. While cathepsin A is known to be the major enzyme hydrolyzing TAF in cells targeted by HIV, including lymphocytes and macrophages, TAF was primarily hydrolyzed by carboxylesterase 1 (CES1) in primary human hepatocytes, with cathepsin A making a small contribution. Following oral administration of TAF to dogs for 7 days, TAF was rapidly absorbed. The appearance of the major metabolite TFV in plasma was accompanied by a rapid decline in circulating TAF. Consistent with the in vitro data, high and persistent levels of TFV-DP were observed in dog livers. Notably, higher liver TFV-DP levels were observed after administration of TAF than those given TDF. These results support the clinical testing of once-daily low-dose TAF for the treatment of HBV infection.

Human immunodeficiency virus (HIV) continues to be a major health concern. AIDS-related deaths (acquired immunodeficiency syndrome) have decreased recently, but chronic liver disease is now a major cause of mortality among HIV patients. Widespread alcohol use is recognized to be a major contributing factor. Tenofovir alafenamide fumarate (TAF), one of the most used HIV drugs, requires hydrolysis followed by phosphorylation to produce tenofovir diphosphate, the ultimate anti-HIV metabolite. Carboxylesterase-1 (CES1), established to hydrolyze TAF, is known to catalyze transesterification in the presence of ethanol. The aim of the study was to test the hypothesis that metabolism-based interactions between TAF and ethanol negatively impact both efficacy and safety of TAF. To test this hypothesis, the metabolism of TAF was determined in human primary hepatocytes and with a large number of human liver samples (S9 fractions) in the presence or absence of ethanol. The metabolism was monitored by LC-MS/MS (liquid chromatography with tandem mass spectrometry) and the level of CES1 or CES2 was determined by Western blotting. Consistent with the hypothesis, TAF underwent transesterification in the presence of ethanol accompanied by decreased hydrolysis. The formation of tenofovir diphosphate (the therapeutically active metabolite) was significantly decreased. In addition, TAF but not its hydrolytic metabolite, was found to increase intracellular lipid retention, and the increase was enhanced by ethanol. These findings conclude that alcohol consumption, beyond commonly accepted poor adherence to HIV medications, directly impacts the efficacy and safety of TAF.

The prodrug tenofovir alafenamide (TAF) is a first-line antiviral agent for the treatment of chronic hepatitis B infection. TAF activation involves multiple steps, and the first step is an ester hydrolysis reaction catalyzed by hydrolases. This study was to determine the contributions of carboxylesterase 1 (CES1) and cathepsin A (CatA) to TAF hydrolysis in the human liver. Our in vitro incubation studies showed that both CatA and CES1 catalyzed TAF hydrolysis in a pH-dependent manner. At their physiological pH environment, the activity of CatA (pH 5.2) was approximately 1,000-fold higher than that of CES1 (pH 7.2). Given that the hepatic protein expression of CatA was approximately 200-fold lower than that of CES1, the contribution of CatA to TAF hydrolysis in the human liver was estimated to be much greater than that of CES1, which is contrary to the previous perception that CES1 is the primary hepatic enzyme hydrolyzing TAF. The findings were further supported by a TAF incubation study with the CatA inhibitor telaprevir and the CES1 inhibitor bis-(p-nitrophenyl) phosphate. Moreover, an in vitro study revealed that the CES1 variant G143E (rs71647871) is a loss-of-function variant for CES1-mediated TAF hydrolysis. In summary, our results suggest that CatA may play a more important role in the hepatic activation of TAF than CES1. Additionally, TAF activation in the liver could be affected by CES1 genetic variation, but the magnitude of impact appears to be limited due to the major contribution of CatA to hepatic TAF activation. Significance Statement Contrary to the general perception that carboxylesterase 1 (CES1) is the major enzyme responsible for tenofovir alafenamide (TAF) hydrolysis in the human liver, the present study demonstrated that cathepsin A (CatA) may play a more significant role in TAF hepatic hydrolysis. Furthermore, the CES1 variant G143E (rs71647871) was found to be a loss-of-function variant for CES1-mediated TAF hydrolysis.

ProTide technology is a powerful tool for the design of nucleoside/nucleotide analog prodrugs. ProTide prodrug design improves cell permeability and enhances intracellular activation. The hydrolysis of the ester bond of a ProTide is a determinant of the intracellular activation efficiency and final antiviral efficacy of the prodrug. The hydrolysis is dictated by the catalytic activity and abundance of activating enzymes. The antiviral agents tenofovir alafenamide (TAF) and sofosbuvir (SBV) are typical ProTides. Both TAF and SBV have also been proposed to treat patients with COVID-19. However, the mechanisms underlying the activation of the two prodrugs in the lung remain inconclusive. In the present study, we profiled the catalytic activity of serine hydrolases in human lung S9 fractions using an activity-based protein profiling assay. We evaluated the hydrolysis of TAF and SBV using human lung and liver S9 fractions and purified enzymes. The results showed that CatA and CES1 were involved in the hydrolysis of the two prodrugs in the human lung. More specifically, CatA exhibited a nearly 4-fold higher hydrolytic activity towards TAF than SBV, whereas the CES1 activity on hydrolyzing TAF was slightly lower than that for SBV. Overall, TAF had a nearly 4-fold higher hydrolysis rate in human lung S9 than SBV. We further analyzed protein expression levels of CatA and CES1 in the human lung, liver, and primary cells of the two tissues using proteomics data extracted from the literature. The relative protein abundance of CatA to CES1 was considerably higher in the human lung and primary human airway epithelial cells than in the human liver and primary human hepatocytes. The findings demonstrated that the high susceptivity of TAF to CatA-mediated hydrolysis resulted in efficient TAF hydrolysis in the human lung, suggesting that CatA could be utilized as a target activating enzyme when designing antiviral ester prodrugs for the treatment of respiratory virus infection.

Tenofovir alafenamide fumarate (TAF) is an oral phosphonoamidate prodrug of the HIV reverse transcriptase nucleotide inhibitor tenofovir (TFV). Previous studies suggested a principal role for the lysosomal serine protease cathepsin A (CatA) in the intracellular activation of TAF. Here we further investigated the role of CatA and other human hydrolases in the metabolism of TAF. Overexpression of CatA or liver carboxylesterase 1 (Ces1) in HEK293T cells increased intracellular TAF hydrolysis 2- and 5-fold, respectively. Knockdown of CatA expression with RNA interference (RNAi) in HeLa cells reduced intracellular TAF metabolism 5-fold. Additionally, the anti-HIV activity and the rate of CatA hydrolysis showed good correlation within a large set of TFV phosphonoamidate prodrugs. The covalent hepatitis C virus (HCV) protease inhibitors (PIs) telaprevir and boceprevir potently inhibited CatA-mediated TAF activation (50% inhibitory concentration [IC50] = 0.27 and 0.16 muM, respectively) in vitro and also reduced its anti-HIV activity in primary human CD4(+) T lymphocytes (21- and 3-fold, respectively) at pharmacologically relevant concentrations. In contrast, there was no inhibition of CatA or any significant effect on anti-HIV activity of TAF observed with cobicistat, noncovalent HIV and HCV PIs, or various prescribed inhibitors of host serine proteases. Collectively, these studies confirm that CatA plays a pivotal role in the intracellular metabolism of TAF, whereas the liver esterase Ces1 likely contributes to the hepatic activation of TAF. Moreover, this work demonstrates that a wide range of viral and host PIs, with the exception of telaprevir and boceprevir, do not interfere with the antiretroviral activity of TAF.

Tenofovir alafenamide (TAF) is a prodrug of tenofovir (TFV) currently in clinical evaluation for treatment for HIV and hepatitis B virus (HBV) infections. Since the target tissue for HBV is the liver, the hepatic delivery and metabolism of TAF in primary human hepatocytes in vitro and in dogs in vivo were evaluated here. Incubation of primary human hepatocytes with TAF resulted in high levels of the pharmacologically active metabolite tenofovir diphosphate (TFV-DP), which persisted in the cell with a half-life of >24 h. In addition to passive permeability, studies of transfected cell lines suggest that the hepatic uptake of TAF is also facilitated by the organic anion-transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3, respectively). In order to inhibit HBV reverse transcriptase, TAF must be converted to the pharmacologically active form, TFV-DP. While cathepsin A is known to be the major enzyme hydrolyzing TAF in cells targeted by HIV, including lymphocytes and macrophages, TAF was primarily hydrolyzed by carboxylesterase 1 (CES1) in primary human hepatocytes, with cathepsin A making a small contribution. Following oral administration of TAF to dogs for 7 days, TAF was rapidly absorbed. The appearance of the major metabolite TFV in plasma was accompanied by a rapid decline in circulating TAF. Consistent with the in vitro data, high and persistent levels of TFV-DP were observed in dog livers. Notably, higher liver TFV-DP levels were observed after administration of TAF than those given TDF. These results support the clinical testing of once-daily low-dose TAF for the treatment of HBV infection.

BACKGROUND: Tenofovir alafenamide (TAF) is a novel investigational prodrug of tenofovir (TFV) that permits enhanced delivery of TFV into peripheral blood mononuclear cells (PBMCs) and lymphatic tissues. A critical step in the intracellular metabolic activation of TAF is mediated by the lysosomal protease cathepsin A (CatA). Here, we investigated CatA levels together with intracellular metabolism and antiretroviral activity of TAF in primary CD4+ T-lymphocytes (CD4s) and monocyte-derived macrophages (MDMs) isolated from a demographically diverse group of blood donors. METHODS: CD4s and MDMs were prepared from fresh PBMCs. CatA levels were quantified in cell extracts by monitoring TAF hydrolysis using HPLC. Intracellular TAF metabolites were quantified by HPLC combined with mass spectrometry. Antiviral activities in activated CD4s and MDMs were determined using HIV-1 single-cycle reporter and p24 antigen production assays, respectively. RESULTS: The levels of CatA and intracellular TAF metabolites differed minimally in CD4s and MDMs among 13 tested donors. TAF was >600-fold and 80-fold more potent than parent TFV in CD4s and MDMs, respectively, and its relative range of antiviral activity across all tested donors was comparable to that of other HIV-1 reverse transcriptase inhibitors, with mean +/-sd (range) EC50 values of 11.0 +/-3.4 (6.6-19.9) nM and 9.7 +/-4.6 (2.5-15.7) nM in CD4s and MDMs, respectively. CONCLUSIONS: These results indicate consistent intracellular metabolism and antiretroviral potency of TAF in relevant target cells of HIV-1 infection across multiple donors of variable gender, age and ethnicity, supporting further clinical investigation of TAF.

9-[(R)-2-[[(S)-[[(S)-1-(Isopropoxycarbonyl)ethyl]amino] phenoxyphosphinyl]-methoxy]propyl]adenine (GS-7340) is an isopropylalaninyl phenyl ester prodrug of the nucleotide HIV reverse transcriptase inhibitor tenofovir (TFV; 9-[(2-phosphonomethoxy)propyl]adenine) exhibiting potent anti-HIV activity and enhanced ability to deliver parent TFV into peripheral blood mononuclear cells (PBMCs) and other lymphatic tissues in vivo. The present study focuses on the intracellular metabolism of GS-7340 and its activation by a variety of cellular hydrolytic enzymes. Incubation of human PBMCs in the presence of GS-7340 indicates that the prodrug is hydrolyzed slightly faster to an intermediate TFV-alanine conjugate (TFV-Ala) in quiescent PBMCs compared with activated cells (0.21 versus 0.16 pmol/min/10(6) cells). In contrast, the conversion of TFV-Ala to TFV and subsequent phosphorylation to TFV-diphosphate occur more rapidly in activated PBMCs. The activity of GS-7340 hydrolase producing TFV-Ala in PBMCs is primarily localized in lysosomes and is sensitive to inhibitors of serine hydrolases. Cathepsin A, a lysosomal serine protease has recently been identified as the primary enzyme activating GS-7340 in human PBMCs. Results from the present study indicate that in addition to cathepsin A, a variety of serine and cysteine proteases cleave GS-7340 and other phosphonoamidate prodrugs of TFV. The substrate preferences displayed by these enzymes toward TFV amidate prodrugs are nearly identical to their preferences displayed against oligopeptide substrates, indicating that GS-7340 and other phosphonoamidate derivatives of TFV should be considered peptidomimetic prodrugs of TFV.