(699.4) Contributions of Cathepsin A and Carboxylesterase 1 to the hydrolysis of Tenofovir Alafenamide in the Human Liver, and the Effect of CES1 Genetic Variation on Tenofovir Alafenamide Hydrolysis

Poster Board Number: B117

Jiapeng Li (University of Michigan), Jian Shi (University of Michigan), Haojie Zhu (University of Michigan)

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 700-1000-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.

Support or Funding Information

This work was partially supported by the National Institutes of Health National Heart, Lung, and Blood Institute [R01 HL126969, Hao-Jie Zhu], the Eunice Kennedy Shriver National Institute of Child Health and Human Development [R01 HD093612, John S. Markowitz and Hao-Jie Zhu].

lt;pgt;This work was partially supported by the National Institutes of Health National Heart, Lung, and Blood Institute [R01 HL126969, Hao-Jie Zhu], the Eunice Kennedy Shriver National Institute of Child Health and Human Development [R01 HD093612, John S. Markowitz and Hao-Jie Zhu].lt;/pgt;





Figure 1. (a) Activation pathway of TAF in the human liver. (b)TAF hydrolysis activity of rhCES1, rhCatA, and HLS9 in MES pH 5.2 assay buffer and Tris pH 7.2 buffer. (c) Effect of telaprevir and BNPP on TAF hydrolysis in HLS9 in MES pH 5.2 assay buffer and Tris pH 7.2 buffer. (d) Effect of the CES1 genetic polymorphism G143E on TAF activation in the S9 fractions of the transfected HEK 293 cells. Data are shown as mean ± S.D., n = 3. *P < 0.05, **P < 0.01 compared to the control.; Table 1. The catalytic activity of recombinant human CatA and CES1 on hydrolyzing TAF and the estimated relative contributions of CatA and CES1 to TAF hydrolysis in HLS9.