Intracellular Activation of Tenofovir Alafenamide and the Effect of Viral and Host Protease Inhibitors.
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 μM, 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.
Gilead, Foster City, California, USA gabriel.birkus@gilead.com.
Gabriel
G
Birkus
Gilead, Foster City, California, USA.
Rujuta A
RA
Bam
Gilead, Foster City, California, USA.
Madeleine
M
Willkom
Gilead, Foster City, California, USA.
Christian R
CR
Frey
Gilead, Foster City, California, USA.
Luong
L
Tsai
Gilead, Foster City, California, USA.
Kirsten M
KM
Stray
Gilead, Foster City, California, USA.
Stephen R
SR
Yant
Gilead, Foster City, California, USA.
Tomas
T
Cihlar
D000225:Adenine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000409:Alanine; D019380:Anti-HIV Agents / Q000378:metabolism* / Q000494:pharmacology; D001711:Biotransformation; D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects* / Q000201:enzymology / Q000821:virology; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism; D043402:Cathepsin A / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000069547:Cobicistat / Q000494:pharmacology; D004347:Drug Interactions; D015870:Gene Expression; D057809:HEK293 Cells; D015497:HIV-1 / Q000187:drug effects / Q000254:growth & development; D006367:HeLa Cells; D054884:Host-Pathogen Interactions; D006801:Humans; D009842:Oligopeptides / Q000494:pharmacology; D061251:Primary Cell Culture; D011355:Prodrugs / Q000378:metabolism* / Q000494:pharmacology; D011392:Proline / Q000031:analogs & derivatives / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D000068698:Tenofovir / Q000378:metabolism* / Q000494:pharmacology / Q000031:analogs & derivatives
D019380:Anti-HIV Agents; D009842:Oligopeptides; D011355:Prodrugs; D034741:RNA, Small Interfering; D015842:Serine Proteinase Inhibitors; C486464:telaprevir; C512204:N-(3-amino-1-(cyclobutylmethyl)-2,3-dioxopropyl)-3-(2-((((1,1-dimethylethyl)amino)carbonyl)amino)-3,3-dimethyl-1-oxobutyl)-6,6-dimethyl-3-azabicyclo(3.1.0)hexan-2-carboxamide; D000068698:Tenofovir; D011392:Proline; D002265:Carboxylic Ester Hydrolases; C474609:CES1 protein, human; C468629:CTSA protein, human; D043402:Cathepsin A; C442442:tenofovir alafenamide; D000225:Adenine; D000069547:Cobicistat; D000409:Alanine