Pharmacogenetic Analysis of Nucleotide Kinases that Metabolize the Antiviral Drug Tenofovir

Tenofovir (TFV) is a nucleotide analog used in HIV pre‐exposure prophylaxis, requiring two phosphorylation events for pharmacological activity. We aimed to determine the nucleotide kinases responsible for TFV activation in peripheral blood mononuclear cells (PBMC), vaginal tissue and colorectal tissue as this is currently unknown. In order to identify relevant kinases, siRNA knockdown was performed in PBMC and tissues, incubated with 10µM TFV and metabolites were measured using mass spectrometry. Adenylate kinase 2 (AK2) knockdown decreased TFV‐monophosphate production to 17 ± 1.4%, 9.5 ± 1.1% and 12 ± 1.5% of control in PBMC, vaginal tissue and colorectal tissue, respectively. Knockdown of muscle pyruvate kinase (PKM) decreased TFV‐diphosphate (TFV‐DP) production to 33 ± 5.8% and 27 ± 4.3% and knockdown of liver and red blood cell pyruvate kinase (PKLR) decreased TFV‐DP production to 78 ± 6.6% and 81 ± 7.4% of control in PBMC and vaginal tissue, respectively. Muscle creatine kinase (CKM) knockdown in colorectal tissue decreased TFV‐DP production to 8 ± 2.9% of control. Subsequently, clinical trial participants taking oral TFV were genotyped for variants in these 4 kinases and results were compared to pharmacokinetic data. Of the 148 previously unreported single nucleotide variants and deletions identified, bioinformatics tools predicted 10 AK2 variants (8% of 142 participants), 12 PKLR variants (7%) and 8 CKM variants (6%) to be deleterious. PKM variants were of unknown consequence. In conclusion, these data demonstrate AK2 to be a key contributor to TFV activation and genetic variation within the kinases AK2, PKM, PKLR and CKM may have a potential impact on TFV pharmacokinetics. Funding: R01 GM103853, UM1 AI068613 and UM1 AI106707