Tenofovir Activation in Brain and Liver is Diminished in Creatine Kinase Brain-Type Knockout Mice

Abstract

<b>Abstract ID 16726</b> <b>Poster Board 383</b> Tenofovir (TFV) is a nucleotide reverse transcriptase inhibitor that is administered orally as a prodrug for the treatment and prevention of HIV infection, as well as the treatment of chronic hepatitis B virus (HBV) infection. TFV must be phosphorylated intracellularly to tenofovir-monophosphate (TFV-MP) then to tenofovir-diphosphate (TFV-DP) to become pharmacologically active. The nucleotide kinases responsible for the activation of TFV remain unknown in many tissues central to viral replication and pathophysiology. In identifying TFV activating enzymes, we can interrogate whether individuals experiencing lower drug efficacy possess mutations in these enzymes. Previously, we have demonstrated that creatine kinase brain-type (CKB) is able to phosphorylate TFV-MP to TFV-DP in&nbsp;vitro. To determine where and to what extent CKB activates TFV in&nbsp;vivo, we established a <i>Ckb</i> knockout mouse model on a C57BL/6J background. Considering that the brain harbors HIV infection and our preliminary mass spectrometry-based proteomic analyses placed CKB in the top ten most abundant proteins in wild-type (WT) mouse brain, we homogenized full brains from 12-week old WT and <i>Ckb</i> knockout mice and incubated the lysates with TFV-MP and a mix of phosphodonors (ATP, phosphocreatine, and phosphoenolpyruvate). TFV metabolites were extracted and detected using ultra-high performance liquid chromatography tandem mass spectrometry (uHPLC-MS). Results showed that <i>Ckb</i> knockout male lysates formed 70.5% less TFV-DP than WT male lysates (n=3 each; p&lt;0.01), and <i>Ckb</i> knockout female lysates formed 77.4% less TFV-DP than WT female lysates (n=4 and 3, respectively; p&lt;0.001). This indicates that CKB is the main kinase contributing to TFV-DP formation in the brain. Next, to investigate the role of CKB in the liver, 10-week old mice were dosed with a TFV prodrug (60mg/kg/day via drinking water) for 14 days. TFV metabolites were extracted from the liver and detected using uHPLC-MS. The ratio of TFV-DP to TFV was used as a metric to define TFV activation within each sample. <i>Ckb</i> knockout male mice (n=14) had an average TFV-DP to TFV ratio 22.8% (p&lt;0.05) less than WT male mice (n=12), indicating CKB contributes to TFV activation in male livers, however, to a lesser extent than in the brain. Additionally, this reveals the presence of other TFV activating kinases in the liver that have not yet been identified or characterized. Interestingly, no significant difference was observed in female livers when comparing TFV activation between genotypes. Further, in both brain and liver analyses WT female mice had significantly less TFV-DP formation than WT male mice (p&lt;0.01). Together, these data suggest sexual dimorphisms may exist in the TFV activation pathway in these tissues. Lastly, to examine the impact of naturally occurring mutations on CKB activity, fifteen recombinantly expressed and purified mutant CKB enzymes were incubated with TFV-MP and phosphocreatine. Eight mutations (C74S, R96P, S128R, R132H, R172P, R236Q, R292Q, and H296R) exhibited a significant reduction in the formation of TFV-DP, ranging from 69.6%-98.6% less TFV-DP formed than WT CKB (p&lt;0.05). In conclusion, we hypothesize that individuals possessing missense mutations that diminish TFV-DP formation may have lower levels of TFV-DP in the brain and liver, decreasing the efficacy of TFV in the treatment of HIV and HBV infections. This work is funded by NIH R01 AG064908.