Impact of Adenylate Kinases and Naturally Occurring Adenylate Kinase 2 Variants on the Phosphorylation of the Anti‐HIV Drug Tenofovir

Abstract

Tenofovir (TFV), an acyclic nucleotide analog of adenosine monophosphate, is used in combination with other antiretroviral drugs for the treatment and prevention of human immunodeficiency virus (HIV) infection. TFV requires two sequential intracellular phosphorylation steps to become pharmacologically active; however, mechanistic studies that investigate TFV phosphorylation by kinases and the impact of naturally occurring variants of these kinases on TFV activation are currently lacking. To this end, we established an assay to measure the activity of adenylate kinase (AK) 2, which our lab has previously demonstrated catalyzes the first phosphorylation of TFV to TFV‐monophosphate (TFV‐MP) in peripheral blood mononuclear cells, towards TFV in vitro. Recombinant human AK2 was incubated with TFV and ATP (the source of phosphate to be transferred to TFV) and ultra‐high performance liquid chromatography tandem mass spectrometry was employed to measure the formation of TFV‐MP and TFV‐diphosphate (TFV‐DP). We found that AK2 indeed phosphorylated TFV to TFV‐MP, however, interestingly, we also observed phosphorylation of TFV‐MP to TFV‐DP by AK2. Previously, our lab has identified several naturally occurring variants of AK2 via sequencing of genomic DNA from clinical trial participants. In order to test whether these variants might impact TFV phosphorylation, we expressed and purified 12 AK2 variants (A8V, V19G, K28R, A52T, A55V, E59K, K62E, E77K, E155K, T194I, I206F, I206V). Of these, V19G, K28R, A55V, K62E, and T194I variants were found to be functionally affected, leading to a 1.5‐, 3.5‐, 1.5‐, 1.5‐, and 2.5‐fold decrease, respectively, in the formation of TFV‐MP compared to wild‐type. Interestingly, the K28R variant was the only variant to exhibit a decrease (5‐fold), in the formation of TFV‐DP compared to wild‐type. To test whether human AKs beyond AK2 also have the ability to phosphorylate TFV, we expressed and purified AK1, AK3, AK4 and AK6. AK1, like AK2, exhibited activity towards TFV resulting in the formation of both TFV‐MP and TFV‐DP. Interestingly, AK3, AK4 and AK6 did not phosphorylate TFV to either TFV‐MP or TFV‐DP; however, upon substitution of ATP with GTP as the phosphate source, AK3 was able to catalyze the phosphorylation of TFV to TFV‐MP. The lack of activity of both AK4 and AK6 towards TFV persisted whether ATP or GTP was employed as a phosphate donor. Taken together, these data reveal that AK2 can carry out both required phosphorylation steps for TFV activation and that genetic variants could compromise TFV phosphorylation, contributing to interindividual variability in response to TFV efficacy. In addition, our findings suggest that despite similarities in endogenous function, AK enzymes can exhibit differential activity towards nucleotide analog drugs.Support or Funding InformationThis research is funded by NIH R01 GM103853, NIH R01 AI128781, and NIH UM1 AI068613.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.