Naturally‐Occurring Variants of Muscle‐Type Creatine Kinase Exhibit Altered Tenofovir Monophosphate Phosphorylation Activity
Authors: Mosher Eric P. , Wade Herschel , Bumpus Namandje N. 
Published: April 2020

Tenofovir (TFV), prescribed as a disoproxil fumarate prodrug, is a component of the only FDA‐approved regimen for the prevention of HIV. TFV is an acyclic nucleotide analog reverse transcriptase inhibitor prodrug that mimics a nucleotide monophosphate in structure and must be phosphorylated twice by intracellular kinases to produce the pharmacologically active TFV‐diphosphate (DP) form. There have been clinical observations of variation in intracellular TFV‐DP levels, along with efficacy of TFV‐based HIV pre‐exposure prophylaxis (PrEP). We hypothesize that interindividual differences in the kinases that phosphorylate TFV may be a cause of these outcomes. We have elucidated the kinases responsible for TFV phosphorylation in tissues important to forming a barrier of protection against HIV infection, including muscle‐type creatine kinase (CKM), which is the primary kinase responsible for the phosphorylation of TFV‐monophosphate (MP) to the active TFV‐DP in colon tissue. In pursuit of understanding interindividual differences in these kinases, we sequenced the CKM gene in over 1000 patient samples and discovered several previously unreported single nucleotide variants, some of which lead to mutations in the CKM amino acid sequence. Using bioinformatic tools, we predicted that several of these mutants may markedly decrease enzyme function toward the endogenous substrate of the enzyme, ADP. We performed molecular docking simulations, using AutoDock, to model the binding of TFV‐MP to CKM and found that TFV‐MP binds to CKM much as does ADP, which TFV‐MP mimics in structure, suggesting that the bioinformatic predictions may also apply to changes in TFV‐MP phosphorylation activity of CKM. Docking results reveal a pocket of active site arginine residues at positions 130, 132, 236, 292, and 320 as likely important to charge stabilization of the phosphates of TFV‐MP. Notably, our sequencing revealed genetic variants producing R130H and R132C mutations that are predicted to decrease enzyme activity. In order to determine the effects of the naturally‐occurring point mutations on CKM activity toward TFV‐MP, wild‐type human CKM and 15 mutants from the genetic variation analysis were recombinantly expressed and purified. Then, in vitro assays were performed by incubating the proteins with TFV‐MP and measuring TFV‐DP formation via ultra‐high performance liquid chromatography tandem mass spectrometry. These results reveal 11 mutations in CKM that significantly decrease TFV‐MP phosphorylation. Three active site mutations, R130H, R132C, and N286I, along with W211R resulted in a decrease in TFV‐DP formation to only 3% of that of wild‐type CKM. Additionally, T35I, R43Q, I92M, H97Y, F169L, and Y173C mutations decreased activity to 14–25% of that of wild‐type. This evidence suggests that CKM genetics may play a role in interindividual variability in TFV activation and potentially the efficacy of TFV in PrEP settings.

Support or Funding Information

This research is funded by NIH R01 AI128781