Naturally Occurring Mutations in Muscle‐Type Creatine Kinase Impact Tenofovir Monophosphate Phosphorylation

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Tenofovir (TFV), administered as a tenofovir disoproxil fumarate prodrug, is a nucleotide analog reverse transcriptase inhibitor used in combination for the treatment and prevention of HIV. TFV requires two sequential phosphorylation events in order to become pharmacologically active. We previously found that in colon tissue, a putative site of HIV infection, the muscle‐type isoenzyme of creatine kinase (CKM) is responsible for the second TFV activating phosphorylation step, converting tenofovir monophosphate (TFV‐MP) to the active tenofovir diphosphate (TFV‐DP). Due to the similarity in structure between TFV‐MP and ADP, this is likely analogous to the endogenous activity of CKM, which regenerates ATP from ADP by phosphoryl transfer from phosphocreatine. In addition, we have previously discovered naturally occurring genetic variants of the CKM gene in humans. Some of these single nucleotide variants are predicted to cause single residue mutations in the translated protein, a fraction of which are predicted by bioinformatics programs to have deleterious effects on enzymatic function. In the present study, molecular docking was employed to investigate the potential effects of these mutations on TFV monophosphate (TFV‐MP) binding and phosphorylation by CKM. These results indicate CKM binds TFV‐MP similar to how it binds its natural substrate, ADP. Molecular docking simulation also predict a cluster of active site arginine residues at positions 130, 132, 236, 292, and 320 are likely important in the charge stabilization of the phosphates of TFV‐MP, which may impact substrate binding and phosphorylation. To directly investigate the impact of the aforementioned mutations on TFV‐MP phosphorylation, CKM wild type and mutant proteins were recombinantly expressed and purified. In vitro assays were then performed to compare the ability of the mutant proteins to convert TFV‐MP to TFV‐DP. Enzyme activity was measured by liquid chromatography‐tandem mass spectrometry using a method that separated and detected the phosphorylated tenofovir metabolites concomitantly. It was found that R130H and R132C mutations decreased enzymatic activity to less than 10% of that of the wild type protein. These results indicate that genetic variation in the CKM gene may impact tenofovir activation, potentially implicating a risk to patients possessing these genetic variants.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.