Naturally Occurring Mutations in Creatine Kinase Brain‐Type Exhibit Decreased Tenofovir Activation In Vitro

Abstract

Pre-exposure prophylaxis regimens contain tenofovir prodrugs that must undergo intracellular cleavage and two phosphorylation steps to become the pharmacologically active metabolite, tenofovir-diphosphate (TFV-DP). TFV-DP is a nucleotide analog that mimics ATP in structure and competitively inhibits HIV reverse transcriptase. It has been shown that creatine kinase muscle-type (CKM) is responsible for the final phosphoryl transfer step in the formation of TFV-DP in colon tissue. A closely related enzyme, creatine kinase brain-type (CKB), is 80% homologous to CKM and is highly expressed in brain, prostate, and the gastrointestinal tract. Thus, we speculate that CKB may be responsible for the activation of tenofovir in these tissues. To investigate this, in vitro activity assays were performed by incubating recombinantly expressed and purified CKB with tenofovir-monophosphate and phosphocreatine. Formation of TFV-DP was detected using ultra-high performance liquid chromatography tandem mass spectrometry. Results showed no significant difference in TFV-DP formation when comparing CKB to CKM. Interestingly, it has been observed that intracellular TFV-DP concentrations are variable among individuals and genetic variation in tenofovir activating enzymes could contribute to this. To examine the impact of genetic variation on CKB activity, fifteen naturally occurring missense mutations wereselected based on their location, predicted impact (SIFT and PolyPhen scores), and residue conservation between creatine kinase enzymes. In vitro activity assays using recombinantly expressed mutant CKB enzymes demonstrated that 10 of the chosen mutations exhibit a statistically significant reduction in the formation of TFV-DP when compared to wild-type CKB. Mutations at active site arginine residues (R132C, R236Q, and R292Q) showed only 1-3% activity. Other notable mutations that exhibited reduced TFV-DP formation include S128R (30.4%) and H296R (5.6%) in the tenofovir-monophosphate binding pocket, R172P (21.3%) at the dimer interface, and C74S (20.1%) and R96P (6.8%) in the phosphocreatine binding pocket. Of the five mutations where no reduction in TFV-DP formation was observed, two are located in the dimer interface and three are located on the exterior of the enzyme. Overall, these data suggest that CKB may be responsible for tenofovir activation in certain tissues and naturally occurring mutations in this enzyme could result in variable intracellular TFV-DP concentrations.