Creatine kinases (CK) are abundant enzymes that play a central role in regulating cellular energy homeostasis. They are particularly important in environments of high energy flux. CK enzymes catalyze the interconversion of phosphocreatine and ADP with creatine and ATP, thereby buffering ATP concentrations during periods of rapid cellular energy expenditure. There are four main CK family members. We have reported that creatine kinase muscle (CKM) plays an important role in the phosphorylation of tenofovir (TFV) in colonic tissues, a putative site of HIV infection. TFV is a nucleotide reverse transcriptase inhibitor that is prescribed as a tenofovir disoproxil or tenofovir alafenamide prodrug in combination with other drugs for the treatment and prevention of HIV infection. TFV must be phosphorylated twice, resulting in the formation of TFV-diphosphate (TFV-DP) in order to becomes pharmacologically active. We have found that CKM carries out the final phosphorylation step in colonic tissue that is required for TFV activation. Of note, genomic analysis that we have performed has revealed at least 15 novel variants predicted to result in a loss or decrease of CKM activity. Studies proposed here will investigate these variants mechanistically by introducing point mutations into CKM in colonic CD4+ T cells and testing the impact on TFV-DP production. Hydrogen-deuterium exchange mass spectrometry will be used to probe the impact of naturally occurring mutations on CKM structure. In addition, we will test whether other CK family members are able to phosphorylate TFV, which could be particularly important when seeking to understand TFV pharmacology in the range of tissues in which TFV is active. Further, we will use a mass spectrometry-based imaging approach to elucidate the distribution of CKM and pharmacologically active TFV-DP in colonic tissue and colonic CD4+ T cells. We will examine this across the life span through the recruitment of healthy participants across a range of ages. In addition, we will probe whether CD4+ T cell activation impacts CKM expression and activity, and/or TFV activation in colonic CD4+ T cells. These studies will provide mechanistic insight into the variables that regulate TFV-DP levels in colonic tissue that can be leveraged in optimizing TFV-based drug regimens, while lending unique insight into CK biochemistry and colonic pharmacology more broadly.