PT  - JOURNAL ARTICLE
AU  - Eric P. Mosher
AU  - Colten D. Eberhard
AU  - Namandje N. Bumpus
TI  - Naturally-Occurring Mutations to Muscle-type Creatine Kinase Impact its Canonical and Pharmacological Activities in a Substrate-dependent Manner in vitro
AID  - 10.1124/molpharm.121.000348
DP  - 2021 Jan 01
TA  - Molecular Pharmacology
PG  - MOLPHARM-AR-2021-000348
4099  - http://molpharm.aspetjournals.org/content/early/2021/09/24/molpharm.121.000348.short
4100  - http://molpharm.aspetjournals.org/content/early/2021/09/24/molpharm.121.000348.full
AB  - Tenofovir (TFV) is a key component of HIV pre-exposure prophylaxis (PrEP). TFV is a nucleotide analog reverse transcriptase inhibitor prodrug that requires two separate phosphorylation reactions by intracellular kinases in order to form the active metabolite, tenofovir-diphosphate (TFV-DP). Muscle-type creatine kinase (CKM) has previously been demonstrated to be the kinase most responsible for the phosphorylation of tenofovir-monophosphate (TFV-MP) to the active metabolite in colon tissue. Due to the importance of CKM in TFV activation, genetic variation in CKM may contribute to inter-individual variability in TFV-DP levels. In the present study, we report ten naturally-occurring CKM mutations that reduced TFV-MP phosphorylation in vitro: T35I, R43Q, I92M, H97Y, R130H, R132C, F169L, Y173C, W211R, and N286I. Interestingly, of these ten, only four - R130H, R132C, W211R, and N286I - reduced both canonical CKM activities: ADP phosphorylation and ATP dephosphorylation. While positions 130, 132, and 286 are located in the active site, the other mutations that resulted in decreased TFV-MP phosphorylation occur elsewhere in the protein structure. Four of these eight mutations - T35I, R43Q, I92M, and W211R - were found to decrease the thermal stability of the protein. Additionally, the W211R mutation was found to impact protein structure both locally and at a distance. These data suggest a substrate-specific effect such that certain mutations are tolerated for canonical activities while being deleterious toward the pharmacological activity of TFV activation, which could influence PrEP outcomes. Significance Statement Muscle-type creatine kinase (CKM) is important to the activation of tenofovir, a key component of HIV prophylaxis. This study demonstrates that naturally-occurring CKM mutations impact enzyme function in a substrate-dependent manner such that some mutations that do not reduce canonical activities lead to reductions in the pharmacologically-relevant activity. This finding at the intersection of drug metabolism and energy metabolism is important to the perspective on pharmacology of other drugs acted on by atypical drug metabolizing enzymes.