# Phosphorylation of Drugs in Colonic Tissue

> **NIH NIH R56** · JOHNS HOPKINS UNIVERSITY · 2022 · $725,624

## Abstract

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.

## Key facts

- **NIH application ID:** 10653625
- **Project number:** 1R56AI161030-01A1
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Craig Walter Hendrix
- **Activity code:** R56 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $725,624
- **Award type:** 1
- **Project period:** 2022-08-01 → 2024-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10653625

## Citation

> US National Institutes of Health, RePORTER application 10653625, Phosphorylation of Drugs in Colonic Tissue (1R56AI161030-01A1). Retrieved via AI Analytics 2026-05-29 from https://api.ai-analytics.org/grant/nih/10653625. Licensed CC0.

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