Transforming Thymidine into a Magnetic Resonance Imaging Probe for Monitoring Gene Expression

Abstract

Synthetic chemistry has revolutionized the understanding of many biological systems. Small compounds that act as agonists and antagonists of proteins, and occasionally as imaging probes, have contributed tremendously to the elucidation of many biological pathways. Nevertheless, the function of thousands of proteins is still elusive, and designing new imaging probes remains a challenge. Through screening and characterization we identified thymidine analog as probe for imaging the expression of the Herpes Simplex Virus type-1 thymidine kinase (HSV1-TK). To detect the probe, we used chemical exchange saturation transfer magnetic resonance imaging (CEST-MRI), in which a dynamic exchange process between an exchangeable proton and the surrounding water protons is used to amplify the desired contrast. Initially, five pyrimidine-based molecules were recognized as putative imaging agents, since their exchangeable imino protons resonate at 5–6ppm from the water proton frequency and their detection is therefore less affected by endogenous CEST contrast or confounded by direct water saturation. Increasing the pKa value of the imino proton by reduction of its 5,6-double bond results in a significant reduction of the exchange rate (kex) between this proton and the water protons. This reduced kex of the dihydropyrimidine nucleosides fulfills the “slow to intermediate regime” condition for generating high CEST-MRI contrast. Consequently, we identified 5-methyl-5,6-dihydrothymidine as the optimal probe and demonstrated its feasibility for in vivo imaging of the HSV1-TK. In light of these findings, this new approach can be generalized for designing specific probes for the in vivo imaging of a variety of proteins and enzymes.

Tuning Phenols with Intra‐Molecular Bond Shifted HYdrogens (IM‐SHY) as diaCEST MRI Contrast Agents

“…1 results in reduced sensitivity at 3 T through saturation transfer. Further shifted labile protons (up to ~ 6 ppm) have been reported in barbituric acid [5] , iopamidol analogues [12] and thymidine analogues [13] , and represent improvements, but as shown by these simulations are still not ideal.…”

Cardiac Chemical Exchange Saturation Transfer MR Imaging Tracking of Cell Survival or Rejection in Mouse Models of Cell Therapy

“…Mice were anesthetized and maintained with 2% isoflurane in oxygen, the pectoral muscles were dissected at the fourth intercostal space, mosquito oxide nanoparticle-labeled cells (4-7) has raised concerns of altered gene expression, tissue retention of particles after cell death, and difficulty in differentiating between labeled cells and tissue necrosis (2,(8)(9)(10)(11)(12)(13). Chemical exchange saturation transfer (CEST) MR imaging is an emerging molecular imaging technique wherein the contrast (change in signal intensity) of either paramagnetic CEST agents (14,15) or CEST-active reporter genes (16)(17)(18) is selectively activated by using radiofrequency irradiation at target specific offset frequencies (19). Subsequently, paramagnetic CEST-labeled cells or CEST MR imaging reporter gene-expressing cells can be visualized without disruption of underlying MR image integrity.…”

“…Subsequently, paramagnetic CEST-labeled cells or CEST MR imaging reporter gene-expressing cells can be visualized without disruption of underlying MR image integrity. Prior CEST MR imaging studies have been conducted to track paramagnetic CEST-labeled cells in subcutaneous tumor models in mice (14,15) and image CEST reporter gene-expressing cells implanted in the rodent brain (16)(17)(18). While CEST MR imaging is suited for cell tracking in cardiac cell therapy, conventional CEST approaches are unsuitable for preclinical cardiac imaging and have been limited to stationary organs and the lungs (20).…”

Gaining Mechanistic Insights into Cell Therapy Using Magnetic Resonance Imaging

“…However, MR spectroscopy does not readily lend itself to imaging, i.e., the ability to localize signals to a small area of tissue with a small number of cells, although it is suitable to characterize large tissue grafts in patients [44]. Thymidine analogs for PET imaging have been described to measure cell proliferation [45], and there is some effort to achieve a similar visualization using MRI [46], but so far, no study has applied these approaches to the imaging of cell therapy. Contrast agent-based approaches have been described to assess the functional status of transplanted cells [47], as well as their differentiation into inhibitory GABAergic neurons [48].…”