Monitoring Enzyme Activity Using a Diamagnetic Chemical Exchange Saturation Transfer Magnetic Resonance Imaging Contrast Agent

Abstract

Chemical Exchange Saturation Transfer (CEST) is a new approach to generate magnetic resonance imaging (MRI) contrast that allows monitoring of protein properties in vivo. In this method, radiofrequency is used to saturate the magnetization of specific protons on a target molecule, where it is then transferred to water protons via chemical exchange and detected using MRI. One advantage of CEST imaging is that the magnetization of the different protons can be specifically saturated at different resonance frequencies. This enables the detection of multiple targets simultaneously in living tissue. We present here a CEST MRI approach to detect the activity of Cytosine Deaminase (CDase), an enzyme that catalyzes the deamination of cytosine to uracil. Our findings suggest that metabolism of two substrates of the enzyme, cytosine and 5-fluorocytosine (5FC), can be detected using saturation pulses targeted specifically to protons at +2 ppm and +2.4 ppm (with respect to water), respectively. Indeed, after deamination by recombinant CDase, the CEST contrast disappears. In addition, expression of the enzyme in three different cell lines, each with different expression levels of CDase, show good agreement with the CDase activity measured with CEST MRI. Consequently, CDase activity was imaged with high-resolution CEST-MRI. These data demonstrate the ability to detect enzyme activity based on proton exchange. Consequently, CEST MRI has the potential to follow the kinetics of multiple enzymes in real-time in living tissue.

Label-free imaging of gelatin-containing hydrogel scaffolds

“…After transfer of their saturation to bulk water, from which the proton MRI signal is derived, the water signal is reduced; hence these exchangeable protons are detected indirectly. CEST imaging is now being recognized as an emerging technique for molecular and cellular MR imaging: it can be used to detect MRI reporter genes [15–17], enzyme activity [18], pH changes associated with cell death [14], and the occurrence of an immune response against foreign body materials [19]. …”

Oxidation-responsive Eu2+/3+-liposomal contrast agent for dual-mode magnetic resonance imaging

“…1 Molecular information can also be obtained with MRI using responsive paramagnetic complexes (contrast agents) that alter water proton signal intensities in response to chemical events. Some contrast agents respond to changes in pH, 2,3 temperature, 4 metal ion concentration, 5 enzyme activity, 6,7 or partial pressure of oxygen, 8 the presence of free radicals, 9 antioxidants, 10 phosphate diesters, 11 singlet oxygen, 12 reduced glutathione and hydrogen peroxide, 13 or oxygen, dithionite, and cysteine. 14 Of particular interest are targets that cause changes in redox behavior because they are associated with cancer, 15 inflammation, 16 and cardiovascular diseases.…”

A CatalyCEST MRI Contrast Agent that Can Simultaneously Detect Two Enzyme Activities

“…In some cases, we and others have developed diamagnetic catalyCEST agents that do not require a potentially toxic metal ion, which facilitates clinical translation. [10,15,16] Despite this progress, catalyCEST MRI has not yet been developed to simultaneously respond to multiple enzyme activities. Therefore, we sought to develop a diamagnetic catalyCEST agent that could detect the activity of two enzymes, and would otherwise not generate CEST MRI contrast without the enzymes or with only one enzyme.…”