Development of a Glutamine-Responsive MRI Contrast Agent

Abstract

Magnetic resonance imaging (MRI) is widely used to visualize disease, and image quality can be improved through use of MRI contrast agents. Currently available agents produce a signal based solely on spatial distribution, but modern metabolic profiling has uncovered a variety of biomarkers for disease. For example, tumors greatly increase their uptake and catabolism of glutamine (Gln), leading to modified local concentration. Our laboratory previously developed a switchable artificial metalloprotein (swArM) platform in which Gln-binding causes a protein conformational change that modifies the physicochemical environment of an installed metallocofactor. Installing MRI-active metallocofactors within swArMs, we present a proof-of-concept approch toward the development of an analyte-responsive MRI contrast agent. To develop these swArMs, we tested several MRI-active metals (Gd3+, Dy3+), chelating ligands (DOTA, DTPA, NOTA), and attachment sites, as well as the impacts of peripheral mutations on the Gln-responsive signal. In each case, metal content was analytically defined, and Gln-binding affinity was determined by isothermal titration calorimetry. Circular dichroism was used to verify that our swArMs could still undergo the conformational change. X-ray diffraction structures of the apo- and holo-swArMs further revealed that the metallocofactor is significantly solvent-exposed in both conformations, with additional interactions with the protein in the holo-state coinciding with the observed increase in T2 relaxivity of ~60% upon Gln-binding.