Development of a glutamine-responsive MRI contrast agent

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 <i>apo-</i> and <i>holo-</i>swArMs further revealed that the metallocofactor is significantly solvent-exposed in both conformations, but exhibits additional interactions with the protein in the <i>holo-</i>state coinciding with the observed increase in <i>T</i> ₂ relaxivity of ∼60% upon Gln-binding.