Discipline: Chemistry and Chemical Sciences
Subcategory: Chemistry (not Biochemistry)
Gabriela Fernandez-Cuervo - University of Arizona
Co-Author(s): Kyle M. Jones and Mark D. Pagel, University of Arizona, Tucson, AZ
Tumors have heterogeneous biomarker expression, including the varying expression levels of antigens. If exploited for targeted therapies, low antigen expression can require an amplification mechanism to deliver sufficient active drug for an efficient treatment. To amplify therapy response, antibody-directed enzyme-activated prodrug therapy (ADEPT) uses enzymes as tools to selectively activate prodrugs in tumor tissue. ADEPT requires sufficient antigen expression, delivery of an antibody-enzyme conjugate, strong enzyme activity, and site-specific produrg delivery. A non-invasive diagnostic method is needed to ensure that these components are in place for successful ADEPT. Our overarching goal is to develop a catalyCEST MRI contrast agent that detects enzyme activity of β-glucuronidase (GUS) that is used in ADEPT.
A catalyCEST agent was synthesized that is a substrate for GUS. The optimized catalyCEST MRI protocol consisted of a CEST-FISP MRI pulse sequence with a 5-sec saturation pulse and 5 μT saturation power. The protocol was repeated at 96 saturation frequencies to generate a CEST spectrum. The imaging results were analyzed using fitting algorithms developed using MATLAB (version 2014b), which applies a spatial Gaussian filter, cubic splines, and Lorentzian line shape fitting of a CEST spectrum. A Hanes-Woolf CEST plot was used to determine exchange rates1. In the presence of GUS, the catalyCEST agent underwent saccharide hydrolysis to produce 4-aminosalicylic acid. This reaction converted the carbamate moiety into a free primary amine, and caused a loss of CEST at 4.25 ppm, showing that this agent can detect enzyme activity. The CEST signal at 9.25 ppm did not change, demonstrating that the CEST at 9.25 ppm was a good internal “control” signal. Proton exchange rate constants of the substrate were 1022 Hz for the carbamate moiety at 4.25 pm, and 799 Hz for the salicylic acid proton at 9.25 ppm. After the enzyme reaction, the chemical exchange rate of the remaining salicylic acid proton at 9.25 ppm was 1048 Hz. These results demonstrate that the catalyCEST agent can detect GUS activity by monitoring the change in CEST at 4.25 ppm. Future studies involve expanding this platform technology to evaluate other important enzymes for reporter gene imaging and targeted therapies, and to perform in vivo studies with mouse models of cancer.
1Randtke EA, et al. Magn. Reson. Med. 2014, 71:1603-1612.
Abstract-GFC.docxFunder Acknowledgement(s): This project has been funded by the Biological Chemistry Program Training Grant: T32 GM008804 and was also supported by NIH R01 CA169774-01. Gabriela Fernandez-Cuervo is a Howard Hughes Medical Institute Gilliam Fellow.
Faculty Advisor: Mark D. Pagel, mpagel@email.arizona.edu