Visualizing enzymatic activity using magnetic resonance molecular imaging

Overexpression of ERK (MAP Kinase) and Pin1 is related to many types of cancer. Two pro-contrast agents, Gd-DOTA-APRTPGGRCKKK and Gd-DOTA-PRTPGGRCKK, which were hypothesized to be enzymatically activated to show greater contrast by overexpressed ERK and Pin1 were synthesized using solid phase peptide synthesis. ERK phosphorylation using 33P labeled ATP showed radioisotope incorporation in Gd-DOTA-APRTPGGRCKK only; Gd-DOTA-PRTPGGRCKK (missing alanine residue) did not undergo enzymatic phosphorylation. Contrast measurements of the Gd-DOTA-APRT(phospho)PGGRCKK showed 28% increase in relaxivity (r1) over Gd-DOTA-APRTPGGRCKK. Gd-DOTA-APRT(phospho)PGGRCKK underwent isomerization with the peptidylprolyl isomerase Pin1. It was initially hypothesized that this conformational change caused by the isomerization would further enhance the contrast by creating a more sterically open Gd coordination environment which would accommodate an inner-sphere water molecule. It was also possible for the isomerized structure to attenuate contrast by blocking the 9th Gd coordination site and preventing inner-sphere water coordination. Isomerization of the Gd-DOTA-APRT(phospho)PGGRCKK with Pin1 was monitored using CD-spectroscopy and contrast measurements were done simultaneously which showed no change in contrast. The pro-contrast agents Gd-DOTA-APRTPGGRCKK and Gd-DOTA-PRTPGGRCKK were tested for activity against other MAPK's, such as p38 and JNK1, and showed negative results.;A novel contrast agent, Gd-DOTA-APRTPGGRCKK, which can directly and specifically monitor ERK activity, has been synthesized. Based on molecular modeling using Charmm27 MM3 we believe that the folding of the peptide upon phosphorylation causes a proline residue to recede away from the Gd coordination sphere, allowing free access to bulk water molecules. Based on literature comparisons we believe that the majority of the contrast enhancement is due to increased outer sphere relaxation rates resulting from phosphate incorporation.;This thesis describes the synthesis and relaxivity measurements of a new contrast agent for the non-invasive evaluation of signal transduction pathways that are up-regulated in cancer cells. This is the first example of a medical imaging agent, in any imaging modality, whose contrast generation or enhancement is responsive to kinase activity. Furthermore, this agent is specific to ERK, showing no activity towards other protein kinases, and can report on ERK signaling cascade within cancer cells with high specificity.