Detection of Enzyme Activity and Inhibition during Studies in Solution, In Vitro and In Vivo with CatalyCEST MRI

Abstract

Purpose The detection of enzyme activities and evaluation of enzyme inhibitors have been challenging with magnetic resonance imaging (MRI). To address this need, we have developed a diamagnetic, nonmetallic contrast agent and a protocol known as catalyCEST MRI that uses chemical exchange saturation transfer (CEST) to detect enzyme activity as well as enzyme inhibition. Procedures We synthesized a diamagnetic MRI contrast agent that has enzyme responsive and enzyme unresponsive CEST signals. We tested the ability of this agent to detect the activity of kallikrein 6 (KLK6) in biochemical solutions, in vitro and in vivo, with and without a KLK6 inhibitor. Results The agent detected KLK6 activity in solution and also detected KLK6 inhibition by antithrombin III. KLK6 activity was detected during in vitro studies with HCT116 colon cancer cells, relative to the detection of almost no activity in a KLK6-knockdown HCT116 cell line and HCT116 cells treated with antithrombin III inhibitor. Finally, strong enzyme activity was detected within an in vivo HCT116 tumor model, while lower enzyme activity was detected in a KLK6 knockdown tumor model and in the HCT116 tumor model treated with antithrombin III inhibitor. In all cases, comparisons of the enzyme responsive and enzyme unresponsive CEST signals were critical for the detection of enzyme activity. Conclusions This study has established that catalyCEST MRI with an exogenous diaCEST agent can evaluate enzyme activity and inhibition in solution, in vitro and in vivo.

Furin-mediated intracellular self-assembly of olsalazine nanoparticles for enhanced magnetic resonance imaging and tumour therapy

“…While MRI produces high spatial resolution images with excellent soft tissue contrast, inherent CEST contrast has been exploited for several compounds, including a series of anti-tumor drugs 42 . Until now, only a few enzymeresponsive CEST MRI agents have been studied in vivo 25,[43][44][45] , with only one pertaining to the study of intracellular protease 43 . Yet, few of these have CEST signal at the large 9.8 ppm frequency shift from water that is present in olsalazine and other salicylates, where the detection has almost no interference from endogenous background signals in vivo.…”

Assessments of tumor metabolism with CEST MRI

“…A variety of other CEST agents have been developed that detect enzyme activity using catalyCEST MRI or similar CEST MRI protocols. CatalyCEST agents have detected proteases such as cathepsins, caspases, matrix metalloproteases, urokinase plasminogen activator, kallikrein 6, and general protease activity . Other agents have cleaved substrates to detect glycoside hydrolases, sulfatases, esterases, and phosphatases .…”

“…Substrates can often be processed by multiple enzymes, which compromises the detection specificity for only one enzyme of interest . Most importantly, high concentrations of the agent of approximately 10 mM are needed for in vivo detection of enzyme activity, which substantially reduces the potential for clinical translation . The need for high concentrations of agent may limit catalyCEST MRI to the detection of extracellular enzymes, because internalization of high agent concentrations can affect cell function or lead to cell death.…”

Recent progress in the imaging detection of enzyme activitiesin vivo

“…111 In the past few years, CEST MRI has been applied to detect the activity of the urokinase plasminogen activator, alkaline phosphatases, sulfatase enzyme, cathepsin, carboxypeptidase, cytosine deaminase, glutamyl transferase, kallikrein and so on. [112][113][114][115][116][117][118][119] These studies, mostly from Pagel's group, utilized different types of diamagnetic or paramagnetic CEST agents to assess enzyme activities. For a typical example, this group detected the in vivo enzyme activity of g-glutamyl transferase (GGT) within mouse models using CEST MRI.…”

“…In a similar way, this group also successfully detected the activity of kallikrein 6 in an in vivo tumor model. 119 Moreover, recent in vivo studies have demonstrated that MRI has a strong potential for the non-invasive, selective monitoring of enzymatic activity. However, only a few related studies have been performed in tumor mouse models, and there is hardly any monograph on MR imaging of enzymes activity in human body.…”