A CatalyCEST MRI Contrast Agent That Detects the Enzyme-Catalyzed Creation of a Covalent Bond

CatalyCEST MRI can detect enzyme activity by employing contrast agents that are detected through chemical exchange saturation transfer (CEST). A CEST agent, Tm-DO3A-cadaverine, has been designed to detect the catalytic activity of transglutaminase (TGase), which creates a covalent bond between the agent and the side chain of a glutamine amino acid residue. CEST appeared at −9.2 ppm after TGase conjugated Tm-DO3A-cadaverine to albumin, which also caused a decrease in CEST from albumin at +4.6 ppm. Studies with model peptides revealed similar appearances and decreases in detectable CEST effects following TGase-catalyzed conjugation of the contrast agent and peptide. The MR frequencies and amplitudes of these CEST effects were dependent on the peptide sequence, which demonstrated the sensitivity of CEST agents to ligand conformations that may be exploited to create more responsive molecular imaging agents. The chemical exchange rates of the substrates and conjugated products were measured by fitting modified Bloch equations to CEST spectra, which demonstrated that changes in exchange rates can also be used to detect the formation of a covalent bond by catalyCEST MRI

Detection of Alkaline Phosphatase Enzyme Activity with a CatalyCEST MRI Biosensor

Responsive CEST MRI biosensors offer good sensitivity and excellent specificity for detection of biomarkers with great potential for clinical translation. We report the application of fosfosal, a phosphorylated form of salicylic acid, for the detection of alkaline phosphatase (AP) enzyme. We detected conversion of fosfosal to salicylic acid in the presence of the enzyme by CEST MRI. Importantly the technique was able to detect AP enzyme expressed in cells in the presence of other cell components, which improves specificity. Various isoforms of the enzyme showed different Michaelis–Menten kinetics and yet these kinetics studies indicated very efficient catalytic rates. Our results with the fosfosal biosensor encourage further in vivo studies

Summary of the K^trans reproducibility measurements.

<p>Summary of the K^trans reproducibility measurements.</p

VIFs from Gd-DTPA (a) and P792 (c).

<p>VIFs were smoothed by temporal spline-fit function for Gd-DTPA (b) and P792 (d).</p

K^trans parametric maps of a single mouse acquired on three different days (Day 1, Day 3 and Day 5) within one week (a); Histogram of K^trans in tumor ROI for all 3 days (b); Difference of histograms (c).

<p>Cumulative histograms of K^trans in tumor ROI on three different imaging days (d).</p

ΔR2* calculated from normal tissue in a single mouse acquired on three different days.

<p>In this case, first- and second- pass circulation of CA injection is observed. Only first –pass circulation was used in DSC analysis.</p

Summary of the rBV reproducibility measurements. Arbitrary units (AU).

<p>Summary of the rBV reproducibility measurements. Arbitrary units (AU).</p

Signal amplitude change of DSC on tumor tissue (left column) and normal tissue (right column).

<p>Three rows represent three separate measurements (top row: Day 1; middle row: Day 3; bottom row: Day 5) for a single animal. The 95% confidence limits for significant changes in signal are shown on each graph (dot line), The bolus was injected at 0 seconds.</p

Diamagnetic Imaging Agents with a Modular Chemical Design for Quantitative Detection of β‑Galactosidase and β‑Glucuronidase Activities with CatalyCEST MRI

Imaging agents for the noninvasive in vivo detection of enzyme activity in preclinical and clinical settings could have fundamental implications in the field of drug discovery. Furthermore, a new class of targeted prodrug treatments takes advantage of high enzyme activity to tailor therapy and improve treatment outcomes. Herein, we report the design and synthesis of new magnetic resonance imaging (MRI) agents that quantitatively detect β-galactosidase and β-glucuronidase activities by measuring changes in chemical exchange saturation transfer (CEST). Based on a modular approach, we incorporated the enzymes’ respective substrates to a salicylate moiety with a chromogenic spacer via a carbamate linkage. This furnished highly selective diamagnetic CEST agents that detected and quantified enzyme activities of glycoside hydrolase enzymes. Michaelis–Menten enzyme kinetics studies were performed by monitoring catalyCEST MRI signals, which were validated with UV–vis assays