Detecting enzyme activities with exogenous MRI contrast agents

Abstract

This review focuses on exogenous magnetic resonance imaging (MRI) contrast agents that are responsive to enzyme activity. Enzymes can catalyze a change in water access, rotational tumbling time, the proximity of a (19)F-labeled ligand, the aggregation state, the proton chemical-exchange rate between the agent and water, or the chemical shift of (19)F, (31)P, (13)C or a labile (1)H of an agent, all of which can be used to detect enzyme activity. The variety of agents attests to the creativity in developing enzyme-responsive MRI contrast agents.

Keywords: CEST MRI; MR spectroscopy; enzymes; imaging agents; relaxation-based MRI.

Figure 1

Detecting enzyme activities through changes in T1 relaxation caused by alterations in water accessibility. Top panel: The cleavage of a galactopyranosyl ring of the T1 contrast agent, EgadMe, causes the inner sphere coordination site of the Gd³⁺ ion to become more accessible to water. Middle panel: A space-filling molecular model illustrates the increased accessibility of the Gd³⁺ ion (magenta) upon cleavage. Bottom Panel: Two living X. laevis embryos were injected with EgadMe, and the embryo shown on the right was also injected with β-galactosidase mRNA. The pseudocolor rendering of MR images shows that the signal strength is 45–65% greater in the embryo containing β-galactosidase mRNA, demonstrating the detection of β-galactosidase activity. Labeled anatomy: e, eye; c, cement gland; s, somite; b, brachial arches. Reproduced with permission from (22).

Figure 2

Detecting enzyme activities through changes in T1 relaxation time caused by alterations in rotational tumbling times. Top Panel: The contrast agent, hydroxytyraminyl-glycylmethylDOTA [D-DOTA(Gd)]; Bottom Panel: Polymerization of the contrast agent by perixodiase slows the rotational tumbling time of the agent, which causes a decrease in the T1 relaxation time of the agent on a per-Gd basis. (C) MR images of the contrast agent at various gadolinium concentrations in the presence of peroxidase (+ Px) or in the absence of peroxidase (− Px) and hydrogen peroxide, demonstrating the detection of peroxidase enzyme activity. Reproduced with permission from (31a).

Figure 3

Detecting enzyme activities via changes in T2 relaxation. Top Panel: A nanoassembly of Gd-based contrast agents that are linked with DEVD peptides can be disaggregated following DEVD peptide cleavage by caspase-3. Bottom Panel: The incubation of the DEVD-linked nanoassembly with caspase-3 showed an increase in T2 relaxation time, caused by disassembly. Incubation of the nanoassembly with the enzyme and a caspase-3 inhibitor further confirmed that this approach detected caspase-3 enzyme activity. Reproduced with permission from (43a).

Figure 4

Detecting enzyme activities with CEST MR methods. (A): The cleavage of a peptidyl ligand of a CEST agent, DEVD-amido-(Tm-DOTA), by caspase-3 converts an amide to an amine. (B): The conjugation a CEST agent, Tm-DO3A-cadaverine, to a protein’s glutamine side chain by TGase converts an amine to an amide. (C): The CEST spectra of DEVD-amido-(Tm-DOTA) before (black) and after (gray) incubation with caspase-3 showed a decrease in CEST at μ̵11 ppm and the appearance of CEST at +8 ppm. This appearance of CEST was further confirmed by deconvoluting the CEST spectrum of the product (dashed line), which confirmed the detection of caspase-3 enzyme activity. (D): The CEST spectra of a mixture of albumin, Tm-DO3A-cadaverine and L-glutathione before (black) and after (gray) incubation with TGase showed a decrease in CEST at +4.6 ppm and the appearance of CEST at −9.2 ppm, which demonstrated detection of TGase activity. Reproduced with permission from (55) and (53).

Figure 5

Detecting enzyme activities with MR spectroscopy. A series of ¹⁹F-NMR spectra were acquired of OFPNPG in PBS (0.1M, pH 7.4, 600mL) before and after addition of β-galactosidase. Rapid hydrolysis of OFPNPG to form OFPNP was monitored by observing a new ¹⁹F-NMR signal for the aglycone, which was used to monitor the enzyme activity. The chemical structures of OFPNPG and OFPNP are shown. Reproduced with permission from (68).