Enzyme-activated MR contrast agents

Last updated

Molecular imaging is broadly defined as the visualization of molecular and cellular processes on either a macro- or microscopic level. Because of its high spatial resolution and ability to noninvasively visualize internal organs, magnetic resonance (MR) imaging is widely believed to be an ideal platform for in vivo molecular imaging. [1] For this reason, MR contrast agents that can detect molecular events are an active field of research. [2] One group of compounds that has shown particular promise is enzyme-activated MR contrast agents.

Contents

Enzyme-activated MR contrast agents are compounds that cause a detectable change in image intensity when in the presence of the active form of a certain enzyme. This makes them useful for in vivo assays of enzyme activity. They are distinguished from current, clinical MR contrast agents that give only anatomical information, [3] such as aqueous gadolinium compounds, by their ability to make molecular processes visible. Enzyme-activated contrast agents are powerful tools for molecular imaging. To date, β-galactosidase-activated contrast agents have attracted the most attention in the literature, although there no theoretical reason that other enzymes could not be used to activate contrast agents. Also, mechanisms other than enzyme activation, such as Ca2+-dependent activation, can theoretically be used. [2]

In general, enzyme-activated agents contain a paramagnetic metal ion which can affect the T1 or T2 relaxation times for nearby water molecules. However, the metal ions are unable to interact with the water until an enzyme-catalyzed reaction takes place. Steric hindrance or coordination with other ions prevents water from accessing the paramagnetic center prior to the enzymatic reaction. [4]

Structure of β-galactosidase-activated contrast agents

The a-series of b-galactosidase-activated MR contrast agents. Prior to cleavage, the sugar blocks water from accessing the gadolinium ion. The active enzyme will cleave the sugar, allowing water to interact with the paramagnetic center. Alpha series.svg
The α-series of β-galactosidase-activated MR contrast agents. Prior to cleavage, the sugar blocks water from accessing the gadolinium ion. The active enzyme will cleave the sugar, allowing water to interact with the paramagnetic center.

Two distinct β-galactosidase-activated contrast agents have been reported. Both consist of a Gd(III) ion complexed with a tetraazamacrocycle. At the N-10 position, a two-carbon chain links the gadolinium-tertaazamacrocycle complex to a molecule of galactose. The galactose is linked to the complex by a β-glycosidic bond at its C-1 position. [4]

The two forms of the contrast agent differ only in the location of a single methyl group. The first class, known as the α-series, has a methyl group bound to the carbon that is α to the tetraazamacrocycle. The other class, called the β-series, has a methyl attached to the β carbon relative to the tetraazamacrocycle. The position of this methyl group is significant for the structure of the agent, and thus determines the mechanism by which the non-active compound shields the Gd(III) ion from interacting with water. The α-series adopts a conformation in which the sugar lies directly over the paramagnetic center, thus sterically prohibitting water from accessing the gadolinium. The β-series, on the other hand, blocks water from the gadolinium by coordinating with a carbonate ion. There is no evidence that the stereochemical orientation of the methyl-bearing carbon affects the either the enzyme-catalyzed cleavage or the ability of the sugar to exclude water from the gadolinium ion. [4]

Studies have shown that the α-series is far more effective at blocking water from the paramagnetic center prior to cleavage. [4] The need to coordinate with a carbonate ion and the lower level of signal suppression inherent to the β-series make the α-series a better candidate for use in research and clinical medicine.

Mechanism of activation

The b-galactosidase-catalyzed reaction that activates the contrast agent. Alpha series rxn.svg
The β-galactosidase-catalyzed reaction that activates the contrast agent.

In a tissue where active β-galactosidase is present, the sugar will be cleaved from the rest of the compound. This permits water to access the paramagnetic center, and causes the magnetic relaxation properties of the surrounding water molecules to change. This change in relaxation times will, in turn, visibly alter the signal intensity of images of the tissue obtained from MR scans.

The mechanism proceeds in the same manner as all other β-galactosidase-catalyzed cleavages. The carboxyl group on a glutamic acid side chain within the enzyme acts as an acid catalyst, hastening the cleavage of the glycosidic bond at the C-1 position in the sugar. This cleavage gives water access to the paramagnetic center. The result of the enzyme-catalyzed reaction is a free galactose molecule and an activated contrast agent.

Uses

There are obvious potential uses to this technology, both in research and clinical medicine.

Basic research

In a research context, the α-series of β-galactosidase-activated MR contrast agents has been used to visualize the development and gene expression of cells in a Xenopus laevis embryo. [5] Researchers injected the agent into both cells of a two-cell stage embryo, and then injected only one of the cells with mRNA for the enzyme. Following a period of growth, they obtained MR images of the embryo that clearly displayed signal enhancement only in the cells derived from the parent cell that had been injected with both the enzyme and the contrast agent.

This study demonstrates the value of whole body, in vivo molecular imaging methods for basic research. Such techniques permit scientists to test for gene activity and enzyme function throughout an organism. In contrast, many existing assays (such as fixation of cells on paraffin wax followed by immunostaining) only permit the analysis of a few cells at a time. These methods kill the cells, thus making time-series studies difficult. They also require the researcher to have identified a tissue of interest before obtaining the cells.

The rise of whole-body molecular imaging methods may permit scientists to see where in an organism an enzyme is active without damaging cells; imaging could be repeated at multiple time points to monitor changes in gene expression or enzyme activity. Similar techniques have attracted considerable interest from researchers studying cancer [3] and cardiovascular disease. [6]

Clinical medicine

The ability to detect tissues that contain the active form of an enzyme at certain time has clear value in medicine. Specific contrast agents that provide enhancement only in the presence of active enzymes could allow doctors to conclusively and noninvasively assay for a wide variety of enzymatic diseases, such as fructose bisphosphatase deficiency. However, such diagnostic tools would require the development of contrast agents specific to the enzyme of interest, and would necessitate the development of methods for delivering the agents to cells (see “Limitations” below).

Limitations

Once the contrast agent has been activated by cleavage of the sugar group, the signal enhancing effects will only diminish if the gadolinium is washed out of the compartment containing it, or if water’s access to the metal group is again inhibited. So, to prevent permanent enhancement of the MR signal, cells must either have a way to export the gadolinium group to the bloodstream, or they must be able to replace the cleaved sugar group. There is no data in the literature indicating that either approach is feasible in vivo, suggesting that these methods may result in permanent signal amplification.

Another challenge is the delivery of the contrast agents to target cells. In the sole paper describing in vivo use of enzyme-activated MR contrast agents, the agent was delivered to embryonic cells via a micropipette. However, the authors of the paper acknowledge that this is not a feasible approach for many research projects, [5] and it presents a clear impediment to clinical use. There is active research in using the cell’s native import machinery to load contrast agents. [7]

Related Research Articles

<span class="mw-page-title-main">Gadolinium</span> Chemical element with atomic number 64 (Gd)

Gadolinium is a chemical element; it has symbol Gd and atomic number 64. Gadolinium is a silvery-white metal when oxidation is removed. It is a malleable and ductile rare-earth element. Gadolinium reacts with atmospheric oxygen or moisture slowly to form a black coating. Gadolinium below its Curie point of 20 °C (68 °F) is ferromagnetic, with an attraction to a magnetic field higher than that of nickel. Above this temperature it is the most paramagnetic element. It is found in nature only in an oxidized form. When separated, it usually has impurities of the other rare earths because of their similar chemical properties.

<span class="mw-page-title-main">Lactase</span> Milk-sugar digesting enzyme

Lactase is an enzyme produced by many organisms and is essential to the complete digestion of whole milk. It breaks down the sugar lactose into its component parts, galactose and glucose. Lactase is found in the brush border of the small intestine of humans and other mammals. People deficient in lactase or lacking functional lactase may experience the symptoms of lactose intolerance after consuming milk products. Microbial β-galactosidase can be purchased as a food supplement and is added to milk to produce "lactose-free" milk products.

<span class="mw-page-title-main">Magnetic resonance imaging</span> Medical imaging technique

Magnetic resonance imaging (MRI) is a medical imaging technique used in radiology to form pictures of the anatomy and the physiological processes inside the body. MRI scanners use strong magnetic fields, magnetic field gradients, and radio waves to generate images of the organs in the body. MRI does not involve X-rays or the use of ionizing radiation, which distinguishes it from computed tomography (CT) and positron emission tomography (PET) scans. MRI is a medical application of nuclear magnetic resonance (NMR) which can also be used for imaging in other NMR applications, such as NMR spectroscopy.

β-Galactosidase Family of glycoside hydrolase enzymes

β-Galactosidase is a glycoside hydrolase enzyme that catalyzes hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides.

<span class="mw-page-title-main">Mannans</span> Polysaccharides formed from mannose

Mannans are polymers containing the sugar mannose as a principal component. They are a type of polysaccharide found in hemicellulose, a major source of biomass found in higher plants such as softwoods. These polymers also typically contain two other sugars, galactose and glucose. They are often branched.

<span class="mw-page-title-main">Tryptophan synthase</span> Class of enzymes

Tryptophan synthase or tryptophan synthetase is an enzyme that catalyzes the final two steps in the biosynthesis of tryptophan. It is commonly found in Eubacteria, Archaebacteria, Protista, Fungi, and Plantae. However, it is absent from Animalia. It is typically found as an α2β2 tetramer. The α subunits catalyze the reversible formation of indole and glyceraldehyde-3-phosphate (G3P) from indole-3-glycerol phosphate (IGP). The β subunits catalyze the irreversible condensation of indole and serine to form tryptophan in a pyridoxal phosphate (PLP) dependent reaction. Each α active site is connected to a β active site by a 25 Ångstrom long hydrophobic channel contained within the enzyme. This facilitates the diffusion of indole formed at α active sites directly to β active sites in a process known as substrate channeling. The active sites of tryptophan synthase are allosterically coupled.

Deoxyribonuclease refers to a group of glycoprotein endonucleases which are enzymes that catalyze the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA. The role of the DNase enzyme in cells includes breaking down extracellular DNA (ecDNA) excreted by apoptosis, necrosis, and neutrophil extracellular traps (NET) of cells to help reduce inflammatory responses that otherwise are elicited. A wide variety of deoxyribonucleases are known and fall into one of two families, which differ in their substrate specificities, chemical mechanisms, and biological functions. Laboratory applications of DNase include purifying proteins when extracted from prokaryotic organisms. Additionally, DNase has been applied as a treatment for diseases that are caused by ecDNA in the blood plasma. Assays of DNase are emerging in the research field as well.

<i>lac</i> operon Set genes encoding proteins and enzymes for lactose metabolism

The lactose operon is an operon required for the transport and metabolism of lactose in E. coli and many other enteric bacteria. Although glucose is the preferred carbon source for most enteric bacteria, the lac operon allows for the effective digestion of lactose when glucose is not available through the activity of β-galactosidase. Gene regulation of the lac operon was the first genetic regulatory mechanism to be understood clearly, so it has become a foremost example of prokaryotic gene regulation. It is often discussed in introductory molecular and cellular biology classes for this reason. This lactose metabolism system was used by François Jacob and Jacques Monod to determine how a biological cell knows which enzyme to synthesize. Their work on the lac operon won them the Nobel Prize in Physiology in 1965.

<span class="mw-page-title-main">Phosphoglucomutase</span> Metabolic enzyme

Phosphoglucomutase is an enzyme that transfers a phosphate group on an α-D-glucose monomer from the 1 to the 6 position in the forward direction or the 6 to the 1 position in the reverse direction.

<span class="mw-page-title-main">Galactokinase</span> Enzyme

Galactokinase is an enzyme (phosphotransferase) that facilitates the phosphorylation of α-D-galactose to galactose 1-phosphate at the expense of one molecule of ATP. Galactokinase catalyzes the second step of the Leloir pathway, a metabolic pathway found in most organisms for the catabolism of α-D-galactose to glucose 1-phosphate. First isolated from mammalian liver, galactokinase has been studied extensively in yeast, archaea, plants, and humans.

<span class="mw-page-title-main">Gadolinium(III) chloride</span> Chemical compound

Gadolinium(III) chloride, also known as gadolinium trichloride, is GdCl3. It is a colorless, hygroscopic, water-soluble solid. The hexahydrate GdCl3∙6H2O is commonly encountered and is sometimes also called gadolinium trichloride. Gd3+ species are of special interest because the ion has the maximum number of unpaired spins possible, at least for known elements. With seven valence electrons and seven available f-orbitals, all seven electrons are unpaired and symmetrically arranged around the metal. The high magnetism and high symmetry combine to make Gd3+ a useful component in NMR spectroscopy and MRI.

α-Galactosidase Enzyme

α-Galactosidase is a glycoside hydrolase enzyme that catalyses the following reaction:

<span class="mw-page-title-main">X-gal</span> Chemical compound

X-gal is an organic compound consisting of galactose linked to a substituted indole. The compound was synthesized by Jerome Horwitz and collaborators in 1964. The formal chemical name is often shortened to less accurate but also less cumbersome phrases such as bromochloroindoxyl galactoside. The X from indoxyl may be the source of the X in the X-gal contraction. X-gal is often used in molecular biology to test for the presence of an enzyme, β-galactosidase, in the place of its usual target, a β-galactoside. It is also used to detect activity of this enzyme in histochemistry and bacteriology. X-gal is one of many indoxyl glycosides and esters that yield insoluble blue compounds similar to indigo dye as a result of enzyme-catalyzed hydrolysis.

<span class="mw-page-title-main">Gadopentetic acid</span> Complex of gadolinium by DTPA

Gadopentetic acid, sold under the brand name Magnevist, is a gadolinium-based MRI contrast agent.

<span class="mw-page-title-main">Gadolinium(III) oxide</span> Chemical compound

Gadolinium(III) oxide (archaically gadolinia) is an inorganic compound with the formula Gd2O3. It is one of the most commonly available forms of the rare-earth element gadolinium, derivatives, of which are potential contrast agents for magnetic resonance imaging.

MRI contrast agents are contrast agents used to improve the visibility of internal body structures in magnetic resonance imaging (MRI). The most commonly used compounds for contrast enhancement are gadolinium-based contrast agents (GBCAs). Such MRI contrast agents shorten the relaxation times of nuclei within body tissues following oral or intravenous administration.

<span class="mw-page-title-main">Inositol-phosphate phosphatase</span> Class of enzymes

The enzyme Inositol phosphate-phosphatase is of the phosphodiesterase family of enzymes. It is involved in the phosphophatidylinositol signaling pathway, which affects a wide array of cell functions, including but not limited to, cell growth, apoptosis, secretion, and information processing. Inhibition of inositol monophosphatase may be key in the action of lithium in treating bipolar disorder, specifically manic depression.

<span class="mw-page-title-main">Val Murray Runge</span> American professor of radiology

Val Murray Runge is an American and Swiss professor of radiology and the editor-in-chief of Investigative Radiology. Runge was one of the early researchers to investigate the use of gadolinium-based contrast agents for magnetic resonance imaging (MRI), giving the first presentation in this field, followed two years later by the first presentation of efficacy. His research also pioneered many early innovations in MRI, including the use of tilted planes and respiratory gating. His publication on multiple sclerosis in 1984 represented the third and largest clinical series investigating the role of MRI in this disease, and the first to show characteristic abnormalities on MRI in patients whose CT was negative.

<span class="mw-page-title-main">Perfusion MRI</span>

Perfusion MRI or perfusion-weighted imaging (PWI) is perfusion scanning by the use of a particular MRI sequence. The acquired data are then post-processed to obtain perfusion maps with different parameters, such as BV, BF, MTT and TTP.

<span class="mw-page-title-main">Boris Rotman</span> Chilean American immunologist–molecular biologist (1924–2021)

Marcos Boris Rotman was a Chilean American immunologist–molecular biologist and professor emeritus of Medical Science at Alpert Medical School of Brown University. He is widely recognized for performing the first single molecule experiments in biology. He died in July 2021 at the age of 96.

References

  1. Rodriguez I, Perez-Rial S, Gonzalez-Jiminez J, et al., Magnetic Resonance Methods and Applications in Pharmaceutical Research. J Pharma Sci, 28 Jan. 2008 (E-publication ahead of print)
  2. 1 2 Meade TJ, Taylor AK, and Bull SR, New magnetic resonance contrast agents as biochemical reporters. Curr Opin Neurobiol 13, pp. 597-602.
  3. 1 2 Welssleder R, and Umar M, Molecular Imaging. Radiology 219, pp. 316-333.
  4. 1 2 3 4 Urbanczyk-Pearson LM, Femia FJ, Smith J, et al., Mechanistic Investigation of β-Galactosidase-Activated MR Contrast Agents. Inorg Chem 48, pp. 56-68
  5. 1 2 Louie AY, Huber MM, Ahrens ET, et al., In vivo visualization of gene expression using magnetic resonance imaging. Nature Biotech 18, pp. 321-25
  6. Aikawa E, Nahrendorf M, Figueiredo JL, et al., Osteogenesis associates with inflammation in early-stage atherosclerosis evaluated by molecular imaging in vivo. Circulation 116, pp. 2841-50.
  7. Kayyem JF, Kumar RM, Fraser SE, et al., Receptor-targeted co-transport of DNA and magnetic resonance contrast agents. Chem & Biol 2, pp. 615-20