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We are developing 129Xe
magnetic resonance
imaging (MRI) biosensors with the potential to detect molecular markers
associated with cancer and other diseases in humans. This follows
on a seminal 2001 PNAS paper by Schultz and co-workers. Xenon gas
is non-toxic, and has been used in many animal and human imaging
studies, particularly of the lungs, where xenon is readily delivered
through inhalation. 129Xe is spin-˝ and its chemical shift is
very sensitive to molecular environment. Laser polarization with
our home-built instrument currently provides 2% polarization,
corresponding to a 2000-fold signal enhancement, and levels over 60%
have recently been reported. Xenon binds cryptophane-A (Figure 4) with
high affinity.
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 Figure 4. Crystal structure of Xe-C8B-CAII
complex.
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| Group members developed an
efficient method for attaching a peptide to the cryptophane, in order
to create Xe biosensors that target specific cancer biomarkers.
Matrix metalloproteinases (MMPs) were targeted, based on the
up-regulation and secretion of these enzymes by many cancer cells. This
represents the first and currently the only example of a 129Xe NMR
biosensor that measures enzyme activity. These strategies are
being optimized to achieve much larger 129Xe NMR chemical shift
changes. In this way, it will be possible to monitor multiple
enzymes simultaneously by hyperpolarized 129Xe NMR, which should
improve early cancer diagnosis. A current thrust of this project is to elucidate how biomolecular interactions perturb the 129Xe NMR chemical shift. We have introduced human carbonic anhydrase (CA) as a single-binding-site enzyme for studying xenon biosensor-protein interactions. A xenon-binding cryptophane was substituted with linkers varying between 6 and 8 bonds to CA-specific p-benzenesulfonamide ligand to yield non-diastereomeric biosensors with a single 129Xe NMR resonance. We are collaborating with Prof. David Christianson’s group to obtain crystal structures of Xe biosensors that bind preferentially to specific isoforms of human carbonic anhydrase. X-ray crystallography recently confirmed binding of the 8-bond-linked biosensor to Zn2+ at the CAII active site, with the cryptophane positioned at the protein channel (Figure 5). Biosensor dissociation constants (Kd = 20-110 nM) were determined by isothermal titration calorimetry (ITC) for isozymes CA I and II. The biosensors complexed with CA yielded “bound” hyperpolarized 129Xe NMR resonances of narrow linewidth at approximately 64 ppm that were shifted between 3.0 and 7.5 ppm downfield upon CA binding. The shortest 6-bond-linker biosensor gave a single “bound” peak when complexed with CAI (70 ppm) and CAII (69 ppm). These isozyme-specific 129Xe NMR chemical shifts clearly differentiated CA I and II. Based on many data, we postulate that confining the cryptophane with a short rigid linker at the nearest protein surface leads to a single “bound” peak, which yields the highest possible signal-to-noise ratio. We believe that xenon biosensors may provide a powerful strategy for diagnosing human diseases characterized by the upregulation of specific CA isozymes and other protein biomarkers. Upregulation of several CA isozymes, including IX and XII, has been implicated in various cancers and glaucoma. New xenon-binding cages have been developed that can be functionalized in high yields for studies in animals, bind xenon with higher affinity, and produce a wider range of 129Xe NMR chemical shifts. A water-soluble cryptophane was synthesized that has a larger association constant for xenon than has been measured for any synthetic or biological compound, KA = 33,000 M-1 + 2000 M-1 at 293 K. To obtain binding data, fluorescence- and isothermal calorimetry-based assays were developed that are much more sensitive and accurate than standard NMR methods. Interestingly, TΔS was determined to equal roughly 2.5 kcal/mol, which we attribute to the desolvation of xenon that occurs during binding of cryptophane in water. Our recent X-ray crystal structure of a cryptophane complexed with xenon, fluorescence lifetime measurements of cryptophane fluorescence quenching by xenon, computational modeling, and 1- and 2-D NMR experiments are helping to provide a better molecular understanding of xenon-cryptophane interactions. The first studies on the cellular uptake of cryptophane were performed by CLSM. Cryptophanes attached to the cell-penetrating TAT peptide or integrin-targeting tetraRGD peptide (Figure 6) were shown to be taken up by a variety of cancer cell lines. Blockage of cell uptake of a tetraRGD-modified cryptophane with a higher affinity cyclic RGD peptide (Figure 5E) or an antibody targeted to the intergrin receptor (Figure 5F) confirmed that cryptophane uptake was integrin-mediated. |
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  Figure 5. Above:
Structure of RGD. Uptake of 2 μM Cy3-labeled RGD targeting αvβ3
integrin in NCI-H1975 (A), HFL-1 (B), and CAPAN-2 (C) cells after 30
min incubation at 37 oC. Red blood cells (D) did not uptake this
compound. Targeting in NCI-H1975 was successfully blocked with 100 μM
cyclic RGDfV peptide (E) and 10 μM anti-αv antibody (F).
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| The long-range goal of Project 2 is to design compounds that localize to cancer cells and provide unique chemical shifts that are associated with binding to target biomolecules. Importantly, the peptide-functionalized cryptophanes were found to be nontoxic to cells in a variety of MTT and cell proliferation assays at the concentrations required for hyperpolarized 129Xe NMR studies. Cell experiments will soon be initiated with a folate-conjugated cryptophane that should exhibit even greater cancer cell specificity. The large chemical shift changes produced in the carbonic anhydrase studies indicate that it should be possible to make “smart” xenon probes that report on their environment (pH, inter- vs. intracellular, intact vs. cleaved, bound vs. unbound) and provide multiplexed, molecular information that will be very useful for early and accurate diagnosis of disease. | |