ABSTRACT
Expression of the chemokine receptor chemokine C-X-C motif receptor 4 (CXCR4) plays an important role in cancer metastasis, in autoimmune diseases, and during stem cell-based repair processes after stroke and myocardial infarction. Previously reported PET imaging agents targeting CXCR4 suffer from either high nonspecific uptake or bind only to the human form of the receptor. The objective of this study was to develop a high-stability 64Cu-labeled small-molecule PET agent for imaging both human and murine CXCR4 chemokine receptors. Methods: Synthesis, radiochemistry, stability and radioligand binding assays were performed for the novel tracer 64Cu-CuCB-bicyclam. In vivo dynamic PET studies were performed on mice bearing U87 (CXCR4 low-expressing) and U87.CXCR4 (human-CXCR4 high-expressing) tumors. Biodistribution and receptor blocking studies were performed on CD1-IGS immunocompetent mice. CXCR4 expression on tumor and liver disaggregates was confirmed using a combination of immunohistochemistry, quantitative polymerase chain reaction, and Western blot. Results:64Cu-CuCB-bicyclam has a high affinity for both the human and the murine variants of the CXCR4 receptor (half-maximal inhibitory concentration, 8 nM [human]/2 nM [murine]) and can be obtained from the parent chelator that has low affinity. In vitro and in vivo studies demonstrate specific uptake in CXCR4-expressing cells that can be blocked by more than 90% using a higher-affinity antagonist, with limited uptake in non-CXCR4-expressing organs and high in vivo stability. The tracer was also able to selectively displace the CXCR4 antagonists AMD3100 and AMD3465 from the liver. Conclusion: The tetraazamacrocyclic small molecule 64Cu-CuCB-bicyclam has been shown to be an imaging agent for the CXCR4 receptor that is likely to be applicable across a range of species. It has high affinity and stability and is suitable for preclinical research in immunocompetent murine models.
Subject(s)
Copper Radioisotopes/chemistry , Receptors, CXCR4/antagonists & inhibitors , Receptors, CXCR4/chemistry , Animals , Benzylamines , Cell Line, Tumor , Chelating Agents/chemistry , Cyclams , Female , Heterocyclic Compounds/chemistry , Humans , Image Processing, Computer-Assisted , Liver/diagnostic imaging , Mice , Mice, Nude , Neoplasm Transplantation , Positron-Emission Tomography , Protein Binding , Pyridines/chemistry , Radiopharmaceuticals/chemistry , Tissue DistributionABSTRACT
UNLABELLED: Deinococcus radiodurans is an aerobic organism with the ability to survive under conditions of high radiation doses or desiccation. As part of its protection system against oxidative stress, this bacterium encodes three monofunctional catalases. The DR1998 catalase belongs to clade 1, and is present at high levels under normal growth conditions. The crystals of DR1998 diffracted very weakly, and the merged diffraction data showed an R sym of 0.308. Its crystal structure was determined and refined to 2.6 Å. The four molecules present in the asymmetric unit form, by crystallographic symmetry, two homotetramers with 222 point-group symmetry. The overall structure of DR1998 is similar to that of other monofunctional catalases, showing higher structural homology with the catalase structures of clade 1. Each monomer shows the typical catalase fold, and contains one heme b in the active site. The heme is coordinated by the proximal ligand Tyr369, and on the heme distal side the essential His81 and Asn159 are hydrogen-bonded to a water molecule. A 25-Å-long channel is the main channel connecting the active site to the external surface. This channel starts with a hydrophobic region from the catalytic heme site, which is followed by a hydrophilic region that begins on Asp139 and expands up to the protein surface. Apart from this channel, an alternative channel, also near the heme active site, is presented and discussed. DATABASE: Coordinates and structure factors have been deposited in the Protein Data Bank in Europe under accession code 4CAB.
Subject(s)
Bacterial Proteins/chemistry , Catalase/chemistry , Deinococcus/enzymology , Amino Acid Sequence , Catalytic Domain , Crystallography, X-Ray , Heme/chemistry , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Molecular Sequence Data , Protein Structure, Quaternary , Protein Structure, SecondaryABSTRACT
The protective mechanisms of Deinococcus radiodurans against primary reactive oxygen species involve nonenzymatic scavengers and a powerful enzymatic antioxidant system including catalases, peroxidases and superoxide dismutases that prevents oxidative damage. Catalase is an enzyme that is responsible for the conversion of H2O2 to O2 and H2O, protecting the organism from the oxidative effect of H2O2. This study reports the purification and crystallization of the DR1998 catalase from D. radiodurans. The crystals diffracted to 2.6 Å resolution and belonged to space group C2221, with unit-cell parameters a = 97.33, b = 311.88, c = 145.63 Å, suggesting that they contain four molecules per asymmetric unit. The initial phases were determined by molecular replacement and the obtained solution shows the typical catalase quaternary structure. A preliminary model of the protein structure has been built and refinement is currently in progress.
