Two genetic reporter systems were developed for multimodality reporter gene imaging of different molecular-genetic processes using fluorescence, bioluminescence (BLI), and nuclear imaging techniques. The eGFP cDNA was fused at the N-terminus with HSV1-tk cDNA bearing a nuclear export signal from MAPKK (NES-HSV1-tk) or with truncation at the N-terminus of the first 45 amino acids (Delta45HSV1-tk) and with firefly luciferase at the C-terminus. A single fusion protein with three functional subunits is formed following transcription and translation from a single open reading frame. The NES-TGL (NES-TGL) or Delta45HSV1-tk/GFP/luciferase (Delta45-TGL) triple-fusion gene cDNAs were cloned into a MoMLV-based retrovirus, which was used for transduction of U87 human glioma cells. The integrity, fluorescence, bioluminescence, and enzymatic activity of the TGL reporter proteins were assessed in vitro. The predicted molecular weight of the fusion proteins (~130 kDa) was confirmed by western blot. The U87-NES-TGL and U87-Delta45-TGL cells had cytoplasmic green fluorescence. The in vitro BLI was 7- and 13-fold higher in U87-NES-TGL and U87-Delta45-TGL cells compared to nontransduced control cells. The Ki of (14)C-FIAU was 0.49+/-0.02, 0.51+/-0.03, and 0.003+/-0.001 ml/min/g in U87-NES-TGL, U87-Delta45-TGL, and wild-type U87 cells, respectively. Multimodality in vivo imaging studies were performed in nu/ nu mice bearing multiple s.c. xenografts established from U87-NES-TGL, U87-Delta45-TGL, and wild-type U87 cells. BLI was performed after administration of d-luciferin (150 mg/kg i.v.). Gamma camera or PET imaging was conducted at 2 h after i.v. administration of [(131)I]FIAU (7.4 MBq/animal) or [(124)I]FIAU (7.4 MBq/animal), respectively. Whole-body fluorescence imaging was performed in parallel with the BLI and radiotracer imaging studies. In vivo BLI and gamma camera imaging showed specific localization of luminescence and radioactivity to the TGL transduced xenografts with background levels of activity in the wild-type xenografts. Tissue sampling yielded values of 0.47%+/-0.08%, 0.86%+/-0.06%, and 0.03%+/-0.01%dose/g [(131)I]FIAU in U87-NES-TGL, U87-Delta45-TGL, and U87 xenografts, respectively. The TGL triple-fusion reporter gene preserves the functional activity of its subunits and is very effective for multimodality imaging. It provides for the seamless transition from fluorescence microscopy and FACS to whole-body bioluminescence imaging, to nuclear (PET, SPET, gamma camera) imaging, and back to in situ fluorescence image analysis.

Targeting kinases is central to drug-based cancer therapy but remains challenging because the drugs often lack specificity, which may cause toxic side effects. Modulating side effects is difficult because kinases are evolutionarily and hence structurally related. The lack of specificity of the anticancer drug imatinib enables it to be used to treat chronic myeloid leukemia, where its target is the Bcr-Abl kinase, as well as a proportion of gastrointestinal stromal tumors (GISTs), where its target is the C-Kit kinase. However, imatinib also has cardiotoxic effects traceable to its impact on the C-Abl kinase. Motivated by this finding, we made a modification to imatinib that hampers Bcr-Abl inhibition; refocuses the impact on the C-Kit kinase; and promotes inhibition of an additional target, JNK, a change that is required to reinforce prevention of cardiotoxicity. We established the molecular blueprint for target discrimination in vitro using spectrophotometric and colorimetric assays and through a phage-displayed kinase screening library. We demonstrated controlled inhibitory impact on C-Kit kinase in human cell lines and established the therapeutic impact of the engineered compound in a novel GIST mouse model, revealing a marked reduction of cardiotoxicity. These findings identify the reengineered imatinib as an agent to treat GISTs with curbed side effects and reveal a bottom-up approach to control drug specificity.

A human-derived intrinsically nonimmunogenic reporter gene was tested for PET imaging of different molecular-genetic processes for potential clinical use. Methods: The human mitochondrial thymidine kinase type 2 (hTK2) reporter gene truncated at the N terminus (DhTK2), alone or fused with green fluorescent protein (GFP), was used for preclinical evaluation in a mouse model. The levels of enzymatic activity of DhTK2 and DhTK2 GFP proteins were assessed using radiotracer accumulation and prodrug activation assays in vitro and in subcutaneous tumors grown from the corresponding cell lines in nude mice. Kinetic analyses of 124 I-29-or 18 F-9-(4-18 F-fluoro-3-hydroxymethylbutyl)guanine (FHBG) uptake in tumors and biodistribution studies were performed. Results: DhTK2 was successfully expressed in the cytoplasm of transduced cells. A new anti-hTK2 monoclonal antibody 8G2 was developed. The levels of FIAU and FEAU accumulation in cells expressing DhTK2 and DhTK2 GFP were at least 10-fold higher than in wildtype cells in vitro and about 6 times higher in vivo. We determined that FEAU is a more specific reporter substrate for DhTK2 than FIAU, whereas FHBG is not phosphorylated by this enzyme. In addition, we showed that DhTK2 transduced cells can be eliminated by treatment with D-arabinofuranosyl-cytosine. Conclusion: We have tested a human-derived reporter gene that is likely to be nonimmunogenic and potentially allows for long-term monitoring of different molecular-genetic processes by nuclear imaging techniques in humans. Using 124 I-FIAU, 18 F-FIAU, or 18 F-FEAU, it should be possible to image DhTK2 reporter gene expression with PET in preclinical and clinical studies.

To optimize the sensitivity of imaging HSV1-tk/GFP reporter gene expression, a series of HSV1-tk/GFP mutants was developed with altered nuclear localization and better cellular enzymatic activity, compared to that of the native HSV1-tk/GFP fusion protein (HSV1-tk/GFP). Several modifications of HSV1-tk/GFP reporter gene were performed, including targeted inactivating mutations in the nuclear localization signal (NLS), the addition of a nuclear export signal (NES), a combination of both mutation types, and a truncation of the first 135 bp of the native hsv1-tk coding sequence containing a "cryptic" testicular promoter and the NLS. A recombinant HSV1-tk/GFP protein and a highly sensitive sandwich enzyme-linked immunosorbent assay for HSV1-tk/GFP were developed to quantitate the amount of reporter gene product in different assays to allow normalization of the data. These different mutations resulted in various degrees of nuclear clearance, predominant cytoplasmic distribution, and increased total cellular enzymatic activity of the HSV1-tk/GFP mutants, compared to native HSV1-tk/GFP when expressed at the same levels. This appears to be the result of improved metabolic bioavailability of cytoplasmically retargeted mutant HSV1-tk/GFP enzymes for reaction with the radiolabeled probe (e.g., FIAU). The analysis of enzymatic properties of different HSV1-tk/GFP mutants using FIAU as a substrate revealed no significant differences from that of the native HSV1-tk/GFP. Improved total cellular enzymatic activity of cytoplasmically retargeted HSV1-tk/GFP mutants observed in vitro was confirmed by noninvasive imaging of transduced subcutaneous tumor xenografts bearing these reporters using [(131)I]FIAU and a gamma-camera.

Cannabinoid receptor 2 (CB2) plays an important role in human physiology and the pathophysiology of different diseases, including neuroinflammation, neurodegeneration, and cancer. Several classes of CB2 receptor ligands, including 2-oxoquinoline derivatives, have been previously reported. We report the synthesis and results of in vitro receptor binding of a focused library of new fluorinated 2-oxoquinoline CB2 ligands. Twelve compounds, 13-16 18, 19, 21-24, 27, and 28 were synthesized in good yields in multiple steps. Human U87 glioma cells expressing either hCB1 (control) or hCB2 were generated via lentiviral transduction. In vitro competitive binding assay was performed using [3H]CP-55,940 in U87hCB1 and U87hCB2 cells. Inhibition constant (Ki) values of compounds 13, 14, 15, 16, 18, 19, 21, 22, 23, 24, 27, and 28 for CB2 were >10000, 2.8, 5.0, 2.4, 22, 0.8, 1.4, >10000, 486, 58, 620, and 2400 nM, respectively, and those for CB1 were >10000 nM. Preliminary in vitro results suggest that six of these compounds may be useful for therapy of neuropathic pain, neuroinflammatory diseases and immune disorders. In addition, compound 19, with its subnanomolar Ki value, could be radiolabeled with 18F and explored for PET imaging of CB2 expression.