Evaluating Acetate Metabolism for Imaging and Targeting in Multiple Myeloma.

PURPOSE: We hypothesized that in multiple myeloma cells (MMC), high membrane biosynthesis will induce acetate uptake in vitro and in vivo Here, we studied acetate metabolism and targeting in MMC in vitro and tested the efficacy of 11C-acetate-positron emission tomography (PET) to detect and quantitatively image myeloma treatment response in vivo EXPERIMENTAL DESIGN: Acetate fate tracking using 13C-edited-1H NMR (nuclear magnetic resonance) was performed to study in vitro acetate uptake and metabolism in MMC. Effects of pharmacological modulation of acetate transport or acetate incorporation into lipids on MMC cell survival and viability were assessed. Preclinical mouse MM models of subcutaneous and bone tumors were evaluated using 11C-acetate-PET/CT imaging and tissue biodistribution. RESULTS: In vitro, NMR showed significant uptake of acetate by MMC and acetate incorporation into intracellular metabolites and membrane lipids. Inhibition of lipid synthesis and acetate transport was toxic to MMC, while sparing resident bone cells or normal B cells. In vivo, 11C-acetate uptake by PET imaging was significantly enhanced in subcutaneous and bone MMC tumors compared with unaffected bone or muscle tissue. Likewise, 11C-acetate uptake was significantly reduced in MM tumors after treatment. CONCLUSIONS: Uptake of acetate from the extracellular environment was enhanced in MMC and was critical to cellular viability. 11C-Acetate-PET detected the presence of myeloma cells in vivo, including uptake in intramedullary bone disease. 11C-Acetate-PET also detected response to therapy in vivo Our data suggested that acetate metabolism and incorporation into lipids was crucial to MM cell biology and that 11C-acetate-PET is a promising imaging modality for MM. Clin Cancer Res; 23(2); 416-29. ©2016 AACR.

[18F]- and [11C]-labeled N-benzyl-isatin sulfonamide analogues as PET tracers for apoptosis: synthesis, radiolabeling mechanism, and in vivo imaging study of apoptosis in Fas-treated mice using [11C]WC-98.

The radiolabeled isatin sulfonamide caspase-3 inhibitor, [18F] 2 (WC-II-89), is a potential PET radiotracer for noninvasive imaging of apoptosis. The radiolabeling mechanism was studied by 13C NMR, ESI/MS, and computational calculations. It was found that the high electrophilicity of the C3 carbonyl group in the isatin ring, which served as a trap for [18F]fluoride, was responsible for the failure of the radiolabeling via nucleophilic substitution of the mesylate group in 7a by [18F]fluoride. Once treated with a strong base, 7a opened the isatin ring completely to form an isatinate intermediate 16, which lost the ability to trap [18F]fluoride, thereby allowing the displacement of the mesylate group to afford the 18F-labeled isatinate 17. [18F] 17 can be converted to isatin [18F] 2 efficiently under acidic conditions. The ring-opening and re-closure of the isatin ring under basic and acidic conditions were confirmed by reversed phase HPLC analysis, ESI/MS and 13C NMR studies. Computational studies of model compounds also support the above proposed mechanism. Similarly, the ring-opening and re-closure method was used successfully in the synthesis of the 11C labeled isatin sulfonamide analogue [11C] 4 (WC-98). A microPET imaging study using [11C] 4 in the Fas liver apoptosis model demonstrated retained activity in the target organ (liver) of the treated mice. Increased caspase-3 activation in the liver was verified by the fluorometric caspase-3 enzyme assay. Therefore, this study provides a useful method for radio-synthesis of isatin derivative radiotracers for PET and SPECT studies, and [11C] 4 is a potential PET radiotracer for noninvasive imaging of apoptosis.

Oxidative cross-linking of tryptophan to glycine restrains matrix metalloproteinase activity: specific structural motifs control protein oxidation.

Matrix metalloproteinases (MMPs) function in homeostatic and repair processes, but unregulated catalysis by these extracellular proteinases leads to the pathological destruction of tissue proteins. An important mechanism for controlling enzyme activity might involve hypochlorous acid (HOCl), a potent oxidant produced by the myeloperoxidase system of phagocytes. We have shown that inactivation of MMP-7 (matrilysin) by HOCl coincides with the formation of a novel oxidation product, WG-4, through modification of adjacent tryptophan and glycine residues and loss of 4 atomic mass units. Here, we use mass spectrometry, UV/visible spectroscopy, hydrogen-deuterium exchange, and NMR spectroscopy to investigate the formation and structure of WG-4. For the initial step, HOCl chlorinates the indole ring of tryptophan. The resulting 3-chloroindolenine generates a previously unknown cyclic indole-amide species, in which tryptophan cross-links to the main chain nitrogen of the adjacent glycine residue to form an aromatic six-membered ring. WG-4 kinks and stiffens the peptide backbone, which may hinder the interaction of substrate with the catalytic pocket of MMP-7. Our observations indicate that specific structural motifs are important for controlling protein modification by oxidants and suggest that pericellular oxidant production by phagocytes might limit MMP activity during inflammation.