Interaction of Halogenated Tyrosine/Phenylalanine Derivatives with Organic Anion Transporter 1 in the Renal Handling of Tumor Imaging Probes.

Halogenated tyrosine/phenylalanine derivatives have been developed for use in tumor imaging and targeted alpha therapy. 3-Fluoro-α-methyl-l-tyrosine (FAMT), targeting amino acid transporter LAT1 (SLC7A5), is a cancer-specific positron emission tomography probe that exhibits high renal accumulation, which is supposed to be mediated by organic anion transporter OAT1 (SLC22A6). In the present study, we investigated the structural requirements of FAMT essential for interaction with OAT1. OAT1 transported FAMT with a K m of 171.9 µM. In structure-activity relationship analyses, removal of either the 3-halogen or 4-hydroxyl group from FAMT or its structural analog 3-iodo-α-methyl-l-tyrosine greatly decreased the interaction with OAT1, reducing the [14C]p-aminohippurate uptake inhibition and the efflux induction. By contrast, the α-methyl group, which is essential for LAT1 specificity, contributed to a lesser degree. In fluorinated tyrosine derivatives, fluorine at any position was accepted by OAT1 when there was a hydroxyl group at the ortho-position, whereas ortho-fluorine was less interactive when a hydroxyl group was at meta- or para-positions. The replacement of the ortho-fluorine with a bulky iodine atom greatly increased the interaction. In in vivo studies, probenecid decreased the renal accumulation (P < 0.001) and urinary excretion (P = 0.0012) of FAMT, whereas the plasma concentration was increased, suggesting the involvement of OAT1-mediated transepithelial organic anion excretion. LAT1-specific 2-fluoro-α-methyltyrosine, which had lower affinity for OAT1, exhibited lower renal accumulation (P = 0.0142) and higher tumor uptake (P = 0.0192) compared with FAMT. These results would provide a basis to design tumor-specific compounds that can avoid renal accumulation for tumor imaging and targeted alpha therapy. SIGNIFICANCE STATEMENT: We revealed the structural characteristics of halogenated tyrosine derivatives essential for interaction with the organic anion transporter responsible for their renal accumulation. We have confirmed that such interactions are important for renal handling and tumor uptake. The critical contribution of hydroxyl and halogen groups and their positions as well as the role of α-methyl group found in the present study may facilitate the development of tumor-specific compounds while avoiding renal accumulation for use in tumor imaging and targeted alpha therapy.

Evidence for Naturally Produced Beauvericins Containing N-Methyl-Tyrosine in Hypocreales Fungi.

Beauvericin is a depsipeptide mycotoxin. The production of several beauvericin analogues has previously been shown among various genera among Hypocreales fungi. This includes so-called beauvenniatins, in which one or more N-methyl-phenylalanine residues is exchanged with other amino acids. In addition, a range of "unnatural" beauvericins has been prepared by a precursor addition to growth medium. Our aim was to get insight into the natural production of beauvericin analogues among different Hypocreales fungi, such as Fusarium and Isaria spp. In addition to beauvericin, we tentatively identified six earlier described analogues in the extracts; these were beauvericin A and/or its structural isomer beauvericin F, beauvericin C, beauvericin J, beauvericin D, and beauvenniatin A. Other analogues contained at least one additional oxygen atom. We show that the additional oxygen atom(s) were due to the presence of one to three N-methyl-tyrosine moieties in the depsipeptide molecules by using different liquid chromatography⁻mass spectrometry-based approaches. In addition, we also tentatively identified a beauvenniatin that contained N-methyl-leucine, which we named beauvenniatin L. This compound has not been reported before. Our data show that N-methyl-tyrosine containing beauvericins may be among the major naturally produced analogues in certain fungal strains.

Isotope effects in the tyrosinase catalysed hydroxylation of l-tyrosine methyl derivatives.

To investigate isotope effects in the hydroxylation of [3',5'-2H2]-α-methyl- and [3',5'-2H2]-N-methyl-l-tyrosine, they were synthesised using acid catalysed isotope exchange at high temperature. The kinetic and solvent deuterium isotope effects on Vmax and Vmax/Km parameters of tyrosinase in its action on methylated derivatives of l-tyrosine were determined using the non-competitive spectrophotometric method. Lineweaver-Burk plots were used to consider the inhibition type of O-methyl-l-tyrosine, revealing that it is an uncompetitive inhibitor of tyrosinase.

Transport of 3-fluoro-L-α-methyl-tyrosine (FAMT) by organic ion transporters explains renal background in [(18)F]FAMT positron emission tomography.

A PET tracer for tumor imaging, 3-(18)F-l-α-methyl-tyrosine ([(18)F]FAMT), has advantages of high cancer-specificity and low physiological background. In clinical studies, FAMT-PET has been proved useful for the detection of malignant tumors and their differentiation from inflammation and benign lesions. The tumor specific uptake of FAMT is due to its high-selectivity to cancer-type amino acid transporter LAT1 among amino acid transporters. In [(18)F]FAMT PET, kidney is the only organ that shows high physiological background. To reveal transporters involved in renal accumulation of FAMT, we have examined [(14)C]FAMT uptake on the organic ion transporters responsible for the uptake into tubular epithelial cells. We have found that OAT1, OAT10 and OCTN2 transport [(14)C]FAMT. The [(14)C]FAMT uptake was inhibited by probenecid, furosemide and ethacrynic acid, consistent with the properties of the transporters. The amino acid uptake inhibitor, 2-amino-2-norbornanecarboxylic acid (BCH), also inhibited the [(14)C]FAMT uptake, whereas OCTN2-mediated [(14)C]FAMT uptake was Na(+)-dependent. We propose that FAMT uptake by OAT1, OAT10 and OCTN2 into tubular epithelial cells could contribute to the renal accumulation of FAMT. The results from this study would provide clues to the treatments to reduce renal background and enhance tumor uptake as well as to designing PET tracers with less renal accumulation.

Specific transport of 3-fluoro-l-α-methyl-tyrosine by LAT1 explains its specificity to malignant tumors in imaging.

3-(18)F-l-α-methyl-tyrosine ([18F]FAMT), a PET probe for tumor imaging, has advantages of high cancer-specificity and lower physiologic background. FAMT-PET has been proved useful in clinical studies for the prediction of prognosis, the assessment of therapy response and the differentiation of malignant tumors from inflammation and benign lesions. The tumor uptake of [18F]FAMT in PET is strongly correlated with the expression of L-type amino acid transporter 1 (LAT1), an isoform of system L upregulated in cancers. In this study, to assess the transporter-mediated mechanisms in FAMT uptake by tumors, we examined amino acid transporters for FAMT transport. We synthesized [14C]FAMT and measured its transport by human amino acid transporters expressed in Xenopus oocytes. The transport of FAMT was compared with that of l-methionine, a well-studied amino acid PET probe. The significance of LAT1 in FAMT uptake by tumor cells was confirmed by siRNA knockdown. Among amino acid transporters, [14C]FAMT was specifically transported by LAT1, whereas l-[14C]methionine was taken up by most of the transporters. Km of LAT1-mediated [14C]FAMT transport was 72.7 µM, similar to that for endogenous substrates. Knockdown of LAT1 resulted in the marked reduction of [14C]FAMT transport in HeLa S3 cells, confirming the contribution of LAT1 in FAMT uptake by tumor cells. FAMT is highly specific to cancer-type amino acid transporter LAT1, which explains the cancer-specific accumulation of [18F]FAMT in PET. This, vice versa, further supports the cancer-specific expression of LAT1. This study has established FAMT as a LAT1-specific molecular probe to monitor the expression of a potential tumor biomarker LAT1.

Synthesis of 6-[(11)C]methyl-m-tyrosine ([(11)C]6MemTyr) for dopamine synthesis imaging in living brain using PET.

A novel PET probe, 6-[(11)C]methyl-m-tyrosine ([(11)C]6MemTyr), was developed for quantitative imaging of presynaptic dopamine (DA) synthesis in the living brain using positron emission tomography (PET). This probe was evaluated by comparison with conventional 6-[(18)F]fluoro-l-dopa ([(18)F]FDOPA). [(11)C]6MemTyr was labeled using rapid Pd(0)-mediated C-[(11)C]methylation with [(11)C]methyl iodide. The synthesis time was only 35min, and its radiochemical yield was 76%, with radiochemical purity of >99%. PET measurements indicated that [(11)C]6MemTyr could image presynaptic DA synthesis in the striatum of living monkey brain, providing much higher contrast between the striatum and the cerebellum than that with [(18)F]FDOPA.

Effects of intratumoral inflammatory process on 18F-FDG uptake: pathologic and comparative study with 18F-fluoro-α-methyltyrosine PET/CT in oral squamous cell carcinoma.

UNLABELLED: The accurate depiction of both biologic and anatomic profiles of tumors has long been a challenge in PET imaging. An inflammation, which is innate in the carcinogenesis of oral squamous cell carcinoma (OSCC), frequently complicates the image analysis because of the limitations of (18)F-FDG and maximum standardized uptake values (SUV(max)). New PET parameters, metabolic tumor volume (MTV) and total lesion glycolysis (TLG), as well as (18)F-fluoro-α-methyltyrosine ((18)F-FAMT), a malignancy-specific amino acid-based PET radiotracer, are considered more comprehensive in tumor image analysis. Here, we showed the substantial effects of the intratumoral inflammatory process on (18)F-FDG uptake and further study the possibility of MTV and TLG to predict both tumor biology (proliferation activity) and anatomy (pathologic tumor volume). METHODS: (18)F-FDG and (18)F-FAMT PET images from 25 OSCC patients were analyzed. SUV(max) on the tumor site was obtained. PET volume computerized-assisted reporting was used to generate a volume of interest to obtain MTV and TLG for (18)F-FDG and total lesion retention (TLR) for (18)F-FAMT. The whole tumor dissected from surgery was measured and sectioned for pathologic analysis of tumor inflammation grade and Ki-67 labeling index. RESULTS: The high SUV(max) of (18)F-FDG was related to the high inflammation grade. The SUV(max )ratio of (18)F-FDG to (18)F-FAMT was higher in inflammatory tumors (P < 0.05) whereas the corresponding value in tumors with a low inflammation grade was kept low. All (18)F-FAMT parameters were correlated with Ki-67 labeling index (P < 0.01). Pathologic tumor volume predicted from MTV of (18)F-FAMT was more accurate (R = 0.90, bias = 3.4 ± 6.42 cm(3), 95% confidence interval = 0.77-6.09 cm(3)) than that of (18)F-FDG (R = 0.77, bias = 8.1 ± 11.17 cm(3), 95% confidence interval = 3.45-12.67 cm(3)). CONCLUSION: (18)F-FDG uptake was overestimated by additional uptake related to the intratumoral inflammatory process, whereas (18)F-FAMT simply accumulated in tumors according to tumor activity as evaluated by Ki-67 labeling index in OSCC.

Characterization of SfmD as a Heme peroxidase that catalyzes the regioselective hydroxylation of 3-methyltyrosine to 3-hydroxy-5-methyltyrosine in saframycin A biosynthesis.

Saframycin A (SFM-A) is a potent antitumor antibiotic that belongs to the tetrahydroisoquinoline family. Biosynthetic studies have revealed that its unique pentacyclic core structure is derived from alanine, glycine, and non-proteinogenic amino acid 3-hydroxy-5-methyl-O-methyltyrosine (3-OH-5-Me-OMe-Tyr). SfmD, a hypothetical protein in the biosynthetic pathway of SFM-A, was hypothesized to be responsible for the generation of the 3-hydroxy group of 3-OH-5-Me-OMe-Tyr based on previously heterologous expression results. We now report the in vitro characterization of SfmD as a novel heme-containing peroxidase that catalyzes the hydroxylation of 3-methyltyrosine to 3-hydroxy-5-methyltyrosine using hydrogen peroxide as the oxidant. In addition, we elucidated the biosynthetic pathway of 3-OH-5-Me-OMe-Tyr by kinetic studies of SfmD in combination with biochemical assays of SfmM2, a methyltransferase within the same pathway. Furthermore, SacD, a counterpart of SfmD involved in safracin B biosynthesis, was also characterized as a heme-containing peroxidase, suggesting that SfmD-like heme-containing peroxidases may be commonly involved in the biosynthesis of SFM-A and its analogs. Finally, we found that the conserved motif HXXXC is crucial for heme binding using comparative UV-Vis and Magnetic Circular Dichroism (MCD) spectra studies of SfmD wild-type and mutants. Together, these findings expand the category of heme-containing peroxidases and set the stage for further mechanistic studies. In addition, this study has critical implications for delineating the biosynthetic pathway of other related tetrahydroisoquinoline family members.

Comparison of cell proliferation, protein, and glucose metabolism in musculoskeletal tumors in a PET study.

¹¹C-choline and ¹8F-FAMT are known to correlate with tumor cell proliferation and amino acid metabolism. We investigated the ability of ¹¹C-Choline and ¹8F-FAMT PET in diagnosis of musculoskeletal tumors in thirty-six patients in comparison of ¹8F-FDG PET. ¹¹C-Choline and ¹8F-FDG PET were positive in all the malignant tumors (n = 13), whereas ¹8F-FAMT was positive in 11 tumors. The mean SUVs for malignant tumors were significantly higher than those for benign lesions in all three tracers imaging. A moderate correlation was found between ¹¹C-Choline and ¹8F-FDG (r = 0.540, P < .05), or ¹8F-FAMT and FDG (r = 0.596, P < .05). The diagnostic sensitivity and specificity for malignancy were 91.7% and 71.4%, respectively, using ¹¹C-choline with a SUV cut-off of 2.69. The sensitivity and specificity of ¹8F-FAMT for malignancy were 66.7% and 85.7%, respectively, using a SUV cut-off of 1.26. For ¹8F-FDG, using a SUV cut-off of 2.77, the sensitivity and specificity were 83.3% and 71.4%, respectively. According to ROC analysis, the ROC curves for ¹¹C-Choline, ¹8F-FAMT, and ¹8F-FDG were 0.855, 0.734, and 0.847, respectively. ¹¹C-Choline PET is superior in the visualization of musculoskeletal tumors with high contrast imaging, whereas the combination of ¹8F-FAMT and ¹8F-FDG PET provides valuable information for the preoperative planning in patients with musculoskeletal tumors.