Fluorination at the 4 position alters the substrate behavior of l-glutamine and l-glutamate: Implications for positron emission tomography of neoplasias

doi:10.1016/j.jfluchem.2016.10.008

Journal of Fluorine Chemistry

Volume 192, Part A, December 2016, Pages 58-67

https://doi.org/10.1016/j.jfluchem.2016.10.008 Get rights and content

Highlights

•
Activity with glutamine transaminase K: Gln ≈ (2S,4S)-4-FGln >> (2S,4R)-4-FGln.
•
Activity with glutamine transaminase l: Gln >> (2S,4R)-4-FGln ≈ (2S,4S)-4-FGln.
•
Activity with glutaminase (short form): Glu > (2S,4S)-4-FGln >> (2S,4R)-4-FGln.
•
Activity with glutamine synthetase: Glu > (2S,4S)-4-FGlu ≈ (2S,4R)-4-FGlu.
•
(2S,4S)-4-F-(3-Fluoropropyl)glutamine is likely largely metabolically inert in vivo.

Abstract

Two 4-fluoro-l-glutamine diastereoisomers [(2S,4R)-4-FGln, (2S,4S)-4-FGln] were previously developed for positron emission tomography. Label uptake into two tumor cell types was greater with [¹⁸F](2S,4R)-4-FGln than with [¹⁸F](2S,4S)-4-FGln. In the present work we investigated the enzymology of two diastereoisomers of 4-FGln, two diastereoisomers of 4-fluoroglutamate (4-FGlu) (potential metabolites of the 4-FGln diastereoisomers) and another fluoro-derivative of l-glutamine [(2S,4S)-4-(3-fluoropropyl)glutamine (FP-Gln)]. The two 4-FGlu diastereoisomers were found to be moderate-to-good substrates relative to l-glutamate of glutamate dehydrogenase, aspartate aminotransferase and alanine aminotransferase. Additionally, alanine aminotransferase was shown to catalyze an unusual γ-elimination reaction with both 4-FGlu diastereoisomers. Both 4-FGlu diastereoisomers were shown to be poor substrates, but strong inhibitors of glutamine synthetase. Both 4-FGln diastereoisomers were shown to be poor substrates compared to l-glutamine of glutamine transaminase L and α-aminoadipate aminotransferase. However, (2S,4R)-4-FGln was found to be a poor substrate of glutamine transaminase K, whereas (2S,4S)-4-FGln was shown to be an excellent substrate. By contrast, FP-Gln was found to be a poor substrate of all enzymes examined. Evidently, substitution of H in position 4 by F in l-glutamine/l-glutamate has moderate-to-profound effects on enzyme-catalyzed reactions. The present results: 1) show that 4-FGln and 4-FGlu diastereoisomers may be useful for studying active site topology of glutamate- and glutamine-utilizing enzymes; 2) provide a framework for understanding possible metabolic transformations in tumors of ¹⁸F-labeled (2S,4R)-4-FGln, (2S,4S)-4-FGln, (2S,4R)-4-FGlu or (2S,4S)-4-FGlu; and 3) show that [¹⁸F]FP-Gln is likely to be much less metabolically active in vivo than are the [¹⁸F]4-FGln diastereoisomers.

Graphical abstract

Metabolism of the 2S,4S- and 2S,4R diastereoisomers of 4-FGln and 4-FGln. The solid arrows indicate the proposed metabolic pathways for the 2S,4S- and 2S,4R diastereoisomers of 4-FGln and 4F-Glu, while the broken arrows indicate the less favored pathways.

Introduction

Positron emission tomography (PET) is frequently used to image aggressive neoplasias and to assess the efficacy of treatment. One important and emerging group of imaging agents for these studies are ¹⁸F-labeled analogues of glutamate and glutamine. These compounds exploit the high requirement for glutamine (glutamine addiction) exhibited by many cancer cell lines to selectively image these cells. The intracellular fate of this pool of glutamine depends on the origins of the neoplastic cells. Following cellular uptake, glutamine is deamidated to glutamate by glutaminase. This pool of glutamate is commonly thought to undergo conversion to 2-oxoglutarate and thereby enter the TCA cycle and support anaplerosis [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12]. By contrast, Tardito et al. demonstrated the glutamine-derived pool of glutamate in glioblastoma cells is either exported or converted to glutamine by glutamine synthetase [13]. Moreover, under conditions of glutamine starvations these cells siphon 2-oxoglutate from the TCA cycle (cataplerosis) to maintain the intracellular glutamine pools of glioblastoma cells. These observations reveal important variations in the metabolism of glutamine and glutamate among neoplastic cells of differing origins. The extremely rapid rates at which glutamate and glutamine are metabolized within cells [14], make the ¹⁸F-labeled fluorinated forms of these amino acids especially attractive as PET probes for the study of tumor cell metabolism.

Kung et al. have developed procedures for the rapid radiochemical synthesis of all four possible ¹⁸F-labeled diastereoisomers of 4-FGln (i.e., 2S,4S-, 2S,4R-, 2R,4S- and 2R,4R-4-FGln) [15], [16], [17] (¹⁸F is a positron emitter with a t_1/2 of 109.8 min). It was reasoned that replacement of an H with an F in the 4 position of l-glutamine will result in labeled glutamine analogues that can be used for PET imaging of glutamine-utilizing tumors [15], [16], [17]. Our previous work with (2S,4R)-4-FGln and (2S,4R)4-FGlu supports this hypothesis [18], but with certain caveats, namely that the rates at which the enzymes utilize (2S,4R)-4-FGln and (2S,4R)4-FGlu as substrates relative to their non-fluorinated counterparts are different [18]. Since glutamine and glutamate serve as substrates for a wide range of enzymes, we sought to extend our earlier studies by examining the substrate behavior of both (2S,4S)-4-FGlu and (2S,4R)-4-FGlu diastereoisomers toward glutamate-utilizing enzymes and both (2S,4S)-4-FGln and (2S,4R)-4-FGln diastereoisomers toward glutamine-utilizing enzymes. Note that 2S- is equivalent to 2-l at carbon 2, whereas 2R- is equivalent to 2d- at carbon 2. Therefore, only the 2S- diastereoisomers (and not the 2R-diastereoisomers) of fluorinated analogues were used in the present studies as likely substrates of l-glutamate- and l-glutamine-utilizing enzymes.

Previous work showed that tumors in rodents metabolize [¹⁸F](2S,4R)-4-FGln in part to ¹⁸F⁻ [17]. Subsequently, the glutamine analogue [¹⁸F](2S,4S)-4-(3-fluoropropyl)glutamine ([¹⁸F]FP-Gln) was synthesized as a tumor PET imaging agent [19]. This compound is unlikely to undergo defluorination in vivo. In the present work we show that FP-Gln is likely to be metabolized very slowly in vivo. We have also uncovered a few remarkable differences in the enzymology between (2S,4S)-4-FGln and (2S,4R)-4-FGln. The present studies provide insights as to the possible metabolic fates in vivo of (2S,4S)-4-FGlu, (2S,4R)-4-FGlu, (2S,4S)-4-FGln and (2S,4R)-4-FGln. In addition, we suggest that these fluorinated compounds will be excellent probes for studying the active site topology of a number of glutamate- and glutamine-utilizing enzymes.

Section snippets

Reagents

(2S,4R)-4-FGln, (2S,4S)-4-FGln, (2S,4R)-4-FGlu and (2S,4S)-4-FGln were prepared using previously published methods [15], [16], [17], [18]. These compounds were dissolved in aqueous solutions by prolonged heating at 37 °C. 2,4-Dinitrophenylhydrazine was purchased from ICN Biomedicals (Irvine, CA) while all other compounds were supplied by Sigma-Aldrich (St. Louis, MO).

Enzymes

Highly purified recombinant human glutamine transaminase K [rhGTK; also known as kynurenine aminotransferase I (KAT I)] [20],

Glutaminase

GAC was incubated with increasing concentrations of l-glutamine (or 4-FGln) and assayed by an end-point procedure. A hyperbolic plot of l-glutamine concentration versus rate of product formation was obtained with an estimated K_m of ∼2 mM (Fig. 1). When (2S,4S)-4-FGln was employed as a substrate, a hyperbolic plot was not obtained and the rate increased linearly up to a concentration of about 50 mM (at which point the rate of the reaction began to approach that observed with high concentrations of

General comments on the use of ¹⁸F-labeled glutamine analogues as PET tumor imaging agents

For a radiotracer to be a good tumor imaging agent, label associated with the tracer must accumulate in the tumor. In this context, it was previously shown that label associated with both [¹⁸F](2S,4R)-4-FGln and [¹⁸F](2S,4S)-4-FGln accumulated in 9L tumor cells and in SF188-Bcl-xL tumor cells in a transport-dependent manner [15]. Over a period of two hours more label accumulated in the tumor cells exposed to [¹⁸F](2S,4R)-4-FGln than with [¹⁸F](2S,4S)-4-FGln [15]. Evidence was presented that

Conclusions

A major finding of the present work is that both diastereoisomers of 4-FGln are relatively poor substrates of AAT/KAT II, GTL/KAT III and GAC. On the other hand, (2S,4S)-4-FGln is an excellent substrate of GTK/KAT I, whereas, (2S,4R)-4-FGln is a relatively poor substrate. It is suggested that (2S,4R)-4-FGln and (2S,4S)-4-FGln may be useful probes for studying the detailed topology of the active sites of glutamine-utilizing enzymes such as glutaminase and glutamine transaminases. These compounds

Conflict of interest

The authors declare no conflict of interest.

Funding

HFK acknowledges funding from CA-164490 from the National Institutes of Health and by a Stand-Up 2 Cancer grant (SU2C).

Author contributions

AJLC, HFK and KP conceived the idea of investigating the enzymology of 4-FGlu and 4-FGln. TMJ, AJLC, EK, JAA, JTP, CS and JWE carried out the enzyme studies. HFK and KP synthesized the 4-FGlu and 4-FGln diastereoisomers used in the current study and JL provided purified samples of GTK/KAT I, AAT/KAT II and GTL/KAT III. AJLC and TMJ wrote the paper with the assistance of JTP.

References (52)

M. Szeliga et al.
Glutamine in neoplastic cells: focus on the expression and roles of glutaminases
Neurochem. Int.
(2009)
J.B. Wang et al.
Targeting mitochondrial glutaminase activity inhibits oncogenic transformation
Cancer Cell
(2010)
W.P. Katt et al.
Glutaminase regulation in cancer cells: a druggable chain of events
Drug Discov. Today
(2014)
A. Emadi et al.
Inhibition of glutaminase selectively suppresses the growth of primary acute myeloid leukemia cells with IDH mutations
Exp. Hematol.
(2014)
A.J.L. Cooper et al.
Comparative enzymology of (2S,4R)4-fluoroglutamine and (2S,4R)4-fluoroglutamate
Comp: Biochem Physiol. B Biochem. Mol. Biol.
(2012)
J.T. Pinto et al.
Kynurenine aminotransferase III and glutamine transaminase L are identical enzymes that have cysteine S-conjugate β-lyase activity and can transaminate L-selenomethionine
J. Biol. Chem.
(2014)
L.A. Lichtenberg et al.
A sensitive fluorometric assay for amino acid oxidases
Anal. Biochem.
(1968)
J.A. Olson
Spectrophotometric measurement of α-keto acid semicarbazones
Arch. Biochem. Biophys.
(1959)
J.T. Pinto et al.
Measurement of sulfur-containing compounds involved in the metabolism and transport of cysteamine and cystamine. Regional differences in cerebral metabolism
J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.
(2009)
H.Z. Kupchik et al.
Assays of glutamine and its aminotransferase with the enol-borate of phenylpyruvate
Arch. Biochem. Biophys.
(1970)

A.J.L. Cooper et al.

Isolation and properties of a new glutamine transaminase from rat kidney

J. Biol. Chem.

(1974)

M. Nakano et al.

Crystalline mammalian L-amino acid oxidase from rat kidney mitochondria

J. Biol. Chem.

(1966)

N.P. Curthoys et al.

Phosphate-independent glutaminase from rat kidney. Partial purification and identity with γ-glutamyltranspeptidase

J. Biol. Chem.

(1975)

J.A.K. Howard et al.

How good is fluorine as a hydrogen bond acceptor?

Tetrahedron

(1996)

J.W. Erickson et al.

Glutaminase: a hot spot for regulation of cancer cell metabolism?

Oncotarget

(2010)

A.J. Levine et al.

The control of the metabolic switch in cancers by oncogenes and tumor suppressor genes

Science

(2010)

S. Suzuki et al.

Phosphate-activated glutaminase (GLS2), a p53-inducible regulator of glutamine metabolism and reactive oxygen species

Proc. Natl. Acad. Sci. U. S. A.

(2010)

W. Hu et al.

Glutaminase 2, a novel p53 target gene regulating energy metabolism and antioxidant function

Proc. Natl. Acad. Sci. U. S. A.

(2010)

A. Chatterjee et al.

Mitochondrial subversion in cancer

Cancer Prev. Res.

(2011)

C.V. Dang et al.

Therapeutic targeting of cancer cell metabolism

J. Mol. Med.

(2011)

M.J. Lukey et al.

The oncogenic transcription factor c-Jun regulates glutaminase expression and sensitizes cells to glutaminase-targeted therapy

Nat. Commun.

(2016)

D.M. Miller et al.

c-Myc and cancer metabolism

Clin. Cancer Res.

(2012)

S. Tardito et al.

Glutamine synthetase activity fuels nucleotide biosynthesis and supports growth of glutamine-restricted glioblastoma

Nat. Cell Biol.

(2015)

A.J.L. Cooper et al.

Central role of glutamate metabolism in the maintenance of nitrogen homeostasis in normal and hyperammonemic brain

Biomolecules

(2016)

W. Qu et al.

Synthesis of optically pure 4-fluoro-glutamines as potential metabolic imaging agents for tumors

J. Am. Chem. Soc.

(2011)

B.P. Lieberman et al.

PET imaging of glutaminolysis in tumors by ¹⁸F-(2S,4R)4-fluoroglutamine

J. Nucl. Med.

(2011)

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The variation in properties of the complexes depends upon the metal ion as well as attached ligands. Moreover, these properties can be altered with changing the substituent on the attached ligands [5,6]. Most of the Cu(II) carboxylates generally show dinuclear paddlewheel type structures with the coordination number 5 or 6.
New dimeric paddlewheel copper(II) mixed ligand complexes were synthesized having general formulae [(py)Cu(L)₄Cu(py)] (1), [(clpy)Cu(L*)₄Cu(clpy)] (2) and [(py)Cu(L°)₄Cu(py)] (3) where HL = 4-bromophenylacetic acid, HL* = phenyl acetic acid, HL° = 2-nitrophenylacetic acid, py = pyridine, clpy = 2-chloropyridine and characterized via FT-IR, powder and single crystal XRD, electrochemical solution studies and electron spin resonance spectroscopy. The resultant structures are dimeric paddlewheel where the carboxylate ligands act as bridges between two copper ions and pyridine acts as apical ligand leading to a square pyramidal geometry around each copper. The purity and crystalline nature of the complexes was checked by powder XRD technique. ESR spectra of the copper(II) complexes gave a sharp peak with g values = 1.443 (1) 1.512 (2) and 1.391 (3). The electrochemical studies showed diffusion controlled processes with diffusion co-efficient D_o values = 3.68 × 10⁻⁶ cm²s⁻¹ (1) and 3.63 × 10⁻⁶ cm²s⁻¹ (3). Moreover, the complex-DNA interaction was studied by viscosity measurement, spectrofluorometry, cyclic voltammetry and UV–Visible spectrophotometry, the latter two yielding binding constant, K_b values = 4.0497 × 10⁴ M⁻¹ and 1.703 × 10⁴ M⁻¹ (1) and 4.117 × 10⁴ M⁻¹ and 1.743 × 10⁴ M⁻¹ (3), respectively. The complexes 1 and 2 showed moderate scavenging activity for the radical 2,2-diphenyl-1-picryl-hydrazil. Micellization behaviour was studied with cationic surfactant CTAB using conductivity meter and UV–Visible spectrophotometer showing that all the complexes have increased solubility with the addition of surfactant. These initial studies heralded good biological potential of the complexes.
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Full length articleFluorination at the 4 position alters the substrate behavior of l-glutamine and l-glutamate: Implications for positron emission tomography of neoplasias

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Reagents

Enzymes

Glutaminase

General comments on the use of 18F-labeled glutamine analogues as PET tumor imaging agents

Conclusions

Conflict of interest

Funding

Author contributions

Neurochem. Int.

Cancer Cell

Drug Discov. Today

Exp. Hematol.

Comp: Biochem Physiol. B Biochem. Mol. Biol.

J. Biol. Chem.

Anal. Biochem.

Arch. Biochem. Biophys.

J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.

Arch. Biochem. Biophys.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Tetrahedron

Glutaminase: a hot spot for regulation of cancer cell metabolism?

Oncotarget

The control of the metabolic switch in cancers by oncogenes and tumor suppressor genes

Science

Phosphate-activated glutaminase (GLS2), a p53-inducible regulator of glutamine metabolism and reactive oxygen species

Proc. Natl. Acad. Sci. U. S. A.

Glutaminase 2, a novel p53 target gene regulating energy metabolism and antioxidant function

Proc. Natl. Acad. Sci. U. S. A.

Mitochondrial subversion in cancer

Cancer Prev. Res.

Therapeutic targeting of cancer cell metabolism

J. Mol. Med.

The oncogenic transcription factor c-Jun regulates glutaminase expression and sensitizes cells to glutaminase-targeted therapy

Nat. Commun.

c-Myc and cancer metabolism

Clin. Cancer Res.

Glutamine synthetase activity fuels nucleotide biosynthesis and supports growth of glutamine-restricted glioblastoma

Nat. Cell Biol.

Central role of glutamate metabolism in the maintenance of nitrogen homeostasis in normal and hyperammonemic brain

Biomolecules

Synthesis of optically pure 4-fluoro-glutamines as potential metabolic imaging agents for tumors

J. Am. Chem. Soc.

PET imaging of glutaminolysis in tumors by 18F-(2S,4R)4-fluoroglutamine

J. Nucl. Med.

Full length article
Fluorination at the 4 position alters the substrate behavior of l-glutamine and l-glutamate: Implications for positron emission tomography of neoplasias

General comments on the use of ¹⁸F-labeled glutamine analogues as PET tumor imaging agents

PET imaging of glutaminolysis in tumors by ¹⁸F-(2S,4R)4-fluoroglutamine