Decreased glutamate transport in acivicin resistant Leishmania tarentolae

Studies of drug resistance in the protozoan parasites of the genus Leishmania have been helpful in revealing biochemical pathways as potential drug targets. The chlorinated glutamine analogue acivicin has shown good activity against Leishmania cells and was shown to target several enzymes containing amidotransferase domains. We selected a Leishmania tarentolae clone for acivicin resistance. The genome of this resistant strain was sequenced and the gene coding for the amidotransferase domain-containing GMP synthase was found to be amplified. Episomal expression of this gene in wild-type L. tarentolae revealed a modest role in acivicin resistance. The most prominent defect observed in the resistant mutant was reduced uptake of glutamate, and through competition experiments we determined that glutamate and acivicin, but not glutamine, share the same transporter. Several amino acid transporters (AATs) were either deleted or mutated in the resistant cells. Some contributed to the acivicin resistance phenotype although none corresponded to the main glutamate transporter. Through sequence analysis one AAT on chromosome 22 corresponded to the main glutamate transporter. Episomal expression of the gene coding for this transporter in the resistant mutant restored glutamate transport and acivicin susceptibility. Its genetic knockout led to reduced glutamate transport and acivicin resistance. We propose that acivicin binds covalently to this transporter and as such leads to decreased transport of glutamate and acivicin thus leading to acivicin resistance.     

Mechanism for acivicin inactivation of triad glutamine amidotransferases

Acivicin [(alphaS,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid] was investigated as an inhibitor of the triad glutamine amidotransferases, IGP synthase and GMP synthetase. Nucleophilic substitution of the chlorine atom in acivicin results in the formation of an imine-thioether adduct at the active site cysteine. Cys 77 was identified as the site of modification in the heterodimeric IGPS from Escherichia coli (HisHF) by tryptic digest and FABMS. Distinctions in the glutaminase domains of IGPS from E. coli, the bifunctional protein from Saccharomyces cerevisiae (HIS7), and E. coli GMPS were revealed by the differential rates of inactivation. While the ammonia-dependent turnover was unaffected by acivicin, the glutamine-dependent reaction was inhibited with unit stoichiometry. In analogy to the conditional glutaminase activity seen in IGPS and GMPS, the rates of inactivation were accelerated > or =25-fold when a nucleotide substrate (or analogue) was present. The specificity (k(inact)/K(i)app) for acivicin is on the same order of magnitude as the natural substrate glutamine in all three enzymes. The (alphaS,5R) diastereomer of acivicin was tested under identical conditions as acivicin and showed little inhibitory effect on the enzymes indicating that acivicin binds in the glutamine reactive site in a specific conformation. The data indicate that acivicin undergoes a glutamine amidotransferase mechanism-based covalent bond formation in the presence of nucleotide substrates or products. Acivicin and its (alphaS,5R) diastereomer were modeled in the glutaminase active site of GMPS and CPS to confirm that the binding orientation of the dihydroisoxazole ring is identical in all three triad glutamine amidotransferases. Stabilization of the imine-thioether intermediate by the oxyanion hole in triad glutamine amidotransferases appears to confer the high degree of specificity for acivicin inhibition and relates to a common mechanism for inactivation.     

Expression, purification, characterization, and in vivo targeting of trypanosome CTP synthetase for treatment of African sleeping sickness

African sleeping sickness is a fatal disease caused by two parasite subspecies: Trypanosoma brucei gambiense and T. b. rhodesiense. We previously reported that trypanosomes have extraordinary low CTP pools compared with mammalian cells. Trypanosomes also lack salvage of cytidine/cytosine making the parasite CTP synthetase a potential target for treatment of the disease. In this study, we have expressed and purified recombinant T. brucei CTP synthetase. The enzyme has a higher K(m) value for UTP than the mammalian CTP synthetase, which in combination with a lower UTP pool may account for the low CTP pool in trypanosomes. The activity of the trypanosome CTP synthetase is irreversibly inhibited by the glutamine analogue acivicin, a drug extensively tested as an antitumor agent. There is a rapid uptake of acivicin in mice both given intraperitoneally and orally by gavage. Daily injection of acivicin in trypanosome-infected mice suppressed the infection up to one month without any significant loss of weight. Experiments with cultured bloodstream T. brucei showed that acivicin is trypanocidal if present at 1 mum concentration for at least 4 days. Therefore, acivicin may qualify as a drug with "desirable" properties, i.e. cure within 7 days, according to the current Target Product Profiles of WHO and DNDi.     

Acivicin with glutaminase regulates proliferation and invasion of human MCF-7 and OAW-42 cells--an in vitro study

Tumor cells intensely utilize glutamine as the major source of respiratory fuel. Glutamine-analogue acivicin inhibits tumor growth and tumor-induced angiogenesis in Ehrlich ascites carcinoma. In the present study, antitumor properties of acivicin in combination with glutaminase enzyme is reported. Acivicin along with E. coli glutaminase synergistically reduced in vitro proliferation and matrigel invasion of human MCF-7 and OAW-42 cells. Effects of single and combined treatments with acivicin and glutaminase on angiogenic factors were also analyzed in these cell lines. Co-administration of the treatment agents inhibits the release of VEGF and MMP-9 by cells in culture supernatant significantly than single agent treatments. The result suggests that combination of acivicin with glutaminase may provide a better therapeutic option than either of them given separately for treating human breast and ovarian cancer. However, further studies are required to be conducted in vivo for its confirmation.     

Acivicin inhibits Crithidia fasciculata growth in a serum-free medium and inactivates carbamoyl-phosphate synthetase II in vivo

1. Crithidia fasciculata was grown in a serum-free medium. 2. Twenty-six hours after addition of 2, 5, 20, 50, and 150 microM acivicin to logarithmically growing organisms, cell counts were decreased to 46, 23, 14, 9.1, and 8.6% of the control, respectively. 3. Guanosine plus cytidine (0.1 mM each) provided complete protection against growth inhibition by 5 microM acivicin. 4. Cells exposed to 10 microM acivicin showed a time-dependent, irreversible inactivation of L-glutamine-dependent carbamoyl-phosphate synthetase II activity; ammonia-dependent synthetase II activity was increased up to 34% of the control. 5. Glutamine (20 mM) protected the enzyme from inactivation in vivo. 6. These results indicate that acivicin acts as an affinity analog of L-glutamine in vivo as it does in vitro.     

A new acivicin prodrug designed for tumor-targeted delivery

Acivicin is an antitumor agent known to inhibit cell growth. A new prodrug 9b of acivicin 10 was synthesized, based on a p-hydroxybenzylcarbamate self-immolative spacer capable to release acivicin under esterase activity. The prodrug includes a maleimide-containing arm for linkage with thiol-containing macromolecules such as antibodies. This molecule is intended for the conception of bioconjugates to target an inactive acivicin precursor to tumor cells, when linked to a monoclonal antibody (mAb) which recognizes a tumor-specific antigen. Prodrug cleavage by plasmatic esterases will then restore the acivicin's activity toward tumor cells. We report here the synthesis and the in vitro characteristics of the prodrug. As expected, its inhibitory activity against the gamma-glutamyl transpeptidase (gamma-GT) enzyme and its cytotoxicity towards HL-60 cells were highly reduced compared to the parent drug. The chemical and plasmatic hydrolysis kinetics of the compound was studied by HPLC. The prodrug is stable, being slowly hydrolyzed in pH 7.6 buffer at 37 degrees C with a half-life of 37 h. It is converted into an active acivicin under the effect of pig liver esterase, and its half-life in human plasma is 3 h. These results indicate this compound may be further used as a prodrug-antibody conjugate, to target acivicin to malignant cells.     

Significance of gamma-glutamyltranspeptidase in exocrine pancreatic amino acid transport.

The exocrine pancreas is rich in gamma-glutamyltranspeptidase (GGT, EC 2.3.2.2) and exhibits high rates of amino acid transport and protein synthesis. The role of the gamma-glutamyl cycle in mediating neutral amino acid transport in the isolated perfused rat pancreas was investigated using acivicin, an inhibitor of GGT, and a rapid dual isotope dilution technique. When treatment in vivo with acivicin (50 mg/kg) was followed 1 h later by continuous perfusion of the isolated pancreas with 10 microM acivicin, GGT levels decreased from 53 +/- 3 IU/g to 4.9 +/- 1.5 IU/g. This marked inhibition of GGT activity was not associated with decreased uptake for either L-alanine or L-glutamine, suggesting that the gamma-glutamyl cycle plays a negligible role in amino acid transport across the basolateral membrane of the pancreatic epithelium.     

Rapid in vivo inactivation by acivicin of CTP synthetase,carbamoyl-phosphate synthetase II,and amidophosphoribosyltransferase in hepatoma

A single injection of the anti-glutamine drug, acivicin (NSC 163501), in tumor-bearing rats in 30 min decreased the activities of amidophosphoribosyltransferase, carbamoyl-phosphate synthetase II and CTP synthetase to 56, 50 and 7% of those of the controls. By 1 hr the activities were down to 32, 13 and 3% and they remained low for 12 hr, after which they slowly returned towards normal range in 72 hr. The decline of the activity of CTP synthetase (a loss of 80% in 10 min) was the most rapid, and the activity only returned to 60% of the controls by 3 days after the acivicin injection. In the hepatoma the concentrations of ATP and UTP changed little, but those of GTP and CTP rapidly decreased, reaching at the lowest point 32 and 2%, respectively, of control values 2 hr after acivicin; concentrations started to rise at 12 hr, reaching normal levels by 48 hr. The drop in enzyme activities preceded the decline in the pools of GTP and CTP. The behavior of enzyme activities and nucleotide concentrations in the host liver had a pattern similar to that in the hepatoma; however, the changes were less extensive than those in the tumor. The differential response between tumor and liver is attributed, in part at least, to the tissue L-glutamine concentration which in the hepatoma (0.5 mM) was 9 times lower than in the liver (4.5 mM). The selectivity of acivicin action in inhibiting glutamine-utilizing enzymes is also demonstrated by the lack of effect on aspartate carbamoyltransferase, a an enzymic activity which resides in the same complex as that of carbamoyl-phosphate synthetase II. The rapid decline in the activities of glutamine-utilizing enzymes is attributed to an in-activation of the enzymes by acivicin which functions as an active site-directed affinity analog of L-glutamine. The rapid modulation of the enzymic phenotype and ribonucleotide concentrations by acivicin provides a useful tool for elucidating the role of enzymic and nucleotide imbalance in the commitment of cancer cells to replication and in the targeting of anticancer chemotherapy.     

Use of acivicin in the determination of rate constants for turnover of rat renal gamma-glutamyltranspeptidase

The specific enzymatic activity of renal gamma-glutamyltranspeptidase is decreased from control levels (0.86 unit-1 mg-1) to minimal values within 2 h postinjection of 100-g rats with acivicin, an irreversible inhibitor of the enzyme. The recovery of transpeptidase specific activity was followed from 2 to 24 h postinjection and the data were used to calculate the absolute rate constants for degradation (kd = 0.47 +/- 0.03 day-1) and synthesis (ks = 0.41 +/- 0.04 unit-1 mg-1 day-1). This corresponds to a half-life for the renal transpeptidase of 1.46 +/- 0.09 days and 99% recovery of the specific activity by 10 days postinjection. Recovery was followed for 14 days and closely approximates this theoretical curve. The data from control experiments designed to test for secondary effects of the drug, acivicin, show that neither the relative rate of synthesis nor apparent rate of degradation for either total protein or gamma-glutamyltranspeptidase is significantly altered by acivicin treatment of rats. The results also show that the acivicin-inhibited transpeptidase is not degraded differently than enzymatically active enzyme. The individual heterodimer subunits also exhibit similar apparent half-lives in both control and treated animals. Thus, recovery of renal gamma-glutamyltranspeptidase specific activity after acivicin treatment can be used in vivo to determine absolute values of ks and kd for this enzyme. These values have not been reported for any other constituent of the renal brush-border membrane.     

Inhibition of gamma-glutamyl transpeptidase decreases amino acid uptake in human keratinocytes in culture

Acivicin inhibits gamma-glutamyl transpeptidase activity in human keratinocytes in culture. Treatment of these cells with acivicin produces a decrease in the uptake of L-[U-14C]alanine, 2-amino-[1-14C]-isobutyrate, L-[U-14C]leucine and 1-aminocyclopentane-1-[14C]carboxylate. D-[U-14C]glucose uptake is not affected by the presence of acivicin. These results support, for the first time in vitro, the hypothesis that the gamma-glutamyl cycle may be involved in amino acid uptake by human cells.     

Crystal structures of Escherichia coli gamma-glutamyltranspeptidase in complex with azaserine and acivicin: novel mechanistic implication for inhibition by glutamine antagonists

γ-Glutamyltranspeptidase (GGT) catalyzes the cleavage of such γ-glutamyl compounds as glutathione, and the transfer of their γ-glutamyl group to water or to other amino acids and peptides. GGT is involved in a number of biological phenomena such as drug resistance and metastasis of cancer cells by detoxification of xenobiotics. Azaserine and acivicin are classical and irreversible inhibitors of GGT, but their binding sites and the inhibition mechanisms remain to be defined. We have determined the crystal structures of GGT from Escherichia coli in complex with azaserine and acivicin at 1.65 Å resolution. Both inhibitors are bound to GGT at its substrate-binding pocket in a manner similar to that observed previously with the γ-glutamyl-enzyme intermediate. They form a covalent bond with the O^γ atom of Thr391, the catalytic residue of GGT. Their α-carboxy and α-amino groups are recognized by extensive hydrogen bonding and charge interactions with the residues that are conserved among GGT orthologs. The two amido nitrogen atoms of Gly483 and Gly484, which form the oxyanion hole, interact with the inhibitors directly or via a water molecule. Notably, in the azaserine complex the carbon atom that forms a covalent bond with Thr391 is sp³-hybridized, suggesting that the carbonyl of azaserine is attacked by Thr391 to form a tetrahedral intermediate, which is stabilized by the oxyanion hole. Furthermore, when acivicin is bound to GGT, a migration of the single and double bonds occurs in its dihydroisoxazole ring. The structural characteristics presented here imply that the unprecedented binding modes of azaserine and acivicin are conserved in all GGTs from bacteria to mammals and give a new insight into the inhibition mechanism of glutamine amidotransferases by these glutamine antagonists.     

Effect of glutamine analogue-acivicin on tumor induced angiogenesis in Ehrlich ascites carcinoma

The inhibition of tumor growth and tumor induced angiogenesis by the glutamine antimetabolite acivicin was evaluated in 6-7 weeks old male Swiss albino mice bearing Ehrlich ascites carcinoma (EAC) transplanted by intraperitoneal (ip) injections of EAC cells. Treatment involving ip injections with two different doses of acivicin (0.05 and 0.41microg/g body weight/day) in saline revealed decrease in tumor volumes and reduced number of blood vessels on peritoneal wall after 10 and 15 days of treatment when compared to control (i.e. injected with saline only). Vascular hyperpermeability was found to be lesser in the treated groups of mice than the control as indicated by the FITC- D and colloidal carbon assay. Serum VEGF level was found to decrease in the drug treated groups both after 10 and 15 days of treatment. The results thus suggest that acivicin may suppress tumoral angiogenesis through regulation of VEGF level.     

Biological activity of neurotrophins is dependent on recruitment of Rac1 to lipid rafts

The Rho family of small GTPases, key regulators of the actin cytoskeleton in eukaryotic cells, is implicated in the control of neuronal morphology. Here, we report that neurotrophin dependent cytoskeletal changes, characteristic of the phenotype of Rac1, in the hippocampal neurons or PC12 cells are inhibited by the disruption of lipid raft integrity. Activation of Rac1 induced by NGF is impaired in cholesterol-depleted PC12 cells. Pretreatment with gammaGTP shifted significant amount of Rac1, presumably in a GTP-bound form, from non-raft to raft fractions. Proper recruitment of activated Rac1 to lipid rafts, structures that represent specialized signaling organelles, is of fundamental importance in determining neurotrophins' bioactivity.     

Interaction of gamma-glutamyl transpeptidase with glutathione involves specific arginine and lysine residues of the heavy subunit.

Gamma-glutamyl transpeptidase, an enzyme of importance in glutathione metabolism, consists of two subunits, one of which (the light subunit, Mr 22,000; residues 380-568; rat kidney) contains residue Thr-523, which selectively interacts with the substrate analog acivicin to form an adduct that is apparently analogous to the gamma-glutamyl enzyme intermediate formed in the normal reaction (Stole, E., Seddon, A. P., Wellner, D., and Meister, A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1706-1709). The present studies indicate that specific arginine and lysine residues of the heavy subunit (Mr 51,000; residues 31-379) participate in catalysis by binding the substrates. Selective labeling studies of the enzyme with [14C]phenylglyoxal showed that Lys-99 and Arg-111 were modified. This appears to be the first instance in which phenylglyoxal was found to react with an enzyme lysine residue. Incorporation of [14C]phenylglyoxal into Lys-99 was decreased in the presence of acceptor site selective compounds. Incorporation into both Lys-99 and Arg-111 was decreased in the presence of glutathione. The findings suggest that Lys-99 and Arg-111 interact, respectively, with the omega- and alpha-carboxyl groups of glutathione. That these putative electrostatic binding sites are on the heavy subunit indicates that both subunits contribute to the active center. Two additional heavy subunit arginine residues become accessible to modification by phenylglyoxal when acivicin is bound, suggesting that interaction with acivicin is associated with a conformational change.     

Glutathione turnover in human cell lines in the presence of agents with glutathione influencing potential with and without acivicin inhibition of gamma-glutamyltranspeptidase

BACKGROUND: We have previously shown that there were great discrepancies between different agents regarding their glutathione stimulating potential and that agents with mainly oxidative effects did not increase concentrations of glutathione in human cell cultures, in contrast to other thiol reactive agents. In order to evaluate whether increased glutathione degradation might be one reason for these discrepancies, we have investigated the effect of different agents with potential influence on glutathione metabolism in human cell cultures with or without acivicin inhibition of gamma-glutamyltranspeptidase (GT), since GT is responsible for the initial degradation of glutathione. METHODS: Intra- and extracellular concentrations of glutathione were investigated in HeLa and hepatoma cell cultures, with and without acivicin inhibition of GT, in the presence of oxidative and electrophilic agents (copper ions, hydrogen peroxide and N-ethylmaleimide), hydroquinone, reducing agents (lipoic acid and N-acetylcysteine), and a thiol reactive metal (mercury ions). RESULTS: There were great discrepancies between the different agents regarding their maximal glutathione response (the sum of the intracellular and the extracellular amount of glutathione) in cell cultures. There was only a small increase in total glutathione in the presence of hydrogen peroxide or N-ethylmaleimide before the cell protein decreased compared to findings with mercury ions, lipoic acid or hydroquinone. In both HeLa and hepatoma cell cultures, there were correlations between the original glutathione amount and the total glutathione amount observed after acivicin inhibition. CONCLUSION: The relatively small increase of glutathione amount in the presence of oxidative and electrophilic agents compared to other thiol reactive agents is not due to increased GT degradation of glutathione.     

Inactivation of the amidotransferase activity of carbamoyl phosphate synthetase by the antibiotic acivicin.

Carbamoyl phosphate synthetase (CPS) from Escherichia coli catalyzes the formation of carbamoyl phosphate from 2 mol of ATP, bicarbonate, and glutamine. CPS was inactivated by the glutamine analog, acivicin. In the presence of ATP and bicarbonate the second-order rate constant for the inactivation of the glutamine-dependent activities was 4.0 x 10(4) m(-1) s(-1). In the absence of ATP and bicarbonate the second-order rate constant for inactivation of CPS was reduced by a factor of 200. The enzyme was protected against inactivation by the inclusion of glutamine in the reaction mixture. The ammonia-dependent activities were unaffected by the incubation of CPS with acivicin. These results are consistent with the covalent labeling of the glutamine-binding site located within the small amidotransferase subunit. The binding of ATP and bicarbonate to the large subunit of CPS must also induce a conformational change within the amidotransferase domain of the small subunit that enhances the nucleophilic character of the thiol group required for glutamine hydrolysis. The acivicin-inhibited enzyme was crystallized, and the three-dimensional structure was determined by x-ray diffraction techniques. The thiol group of Cys-269 was covalently attached to the dihydroisoxazole ring of acivicin with the displacement of a chloride ion.     

Effects of acivicin and dichloroallyl lawsone upon pyrimidine biosynthesis in mouse L1210 leukemia cells

Acivicin (NSC 163501) and dichloroallyl lawsone (NSC 126771) are potent inhibitors of nucleotide biosynthesis with consequent anti-cancer activity against certain experimental tumors. To determine in detail the metabolic events induced by each inhibitor, we have devised a new two-dimensional chromatographic procedure for measurement of the concentrations of all pyrimidine intermediates and some purine nucleotides from 100 microliter of an extract of cells grown in the presence of [14C]bicarbonate. Addition of acivicin (25 microM) to mouse L1210 leukemia cells causes severe depletion in the cellular levels of CTP and GTP, accumulation of uridine nucleotides, and abrupt but transient increases in the concentrations of the early intermediates of both the pyrimidine and purine pathways. Addition of dichloroallyl lawsone (25 microM) results in a rapid depletion of uridine and cytidine nucleotides; carbamyl aspartate and dihydroorotate accumulate to high levels in an equilibrium ratio of 20.5:1, and orotate, orotidine, and UMP increase transiently before decreasing to levels approaching their original steady states. The predominant inhibitory effects of acivicin are upon the reactions UTP----CTP and XMP----GMP, but there is also an initial transient activation of both the pyrimidine and purine pathways by acivicin. The data obtained with dichloroallyl lawsone are consistent with inhibition of the conversion of UMP----UDP initially followed by potent inhibition of dihydroorotate----orotate.     

Reanalysis of the involvement of gamma-glutamyl transpeptidase in the cell activation process.

The inhibitor of gamma-glutamyl transpeptidase (gamma-GT) acivicin modulates cellular responses including growth, myeloid maturation and apoptosis. Whether these effects result from the inhibition of gamma-GT enzyme activity remains unclear. We compared the cellular effects of acivicin against a more potent and specific inhibitor of gamma-GT (L-2-amino-4-boronobutanoic acid (L-ABBA)) in gamma-GT-negative (B lymphoblastoid Ramos) and gamma-GT-positive (myelomonocytic HL-60, gamma-GT-transfected Ramos) cell lines. Under non-oxidative stress conditions, acivicin-induced cell growth arrest, apoptosis and macrophage maturation occurred independent of gamma-GT while L-ABBA did not influence any of these processes. Acivicin triggered tyrosine phosphorylation and increased nuclear factor kappaB activity. Further insight into the role of gamma-GT in cellular processes is needed.     

The role of glutamine and glucose analogues in metabolic inhibition of human myeloid leukaemia In vitro

• 1.1.Glutamine analogues l-[alphaS,5S]-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin) and 6-diazo-5-oxo-l-norleucine (DON) have been shown to possess cytotoxic activity against a wide variety of animal and human xenografted solid tumours, however their potential in man has been limited by toxicity.
• 2.2.We have analysed the effects of acivicin and DON on glutamine utilization to determine whether the reason for the disappointing therapeutic profile is solely due to the inefficient inhibition of glutamine metabolism.
• 3.3.Human myeloid leukaemic cells treated with acivicin inhibited ribonucleotide biosynthesis but not energy production via glutaminolysis and had little effect on viability, whereas treatment with DON inhibited both ribonucleotide biosynthesis and glutamine oxidation and resulted in reduced viability.
• 4.4.Treatment of the myeloid leukaemic cells with the glucose analogue 2-deoxy-d-glucose in addition to DON potentiated the inhibition of de novo nucleotide biosynthesis, glutaminolysis and glycolysis, and caused a further reduction in cell viability.
• 5.5.These results provide further support for the essential role of glutamine in cellular metabolism, and indicate that use of the glutamine analogue DON in the treatment of acute myeloid leukaemia may be more clinically effective if used in combination with 2-deoxy-d-glucose.