Harry A. Milman

Don, conv and donv--I. Inhibition of L-asparagine synthetase in vitro.

Abstract The inhibition in vitro of l -asparagine synthetase ( l -glutamine hydrolyzing, EC 6.3.5.4) from leukemia 5178Y rendered resistant to l -asparaginase (L5178Y/AR), and from mouse pancreas by the ketoamino acids DON ( l -DON; 6-diazo-5-oxo- l -norleucine), CONV ( l -CONV; 2-amino-5-chlorolevulinic acid; 5-chloro-4-oxo- l -norvaline) and DONV ( l -DONV; 5-diazo-4-oxo- l -norvaline) was investigated using both l -glutamine and ammonium chloride as substrates. At a concentration of 1 mM, DON and CONV almost completely inhibited the utilization in vitro of l -glutamine by l -asparagine synthetase of L5178Y/AR and of mouse pancreas, whereas DONV inhibited both enzymes only by 50 per cent. DON, CONV and DONV did not affect the utilization in vitro of ammonium chloride by l -asparagine synthetase of L5178Y/AR, while DON and CONV modestly inhibited the utilization of ammonium chloride by the pancreatic enzyme. The inhibition produce by DONV was fully and rapidly reversed by dialysis, whereas that produced by DON and CONV was essentially irreversible. The utilization in vitro of l -glutamine by eight fetal rat liver amidotransferases was strongly inhibited by DON and CONV, while DONV exerted modest inhibition on only two of these enzymes. In a survey of other enzymes which use l -asparagine and l -glutamine as substrates, DONV was shown to be the best inhibitor of l -asparagine-utilizing enzymes, DON was the best inhibitor of enzymes utilizing l -glutamine, and CONV affected both groups of enzymes to a variable degree. DON irreversibly inhibited l -asparaginase from Erwinia carotovora (EC 3.5.1.1). This enzyme also was found to catalyze the decomposition of DON. From these findings and also from kinetic studies, it is clear that all three ketoamino acids are capable of behaving as antagonists of l -glutamine in vitro . DON is the most universally active antagonist of this amino acid, followed by CONV, and last by DONV. Similarly, all three agents can function as l -asparagine antagonists under appropriate conditions.

DON, CONV and DONV—III. Pharmacologic and toxicologic studies

Abstract The pharmacologie, toxicologic and oncolytic properties of the ketoamino acids DON ( l -DON; 6-diazo-5-oxo- l -norleucine), CONV ( l -CONV; 5-chloro-4-oxo- l -norvaline; 2-amino-5-chloro-levulinic acid) and DONV ( l -DONV; 5-diazo-4-oxo- l -norvaline) were examined. DON was found to be the most active therapeutic agent of the three drugs against leukemia 1210 and also the most potent cytocidal agent against KB tumor cells in culture. The acute ld 50 values of the agents were dissimilar: CONV was the most toxic drug of the three after single intraperitoneal injections, and DONV the least toxic. Only DON showed evidence of prominent cumulative toxicity. In studies with isolated cells of leukemia 5178Y rendered resistant to l -asparaginase (L5178Y/AR), all three agents appeared to compete both with l -asparagine and with l -glutamine for transport into the cell. DONV competed most effectively with l -glutamine and CONV most effectively with l -asparagine. In mice, all three drugs were cleared from the plasma and excreted into the urine at a rapid rate. None was bound to the proteins of mouse plasma. After an intraperitoneal injection of 100 mg/kg, the concentration of DONV in the pancreas was approximately ten times that of CONV or DON; after comparable intravenous injections, only DONV could be identified in this tissue. Although the metabolism of all three ketoamino acids was found to be minor in degree, evidence is presented that they can be degraded in vitro by organ homogenates and also that purified enzymes can catalyze their transamination. In addition, DON was a good substrate for renal γ-glutamyl transferase (EC 2.3.2.2). In the case of DONV, some conversion to CO 2 by isolated tumor cells also was observed. From these and previous studies it is concluded that, of these analogs of l -glutamine and l -asparagine, DON is the most “ l -glutamine-like” agent of the three, DONV the most “ l -asparagine-like,” while CONV has important attributes of both amino acids.

Regulators of the metabolism of L-asparagine: a search for endogenous inhibitors.

Abstract 1. 1. A study of over 400 intermediary metabolites and naturally occurring macromolecules has been carried out in an attempt to identify molecules capable of regulating the metabolism of l -asparagine. 2. 2. Test systems consisting of mouse hepatic and testicular l -asparaginase, as well as, mouse pancreatic and tumoral l -asparagine synthetase, were used. 3. 3. Selective inhibitors have been categorized as—trace elements; amino acids, analogs and peptides; nucleic acid bases, nucleosides and nucleotides; aldehydes, lipids, porphyrins and pigments; vitamins and coenzymes; proteinaceous materials; citric acid cycle intermediates and related molecules; carbohydrates; and miscellaneous agents. 4. 4. An attempt has been made to extrapolate these in vitro studies to the regulation of the metabolism of l -asparagine in vivo.

Maleimide—Biochemical, pharmacologic and toxicologic studies: Interaction with fl-asparagine metabolism☆

Abstract The effect of maleimide on the metabolism of l -asparagine has been examined in vitro and in viro . In vitro , maleimide is a potent and irreversible inhibitor of the l -asparagine synthetase [ l -glutamine hydrolyzing. (EC 6.3.5.4)] from murine leukemia 5178Y/AR and from murine pancreas; this inhibition is accomplished via sulfhydryl blockade and can be prevented by suitable thiols. In vivo , maleimide was highly irritating locally, producing peritonitis and phlebitis after intraperitoneal and intravenous injection, respectively. The ld 50 in the mouse was 9 mg/kg by the intravenous and intraperitoneal routes; renal, hepatic, neurologic and hematologic toxicities were the principal effects of the drug in this species. l -Asparagine did not alter the lethal effects of maleimide. Maleimide at a dose of 6 mg/kg intraperitoneally did not begin to inhibit pancreatic or tumoral l -asparagine synthetase until 24 hr after its administration: maximum inhibition was delayed until 48–72 hr after dosing. Although maleimide was found to be a potent inhibitor of hepatic l -asparaginase (EC 3.5.1.1) and l -asparagine transaminase (EC 2.6.1.14) in vitro , at no time did the agent inhibit these enzymes in vivo . Additionally, maleimide failed to inhibit protein and nucleic acid synthesis in pancreas and liver, although it did exert a transient repressive effect on these processes in subcutaneous L5178Y/AR tumor. The drug was a potent cytotoxin to 12L1210 cells in culture, causing partial arrest in the G 2 phase of the cell cycle as well as general slowing of progression through the cell cycle. No therapeutic action was produced vs this tumor growing in the peritoneum of mice nor was the drug effective vs subcutaneous L5178Y or L5178Y/AR. However, when maleimide was used to wash the surgical wound created by extirpation of Lewis Lung Carcinoma growing in the muscles of the hind limb, the drug did inhibit local recurrence of tumor, most notably when used in conjunction with parenteral cyclophosphamide. It is concluded that maleimide is an inhibitor, in vitro , of the enzymes of l -asparagine metabolism because of its ability to form covalent bonds with critical sulfhydryl compounds.

Inhibition of L asparagine synthetase by mucochloric and mucobromic acids

Mucochloric and mucobromic acids are powerful inhibitors of tumoral and pancreatic L-asparagine synthetases. Two nitrogen donors, L-glutamine and ammonia, can be used by these enzymes; at a concentration of 1 mmol/l, mucochloric and mucobromic acids preferentially inhibit the utilization of ammonia as opposed to L-glutamine in vitro. Using the tumoral enzyme, kinetic analysis revealed that mucochloric acid produced inhibition which was apparently noncompetitive with ammonia but competitive with L-glutamine. In molar excess, L-glutamine and dithiothreitol effectively antagonized such inhibition; dialysis, however, failed to reverse established inhibition. These findings, suggest that the drugs operate by covalent attachment to crucial sulfhydryl functions on the enzyme.

A radiometric method for the measurement of L-asparagine synthetase activity: Comparison with available methods

Abstract 1. 1. Four variations of a radiometric method for the measurement of L -asparagine synthetase (EC 6.3.5.4) are compared. Each makes use of bacterial L -asparaginase (EC 3.5.1.1) to hydrolyze newly synthesized L -[4-14C]asparagine to L -[4-14C]aspartate which subsequently is transaminated with 2-oxoglutarate by L -glutamate-oxaloacetate transaminase (EC 2.6.1.1) to [4-14C]oxaloacetate—a compound readily 4-decarboxylated by zinc ions. 2. 2. The advantages of each method in a particular experimental setting arc discussed from the standpoint of speed and convenience. 3. 3. Data also are presented on the reliability and applicability of these techniques. 4. 4. Lastly, the modifications are compared comprehensively with other techniques for measuring L -asparagine synthetase activity

A radiometric method for the measurement of l-asparaginase at concentrations below 1 × 10−5 I.U./ml: Technique and application

A radiometric method for the measurement of low levels of l-asparaginase activity (EC 3.5.1.1) has been devised. This technique uses a protracted incubation at 37°C to magnify amidohydrolytic activity. During this time, in most of the cases examined, l-[U-14C]asparagine is hydrolyzed to l-[U-14C]aspartic acid in a linear way; the l-[U-14C]aspartic acid so generated is transaminated with α-ketoglutaric acid by l-glutamic acid oxaloacetate transaminase (EC 2.6.1.1) and the [U-14C]oxaloacetic acid so formed is β-decarboxylated by Zn2+ at pH 5.0. Using this procedure, low levels of l-asparaginase have been detected in the serum of the chicken, horse, and ox. Use also has been made of maleimide, which inhibits mammalian l-asparaginase without affecting the enzymes from bacterial sources, to discount the possibility that bacterial contamination of mammalian samples was responsible for the activity seen. When a survey was conducted of the distribution of l-asparaginase in the organs of Mus musculus, testis was found to contain surprisingly high levels of the analogous hydrolase from liver. The applicability of this technique to the measurement of feeble activities of l-asparaginase leeched from Dacron tubing to which l-asparaginase has been covalently bound, also has been demonstrated.

Ethacrynic acid-an inhibitor of l-asparagine synthetase

Abstract Ethacrynic acid, a clinically useful diuretic, has been shown to inhibit l -asparagine synthetase from leukemia 5178Y resistant to l -asparaginase (L5178Y/AR) in vitro. This inhibition is thought to involve the formation of an adduct between ethacrynic acid and sulfhydryl functions on the enzyme; the adduct is not readily reversible even when another thiol, such as dithiothreitol, is used to displace the acid. A series of analogs of ethacrynic acid were examined as inhibitors of l -asparagine synthetase. Only one proved to be superior to the title compound; it was 2 2-(1-carboxymethoxy-4-chloro-2-naphthyl)3,6-diethyl-6-(1-carboxymethoxy-4-chloro-2-naphthoyl)-5,6-dihydro-[4H]-pyran (Compound D). There was no correlation between the diuretic or natriuretic potency of the series of compounds examined and their ability to inhibit l -asparagine synthetase activity. There was also no correlation between the ability of these agents to inhibit l -asparaginase from Dasyprocta agouti (another sulfhydryl enzyme) and l -asparagine synthetase from L5178Y/AR. In every case, ethacrynic acid and its analogs interrupted the utilization of ammonia by l -asparagine synthetase to a greater degree than the utilization of l -glutamine. In vivo, the inhibition by l -asparagine synthetase from L5178Y/AR by ethacrynic acid was feeble, while the analogous enzyme from pancreas was inhibited to a significant degree.

Reduction of the severity of nephropathy in aging Fischer 344 rats treated with analogs of arylsulfonylurea

Abstract Fischer 344 rats of both sexes were fed diets containing or lacking tolazamide, tolbutamide, chlorpropamide, acetohexamide, or phenformin for 78 or 103 weeks. A complete necropsy was performed on each rat and several tissues were examined histologically. There was a reduction of the severity of nephropathy in aging rats of both sexes treated with the analogs of arylsulfonylurea, but not in those treated with phenformin. Although the analogs of arylsulfonylurea and phenformin, a biguanide, decreased the mean body weight of rats in most of the test groups compared to controls, the length of survival was not affected. Thus, the reduction in the severity of this kidney lesion in rats treated with the analogs of arylsulfonylurea, but not with phenformin, may not be due to differences in growth or survival.

Inhibition of l-asparagine synthetase by mineral cations☆

The inhibition of l-asparagine synthetase from Leukemia 5178YAR and mouse pancreas by mineral cations was examined. Of the 24 agents tested in vitro, 5 (cadmium chloride, cupric chloride, mercuric chloride, mercurous chloride, and zinc chloride) inhibited the utilization of l-glutamine and NH4Cl by the enzymes by nearly 90% at a concentration of 1 mm; 4 other compounds (calcium chloride, cobaltous chloride, cuprous chloride, and manganous chloride) inhibited the utilization of l-glutamine and NH4Cl by the synthetases by more than 50%. The in vitro inhibition of l-asparagine synthetase by zinc chloride, a prototypical example of this class of compounds, was partially reversed (approximately 55%) by dialysis. Moreover, the compound inhibited the l-glutaminase activity of partially purified l-asparagine synthetase from mouse pancreas by 70% at a concentration of 3 mm. In vivo, zinc chloride inhibited preferentially the pancreatic enzyme with no significant inhibition of the enzyme in tumor when administered ip as a single injection (100 mg/kg) or when given daily for 5 days (20 mg/kg) to BDF1 mice bearing subcutaneous implants of Leukemia 5178YAR; the concentration of l-asparagine in tumor or pancreas was not unduly affected by either treatment regimen. That the preferential effect on enzyme activity may be more apparent than real is suggested by the observed pancreatitis produced by this agent. It can be concluded that while zinc chloride may not be a suitable agent for overcoming the state of resistance to l-asparaginase therapy in acute lymphocytic leukemia, its toxic effects on the exocrine pancreas are of importance and warrant further investigation.