Hiremagalur N. Jayaram

Studies on the mechanism of action of 2-β-D-ribofuranosylthiazole-4-carboxamide. V: Factors governing the response of murine tumors to tiazofurin

The pharmacological effects and metabolism of tiazofurin have been compared in the six transplantable tumors comprising the NCI rodent tumor panel, viz. the P388 leukemia (S); the L1210 leukemia (S); the Lewis lung carcinoma (S); the B16 melanoma (R); the colon 38 carcinoma (R); and the M5076 sarcoma (R), where (S) denotes sensitivity and (R) resistance to tiazofurin. In addition, a variant of the P388 leukemia rendered resistant to the drug in vitro, and maintaining stable resistance in vivo, P388/TR, was also studied. Intraperitoneal administration of tiazofurin (100 mg/kg) resulted in a 3- to 30-fold greater accumulation of thiazole-4-carboxamide adenine dinucleotide (TAD), the proposed active metabolite of the drug in S versus R lines. In general, levels of TAD, percent inhibition of IMP dehydrogenase (mean 40% in S versus 10% in R), depression in the concentration of guanosine nucleotides, (50% in S versus 20% in R) and percent elevation of levels of IMP (500% in S versus 60% in R) correlated well with sensitivity or resistance. However, the B16 melanoma, although resistant to tiazofurin treatment, showed certain biochemical features characteristic of an S line. The sensitive and resistant tumors displayed comparable abilities to phosphorylate tiazofurin, but there was significant depression only in the R lines of the pyrophosphorylase which converts tiazofurin-5-monophosphate to TAD (mean 78 nmoles/mg protein/hr in S versus 22 nmoles/mg protein/hr in R). The naturally resistant tumors were also found to exhibit a greater ability to degrade synthetic TAD than the sensitive lines (mean 102 nmoles/mg protein/hr in R versus 29 nmoles/mg protein/hr in S lines). The state of sensitivity or resistance could not be attributed to the basal levels of IMP dehydrogenase, to the specific activities of the enzymes of purine salvage, or to the basal concentration of purine and pyrimidine nucleotides. Moreover, treatment with tiazofurin did not influence the enzymes of TAD synthesis or of purine salvage.

Studies on the mechanism of action of tiazofurin metabolism to an analog of NAD with potent IMP dehydrogenase-inhibitory activity.

Following the parenteral administration of tiazofurin, 2-beta D-ribofuranosylthiazole-4-carboxamide (thiazole nucleoside, TR), a potent but reversible inhibitor of IMP dehydrogenase is generated in subcutaneous nodules of the P388 leukemia. The compound responsible for this effect has been isolated from homogenates of the tumor by ion-exchange HPLC, and its presence monitored by enzyme-inhibition assay. The inhibitor has also been prepared by incubation of tiazofurin with P388 cells in culture. Chromatographically, the inhibitory principle exhibits a moderately strong set negative charge at pH 3, and elutes in the general vicinity of the nucleoside-5-diphosphates; its absorption maximum in aqueous solution (pH 7) lies at 252 nm. Exposure of the molecule to snake-venom phosphodiesterase or to nucleotide pyrophosphatase destroys its inhibitory potency, whereas other phosphodiesterases are either less effective or inert. Since these results suggested that the anabolite might be a dinucleotide with a phosphodiester linkage of the kind found in NAD, attempts were made to synthesize such an analogue from the 5-monophosphate of thiazole nucleoside and ATP-Mg2+, using a purified preparation of NAD pyrophosphorylase; modest yields were obtained of a compound with chromatographic, spectral and enzyme-inhibitory properties identical to those of the material isolated from P388 tumor nodules. This enzyme-synthesized material was radioactive when [3H]ATP was used as cosubstrate, and yielded both AMP and thiazole nucleoside-5-monophosphate on treatment with phosphodiesterase. It resisted attack by NAD glycohydrolase. An apparently identical dinucleotide was also synthesized chemically by means of the Khorana condensation. Mass spectral analysis and nuclear magnetic resonance studies with homogeneous preparations of both the enzymically and chemically synthesized compound were compatible with its being a dinucleotide in which the nicotinamide of NAD has been replaced by thiazole-4-carboxamide. Versus IMP dehydrogenase, the dinucleotide exhibited a K1 of approximately 2 X 10(-7) M and was non-competitive with NAD as the variable substrate. Other NAD utilizing enzymes, including representative dehydrogenases and poly ADP ribose polymerase, were, by comparison to mammalian IMPD, resistant to inhibition by TAD. The properties of this novel dinucleotide are compared and contrasted with those of analogs of NAD containing modifications in the pyridine, adenine or ribofuranose rings, as well as in the pyrophosphate bridge.

Studies on the mechanism of action of 2-β-d-ribofuranosylthiazole-4-carboxamide (NSC 286193)-II. Relationship between dose level and biochemical effects in P388 leukemia in vivo

Administration of the novel thiazole C-nucleoside, 2-beta-D-ribofuranosylthiazole-4-carboxamide (NSC 286193), to BDF1 mice bearing subcutaneous implants of P388 leukemia provoked a sharp depression in the concentration of intratumoral guanine nucleotides and a correspondingly large expansion of the IMP pools. Measurements of IMP dehydrogenase in the tumors of treated mice revealed that this enzyme was inhibited in a dose-responsive way, with approximately 50% inhibition engendered by the administration of the drug at a dose of 25 mg/kg and greater than 90% inhibition by all doses greater than 100 mg/kg. The inhibition of enzyme activity, seen after a dose of 250 mg/kg, reached a maximum 120 min after treatment and had subsided substantially 8 hr after dosing; by 24 hr. enzyme activity was fully restored. These results, coupled with the observation that the antitumor activity of the drug could be prevented in large part by the simultaneous administration of guanosine, support the conclusion that 2-beta-D-ribofuranosylthiazole-4-carboxamide, after anabolism, exerts its antineoplastic effects via a state of guanine nucleotide depletion. In extracts of the tumors of mice given parenteral injections of the thiazole nucleoside, a potent dialyzable inhibitor of IMP dehydrogenase was demonstrable: its concentration fluctuated in parallel with enzyme inhibition. Although the chemical identity of the proximate inhibitory species has yet to be established, it is concluded on kinetic grounds that it is neither the native nucleoside nor its 5-monophosphate.

Synergistic effect of 5-fluorouracil and N-(phosphonacetyl)-l-aspartate on cell growth and ribonucleic acid synthesis in a human mammary carcinoma

Abstract The biological effects of N-( phosphonacetyl )- l - aspartate (PALA) and 5-fluorouracil (5-FU) were examined singly, and in combination, on the growth of a human mammary carcinoma (MDA) cell line in culture. All combinations of 5-FU (2.5 × 10 −7 to 1.5 × 10 −5 M) and PALA (6.0 × 10 −5 to 3.6 × 10 −3 M) resulted in synergistic inhibition of cell growth as revealed by a 50 per cent isobologram.To examine the biochemical basis for the synergism, measurements of the incorporation of [ 3 H]-5-FU into total non-poly(A)- and poly(A)-RNA, and of the simultaneous incorporation of [ 14 C]deoxyguanosine and [ 3 H]deoxyuridine into DNA, were determined. The combination of 3.7 × 10 −5 M PALA and 1 × 10 −6 M 5-FU produced 65–85 per cent inhibition of cell growth after continuous treatment for 13 days. Treatment of the cells for 3 or 24 hr with the same drug regimen produced approximately a 170 per cent increase in the incorporation of 1 × 10 −6 M [ 3 H]-5-FU into poly(A)RNA in comparison to [ 3 H]-5-FU treatment alone; exposure for 24 hr to 3.7 × 10 −5 M PALA and 1 × 10 −6 M [ 3 H]-5-FU resulted in a 285 per cent increase in the incorporation of [ 3 H]-5-FU into non-poly(A)RNA. The incorporation of either [ 14 C]deoxyguanosine or [ 3 H]deoxyuridine into DNA was not inhibited by this drug regimen; however, the incorporation of [ 3 H]deoxyuridine into DNA was elevated significantly upon 12 or 24 hr of exposure to PALA alone. PALA and 5-FU treatment resulted in a 75 per cent reduction in the concentration of UTP and no change in the concentration of 5-fluorouridine-5′triphosphate 5-FUTP) versus 5-FU treatment alone. Thus, the proportion of 5-FUTP in the total 5FUTP + UTP pool was enhanced more than 3-fold by the combination regimen. These results indicate that the synergistic effect of the combination of PALA and 5-FU on the growth of MDA cells correlates with an increased proportion of 5-FUTP in the pyrimidine nucleotide pool and, consequently, with an enhanced incorporation of 5-FU into RNA, but not with inhibition of DNA synthesis.

Relationships between the cytotoxicity of tiazofurin and its metabolism by cultured human lung cancer cells.

The antitumor activity of the antineoplastic agent, tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide), has previously been shown to require intracellular anabolism of the drug to a nicotinamide adenine dinucleotide (NAD) analog (2-beta-D-ribofuranosylthiazole-4-carboxamide adenine dinucleotide or tiazofurin adenine dinucleotide), which then acts as a potent inhibitor of the target enzyme inosine monophosphate (IMP) dehydrogenase. Inhibition of the latter enzyme in turn brings about a profound depletion of intracellular guanosine nucleotides essential for tumor cell growth and replication. In the present study, the cytotoxicity and metabolism of tiazofurin have been examined in six human lung cancer cell lines. At the pharmacologically attainable drug concentration of 100 microM, colony survival was less than 1.5% in three cell lines (sensitive), while survival in the remaining three was greater than 50% (resistant). The metabolism of tritiated tiazofurin was examined at concentrations ranging from 0.5 to 100 microM following both brief (6 h) and protracted (14 d) exposures. The sensitive lines accumulated concentrations of tiazofurin adenine dinucleotide that were approximately 10 times those achieved by the resistant lines at both time points. We also observed tendencies for the sensitive cell lines to exhibit: (a) higher specific activities of NAD pyrophosphorylase, the enzyme required for the synthesis of tiazofurin adenine dinucleotide, (b) significantly lower levels of a phosphodiesterase which degrades the latter dinucleotide, (c) greater inhibition of the target enzyme IMP dehydrogenase, and (d) greater depressions of guanosine nucleotide pools after drug treatment. By contrast, the basal levels of IMP dehydrogenase and purine nucleotides in these six lines did not correlate in any obvious way with their responsiveness or resistance. The accumulation and monophosphorylation of parent drug were also not prognostic variables. These studies thus suggest that the extent of accumulation of tiazofurin adenine dinucleotide, as regulated by its synthetic and degradative enzyme activities, is the single most predictive determinant of the responsiveness of cultured human lung tumor cells to tiazofurin.

A simple and rapid method for the estimation of l-asparaginase in chromatographic and electrophoretic effluents: Comparison with other methods

Abstract Four methods for the measurement of l -asparaginase have been compared with a new modification of an enzymatic, spectrophotometric technique utilizing l -glutamate oxaloacetate transaminase and malate dehydrogenase. This last technique uses disposable polycarbonate tubes both as reaction vessels and as cuvettes. Only a single addition of reagents and a single reading of absorbance is required. The simplicity of this technique is especially helpful when the prompt analysis of many samples of l -asparaginase is desired.

the metabolism of l-asparagine in asparagus officinalis

Abstract 1. 1. l -Asparagine is present at a concentration ranging from 0.004–0.04 M in Asparagus officinalis . 2. 2. Sensitive radiometric techniques have been used to examine enzymes responsible for the synthesis and metabolism of l -asparagine in Asparagus . 3. 3. l -Asparagine synthetase was undetectable but p-cyano-L-alanine synthase was abundant. 4. 4. Additionally, β-cyano- l -alanine hydratase, several l -asparagine transaminases and a feeble, possibly exogenous, l -asparaginase were also detectable. 5. 5. Attempts to measure the substrates of β-cyano- l -alanine synthase in freeze-clamped specimens indicated that both l -eysteine and cyanide were present at concentrations well below the K m of the enzyme for them. 6. 6. Studies to demonstrate alternate routes for the biosynthesis of l -asparagine failed; thus, β-cyano- l -alanine synthase remains the most plausible candidate as the primary participant in the biosynthesis of l -asparagine in Asparagus .

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.

Metabolites of alanosine, an antitumor antibiotic

Abstract The metabolism of alanosine. dl -2-amino-3-( N -hydroxy, N -nitrosamino) propionic acid (NSC-143647), a new antitumor antibiotic, was studied in mice, rats, monkeys and dogs. Urine is the principal excretory vehicle for the drug in these four species. In rats, unchanged alanosine is the principal excretory product. In the other species, a second, more acidic component accounts for the major fraction of the drug-derived radioactivity in urine; this product retains the characteristic u.v. spectrum and both carbons, 1 and 3, of alanosine: it is chromatographically and spectrally indistinguishable from the compound generated by the action of NADH and malate dehydrogenase on the product resulting from the incubation of l -alanosine with l -glutamate oxaloacetate transaminase (GOT) (EC 2.6.1.1) and α-ketoglutarate. On the basis of this evidence, this metabolite is concluded to be the α-hydroxy counterpart of l -alanosine. The antibiotic was susceptible to transamination in vitro by extract of organs rich in GOT; heart was pre-eminent in this regard, and α-ketoglutaric acid was found to be the preferred α-keto partner in the reaction. Crystalline GOT catalyzed an identical reaction in vitro , and the product, like oxaloacetic acid, was susceptible to enzymatic condensation with acetyl CoA, in the presence of citrate synthase. Inability to detect the -α- keto analogue of alanosine. 2-oxo-3-( N -hydroxy, N -nitrosamino) propionic acid, in tissues and excreta is attributed to the facile decomposition of this metabolite in vivo . In vitro , alanosine was susceptible to decarboxylation by homogenates of mouse brain and by purified l -glutamate decarboxylase (EC 4.1.1.15) from Escherichia coli . No evidence could be adduced for denitrosation of the antibiotic nor for reduction of the nitroso function in a system containing hepatic microsomes and NADPH. However, l -amino acid oxidase (EC 1.4.3.2) and high concentrations of pyridoxal phosphate catalyzed the deamination of alanosine at alkaline pH. In confirmation of the observations of Hurlbert et al , ¦ Proc. Am. Ass. Cancer Res. 18 , 234,(1977)¦. alanosine was found to be used by phosphoribosylaminoimi-dazole-succinocarboxamide synthetase (EC 6.3.2.6) as a fraudulent substrate. Also observed was the condensation of alanosine with IMP. catalyzed by a partially purified preparation of adenylosuccinate synthetase (EC 6.3.4.4) from rabbit muscle; this anabolite exhibited chromatographic properties quite similar to adenylosuccinic acid. Inasmuch as a substantial percentage of the administered dose of alanosine was found to associate with carcass-macromolecules for protracted periods, attempts were made to determine the basis for this fate. Equivalent labeling was produced irrespective of whether dl -[1- 14 C] or dl -[3- 14 C] alanosine was the injectate, so that reutilization of metabolically generated [ 14 C]O 2 is not likely to explain macromolecular retention of the antibiotic. In vitro , no esterification of alanosine to tRNA was observed, but the drug did bind to tRNA in an ATP-independent reaction. In vivo , ten times more dl -[3- 14 C] alanosine was incorporated into the hemoglobin of animals recovering from phenylhydrazine anemia than was observed in their saline-treated counterparts. Isolated reticulocytes incorporated only minor amounts of purified dl -[I- 14 C] alanosine into their molecules; this process was insensitive to inhibition by cycloheximide. In vitro , alanosine (used in lieu of l -aspartic acid) neither supported nor inhibited globin synthesis by rabbit reticulocyte lysates. These results leave unsettled the question of whether macromolecular association of alanosine reflects incorporation or affiliation.

Effect of l-glutamine antagonists on 5-phosphoribosyl 1-pyrophosphate levels in p388 leukemia and in murine colon adenocarcinomas in vivo

The intratumoral content of 5-phosphoribosyl 1-pyrophosphate (PRPP) and the activity of the enzymes anabolizing and catabolizing the sugar phosphate were determined following i.p. administration of an LD10 dose of an L-glutamine antagonist or saline to tumor-bearing animals. Elevation of PRPP pool size following administration of L-[alpha S,5S]-alpha-amino-3-chloro-4,5-dihydro-5-isopazoleacetic acid (NSC-163501) (AT-125) was maximal at 8 hr and returned to pretreatment levels by 24 hr. In P388 leukemia, dose for dose, at 4 hr, 6-diazo-5-oxo-L-norleucine (NSC-7365) (DON) was the most potent of the L-glutamine antagonists in elevating basal PRPP pool size (50% above control) followed by AT-125 and azaserine, 300 and 100% above control respectively. Moreover, such augmentation in PRPP pool size preferentially affected P388 tumor rather than the small intestine. Following i.p. administration of LD10 doses of AT-125, DON and azaserine, the specific activities of PRPP anabolizing and catabolizing enzymes were determined. A significant inhibition of PRPP amidotransferase was demonstrated with DON and AT-125 (P less than 0.05), and no inhibition with azaserine. A similar modulation of PRPP pool size demonstrated in vivo following administration of 250 mg/kg of ART-125 in mice bearing colonic adenocarcinoma lines. It was suggested that a significant increase of PRPP pool size might cause the possible synergism of a selected L-glutamine antagonist and 5-fluorouracil as reported after the appropriately scheduled administration of methotrexate and 5-fluorouracil.