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Biochemical Differences Among Four Inosinate Dehydrogenase Inhibitors

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Biochemical Differences Among Four Inosinate Dehydrogenase Inhibitors [CANCER RESEARCH 45.5512-5520, November 1985] Biochemical Differences among Four Inosinate Dehydrogenase Inhibitors, Mycophenolic Acid, Ribavirin, Tiazofurin, and Selenazofurin, Studied in Mouse Lymphoma Cell Culture1 Huey-Jane Lee, Katarzyna Pawlak, Binh T. Nguyen, Roland K. Robins, and Wolfgang Sadee2 Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, California 94143 [H-J. L, K. P., B. T. N., W. S.], and Cancer Research Center, Department of Chemistry, Brigham Young University, Provo, Utah 84602 [R. K. R.J ABSTRACT 1.2.1.14) catalyzes the conversion of IMP to xanthylate, and it represents a key enzyme in the biosynthesis of guanine nucleo- The mechanism of the cellular toxicity of four ¡nosinatedehy- tides. The activity of IMP dehydrogenase is positively linked to drogenase (IMP-DH) inhibitors with different antitumor and anti cellular transformation and tumor progression; therefore, this viral pharmacological profiles was investigated in mouse lym- enzyme represents a promising target of cancer chemotherapy phoma (S-49) cell culture. Drug effects on cell growth, nucleotide (1-3). Inhibition of IMP dehydrogenase results in the depletion pools, and Ã’NA and RNA synthesis were measured in the of cellular guanine nucleotides by blocking their de novo synthe presence and absence of guanine salvage supplies. Both guanine and guanosine were capable of bypassing the IMP-DH block, sis (4). Guanine nucleotides are required as substrates, activa while they also demonstrated some growth-inhibitory effects tors, or regulators in many pathways of cellular metabolism, including DNA, RNA, and protein synthesis (2). when added alone in high concentrations. All four drugs reduced Mycophenolic acid (5), ribavirin (6), selenazofurin (7, 8), and cellular guanosine triphosphate levels and caused secondary tiazofurin (9-11) (Chart 1) are all thought to exert toxic effects changes of the undine, cytidine, and adenosine triphosphate on mammalian cells primarily via IMP dehydrogenase inhibition, pools that were similar among the four drugs. However, several drug effects in addition to IMP-DH inhibition were observed since the toxicities of these drugs are largely preventable by an except with mycophenolic acid which may represent a pure IMP- exogenous supply of guanine nucleotides via the salvage path DH inhibitor. Both tiazofurin and selenazofurin interfered with the ways. Mycophenolic acid represents a prototype IMP dehydro uptake and/or metabolism of undine and thymidine tracers; genase inhibitor (5), with no other biochemical effects noted. It shows weak to moderate antitumor, antifungal, and immunosup- however, this effect appeared not to contribute to their cellular toxicity m vitro. Moreover, selenazofurin and tiazofurin impaired pressive activities (12). Despite the similar major mechanism of the utilization of exogenous guanine salvage supplies for DNA cellular toxicity, it was shown that ribavirin (13,14) represents a and RNA synthesis, and guanine was particularly ineffective in potent antiviral agent currently in clinical use with low antitumor reversing the toxic effects of tiazofurin on cell growth. This finding effects, while tiazofurin which is undergoing clinical trial (Phase is important in view of the available guanine salvage supplies in II) as an antitumor agent has low antiviral activities but acts as a vivo. Since tiazofurin, selenazofurin, and their known metabolites potent anticancer agent in certain animal tumor models (15). failed to inhibit hypoxanthine-guanine-phosphoribosyl transfer- Selenazofurin, a seleno analogue of tiazofurin, is highly effective ase, guanosine monophosphate kinase, and guanosine diphos- against both viral infections and animal tumors (16, 17); it is phate kinase in cell extracts or permeabilized cells, these drugs approximately 5 to 10 times more potent as an antitumor agent may interfere with salvage transport across cellular membranes. than tiazofurin (18). The toxic effects of mycophenolic acid and ribavirin were similarly At least two hypotheses can be proposed for the different reversed by salvage supplies of up to 200 UM guanine, which pharmacological effects of these four agents, (a) Differences in suggests that ribavirin primarily acts as an IMP-DH inhibitor under their pharmacokinetics could cause different exposure times that these conditions. This result could explain the rather low antitu- may be critical to the therapeutic outcome, (o) Some of these mor efficacy of both mycophenolic acid and ribavirin in vivo. agents produce biochemical effects at therapeutic concentra However, increasing the guanine salvage supply in the medium tions that are unrelated to IMP dehydrogenase inhibition. This above 200 ^M further reversed the toxic effects of mycophenolic paper addresses the second question by comparing the effects acid to maximum rescue, while it increased the toxicity of ribavirin of each agent on cell growth, cellular nucleotide pools, and DNA (300 UM). This finding suggests the presence of a toxic mecha and RNA synthesis of mouse S-49 lymphoma cells. These stud nism of ribavirin at higher concentrations that is dependent upon ies were performed in the presence and absence of guanine the presence of guanine supplies sufficient to fully overcome the salvage supplies that bypass the IMP dehydrogenase block. The IMP-DH inhibition. This study documents that each antimetabo- results reveal several biochemical differences among the four lite displays a unique spectrum of activities with multiple toxic IMP dehydrogenase inhibitors. targets. INTRODUCTION MATERIALS AND METHODS Inosinate dehydrogenase (IMP:NAD oxidoreductase, EC Reagents and Apparatus. Mycophenolic acid was provided by Eli Lilly and Co., Indianapolis, IN. Ribavirin (1-0-D-ribofuranosyl-1,2,4-tria- 1This research was supported by USPHS Grants CA-27866. CA 34304. and zole-3-carboxamide), ribavirin-5'-monophosphate, selenazofurin (2-/ÃŽ- CA-34384 from the National Cancer Institute. 2 To whom requests for reprints should be addressed. D-ribofuranosylselenazole-4-carboxamide), setenazofurin-5'-monophos- Received 9/10/84; revised 7/3/85; accepted 7/8/85. phate, tiazofurin (2-/Õ-D-ribofuranosylthiazole-4-carboxamide), tiazofurin- CANCER RESEARCH VOL. 45 NOVEMBER 1985 5512 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1985 American Association for Cancer Research. """•'W"«T-".'-." INOSINATE DEHYDROGENASE INHIBITION 0.5 M KCI solution with a linear gradient of 0.01 M NH<H2PO4 (pH 3.6) increasing to 0.8 M NH4H2P04 over 15 min (24). Eluent fractions were collected and analyzed by liquid scintillation counting when 3H tracers HOOC-CH r CH rC-CH-CH were used. Drug effects on pool sizes and tracer metabolism were usually determined over a 4-h incubation. CH, HjC Tracer Incorporation into RNA and DMA. Cells were incubated for 4 h in complete medium with various concentrations of the chemical agents OH OH to be tested. [mef/)y/-3H]Thymidine or [5-3H]uridine was added to the cell MycophenolicAcid Tiazofurin suspension 30 min prior to harvest. The acid-insoluble cell pellet was precipitated by adding perchloric acid, and radioactivity was measured by liquid scintillation counting as previously described (21). Absolute DMA and RNA Synthesis Rates. Absolute rates of DNA and RNA synthesis were estimated by the incorporation into acid- insoluble material of [8-14C]guanine diluted with 50 to 400 ^M unlabeled guanine, after correction for the specific [14C]guanine activity in the medium. In the presence of high concentrations of the IMP dehydrogen ase inhibitors, exogenous guanine should represent the predominant HOCH2 guanine source for RNA and DNA. [8-14C]Guanine (1 nCi/ml), unlabeled guanine (50 to 400 MM),and the drugs were added to the cell medium 4 h prior to harvest. The acid-insoluble cell pellet was washed with water and then suspended overnight in 0.3 N KOH at room temperature to OH hydrolyze RNA to free ribonucleotides as described previously (21). Selenazofurin Acidification with 0.4 N HCIO4 precipitated DNA, while free ribonucleo Ribavirin tides derived from RNA remained in the supernatant. The radioactivity Chart 1. Chemical structures of mycophenolic acid, ribavirin, tiazofurin, and of the supernatant and pellet was measured by liquid scintillation count selenazofurin. ing. In order to assure that the specific activities of endogenous guanine 5'-monophosphate, and thiazole-4-carboxamide adenine dinudeotide nucleotide pools reach the same specific 14C activity as that of the (tiazofurin-NAD) were synthesized in the laboratory of Dr. Roland K. [14C]guanine in the medium, a requisite for the use of this approach to Robins. Guanine, guanosine, ATP, GMP, GDP, and L-a-lysophosphati- determine absolute nucleic acid synthesis rates, the specific 14Cactivity dylcholine (i_-«-lysolecithin)type I were purchased from Sigma, St. Louis, of the cellular GTP pool was determined by HPLC and liquid scintillation MO. All the other chemical reagents were of analytical grade. [8-14C]- counting. Cells were incubated for 4 h with 10 UM mycophenolic acid, Guanine HCI (54.4 mCi/mmol), [mef/iy/-3H]thymidine (80 Ci/mmol), and 300 /jM ribavirin, 300 /IM tiazofurin, or 40 /¿Mselenazofurin plus 100, [5-3H]uridine (23 Ci/mmol) were provided by ICN, Irvine, CA. [8,5'-3H]- 200, or 400 MM[14C]guanine. In each case, the specific [14C]GTP activity Guanosine 5'-monophosphate (38 Ci/mmol) was obtained from NEN, was identical to that of [14C]guanine in the medium (within experimental Boston,
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