Studies with Benzamide Riboside, a Recent Inhibitor of Inosine 5

Chapter 12

Downloaded by PENNSYLVANIA STATE UNIV on July 27, 2012 | http://pubs.acs.org Publication Date: March 3, 2003 | doi: 10.1021/bk-2003-0839.ch012

Studies with BenzamideRiboside,a Recent Inhibitor of Inosine5'-MonophosphateDehydrogenase 1

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Hiremagalur N . Jayaram , Joel A . Yalowitz , Georg Thomas Szekeres , Karsten Krohn , and Krzysztof W . 2

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Krupitza , Pankiewicz

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Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Drive,MS4053, Indianapolis, IN 46202-5122 University of Vienna, Wahringer Gurtel 18-20, A-1090 Vienna, Austria Division of Organic Chemistry, Universitat Gesamthochschule Paderborn, Paderborn 1621-4790, Germany Pharmasset, Inc., 1860 Montreal Road, Tucker, GA 30084 2

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Benzamide riboside was synthesized as a potential inhibitor of poly(ADP-ribose)polymerase (PARP). However, it was a weak inhibitor of PARP, but instead an extremely potent inhibitor of cell proliferation. We demonstrated that it was a selective inhibitor of inosine 5'-monophosphate dehydrogenase (IMPDH). Benzamide riboside is converted intracellularly to an analogue of N A D , BAD (benzamide adenine dinucleotide) that is a potent inhibitor of I M P D H . We characterized the formation of B A D in cells, and it was followed by the chemical synthesis of B A D and its non-hydrolyzable methylene bis(phosphonate) analogues. On a molar basis, benzamide riboside exhibits more potent antitumor activity than tiazofurin, the first inhibitor of I M P D H that we had previously demonstrated to be acting through its N A D analogue, TAD (thiazole-4-carboxamide adenine dinucleotide). In the National Cancer Institute's panel of 60 tumors, benzamide riboside showed selective antitumor

© 2003 American Chemical Society

In Inosine Monophosphate Dehydrogenase; Pankiewicz, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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activity against human central nervous system tumors. In addition, benzamide riboside induces apoptosis in human ovarian carcinoma N.1 cells, a property not shared by tiazofurin. Induction of apoptosis by benzamide riboside is associated with a down-regulation of Cdc25A, a cell cycle specific phosphatase. New and potent analogues of benzamide riboside and its active metabolites are being synthesized to find specific inhibitors for I M P D H Type II, expression of which is up-regulated in tumor cells. I M P D H inhibitors are not only valuable as chemotherapeutic agents but also as probes for investigating biochemical functions of guanylates in intact cells.

Poly(ADP-ribose) polymerase [PARP] utilizes N A D as a substrate for the formation of poly(ADP-ribose)polymers that are utilized as a substrate for chromatin modification. In this process, protein-bound homopolymers are rapidly formed in response to D N A damage ' . Therefore, action of agents (such as U V , alkylating agents, glucocorticoids, etc.) that induces D N A fragmentation stimulates the formation of protein bound poly(ADP-ribose) chains " . Nicotinamide and its analogues are known to potentiate cytotoxicity by inhibiting P A R P , thereby increasing the strand breaks induced by D N A alkylation . Although benzamide and related compounds are good in vitro inhibitors of P A R P , they are known to be neurotoxic in vivo and thus difficult to use for cancer chemotherapy . To overcome toxicity problem, Krohn and his associates designed B R as an inhibitor of P A R P . However, their studies suggested that B R was a poor inhibitor of P A R P . Inosine 5'-monophosphate dehydrogenase (IMPDH) oxidizes I M P to form X M P and in this process N A D accepts the electron. Thus, N A D and I M P are substrates for I M P D H . Our studies had demonstrated that tiazofurin by being converted into an analogue of N A D , T A D (thiazole-4-carboxamide adenine dinucleotide) was mimicking N A D and thus inhibited I M P D H utilization of N A D for the reaction " . Inhibition of I M P D H resulted in decreased guanylate levels including G T P and dGTP. T A D can also be hydrolyzed by a phosphodiesterase in resistant cells to tiazofurin 5'-monophosphate and A M P . Further studies showed that the tiazofurin analogue selenazofurin, wherein selenium replaces sulfur on the thiazole ring, also acts as a potent I M P D H inhibitor by forming an analogue of N A D , S A D (selenazole-4-carboxamide adenine dinucleotide) . Selenazofurin was 5-fold more cytotoxic to leukemic cells than tiazofurin, but was also more toxic to mice. Marquez and his associates chemically synthesized T A D and S A D . 1 2

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1 2

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0.288 0.489

0.618 0.163 0.382

0.268 0.310 0.218 0.510

0.614 0.264 0.178 0.150

0.832

0.273

0.323

0.254

0.503

SR

6-MP

3-DAG

ATH

PYF

0.165

1.000

ATH

1.000

PYF

TR, Tiazofurin; BR, Benzamide riboside; SR, Selenazofurin; 6-MP, 6-Mercaptopurine; 3-DAG, 3-Deazaguanine; ATH, Aminothiadiazole; and PYF, Pyrazofurin

0.259

1.000

L000

1.000

1.000

3-DAG

0.656

6-MP

BR

SR

1.000

BR

TR

TR

T A B L E I. C O M P A R E ANALYSIS O F TIAZOFURIN, BENZAMIDE RIBOSIDE, AND OTHER IMPDH INHIBITORS


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COMPARE analysis to assign mechanism of action for benzamide riboside As part of the screening program at the National Cancer Institute, compounds were tested for cytotoxicity against 60 different human tumor cell lines . The data resulting from these tests were evaluated by utilizing a computer program, C O M P A R E developed by Dr. Kenneth Paull and his associates . This program creates pair-wise correlations of the cell line data for a probe or "seed" compound with the corresponding cell line information for other compounds, permitting the estimation of the in vitro biochemical mechanisms of action of the "seed" compound. When tiazofurin (TR) was used as "seed" and the C O M P A R E analysis was performed, the correlation coefficients with B R (benzamide riboside) and SR (selenazofurin) were close to TR, suggesting that the two compounds might act through the same mechanism as T R . As provided in Table I, it is evident that although other agents (6mercaptopurine, 3-deazaguanine and aminothiadiazole) exhibit I M P D H inhibitory activity, they do not have close correlation coefficients. Pyrazofurin, 15


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T A B L E II. RELATIONSHIP OF INHIBITION O F IMPDH WITH BENZAMIDE RIBOSIDE CONCENTRATION IMPDH Activity (Percent of control) 100 90 63* 48* 7*

BR Concentration (JJM) 0 10 25 50 100

^Significantly different compared to saline controls (p < 0.05) 7

Human myelogenous leukemia K562 cells (1 x 10 cells/10 ml) were incubated with saline or indicated concentrations of BR for 2 hr at 37°C, and cells were harvested and IMPDH activity determined as cited . Under the conditions of the assay, 17 ± 1.9 nmol of XMP/h/mg protein was formed in the cells incubated with saline. 18

an unrelated compound (an inhibitor of pyrimidine nucleotide biosynthesis) is included to demonstrate that it does not have any close resemblance in its correlation coefficient with I M P D H inhibitors. Therefore, it was of interest to


235 conduct comparative examination of biochemical actions of TR, SR and B R in cancer cells.

Relationship of benzamide riboside and guanylate metabolism We found that benzamide riboside was also metabolized in sensitive tumor cells to its 5'-monophosphate and then converted to its active metabolite, B A D (benzamide adenine dinucleotide) . B A D inhibits I M P D H resulting in reduced guanylate synthesis. Relationship of the inhibition of I M P D H in human myelogenous leukemia K562 cells following incubation with graded concentrations of benzamide riboside is shown in Table I I This table demonstrates that there is a relationship between benzamide riboside dose and I M P D H inhibition in these human leukemic cells. I M P D H inhibition should be associated with a decrease in guanylate nucleotide concentration in these cells. To ascertain this fact, we incubated K562 cells with benzamide riboside and examined the nucleotide pool sizes by high pressure liquid chromatography (HPLC). The results presented in Table III shows that guanylate (GMP and GTP) concentration was decreased without affecting adenylate concentration. Concurrently, I M P pools were elevated, suggesting that guanylate depletion was due to non-utilization of I M P for guanylate synthesis. We have always noticed that at the initial time points up to 2 hr, GDP levels were not influenced by I M P D H inhibitor action.


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T A B L E III. INFLUENCE OF BENZAMIDE RIBOSIDE T R E A T M E N T ON T H E L E V E L S OF RIBONUCLEOTIDES IN H U M A N MYELOGENOUS L E U K E M I A K562 C E L L S Ribonucleotide NAD IMP GMP GDP GTP AMP ADP ATP

Control Concentration (nmol/g cells) 702.3 ± 24.6 10.5 ± 0 . 5 7.8 ± 7 . 8 62.0 ± 3.4 1199.4 ± 4 2 . 9 52.8 ± 2.2 298.8 ± 9.8 6023.1 ± 2 0 1 . 1

BR Treated (% of Control) 87 545* 0* 112 49* 124 157* 99

*Significantly different compared to saline controls (p < 0.05)


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Human myelogenous leukemia K562 cells (1 x 10 cells/10 ml) were incubated with saline or 10 uM BR for 2 hr at 37°C. Cells were harvested and nucleotide levels were determined as cited . 17

The biochemical effects exerted by benzamide riboside on ribonucleotide pools were abrogated by replenishing guanylate concentrations with the addition of guanosine . We examined the influence of benzamide riboside on deoxynucleotide concentration in K562 cells. This study provided in Table I V (taken from Reference 18) demonstrated a dose-related decrease in dGTP levels . The concentrations of dCTP and dTTP were not altered due to treatment. The level of dATP was not affected when treated with 1 p M B R , whereas at higher concentrations of B R there was a decrease in dATP concentration .


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T A B L E IV. E F F E C T O F BENZAMIDE RIBOSIDE ON 2'DEOXYRIBONUCLEOTIDE POOLS IN H U M A N M Y E L O G E N O U S L E U K E M I A K562 C E L L S 1

Control Concentration (pmol/10 cells) 5.3 ± 0.4 12.3 ± 1 . 2 47.4 ± 1 . 9 8.2 ± 0 . 1

Nucleotides DCTP DTTP DATP DGTP x

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Percent of control 1 /M 95 91 84 82

2.5 /JM 82 107 77 60*

5 fjM 96 116 71* 49*

Data reproduced from Ref. 18.

*Significantly different from Control values (p

Studies with Benzamide Riboside, a Recent Inhibitor of Inosine 5

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