Inhibition of Polyamine Metabolism. Biological Significance and Basis for new Therapies

374 Pages·1987·10.329 MB·English

by  Peter McCann (Eds.)

INHIBITION
OF POLYAMINE
METABOLISM
Biological Significance and
Basis for New Therapies
Edited by
Peter P. McCann
Merrell Dow Research Institute
Cincinnati, Ohio
Anthony E. Pegg
Department of Physiology and Cancer Research Center
Pennsylvania State University Medical School
Milton S. Hershey Medical Center
Hershey, Pennsylvania
Albert Sjoerdsma
Merrell Dow Research Institute
Cincinnati, Ohio
mi
ACADEMIC PRESS, INC.
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United Kingdom Edition published by
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Library of Congress Cataloging in Publication Data
Inhibition of polyamine metabolism.
Includes index.
1. Polyamines—Synthesis—Inhibitors—Physiological
effect. 2. Polyamines—Synthesis—Inhibitors—Therapeutic
use. 3. Polyamines—Metabolism —Regulation. I. McCann,
Peter P. II. Pegg, Anthony E. III. Sjoerdsma, Albert,
Date . [DNLM: 1. Polyamines-metabolism.
QU 60 155]
QP801.P638I55 1987 574.2Ί92 86-32147
ISBN 0-12-481835-8 (alk. paper)
PRINTED IN THE UNITED STATES OF AMERICA
87 88 89 90 9 8 7 6 5 4 3 2 1
Preface
Research into the biochemistry and cellular physiology of polyamines
has been a steadily growing field over the last twenty years. Progress
during this period and overviews of the area have been presented in a
number of books and a much larger number of review articles. Although
the function of polyamines is still not well understood at the molecular
level, there is a great deal of information which attests to the importance
of polyamines in cellular function. More recently, there has been a grow
ing awareness that the polyamine biosynthetic pathway provides a useful
target for the design of inhibitors which have value as pharmacological
agents. The availability of such inhibitors has led to a rapid increase in
knowledge of the importance of polyamines in a wide variety of living
organisms, and there is now convincing evidence of clinical utility of
these inhibitors in certain situations. In view of the ubiquitous distribution
of polyamines and their key role in a variety of cellular processes, it
seems likely that further uses for these inhibitors as chemotherapeutic
agents and in the treatment of plant and animal pathology may be ex
pected.
The large increase in information on the inhibition of polyamine metab
olism has led to a need for a detailed summary of knowledge in this area.
The purpose of the present volume is to provide an overview of the field
concerning polyamine biosynthesis inhibitors at the present time. Chap
ters are included which describe the compounds which are currently in
use; the rationale for the design of such inhibitors; their synthesis and
action; their biological effects on mammalian cells and tissues, plants, and
microorganisms including protozoal parasites; and a review of the present
clinical experience with these inhibitors. These chapters also demonstrate
clearly the major beneficial effect which the wide availability of these
inhibitors has had on the field of polyamine research in general. It is
hoped that the presentation of this material in a single volume will stimu
late further research on both the basic and applied aspects of polyamines.
The editors are most grateful to the authors of the individual chapters for
xi
xii Preface
the care with which their topic has been covered, the comprehensive
nature of their contributions within the space available, and submitting
the material in a timely manner.
Peter P. McCann
Anthony E. Pegg
Albert Sjoerdsma
Introduction: Polyamine Metabolism
All cells contain significant amounts of the polyamines, putrescine (a
diamine), spermidine, and spermine. Although the physiological function
of these amines is still not well understood at the molecular level, many
studies summarized in the following chapters have shown that their con
centration is highly regulated and that normal cellular growth and differ
entiation require polyamines. Many bacteria and protozoa contain putres
cine and spermidine as their only polyamines while mammalian cells,
yeast, other fungi, and other protozoa synthesize all three polyamines.
This introduction outlines the general metabolic reactions responsible for
the maintenance of polyamine content.
The general pathway for polyamine biosynthesis is shown in Fig. 1.
Detailed description of the metabolic reactions responsible for polyamine
synthesis are given in the various subsequent chapters. Many microor
ganisms and higher plants are able to produce putrescine from agmatine
produced by decarboxylation of arginine, but all mammalian cells, lower
eukaryotes including fungi, and most probably protozoa lack arginine
decarboxylase. In these species, therefore, the only route to putrescine is
via the enzyme ornithine decarboxylase. In contrast, plants and many
bacteria are able to synthesize putrescine by at least two routes, either
directly from ornithine, through the activity of ornithine decarboxylase,
or indirectly, via arginine and arginine decarboxylase and one or two
intermediates, i.e., agmatine and in some instances N-carbamoyl putres
cine.
In mammalian cells, ornithine, the substrate for ornithine decarboxy
lase, is available from the plasma and can also be formed within the cell
from arginine by the action of arginase. It is possible that arginase, which
is much more widely distributed than other enzymes of the urea cycle, is
present in extrahepatic tissues to ensure the availability of ornithine for
polyamine production. Arginase can, therefore, be thought of as an initial
step in the polyamine biosynthetic pathway.
Both ornithine decarboxylase and arginine decarboxylase are pyridoxal
phosphate-dependent enzymes. Ornithine decarboxylase is present in
xiii
xiv Introduction: Polyamine Metabolism
very small amounts in quiescent cells, and its activity can be increased
many fold within a few hours of exposure to trophic stimuli. Such stimuli
include hormones, drugs, tissue regeneration, and growth factors. Even
after such stimulation, ornithine decarboxylase is only a very small frac
tion of the total cellular protein. There is evidence for a macromolecular
inhibitor of ornithine decarboxylase that may regulate the activity of the
enzyme under some circumstances.
To convert putrescine into spermidine, an aminopropyl group must be
added. This aminopropyl moiety is derived from methionine, which is first
converted into 5-adenosylmethionine and is then decarboxylated. The
resulting decarboxy-5-adenosylmethionine is used as an aminopropyl
donor in a manner analogous to the use of 5-adenosylmethionine itself as
a methyl donor. Once it has been decarboxylated, 5-adenosylmethionine
is committed to polyamine synthesis because the decarboxylated form is
virtually inactive as a methyltransferase substrate. Therefore, the produc
tion of decarboxy-5-adenosylmethionine is kept low and constitutes the
rate-limiting factor in spermidine formation. Mammalian 5-adeno-
sylmethionine decarboxylase is activated by putrescine and repressed by
spermidine, linking the supply of decarboxy-5-adenosylmethionine to the
need for spermidine and the availability of the other substrate (putrescine)
for spermidine synthesis (Fig. 1). 5-adenosylmethionine decarboxylase
(which has an enzyme-bound pyruvate as a cofactor) resembles ornithine
decarboxylase in that it is also present in mammalian tissues in very small
amounts and that its activity is regulated by many hormones and other
growth-promoting stimuli.
Nonmammalian 5-adenosylmethionine decarboxylases also have a co-
valently attached pyruvate group but differ in other ways. 5-Adeno
sylmethionine decarboxylase from bacteria and plants are activated by
Mg2"1", unlike 5-adenosylmethionine decarboxylases from yeast and other
fungi which resemble the mammalian enzyme in being strongly activated
by putrescine. Protozoal 5-adenosylmethionine decarboxylases on the
other hand appear to be both Mg2+ insensitive and relatively putrescine
insensitive.
The transfer of the aminopropyl group from decarboxy-5-adeno-
sylmethionine to putrescine is catalyzed by spermidine synthase. Another
aminopropyl group is needed to convert spermidine into spermine, and
this also comes from decarboxy-5-adenosylmethionine in a reaction cata
lyzed by a second aminopropyltransferase termed spermine synthase.
Despite the similarity between these reactions, spermidine synthase and
spermine synthase are discrete enzymes each specific for its own particu
lar substrate. The aminopropyltransferases are present in many cells in
amounts much greater than the decarboxylases and are thought to be
Introduction: Polyamine Metabolism xv
ARGINASE
S-ADENOSYLMETHIONINE ORNITHINE ARGININE
I
tI t
ORNITHINE ARGININE
S-ADENOSYLMETHIONINE DECARBOXYlASE DECARBOXYlASE
DECARBOXYLASE
AGMATINASE
PUTRESCINE ~ or- AGMATINE
AGMATINE DEIMINASE
H2N~NH2
DECARBOXY
S-ADENOSYLMETHIONINE
\60LYAMINE
\XIDASE
AMINOPROPYL SSPYENRTMHIADSINEE N'-ACETYLSPERMIDINE
TRANSFER
POLYAMINE
) ACETYlASE
METHYL SPERMIDINE
THIOADENOSINE
H2N~N~NH2
H \POLYAMINE
OXIDASE
SPERMINE N'-ACETYLSPERMINE
SYNTHASE
POLYAMINE
ACETYLASE
)
SPERMINE
H2N~N~~~NH2
H
Fig. 1. Schematic overview of polyamine metabolism.
regulated by the availability of their substrates, particularly the amino
propyl donor, decarboxy-S-adenosylmethionine.
The other product of aminopropyltransferase reactions is 5'-methyl
thioadenosine. Although this nucleoside is produced in stoichiometric
amounts with the polyamines, its concentration in the cell is very low due
to rapiddegradationby aphosphorylaseproducingadenineand5'-methyl
thioribose-l-phosphate. The adenine is then converted to 5'-AMP by
action ofadenine phosphoribosyltransferase and the 5'-methylthioribose
l-phosphate is converted back to methionine in a reaction that conserves
the rnethylthiogroup and all but the C-l ofthe carbon atoms ofthis sugar.
Therefore, all of the S-adenosylmethionine molecule not used for poly
amine production is effectively salvaged. 5'-Methylthioadenosine phos
phorylase is a widely distributed enzyme that is present in all normal
tissues examined in amounts sufficient to maintain 5'-methylthioadeno-
xvi Introduction: Polyamine Metabolism
sine at very low levels. Certain mammalian tumor cell lines including
some derived from humans have lost this enzyme, and these appear to
excrete 5-methylthioadenosine to reduce the intracellular content of this
nucleoside. It cannot be ruled out that 5'-methylthioadenosine (which has
a number of effects on cellular physiology when applied exogenously)
rather than the polyamines is the critical product of the pathway in Fig. 1,
but the rapid degradation of more than 99% of this product argues against
this possibility.
The aminopropyltransferase reactions which form spermidine and sper
mine are effectively irreversible but these polyamines can be converted
back into putrescine by the combined actions of two enzymes, spermi-
dine/spermine-A^-acetytransferase (polyamine acetylase) and polyamine
oxidase (FAD dependent) (Fig. 1). Polyamine acetylase catalyzes the
conversion of spermine into A^-acetylspermine which is then degraded by
polyamine oxidase to form spermidine and 3-acetamidopropanal. Simi
larly, spermidine is a substrate for polyamine acetylase which forms Nl-
acetylspermidine and this is split by polyamine oxidase to form putrescine
and 3-acetamidopropanal. The acetylase/oxidase system and either of the
aminopropyltransferases can therefore be regarded as forming a cycle
which leads to the production of 5'-methylthioadenosine from decarboxy-
5-adenosylmethionine. The importance of these cycles and the extent to
which they occur in unstimulated cells are unknown at present.
The regulation of the polyamine biosynthesis and interconversion path
way shown in Fig. 1 is accomplished by changes in the activity of three of
these enzymes: ornithine decarboxylase, 5-adenosylmethionine decar
boxylase, and polyamine acetylase. The other enzymes (spermidine syn
thase, spermine synthase, and polyamine oxidase) appear to be regulated
via the availability of their limiting substrates which are the decarboxyl
ated and the acetylated polyamine derivatives. Polyamine concentration
may also be affected by degradation of putrescine by diamine and mo
noamine oxidase. Ornithine decarboxylase, 5-adenosylmethionine decar
boxylase, and polyamine acetylase can undergo large changes in their
activity which appear to be brought about by alterations in the amount of
enzyme protein present. All three of these enzymes have an extremely
short half-life, which enables a new level of enzyme protein to be
reached very rapidly after the application of an appropriate stimulus.
Peter P. McCann
Anthony E. Pegg
Albert Sjoerdsma
1
Inhibition of Basic Amino Acid
Decarboxylases Involved in
Polyamine Biosynthesis
PHILIPPE BEY,* CHARLES DANZIN,t
AND MICHEL JUNGt
Merrell Dow Research Institute
*2110 East Galbraith Road
Cincinnati, Ohio 45215
fMerrell Dow Research Institute
Strasbourg Centre
16 rue a" Ankara
y
F 67084 Strasbourg Cedex, France
I. INTRODUCTION
After ornithine decarboxylase (ODC) was established to be one of the
rate-limiting enzymes in the biosynthesis of polyamines in eucaryotes,
and Russell and Synder (1968) had reported an extraordinary stimulation
and decay of ODC activity in regenerating rat liver and in other situations
of active cellular and tissue growth, the design and synthesis of specific
inhibitors of ODC became a logical approach to unravel the physiological
role of polyamines and to control rapid cell proliferation. The first publi­
cation dealing with inhibitors of ODC appeared in 1972 (Skinner and
Johansson). This chapter is intended to discuss the extensive work since
that date aimed at the inactivation of ODC, focusing on the mechanistic
considerations which eventually led to the design and synthesis of potent
ODC inhibitors acting directly at the enzyme active site. Extension of this
work to the inhibition of arginine decarboxylase (ADC) and lysine decar­
boxylase (LDC), two other α-amino acid decarboxylases involved in the
ι
INHIBITION OF POLYAMINE METABOLISM Copyright © 1987 by Academic Press, Inc.
All rights of reproduction in any form reserved.
2 Philippe Bey et al.
biosynthesis of polyamines in plants and bacteria (for a recent review, see
Tabor and Tabor, 1984), will also be briefly reported. Comprehensive
reviews on ODC and its inhibition have appeared regularly in the litera­
ture (Williams-Ashman et al., 1976; Stevens and Stevens, 1980; Pegg and
Williams-Ashman, 1981; Pegg and McCann, 1982; Pegg, 1986). Not cov­
ered in this chapter are agents which control in vivo ODC activity at the
transcriptional, posttranscriptional, or posttranslational level. It is now
firmly established that in vivo the polyamines can control ODC activity.
The search for unnatural polyamine analogs capable of decreasing ODC
activity in vivo, but devoid of the physiological properties of the natural
polyamines, has been and continues to be a line of active research in
many laboratories.
II. PROPERTIES OF ODC
The first ODC activity was detected in an extract of Escherichia coli
(Gale, 1946). Later it was recognized that E. coli strains can contain two
distinct enzymes capable of catalyzing the decarboxylation of ornithine;
these were classified as the biodegradative and the biosynthetic enzymes
(for a review see Morris and Fillingham, 1974). The biodegradative ODC
is active only at low pH and appears to be involved in the regulation of the
pH of the medium, whereas the biosynthetic ODC is responsible for the
synthesis of putrescine and, ultimately, spermidine. In contrast to the
biodegradative enzyme, which is found only in about 1 out of 10 E. coli
strains, the presence of the biosynthetic ODC has been confirmed practi­
cally in all strains of E. coli and also in other unicellular organisms,
including the lower ones such as Physamm polycephalum (Mitchell and
Carter, 1977) and Euglena gracilia (Lafarge-Frayssinet et al., 1978).
The biodegradative ODC has been purified to homogeneity (Applebaum
et al., 1977) and the biosynthetic enzyme has been obtained with a purity
of about 85% as judged by polyacrylamide gel electrophoresis (Apple­
baum et al., 1977). Both enzymes are dimers of 80,000 to 82,000
molecular weight and exhibit similar kinetic properties. The biosynthetic
ODC has a K for ornithine of 9.6 mM and a specific activity of 6000 μ,πιοί
m
of C0 liberated per milligram of protein per hour, compared with 3.6 mM
2
and 7800 μ,πιοί of C0 per milligram of protein per hour for biodegradative
2
ODC. The two enzymes, however, differ markedly in their pH profile and
in their activation patterns by nucleotides. The optimal pH of biodegrada­
tive ODC is 6.9, whereas it is 8.1 for the biosynthetic enzyme. Both
enzymes are activated by GTP, but CTP, UTP, and GMP have opposite
effects on the activity of the two enzymes. The K and V are strongly
m max
influenced by the presence of nucleotides.