Polyamines and Their Role in Human Disease
Polyamine biosynthetic enzymes as drug targets
in parasitic protozoa
O. Heby*1 , S.C. Roberts† and B. Ullman†
*Department of Molecular Biology, Umeå University, SE-901 87 Umeå, Sweden, and †Department of Biochemistry and Molecular Biology, Oregon Health &
Science University, Portland, OR 97239-3098, U.S.A.
Abstract
Molecular, biochemical and genetic characterization of ornithine decarboxylase, S-adenosylmethionine
decarboxylase and spermidine synthase establishes that these polyamine-biosynthetic enzymes are
essential for growth and survival of the agents that cause African sleeping sickness, Chagas’ disease,
leishmaniasis and malaria. These enzymes exhibit features that differ significantly between the parasites
and the human host. Therefore it is conceivable that exploitation of such differences can lead to the design
of new inhibitors that will selectively kill the parasites while exerting minimal, or at least tolerable, effects
on the parasite-infected patient.
Introduction
Parasitic protozoa continue to beleaguer and kill millions
of people in the subtropical and tropical regions of the
world. However, some new inroads towards improved
treatments of malaria, leishmaniasis, Chagas’ disease and
African sleeping sickness, diseases caused by protozoan
parasites, have been made. One pathway that has been
exploited successfully in the treatment of parasitic diseases
is that of polyamine biosynthesis (Figure 1). Treatment with
D,L-α-difluoromethylornithine (DFMO), an inhibitor of the
first enzyme in the polyamine biosynthetic pathway, was
found to cure mice infected with Trypanosoma brucei brucei,
a parasite that is closely related to the trypanosomes that
cause sleeping sickness in humans [1]. DFMO treatment
was subsequently found to eradicate Trypanosoma brucei
gambiense infections in patients with West African sleeping
sickness, even after central nervous system involvment [2].
Other protozoan parasites also depend on polyamines for
growth and survival. Because the polyamine biosynthetic
enzymes of the parasites seem to exhibit features that are
significantly different from those of the human host and also
because some enzymes that use polyamines as substrates are
unique to the parasites, it is conceivable that exploitation of
this pathway could lead to the design of new antiparasitic
drugs that will selectively kill the parasites.
Polyamine biosynthetic enzymes and
enzyme-activated inhibitors
Ornithine decarboxylase (ODC) catalyses the first step in
the polyamine biosynthetic pathway, the decarboxylation of
Key words: African sleeping sickness, Chagas’ disease, leishmaniasis, malaria, polyamine.
Abbreviations used: AbeAdo, 5 -{[(Z)-4-amino-2-butenyl]methylamino}-5 -deoxyadenosine;
AdoMet, S-adenosylmethionine; AdoMetDC, S-adenosylmethionine decarboxylase; dcAdoMet,
decarboxylated S-adenosylmethionine; DFMO, d,l-α-difluoromethylornithine; ODC, ornithine
decarboxylase; MGBG, methylglyoxal-bis(guanylhydrazone).
1
To whom correspondence should be addressed (e-mail [email protected]).
ornithine to putrescine (Figure 1). S-Adenosylmethionine
decarboxylase (AdoMetDC) generates decarboxylated
S-adenosylmethionine (dcAdoMet), which serves as the
aminopropyl group donor for spermidine and spermine
synthesis. The latter reactions are catalysed by spermidine
synthase and spermine synthase, respectively (Figure 1A).
Prokaryotes and lower eukaryotes, including the protozoan
parasites, either lack or appear to lack spermine synthase
(Figures 1B–1D). Nevertheless, some parasites contain
spermine, most likely as a result of uptake from their host.
So-called ‘suicide inhibitors’ have been developed for all
four biosynthetic enzymes (see [3] and references therein).
Their mechanism of action is based on activation by the
target enzyme, which ensures high specificity for each of
these irreversible inhibitors. Other types of drugs, which
interfere with the functions of polyamines, are the structural
analogues.
African sleeping sickness (African
trypanosomiasis)
The causative agent of African sleeping sickness is T. brucei, a
protozoan transmitted to humans in the equatorial region of
Africa by the bite of flies of the genus Glossina. The infective
trypomastigotes, formed from epimastigotes in the salivary
glands of the tsetse fly, find their way from the wound created
by the bite into blood, lymph and eventually cerebrospinal
fluid.
DFMO is effective against both early- and late-stage West
African sleeping sickness caused by T. b. gambiense [2].
Unfortunately, the East African form of the disease, caused by
Trypanosoma brucei rhodesiense, is largely unaffected by the
drug. The reason for this difference has not been clarified
but may be a function of the more rapid progression of
the latter disease. While the high efficacy of DFMO against
T. b. gambiense infections in humans may have multiple
C 2003
Biochemical Society
415
416
Biochemical Society Transactions (2003) Volume 31, part 2
Figure 1 Enzymes involved in the regulation of polyamine levels in parasitic protozoa and in human host cells
(A) In human cells, the decarboxylases ODC and AdoMetDC are highly regulated, and have very short half-lives (<1
h), whereas the synthases SpdS and SpmS are constitutively expressed. SAT and PAO provide a pathway for backconversion from spermine to spermidine to putrescine. A transport system (T) can regulate the intracellular polyamine
levels. (B) In Leishmania donovani and in T. brucei, ODC and AdoMetDC have long half-lives (>6 h), and SpmS is lacking.
These organisms conjugate spermidine and glutathione (GSH), using two enzymes (GSS and TryS) to form trypanothione,
which is involved in defence against chemical and oxidant stress. L. donovani can survive on exogenous polyamines.
(C) Trypanosoma cruzi lacks ODC and SpmS and depends on efficient putrescine uptake and AdoMetDC for spermidine
synthesis. A single enzyme (TryS) catalyses the formation of trypanothione. (D) Plasmodium falciparum has a bifunctional
AdoMetDC-ODC and lacks SpmS as well as the pathway to trypanothione. Abbreviations: AdoMet, S-adenosylmethionine;
AdoMetDC, S-adenosylmethionine decarboxylase; dcAdoMet, decarboxylated AdoMet; GSH, glutathione; GSS, glutathionylspermidine synthase; MTA, 5 -methylthioadenosine; Orn, ornithine; ODC, ornithine decarboxylase; SAT, spermidine/
spermine-N1 -acetyltransferase; PAO, polyamine oxidase; ROS, reactive oxygen species; SpdS, spermidine synthase;
SpmS, spermine synthase; T, polyamine-transport system; TryR, trypanothione reductase; TryS, trypanothione synthase.
causes, the primary basis is probably the dramatic discrepancy
in stability between the host and parasite ODC enzymes [4].
The parasitic ODC is quite stable (t1/2 > 6 h) whereas the
human enzyme turns over rapidly (t1/2 < 1 h). This implies
that newly synthesized, active ODC rapidly replaces DFMOinactivated ODC in the human host but not in the parasite
where the turnover rate of the enzyme is slow. The C-terminal
region of human ODC appears to be essential for the rapid
degradation of the protein. Since there is no corresponding
domain in T. brucei ODC, the stability of this protein has been
C 2003
Biochemical Society
attributed to the lack of this C-terminal domain, i.e. removal
of a degradation signal [4]. However, the finding that ODC of
another trypanosomatid, Crithidia fasciculata, turns over
rapidly despite lacking the corresponding domain, indicates
that other motifs on the protein may mediate the targeting of
ODC for rapid degradation [5].
The reduced production of putrescine and spermidine in
T. brucei apparently cannot be compensated for by uptake
of polyamines from the blood of the host, either because of
the inefficient polyamine-transport system of this parasite
Polyamines and Their Role in Human Disease
or the low levels of polyamines available to the parasite in the
blood (Figure 1B). However, other trypanosomatids, such as
Trypanosoma cruzi, appear to be absolutely dependent upon
polyamine uptake for their growth and survival and have developed a very efficient putrescine-uptake system (Figure 1C)
[6]. T. cruzi also invade host cells where the polyamine
concentration is significantly higher than in the serum.
Because of the DFMO-mediated depletion of intracellular
spermidine, the parasites cannot synthesize trypanothione,
a conjugate of spermidine and glutathione that is unique
to trypanosomes and Leishmania (Figure 1B) [7]. Trypanothione is a reducing agent with many protective and
regulatory functions, and consequently its depletion in
DFMO-treated parasites is detrimental to the parasite.
T. b. brucei strains lacking both wild-type ODC alleles
have been created by double-targeted gene replacement [8].
These ∆odc-null mutants require exogenous putrescine for
proliferation. When injected into mice, a bloodstream form
of the ∆odc mutant was unable to multiply and was quickly
cleared from the blood, presumably because of the low levels
of polyamines available to the parasites in the blood [9].
Irreversible inhibition of AdoMetDC, using 5 -{[(Z)-4amino-2-butenyl]methylamino}-5 -deoxyadenosine (MDL
73811; AbeAdo), a structural analogue of dcAdoMet, was
found to be very effective against T. b. brucei infections
in mice and rats [10]. Since the trypanosomes disappeared
from the bloodstream before polyamine depletion, the
anti-trypanosomal effect was attributed to a dramatic
increase in the level of S-adenosylmethionine (AdoMet),
the universal methyl donor, that correlated with the decrease
in parasitaemia. This accumulation of AdoMet may cause
aberrant methylation, which in turn may interfere with
growth and survival of the parasite. AbeAdo was much more
potent than DFMO, and combinations of the two drugs were
synergistic, even curing mice infected with clinical isolates of
T. b. rhodesiense [11]. These data indicate that by inhibiting
both ODC and AdoMetDC, it may be possible to achieve
effective therapy against East African sleeping sickness, even
drug-refractory and late-stage forms.
Chagas’ disease (American
trypanosomiasis)
Chagas’ disease is a severely debilitating infection that is
distributed widely in rural zones of Latin America. The
causative agent is T. cruzi, transmitted by blood-sucking
insects of the subfamily Triatominae. The infective trypomastigotes, present in the faeces of the bug, enter the bite
wound and migrate to various tissues, where they become
intracellular replicating amastigotes. Other routes of transmission include blood transfusion or organ transplantation
from an infected donor, as well as congenital transmission from an infected mother.
T. cruzi lacks ODC and therefore cannot synthesize putrescine de novo (Figure 1C) [12,13]. Although there is some
evidence to suggest that T. cruzi can generate putrescine from
arginine via agmatine [14], it seems more likely that the
parasite relies on putrescine uptake and AdoMetDC for
spermidine synthesis [7,12,13]. In fact, putrescine uptake is
10–50-fold higher in T. cruzi than in Leishmania mexicana and
C. fasciculata [6]. Lack of ODC and the inability to synthesize
putrescine is consistent with the following findings : (i) that
T. cruzi is dependent on uptake of diamines [15], (ii) that
DFMO is completely inactive against all stages of the parasite,
(iii) that expression of a foreign ODC gene in T. cruzi
overcomes the requirement of exogenous polyamines for
growth [15] and (iv) that DFMO treatment causes growth
arrest of T. cruzi that express a foreign ODC [16].
Methylglyoxal-bis(guanylhydrazone) (MGBG), a potent
inhibitor of human AdoMetDC, is a poor inhibitor of the
T. cruzi enzyme [12]. Likewise, some aromatic derivatives of
MGBG, which show trypanocidal effects against T. b. brucei
and T. b. rhodesiense, are inactive against T. cruzi amastigotes
in murine macrophages in vitro [17]. Nevertheless, the
differential sensitivity to MGBG suggests that the human and
T. cruzi AdoMetDCs are sufficiently dissimilar to warrant the
design of structure-based compounds that might selectively
interfere with growth and survival of the parasite without
seriously affecting the human host. Thus far, only the crystal
structure of human AdoMetDC has been determined [18].
Considering the efficiency of the polyamine transporter(s)
in T. cruzi, it seems unlikely that the intracellular amastigote
form of the parasite can be killed by using inhibitors of
AdoMetDC or spermidine synthase. The host cells constitute
a rich source of polyamines, and the polyamine transporter(s)
of the parasite probably has to be blocked to confer a
polyamine-deficient state. Of further interest is the fact
that in T. cruzi a single enzyme catalyses the formation of
trypanothione from spermidine and glutathione (Figure 1C)
[19].
Visceral leishmaniasis (Kala-azar)
Leishmania donovani and Leishmania infantum are causative
agents of visceral leishmaniasis, a devastating and often fatal
disease in humans. The parasites exhibit a digenetic life cycle;
the motile, extracellular promastigote resides in a sandfly
vector of the genus Phlebotomus, and the non-motile, intracellular amastigote propagates within the phagolysosomes
of macrophages in the human host.
DFMO, as well as several other fluorinated ornithine
analogues, are cytotoxic not only to T. brucei spp. but also to
L. donovani [20] and L. infantum [21] promastigotes. As in
the case of trypanosomal ODC [4], leishmanial ODC exhibits
a long half-life (>20 h) that is attributable to the lack of a
C-terminal extension [22].
To investigate the importance of ODC, AdoMetDC and
spermidine synthase (Figure 1B) in L. donovani, the genes
encoding these enzymes were cloned and sequenced [22–
25]. The corresponding null mutants (∆odc, ∆adometdc and
∆spds) were created in promastigotes by double-targeted
gene replacement [23–25]. The ∆odc-knockout strain lacking
both ODC alleles was incapable of growth in polyaminedeficient medium [23]. This auxotrophy could be overcome
C 2003
Biochemical Society
417
418
Biochemical Society Transactions (2003) Volume 31, part 2
by addition of putrescine or spermidine, but not spermine.
Putrescine restored the intracellular pools of both putrescine
and spermidine, but spermidine was not converted back into
putrescine. These findings indicate that spermidine alone is
sufficient to meet the polyamine requirement for growth
and that L. donovani lacks the polyamine back-conversion
pathway (cf. Figures 1A and 1B) [23].
∆adometdc and ∆spds mutants were also incapable of
growth in medium without polyamines [24,25]. Auxotrophy
could be overcome by spermidine but not by putrescine
or spermine. AdoMetDC overexpression, resulting from
transfection and amplification of an episomal AdoMetDC
construct, abrogated polyamine auxotrophy in the
AdoMetDC-deficient mutants and alleviated the toxic effects
of AbeAdo in wild-type parasites [24]. The stability of
L. donovani AdoMetDC (t1/2 > 24 h) and the lability of the
human enzyme suggests that irreversible inhibitors of
AdoMetDC may also be able to eradicate leishmanial
infections, depending upon the precise polyamine content
of the intracellular milieu in which the parasites reside. Since
spermidine synthase is a stable enzyme in both L. donovani
and human cells [25], inhibitors of both enzymes are likely
to affect parasite and host cells similarly, unless structural
differences can be exploited to selectively target the parasite
enzyme.
Malaria
Malaria, which is caused by protozoan parasites of the genus
Plasmodium, affects up to 500 million individuals and kills
over 1 million people every year, mainly in sub-Saharan
Africa. These parasites require two hosts : the Anopheles
mosquito for the sexual reproductive stages, and humans for
the asexual reproductive stages. The infectious sporozoites
are injected by the mosquito into blood vessels in the skin.
They invade hepatocytes and begin the exoerythrocytic cycle,
which generates thousands of merozoites (schizogony). After
rupture of the hepatocyte, the merozoites enter the bloodstream and begin the erythrocytic cycle by invading red blood
cells, forming trophozoites. Replication in the erythrocyte
(schizogony) culminates in rupture and release of a small
number of merozoites, which initiate another cycle of replication by infecting other erythrocytes.
The life cycle of Plasmodium can be interrupted at
several stages by treatment with ODC inhibitors. When
administered to the mosquito vector, DFMO inhibits the
sporogonous cycle of Plasmodium berghei, and mice bitten
by the mosquitoes do not contract malaria [26]. DFMO also
blocks exoerythrocytic schizogony and limits erythrocytic
schizogony of P. berghei in mice [27]. Several ODC inhibitors
inhibit erythrocytic schizogony of Plasmodium falciparum
in vitro. In human P. falciparum-infected erythrocytes,
DFMO inhibits growth and maturation of the intracellular
parasite at the trophozoite stage [28]. Irreversible inhibition
of AdoMetDC with AbeAdo causes similar effects against
chloroquine-resistant strains of P. falciparum [29].
C 2003
Biochemical Society
When the polyamine biosynthetic enzymes from P. falciparum, the deadliest malaria parasite, were studied at the
molecular level, it was demonstrated that the ODC and
AdoMetDC enzymes were components of a bifunctional
protein (Figure 1D) [30,31]. The N-terminal region of
the newly synthesized protein contains an AdoMetDC
proenzyme, which cleaves itself to produce an active enzyme
with a catalytically essential pyruvoyl residue at the new
N-terminus. The AdoMet domain is connected to the
C-terminal ODC domain through a hinge domain. The
mature enzyme is a heterotetrameric complex, containing two
sets of the cleavage products. The significance of the bifunctional nature of this protein remains obscure, but the unique
organization of AdoMetDC and ODC may offer the possibility of therapeutic intervention, particularly because of
regulatory differences between the enzymes of the parasite
and the human host cells [32].
Conclusions
Analyses of knockout strains of T. b. brucei and L. donovani
lacking ODC, AdoMetDC or spermidine synthase clearly
show that these parasites cannot survive unless they can access
sufficient amounts of exogenous putrescine or spermidine.
Wild-type T. cruzi, which are naturally ODC-deficient,
also depend on exogenous putrescine or spermidine for
their survival. Trypanocidal activities of inhibitors of the
polyamine biosynthetic enzymes further underscore the
relevance of this pathway as a therapeutic target. As the threedimensional structures for these enzymes become known it
may be possible to design structure-based inhibitors that will
selectively kill the parasites while exerting minimal or at least
tolerable effects on the parasite-infected patient. However,
intracellular parasites may not be amenable to drug-mediated
polyamine depletion, unless polyamine-uptake mechanisms
can be blocked.
The authors’ laboratories are supported by grants from the J.C. Kempe
Memorial Foundation, the Royal Physiographic Society in Lund,
the Swedish Natural Science Research Council, the Royal Swedish
Academy of Sciences and the National Institute of Allergy and
Infectious Disease (AI10096 and AI41622) at the National Institutes
of Health (Bethesda, MD, U.S.A.).
References
1 Bacchi, C.J., Nathan, H.C., Hutner, S.H., McCann, P.P. and Sjoerdsma, A.
(1980) Science 210, 332–334
2 Van Nieuwenhove, S., Schechter, P.J., Declercq, J., Boné, G., Burke, J. and
Sjoerdsma, A. (1985) Trans. R. Soc. Trop. Med. Hyg. 79, 692–698
3 Cohen, S.S. (1998) A Guide to the Polyamines, Oxford University Press,
New York
4 Phillips, M.A., Coffino, P. and Wang, C.C. (1987) J. Biol. Chem. 262,
8721–8727
5 Svensson, F., Ceriani, C., Wallström, E.L., Kockum, I., Algranati, I.D.,
Heby, O. and Persson, L. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 397–402
6 González, N.S., Ceriani, C. and Algranati, I.D. (1992) Biochem. Biophys.
Res. Commun. 188, 120–128
Polyamines and Their Role in Human Disease
7 Fairlamb, A.H. and Le Quesne, S.A. (1997) in Trypanosomiasis and
Leishmaniasis (Hide, G., Mottram, J.C., Coombs, G.H. and Holmes, P.H.,
eds.), pp. 149–161, CAB International, Oxon, UK
8 Li, F., Hua, S.-b., Wang, C.C. and Gottesdiener, K.M. (1996) Mol. Biochem.
Parasitol. 78, 227–236
9 Li, F., Hua, S.-b., Wang, C.C. and Gottesdiener, K.M. (1996) Exp. Parasitol.
88, 255–257
10 Byers, T.L., Bush, T.L., McCann, P.P. and Bitonti, A.J. (1991) Biochem. J.
274, 527–533
11 Bacchi, C.J., Nathan, H.C., Yarlett, N., Goldberg, B., McCann, P.P.,
Bitonti, A.J. and Sjoerdsma, A. (1992) Antimicrob. Agents Chemother.
36, 2736–2740
◦
12 Persson, K., A slund, L., Grahn, B., Hanke, J. and Heby, O. (1998)
Biochem. J. 333, 527–537
13 Kinch, L.N., Scott, J.R., Ullman, B. and Phillips, M.A. (1999) Mol. Biochem.
Parasitol. 101, 1–11
14 Majumder, S., Wirth, J.J., Bitonti, A.J., McCann, P.P. and Kierszenbaum, F.
(1992) J. Parasitol. 78, 371–374
15 Carrillo, C., Cejas, S., González, N.S. and Algranati, I.D. (1999) FEBS Lett.
454, 192–196
16 Carrillo, C., Cejas, S., Cortes, M., Ceriani, C., Huber, A., González, N.S. and
Algranati, I.D. (2000) Biochem. Biophys. Res. Commun. 279, 663–668
17 Brun, R., Bühler, Y., Sandmeier, U., Kaminsky, R., Bacchi, C.J., Rattendi, D.,
Lane, S., Croft, S.L., Snowdon, D., Yardley, V., Caravatti, G., Frei, J.,
Stanek, J. and Mett, H. (1996) Antimicrob. Agents Chemother. 40,
1442–1447
18 Ekstrom, J.L., Mathews, I.I., Stanley, B.A., Pegg, A.E. and Ealick, S.E.
(1999) Structure Fold. Des. 7, 583–595
19 Oza, S.L., Tetaud, E., Ariyanayagam, M.R., Warnon, S.S. and Fairlamb, A.H.
(2002) J. Biol. Chem. 277, 35853–35861
20 Kaur, K., Emmett, K., McCann, P.P., Sjoerdsma, A. and Ullman, B. (1986)
J. Protozool. 33, 518–521
21 Reguera, R.M., Fouce, R.B., Cubria, J.C., Bujidos, M.L. and Ordonez, D.
(1995) Life. Sci. 56, 223–230
22 Hanson, S., Adelman, J. and Ullman, B. (1992) J. Biol. Chem. 267,
2350–2359
23 Jiang, Y., Roberts, S.C., Jardim, A., Carter, N.S., Shih, S., Ariyanayagam, M.,
Fairlamb, A.H. and Ullman, B. (1999) J. Biol. Chem. 274, 3781–3788
24 Roberts, S.C., Scott, J., Gasteier, J.E., Jiang, Y., Brooks, B., Jardim, A.,
Carter, N.S., Heby, O. and Ullman, B. (2002) J. Biol. Chem. 277,
5902–5909
25 Roberts, S.C., Jiang, Y., Jardim, A., Carter, N.S., Heby, O. and Ullman, B.
(2001) Mol. Biochem. Parasitol. 115, 217–226
26 Gillet, J.M., Charlier, J., Boné, G. and Mulamba, P.L. (1983) Exp. Parasitol.
56, 190–193
27 Bitonti, A.J., McCann, P.P. and Sjoerdsma, A. (1987) Exp. Parasitol. 64,
237–243
28 Whaun, J.M. and Brown, N.D. (1985) J. Pharmacol. Exp. Ther. 233,
507–511
29 Wright, P.S., Byers, T.L., Cross-Doersen, D.E., McCann, P.P. and Bitonti, A.J.
(1991) Biochem. Pharmacol. 41, 1713–1718
30 Müller, S., Da’dara, A., Lüersen, K., Wrenger, C., Das Gupta, R.,
Madhubala, R. and Walter, R.D. (2000) J. Biol. Chem. 275, 8097–8102
31 Wrenger, C., Lüersen, K., Krause, T., Müller, S. and Walter, R.D. (2001)
J. Biol. Chem. 276, 29651–29656
32 Müller, S., Coombs, G.H. and Walter, R.D. (2001) Trends Parasitol. 17,
242–249
Received 25 November 2002
C 2003
Biochemical Society
419