18 Parasitology Today, vol. 4, no. I, 1988

Polyamine Metabolism of Filaria and Allied Parasites

R.D, WaLter

Putrescine and the polyamines spermidine and spermine occur both in prokaroytes and in eukaryotes where they seem intimately involved in regulatory processes of cellular growth and differentiation. They seem to play an important role related to the biosynthesis of nucleic acids and proteins, although at the molecular level their precise function remains unclear. In general, prokaryotes utilize putrescine and spermidine while eukaryotes tend to have higher concentrations of spermidine and spermine compared to putrescinel-3.

D/fferences in polyamine metabolism between parasites and their hosts suggest several potential targets for chemotherapeutic attack As Roll Walter discusses here, such approaches have already been exploited for African trypanosomes and also offer some leads for the chemotherapy of helminth infections.

During recent years, the polyamine metabolism of parasites has attracted increasing interest as a lead for chemotherapeutic development. In Afri- can trypanosomes, the initial enzyme in the polyamine biosynthetic pathway- ornithine decarboxylase - has already been exploited as a target for chemotherapy of sleeping sickness by using difluoromethylornithine (DFMO)- an irreversible inhibitor of this enzyme. Indeed, DFMO has now been shown effective in vitro and in vivo against various trypanosomes, malaria and leishmania ¢-I°. In addition, the older trypanocidal drugs berenil and pentamidine were recently shown to interact with S-adenosyl- methionine decarboxylase - another reg- ulatory enzyme involved in the synthesis of polyamines I 1,12 Inhibition of S-adenosyl- methionine decarboxylase may therefore play a role in the therapeutic efficacy of both compounds for the treatment of protozoal infections.

Trypanosomes are particularly vulner- able to a blockade of polyamine biosyn- thesis, since they depend on a novel and unique trypanothione system for the removal of toxic hydrogen peroxide and for the maintenance of intracellular thiols in the redox state 13. Analogous to glutathione peroxidase and reduct~e from other organisms, trypanosomes pos- sess a trypanothione peroxidase and reductase cycle using trypanothione - a spermidine-glutathione conjugate - as cofactorl3-16.

Polyamine Metabolism in Helminths

The polyamine metabolism of hel- minths has attracted less interest, although in filarial worms some peculiarities have

recently been reported which merit con- sideration as chemotherapeutic targets. For example, Onchocerca volvulus appears to lack ornithine and arginine decar- boxylase ~7. This suggests that both path- ways for the synthesis of putrescine are absent - decarboxylation of ornithine as well as decarboxylation of arginine to ag- matine, subsequently converted to putres- cine and urea. So far no detectable or- nithine decarboxylase activity has been found in preparations ofO. volvulus and Di- rof~laria irnmitis, nor in Ascaris suum or the lung fluke Paragonimus uterobilateralis. In addition, maintenance of O. volvulus and D. immitis in culture medium supplemented with radiolabelled ornithine and DFMO led neither to putrescine synthesis as mea- sured by l4CO2 production nor to changes in the distribution pattern of polyamines. Also, treatment of filarial infected animals by DFMO showed no effect on survival and production of microfilariae (FL Walter, unpublished). An earlier report on orni- thine degradation in A. suun by ornithine decarboxylase could not be confirmed 18.

Degradation of ornithine in A. suum and P. uterobilateralis appears to pro- ceed via transamination by ornithine: 2- oxo-acid aminotransferase to glutamate semialdehyde, subsequently converted to praline or catabolized via glutamic acid. In viva treatment of lung flukes with 4-aminohex-5-ynoic acid 19 or preincu- bation of extracts from A. suum and P. uterobilateralis with this irreversible inhibitor of ornithine aminotransferase completely abolished the degradation of ornithine (R. Walter, unpublished).

Uptake and Interconversion of Polyamines

Since they are unable to synthesize

putrescine, filarial worms depend on their host for a supply of polyamines. Thus, uptake mechanisms and enzymes which are involved in the interconver- sion of polyamines- insofar as they show parasite-specific peculiarities - might be exploited as targets for chemothera- peutic attack.

Spermidine and spermine, and trace amounts of putrescine, have been de- tected from various helminths 2°. In filarial worms, spermine was found as the main component, followed by spermidine, whereas putrescine levels were much lower t7. This distribution pattern resem- bles that in mammalian tissues, whereas in lower eukaryotes such as culture forms of Leishmania donovani, Crithidia facsiculata, Trypanosoma mega and T. brucei, spermidine could either not be detected or, as in Plasmodium falciparum, occurs in trace amounts 2t-23.

Based on results from experiments with polyamine uptake, interconversion and excretion, a scheme has been suggested for the polyamine metabolism in fllarial worms (Fig. I) 17. However, only some of the enzymes involved in the synthetic and reverse pathway have yet been demonstrated. Adults ofO. volvulus and D. immitis were maintained in culture medium supplemented with labelled put- rescine, spermidine and spermine. When putrescine is supplied to the medium it is taken up by the parasites and subsequently degraded to N-acetyl- putrescine or used for synthesis of spermidine and spermine. Uptake of spermidine and spermine results in the occurrence of spermine, spermidine, putrescine and N-acetylated derivatives within the worms. Degradation products from added putrescine, spermidine and spermine excreted into the spent med- ium were mainly N-acetylputrescine and putrescine. DFMO added to the culture medium did not lead to any changes of the polyamine distribution pattern in O. volvulus but the addition of methylglyoxal bis(guanylhydrazone, MGBG), a tight binding inhibitor of S-adenosyl- methionine decarboxylase, resulted in reduced synthesis of spermidine and spermine. These results are in agree- ment with the existence of two path- ways for synthesis and interconversion of polyamines in filarial worms.

(~)1988, Elsevier PublLcat~ons~ Cambridge 0q 694758/88150200

Parasitology Today, voL 4, no. I, 1988 19

Synthesis of Spermidine and Spermine

In filariae, spermidine and spermine synthesis begins with putrescine rather than ornithine as in trypanosomes. The rate-limiting enzyme within this pathway is the S-adenosylmethionine decar- boxylase, which provides the amino- propyl group for the reactions catalysed by spermidine and spermine synthase. S- adenosylmethionine decarboxylase has been demonstrated in O. volvulus and A. suum 24. The activity of this enzyme from nematodes is stimulated by putrescine, This activation, which has been reported from S-adenosylmethionine decarboxyl- ase of mammalian tissue, yeast, and pro- tozoa, has been proposed as an import- ant regulatory mechanism which links the supply of decarboxylated S-adeno- sylmethionine to that of putrescine - favouring the synthesis of spermidine and spermine 2s. The much higher con- centrations of spermidine and spermine compared to that of putrescine in O. volvulus and mammalian tissue are in agreement with this hypothesis. The S- adenosylmethionine decarboxylase from nematodes seems to resemble the iso- functional enzyme from mammals, whereas the enzyme from bacteria depends on Mg 2+ for activity while those from Tetrahymena pyriformis and Physarum polycepholum are, not affected by cations 2s.

With respect to inhibition of S- adenosylmethionine decarboxylase by berenil, pentamidine and methylglyoxal his (guanylhydrazone), ther~ are few dif- ferences between the enzyme from nematodes and that from host tissues, with one exception. The type of inhibi- tion by berenil was reported to be irreversible for the S-adenosylmeth- ionine decarboxylase from rat liver II and T. brucei 12, whereas in the case of A. suum 24 and Acanthamoeba culbertsoni 26 the inhibition is reversible. In addition, there is some evidence for structural dif- ferences between the enzyme from mammals and nematodes. The molecu- lar weight of the S-adenosylmethionine decarboxylase from A. suum was deter- mined as 220 000 daltons compared with 65-70 000 daltons for the mamma- lian enzyme 24,25.

There is evidence for the occurrence of aminopropyl-transferase activity in O. volvulus and D. immitis by experiments which demonstrated uptake of putres- cine and its conversion to spermidine and spermine 17. However, spermidine and spermine synthases have not yet been isolated and characterized from filarial worms.

~ r A c e t y l CoA ~ > .:> dc SAM I \

4 NAc Spd • .-

• , ,-. . . . I ~ A c e t y l CoA I / - I perm alne I / =

?CSMTM pIT2 143" I NAcSpm I /=

Fig. I. Proposed scheme for uptake, interconversion and excretion of polyamines in filarial worms. Enzymes: I - spermidine synthase; 2 - spermine synthase; 3 - spermine/spermidine-N I-acetyl- transferase; 4 - polyamine oxidase; 5 - putrescine-N I-acetyltransferase. Abbreviations: dcSAM - decarboxylated S-adenosylmethionine; M TA - 5 I-methylthioadenosine; NAc Put- N I-acetylput- rescine; NAc Spd- Ni-acetylspermidine; NAc Spin - N I-acetylspermine.

Degradation of Polyamines

A functional reverse pathway from spermine via spermidine and N-acety- lated derivatives to putrescine, by which putrescine can be reused for the syn- thesis of spermidine and spermine (as re- ported from brain tissues 27) has been proposed for O. volvulus and D. immitis t7,

Addition of labelled spermine to fila- rial worms maintained in culture medium resulted in uptake and conversion to N- acetyl spermine, spermidine, N-acetyl spermidine, putrescine and N-acetyl put- rescine. Spermine and spermidine prob- ably represent the main supply by the host because the level of putrescine in mammalian tissues is much lower. By the reverse pathway, the filarial worms con- trol and reduce the level of higher polyamines and supply putrescine for re- cycling via the synthetic pathway. In addi- tion, there is some evidence that filarial worms use oxidation and probably acetylation to putrescine and N-acetyl putrescine, for breakdown and excre- tion of excessive polyamines. The sper- mine/spermidine N-acetyltransferase is known from mammalian tissues as the rate-limiting step in the reverse pathway; regulation of its activity depends, as in the

case of ornithine decarboxylase and S- adenosylmethionine decarboxylase, on a short half-life and synthesis of the en- zyme. Activities of spermine/spermidine N-acetyltransferase as well as of putres- cine N-acetyltransferase have been iden- tified in A. suum (R.M. Wittich and R. Walter, unpublished). Polyamine oxidase activity has also been shown in A. suum (S. M011er and R. Walter, unpub- lished) but the enzyme has not yet been isolated and characterized. Excretion of N-acetyl putrescine as the main product of polyamine catabolism in filaria, as well as the high activity of putrescine N- acetyltransferase in A. suum, demon- strate the importance of the reverse pathway for degradation of polyamines.

Polyamine Inhibition?

Despite recent research, there re- main many gaps in our understanding of the polyamine metabolism in filarial worms and other helminths which have to be filled before recommendations can be given for rational approaches to chemotherapy. Firstly, it should be de- monstrated and confirmed for helminths

20 Parasitology Today, vol. 4, no. I, 1988

that blockade and disturbance of polyamine synthesis and distribution can lead to death of the worms. DFMO and MGBG, both of which are potent in- hibitors of polyamine synthesis in mam- malian cells, do not significantly affect fila- rial worms. The absence of a biological effect by DFMO is due to the lack of its target ornithine decarboxylase; that of MGBG may be due to a less important role of the synthetic pathway compared to the interconversion pathway for polyamine synthesis and distribution in nematodes, These results also cast doubt on the signification of S- adenosyl- methionine decarboxylase as a target for design and development of enzyme in- hibitors as antifllarial drugs. On the other hand, b e r e n i l - known as a potent in- hibitor of S-adenosylmethionine decar- boxylase as well as of enzymes involved in the interconversion pathway - does affect the viability of Brugia pahangi (R. Walter, unpublished),

Considering the absolute dependence of filarial worms on uptake of polyamines and the obviously decisive role of the interconversion pathway for maintaining adequate and crucial levels of spermine, spermidine and putrescine, the most rewarding avenues for research would appear to be on uptake mechanisms as well as on polyamine oxidase and spermine/spermidine N- acetyltransferase. In addition, the role of putrescine Noacetyltransferase in the excretion of excessive and degraded polyamines as well as some novel fea- tures of this enzyme, may also offer a possible target for chemotherapeutic attack,

References I Tabor, C,W. and Tabor, H. (1984)Annu. Rev.

Biochem. 53, 749-790 2 Grillo, M.A. (I 985) Int.]. Biochem. 17,943-948 3 Pegg, A.E. (1986) Biochem.]. 234, 249-262 4 Bacchi, C.J., Nathan, H.N., Huttner, SN.,

McCann, P.P. and Sjoerdsma, A. (1980)Science 210, 332-334

5 Bacchi, C.J. et aL (1983) Mol. Biochem. Parasitol. 7, 209-224

6 Sjoerdsma, A. and Schechter, P.J. (I 984) Clin. Pharmacol. Ther. 35,287-300

7 Hollingdale, M.R., McCann, P.P. and Sjoerdsma, A.(1985)Exp. Parasitol, 60, II I- I 17

8 Whaun, J. and Brown, N.D. (1985) Pharmacol. Exp. Ther. 233, 507-51 I

9 Giffin, B.F., McCann, P,P., Bitonti, A.J. and Bacchi, C.J. (I 986)]. ProtozoaL 33,238-243

10 Kaur, K,, Emmet, K., McCann, P.P., Sjoerdsma, A. and UIImann, B. (I 986)]. Protazool. 33, 518- 521

II Karvonen, E., Kauppinen, L., Partanen, T. and P6sO, H, (I 985) Biochem.], 23 I, 165-169

12 Bitonti, AJ., Dumont, J.A, and McCann, P.P. (1986) Biachem. J. 237, 685489

13 Fairlamb, A.H., Blackburn, P., Ulrich, P., Chait, B.T. and Cerami, A. (1985) Science 227, 1485- 1487

14 Henderson, G.B. and Fairlamb, A.H. (1987) ParasitoL Today 3, 312-315

15 Henderson, G.B., Fair[amb, A.H. and Cerami, A. (1987) Mol Biochem ParasitoL 24, 39-45

16 Fairlamb, A.H., Henderson, G.B., Bacchi, C.J. and Cerami, A. (1987)Mol. Biochem Parasitol, 24, 185-191

17 Wittich, FLM., Kilian, H.D. and Walter, R.D. (1987) Mol. Biochem. Parasitol. 24, 155-162

18 Walter, R.D,, Ossikovski, E. and K~nigk, E. (I 984) Proc. Int. Canf on Polyamines, Budapest, p.82 (Abstract)

19 Jung, M.J. and Seller, N. (1978)J. Biol. Chem 253,7431-7438

20 Srivastava, D.K., Roy, T.K. and Shukla, O.P. (1980) Ind.J. ParasitoL 4, 187-189

21 Bacchi, C.J., Lipschik, G.Y. and Nathan, H.C. ( 1977)J. Bacterial. 13 I, 65746 I

22 Morrow, C.D., Flory, B. and Krassner, S.M.

Origin of Falciparum

Sir- May I reply briefly to Professor Bruce-Chwatt's comments I on my original note about the origin of the name falciparum?

Welch, in the article on Malaria published in 1897, commented as follows 2.

"The name Haematozoon falciforme suggested by Antolisei and Angelini is objectionable, as it implies that the shape is always falciform, and is applicable only to the crescent forms. The adjective 'falciparum' (falx, 'sickle', parire, 'to bring forth', 'to produce'), on the other hand, indicates that the property of forming crescents is a

Industrial Approaches to Tropical Diseases

Sir- The recent decision by the Wellcome Foundation to limit its future support for research on the chemotherapy of tropical diseases to malaria effectively brings to an end an era which began with the establishment of The Wellcome Tropical Research Laboratories in 1913. The decision also focuses attention on the precarious relationship between the pharmaceutical industry and tropical diseases,

The basis of the problem is not new. Most people requiring treatment are often too poor to afford the drugs - as are their governments - yet profit is the only catalyst for the pharmaceutical industry to create drugs and costs of up to £1 O0 million per drug are often quoted, (This figure includes all costs for drug failures and overheads; a more relevant figure for the research and development for a single drug is probably £5-I 0 million.) Moreover, the short patent life to recoup costs and make a profit is often eroded by 'pirate' manufacture of the products by drug companies in other parts of the world - exacerbating the move of Western multinationals away from tropical diseases.

(1980) Camp. Biochem. Physiol., Ser. B 66, 307- 311

23 Rathaur, S. and Walter, FLD. (1987) ~xp Parasitol. 63, 227-232

24 Rathaur, S., Wittich, FLM. and Walter, FLD. (1987) Trap, Meal. ParasitoL 38 (Suppl.), 71-72

25 Pegg, A.E. (I 984) Cell Biochem. Function 213, 11-15

26 Gupta, S., Shukta, O,P. and Walter, FLD. (I 987) Mol. Biochem ParasitoL 23,247-252

27 Seller, N. and Bolkenius, F.N. (1985) Neurochem. Res. I 0, 529-544

RolfWalter is at the Abteilung Biochemie, Bern- hard Nocht Institut fQr Schiffs und Tropenkran- kheiten, D-2000 Hamburg 4, FR Germany.

distinctive character of the organism .. / '

Thus Welch derived the term from the verb pario, parire, not the adjective par. (Pareo was a misprint.) To qualify the Greek neuter noun Haematozoon requires a Latin gerundive, falcipariendum, meaning 'sickle-producing' or 'sickle-bearing'. Perhaps Welch abbreviated for convenience, but we shall probably never know.

A.J. Knell Wellcome Tropical Institute 200 Euston Road London NW I 2BQ, UK.

References I Bruce-Chwatt, L J. (1987) Parasitology Today 3,

p, 252 2 Welch, W.H. (1897) in A System of Practical

Medicine by American Authors (ed. Loomis & Thompson) Lea Bros & Co., New York

The complexity of parasites, difficulties of maintenance in laboratories, and poor in vitro and in viva models, have in the past contributed to the high costs in this area. However, there are signs that the industry is concerned about the situation, and the level of involvement and provision of concessionary prices have been discussed 1,2. Some companies are currently providing active support for the development of specific drugs - f o r example, Merrell Dow with difluoromethylornithine (DFMO) for African human trypanosomiasis 3 and Merck, Sharpe and Dohme with ivermectin for onchocerciasis 4. The industry has also had a part in the development of antischistosomal drugs, although several of the drugs were originally developed for the profitable veterinary market and the 1979 figure for the worldwide funding of research on schistosomiasis was only US$0.04 per infected person s . But despite these highlights, the overriding trend remains that more drug companies are reducing their commitment to tropical diseases.

The problems are not insoluble but commercial difficulties remain because of the necessary involvement of the pharmaceutical industry, which alone possesses the expertise and experience for drug development. Malaria retains the possibility of profit through the prophylactic market,