Ribonucleotide Reductase: A New Target for Antiparasite Therapies

Reviews 20 Singh, S.B. et al. (1996) Apicidins: novel cyclic tetrapeptides as coccidiostats and antimalarial agents from Fusarium pallidoroseum. Tetrahedron Lett. 37, 8077–8080 21 Keithly, J.S. et al. (1997) Polyamine biosynthesis in Cryptosporidium parvum and its implications for chemotherapy. Mol. Biochem. Parasitol. 88, 35–42 22 Rehg, J.E. (1991) Anticryptosporidial activity is associated with specific sulphonamides in immunosuppressed rats. J. Parasitol. 77, 238–240 23 Vásquez, J.R. et al. (1996) Potential antifolate resistance determinants and genotypic variation in the bifunctional dihydrofolate reductase-thymidylate synthase gene from human and bovine isolates of Cryptosporidium parvum. Mol. Biochem. Parasitol. 79, 153–165 24 Georgiev, V.S. (1994) Management of toxoplasmosis. Drugs 48, 179–188 25 Rohlman, V.C. (1993) Therapy with atovaquone for Cryptosporidium parvum infection in neonatal severe combined immunodeficiency mice. J. Infect. Dis. 168, 258–260 26 McFadden, G.I. et al. (1996) Plastid in human parasites. Nature 381, 482 27 Kohler, S. et al. (1997) A plastid of probable green algal origin in apicomplexan parasites. Science 275, 1485–1489 28 Soldatl, D. (1999) The apicoplast as a potential therapeutic target in Toxoplasma and other apicoplexan parasites. Parasitol. Today 15, 5–7 29 Roos, D.S. (1999) The apicoplast as a potential therapeutic target in Toxoplasma and other apicoplexan parasites: some additional thoughts. Parasitol. Today 15, 41 30 Wilson, R.J.M. and Williamson, D.H. (1997) Extrachromosomal DNA in the Apicomplexa. Microbiol. Mol. Biol. Rev. 61, 1–16 31 Wilson, R.J.M. (1998) in Evolutionary Relationships among Protozoa (Coombs, G.H. et al., eds), pp 293–303, Kluwer 32 Waller, R.F. et al. (1998) Nuclear-encoded proteins target to the plastid in Toxoplasma gondii and Plasmodium falciparum. Proc. Natl. Acad. Sci. U. S. A. 95, 12352–12357 33 Beckers, C.J.M. et al. (1995) Inhibition of cytoplasmic and organellar protein synthesis in Toxoplasma gondii: implications for the target of macrolide antibiotics. J. Clin. Invest. 95, 367–376 34 Fichera, M.E., Bhopale, M.K. and Roos, D.S. (1995) In vitro assays elucidate peculiar kinetics of clindamycin action against Toxoplasma gondii. Antimicrob. Agents Chemother. 39, 1530–1537 35 Fichera, M.E. and Roos, D.S. (1997) A plastid organelle as a drug target in apicomplexan parasites. Nature 390, 407–409

36 Hackstein, J.H.P. et al. (1995) Parasitic apicomplexans harbour a chlorophyll a–D1 complex, the potential target for therapeutic triazines. Parasitol. Res. 81, 207–216 37 Roberts, F. et al. (1998) Evidence for the shikimate pathway in apicomplexan parasites. Nature 393, 801–805 38 Keeling, P.J. et al. (1999) Shikimate pathway in apicomplexan pathways. Nature 397, 219–220 39 Roberts, C.W. et al. (1999) Shikimate pathway in apicomplexan pathways: reply. Nature 397, 220 40 Kishore, G.M. and Shah, D.M. (1988) Amino acid biosynthesis inhibitors as herbicides. Annu. Rev. Biochem. 57, 627–663 41 Davies, G.M. et al. (1994) (6S)-6-Fluoroshikimic acid, an antibacterial agent acting on the aromatic biosynthetic pathway. Antimicrob. Agents Chemother. 38, 403–406 42 Gonzalez-Bello, C. et al. (1998) Synthesis of 2-bromo- and 2-fluoro3-dehydroshikimic acids and 2-bromo- and 2-fluoroshikimic acids using synthetic and enzymatic approaches. J. Org. Chem. 63, 1591–1597 43 Pfefferkorn, E.R., Eckel, M.E. and McAdams, E. (1988) Toxoplasma gondii: in vivo and in vitro studies of a mutant resistant to arprinocid-N-oxide. Exp. Parasitol. 65, 282–289 44 Wang, C.C., Simashkevich, P.M. and Stotish, R.L. (1979) Mode of anticoccidial action of arprinocid. Biochem. Pharmacol. 28, 2241–2248 45 Doyle, P.S., Kanaani, J. and Wang, C.C. (1998) Hypoxanthine, guanine, xanthine phosphoribosyltransferase activity in Cryptosporidium parvum. Exp. Parasitol. 89, 9–15 46 Krauth-Siegel, R.L and Coombs, G.H. Enzymes of parasite thiol metabolism as drug targets. Parasitol. Today (in press) 47 Fayer, R. and Fetterer, R. (1995). Activity of benzimidazoles against cryptosporidiosis in neonatal BALB/c mice. J. Parasitol. 81, 794–795 48 Katiyar, S.K. et al. (1994) Antiprotozoal activities of benzimidazoles and correlations with b-tubulin sequence. Antimicrob. Agents Chemother. 38, 2086–2090 49 Arrowood, M.J. et al. (1996) In vitro anticryptosporidial activity of dinitroaniline herbicides. FEMS Microbiol. Lett. 136, 245–249 50 Benbow, J.W. et al. (1998) Synthesis and evaluation of dinitroanilines for treatment of cryptosporidiosis. Antimicrob. Agents Chemother. 42, 339–343 51 Perkins, M.E., Wu, T.W. and Le Blancq, S.M. (1998) Cyclosporin analogs inhibit in vitro growth of Cryptosporidium parvum. Antimicrob. Agents Chemother. 42, 843–848 52 Tzipori, S. (1998) Cryptosporidiosis: laboratory investigations and chemotherapy. Adv. Parasitol. 40, 187–221

Focus

Ribonucleotide Reductase: A New Target for Antiparasite Therapies G.M. Ingram and J.H. Kinnaird New treatments are required urgently for the control of parasitic protozoan diseases of humans and animals. One approach is the development of subunit vaccines; the other focuses on identifying and exploiting specific differences in essential functions between the host and parasite. One enzyme currently attracting attention is ribonucleotide reductase, an essential component in the biosynthesis of DNA. In this article, Geoffrey Ingram and Jane Kinnaird examine differences between the host and parasite enzymes and assess possible means of therapeutic intervention.

Geoffrey Ingram is at the Department of Disease Control, Graduate School of Veterinary Medicine, University of Hokkaido, Sapporo 060-0818, Japan. Jane Kinnaird is at the Department of Veterinary Parasitology, University of Glasgow, Bearsden Road, Glasgow, UK G61 1QH. Tel: +81 11 706 5216, Fax: +81 11 709 7198, e-mail: [email protected] 338

Current chemotherapeutics used in the treatment of protozoan diseases are undermined by the emergence of parasite and vector resistance to drugs and by the high toxicity of some drugs1. Several new targets for intervention have been identified and one promising enzyme is ribonucleotide reductase (RNR). By studying the differences in the biochemistry and protein structure of parasitic protozoa, rational targets for chemotherapy can be selected. However, Wang2 has sounded a note of caution about the number of parasitic enzymes identified as possible chemotherapeutic targets and has suggested that candidates should be assessed carefully for their suitability as targets before commitment to longterm studies on the development of specific inhibitors. RNR has now been isolated from a number of protozoan parasites and detailed studies have suggested that it may hold considerable promise as a therapeutic target. Here, we outline reasons why we believe that

0169-4758/99/$ – see front matter © 1999 Elsevier Science. All rights reserved. PII: S0169-4758(99)01478-7

Parasitology Today, vol. 15, no. 8, 1999

Focus RNR stands a good chance of eventually making the transition from laboratory focus to field target.

dGTP +

gene, the second R2 gene can be induced by DNA damage but the two R2 genes have non-overlapping functions and cannot substitute for one another even when overexpressed11. However, there is no evidence for more than one gene for each RNR subunit in higher eukaryotes.

ADP dADP The activity and regulation of RNR RNR, which has been shown by dTTP mutation to be essential for cell vi+ ability in yeast3, plays a central role in the biosynthesis of DNA, convertParasite RNR protein GDP dGDP ing ribonucleoside diphosphates into Genes encoding RNR subunits the corresponding deoxyribonucleofrom six different protozoan parasites ATP/dATP* tides (dNTPs) by the reduction of have been isolated and characterized carbon-2 on the ribose ring. dNTPs to varying degrees. These include + are almost exclusively used in the bioTheileria annulata R1 (G. Ingram and CDP dCDP synthesis of DNA and hence the enJ. Kinnaird, unpublished), Plasmodium zyme is subject to intricate regulatory falciparum R1 and R2 (Refs 12,13), mechanisms4. The mammalian enToxoplasma gondii R1 and R2 (K. Kim ATP/dATP zyme has an unusual regulatory pathet al., Abstract*), Leishmania mexicana + way in that both the activity and subR2 (Ref. 14), Cryptosporidium parvum strate specificity are regulated by the R1 (R. Balakrishnan et al., unpubUDP dUDP binding of effector molecules. The lished; submitted EMBL database, Fig. 1. Regulation of ribonucleotide reactivity site affects overall enzyme Accession Number AF043243) and ductase. This figure summarizes the activity and binds either ATP, which Trypanosoma brucei R1 and R2 (Ref. allosteric interactions controlling the refurther activates the enzyme depend15). In all the protozoan R1 subunits duction of ribonucleoside diphosphates. ing on the effector bound to the specithe active site cysteines are conserved, *NB dATP is a positive effector at high ficity site, or dATP, which inactivates as are the electron transfer cysteines concentrations and a negative effector it. The affinity of dATP for the speciand the two tyrosine residues inat lower concentrations. ficity site is greater than its affinity for volved in the proposed electron transthe activity site; thus, at low concenport pathway7 (Fig. 2a). The degree trations dATP is a positive effector for CDP reduction but of conservation of these functionally important residues at high concentrations it exerts a negative effect, owing shows that the parasitic protozoan RNR enzymes use to binding to the activity site. Substrate specificity can be the same free radical chemistry to reduce the ribose ring altered depending upon the effector molecule bound as is seen in higher eukaryotic enzymes. In general, there (ATP, dATP, dTTP or dGTP). If ATP or dATP is bound, is good but not absolute conservation of the surrounding the reduction of UDP and CDP is stimulated; however, amino acid residues between the protozoa and higher if dTTP or dGTP is bound, the reduction of GDP and ADP eukaryotes. However, there are also some noticeable is favoured (Fig. 1). This elaborate mechanism ensures a differences in the amino acid sequences between them, balanced supply of all four dNTPs for DNA synthesis. particularly towards the C-terminus in the R1 subunit The chemical reduction of a ribonucleotide must be sequence, where there are insertions present in the P. falciparum, T. annulata and T. brucei subunits, although initiated by an organic free radical and different means the functional significance of these regions is unknown. of achieving this have evolved, with at least three difThe R2 subunit has been less well characterized in ferent classes of RNR known to date5. The evolution of the different classes of RNR may have been driven by the protozoan parasites. Residues that are proposed to form emergence of atmospheric oxygen. All three classes use the electron transport pathway are conserved, as are the same free radical chemistry, albeit employing a difthe iron ligands, the free radical generating residue and ferent mechanism to generate the free radical (reviewed those radicals forming a hydrophobic pocket around in Ref. 6). Virtually all eukaryotes possess a class I RNR the radical. All protozoan R2 subunits studied to date enzyme, and this review will consider only members of are about 50 residues shorter in the N-terminal regions class I. This class is characterized by heterodimeric enthan those of higher eukaryotic R2s. The importance of zymes of the a2b2 type. The large R1 (a) subunit, encoded this is not clear although this region does not have a by RNR1, has one substrate-binding site with redox active functional role16. thiols for ribose reduction and two separate allosteric sites. The small R2 (b) subunit, encoded by RNR2, has Parasite RNR regulation a tyrosyl radical generated by a ferric iron [Fe3+] centre. Hofer and colleagues15 showed that the T. brucei RNR The radical is believed to link with the R1 active site via allosteric regulation of CDP reduction differs from that of a hydrogen-bonded electron transport chain7, although the mammalian enzymes. Here, dATP cannot inhibit the crystallographic structure of the holoenzyme R1–R2 CDP reduction, even at saturating concentrations, where complex remains to be characterized. Two R1 genes have all activity and specificity sites would be occupied, a been identified in Saccharomyces cerevisiae, RNR1 and situation found also in RNR from the Herpesviridiae RNR3 (Ref. 8). family and Mycobacterium tuberculosis17. Trypanosoma Expression of RNR1 is largely cell cycle regulated, brucei RNR reduces CDP more efficiently in the presbut RNR3 is induced by DNA damage and is under the ence of dTTP or dGTP, whereas in higher eukaryotic control of a different promoter system to RNR1 (Ref. 9). More recently, a second R2 gene has been isolated (RNR4) * Regulation of cell cycle gene expression in Toxoplasma. Woods Hole Molecular encoding another small subunit10,11. Like the RNR3 Parasitology Meeting, Abstract 251, September 1996 Parasitology Today, vol. 15, no. 8, 1999

339

Focus a Block 1 Cys 218 Escherichia coli

TRQFSSCVLI

Theileria annulata

HPQMSSCFLL

Plasmodium falciparum

RPQMSSCFLL

Trypanosoma brucei

FPQMSSCFLV

Cryptosporidium parvum

RPQMSSCFLL

Human

PRQLSSCFLL

Block 2 Cys 439 Escherichia coli

RGSNLCLEIA

Theileria annulata

KSSNLCCEIV

Plasmodium falciparum

KCSNLCCEII

Trypanosoma brucei

KCSNLCTEIV

Cryptosporidium parvum

VSSNLCTEII

Human

KCSNLCCEIV

Block 3 Cys 462 Escherichia coli

GEIALCTLSAFNL

Theileria annulata

DEVAVCNLASVAL

Plasmodium falciparum

DEVAVCNLASIAL

Trypanosoma brucei

DEVAVCNLASIAL

Cryptosporidium parvum

DEVANCNLASIAL

Human

DEVAVCNLASIAL

Block 4 Cys 759 Escherichia coli

CKI*

Theileria annulata

CSS*

Plasmodium falciparum

CGS*

Trypanosoma brucei

CGS*

Cryptosporidium parvum

CMM*

Human

CGS*

Block 5 Tys 730/731 Escherichia coli

GVKT.LYYQNTRDG

Theileria annulata

GVKTGVYYLRTQPA

Plasmodium falciparum

GVKTGAYYLRTQAA

Trypanosoma brucei

GVKTGMYYLRSQAA

Cryptosporidium parvum

GVKTGMYYLRTQSA

Human

GVKTGMYYLRTQAA

b Region implicated in dTTP binding Escherichia coli

294SQGGV.RGG300

Mammals

DQGGNKRPG

Yeast

DQGGNKRPG

Protozoa

DQGGGKRKG

Fig. 2. Regions conserved in thioyl transfer reaction of the large subunit (a). This figure shows the conservation of several cysteine residues known to be involved in the reaction mechanism of the Escherichia coli ribonucleotide reductase and the conservation of the two tyrosine residues in the proposed electron transport pathway. A star denotes a translation stop codon. Numbering refers to the E. coli amino acid sequence. A region implicated in dTTP binding with the replacement residues (indicated in bold) being conserved in all the protozoan R1 subunits sequenced to date is shown in (b). 340

RNRs it is ATP or dATP bound to the specificity site that are the best effectors for CDP reduction. So far, only CDP reduction has been studied and work is now being carried out to determine the regulation of UDP, GDP and ADP reduction15. It is likely that this differential regulation may be found in other protozoan parasites, although this is only speculative at this stage. Evidence for this prediction comes from the conservation of a region, absolutely conserved from yeast to mammals and implicated in dTTP binding (Fig. 2b). Despite the evolutionary divergence between some of the protozoa18, this motif is identical in all parasite R1 sequences and has two non-conservative substitutions compared with other eukaryotes. Further evidence for mechanistic or regulatory differences between T. brucei and mammalian RNRs is shown by experimental mixtures of recombinant mouse and T. brucei R1–R2 subunits, all of which were totally inactive. The fact that parasites undergo repeated cycles of replication and differentiation throughout their life cycles might mean that the traditional style of allosteric control evolved by higher eukaryotes may be unsuitable for a parasitic life style. Even at the basic level of cell cycle control19,20, there are important differences between the parasitic protozoa and their mammalian hosts, which are probably reflected also at the level of DNA replication and metabolism. The P. falciparum genome is extremely AT rich, and it is feasible that differences in allosteric control exist because of this. Indeed, it could be argued that there would need to be some mechanism built into Plasmodium RNR to reduce feedback sensitivity to ATP and TTP, in order to maintain a high output of dATP and dTTP for replication. Future studies of the allosteric regulation of the P. falciparum holoenzyme should be very interesting. Transcription of the P. falciparum RNR subunit genes during the cell cycle also shows a pattern distinct from that of the higher eukaryotes, as the R2 transcript appears earlier and persists for longer than the R1 transcript12,13, whereas in mammals the levels of both transcripts rise in parallel as cells progress through the S phase and then decline as they progress through the G2 and M phases21, with the peak of enzyme activity occurring during the S phase of the cell cycle. This cell cycle-specific activity is regulated by the S phase-specific synthesis and breakdown of the R2 protein, which has a much shorter halflife than the R1 protein22. It has not yet been shown whether RNR transcript levels are related to protein turnover in Plasmodium. In addition, the molecular mechanisms governing this differential regulation of P. falciparum RNR gene expression have not been studied yet. Inhibition strategies for parasite RNRs There are ever-increasing ways to target specific parasite enzymes including protein inhibitors, ribozymes, antisense oligonucleotides (AS-ODNs) and substrate inhibitors. In other fields, most notably cancer chemotherapy, many compounds have been developed to inhibit RNR. These compounds include iron chelators, radical scavengers and nucleoside analogues23. Peptidomimetic inhibitors. Perhaps of most relevance to parasitologists, in terms of being able to inhibit the parasite enzyme specifically, was the finding that a nonapeptide corresponding to final residues in the Cterminus of the Herpes simplex virus R2 subunit was able to inhibit enzyme activity by interfering with subunit association24. Further work showed that this inhibition Parasitology Today, vol. 15, no. 8, 1999

Focus Organism

R2 C-terminal sequence

Human

FTLDADF

Mouse

FTLDADF

Saccharomyces cerevisiae

FTFNEDF

Schizosaccharomyces pombe

FTIDEDF

Plasmodium falciparum

FCLNTEF

Trypanosoma brucei

FSLDADF

Leishmania mexicana

FSLSEDF

Herpes simplex GAVVNDL Fig. 3. C-terminal region of the R2 subunit responsible for binding to the R1 subunit. As shown for the Herpes simplex virus ribonucleotide reductase (RNR), if host and parasite sequences are sufficiently different then peptidomimetic inhibitors can be targeted effectively against this region to disrupt RNR subunit assembly.

was effective in vivo and, by using chemical modification, that it was possible to increase the potency of the peptidomimetic inhibition by a factor of 200000, mainly by substitution of methyl and amide groups by sterically bulky side chains, such as benzene and imidazole rings, to decrease the binding coefficient between inhibitor and target25. Nuclear magnetic resonance studies have shown that this might be a common approach that could be used in the design of class I RNR inhibitors26. Peptidomimetic inhibitors may not be of use in trypanosomes, as there is little variation between trypanosome RNRs and those of higher eukaryotes at the C-terminal R2 sequence known to be involved in R1–R2 subunit association (Fig. 3). Indeed, it has been demonstrated that a peptide corresponding to this sequence in the C-terminus of mouse R2, which is an effective inhibitor of mouse RNR, can inhibit recombinant T. brucei RNR to a similar extent15. However, the corresponding motif in P. falciparum is significantly divergent from the mammalian host, with three non-conservative amino acid substitutions12,13, which suggests that this approach may be valid for the apicomplexan parasites. Allosteric inhibitors.The important finding that the allosteric regulation of RNR in T. brucei differs from that of the mammalian enzymes allows for optimism that suicide inhibitors can be targeted specifically against the parasite enzyme. Among protozoan parasite R1 sequences, conservation of several amino acid substitutions in a region recognized as being involved in dTTP binding implies this unusual allosteric regulation is a common feature of parasite RNRs. If this is so, it might be possible to design a suicide substrate inhibitor or a nucleoside analogue that, alone or in combination with a specific allosteric effector, would be more toxic to the parasite enzyme than to the host. This novel strategy might be effective against all the protozoan parasites. Ribozymes. Ribozymes encode a sequence that can cleave the complementary mRNA. For example, a ribozyme has been designed against the carbamoyl phosphate II synthetase enzyme in P. falciparum27. The parasite transcript contains two large inserts not present in the mammalian mRNA, thus ensuring target specificity. Ribozymes could be used against any enzyme whose function is indispensable to the parasite if specific potential target regions are present, thus negating any possible interference with the host mRNAs. Parasite RNR mRNA molecules fulfil these criteria because they contain sequences that are absent from host mRNAs, which Parasitology Today, vol. 15, no. 8, 1999

might enable ribozymes to be designed against these transcripts. Examples of such sequences are the RNA sequences encoding the specific insertions identified at the C-terminus of P. falciparum, T. annulata and T. brucei R1 subunits. Antisense oligonucleotides. Barker and colleagues28 showed that AS-ODNs at concentrations below 0.5 mM could be used against a wide range of P. falciparum genes, such as dihydrofolate reductase, RNR and triose phosphate isomerase, to achieve target-specific inhibition of parasite growth. Although these effects were not potent, these results are interesting from the point of view that these molecules could be a valuable tool for probing parasite gene function in culture. The AS-ODN uptake in vitro was specific to the infected erythrocyte, which might indicate a possible future role as a therapeutic agent, although the effects in vivo are likely to be much more complex. However, more work is needed to maximize the potential of AS-ODNs, as they exhibit little sequence-specific inhibition at concentrations above 1 mM. AS-ODNs also hold promise for chemotherapeutic targeting of the unique mini-exon sequence of the trypanosomatids29 and no doubt future research will improve the nuclease resistance and uptake efficiency of AS-ODNs. Conclusion Sequence differences between mammalian and parasite RNRs indicate that this essential enzyme is a potentially useful target for inhibition by currently developing technologies. The mechanism of allosteric regulation used by the T. brucei RNR will soon be fully understood and will be examined in other protozoans to determine if the mechanism is common to all. If so, then this begs the questions as to how and why did this mechanism evolve? Future work on the regulation and turnover of the subunit mRNAs will determine the feasibility of ribozymes and AS-ODNs as therapeutics. It will be possible to test substrate inhibitors against the T. brucei RNR and, given the expertise available in the design of such inhibitors as cancer chemotherapeutics, this may make the task simpler. The next few years may well see RNR come to be the target of choice for the new generation of antiprotozoan therapies. References 1 Gutteridge, W. (1993) in Modern Parasitology (2nd edn) (Cox, F.E.G., ed.), pp 219–242 Blackwell Scientific Publications 2 Wang, C.C. (1997) Validating targets for anti-parasite therapy. Parasitology 114, S31–S45 3 Sarabia, M. et al. (1993) The cell cycle genes cdc22 and suc221 of the fission yeast Schizosaccharomyces pombe encode the large and small subunits of ribonucleotide reductase. Mol. Gen. Genet. 238, 241–251 4 Thelander, L. and Reichard, P. (1979) Ribonucleotide reductases. Annu. Rev. Biochem. 48, 133–158 5 Harder, J. (1993) Ribonucleotide reductases and their occurrence in microorganisms: a link to the RNA/DNA transition. FEMS Microbiol. Rev. 12, 273–292 6 Reichard, P. (1997) The evolution of ribonucleotide reductase. Trends Biochem. Sci. 22, 81–85 7 Eklund, H. et al. (1997) Ribonucleotide reductase – structural studies of a radical enzyme. Biol. Chem. 378, 821–825 8 Elledge, S.J. and Davis, R.W.D. (1990) Two genes differentially regulated in the cell cycle and by DNA damaging agents encode alternative regulatory subunits of ribonucleotide reductase. Genes Dev. 4, 740–751 9 Johnston, L.H. and Johnston, A.L. (1995) The DNA repair genes RAD54 and UNG1 are cell cycle regulated in budding yeast but MCB promoter elements have no essential role in DNA damage response. Nucleic Acids Res. 23, 2147–2152

341

Focus 10 Wang, P. et al. (1997) Rnr4p, a novel ribonucleotide reductase small subunit protein. Mol. Cell. Biol. 17, 6114–6121 11 Huang, M. and Elledge, S.J. (1997) Identification of RNR4, encoding a second essential small subunit of RR in Saccharomyces cerevisiae. Mol. Cell. Biol. 17, 6105–6113 12 Rubin, H. et al. (1993) Cloning, sequence determination and regulation of the ribonucleotide reductase subunits from Plasmodium falciparum – a target for anti-malarial therapy. Proc. Natl. Acad. Sci. U. S. A. 90, 9280–9284 13 Chakrabarti, D. et al. (1993) Cloning and characterization of the subunit genes of ribonucleotide reductase, a cell cycle regulated enzyme from Plasmodium falciparum. Proc. Natl. Acad. Sci. U. S. A. 90, 12020–12024 14 Lye, L.F. et al. (1997) Cloning and functional analysis of the ribonucleotide reductase gene small subunit from hydroxyurearesistant Leishmania mexicana amazonensis. Mol. Biochem. Parasitol. 90, 353–358 15 Hofer, A. et al. (1997) Cloning and characterization of the R1 and R2 subunits of ribonucleotide reductase from Trypanosoma brucei. Proc. Natl. Acad. Sci. U. S. A. 94, 6959–6964 16 Mann, G.J. et al. (1991) Purification and characterisation of recombinant mouse and Herpes simplex virus ribonucleotide reductase R2 subunit. Biochemistry 30, 1939–1947 17 Yang, F. et al. (1997) Characterisation of two genes encoding the Mycobacterium tuberculosis ribonucleotide reductase small subunit. J. Bacteriol. 179, 6408–6415 18 Cavalier-Smith, T. (1993) Kingdom protozoa and its 18 phyla. Microbiol. Rev. 57, 953–994

19 Wong, J.T. (1996) Protozoan cell cycle control. Biol. Signals 5, 301–308 20 Leete, T.H. and Rubin, H. (1996) Malaria and the cell cycle. Parasitol. Today 12, 442–444 21 Bjorklund, S. et al. (1993) Structure and promoter characterization of the gene encoding the large subunit (R1 protein) of mouse ribonucleotide reductase. Proc. Natl. Acad. Sci. U. S. A. 90, 11323–11326 22 Bjorklund, S. et al. (1990) S phase specific expression of mammalian ribonucleotide reductase R1 and reductase R2 subunit messenger RNAs. Biochemistry 29, 5452–5458 23 Nocentini, G. (1996) Ribonucleotide reductase inhibitors – new strategies for cancer chemotherapeutics. Crit. Rev. Oncol. Hematol. 22, 89–126 24 Dutia, B.M. et al. (1986) Specific inhibition of herpes virus ribonucleotide reductase by synthetic peptides. Nature 321, 439–441 25 Luizzi, M. et al. (1994) A potent peptidomimetic inhibitor of HSV ribonucleotide reductase with antiviral activity in vivo. Nature 372, 695–697 26 Fisher A., Laub, P. and Cooperman, B. (1995) NMR structure of an inhibitory R2 C-terminal peptide bound to mouse ribonucleotide reductase R1 subunit. Nat. Struct. Biol. 2, 951–955 27 Flores, M. et al. (1997) Inhibition of Plasmodium falciparum proliferation in vitro using ribozymes. J. Biol. Chem. 272, 16940–16945 28 Barker, R.H. et al. (1996) Inhibition of Plasmodium falciparum malaria using anti-sense oligonucleotides. Proc. Natl. Acad. Sci. U. S. A. 93, 514–518 29 Toulme, J. et al. (1997) Control of gene expression in viruses and protozoan parasites by anti-sense oligonucleotides. Parasitology 114, S45–S59

The Human Immune Response during Cutaneous Leishmaniasis: NO Problem M.D. Mossalayi, M. Arock, D. Mazier, P. Vincendeau and I. Vouldoukis During some helminth infections, increased expression of the low-affinity receptor for IgE (CD23/FceRII) by macrophages and/or increased levels of plasma IgE have been seen, but their role in host protection or disease progression remains unclear. Recently, crosslinking of CD23 was shown to promote intracellular killing of Leishmania parasites in human macrophages, a phenomenon involving the production of tumor necrosis factor a and nitric oxide (NO). Based upon various in vitro and in vivo studies of human cutaneous leishmaniasis, Djavad Mossalayi, Michel Arock, Dominique Mazier, Philipe Vincendeau and Ioannis Vouldoukis here propose a model for an immune response that involves CD23– IgE-mediated NO release during protection, as well as during active cutaneous leishmaniasis. The roles of immune cells and their various mediators in healing or progression of leishmaniasis remain unclear. Macrophage-derived cytokines and nitric oxide (NO) kill Leishmania major in murine cells1–3. In human macrophages, leishmanicidal activity was observed in L. major- or L. infantum-infected cells following their in M. Djavad Mossalayi and Michel Arock are at the Hematology Laboratory, Faculty of Pharmacy Paris V, 4 Avenue de l’Observatoire, 75006 Paris, France. Dominique Mazier and Ioannis Vouldoukis are at Inserm U313, Pitié-Salpêtrière Hospital, Paris, France. Phillipe Vincendeau is at the Parasitology Laboratory, Bordeaux 2 University, Bordeaux, France. Tel: +33 1 53 73 97 12, Fax: +33 1 40 46 96 55, e-mail: Djavad.Mossalayi@ umr5540.u-bordeaux2.fr 342

vitro activation by various factors4, including interferon g (IFN-g), or crosslinking of the CD23/FceRII activation antigen5. In an attempt to clarify the role of IgE, CD23 and proinflammatory factors in human Leishmania infection, we compared the in vivo expression of various mediators, as reported by different groups, with their effect on the in vitro leishmanicidal activity of human macrophages. These findings provide a model whereby an immune network that includes T cells and macrophages, as well as B cells, mediates pathogen killing or disease chronicity through targeting cytokine and NO generation by infected macrophages. Experimental Leishmania infection The role of T cells and their cytokines in determining disease outcome in leishmaniasis has been investigated extensively3,6,7. In mice, the inability to control leishmaniasis has been correlated with the absence of IFN-g production by parasite-specific T cells and their failure to activate macrophages to destroy intracellular amastigotes3,6. Macrophage-derived tumor necrosis factor a (TNF-a), the oxidative burst, and/or functional inducible NO synthase (iNOS or type II NOS) seem to be essential for parasite elimination by these cells8. The human immune response against Leishmania infection has been documented by various authors4,7. Experimental models clearly indicate a role for specific Leishmania antigens in inducing immune responses from both CD41 and CD81 T cells4,9–12. IFN-g, lipopolysaccharides and/or TNF-a4,5,7,13,14 can enhance the in vitro

0169-4758/99/$ – see front matter © 1999 Elsevier Science. All rights reserved. PII: S0169-4758(99)01477-5

Parasitology Today, vol. 15, no. 8, 1999