Inhibitors of Dihydrofolate Reductase in Leishmania and Trypanosomes

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Biochimica et Biophysica Acta 1587 (2002) 249–257 www.bba-direct.com Review Inhibitors of dihydrofolate reductase in leishmania and trypanosomes

Ian H. Gilbert*

Welsh School of Pharmacy, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff, CF10 3XF, UK Received 24 January 2002; accepted 24 January 2002

Abstract

The protozoan diseases leishmaniasis, Chagas’ disease and African trypanosomiasis are major health problems in many countries, particularly developing countries, and there are few drugs available to treat these diseases. Dihydrofolate reductase (DHFR) inhibitors have been used successfully in the treatment of a number of other diseases such as cancer, malaria and bacterial infections; however they have not been used for the treatment of these diseases. This article summarises studies on leishmanial and trypanosomal DHFR inhibitor development and evaluation. Possible mechanisms of resistance to DHFR inhibitors are also discussed. D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Dihydrofolate reductase; Trypanosoma cruzi; Trypanosoma brucei; Leishmania

1. Introduction enylated to form methylene-tetrahydrofolate, which then methylates deoxyuridine monophosphate (dUMP) to give Leishmaniasis, African trypanosomiasis (sleeping sick- TMP in a reaction catalysed by thymidylate synthase (TS) ness) and Chagas’ disease are major causes of mortality and (Fig. 1). During this reaction, methylene-tetrahydrofolate is cause much economic hardship, particularly in the develop- converted back to dihydrofolate, completing the cycle. There- ing world. The World Health Organisation estimates that fore, inhibition of DHFR prevents biosynthesis of thymidine, millions of people are infected, or are at risk, from these and as a consequence, DNA biosynthesis. In addition, inhib- diseases [1]. In addition, leishmaniasis is increasingly a ition of DHFR probably leads to a buildup in levels of dUMP complication with AIDS patients. Unfortunately, there are and hence to a biosynthetic precursor, deoxyuridine triphos- few drugs available to treat these diseases and most of these phate [4]. High levels of deoxyuridine triphosphate lead to suffer from poor clinical efficacy, unwanted effects, and incorporation of uracil into DNA to levels beyond which the require parenteral administration, which is not appropriate in DNA repair enzymes (uracil-DNA-glycosylase) can cope, rural areas. The situation is made more serious by reports of leading to cell death. treatment failures of the existing drugs, for example, mel- arsoprol for the treatment of African trypanosomiasis. Dihydrofolate reductase (DHFR) is a key enzyme in folate 2. Background to the diseases metabolism and, therefore, in the production of thymidine [2,3]. Its role in thymidine biosynthesis is the reduction of The parasites that cause leishmaniasis, African trypano- dihydrofolate to tetrahydrofolate using the cofactor NADPH somiasis and Chagas’ disease are protozoan parasites of the (Fig. 1). Following this reduction, tetrahydrofolate is meth- order kinetoplastida [5,6]. They are all transmitted to the human host by various vectors (see below) and have complex life cycles; each stage of which has different Abbreviations: DHFR, dihydrofolate reductase; DHFR-TS, dihydrofolate reductase-thymidylate synthase; dUMP, deoxyuridine monophosphate; morphologies and different metabolic properties. The para-

EC50, the effective concentration of a compound to produce a therapeutic sites are phylogenetically related and share many similar- effect in 50% of the sample; IC50, the concentration of compound which ities, but there are also significant differences between them. gives 50% inhibition of an enzyme; QSAR, quantitative structure activity Leishmaniasis is caused by various species and subspe- relationship; TMP, thymidine monophosphate; TD50, the effective concen- cies of Leishmania, of which the most important include tration of a compound to kill 50% of the host cells; TS, thymidylate synthase subspecies of Leishmania donovani, Leishmania tropica, * Tel.: +44-29-2087-5800; fax: +44-29-2087-4149. Leishmania mexicana and Leishmania braziliensis. These E-mail address: [email protected] (I.H. Gilbert). cause a variety of diseases in humans. The diseases found

Fig. 1. The reaction carried out by DHFR: the reduction of dihydrofolate to tetrahydrofolate (shown in the box); and the role of this reaction in the folate- mediated production of TMP from dUMP.

are: cutaneous leishmaniasis, which can vary from small an epimastigote and in the human host as an intracellular self-healing skin lesions to a more widespread and disfigur- amastigote. The disease causes a chronic infection in ing condition; mucocutaneous leishmaniasis, which leads to humans, with particular effects on the heart, colon, oeso- highly disfiguring lesions of the mouth, nose and throat; and phagus and peripheral nervous system. There is a high death visceral leishmaniasis, which is a systemic infection that is rate amongst children. usually fatal if not treated, with the parasites multiplying within macrophages, giving rise to damage of the liver, spleen, bone marrow and lymph nodes. The parasite is 3. The parasite enzyme found in the vector stage (the sandfly) as the promastigote and in the human host as an amastigote in acidic phagoly- 3.1. Characterisation somes within macrophages. African trypanosomiasis in humans is caused by two DHFR has been cloned and over-expressed from Leish- subspecies of Trypanosoma brucei: T. b. rhodesiense and T. mania major, [7,8] T. cruzi [9] and T. brucei [10]. These b. gambiense. The parasites live in the bloodstream and enzymes show high sequence identity, especially the T. invade the central nervous system giving rise to the neuro- brucei and T. cruzi enzymes (Table 1). The enzyme is actually logical symptoms of African trypanosomiasis and eventu- a bi-functional dimeric enzyme linked to TS (Fig. 2). This is a ally to death unless the disease is treated. The human form common occurrence amongst protozoa, in contrast to the of the life cycle is the trypomastigote and the vector stage situation in mammalian and bacterial cells where the enzyme (tsetse fly) is the epimastigote. is a monomer [11]. Gene knockout experiments have shown Chagas’ disease is caused by the parasite Trypanosoma the enzyme to be essential in Leishmania, at least in the cruzi. The parasite is found in the vector (reduviid bug) as promastigote stage of the life cycle [12]. I.H. Gilbert / Biochimica et Biophysica Acta 1587 (2002) 249–257 251

Table 1 Sequence alignment (percentage identity) of DHFR from different organisms L. major T. cruzi T. brucei E. coli Human 26 27 26 27 L. major 50 46 24 T. cruzi 58 26 T. brucei 23 Modified from Ref. [14] with permission.

3.2. Structural data

The enzyme from L. major has been crystallised by Knighton et al. [13] and homology models have been produced of the T. brucei and T. cruzi enzymes [14]. Detailed analysis [14] and comparison of the structures of the L. major, human and bacterial enzymes, and the homology models of the T. brucei and T. cruzi enzymes, suggests the following: . The L. major, T. brucei and T. cruzi enzymes show high Fig. 3. Part of the active site of the L. major DHFR. This figure shows the structural similarity. location of methotrexate and those residues in the active site of L. major . The three protozoan enzymes show structural differ- DHFR which are not homologous to the corresponding residues in human ences from the human and bacterial enzymes. DHFR (Glu43, Ser44, Met53, Lys57, Phe91). . The three-dimensional shapes of the parasite enzyme folate binding sites are slightly different to the corresponding human active sites. Points of particular difference are: a slightly larger binding pocket in the region where the Knighton et al. [13] studied the crystal structure of the bi- benzamide moiety of the substrate binds in the case of the functional DHFR-TS enzyme and concluded that there is trypanosomal and leishmanial enzymes; and a small tunnel substrate channelling between the DHFR and TS domains of under the pteridine ring in the case of the trypanosomal the enzyme, by a positively charged electrostatic channel on enzymes. the protein surface, which interacts with the negatively . Differences in residues in the active sites (Table 2, Fig. charged substrate. Liang and Anderson [15] have done 3). detailed kinetic analysis, which supports this model and, It may be possible to exploit these differences between the in addition, they suggest that there may be interaction parasite and human enzymes to design selective inhibitors. between the DHFR and TS domains.

Fig. 2. A representation of the crystal structure of L. major DHFR-TS prepared by Web-lab Viewer. The enzyme is a symmetrical dimer of TS and DHFR. 252 I.H. Gilbert / Biochimica et Biophysica Acta 1587 (2002) 249–257

4. Inhibitors of parasite DHFR Methotrexate, a potent inhibitor of DHFR, is 8-fold selective for the L. major DHFR, and also showed activity 4.1. Trimethoprim, cycloguanil, pyrimethamine and metho- against L. major promastigotes (EC50 = 0.3 AM) [12]. Scott trexate: known DHFR inhibitors et al. [21] investigated the activity of methotrexate against promastigotes from various species of Leishmania (L. Classical anti-microbial DHFR inhibitors, such as pyr- major, L. donovani, L. m. mexicana) and found L. major imethamine and trimethoprim (Fig. 4) are, in general, not most susceptible. Methotrexate has been reported as a potent very selective for the analogous leishmanial and trypanoso- inhibitor of the T. cruzi enzyme (Ki = 0.038 nM), but it is mal enzymes. Thus, trimethoprim (Ki = 0.12 AM), pyrimeth- poorly active against the amastigotes (EC50 =9.2 AM) amine (Ki = 0.25 AM) and cycloguanil (Ki = 5.8 AM) are [18,22]. only weakly active against L. major DHFR. Pyrimethamine is weakly selective for the human, relative to the parasite 4.2. Novel DHFR inhibitors enzyme (1- to 2-fold). Trimethoprim has been reported as weakly selective for either the L. major or human enzyme. Relatively little has been reported on the development of There is weak inhibition and selectivity of trimethoprim specific inhibitors of leishmanial and trypanosome DHFR. (Ki =1 AM, selectivity 1- to 30-fold) and pyrimethamine Coombs’ group [21,23] investigated some 5-substituted (Ki = 0.1 AM, no selectivity) for the T. cruzi enzyme. In the 2,4-diaminopyrimidines that were good inhibitors of L. m. case of T. brucei, trimethoprim (Ki = 0.01 AM, selectivity mexicana DHFR in crude extracts of the enzyme 134- to 600-fold) and pyrimethamine (Ki = 0.01 AM, selec- (IC50 = 0.2–2 AM) and showed activity against the promas- tivity 10-fold) were more selective for the parasite enzyme tigotes (EC50 =12–24AM) (Fig. 5). One of the compounds [10,16–18]. was studied further in amastigotes, where it showed some Trimethoprim, pyrimethamine and cycloguanil are activity at concentrations below that at which it killed the weakly active against L. major promastigotes (EC50 = 175, host macrophages. However, in a mouse study, the com- 32, > 500 AM, respectively) and L. donovani amastigotes pound was too toxic to warrant further study. (EC50 = 160, 34, >100 AM, respectively) [16,19]. In rodent Quantitative structure activity relationship (QSAR) anal- models of leishmaniasis, Peters et al. [20] reported that ysis was carried out on the inhibition of L. major [24] and activity was species-specific; trimethoprim exhibited some human [25] DHFR for a series of triazines of the general activity against infection by L. major, cycloguanil had some structures shown in Fig. 6(a),(b). Compounds with lip- activity against infection by Leishmania infantum, while ophilic substituents (X) showed greater activity than com- pyrimethamine exhibited poor activity. pounds with polar substituents. However, few compounds

Fig. 4. Some clinically used DHFR inhibitors: pyrimethamine and cycloguanil as anti-malarials; trimethoprim as anti-bacterial; and methotrexate as anti-cancer. I.H. Gilbert / Biochimica et Biophysica Acta 1587 (2002) 249–257 253

Fig. 5. A series of 2,4-diaminopyrimidines showing activity against Leishmania DHFR [21,23]. showed selectivity for the Leishmania enzyme over the the L. major DHFR and EC50 against L. major promasti- human enzyme, notable exceptions being when X = nonyl, gotes of 5.6 AM and L. donovani amastigotes of 4.6 AM. dodecyl and CH2O-1-napthyl. Some of the compounds were A detailed structure-activity study on these alkyl-substi- assayed for activity against L. major promastigotes and tuted 5-benzyl-2,4-diaminopyrimidines (Fig. 6(c)) was showed activities varying from 1 to 500 AM. undertaken by Chowdhury et al. [17]. They varied the Sirawaraporn et al. [16] studied a series of substituted 5- length of the substituent chain from hydrogen to decyl benzyl-2,4-diaminopyrimidines, some of which showed (n =0ton = 10) and also extended the study to include both selectivity for the L. major DHFR in enzyme assays and T. cruzi and T. brucei. Optimum activity and selectivity moderate activity against L. major promastigotes and L. against L. major, T. cruzi and T. brucei DHFR was found for donovani amastigotes. The most active and selective com- compounds with a chain length of 2–6 carbon atoms. The pound in the enzyme assay experiments was the 8-octyloxy maximum selectivities of these compounds, compared to the derivative (n = 8, Fig. 6(c)), with a selectivity of 130-fold for human enzyme, were 20-, 5- and 250-fold, respectively, and inhibition constants were 0.13, 0.22 and 0.01 AM, respectively. These compounds were modelled in the active sites of the L. major and human enzymes. In the L. major enzyme, the alkyl chain of the substituent appears to interact with a phenylalanine residue in the enzyme active site. This interaction appears to be optimal for a chain length of 4–6 carbon atoms. As the alkyl chain length increases further, the alkyl substituent appears to move away from the Phe residue, decreasing the interaction and offering a possible explanation for the optimal activity. This model can also explain the selectivity of the compounds. In the human enzyme, the phenylalanine is replaced by asparagine (Table 2) [14], which does not interact with the alkyl chain.

Table 2 Differences in residues in the folate binding sites of human and trypanosomal and leishmanial DHFR Human L. major T. cruzi T. brucei Interaction with Residue residue residue residue folate Gly20 Glu43 Arg39 Gly45 pteridine/NADPH Asp21 Ser44 Ser40 Thr46 pteridine/NADPH Phe31 Met53 Met49 Met55 pteridine/benzamidine/ glutamate Gln35 Lys57 Arg53 Arg59 glutamate Fig. 6. Compounds (a) and (b) are the basic structures used for QSAR Asn64 Phe91 Phe88 Phe94 glutamate analysis by Booth et al. [24] and Hathaway et al. [25]. Compound (c) is the The portion of the substrate with which residues interact is indicated as basic structure studied by Sirawaraporn et al. [16] and Chowdhury et al. pteridine, benzamidine, and glutamate. Modified from Ref. [14] with [17]. permission. 254 I.H. Gilbert / Biochimica et Biophysica Acta 1587 (2002) 249–257

Fig. 7. Examples of 2,4-diaminoquinazolines tested by Berman et al. [26]. The activities against the enzyme, L. major amastigotes cultured in macrophages and cellular toxicity values (against macrophages) are given for the most active compounds.

Fig. 8. Quinazolines and 2,4-diaminopyrimidines showing activity against L. major DHFR [12].

Fig. 9. Differences in the active site between the human and T. cruzi enzymes used by Zuccotto et al. [22] to design novel inhibitors. I.H. Gilbert / Biochimica et Biophysica Acta 1587 (2002) 249–257 255

macrophages. However, there was little correlation between inhibition of DHFR and activity against the L. major amastigotes, suggesting an alternate mode(s) of action, other than or in addition to inhibition of DHFR, may be operating with these compounds. In the case of T. cruzi, some 2,4- diaminoquinazolines showed good activity against the amastigote form [27,28] and were able to prolong the life of infected mice, but not to give a radical cure [29]. Hardy et al. [12] screened a set of pteridines, quinazolines and pyrimidines against L. major and human DHFR and against L. major promastigotes (for representative structures see Fig. 8). Generally, compounds that showed good inhibition of DHFR were active against the L. major promastigotes and conversely those which showed poor activity against the DHFR were also poorly active against the promastigotes. Some of the compounds also showed reasonable levels of selectivity for the L. major enzyme over the human enzyme; the most potent and selective compounds were some quinazolines and a pyrimidine (Fig. 8). However, some of these compounds were also active in L. major promastigotes that lacked the enzyme DHFR-TS Fig. 10. Examples of novel inhibitors of L. major (a) and T. cruzi (b) DHFR identified by database mining using DOCK 3.5 [30,31]. suggesting that they might have (an) additional enzymatic targets within the cell. Zuccotto et al. [22] used differences in the structures of the These substituted 5-benzyl-2,4-diaminopyrimidines T. cruzi and human DHFRs to design a series of selective showed in vitro activity against T. cruzi amastigotes and inhibitors of the T. cruzi enzyme. In the human enzyme, the T. brucei trypomastigotes, with the best activity being found channel linking the folate to the NADPH binding sites for chain lengths of 6–10 carbon atoms (Fig. 6(c)). The contains the sequence Gly20–Asp21, which is overall neg- most active compounds gave 100% inhibition of growth of atively charged. In contrast, the T. cruzi enzyme contains the T. cruzi amastigotes at 3 AM and an EC50 of 1 AM against T. sequence Arg39–Ser40, which is overall positively charged brucei trypomastigotes. The chain length for optimum (Table 2). Modelling suggested substitution of a negatively activity against the intact parasites (6–10 carbon atoms) charged substituent on methotrexate should lead to repulsion does not correspond to that for optimum activity against the with Asp21 in the case of the human enzyme and possibly enzyme (2–6 carbon atoms). This suggests that inhibition of attraction in the case of the T. cruzi enzyme (Fig. 9). The DHFR is not the only factor for activity against the parasite. compounds did appear to have increased selectivity for the The compounds were generally inactive against L. infantum parasite enzyme but unfortunately showed weak in vitro amastigotes. In rodent models of African trypanosomiasis, activity against T. cruzi amastigotes. several of the 5-benzyl-2,4-diaminopyrimidines showed a An approach to discover novel parasite DHFR inhibitors marginal effect, while against a rodent model of Chagas’ using database mining has been reported by Chowdhury et disease, the compounds were inactive. al. [30] and Zuccotto et al. [31] who used the programme Some 2,4-diaminoquinazolines investigated by Berman DOCK 3.5 [32] to search the Cambridge Structural Data- et al. [26] showed reasonable inhibition of DHFR isolated base. The goal of their research was to find DHFR inhibitors from L. mexicana promastigotes (Fig. 7). The compounds that were not based on the 2,4-diaminopyrimidine motif. also showed very potent activity against L. major amasti- Chowdhury et al. [30] used the L. major active site for gotes cultured in human macrophages (ED50 =0.04–25 docking and identified compounds with weak activity nM), and a large therapeutic index compared to uninfected against both the enzyme and parasite (EC50 =20–60 AM).

Fig. 11. Reduction of biopterin by ptr1. 256 I.H. Gilbert / Biochimica et Biophysica Acta 1587 (2002) 249–257

It is not known whether this anti-parasitic activity is due to Acknowledgements inhibition of DHFR (Fig. 10(a)). Zuccotto et al. [31] found one particular compound that had activity against T. cruzi I would like to acknowledge the following people who DHFR (Fig. 10(b)) and also showed good activity against T. have worked on design and synthesis of inhibitors of DHFR brucei trypomastigotes (EC50 = 3.6 AM). in my research group (Shafinaz F. Chowdhury, Raffaella Di Lucrezia, Didier Pez, Richard Weaver, Fabio Zuccotto) and my collaborators (Victor Bernier Villamor, Reto Brun, 5. Resistance Simon L. Croft, Dolores Gonzalez Pacanowska, Jonathan Goodman, Ramon Hurtardo Guerrero, Isabel Leal, Louis There are a number of possible mechanisms of resistance Maes, Luis M. Ruiz Perez). I would also like to acknowl- to DHFR inhibitors in Leishmania which include over- edge the following organisations who have contributed expression of the enzyme DHFR-TS and over-expression funding: The British Council/Overseas Development of the enzyme ptr1. Agency, Acciones Integradas, the Welsh School of Phar- ptr1 is an enzyme involved in the reduction of biopterin to macy, the Welsh Scheme for the Development of Health and dihydrobiopterin and terahydrobiopterin (Fig. 11) [33,34]. It Social Research, the World Health Organisation Special has been shown that this enzyme can also reduce dihydrofo- Programme for Research and Training in Tropical Diseases. late to tetrahydrofolate, although the enzyme is structurally I would also like to acknowledge Dr. Suzanne Duce and Dr. unrelated to DHFR. Under normal conditions, the majority of Claire Simons for reading this manuscript. reduction of dihydrofolate to tetrahydrofolate is carried out by DHFR. However, should ptr1 be over-expressed (which has been demonstrated in laboratory models of resistance References [35]), then this enzyme could substitute, albeit much less efficiently, for DHFR [36]. It has been shown that while it is [1] http://www.who.int. [2] J.M. Blaney, C. Hansch, C. Silipo, A. Vittoria, Chem. Rev. 84 (1984) probable that ptr1 could not fully replace the role of DHFR, it 333–407. is important in reducing the sensitivity of Leishmania to [3] C.D. Selassie, T.E. Klein, Comparative QSAR, Taylor & Francis, antifolates [37]. Compounds that inhibit both DHFR and ptr1 London, 1997. may make prospects as drug candidates [12]. While there are [4] E.M. McIntosh, R.H. Haynes, Acta Biochim. Pol. 44 (1997) 159– differences in substrate specificity between DHFR and ptr1, 172. [5] D.M. James, H.M. Gilles (Eds.), Human Antiparasitic Drugs: Phar- some compounds inhibit both well. The presence of ptr1 has macology and Usage, Wiley, Chichester, 1985. been shown in L. major promastigotes and more recently in L. [6] http://www.who.int/tdr. mexicana amastigotes [38]. This contrasts to the situation in [7] T.D. Meek, E.P. Garvey, D.V. Santi, Biochemistry 24 (1985) 678– T. cruzi where levels of ptr1 are not detectable in the 686. amastigote stage, although the enzyme is expressed in the [8] R. Grumont, W. Sirawaraporn, D.V. Santi, Biochemistry 27 (1988) 3776–3784. epimastigote (vector) form [39]. [9] P. Reche, R. Arrebola, A. Olmo, D.V. Santi, D. Gonzalez Pacanowska, A recent publication by Beverley’s group [40] showed L.M. Ruiz Perez, Mol. Biochem. Parasitol. 65 (1994) 247–258. that ptr1 knockouts are in fact more virulent against mouse [10] F. Gamarro, P.L. Yu, J. Zhao, U. Edman, P.J. Greene, D. Santi, Mol. models of leishmaniasis than the wild-type, and they sug- Biochem. 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