JAC Advance Access originally published online on April 14, 2006
Journal of Antimicrobial Chemotherapy 2006 57(6):1043-1054; doi:10.1093/jac/dkl104
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Reviews |
The past, present and future of antifolates in the treatment of Plasmodium falciparum infection
1 Kenya Medical Research Institute (KEMRI)/Wellcome Trust Collaborative Research Program PO Box 230, 80108, Kilifi, Kenya 2 Department of Pharmacology and Therapeutics, University of Liverpool Liverpool L69 3BX, UK 3 Liverpool School of Tropical Medicine Pembroke Place, Liverpool L53QA5, UK
*Correspondence address. Kenya Medical Research Institute (KEMRI)/Wellcome Trust Collaborative Research Program, PO Box 230, 80108, Kilifi, Kenya. Tel: +254-41-522535/522063/525043; Fax: +254-41-522390; E-mail: anzila{at}kilifi.mimcom.net
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Abstract |
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Top
Abstract
1. Historic perspective of...Chemotherapy remains the most important means of controlling malaria, one of the deadliest infectious parasitic diseases in the world. Antimalarial antifolates have been central for prophylaxis and treatment of malaria. This drug family was discovered in the 1940s, during the Second World War, and molecules that are currently in clinical use were discovered at that time. Since the 1940s, no new antimalarial antifolates have been developed that have reached Phase I/II stages. Limited work has been carried out to exploit the inhibition of the malaria folate pathway as a means of discovering new drugs. In this review, work carried out on antimalarial antifolates since the 1940s up to the present time is discussed in terms of discovery, clinical use, mode of action and mechanism of resistance. New concepts have been presented to improve antimalarial antifolate in vivo efficacy and to identify potent new antifolate agents.
Keywords: malaria , antimalarials , folate , dihydrofolate reductase , dihydropteroate synthase
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1. Historic perspective of folate discovery |
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The identification and development of antifolates as therapeutic agents derived from the understanding of the role of folate derivatives in humans. Thus, before discussing antifolate drugs, work that led to the discovery of folate is summarized.
The discovery of the folate compound is associated with the understanding of anaemia complications. By the 1930s, evidence showed that some forms of anaemia, mainly megaloblastic anaemia, could be reversed by the addition of yeast or liver extract, leading to the hypothesis that a specific factor may be associated with anaemia.1 This factor was given the name folic acid in 1941 when it was isolated from spinach leaves by Mitchell et al.2 The word folate is derived from ‘folium’, meaning ‘leaf’ in Latin. Two years later, the chemical structure of folate was resolved, and it was synthesized in pure crystalline form.3 Soon after, this compound proved to be effective in the treatment of several types of anaemia.4,5
Folic acid is composed of a pteridine ring, para-aminobenzoic acid (pABA) and glutamate; the compound is also called pteroyl glutamic acid (PGA). Soon after its synthesis, several reports demonstrated that, in fact, it is not a naturally occurring folate. Natural folate differs from folic acid in three aspects: (i) reduction to di- or tetrahydro forms of the pteridine rings at positions 7,8 and 5,6; (ii) presence of additional glutamate residues, leading to the formation of polyglutamated derivatives; and (iii) the presence of an additional single carbon unit attached to the N5 or N5 nitrogen atom: methyl (CH3), formyl (CHO), methylene (=CH2) and methenyl (=CH+). Folic acid is now used to denote the fully oxidized compound, and the term ‘folate’ is a generic term including folic acid and all naturally occurring folates. The chemical structure of folic acid is shown in Figure 1, and Figure 2 describes the relationship between folate derivatives.
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2. Genesis of the use of antifolates against human disease |
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The development of antifolate agents derived from efforts to treat leukaemia. The observation of serum folate deficiency among patients with acute leukaemia prompted some investigators in the early 1940s to postulate that acute leukaemia might be the result of deficiency of folate. Specifically, megaloblasts resembling leukaemic blasts predominate in the bone marrow of folate-deficient patients. The availability of folic acid prompted investigators to treat leukaemic patients with folate. However, the result indicated that administration of this substance was not only ineffective but even accelerated the course of the disease.6 This study showed that folic acid was an essential factor for the spread of leukaemia. Thus, the inhibition of the availability of this factor could lead to anti-proliferative property, by creating a folate deficiency status. This hypothesis led to the synthesis of the first folate analogue, aminopterin or 4-amino-pteroyl-glutamic acid (Figure 1). The clinical trial of aminopterin produced temporary remissions in 5 out of 16 patients with acute leukaemia.6 This demonstration was a landmark in cancer chemotherapy: it provided, for the first time, evidence that an antimetabolite could be an effective antineoplastic agent.
The high toxicity of aminopterin led to the development of methotrexate or 4-amino-10-methyl PGA or amethopterin (Figure 1). Methotrexate was found to have a more favourable therapeutic index than aminopterin, and thus for the last 50 years methotrexate has supplanted aminopterin in the clinic.7 The success of this drug has sustained the search for other antifolate agents. Currently, the disruption of the folate pathway is one of the main arms for the treatment of tumour diseases in humans.7
The success of antifolates in the treatment of tumours also led to the use of this class of drugs in the treatment of other rapidly dividing cells such as bacteria and parasites. This review focuses on the use of antifolates against Plasmodium falciparum infection. Additional information on the same topic can be found in a previous publication.8 Those interested in the use of antifolates against bacteria are referred to excellent reviews reported elsewhere.9–12
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3. Antifolate antimalarial agents |
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Antifolate agents used in the treatment of malarial infection are subdivided into two classes: inhibitors of dihydropteroate synthase (DHPS), known as class I antifolates, and inhibitors of dihydrofolate reductase (DHFR), the class II antifolates. The combination of DHFR and DHPS inhibitors is synergistic, hence their use in combination in the treatment of malaria. In this section, the discovery of anti-DHFR and anti-DHPS is discussed separately, and how they have been used in combination is summarized. Discussion is limited to antifolates that have reached at least Phase I trials in humans.
3.1. Inhibitors of DHFR
3.1.1. Proguanil. Proguanil was the first reported antimalarial antifolate agent and was discovered as a result of an intensive British research programme, led by Imperial Chemical Industries (ICI) on synthetic antimalarials, which started during the Second World War. The first report on proguanil was in 1945, and the drug was found to be more active than quinine against avian malaria and to have a better therapeutic index13 in animal models, prompting its use in humans. After this discovery, studies demonstrated that, in fact, proguanil is a prodrug and metabolizes to its triazine form chlorcycloguanil, an inhibitor of the parasite DHFR14 (Figure 3). This drug has been used alone as a prophylactic agent against malaria or in combination with chloroquine.15–20 Proguanil has recently been combined with atovaquone, an inhibitor of electron-transport to the cytochrome bc1 complex (coenzyme Q); this combination, known as Malarone®, is synergistic and is used as a prophylactic agent against malaria.21 Though proguanil can be converted in vivo into cycloguanil, an inhibitor of DHFR, proguanil and atovaquone are the active agents and the mechanism of synergy between these drugs is still not well understood.
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The potency of proguanil led to the search for its analogues. These studies have demonstrated that chlorination of the phenyl ring and the increase in the linker between the phenyl ring and the diaminopyrimidine ring increase the potency of this class of antifolates (Figure 3). These studies led to the discovery of chlorproguanil, clociguanil (BRL 50216) and BRL 6231 (WR 99210).
3.1.2. Chlorproguanil. Chlorproguanil is generated by the chlorination of proguanil (on the phenyl ring) (Figure 3). Similar to proguanil, chlorproguanil is metabolized to chlorcycloguanil, the active metabolite. Chlorproguanil was recommended for prophylaxis but has not been used as much as proguanil.18–20 Because of its higher potency compared with proguanil, chlorproguanil was recommended for prophylaxis at a lower dose. However, a study has demonstrated the inadequacy of the recommended dose to provide prophylactic protection.22 This antifolate has now been combined with dapsone as an antimalarial antifolate combination (section 3.3.4).
3.1.3. Clociguanil (BRL 50216). Clociguanil is an analogue of chlorcycloguanil with a methylene-oxy linker between the triazine and the phenyl ring (Figure 3). Clinical evaluation of clociguanil has been conducted in Africa, and the results showed that clociguanil was a good antimalarial. However, this drug has an erratic bioavailability and its short acting property was not seen as an advantage at that time. Thus, the drug was abandoned, since it did not offer an advantage over pyrimethamine and proguanil.23,24
3.1.4. BRL 6231 (WR 99210). WR 99210 has three chlorine atoms on the phenyl ring and a longer aliphatic linker (Figure 3). As with clociguanil, studies in animal models have demonstrated the low bioavailability of WR 99210, preventing its development as an antimalarial agent.25 In fact, all the antifolates cycloguanil, chlorcycloguanil, clociguanil and WR 99210 are poorly bioavailable, and their in vivo use requires their transformation to a non-cyclic biguanide or triazine ring in the form of a pro-drug, as is the case with proguanil and chlorproguanil. Thus, based on the cyclization of the latter two antifolates, a pro-drug of WR 99210 has been developed (see section 5.1).
3.1.5. Pyrimethamine. Pyrimethamine belongs to the 2,4-diaminopyrimidine derivative family. The interest in the antimalarial activity of this family of compounds was sparked in the late 1940s when they were synthesized and tested as analogues of folic acid in the treatment of tumours.26 Falco et al.27 observed that the structures of these compounds and proguanil were similar (Figure 3) and hypothesized that 2,4-diaminopyrimidine could have antimalarial activity. The screening of their antimalarial activity led to the identification of pyrimethamine. This has been the most widely used antimalarial antifolate agent so far. It is used in combination with sulfadoxine or sulfalene (see section 3.3.1), and to a lesser extent it has been used in monotherapy. The drug is known as Daraprim®.
All these antifolates have a higher affinity of binding with P. falciparum than human DHFR. It was accepted that differences of binding affinity account for their good therapeutic index. However, a report indicated that the malaria parasite and its host differ fundamentally in the way that translation of DHFR-TS is regulated, such that the parasite enzyme is less readily replenished when targeted by anti-DHFR inhibitors.28 Nevertheless, this observation needs to be confirmed since a recent study did not support these findings.29 Thus, the antifolate antimalarial selectivity is still an open question.
3.2. DHPS inhibitors
The discovery that sulfadrugs block the synthesis of de novo folate synthesis led to the use of this class of compounds as antimalarial agents since the parasite can synthesize folate de novo. These sulfadrugs belong to two families: sulphonamide and sulphone, and their structures are presented in Figure 4. In the past, attempts were made to use these drugs alone as antimalarial agents.30 However, this approach was abandoned because of their low efficacy and unacceptable toxicity.
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The interest in this class of antifolates was fostered when it was demonstrated that they synergized with anti-DHFR, thus explaining their use as components in antifolate combinations. Among these anti-DHPS drugs, dapsone is the most potent DHPS inhibitor of malaria ever described. A summary of the discovery of this sulfadrug is now presented.
Dapsone was synthesized at the beginning of the 20th century, in 1908, as a result of a search for molecules to produce azo dyes.31 This compound was not tested as an antimicrobial until the late 1930s, when Buttle et al.32 and Fourneau et al.33 assessed its chemotherapeutic effects against bacteria cells. This sulphone-based agent was found to suppress the growth of various pathogenic agents including mycobacteria and the malaria parasite. It has been used in monotherapy in the past for the treatment of malaria (both falciparum and vivax). However, because of its limited efficacy and high toxicity, development of this drug was abandoned.34,35 Dapsone was combined with pyrimethamine as Maloprim® (section 3.3.1), and it is now used in combination with chlorproguanil for the treatment of malaria (section 3.3.4).
Dapsone is also used in the treatment of other infectious diseases, mainly leprosy and36 pneumocystic pneumonia,37 and in the treatment of dermatological inflammatory diseases such as dermatitis herpetiformis.38 Dapsone is currently the most widely used anti-DHPS agent in human chemotherapy.
3.3. Drugs used for malaria treatment
3.3.1. Pyrimethamine and sulfadrug combinations. The literature is replete with reports on clinical trials of anti-DHFR/anti-DHPS in the treatment of malaria. Pyrimethamine, a long acting drug, has been combined with the following sulfadrugs: sulfadiazine, sulfametopyrazine, sulfamethoxine, sulfadimethoxine, sulfaphenazole and sulfisoxazole.39–41 However, none of the aforementioned combinations has been developed further as antimalarial agents. Instead, pyrimethamine was combined with sulfadoxine, which is known as Fansidar®, and combined with sulfalene under the name of Metakelfin®. These two sulfadrugs have a long elimination profile, like pyrimethamine, with half-lives >80 h.42 On the other hand, pyrimethamine has been combined with a short acting sulfadrug, dapsone, under the name of Maloprim®. The half-life of dapsone is around 24 h,43 and, as a result, from the second day of treatment, the synergic property of the combination is substantially reduced, decreasing the drug efficacy, and explaining the relatively low efficacy of this drug combination.44
Fansidar® has been extensively used as an antimalarial in most malaria endemic areas. Though the efficacy of Fansidar® and Metakelfin® is comparable,45 Fansidar® has been more widely used than Metakelfin®. Information on the clinical use of these drugs can be found elsewhere.45
3.3.2. Trimethoprim and sulfadrugs. It is of interest to note that attempts were made in the past to use the anti-DHFR antibacterial agent trimethoprim in combination with sulfadrugs to treat malaria. Trimethoprim was combined with sulfamethoxazole (this is the antibacterial combination known as Septrim® or Bactrim®, co-trimoxazole), sulfalene and sulfametopyrazine.45 However, since the use of trimethoprim did not present any advantage over pyrimethamine, trimethoprim was discouraged as an antimalarial. Trimethoprim/sulfamethoxazole has been recommended as an antibacterial prophylactic agent in patients infected with HIV. Since malaria is endemic in many areas where HIV is prevalent, there is a renewed interest in the antimalarial properties of this combination. Clinical studies have demonstrated that this drug remains efficacious against P. falciparum infections in areas where resistance to the combination pyrimethamine/sulfadoxine is still low.46–50 Concerns have also been raised regarding the possibility of selection of trimethoprim/sulfamethoxazole-resistant Plasmodium parasites. Such parasites could be less sensitive to pyrimethamine/sulfadoxine since both drugs target the same enzymes (DHFR and DHPS). However, the possible selection of trimethoprim/sulfamethoxazole-resistant Plasmodium parasites is still a matter of debate, and more studies are needed to clarify this issue.46
3.3.3. Proguanil/dapsone. Proguanil has been used in combination with dapsone for prophylaxis and treatment.45,51–53 Recently, proguanil/dapsone has been combined with artesunate;54 however, because of the high potency of chlorcycloguanil (active metabolite of chlorproguanil) over cycloguanil (active metabolite of proguanil), proguanil/dapsone offers no clinical advantage over chlorproguanil/dapsone55,56 (see also section 3.3.4).
3.3.4. Newly developed antifolate combination: chlorproguanil/dapsone (Lapdap®). Lapdap® is a combination of existing old drugs, chlorproguanil and dapsone. This combination was identified by the Wellcome Trust laboratory in Nairobi, as a result of the studies that aimed at understanding the mechanism of pyrimethamine/sulfadoxine resistance. Our group has demonstrated that the rapid selection of pyrimethamine resistance is associated with the long half-life of this drug, based on the assessment of the in vitro chemo-sensitivity profile and the comparison of molecular markers of pyrimethamine/sulfadoxine resistance in isolates collected before and after pyrimethamine/sulfadoxine treatment.42,57 Resistant parasites are selected during the period when the in vivo concentrations of pyrimethamine are below the concentration required for therapeutic effectiveness: because of its long half-life, pyrimethamine exerts strong selective pressure for mutations in its target gene, DHFR.42,57,58 To minimize this selection, the short acting antifolate combination chlorproguanil/dapsone was identified and developed as an antimalarial.43,56 In vitro analyses have demonstrated that chlorcycloguanil, the active metabolite of chlorproguanil, and dapsone are more potent than pyrimethamine and sulfadoxine, respectively.55,59 In vivo, chlorproguanil/dapsone is efficacious in treating malaria60 and it retains activity against pyrimethamine/sulfadoxine-resistant parasites.61 It is now established that this new combination, because it is a short acting drug, selects less efficiently for resistance than pyrimethamine/sulfadoxine.57,58 Thus, this new drug is likely to have a longer, useful therapeutic lifespan. Chlorproguanil/dapsone is now available and is part of the antimalarial armamentarium in Africa. Patients with glycose-6-phosphate deficiency (G6PD) are at a higher risk of developing methaemoglobinaemia, as a result of dapsone toxicity. However, at the population level, the impact of this deficiency on the toxicity of chlorproguanil/dapsone is still unknown. Pharmaco-vigilance based studies of chlorproguanil/dapsone are underway to address this issue. In addition, an artemisinin-based combination of chlorproguanil/dapsone with artesunate (CDA) is being developed. It has already reached Phase II, and Phase III clinical development is about to start.62 All these studies will also increase our understanding of the safety of this drug.
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4. Antifolate drug resistance |
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4.1. In vitro resistance to antifolates
Earlier studies, carried out on laboratory-reference isolates, demonstrated that pyrimethamine resistance is associated with point mutations in the dhfr domain of the dhfr-ts gene.63–65 The first step of pyrimethamine resistance arises with the mutation of amino acid Ser to Asn at codon 108 of dhfr. Ancillary mutations of Asn to Ile at codon 51 and of Cys to Arg at codon 59 are associated with a progressive increase in resistance. More than a 1000-fold increase in pyrimethamine resistance occurs when a fourth point mutation, Ile to Leu at codon 164, is added.64,66 These in vitro data have been supported by assays of the purified enzymes67,68 and by the analysis of the association between point mutations in DHFR and the in vitro susceptibility of falciparum parasites collected from different malaria endemic areas.59
Cycloguanil resistance is associated with point mutation at Ala to Val at codon 16 and Ser to Thr at codon 108; however, point mutations associated with pyrimethamine resistance also confer substantial cross-resistance to cycloguanil.64,65 Analyses of field isolates have shown that the presence of Val-16 and Thr-108 mutations is restricted in samples from South America.69,70 One of the reasons for this restrictive distribution is that this drug has been more extensively used in this area than in Africa and south-east Asia.
The dhps gene has been less intensively studied than dhfr, but the predominant mode of sulfadoxine resistance also appears to be point mutations in the dhps domain of the dhps-pppk gene. Changes in five different amino acids have been observed in P. falciparum in laboratory reference isolates: Ser-436 to Ala or Phe (S436A/F), Ala-437 to Gly (A437G), Lys-540 to Glu (K540E), Ala-581 to Gly (A581G) and Ala-613 to Ser or Thr (A613S/T).71–74 Although not all of the observed mutations have been tested in isolation for their effect on parasite resistance, all of those tested do increase resistance to sulfadoxine, to a number of other sulphonamides and to the sulphone dapsone.73 Excellent reviews have covered this topic, and we refer the readers to consult the publications by Sibley et al. 70 and Gregson and Plowe.75
4.2. In vivo resistance to the combination pyrimethamine/sulfadoxine (Fansidar®)
Extensive studies have been carried out to determine dhfr and dhps genotypes that could predict resistance to pyrimethamine/sulfadoxine. The result of these analyses demonstrate that pyrimethamine/sulfadoxine resistance is attributable to parasites that carry point mutations at codons 108, 51 and 59 of dhfr, and resistance is augmented by point mutations at codons 437 and/or 540 or 437 and/or 581 of the dhps gene.70,75 High levels of pyrimethamine/sulfadoxine resistance are associated with the selection of mutation at codon 164 of dhfr. Interestingly, this mutation is not commonly found in Africa. Using pharmacokinetic-pharmacodynamic information, our group has demonstrated that the selection of this 164 point mutation would be associated with a decrease in the efficacy of the newly developed antifolate Lapdap®.56 In support of this hypothesis, a clinical trial of Lapdap® in south-east Asia, where this mutation is commonly found, showed that it was ineffective in the treatment of uncomplicated malaria.76 Thus, there is a concern that the continuous use of pyrimethamine/sulfadoxine would select for this mutation in Africa, thus compromising the usefulness of Lapdap®. However, the existence of this 164 point mutation in Africa has been a matter of debate.77–80 A recent study reports the presence of this mutation in P. falciparum isolates from Malawi, using real-time PCR analysis, an indication that this mutation is being selected in Africa, in agreement with an early observation for P. falciparum isolates from Tanzania.77 It now remains to be seen whether this mutation will spread across Africa and also how it will affect the efficacy of Lapdap®.
It is important to note that the correlation of these genotypes with drug efficacy is a population phenomenon. The genotype of the parasite is only one determinant of the outcome of drug treatment in an individual patient. The complexity of the parasite infection, the nutritional status and the immune response of the patient are all important in determining whether a patient will clear his or her infection. However, when parasites with triple mutant DHFR and double or more mutant DHPS alleles are common in a region, the efficacy of pyrimethamine/sulfadoxine is compromised.70,75
4.3. New tools to study the mechanism of antifolate resistance in malaria
The current methods of genotyping Plasmodium dhfr and dhps are based on gene sequencing or the detection of point mutations by PCR–RFLP, PCR-allele specific oligonucleotide or sequencing techniques. In many malaria endemic sites, most falciparum infections are polyclonal, and the sensitivity of the standard approach is not high enough to allow for the detection of alleles that are present at low levels (<10%) in an isolate. As a result, rare resistant alleles cannot be detected. If molecular analyses are to be useful as an early warning for the emergence of highly resistant alleles, such alleles should be detected before their spread. To address this point, a yeast complementation approach has been developed, based on the expression of plasmodium DHFR genes in Saccharomyces cerevisiae (yeast cells) followed by the selection of cells expressing these highly resistant alleles.77,81,82 For instance, the application of this new technique has allowed the detection of quadruple mutants (164-Leu) in a few isolates in Africa; yet this mutation was not detected by the standard genotyping approach.77
Similarly, an Escherichia coli complementation system has been developed. The system is based on the transformation of E. coli with pf-dhfr alleles that have been generated by random mutagenesis.83 The endogenous bacteria DHFR is selectively inhibited by trimethoprim and this activity is complemented by pf-DHFR enzyme. By selecting pyrimethamine-resistant alleles using this approach, the authors have identified the same pf-dhfr alleles that occur in nature, in addition to detecting new alleles.83
These approaches (yeast and bacteria) can also be used to select in vitro DHFR-resistant alleles against antifolates that are not yet being used in the clinic. For instance, DHFR-resistant alleles against chlorcycloguanil and the experimental drug SO3 have been selected in vitro.83–85 In the case of chlorcycloguanil, now Lapdap® is in clinical use, it would be interesting to see whether these in vitro selected chlorcycloguanil-resistant alleles can be found in naturally occurring resistant isolates. Another interesting application of these new approaches is their use as screening systems for antimalarial agents.83–86 However, because of the limitation of yeast (and to a lesser extent, bacteria) to transport compounds, this technique could be restricted to highly lipophilic compounds.
4.4. Inhibition of folate salvage pathway: a potential target to increase antifolate activity
The malaria parasite can both salvage folate and synthesize it de novo. Both pathways increase the availability of folate in the cells. The potency of antimalarial sulfadrugs is due to the inhibition of de novo folate synthesis through the blockade of DHPS. Studies have clearly demonstrated that the addition of folate derivatives (folic acid or folinic acid) decreases the activity of antifolate drugs in vitro and in vivo.87–90 Likewise, the lowering of folate concentration in in vitro culture medium enhances the activity of antifolate antimalarial agents.56,91 This clearly shows that folate uptake makes a significant contribution to antifolate drug efficacy. Therefore, the inhibition of this salvage pathway could provide a rationale for the development of agents that could potentiate the activity of antifolate antimalarials.
The potential for such a strategy has been highlighted by investigations in the field of cancer chemotherapy. One of the mechanisms of methotrexate resistance in tumours is the overexpression of multidrug resistance associated proteins (MRP) which enhance methotrexate efflux from the cells.92 More importantly, inhibitors of MRPs, such as the uricosuric agent probenecid (PBN), can inhibit drug efflux via MRP, in mammalian cells,92 thereby reversing methotrexate resistance. In addition, PBN can inhibit folate uptake via a direct interaction with endogenous folate derivative transporters in mammals.92
We recently demonstrated that PBN, at a concentration of <100 µM, which is readily achievable in vivo in humans (http://www.medscape.com), increases substantially the activity of antimalarial anti-DHFR pyrimethamine, chlorcycloguanil and the anti-DHPS sulfadoxine and dapsone against sensitive and multidrug-resistant P. falciparum isolates. This ‘chemosensitization’ (or increase in antifolate activity) was also associated with a decrease in folate uptake into the parasite, explaining why PBN increases antifolate activity.93,94 We thus hypothesized that PBN could be used as a potentiator of antimalarial antifolates.93,94 Recently, this hypothesis has been borne out by clinical trials demonstrating that the use of PBN significantly increases the efficacy of the antifolate pyrimethamine/sulfadoxine in African children suffering from P. falciparum infection.95–97 This information indicates that inhibitors of the anion transporters could be of clinical importance in the treatment of malaria with antifolates, as it has now been proven in cancer treatment.98
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5. Antifolates in the pipeline |
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A number of new antifolate agents, based on pyrimidines and triazine structure and their potency against the pyrimethamine-resistant parasite, are being developed as potential antimalarials.
5.1. Diaminotriazine analogues of WR 99210
As described in section 3.1.4, WR 99210 could not be developed as an antimalarial because of its poor bioavailability and gastrointestinal intolerance.99 Based on the conversion of proguanil into cycloguanil, a biguanide precursor of WR 99210, PS-15, has been developed; this compound has better bioavailability properties and showed more potency than WR 99210 in an in vivo model.25,99–101 These observations have led to the development of analogues of PS-15, and some of these compounds are at a late stage in preclinical studies.102
5.2. Analogues of pyrimidine and cycloguanil
A group based in Thailand (Biotec Thailand) has conducted a research programme based on the synthesis and assessment of the antimalarial activity of analogues of pyrimethamine and cycloguanil.103 These studies have led to the identification of lead compounds whose development as antimalarials is supported by MMV (Medicines for Malaria Venture)104 (see also section 6.1).
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6. Future perspective |
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6.1. Screening programmes: crystal structure of DHFR
In the past, several attempts have been made to determine the structure of Plasmodium DHFR; however, all efforts have proved unfruitful because of the difficulty in obtaining crystals of high quality from parasite DHFR or DHFR-TS.105 This limitation has recently been overcome and the three-dimensional structure of P. falciparum DHFR has now been resolved by a group based in Thailand.105,106 This group has carried out extensive work based on predictive and crystallographic structures of DHFR protein, and on dhfr gene mutagenesis experiments to define the nature of interaction of amino acid residues in the active site of DHFR with key parts of effective inhibitor molecules, using pyrimethamine, cycloguanil and WR 99210 as reference drugs. These studies have led to the identification of the following features that are critical for the activity of inhibitors: hydrogen bonding of inhibitors with the carboxyl oxygens of the amino acid aspartate at codon 54 (Asp-54) and with the backbone of oxygens of the DHFR residue Ile-64; the length and flexibility of the side chain of the inhibitor, which should minimize the steric obstruction between the inhibitor and the Asn-108 mutated codon of DHFR; and the free space between the inhibitor and the Ala-16 residue, a codon associated with cycloguanil resistance.83,103,106–113 Based on this information, a number of inhibitors have been designed and tested against wild-type and mutant pf-DHFR enzymes. In line with their prediction, the authors have identified many inhibitors with interesting in vitro antimalarial activity and some of them have proven promising in vivo, in an animal model.103,109,114
In addition, many other groups, including ours, are carrying out in vitro screening to identify potent DHFR inhibitors.86,115–121
6.2. Targeting other folate enzymes
All antimalarial antifolates in clinical use or at the experimental stage target DHFR only or, less frequently, DHPS; yet, observations of the mammalian folate pathway and experience in cancer research indicate that other enzymes could represent good targets. For instance, currently, most antifolate anticancer agents used in clinical trials do not target DHFR; they target thymidylate synthase (TS) and folate based-purine-synthesis enzymes [5'-phosphoribosylglycinamide transformylase (GART) and 5'-phosphoribosyl-5-amino-4-imidazolecarboxamide formyltransferase (AICARFT)],7 and several folate enzymes [serine-hydroxy-methyl-transferase, folylpolyglutamate synthase (FPGS), methionyl-tRNA formyltransferase] are being investigated as potential targets against tumour cells.122,123 Many of these anticancer enzyme-targets have counterparts in P. falciparum; thus, they also have potential to become antimalarial targets. In a recent series of reviews, we have discussed and provided information on how some of these folate enzymes could be exploited as targets against P. falciparum.122,123
6.3. Use of the analogues of folate precursor as antimalarial agents
Sulfadrugs are analogues of pABA, and it is generally accepted that sulfadrugs are competitive inhibitors of DHPS. However, we demonstrated that 2-amino-4-hydroxy-6-hydroxymethyldihydro-pteridine pyrophosphate (HMDP-PP) can condense with sulfadoxine (an inhibitor of the enzyme dihydrofolate synthase) to generate an HMDP-sulfadoxine adduct, and that this adduct is toxic to the parasite;124 confirmation of an earlier study carried out in P. falciparum.125
Aminopterin and methotrexate are potent inhibitors of virtually all DHFR enzymes, including those of humans, and both drugs are used in the treatment of diverse malignancies.126 In vitro studies have shown that aminopterin and methotrexate are also potent inhibitors of P. falciparum growth.127–129 However, these compounds cannot be used directly to treat malaria because of their narrow therapeutic indices and the resulting life-threatening toxicity to the human host.
Based on the parasite's ability to metabolize analogues of folate precursors, we have hypothesized that precursors of methotrexate or aminopterin might be used to safely synthesize these potent inhibitors within the parasite cells. By this logic, when supplied with 2,4-diamino-6-hydroxymethyl-pteridine (DAP), 2,4-diaminopteroic acid (DAPA) and 2,4 diamino-N10-methyl-pteroic acid (DAMPA) (Figure 5), the parasite would synthesize aminopterin (from DAP, DAPA) and methotrexate (from DAMPA) de novo. We have established that DAP, DAPA and DAMPA bear antimalarial activities.130 Thus, if proven true, precursors of folate analogues could be used as a rationale to develop a new family of antifolate agents.
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7. Concluding remarks |
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The first antifolate antimalarial, proguanil, was discovered in the 1940s, during the Second World War. Following this discovery, chlorproguanil and pyrimethamine were identified as potent antimalarial agents. Fifty years later, these three drugs are still the mainstay antifolate drugs used in the treatment and prevention of malaria. For instance, the current most potent prophylactic agent is the combination of proguanil and atovaquone.21 Pyrimethamine/sulfadoxine has been the drug of choice used in malaria treatment throughout Africa, and it is still part of the antimalarial armamentarium in western parts of Africa.70 Chlorproguanil has recently been combined with dapsone, the oldest synthesized antifolate agent.60 The inhibition of malaria DHFR has been established to be effective in blocking P. falciparum growth since the 1940s; however, it is a surprise to note that no new antifolate has been discovered that has reached the clinical stage since that time.
The observation of work on antifolates in the treatment of tumours is informative and edifying. Methotrexate, a potent inhibitor of human DHFR—which was discovered at almost the same time as the antimalarial antifolate proguanil—has been the most widely used single cancer therapeutic agent. The success of methotrexate has led to sustained efforts to understand folate metabolism more completely and to develop new strategies for its disruption. Currently, five different enzymes of the folate pathway, DHFR, TS, FPGS, GART and AICARFT, are targets of anticancer drugs, and other folate enzymes are being evaluated as potential anticancer targets. Some of these anticancer folate targets have counterparts in P. falciparum.122,123 However, no work has been done to test the validity of the inhibition of these folate enzymes, except TS.131–136 In the case of TS, the strong similarity of the human and parasite enzymes was thought to be an impediment to the development of antimalarial TS. However, this cross-inhibition could be overcome by the use of pyrimidine nucleotides as adjuvant therapeutic agents, since the host cell can efficiently salvage them, but the parasite cannot, by-passing then the effect of the anti-TS compound against human cells.137 The mammalian folate pathway has provided several targets against tumour cells, and some of these targets could also be of critical value in the development of antimalarial agents.
The malaria parasite has a unique feature of being able to salvage exogenous folate derivatives and synthesize them de novo. The concentration of the exogenous folate in the medium has an impact on the activity of antifolates. The higher the amount of folate, the lower the activity of antifolates. Our group has recently established that inhibitors of anion transporters, such as probenecid, could lower the salvage of the exogenous folate, increasing the activity of antifolates in vitro,93,94 and these findings have also been confirmed in vivo.95–97 This is an interesting observation that could have clinical implications in the treatment of malaria if exploited: there now exists a possibility of increasing the efficacy of the antimalarial antifolates in vivo.
One of the striking differences between P. falciparum and mammalian folate is that the parasite can synthesize folate de novo but the mammalian cells cannot. This feature has been known for more than 50 years and has been exploited for the use of sulfadrugs in combination with anti-DHFR. We have recently shown that this de novo folate pathway could be exploited to identify a new family of antifolates, where precursors of folate analogues could be used as prodrugs against malaria.130 Since the host (mammalian cells) does not synthesize folate de novo, this approach, if proven true, could be used to identify active and safe compounds against malaria.
The unravelling of the malaria genome information has heralded a new era full of hope and expectation, where new targets against the malaria parasite could be discovered more quickly, which would lead to a rapid development of antimalarials. Efforts are now centred on target identification and target validation.138 While such effort should be pursued to identify good drug targets, we should also question why targets that have already been proven to be good have not been exploited to deliver new antimalarial drugs. The inhibition of the folate pathway is one example. Experience of folate inhibition as a means of treating cancer cells indicates that the malaria folate pathway, if exploited, could provide a good platform for the identification of antimalarial agents.
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Transparency declarations |
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None to declare.
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Acknowledgements |
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I thank the director of the Kenya Medical Research Institute for permission to publish these data. This work was supported by the European Developing Countries Clinical Trials Partnership (EDTCP) and KEMRI/Wellcome Trust Research Program, Kenya. A. N. is an EDTCP senior fellow.
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