Current Drug Targets - Infectious Disorders - Volume 4, Issue 1, 2004

Drug resistance is one of the major factors contributing to the resurgence of malaria, especially resistance to the most affordable drugs such as chloroquine and Fansidar™, a combination drug of pyrimethamine and sulfadoxine. Understanding the mechanisms of such resistance and developing new treatments, including new drugs, are urgently needed. Great progress has been made recently in studying the mechanisms of drug action and drug resistance in malaria parasites, particularly in Plasmodium falciparum. These efforts are highlighted by the demonstration of mutations in the parasite dihydrofolate reductase and dihydropteroate synthase genes conferring resistance to pyrimethamine and sulfadoxine, respectively, and by the recent discovery of mutations in the gene coding for a putative transporter, PfCRT, conferring resistance to chloroquine. Mutations in a homologue of a human multiple-drug-resistant gene, pfmdr1, have also been shown to be associated with responses to multiple drugs. However, except in the case of resistance to antifolate drugs, the mechanisms of action and resistance to most drugs currently in use are essentially unknown or are being debated. Additionally, novel mechanisms of resistance exist in different malaria parasites, complicating the process of developing new drugs and treatment strategies. Here we summarise the progress made in drug resistance research in malaria parasites over the past 20 years, emphasising the most recent developments in the genetics of drug resistance.

Malaria parasites possess three genomes: the nuclear chromosomes, the mitochondrial genome, and the plastid genome. Realization that the parasites contain a plastid remnant with its own genome has created much excitement not only from a basic biological point of view but also from the prospects for developing new antimalarial drugs. Both the mitochondrial and the plastid genomes are the smallest examples of their kind known to date. The plastid appears to be derived from an ancestral secondary endosymbiotic event. Interestingly, the main functions usually associated with a mitochondrion and a plastid, i.e. oxidative phosphorylation and photosynthesis, do not appear to be conserved in malaria parasites. Completion of the parasite genome sequence has provided the opportunity to assess functions assigned to these highly derivatized organelles. It is clear that these organelles serve vital functions since interference with their activity is incompatible with parasite growth. A number of antimalarial compounds target functions of either the mitochondrion or the plastid. This review will survey our current understanding of mitochondrial and plastid functions with a view to identify processes that are or have a potential to be targets for antimalarial drugs.

The creation of databases that make enormous and diverse amounts of information available, the coding of algorithms that allow the collection and investigation of these data and the wide availability of desktop computers capable of handling the data and running the algorithms have set the stage for innovative approaches to drug target identification. Here we review the main currents in this new field, providing an overview of some of the databases and software used to generate and shorten the lists of potential drug targets using in silico methods. As a case study, we look at the identification and investigation of malarial proteases as therapeutic targets in the Plasmodium spp. parasites.

Ever increasing drug resistance by Plasmodium falciparum, the most virulent of human malaria parasites, is creating new challenges in malaria chemotherapy. The entire genome sequences of P. falciparum and the rodent malaria parasite, P. yoelii yoelii are now available. Extensive genome sequence data from other Plasmodium species including another important human malaria parasite, P. vivax are also available. Powerful research techniques coupled to genomic resources are needed to help identify new drug and vaccine targets against malaria. Applied to Plasmodium, proteomics combines high-resolution protein or peptide separation with mass spectrometry and computer software to rapidly identify large numbers of proteins expressed from various stages of parasite development. Proteomic methods can be applied to study sub-cellular localization, cell function, organelle composition, changes in protein expression patterns in response to drug exposure, drug-protein binding and validation of data from genomic annotation and transcript expression studies. Recent high-throughput proteomic approaches have provided a wealth of protein expression data on P. falciparum, while smaller-scale studies examining specific drug-related hypotheses are also appearing. Of particular interest is the study of mechanisms of action and resistance of drugs such as the quinolines, whose targets currently may not be predictable from genomic data. Coupling the Plasmodium sequence data with bioinformatics, proteomics and RNA transcript expression profiling opens unprecedented opportunities for exploring new malaria control strategies. This review will focus on pharmacological research in malaria and other intracellular parasites using proteomic techniques, emphasizing resources and strategies available for Plasmodium.

The completed Plasmodium falciparum genome sequence poses a significant challenge: how do we bring this wealth of data to bear against the steady march of malaria parasites towards multiple-drug resistance? Studies of parasite drug resistance have until now focused on a qualitative, single-gene concept of resistance determination; however, the emergence of powerful genomics tools insists that these questions be rephrased. It is now possible to address the true genetic complexity underlying quantitative drug sensitivities. Quantitative trait loci (QTL) mapping is an effective tool for tracking multi-gene traits by partitioning genetic effects that influence these traits into specific genomic regions. A cross between two parasite clones captures allele combinations that have segregated into progeny clones that display varying sensitivity to drugs. The specific allele forms and their combinations contributed by each parent are, in effect, genetic signatures of their unique evolutionary histories. In addition to resistance genes, per se, a drug resistant parasite carries coevolved gene combinations comprising a genetic background of drug resistance. Research into drug resistance necessarily has been directed at specific genes and mechanisms favored by a priori knowledge and assumptions about how resistance works. QTL mapping, by superimposing real biological phenotypes on genome sequence, structural polymorphisms, and gene expression data, can provide an alternative, unbiased view of the network of gene actions that build a complex phenotype. Through an integrated approach, studies can move beyond the search for markers of resistance to instead characterize the predisposition of parasites to develop new resistances and cross-resistances.

When alleles conferring drug resistance spread through a population of malaria parasites, they leave characteristic “scars” in the parasite genome. Flanking neutral polymorphisms “hitchhike” to high frequency with the resistance mutation, generating deep valleys of reduced variation and broad swathes of elevated linkage disequilibrium around the resistance locus. We can systematically search the genome for these scars by genotyping polymorphic marker loci at intervals throughout the genome of P. falciparum, and use them as signposts for locating drug resistance genes. In this review I outline the rational behind this approach to genetic mapping. I describe key features of P. falciparum population biology, such as recombination rate, inbreeding, and selection intensity that influence the size of genomic regions affected by selection and the choice of study population. I discuss suitable genetic markers, study designs, and statistical approaches to data analysis. Finally, to demonstrate the utility of the approach I describe two proof-of-principle studies documenting patterns of genetic variability around known drug resistance genes.

Aside from its profound clinical importance, the human malaria parasite, P. falciparum, is intrinsically fascinating to the cell biologist because it resides within the mature red blood cell (RBC). Because the inert RBC cannot otherwise provide sufficient amounts of key substrates to fuel the vigorous parasite metabolism, the parasite must have specialized adaptations for getting these solutes into the RBC and scavenging them from RBC cytosol. Two unusual ion channels have recently been identified within the infected RBC complex; these channels likely function in a sequential diffusive pathway of nutrient acquisition by the intracellular parasite. If so, they present novel opportunities for identification of ion channel inhibitors that may be useful antimalarial compounds.