Malaria a_ects more than 40% of the worlds population, and is accountable for between 1 and 2 million deaths per year. About four-_fths of these are in Africa . The infection is caused by the protazoan Plasmodium, which is transmitted between humans by the bite of the female mosquito Anopheles. Four malarial species are capable of infecting humans Plasmodium malariae, P. vivax, P. ovale and P.falciparum, the latter causing the most severe form of the disease .
The life cycle of the malaria parasite is divided between the two hosts. Within the human host the cycle can be further divided into the liver stage and the blood stage. The malaria life cycle begins when the infected female mosquito bites the human host. During this process blood is withdrawn from the host and sporozoite-containing saliva injected into the skin capillaries. Sporozoites migrate to blood vessels, where blood circulation transports the sporozoites to the liver, initiating the human liver stage of the life cycle (Figure 0.1 A). Sporozoites infect hepatocytes and mature into schizonts. Within the schizont rapid nuclear division takes place producing up to 30 000 exo-erythrocytic merozoites. The schizont ruptures, releasing the merozoites, which are now free to infect erythrocytes. The release signalling the start of the asexual blood stage (Figure 0.1 B). The cycle is characterised by the formation of trophozoites, which mature to form erythrocytic schizonts .With the rupturing of the erythrocyte merozoites are released from the erythrocytic schizonts which can invade other erythrocytes . During the blood cycle not all parasitic cells develop into meroziotes. Some will develop into dormant male and female gametocytes (Figure 0.1 C). During a blood meal by the female Anopheles mosquito, these dormant gametocytes are ingested. Once ingested, they are activated and fertilisation takes place when the male gamete penetrates the female gamete, resulting in the formation of a diploid zygote. Prior to the formation of a motile ookinete, which will penetrate the midgut forming an oocyst, the zygote undergoes a single round of meiotic division. The ookinete will mature to form a oocysts, that releases sporozoites that migrate to the salivary gland ready to be passed on to the human host, completing the cycle . Pathology of the disease is mostly caused during the blood cycle of the parasite; it is also the stage of the life cycle where the parasite is most susceptible to antimalarial drugs. For most of the quinoline based antimalarial act exclusively on the blood stage . The _rst cure for malaria was made from granulated bark of the cinchona tree. These extract contained quinoline alkaloid extracts, and was distributed widely in Europe .
Variation the the quality of the bark and also the extracts served as a driving force for chemist to isolate the active ingredients, but this also proved to have its obstacles since the synthetics preparation was to complex for commercial preparation. Plantations were established in Java. With the onset of World War I and World War II the production was either to low or availability entirely discontinued due to occupation by enemy forces. In turn the low availability was the _rst driving force for development of alternative antimalarial drugs, with chloroquine (a 4 Aminoquinolines based drug) and pyrimethamine replacing quinine [5, 7].
The second greatest force with regards to development of antimalarial drug was one again driven by was. Chloroquine resistance developed during the Vietnam War, posin a major problem for soldiers stationed in these areas. The Walter Reed Army Institute for Research, started a large scale screening proses for new antimalarial drugs. Of the thousands of compounds screened only a small amount showed possible action against malaria. One of the drugs found to have an antimalarial e_ect was Meoquine, which forms part of the quinolinemethanols. It is certain that the biggest driving force behind antimalarial drug development was in most cases war related. Considering that since the Vietnam war research has been greatly neglected until the great surge in in parasites showing resistance to the drug and the e_ect of these drug resistance on world population .
In order to understand the complexity of these drugs and the development of resistance is is important to look at both the mechanism of action of these antimalarial as an agent to eradicate the parasite, and also the mechanisms developed by the organism to overcome the e_ect of these antimalarial. The understanding of the mechanism of action of the existing drugs, could in e_ect provide a indication as to where to target the parasite with new drugs development. Regrettably there is a de_ciency current understanding in either the mechanism of action or resistance.
Mechanisms of action for antimalarials
When studding resistance to drugs, one should _rst look at the mechanism of action of the current antimalarial drugs. Despite the amount of progress that has been made in this _eld, some aspects still defy our current understanding, taking into account the number of proposed models of action that has been put forward with regard to these drugs.
Aminoquinolines - Chloroquine
To obtain amino acids, essential for parasitic growth, hemoglobin is obtained from the cytosol of the erythrocyte via endocytosis. Hemoglobin is degraded within the digestive vacuole of the parasite by a series of proteases. The breakdown of hemoglobin, produces haem (ferriprotoporphyrin IX). Haem a toxic and soluble molecule. The parasite does not possess the ability to degrade this toxic molecule. To overcome this shortfall the haem is polymerised into nontoxic insoluble crystals of hemozion [8, 9].
It is proposed that the primary mechanism of chloroquine action interferes with the production of the hemozion crystals. The accumulated chloroquine limit hemozion production by forming a tight complex with the haem molecules, the haem-chloroquine complex. The heme-chloroquine complex is highly toxic (Figure 0.2). The formation of this complex prevents further polymerisation of heme to form crystals. Without polymerisation of heme to hemozion it leads to heme and heme-chloroquine buildup. Action of the toxic haem-chloroquine complex and unbound haem, results in cell lysis and ultimately parasitic cell death. Fundamentally the parasite is poisoned by its own metabolic waist [10, 11, 12].
Chloroquine is only active during the blood stage (Figure 0.1 A), narrowing the scope of possible chloroquine targets. Chloroquine action is dependent on accumulation within the vacuole. Chloroquine has a unique characteristic,in that concentration buildup outside the parasitic cell is very low compared to the the concentration buildup inside the digestive vacuole of the malaria parasite during the blood cycle . One explanation for chloroquine accumulation within the digestive vacuole, follows an ion-trapping mechanism (Figure 0.2). This mechanism is based on the fact that chloroquine is a weak base [14, 15]. The unprotonated form of chloroquine crosses the membrane, moving down the pH gradient and accumulates in the acidic digestive vacuole of the parasite. The acidic surroundings protonates the drug. The protonated form of chloroquine (CQ2+), is trapped since it is now membrane impermeable .
A second study proposes an alternative for chloroquine accumulation. This model proposes a carrier mediated import system. A proposed plasmodial Na+/H+ exchanger acts as an chloroquine importer . Chloroquine binds to the Na+ antiporter side of the Na+/H+ exchanger and is exchanged for protons. One proposed mechanism of resistance is motivated by possible changes in this mechanism .
Several other mechanisms have also been proposed based on speci_c actions of chloroquine on other related mechanisms or close relation to metabolic actions of the parasite. During the degradation of hemoglobin there is a production of reactive oxygen species, which accounts for oxidative stems. Within most cells antioxidant are used to protect the cell from these compounds. The malaria parasite has been shown to be sensitive to oxidative stress. Furthermore haem displays some catalase activities which would reduce the oxidative stress. However haem also has peroxidase activity that will increase the oxidative stress. It has been noted that chloroquine seems to be an e_ective inhibitor of the catalase activity of haem, that would in turn increase the oxidative stress by increasing the e_ect of reactive oxygen species .
Quinolinemethanols - Quinine and Meoquine
As with chloroquine the primary interaction of the drug appears to be during the blood stage of the parasite life cycle. However the antimalarial mechanism is still poorly understood. Some studies show evidence that quinine mechanisms might be similar to that of chloroquine. This has stem from the observation that quinine can compete with chloroquine for haem binding. Also there is an observed change in the digestive vacuole which resemble the changes observed in the parasitic digestive vacuole after treatment with the 4 aminoquinoline - chloroquine. However the accumulation of meoquine and quinine due to ow down the pH gradient is somewhat challenged, if considered that meoquine and quinine are both much weaker bases than chloroquine. When considering the binding of meoquine and quinine to haem is is also weaker than that of chloroquine, thus challenging the model that haem binding being the major target for these drugs. An alternative explanation for quinine and meoquine accumulation states that it might be due to a carrier mediated transport system [19, 20].
Mechanisms of resistance against antimalarials
The partial understanding of the mechanism of action has led to some clues to how the parasite has developed resistance to the drug. Since there is still a limited understanding of the mechanism of action of the antimalarial drugs, it has led to a similar problem with the study on the mechanism of resistance. Resistance against antimalarial drug seems to be related to speci_c drugs, and several models have been proposed on how resistance is developed, and the speci_c mechanism related to to these models.
These models focus mainly on the current antimalarial drugs, but with the search for new drug targets, studies have also been done to identify how the parasite might develop resistance against new drug targets.
Resistance to chloroquine
Chloroquine resistance seems to be associated with the increase distribution of the drug as an antimalarial. When the phenotype for resistance is established within a population it tends to be stable, expressing the phenotype even in the absence of the drug. Multiple mutation in several genes might have been needed to obtain the phenotype if one considers the length of time it took for resistance to develop [13, 21].
The chloroquine resistant (CQR) parasite phenotype is identi_ed by the reduced level of chloroquine accumulation in the digestive vacuole [12, 22]. This poses the possibility that resistance is due to reduced amount of chloroquine binding to haem and not necessarily an alteration in the drug target . The question now arises on how the reduction of chloroquine accumulation is accomplished?
One possible mechanism to explain this came from the notion that there might be a di_erence in the pH of the digestive vacuole of the resistant parasite compared to that of the chloroquine sensitive (CQS) parasite. This builds on the mechanism of action of chloroquine where the drug is transported into the digestive vacuole by the ion-trapping model (Figure 0.3 A). This di_erence in pH, accounting for a change in ion-trapping, would lead to lower accumulation. However recent studies have shown that the di_erence in pH between the CQR and CQS strains is not signi_cant enough to account as the only possible mechanism for resistance . Other studies have show that there is a change in the pH of digestive vacuole of the CQR compared to that of CQS .
A second proposed mechanism was hypothesised after the observation by Krogstad et al , which reported that chloroquine ow was much faster out of CQR parasites than from a CQS parasite using a verapamil blockable route. Taking into account that verapamil is known to block P-glycoprotein-mediated e_ux it was proposed, that the e_ux of chloroquine by CQR was due to the plasmodial P-glycoprotein. This e_ux hypothesis, states that an increased chloroquine e_ux leads to a decrease in chloroquine accumulation within the digestive vacuole [25, 26].
Multiform resistance occurs when a cell line becomes resistant to a range of structurally unrelated drugs. Reversal of chloroquine resistance can be seen in vivo, when there is a co-administration of compounds. These compounds also show modulation in human tumour cell lines. This discovery led to the suggestion of an alternative multidrug resistant phenotype. Ampli_cation studies con_rmed this with the discovery of two related genes pfmdr1 and pfmdr2 [27, 28].The gene product of pfmdr1 and pfmdr2 being Pgh1.This study led to the discovery of the vacuole membrane protein Pgh1 . Expression of the mdr phenotype, have been shown to be linked to increased levels of resistance. Other studies show that resistance to chloroquine might be linked to the pfmdr1 gene product,Pgh1 . Studies have also revealed that overexpression of the gene product actually increase sensitivity to the drug ,which di_er to most studies which indicate that overexpression decreases the sensitivity to the drug  . With the mdr phenotype shown as being dependant on ATP hydrolysis, it supports the idea that P-glycoprotein may act in an energy depended fashion .
With the discovery of a DNA segment on chromosome 7 that was linked to CQR, attention shifted to the gene that encoded for a transmembrane protein of the digestive vacuole, termed P. falciparum chloroquine resistant transporter (pfcrt). PfCRT with a predicted ten transmembrane domains that is located on the digestive vacuole, seems to be the major determinant of chloroquine resistance . Within the pfcrt, twenty point mutation have been observed. The most characteristic point mutations regarding chloroquine resistance is accounted for by a substitution of threonine (T) for lysine (K) at position 76. Various studies have shown that chloroquine resistance is dependent on this pfcrt mutation. PfCRT has a greater association with chloroquine resistance than pfmdr1,nonetheless a combination of pfcrt and pfmdr1 together result in higher levels of chloroquine resistance .
Models of chloroquine resistance assisted by PfCRT
PfCRT assisted resistance might result from a from a drug e_ux, this mechanism requires either a channel or a transporter. From this two models of possible mechanisms have evolved, one relating to a transporter and the other to a channel. The _rst proposed model, where PfCRT on the digestive vacuole plays a role is one where PfCRT functions as a channel. This mechanism enables protonated CQ (CQ+) and (CQ2+) to ow out of the food vacuole down its electrochemical gradient. The loss of the positive charges that is the result of the K76T, might account for the ability of the drug to move from the digestive vacuole back into the cytoplasm (Figure 0.3 D) [33, 34]. The digestive vacuole membrane potential might drive chloroquine out of this organelle in chloroquine resistant strains. In chloroquine sensitive parasites, the channel might be blocked by the positively charged lysine within PfCRT. A channel for protonated chloroquine provides an acceptable model for the low accumulation of chloroquine in the CQR parasites.
The transporter model where PfCRT on the digestive vacuole play a role, correlates with the notion that the mdr phenotype is dependent an ATP hydrolysis (Figure 0.3 B), (Figure 0.3 C). The energy obtained from the hydrolysis might be used to transport the protonated CQ (CQ+) and (CQ2+) out of the digestive vacuole. The transport of chloroquine is either done directly via the mutated PfCRT and ATP hydrolysis, or indirectly where PfCRT transport of drug is due to electrochemical gradient maintained by a digestive vacuole proton pump which is also dependent on ATP hydrolysis. In the indirect ATP dependent transporter models the digestive vacuole proton pump drives facilitated di_usion which can be explained why the protonated form of chloroquine leaves the digestive vacuole. Here, energy is proposed to drive and to maintain the concentration gradient of protonated chloroquine, rather than being directly coupled to drug movement. This might be due to the K76T mutation within pfcrt converting the exchange carrier to a transporting carrier.
Both the channel and energy-coupled transport models shows that PfCRT is directly involved in chloroquine movement. The main di_erence of these two models and the mechanism relating to drug movement, follows that in the transport model the protein actively e_uxes the drug, and in the channel model, protonated chloroquine passively leaks out of the digestive vacuole.
Resistance to quinoline
In areas where chloroquine resistance is high, treatment has been mostly replaced by quinine. Quinine resistance is not as wide spread as that of chloroquine, but there is a observed decrease in sensitivity as the use of the drug is increasing . In most cases meoquine resistance has a strong correlation with the pfmdr1, this is due to the fact that ampli_cation and overexpression of the gene product seems to correlation with meoquine resistance . There might also be a similar mechanism for quinine since resistance of the parasite shows correlations to that of meoquine resistance . Resistance of the drugs is not reversed the administration of the drug verapamil, as is seen in chloroquine resistance which indicates that that the two mechanisms follow somewhat di_erent methods.Other studies have shown that a decrease in the copy number pfmdr1, increases the susceptibility of the parasite to these drugs . For resistance against these drugs the major focus seems to be the copy number of the pfmdr1 gene. Where in increase copy number showed increased resistance to meoquine, and a decrease copy number showed heighten susceptibility to not only meoquine, but also other related quinoline drugs. [12, 37].
Development of new drug targets
When identifying possible new drug target one should not only focus on the action of antimalarial drugs, but also on possible targets of the parasite metabolism. Much research has been done in the study of metabolic pathways and the extension of these as possible targets for drugs. It is these pathways that could serve as identi_cation of drug targets. Glycolysis has been shown to be an essential energy-producing pathway in malaria, with lactate being the primary catabolic product of anaerobic glycolysis. It has also been show that the antimalarial drug, chloroquine interacts with lactate dehydrogenase in the parasite (PfLDH). PfLDH is not a_ected by chloroquine alone, indicating that PfLDH is not the direct target of the antimalarial. Also PfLDH has been shown to be very sensitive to haem, a product of parasite metabolism. Since chloroquine forms the chloroquine-haem complex when accumulated in the digestive vacuole, it might actually protect the PfLDH from the inhibitory e_ect haem . PfLDH is responsible for NAD+ NADH recycling, which might inhibit ATP production. This makes the enzyme a good target for a drug . Malaria protein structure studies could also provide novel targets for drugs. An increased understanding of the structure, could be used in drug design .
The understanding of the mechanism of action plays a vital role, for it does not only help us to understand the metabolic action of the parasite but also help us to identify possible drug target. The action of the various drugs on the parasite, help one to identify areas where targeting of drugs would be best suited. It is also important to look at alternative targets. In order to focus on possible alternatives, the span must be increased and must not only be focus on the current drugs actions, but also possible new compound interaction. The current view on the mechanism of drug action is incomplete. This is only due to the fact that there is a still a great amount of research that must be done in order to reach a consensus on the mechanism, as it seem that for every possible proposal of a mechanism there is inadvertently an opposing mechanism. It might be in itself, this gap that is the greatest driving force for developing a greater understanding of the mechanism of these drugs.
The driving force behind antimalarial drug development is three pronged. Firstly the treatment of the disease. When one considers the impact of malaria, and the amount of deaths accounted for by malaria. Infections by malaria cannot be considered as inconse- quential.
Secondly by the need for more e_ective drugs as the parasite builds up resistance against the drug. In order to produce drugs that are more e_ective against malaria, there is a great need to not only understand the mechanism of the currently used drug, but also understanding of how the parasite acts to overcome the e_ect of the drugs. On the other hand when one considers the history of man with malaria, we were ones before so brave as to think that we have eradicated the problem. It is due to this gap in research has, to some extend led as to the revelation that continuous research within this _eld must be considered vital.
Finally, the need for funding to support research for more e_ective drugs. It is para- doxical to think that is was in fact the contribution of Wold War I and II, which supplied the greatest amount of research into antimalarial drugs. Although extensive work has been done in the last couple of years with the great proliferation of drug resistance, we are yet far from completely understanding both the mechanisms involved with action of antimalarial drugs and the resistance of the parasite against these drugs. This can be seen from the In order to win the war against malaria we must increase our arsenal of knowledge of ways to overcome drug resistance