Bloodsucker rising

The sun rises to greet another hot and humid morning at the encampment near the pond. As the haze slowly lifts from the still waters, the children of the camp begin to stir and skitter near the water’s edge, like so many small insects. Before one young boy can swat at the mosquito gorging on his arm, it flies off, taking with it a healthy dose of life-giving blood and leaving behind a small present in return. Within the boy’s bloodstream, single-celled creatures swim toward his liver, looking to slake their own blood lust. Within weeks, the boy’s body will be wracked with fever, fatigue, diarrhea—another victim of malaria.

But the boy is not just another statistic among the myriad people living in tropical climes stricken with the disease. The 11-year-old victim, struck down in the summer of 1999, was a resident of Suffolk County, NY. Neither the boy nor his family had traveled to malaria-endemic areas, and health officials were unable to identify other infected people in the area near the camp.

For almost 300 years, malaria was endemic to the United States. It was only through the wide use of pesticides such as DDT that, by the 1950s, malaria was finally brought under control. And although there have been only 60 U.S. cases reported in the past 30 years—most acquired abroad—global travel, recent experiences with West Nile virus, and the mounting evidence of global warming suggest that locally acquired malaria may become a bigger problem. Add to this the rising incidence of drug resistance in the malarial parasite population, and the Northern Hemisphere had better begin to prepare itself for a new onslaught of the disease.

Epidemiology
Worldwide, 300–500 million cases of malaria, transmitted by anopheline mosquitoes, are reported annually and result in 1–2 million deaths, mostly of children under the age of 5—almost one child every 30 s. The World Health Organization (WHO) estimates that 2.4 billion people, 40% of the planet’s population, are infected with Plasmodium species parasites. Almost 90% of the cases occur in sub-Saharan Africa, where its annual economic toll may exceed $2 billion—1 to 5%of the continent’s GDP.

Predominantly a disease of rural communities, malaria’s economic burden is particularly heavy because of increased incidence around harvest time, when families earn most of their annual income. Also, as malaria hits children the hardest, it keeps the young from attending school, further slowing their progress. Finally, as urban populations grow and encroach upon farms and forests, the incidence of malaria in cities is rising and can spread quickly among densely packed city dwellers.

As the New York case indicates, malaria is reaching outside of its traditionalequatorial boundaries. Both the rise of chloroquine-resistant parasites and concerns over the use of insecticides like DDT have hampered efforts to control malaria in the developing world. Given the relative ease with which people travel from region to region, these factors have increased the incidence of the infection further north.

So where does this leave us, when the most prominent prophylactics and treatments are becoming less effective with each passing month?To understand the ways in which researchers are tackling the problem of malaria, it is important to have a good understanding of the life cycle of the malarial parasite.

Life cycle
Plasmodia go through several stages of development as they pass through mammalian and mosquito hosts. During a blood meal on an infected person, an anopheline mosquito ingests a population of malarial parasites in the form of gametocytes. While in the mosquito’s gut, the parasites reproduce to generate sporozoites, which travel to the insect’s salivary glands. During the mosquito’s next blood meal, sporozoites enter the victim’s bloodstream and make their way to the liver. Here, the parasites reproduce to generate thousands of merozoites, which pass back into the bloodstream and infect red blood cells (RBCs). The merozoite sits within a vacuole in the RBC, using proteases to digest hemoglobin, and generates more merozoites. These progeny then enterthe bloodstream to infect more RBCs. Eventually, the merozoites generate gametocytes that are picked up by another mosquito.

Given the seemingly limitless range of potential targets—surface antigens, metabolic steps—one would think that there would be no end to the number of treatment methods available to health workers around the world. Unfortunately, this is not the case. Although researchers have had a broad understanding of the parasite’s life cycle, the biochemical details have eluded them. The tide seems to be turning, however, as researchers examine the parasites and hosts more closely at societal, genetic, and biochemical levels.And, if the recent literature is any example, malaria may yet be eradicated worldwide as it once was in the United States.

Society
As the fallout after the rampant spraying of DDT subsided and chemical companies developed safer compounds to challenge malarial vectors, it became common practice to spray the interior of homes in malaria-endemic areas. Further, insecticide-treated nets to cover the sleeping areas became popular—baited traps that lure mosquitoes onto a toxic surface. Unfortunately, because of the expense of annual spraying and treated netting, these methods are of limited use.To get around the challenges, some researchers have started to take advantage of the fact that humans are not the only victims of mosquito attacks.

Researchers recently reported on a three-year study in which cattle that live among the residents of northwest Pakistan were sponged with the insecticide deltamethrin. The benefits were immediate and significant: Ticks were eliminated, the cattle gained weight, and milk production increased by more than 10%. Mosquito populations dropped almost 50%, and the incidence of malaria in humans dropped 30–55% in the treated populations, suggesting that highly targeted insecticide use might address some of the vector-related issues.

At the same time, other researchers are looking for ways to limit the effects of malaria on infected individuals, and potentially for a cure.

Genetics
Given the recent spate of genome projects, Plasmodium was not to be outdone. Researchers at several locations have been working on cataloging the sequences contained in the 14 chromosomes of the parasite, depositing the information at the Plasmodium Genome Resource (www.plasmodb.org). To date, researchers have completely sequenced two chromosomes and have a handle on several others. With the complete genome, researchers will be able to tackle the problem of malaria from a variety of angles.

Researchers have already been able to identify specific markers associated with resistance to chloroquine treatment (more on the markers below).Using a PCR-based test, clinicians can quickly diagnose by what strain of parasite an individual has been infected and thus more easily select the appropriate treatment option.

Although vaccines are being developed at a variety of centers around the world (Table 1), coordinated in part by the Malaria Vaccine Initiative (www.malariavaccine.org), the putative vaccines fall into two categories: those designed to prevent clinical manifestations of malaria, and those designed to limit death and disease. The first group attempts to prevent the infection of the liver or RBCs by immunizing the body against proteins found on the sporozoites (Figure 1). The second group targets infected RBCs (presenting malaria-specific antigens) or the merozoites.

Biochemistry

Figure 1. Vaccine targets. Throughout its life cycle, Plasmodia express a variety of proteins on its surface or on that of the cells it infects, providing myriad targets for an immunological assault.

The task of attacking malaria biochemically has traditionally fallen to compounds that are members of the quinine family, but the rising resistance to chemicals like chloroquine has resulted in a need to discover new directions of attack. In part, this push has been led by organizations such as the Medicines for Malaria Venture (www.mmv.org) and WHO. Although the number of steps in the parasite life cycle makes it difficult to stop by methods such as vaccination, it does set the stage for a multiplicity of potential chemotherapeutic targets.

Before the sporozoites can be transmitted from the mosquito to the human bloodstream, they must pass from the insect’s gut to its salivary glands. Researchers at Johns Hopkins University (Baltimore) recently identified two proteins specific to these glands in female mosquitoes. The group generated monoclonal antibodies (mAbs) against the proteins and discovered that feeding the mAbs for one of the proteins to mosquitoes inhibited the invasion of sporozoites into the salivary glands by almost 75%. Although these experiments were performed with mAbs, the researchers speculate that the same or better results may be achieved with polyclonal antibodies against a range of mosquito-specific proteins.

Another popular target, and the one where chloroquine plays its role, is the digestion of hemoglobin by the parasite. As a nutrient source, the parasite ingests hemoglobin into the food vacuole (FV) and uses proteases to break it into amino acids, resulting in free hemeas a toxic byproduct. Within the FV, the hemeaggregates, forming an inert product called haemozoin. Drugs like chloroquine and artemisinin—from a Chinese herb—inhibit this aggregation process, thereby creating a toxic buildup of heme.Recent research, however, suggests that the parasites have been able to get around this stumbling block by altering the pH conditions of the FV. The researchers discovered that mutations in a gene called pfcrt correlate with the incidence of chloroquine resistance. The gene appears to encode a transmembrane protein that sits in the lysosomal membrane and may facilitate the transport of organic cations across the membrane, resulting in a pH drop within the FV. The researchers speculate that the acidification may affect the ability of chloroquine to disrupt hemeaggregation, thus pushing the equilibrium toward haemozoin. Although this spells trouble for the use of chloroquine or other aggregation-promoting compounds, an improved understanding of the mechanism of resistance opens the door to new treatments.

Other researchers have taken one step back from the formation of haemozoin and are instead targeting the proteases that degrade the hemoglobin. Researchers at the University of California, San Francisco, are developing a series of compounds based on peptidyl fluoromethyl ketones and vinyl sulfones to target falcipain, a cysteine protease involved in hemoglobin digestion. Initial studies using inhibitors parenterally administered to mice showed very promising results, with almost 80% of the mice being cured of malaria. But this administration route is not widely applicable, so the group concentrated its efforts on an oral route. Although the results with the fluoromethyl ketones were only moderately promising—showing slower disease progression—one vinyl sulfone inhibitor showed a 40% cure rate and a marked delay in disease progression in the remaining mice, suggesting that these compounds are strong lead candidates for further study.

Another area in which protease inhibitors may prove effective is in the release of infective merozoites from RBCs. As the parasites exit the RBC, they do so as a group of 16–32 merozoites wrapped in a membrane. It has been known for some time that proteases are involved in the subsequent release from the membrane, but the specific mechanism has been a mystery. In a study at the Howard Hughes Medical Institute (St. Louis, MO), researchers inhibited this release using the cysteine protease inhibitor E64 and were able to determine that the membrane was distinct from that of the RBC. Instead, the merozoites were trapped within the parasitophorous vacuolar membrane (PVM), rendering them unable to infect other RBCs. Although the researchers were unable to explain the role of the protease in the release of the merozoites, the existence of the PVM-encapsulation process does offer yet another avenue of attack, whether through further protease inhibitor studies or other membrane-associating drugs.

Of course, these examples only represent the tip of the proboscis, as it were (Table 2), as researchers continue to look for ways around drug resistance (e.g., ferrochloroquines) and to develop new compounds from natural sources (e.g., manzamines).

Regardless of how the problem is addressed, malaria is ripe to become an even bigger problem in the near future, and it is in the best interests of the Western world to focus its pharmaceutical crosshairs on a challenge that will not back down easily.

Further reading

• Brennan, J. D. G.; et al. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 13,859–13,864.
• Cowman, A. F. Intl. J. Parasitol. 2001, 31, 871–878.
• Djimdé, A.; et al. New Engl. J. Med. 2001, 344, 257–263.
• Doolan, D. L.; Hoffman, S. L. Intl. J. Parasitol. 2001, 31, 753–762.
• Persidis, A. Nature Biotechnol. 2000, 18, 111–112.
• Salmon, B. L.; Oksman, A.; Goldberg, D. E. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 271–276.
• The Plasmodium Genome Database Collaborative. Nucleic Acids Res. 2001, 29, 66–69.

Randall C. Willis is an assistant editor of Modern Drug Discovery. Send your comments or questions regarding this article to mdd@acs.org or the Editorial Office by fax at 202-776-8166 or by post at 1155 16th Street, NW; Washington, DC 20036.