Malaria is a serious illness that affects hundreds of millions of people worldwide and kills 700,000-2.7 million people each year. Malaria is caused by protozoan parasites that are transmitted from person to person via the Anopheles mosquito (United States Center for Disease Control, Malaria Website; World Health Organization, Global Malaria Programme), or in some cases in endemic areas, by allogeneic blood transfusion from an infected donor (Marcucci et al., “Allogeneic Blood Transfusions Benefit, Risks and Clinical Indications in Countries With a Low or High Human Development Index,” Br. Med. Bull. 70:15-28 (2004)). Five species of apicomplexan protozoa can infect and cause malaria in humans: Plasmodium falciparum, P. vivax, P. ovale, P. malaria, and P. knowlesi. The disease is characterized by cyclical episodes of high fever, chills, severe pain, and nausea. In severe cases, caused by P. falciparum infection, patients present with brain ischemia (“cerebral malaria”) and other end-organ damage. Children and pregnant women are highly susceptible to this form of malaria, which, without immediate and aggressive treatment, is often fatal.
Plasmodium species have a very complex lifecycle, involving sexual reproduction (gamete fusion), asexual replication (oocysts and schizonts), invasive stages (sporozoites and merozoites), and two different hosts (mosquito and mammal). Plasmodium ookinetes differentiate into oocysts within the Anopheles mosquito midgut. Sporozoites develop within and bud off of the oocysts. They are then carried by the mosquito's hemolymph to the salivary glands, where they invade the cells of the salivary ducts. When the infected mosquito bites a human, the sporozoites are injected into his or her skin, through which they migrate until they are able to penetrate a blood vessel and enter the bloodstream. The sporozoites travel to the liver where they invade hepatocytes and develop into merozoites—the invasive form of the Plasmodium blood stages (Kappe et al., “Plasmodium Sporozoite Molecular Cell Biology,” Annu. Rev. Cell Dev. Biol. 20:29-59 (2004)).
When the infected hepatocytes rupture, thousands of merozoites are released into the blood and invade erythrocytes, where they proceed through a “ring” form and a trophozoite stage. During these stages, the parasite feeds off of the surrounding red blood cell, ingesting its hemoglobin along with portions of the erythrocyte's cytosol. Parasite proteins released into the host cell induce marked changes to the structure and adhesive properties of the infected red blood cell membrane, causing other cells to clump around them. These erythrocyte “rosettes” adhere to the walls of deep visceral blood vessels, preventing their destruction by the liver and spleen (Bannister and Mitchell, “The Ins, Outs and Roundabouts of Malaria,” Trends Parasitol. 19:209-213 (2003); Bannister et al., “A Brief Illustrated Guide to the Ultrastructure of Plasmodium falciparum Asexual Blood Stages,” Parasitol. Today 16:427-433 (2000)). After 36, 48, or 72 hours—depending on the Plasmodium species—within the red blood cell, the parasite undergoes schizogony (asexual replication). The schizont-infected cells eventually rupture, releasing many new merozoites that can either re-invade other erythrocytes or develop into gametes—the parasite's sexual stage—which can be ingested by another mosquito during a blood meal. Within the mosquito, the gametes fuse and differentiate into ookinetes, and the cycle of infection repeats (Kappe et al., “Plasmodium Sporozoite Molecular Cell Biology,” Annu. Rev. Cell Dev. Biol. 20:29-59 (2004)).
The parasite's blood stages are primarily responsible for the mortality of the disease—the mass rupture of red blood cells causes the painful symptoms of malaria and subsequent anemia. The complications of cerebral malaria ensue when ruptured cells occlude blood vessels supplying the brain or other organs, and the obstruction of placental vasculature contributes to the high mortality rate among pregnant patients (Kappe et al., “Plasmodium Sporozoite Molecular Cell Biology,” Annu. Rev. Cell Dev. Biol. 20:29-59 (2004); Bannister & Mitchell, “The Ins, Outs and Roundabouts of Malaria,” Trends Parasitol. 19:209-213 (2003); Bannister et al., “A Brief Illustrated Guide to the Ultrastructure of Plasmodium falciparum Asexual Blood Stages,” Parasitol. Today 16:427-433 (2000)).
Two Plasmodium species, P. vivax and P. ovale, also exhibit a hynozoite stage which can remain dormant in the liver for long periods of time, resulting in a relapse of the illness weeks or even years after the initial infection.
Plasmodium sporozoites are characterized by an outer pellicle composed of a double-layer inner membrane complex (IMC) surrounded by a plasma membrane (PM) and supported by a microtubule network. An actin-myosin motor lies between the IMC and PM, and is attached to transmembrane adhesive proteins that connect the interior of the parasite cell to receptors, proteoglycans, glycosaminoglycans, and glycoproteins on the host cell's surface. These adhesins are secreted through specialized structures called micronemes on the apical end of the organism. The force transduced by the actin-myosin motor results in the backward redistribution of the adhesive proteins on the parasite's surface. This movement propels sporozoites forward with a spiral-shaped trajectory. When the adhesive proteins reach the posterior end of the cell, they are shed from the zoite membrane, which—at least for sporozoites—allows the parasite's path to be visualized with fluorescently-tagged antibodies to this “protein trail” (Kappe et al., “Apicomplexan Gliding Motility and Host Cell Invasion: Overhauling the Motor Model,” Trends Parasitol. 20:13-16 (2004)).
While merozoites have not been observed to glide along a surface in the same way sporozoites do, they appear to utilize the same motor machinery to propel themselves into red blood cells (Baum et al., “A Conserved Molecular Motor Drives Cell Invasion and Gliding Motility Across Malaria Life Cycle Stages and Other Apicomplexan Parasites,” J. Biol. Chem. 281:5197-5208 (2006); Pinder et al., “Motile Systems in Malaria Merozoites: How is the Red Blood Cell Invaded?” Parasitol. Today 16:240-245 (2000); Pinder et al., “Actomyosin Motor in the Merozoite of the Malaria Parasite, Plasmodium falciparum: Implications for Red Cell Invasion,” J. Cell. Sci. 111:1831-1839 (1998)). When Plasmodium merozoites invade host cells, their surface adhesive proteins engage host membrane receptors and form a moving junction between the host cell membrane and the parasite, characterized by a thickened ring within the host cell membrane. The parasite then enters the cell, at which point its surface proteins are cleaved, allowing it to separate from the host cell membrane at its posterior end. The subsequent resealing of the cell membrane generates a specialized “parasitophorous” vacuole within the host cell where parasite differentiation and multiplication occur (Kappe et al., “Plasmodium Sporozoite Molecular Cell Biology,” Annu. Rev. Cell Dev. Biol. 20:29-59 (2004); Baum et al., “A Conserved Molecular Motor Drives Cell Invasion and Gliding Motility Across Malaria Life Cycle Stages and Other Apicomplexan Parasites,” J. Biol. Chem. 281:5197-5208 (2006); Pinder et al., “Motile Systems in Malaria Merozoites: How is the Red Blood Cell Invaded?” Parasitol. Today 16:240-245 (2000); Pinder et al., “Actomyosin Motor in the Merozoite of the Malaria Parasite, Plasmodium falciparum: Implications for Red Cell Invasion,” J. Cell. Sci. 111:1831-1839 (1998); Menard, “The Journey of the Malaria Sporozoite Through Its Hosts: Two Parasite Proteins Lead the Way,” Microbes Infect. 2:633-642 (2000)).
Many of the proteins involved in this form of movement and invasion have been elucidated (Kappe et al., “Plasmodium Sporozoite Molecular Cell Biology,” Annu. Rev. Cell Dev. Biol. 20:29-59 (2004); Baum et al., “A Conserved Molecular Motor Drives Cell Invasion and Gliding Motility Across Malaria Life Cycle Stages and Other Apicomplexan Parasites,” J. Biol. Chem. 281:5197-5208 (2006); Buscaglia et al., “Sites of Interaction Between Aldolase and Thrombospondin-Related Anonymous Protein in Plasmodium,” Mol. Biol. Cell. 14:4947-4957 (2003); Kappe et al., “Apicomplexan Gliding Motility and Host Cell Invasion: Overhauling the Motor Model,” Trends Parasitol. 20:13-16 (2004); Matuschewski et al., “Plasmodium Sporozoite Invasion Into Insect and Mammalian Cells is Directed by the Same Dual Binding System,” Embo J. 21:1597-1606 (2002); Menard, “The Journey of the Malaria Sporozoite Through Its Hosts: Two Parasite Proteins Lead the Way,” Microbes Infect. 2:633-642 (2000); Mota & Rodriguez, “Invasion of Mammalian Host Cells by Plasmodium Sporozoites,” Bioessays 24:149-156 (2002); Muller et al., “Thrombospondin Related Anonymous Protein (TRAP) of Plasmodium falciparum in Parasite-Host Cell Interactions,” Parassitologia 35 Suppl:69-72 (1993); Sultan, “Molecular Mechanisms of Malaria Sporozoite Motility and Invasion of Host Cells,” Int. Microbiol. 2:155-160 (1999); Sultan et al., “TRAP is Necessary for Gliding Motility and Infectivity of Plasmodium Sporozoites,” Cell 90:511-522 (1997); Kappe et al., “Conservation of a Gliding Motility and Cell Invasion Machinery in Apicomplexan Parasites,” J. Cell. Biol. 147:937-944 (1999); Green et al., “The MTIP-Myosin A Complex in Blood Stage Malaria Parasites,” J. Mol. Biol. 355:933-941 (2006); Jewett & Sibley, “Aldolase Forms a Bridge Between Cell Surface Adhesins and the Actin Cytoskeleton in Apicomplexan Parasites,” Mol. Cell. 11:885-894 (2003); Yuda et al., “Structure and Expression of an Adhesive Protein-Like Molecule of Mosquito Invasive-Stage Malarial Parasite,” J. Exp. Med. 189:1947-1952 (1999)). While features of the motor complex were originally resolved in Plasmodium sporozoites, and in tachyzoites of the related apicomplexan parasite, Toxoplasma gondii, most of its primary components have since been characterized in Plasmodium merozoites as well. In the currently prevailing model, the actual motor consists of F-actin (Dobrowolski and Sibley, “Toxoplasma Invasion of Mammalian Cells is Powered by the Actin Cytoskeleton of the Parasite,” Cell 84:933-939 (1996)) and myosin A (MyoA) (Bannister and Mitchell, “The Ins, Outs and Roundabouts of Malaria,” Trends Parasitol. 19:209-213 (2003); Dobrowolski et al., “Participation of Myosin in Gliding Motility and Host Cell Invasion by Toxoplasma gondii,” Mol. Microbiol. 26:163-173. (1997); Hettmann et al., “A Dibasic Motif in the Tail of a Class XIV Apicomplexan Myosin is an Essential Determinant of Plasma Membrane Localization,” Mol. Biol. Cell 11:1385-1400 (2000); Heintzelman & Schwartzman, “A Novel Class of Unconventional Myosins From Toxoplasma gondii,” J. Mol. Biol. 271:139-146 (1997)), and is anchored to the IMC via MyoA tail-interacting protein (MTIP) (Bannister. & Mitchell, “The Ins, Outs and Roundabouts of Malaria,” Trends Parasitol. 19:209-213 (2003); Bergman et al., “Myosin A Tail Domain Interacting Protein (MTIP) Localizes to the Inner Membrane Complex of Plasmodium Sporozoites,” J. Cell. Sci. 116:39-49 (2003)), glideosome associated protein 45 (GAP45), and the integral membrane glycoprotein, GAP50 (Baum et al., “A Conserved Molecular Motor Drives Cell Invasion and Gliding Motility Across Malaria Life Cycle Stages and Other Apicomplexan Parasites,” J. Biol. Chem. 281:5197-5208 (2006); Gaskins et al., “Identification of the Membrane Receptor of a Class XIV Myosin in Toxoplasma gondii,” J. Cell. Biol. 165:383-393 (2004)). Aldolase tetramers link F-actin to the transmembrane adhesin, thrombospondin-related anonymous protein (TRAP), in sporozoites (Buscaglia et al., “Sites of Interaction Between Aldolase and Thrombospondin-Related Anonymous Protein in Plasmodium,” Mol. Biol. Cell. 14:4947-4957 (2003); Buscaglia et al., “Modeling the Interaction Between Aldolase and the Thrombospondin-Related Anonymous Protein, a Key Connection of the Malaria Parasite Invasion Machinery,” Proteins 66:528-537 (2007); Bosch et al., “Aldolase Provides an Unusual Binding Site for Thrombospondin-Related Anonymous Protein in the Invasion Machinery of the Malaria Parasite,” Proc. Nat'l. Acad. Sci. USA 104(17):7015-20 (2007)), or to its homolog, MTRAP, in merozoites (Baum et al., “A Conserved Molecular Motor Drives Cell Invasion and Gliding Motility Across Malaria Life Cycle Stages and Other Apicomplexan Parasites,” J. Biol. Chem. 281:5197-5208 (2006)). Aldolase is therefore a critical linking molecule in the molecular motor that powers the gliding motility capability of the parasite. As Plasmodium Aldolase is highly similar to human Aldolase, and as specificity for Plasmodium Aldolase over human Aldolase is required for any potential drug targeting this site, drug development targeted at the Aldolase component of the glideosome is considered challenging to the point of non-obviousness to a reasonable practitioner of the art.
Several effective treatments for malaria exist, including chloroquine, primaquine, sulfadoxine-pyrimethamine, doxycycline, artesunate, and others (Rosenthal, “Antiprotozoal Drugs. in Basic & Clinical Pharmacology (ed. Katzung, B. G.) Lange Medical Books/McGraw Hill:New York, pp. 864-885 (2004)). However, many of these drugs have potentially serious side affects and/or are prohibitively expensive. Quinine and quinidine commonly illicit cinchonism, which in some cases can be severe (Katzung, Basic and Clinical Pharmacology, Appleton & Lange:Norwalk, Conn., (2004)). Many anti-malarials can produce hemolysis in patients with glucose 6-phosphate dehydrogenase deficiencies (Katzung, Basic and Clinical Pharmacology, Appleton & Lange:Norwalk, Conn., (2004)), which due to its conference of some degree of malaria resistance, is commonly found in malaria endemic regions (Kwiatkowski, “How Malaria Has Affected the Human Genome and What Human Genetics Can Teach Us About Malaria,” Am. J. Hum. Genet. 77:171-192 (2005)). Some anti-malarial agents have also been associated with rare cases of neuropsychiatric toxicities (Katzung, Basic and Clinical Pharmacology, Appleton & Lange: Norwalk, Conn., (2004)). Additionally, parasite resistance to these drugs is growing rapidly, especially in areas lacking adequate medical infrastructures and support networks (United States Center for Disease Control Malaria Website; World Health Organization Global Malaria Programme). On the other hand, artemisinin-based combination therapies (ACTs), while still highly effective in most regions, are cumbersome to produce and are often too expensive for patients in endemic areas (Enserink, “Combating Malaria. Malaria Treatment: ACT Two,” Science 318:560-563 (2007)). Reports of parasite resistance to ACTs—the current gold-standard for malaria treatment—have begun to emerge as well (Noedl et al., “Evidence of Artemisinin-Resistant Malaria in Western Cambodia,” N. Engl. J. Med. 359:2619-2620 (2008)). This is especially alarming as there are very few other drugs in the malaria pipeline. There is clearly a critical need for the discovery of novel drug targets and reagents to combat this disease.
Most anti-malarials in clinical use target the parasite's haem (quinilones: chloroquine, quinine, amodiaquine, mefloquine, and halofantrine) or folate (antifolates: sulphadoxine-pyrimethine) metabolism pathways, while others interfere with mitochondrial electron-transport via cytochrome C inhibition (atavaquone, proguanil). Additionally, some common antibiotics are effective against malaria by inhibiting prokaryote-like protein synthesis within the malarial apicoplast (tetracycline, doxycycline, and clindamycin). Newer artemisin derivatives (artemether, arteether, artesunate) are believed to rapidly kill merozoites and gametocytes via free-radical generation (Ridley, “Medical Need, Scientific Opportunity and the Drive for Antimalarial Drugs,” Nature 415:686-693 (2002); Wells et al., “New Medicines to Improve Control and Contribute to the Eradication of Malaria,” Nat. Rev. Drug Discov. 8:879-891 (2009)).
Considering the global impact of malarial disease, and the years of research and billions of dollars poured into attempts to eradicate it, the above list of treatments and therapeutic targets is alarmingly short. There are clearly many more pathways, mechanisms, and biological features of Plasmodium that have yet to be targeted for anti-malarial drug design. The glideosome is thus established as a promising target, and a drug inhibiting the glideosome would be new and useful. Moreover, cross species conservation of the glidoesome might have the potential to be valuable for economical reasons preventing livestock diseases in cattle (Babesia) or chicken (Eimeria).
Importantly, there are precious few drugs in clinical use that are effective against the exo-erythrocytic stages of the parasite, making the prevention of transmission and disease relapse due to dormant liver-stage hypnozoites especially difficult. Without the ability to effectively prevent initial infections by malaria sporozoites through vaccination or prophylactic drug therapy, it is unlikely to achieve complete global eradication of this deadly disease (Mazier et al., “A Pre-Emptive Strike Against Malaria's Stealthy Hepatic Forms,” Nat. Rev. Drug Discov. 8:854-864 (2009)).
The present invention is directed to overcoming these and other deficiencies in the art.
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USPTO Backgrounds