Plant-like pathway in parasites provides new treatment targets

Effective new ways to inhibit parasites that cause malaria, toxoplasmosis and cryptosporidiosis

June 25, 1998

Researchers from the United States and the United Kingdom have found that a group of parasites responsible for several devastating diseases, including malaria, share a metabolic pathway essential for survival in many plants, fungi, and bacteria--but not found in mammals. This finding provides many new targets for anti-parasitic medications, including the possibility of medicines related to currently available herbicides.

In the June 25, 1998 issue of Nature, the researchers demonstrate that the herbicide glyphosate (Roundup), which interferes with the sixth of the initial seven enzymes in the plant-like pathway, inhibits in the test tube the growth of the parasites that cause malaria, toxoplasmosis, and crytosporidiosis.

They also show that adding the herbicide to sub-therapeutic doses of a common anti-microbial medication can protect mice from otherwise fatal infections with Toxoplasma gondii. Similar tests against related parasites are planned.

"We urgently need new and better medicines to treat these extremely common diseases caused by parasites," said team leader Rima McLeod, MD, the Jules and Doris Stein Research to Prevent Blindness Professor of Ophthalmology and Visual Sciences at the University of Chicago. Some of these diseases are untreatable at present because no known medicines inhibit them and others are caused by parasites that have developed resistance to available medicine. "These studies provide new, rational targets for development of novel treatments as well as several well-studied compounds that can interfere with this pathway."

The parasites that rely on this chemical cascade are members of the phylum Apicomplexa, which includes many important human and animal pathogens, such as:

• Malaria parasites (Plasmodium falciparum)
• Plasmodium: causes malaria, the single most important tropical disease in the world. (Four of the more than 50 species infect humans.) Malaria affects an estimated 300 million people annually and kills nearly 3 million children each year, one every 11 seconds. Drug-resistant strains of malaria are increasingly common.
• Toxoplasmic eye lesion: Toxoplasma gondii: causes toxoplasmosis. T. gondii infection, extremely common throughout the world, including industrialized countries, is spread by cats and is acquired from eating meat that has not been cooked to "well done." Although infection is usually harmless, it can cause devastating problems for those with weakened immune systems or when transmitted from mother to unborn child. About 3,000 infants are born each year in the U.S. with this disease. Manifestations include severe eye damage and mental retardation. Neonatal infections can be fatal if not detected and successfully treated at birth. The cost of caring for these children is estimated to exceed $500 million per year. No medications treat the latent life-cycle stages of the T. gondii parasite.
• Crytosporidia in intestine, scanning electronmicrograph.
• Cryptosporidium parvum: causes severe diarrhea in those with weakened immune systems and is a common infection in AIDS patients. A recent Cryptosporidiosis outbreak was traced to the Milwaukee, Wisconsin water system.
• Eimeria: a serious problem in poultry, causing losses on the order of $80 million per year.

About six years ago, researchers discovered that an unusual organelle of these parasites, called a plastid, had some striking resemblances to chloroplasts of plants. Plant plastids are the site for many essential biochemical functions, including the synthesis of compounds necessary for life such as certain amino acids and folate, which animals get from eating plants.

The plant-like shikimate pathway, which Dr. McLeod's team found in these parasites, consists of a series of seven biochemical reactions catalyzed by seven enzymes. This pathway results in the formation of: chorismate, an essential building block of folate; certain amino acids; and ubiquinone, a molecule used in energy generation.

Dr. McLeod's team showed that glyphosate, by interfering with just one of those enzymes, could block the production of folate, inhibiting parasite growth and survival. Glyphosate proved effective against malaria strains that were resistant to an anti-malarial medicine, pyrimethamine, which interrupts folate processing at a different point. To confirm the finding, they demonstrated that these folate-starved parasites could be rescued, in the test tube, by giving them folate.

Using biochemical, genetic and chemotherapeutic techniques, McLeod's team demonstrated that these parasites all utilized the shikimate pathway. They isolated and sequenced the genes for one of the shikimate pathway enzymes from Toxoplasma and a malaria parasite and demonstrated the presence of four of the seven enzymes in parasites extracts.

Finally, they tested glyphosate in mice infected with lethal doses of T. gondii. Doses of glyphosate or pyrimethamine that could not protect the mice when used alone rescued infected mice when used in combination, even when the mice were allowed to eat diets with folate. "Since resistance to antimicrobial agents is a major problem in the treatment of malaria, we were pleased to find that combining medications in a logical way, to inhibit multiple enzymes in the pathway, enhanced their effect," said Dr. McLeod. "This emphasized the potential value of concomitantly targeting alternative enzymes along the same pathway."

Thanks to decades of herbicide research, the pathway is already well studied and several inhibitory compounds are available. The researchers have also begun looking for compounds that affect other enzymes and substrates in the pathway in novel ways.

In addition to the seven shikimate-pathway enzymes, these findings suggest that the enzymes responsible for other biochemical pathways--which branch from the shikimate pathway--as well as other plant-like metabolic pathways, could also become drug targets.

Medications that target the shikimate pathway may also prove valuable against other disease-causing bacteria or fungi that rely on the pathway, such as: Mycobacterium tuberculosis, which causes tuberculosis; Staphylococcus aureus, a common and increasingly drug-resistant cause of serious post-operative infections; and Pneumocystis carinii, the most frequent cause of pneumonia in patients with AIDS.

"Despite 50 years of intense research into anti-microbials, rationally selecting plant-like metabolic pathways to identify targets in Apicomplexans has not been tried previously," commented Dr. McLeod. Her team sought targets that were part of interrelated metabolic pathways present in lower species, but not present or very different in humans--a novel paradigm for rational drug discovery.

"The urgent need for better medications combined with recent recognition of similarities of Apicomplexan parasites and plants provided the rationale for the initial experiments," she added, "followed by multi-institutional collaborations of infectious disease specialists, parasitologists, biochemists, plant molecular biologists, and John Coggins, PhD, of Glasgow University--one of the world's experts in the shikimate pathway."

Additional authors of the paper include co-first authors Fiona Roberts, MD, and Craig Roberts, MD, who were research fellows in McLeod's laboratory and are now at the Universities of Glasgow and Strathclyde, Glasgow, Scotland; Jennifer Johnson, also working in McLeod's laboratory; Graham Coombs, PhD, and Tino Krell, PhD, University of Glasgow; Dennis Kyle, PhD, and Wil Milhous, MD, Walter Reed Army Institute of Research in Washington, DC; Saul Tzipori, DVM., Tufts University, Boston; David Ferguson, PhD, Oxford University; and Debopam Chakrabarti, PhD, University of Central Florida, Orlando.

Funding support came from several sources including the National Institutes of Health, the World Health Organization, the Toxoplasmosis Research Institute, the Research to Prevent Blindness Foundation, the Wellcome Trust, a Glaxo-Jack Lectureship, the Michael Reese Physicians Research and Education Foundation, the Fulbright Scholars Program, and the European Union.

Summary:
The parasites responsible for malaria, toxoplasmosis, and several other diseases share a metabolic pathway essential for survival in plants but not found in mammals, making it an ideal treatment target. Researchers demonstrate that an herbicide inhibits growth of these parasites in tissue culture and protects mice from fatal parasitic infections.