Researchers find two new targets to thwart malaria's resistance to drugs

mosquito

Malaria, a confoundingly clever global killer, can evade treatment by developing resistance to drugs, but now a team of researchers at the School of Medicine has identified two new therapeutic targets.

The team, led by Matthew Bogyo, PhD, elucidated a method that could keep malaria parasites sequestered in blood cells of infected people long enough that the parasites die before they can wreak havoc on the body.

The team identified two enzymes that enable malaria parasites to escape host blood cells, and also found compounds that block those enzymes, which could block the parasites from spreading. The research was published Feb. 3 in the advance online issue of Nature Chemical Biology.

'The bottom line is that to combat malaria effectively, we are going to have to keep launching multiple classes of new drugs with different mechanisms of action if we want to prevent resistance,' said Bogyo, assistant professor of pathology, who has been applying his expertise in enzyme chemistry to malaria for the last eight years. 'We need to come up with strategies that affect how it survives in the host.'

Malaria strikes half a billion people annually, according to the World Health Organization. At least a million of those stricken will die each year.

The culprits that cause malaria are microscopic, one-celled parasites called plasmodia that live in the saliva of certain types of mosquitoes. Shaped like tiny worms, even a single plasmodium released through the saliva of an infected mosquito is enough to kill someone.

Once an infected mosquito bites a person, the parasites journey to the liver, where they hunker down for a couple of weeks. They replicate themselves thousands of times and then burst the depleted liver cells to release the second stage of the parasites into the bloodstream. This form of the parasite immediately invades the red blood cells, where they replicate again, burst out and infect new blood cells. Symptoms at this stage include anemia, fever, chills and, in severe cases, coma and death.

Malaria researchers have spent a lot of time trying to figure out how the parasites invade cells to establish an infection, but relatively little is known about how the parasites emerge from an infected cell, said Bogyo. This process is critical for the infection to continue, so Bogyo's group reasoned that agents that could block the rupture might be valuable as anti-malarial agents.

The researchers began with what was known: The release from host red blood cells depends on enzymes in the parasite called proteases, which chew up proteins at specific sites. If these proteases are blocked, that stops the release of the malaria parasites. 'But no one really knew which proteases were responsible,' said Bogyo, the senior author of the study.

To find out, they screened approximately 2,000 compounds from a collection of protease inhibitors gathered from their own lab as well as chemistry labs across the country. Bogyo's collection efforts included some compounds that had been sitting in labs for nearly 30 years, created for long-ago published studies.

The result is a 'library' of protease inhibitors that can be used to screen for these compounds' effects on various biological systems. As the first test of their library, they looked at how each of the compounds affects the life stages of the malaria parasites in blood cells.

Bogyo's lab is the only basic research lab at Stanford working directly with malaria cultures. 'People always ask me if I'm going to get malaria,' Bogyo said. It is safe, he explained, because his team works with the parasite's blood stage, which is not an efficient transporter of the infection - unlike the highly infectious stage in mosquito saliva.

In the lab, the researchers grow the parasites in culture dishes, nourished by human blood from the Stanford Blood Center. Under these conditions, the parasites simultaneously move together through their life cycles. It is possible to determine if any added chemical affects their progression through the stages.

From this screening, they identified several potentially useful compounds, including some that completely blocked the rupture of blood cells, thus trapping the malaria parasites inside where they perished within 48 hours.

The study's first author, Shirin Arastu-Kapur, PhD, a postdoctoral scholar in Bogyo's lab, used the promising compounds to identify the enzymatic pathways involved, concluding that two different proteases are at work. Both of them - subtilisin-like serine protease 1 and dipeptidyl peptidase 3 - work through different mechanisms to process proteins required for cell rupture.

'Finding hits that target two different enzymes that seem to be involved in the same pathway turned out to be an exciting finding with real implications for drug development,' said Bogyo. Using compounds that target distinct proteases involved in the same biological pathway might be a good way to prevent resistance, as it is harder for parasites to surmount two barriers than just one.

Arastu-Kapur has begun testing the two compounds in mouse models of malaria, as well as in insect stages, to determine if they could be developed into useful drugs that are simple and cheap to produce. She and Bogyo are hopeful because there are already a number of these types of drugs - based on small molecule inhibitors of proteases - currently in use for treating a variety of human diseases, including hypertension and cancer.

This work was funded by the Kinship Foundation as part of the Searle Scholars program, Burroughs Wellcome Trust, the National Institutes of Health, the National Science Foundation and the American Society for Microbiology.

Others from Stanford who contributed to this study are: graduate students Elizabeth Ponder and Carolyn Phillips; research technician Ursa Fonovic, and postdoctoral scholars Fang Yuan, PhD, and Marko Fonovic, PhD.


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