An international study, involving researchers from Griffith University’s Eskitis Institute, has discovered a molecule which could form the basis of powerful new anti-malaria drugs.
Professor Vicky Avery from Griffith University’s Eskitis Institute is co-author of the paper “Quinolone-3-Diarylethers: a new class of drugs for a new era of malaria eradication” which has been published in the journal Science Translational Medicine.
“The 4(1H)-quinolone-3- diarylethers are selective potent inhibitors of the parasite mitochondrial cytochrome bc1 complex,” Professor Avery said.
“These compounds are highly active against the types of malaria parasites which infect humans, Plasmodium falciparum and Plasmodium vivax,” she said.
“What is really exciting about this study is that a new class of drugs based on the 4(1H)- quinolone-3- diarylethers would target the malaria parasite at different stages of its lifecycle.”
This provides the potential to not only kill the parasite in people who are infected, thus treating the clinical symptoms of the disease, but also to reduce transmission rates.
“Just one of these properties would be of great benefit but to achieve both would really make a difference in reducing the disease burden on developing nations,” Professor Avery said.
“There is also the real possibility that we could begin to impact on the incidence and spread of malaria, bringing us closer to the ultimate goal of wiping out malaria altogether.”
The selected preclinical candidate compound, ELQ-300, has been demonstrated to be very effective at blocking transmission in the mouse models.
There is a further benefit in that the predicted dosage in patients would be very low and it’s expected that ELQ-300, which has a long half-life, would provide significant protection.
The development of a new chemical class of anti-malarial drugs is very timely as the parasite is becoming increasing resistant to currently available treatments.
Eskitis Director Professor Ronald J Quinn AM said “I congratulate Professor Avery on her contribution to the discovery of this new class of anti-malarials. This is an exciting discovery that closely aligns with the Institute’s focus on global health and fighting diseases that burden the developing world. We are continuing to take the fight to malaria along a number of fronts, including targeting its many life cycle stages.”
Journal reference: Science Translational Medicine
A. Nilsen, A. N. LaCrue, K. L. White, I. P. Forquer, R. M. Cross, J. Marfurt, M. W. Mather, M. J. Delves, D. M. Shackleford, F. E. Saenz, J. M. Morrisey, J. Steuten, T. Mutka, Y. Li, G. Wirjanata, E. Ryan, S. Duffy, J. X. Kelly, B. F. Sebayang, A.-M. Zeeman, R. Noviyanti, R. E. Sinden, C. H. Kocken, R. N. Price, V. M. Avery, I. Angulo-Barturen, M. B. Jiménez-Díaz, S. Ferrer, E. Herreros, L. M. Sanz, F.-J. Gamo, I. Bathurst, J. N. Burrows, P. Siegl, R. K. Guy, R. W. Winter, A. B. Vaidya, S. A. Charman, D. E. Kyle, R. Manetsch, M. K. Riscoe, Quinolone-3-Diarylethers: A New Class of Antimalarial Drug. Sci. Transl. Med. 5, 177ra37 (2013).
Provided by: Griffith University
Biologists at the University of California, San Diego have succeeded in engineering algae to produce potential candidates for a vaccine that would prevent transmission of the parasite that causes malaria, an achievement that could pave the way for the development of an inexpensive way to protect billions of people from one of the world’s most prevalent and debilitating diseases. Initial proof-of-principle experiments suggest that such a vaccine could prevent malaria transmission.
(up) The edible algae Chlamydomonas, seen here at UC San Diego, can be grown in ponds anywhere in the world. (Credit: SD-CAB)
Malaria is a mosquito-borne disease caused by infection with protozoan parasites from the genus Plasmodium. It affects more than 225 million people worldwide in tropical and subtropical regions, resulting in fever, headaches and in severe cases coma and death. While a variety of often costly antimalarial medications are available to travelers in those regions to protect against infections, a vaccine offering a high level of protection from the disease does not yet exist.
The use of algae to produce malaria proteins that elicited antibodies against Plasmodium falciparum in laboratory mice and prevented malaria transmission was published May 16 in the online, open-access journal PLoS ONE. The development resulted from an unusual interdisciplinary collaboration between two groups of biologists at UC San Diego — one from the Division of Biological Sciences and San Diego Center for Algae Biotechnology, which had been engineering algae to produce bio-products and biofuels, and another from the Center for Tropical Medicine and Emerging Infectious Diseases in the School of Medicine that is working to develop ways to diagnose, prevent and treat malaria.
Part of the difficulty in creating a vaccine against malaria is that it requires a system that can produce complex, three-dimensional proteins that resemble those made by the parasite, thus eliciting antibodies that disrupt malaria transmission. Most vaccines created by engineered bacteria are relatively simple proteins that stimulate the body’s immune system to produce antibodies against bacterial invaders. More complex proteins can be produced, but this requires an expensive process using mammalian cell cultures, and the proteins those cells produce are coated with sugars due to a chemical process called glycosylation.
“Malaria is caused by a parasite that makes complex proteins, but for whatever reason this parasite doesn’t put sugars on those proteins,” said Stephen Mayfield, a professor of biology at UC San Diego who headed the research effort. “If you have a protein covered with sugars and you inject it into somebody as a vaccine, the tendency is to make antibodies against the sugars, not the amino acid backbone of the protein from the invading organism you want to inhibit. Researchers have made vaccines without these sugars in bacteria and then tried to refold them into the correct three-dimensional configuration, but that’s an expensive proposition and it doesn’t work very well.”
Instead, the biologists looked to produce their proteins with the help of an edible green alga, Chlamydomonas reinhardtii, used widely in research laboratories as a genetic model organism, much like the fruit fly Drosophila and the bacterium E. coli. Two years ago, a UC San Diego team of biologists headed by Mayfield, who is also the director of the San Diego Center for Algae Biotechnology, a research consortium seeking to develop transportation fuels from algae, published a landmark study demonstrating that many complex human therapeutic proteins, such as monoclonal antibodies and growth hormones, could be produced by Chlamydomonas.
That got James Gregory, a postdoctoral researcher in Mayfield’s laboratory, wondering if a complex protein to protect against the malarial parasite could also be produced by Chlamydomonas. Two billion people live in regions where malaria is present, making the delivery of a malarial vaccine a costly and logistically difficult proposition, especially when that vaccine is expensive to produce. So the UC San Diego biologists set out to determine if this alga, an organism that can produce complex proteins very cheaply, could produce malaria proteins that would inhibit infections from malaria.
“It’s too costly to vaccinate two billion people using current technologies,” explained Mayfield. “Realistically, the only way a malaria vaccine will ever be used is if it can be produced at a fraction of the cost of current vaccines. Algae have this potential because you can grow algae any place on the planet in ponds or even in bathtubs.”
Collaborating with Joseph Vinetz, a professor of medicine at UC San Diego and a leading expert in tropical diseases who has been working on developing vaccines against malaria, the researchers showed that the proteins produced by the algae, when injected into laboratory mice, made antibodies that blocked malaria transmission from mosquitoes.
“It’s hard to say if these proteins are perfect, but the antibodies to our algae-produced protein recognize the native proteins in malaria and, inside the mosquito, block the development of the malaria parasite so that the mosquito can’t transmit the disease,” said Gregory.
“This paper tells us two things: The proteins that we made here are viable vaccine candidates and that we at least have the opportunity to produce enough of this vaccine that we can think about inoculating two billion people,” said Mayfield. “In no other system could you even begin to think about that.”
The scientists, who filed a patent application on their discovery, said the next steps are to see if these algae proteins work to protect humans from malaria and then to determine if they can modify the proteins to elicit the same antibody response when the algae are eaten rather than injected.
Other UC San Diego scientists involved in the discovery were Fengwu Li from Vinetz’s laboratory and biologists Lauren Tomosada, Chesa Cox and Aaron Topol from Mayfield’s group. The basic technology that led to the development was supported by the Skaggs family. The research was supported by grants from the National Institute of Allergy and Infectious Diseases and the San Diego Foundation. The California Energy Commission supported work on recombinant protein production for biofuels use, and this technology helped enabled these studies.
Note: Materials may be edited for content and length. For further information, please contact the source cited above.
Gregory JA, Li F, Tomosada LM, Cox CJ, Topol AB, et al.Algae-Produced Pfs25 Elicits Antibodies That Inhibit Malaria Transmission. PLoS ONE, 2012 DOI:10.1371/journal.pone.0037179
A biology lab at Washington University has just cracked the structure and function of a protein that plays a key role in the life of a parasite that killed 655,000 people in 2010.
A cartoon based on the electron density map makes it easier to see the protein’s structure and figure out how it works. The enzyme’s job is to add a methyl group — three times — to a starting molecule as part of a process for making cell membranes. In this cartoon, the phosphate is a stand-in for the starting molecule and the green molecule is the one that donates a methyl group. Both are positioned in the active site of the enzyme, the pocket where the chemistry takes place. (Credit: Jez)
The protein is an enzyme that Plasmodium falciparum, the protozoan that causes the most lethal form of malaria, uses to make cell membrane.
The protozoan cannot survive without this enzyme, but even though the enzyme has many lookalikes in other organisms, people do not make it. Together these characteristics make the enzyme an ideal target for new antimalarial drugs.
The research was published in the January 6 issue of the Journal of Biological Chemistry (JBC) as “Paper of the Week” for that issue.
The work also will be featured in ASBMB Today (the newsletter of the American Society for Biological Molecular Biology, which publishes JBC).
Sweating the cold room
The protein’s structure might have remained an enigma, had it not been the “unreasonable optimism” of Joseph Jez, PhD, associate professor of biology in Arts & Sciences, which carried his team through a six-year-long obstacle course of failures and setbacks.
“What my lab does is crystallize proteins so that we can see what they look like in three dimensions,” Jez says. “The idea is that if we know a protein’s structure, it will be easier to design chemicals that would target the protein’s active site and shut it down,” Jez says.
The latest discovery is the culmination of a project that began years before when Jez was working at the Danforth Plant Science Center in St. Louis and collaborating with scientists at the local biotech startup Divergence. “At the time, C. elegans had just been sequenced and the Divergence scientists were looking at using it as an easy model to work out the biochemistry of parasitic nematodes,” Jez says.
C. elegans is a free-living nematode, or microscopic roundworm, but many nematodes are parasitic and cause disease in plants, livestock and people.
During this project, Lavanya Palavalli, a summer intern working with Jez, crystallized the C. elegans version of the enzyme. The job of the enzyme, phosphoethanolamine methyltransferase, thankfully abbreviated to PMT, is to add methyl groups to a starting molecule, phosophoethanolamine.
“When Soon Goo Lee later took up the project,” says Jez, “the plan was to try to grow better crystals of the C. elegans protein, ones good enough to get readable X-ray diffraction patterns.
Two years later, the crystals were looking better but still not good enough.
So Jez suggested that Lee go after homologous (look-alike) proteins in other organisms. “Even though the proteins are homologous, each has a different amino acid sequence and so will behave differently in the crystallizations,” Jez says. “Lee went from working with two C. elegans proteins to three plant proteins, two other nematode proteins and then the Plasmodium protein,” Jez says.
“He took all six of those PMT versions into the crystallization trials to maximize his odds,” Jez says.
“To crystallize a protein,” Jez says, “we put a solution of a salt or something else that might work as a desiccant in the bottom of a small well. And then we put a drop of our liquid protein on a microscope cover slip and flip it over the top of the well, so the drop of protein is hanging upside down in the well.”
“What we’re trying to do is to slowly withdraw water from the protein. It’s exactly like making rock candy, only in that case, the string hanging into the jar of sugar solution helps to withdraw water,” he says.
The difference is that sugar wants to form crystals and proteins are reluctant to do so.
“There are 24 wells to a tray, and we usually screen 500 wells per protein at first,” Jez says. “Lee had eight proteins and so his first pass was to screen 4,000 conditions. And then he had to try different combinations of ligands to the proteins and crystallize those. This is why it took a few years to finally get where he needed to go.”
The scientists need crystals — preferably nice, big ones — to stick in the path of an X-ray beam at Argonne National Laboratory in Chicago. (If the crystal is a good one, and all the atoms are lined up in a repeating array, the scattered X-rays will produce a clear pattern of spots.)
Embedded in that pattern is the mathematical information needed to back-calculate to the position of the atoms in the protein, a process a bit like throwing a handful of pebbles in a lake and then calculating where they landed by the pattern of waves arriving at the shoreline.
Lee got the PMT from Haemonchus contortus to crystallize first, but there were technical issues with the diffraction pattern that would have made solving it technically and computationally very demanding.
“When the Plasmodium enzyme finally crystallized, Soon got four crystals kind of stacked on top of each other and each of them was paper thin,” Jez says.
“I never thought it would work, but we took them to Argonne anyway and he actually did surgery under the microscope and cracked off a little tiny piece of it.”
To everyone’s surprise, he got a clean diffraction pattern from the crystal. “Because the Plasmodium enzyme was the smallest one and the easiest to work on, we pushed that one first,” Jez says.
The moment of truth
“Once we had a Plasmodium crystal that was diffracting really well, we could try back-calculating to see whether we could extract the atom positions from the data,” Jez says.
After the computer finished its calculations, Lee clicked a mouse button to see the results, which would reveal whether his years of work finally would pay off.
When Lee clicked the mouse, he got an electron density map in exceptionally sharp focus.
“When you see a map like that, it’s like suddenly the wind has kicked up and you’re sailing free,” Jez says, “because there’s this moment, like, before you click that button, no one has ever seen how this protein is put together in three dimensions. You’re the first person to ever see it.
“The irony of it is we got such good quality diffraction pattern and electron density maps off such an ugly crystal,” he says.
Lock and load
“Once you have the electron density map, the task is to build a structure that matches the amino acid sequence of the protein,” Jez says.
“The first thing you do is put in the amino acid backbones and connect them together to form a chain. It’s like having a long thread, each inch of which is an amino acid, and your job is to take that thread and move it in three dimensions through that electron density map.”
The next step is to add the side chains that make one amino acid different from another, Jez says. “The amino acid sequence is known,” he says. “Your goal is to match the way you string together the amino acids in the electron density map to that sequence.”
“Once you have the overall structure, you can start to figure out how the enzyme works. The PMT enzyme is trying to join two molecules,” Jez says. “To do that, it has to lock them in place so that the chemistry can happen, and then it has to let go of them.
“We think the protein has a lid that opens and closes,” he says. “The active site stays open until the substrates enter, and then the lid clamps down, and when it clamps down it actually puts the substrates together.”
Calling Bill and Melinda Gates
Not only do infections by Plasmodium falciparum cause the most severe form of malaria, about 40 percent of the human population lives in areas where the parasite is endemic. Moreover, drugs that used to be effective against malaria are beginning to fail, in part because widespread drug counterfeiting has led to resistance.
New anti-malarial drugs are desperately needed, and the PMT protein is an ideal target. If PMT is disabled, the protozoan can’t make cell membranes and it dies. Moreover, a drug that would kill Plasmodium might have minimal side effects on patients.
Although the process of identifying compounds that would target PMT is in the early stages, a handful of anti-parasitical compounds used to treat diseases are known to block PMT as well.
As for Lee, he has had a hard go of it, but now things are breaking his way. Plasmodium PMT is giving up its secrets, and the plant and nematode PMTs are coming along as well.
When he clicked the mouse button and a clean electron density map came up, he says, it was like seeing “the light at the end of a five-year-long tunnel.”
S. G. Lee, Y. Kim, T. D. Alpert, A. Nagata, J. M. Jez.Structure and Reaction Mechanism of Phosphoethanolamine Methyltransferase from the Malaria Parasite Plasmodium falciparum: AN ANTIPARASITIC DRUG TARGET. Journal of Biological Chemistry, 2011; 287 (2): 1426 DOI:10.1074/jbc.M111.315267
Ahead of World Malaria Day (25 April), EU-funded researchers have discovered that drugs originally designed to inhibit the growth of cancer cells can also kill the parasite that causes malaria. They believe this discovery could open up a new strategy for combating this deadly disease, which, according to World Health Organisation statistics, infected around 225 million and killed nearly 800,000 people worldwide in 2009.
Efforts to find a treatment have so far been hampered by the parasite’s ability to quickly develop drug resistance. The research involved four projects funded by the EU (ANTIMAL, BIOMALPAR, MALSIG and EVIMALAR) and was led by laboratories in the UK, France and Switzerland with partners from Belgium, Germany, Denmark, Greece, Spain, Italy, Netherlands, Portugal, and Sweden, along with many developing nations severely affected by malaria.
Research, Innovation and Science Commissioner Máire Geoghegan-Quinn said: “This discovery could lead to an effective anti-malaria treatment that would save millions of lives and transform countless others. This demonstrates yet again the added value both of EU-funded research and innovation in general and of collaboration with researchers in developing countries in particular. The ultimate goal is the complete eradication of the global scourge of malaria and collaborative work across many borders is the only way of confronting such global challenges effectively.”
Cancer drugs to kill malaria parasite
Malaria is caused by a parasite called Plasmodium, which is transmitted via the bites of infected mosquitoes. In the human body, the parasites reproduce in the liver, and then infect and multiply in red blood cells. Joint research led by EU-funded laboratories at the Inserm-EPFL Joint Laboratory, Lausanne, (Switzerland/France), Wellcome Trust Centre for Molecular parasitology, University of Glasgow (Scotland), and Bern University (Switzerland) showed that, in order to proliferate, the malaria parasite depends upon a signalling pathway present in the host’s liver cells and in red blood cells. They demonstrated that the parasite hijacks the kinases (enzymes) that are active in human cells, to serve its own purposes. When the research team used cancer chemotherapy drugs called kinase inhibitors to treat red blood cells infected with malaria , the parasite was stopped in its tracks.
A new strategy opens up
Until now the malaria parasite has managed to avoid control by rapidly developing drug resistance through mutations and hiding from the immune system inside liver and red blood cells in the body of the host, where it proliferates. The discovery that the parasite needs to hijack some enzymes from the cell it lives in opens up a whole new strategy for fighting the disease. Instead of targeting the parasite itself, the idea is to make the host cell environment useless to it, by blocking the kinases in the cell. This strategy deprives the parasite of a major modus operandi for development of drug resistance.
Several kinase-inhibiting chemotherapy drugs are already used clinically in cancer therapy, and many more have already passed phase-I and phase II clinical trials. Even though these drugs have toxic side-effects, they are still being used over extended periods for cancer treatment. In the case of malaria, which would require a shorter treatment period, the problem of toxicity would be less acute. Researchers are proposing therefore that these drugs should be evaluated immediately for anti-malarial properties, drastically reducing the time and cost required to put this new malaria-fighting strategy into practice.
The next steps will include mobilising public and industrial partners to verify the efficacy of kinase inhibitors in malaria patients and to adjust the dose through clinical trials, before the new treatments can be authorised and made available to malaria patients worldwide.
- Audrey Sicard, Jean-Philippe Semblat, Caroline Doerig, Romain Hamelin, Marc Moniatte, Dominique Dorin-Semblat, Julie A. Spicer, Anubhav Srivastava, Silke Retzlaff, Volker Heussler, Andrew P. Waters, Christian Doerig. Activation of a PAK-MEK signalling pathway in malaria parasite-infected erythrocytes. Cellular Microbiology, 2011; DOI:10.1111/j.1462-5822.2011.01582.x
- The above story is reprinted (with editorial adaptations by ScienceDaily staff) from materials provided by European Commission, Research & Innovation DG, viaAlphaGalileo.