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| Malaria parasites infecting a red blood cell. Image from CDC. |
Today's article comes from Science World Report; it's about the findings of a research paper just published in The Proceedings of the National Academy of Sciences regarding a potential new drug for the treatment of malaria. They found that treating the malaria parasites with a compound named (+)-SJ733 caused the parasites to be unable to hide from the infected host's immune system. This resulted in rapid recovery from infection.
According to the news article, the new potential drug allows the immune system to clear 80% of the parasites from the body in 24 hours, and all of them by 48 hours from the beginning of treatment. Given these promising results researchers are working to begin safety trials in humans and are hoping that within the next few years this drug will prove safe, effective, and relatively inexpensive. If so this will be a big advance for the treatment of malaria, which is currently problematic due to the high rate of dangerous side-effects and the evolution of drug resistant strains of malaria.
Malaria is caused by infection with a Plasmodium: specifically P. falciparum, P. malariae, P. ovale, P. vivax or P. knowlesi. These organism are protists; single-celled microorganisms similar to amoebae. They are not bacteria. Though both protists and bacteria are single-celled organisms, protists are eukarytoic while bacteria are prokarytoic. This means that the protists' cell is very much like our own cells, with a nucleus and other membrane bound organelles.
These Plasmodium parasites have a complex two-host life cycle. The two hosts are mosquitoes and warm-blooded vertebrates (including humans). Plasmodium reproduce both sexually and asexually, sexual reproduction and infection spread require the mosquito. When the mosquito bites someone infected with malaria, the blood meal contains the gametes (think sperm and eggs) of the Plasmodium; these gametes can only fuse to form a zygote in the gut of the mosquito. These zygotes then develop into sporozoites that can migrate from the mosquito gut to the salivary glands. When the mosquito bites her next victim (only female mosquitoes bite), the sporozoites are able to move out of the mosquito and into their new host. Let's assume it is a human that was bitten and infected with the malaria-causing Plasmodium sporozoites; the sporozoites are deposited into the blood stream and from there migrate to the liver. There they invade the liver cells, living and asexually reproducing hidden from the immune system. While hiding in the liver, the sporozoites produce thousands of merozoites. Merozoites and sporozoites are genetically identical, but have different appearance and behavior due to differently expressing the same genes. The merozoites don't stay in the liver, they infect passing red blood cells and are carried throughout the body; in the blood cells the parasites can hide from the immune system. While in the red blood cells most of the merozoites continue to reproduce asexually. After a few replication cycles the blood cell is so full of parasites that it literally bursts! The released merozoites then each attack a new blood cell and the cycle continues. A few of the blood-stream merozoites begin sexual reproduction by forming gametes instead of asexually reproduced clones; these gametes can be taken up by a mosquito if the infected person is bitten. This continues the life cycle and the spread of the malaria parasites.
The symptoms of malaria infection are similar to flu at first, starting with headache, fever, shivering, joint pain, and vomiting. These symptoms are so common in the early stages of serious illness (malaria, ebola, menegitis, etc) because they are the symptoms of your body fighting an infection. As a malaria infection progresses hemolytic anemia, jaundice, hemoglobin in the urine, retinal damage, and convulsions begin. Most of these are related to the way the parasites destroy red blood cells as they reproduce. The destruction of blood cells causes anemia and the body's attempt to clean up the damage causes hemoglobin in the urine and kidney damage. If the infection is left untreated a pattern will often develop where the flu-like symptoms come and go on a 2-4 day cycle. This is because the immune system can only attack in the small interval of time between when the blood cells burst and when the parasites hide in new cells; once re-hidden it will be a few days before the parasites have undergone enough rounds of reproduction to burst their blood cell hiding places.
Many drugs have been developed to treat malaria. The first, quinine, is still in use today. If that sounds familiar then that's because quinine is what gives tonic water it's distinct taste. Originally tonic water (with or without gin) was taken to prevent malaria. Drugs available to treat malaria today include quinine, chloroquine, amodiaquine, pyrimethamine, proguanil, sulfadoxine and sulfamethoxypyridazine, mefloquine, atovaquone, primaquine, artemisinin and derivatives, halofantrine, doxycycline, and clindamycin. However, much like the case with antibiotics used to treat bacterial infection, heavy uses and incomplete treatment regimes (often due to cost or side effects) has resulted in many cases of malaria that are difficult to treat due to evolved drug resistance. The standard of care now dictates that malaria patients be given one of a few different standard combinations of drugs to best treat the infection and prevent resistance from worsening. This increases the cost of treatment and the likelihood of side effects, some of which can be life threatening. Because of these problems with treatment and the number of people infected every year (220 million in 2010), much research has been put into finding better drugs to treat malaria.
So what did the authors of The Proceedings of the National Academy of Sciences paper find out about the possible antimalarial (+)-SJ733?
Previous work by researchers had involved making many different dihydroisoquinolone-type molecules and using large-scale screening methods to see if any of these compounds inhibited important enzymes in Plasmodium. This is an iterative process known as intelligent drug design. When researchers found a compound that inhibited an important enzyme even a little bit, they would make many new compounds that were very similar and try each of those. Each iteration would, ideally, bring researchers closer to a chemical compound worth investigating as a potential drug. This process led researchers to the compound named SJ733, which is asymmetrical and comes in two racemers: right (+) and left (-). Further research then showed that the right-handed version of SJ733, (+)-SJ733, was the most effective.
This work builds on that previous work to better understand if SJ733 works in vivo (i.e. in a living system such as a human or mouse) and how it works. First, researchers needed to know if SJ733 was safe for the host/patient. To study this, the first thing they did was test the drug on many different mammalian cell lines. These are cells that have been removed from their organism and encouraged to grow in a petri dish (or similar). The researchers did not find any negative effects of treating mammalian cells with SJ733. Next they gave various doses of the drug to mice, rats, and dogs. They tried giving the drug orally and intravenously. The animals were watched for side effects and monitored for how long the drug persisted in their blood after being administered (pharmokinetics). Researchers found the drug had a half life of about 12 hours in the blood stream, and that even with a dose as high as 240 mg per kg of body weight the animals did not seem to suffer any major or lasting problems (body condition, blood chemistry, and behavior were monitored). The highest dose did cause minor decrease in hemocrit (blood iron) that resolved within 7 days of ending drug exposure, but the hemocrit did not drop below the normal range.
After determining the safety and pharmokinetics of SJ733, the researchers tested the in vivo ability of the drug to cure malaria. They gave mice malaria and then gave them various doses of SJ733 or (+)-SJ733 and then tested the mice's blood to see if they had been cured. This treatment was compared to several other drugs already on the market for humans. Any mice that were not cured were euthanized. The researchers found a dose of 10 mg/kg of body weight over 4 days was sufficient to clear the infection from the mice. This is 24 times less than the maximum safety-tested dose of the drug. A large gap between therapeutic dose and toxic dose is a very good thing in a drug!
Next researchers wanted to understand how SJ733 was treating malaria. To do this the researchers grew P. falciparum in culture laced with a small amount of SJ733. After many generations of sub-lethal exposure to the chemical, resistance evolved. Researchers repeated this experiment several times, until they had six independent SJ733-resistant strains of parasites. They then sequenced the whole genome of each of these strains and compared them to each other and a reference genome. This allowed the researchers to identify the genetic differences between the resistant and susceptible (reference) strains. The one gene that was mutated in all six of the resistant strains (compared to the susceptible reference strain) was PfATP4. This gene encodes a protein that uses ATP (cellular energy) to move sodium ions out of the cell against their concentration gradient. The mutations all changed the shape of the inside of the sodium ion channel. In addition to conferring resistance to SJ733, these mutations also made the parasites less able to move sodium and thus slower growing than the wild-type
The researchers then took a careful look at malaria infected blood cells after treatment with SJ733. They found that when exposed to SJ733 the parasites would swell up, likely because they couldn't remove excess sodium (we've all been there!), and stop growing. Some parasites burst! They also noted that the infected blood cells swelled and started to undergo eryptosis (cell suicide in a sense). This would help the immune system clear the infection naturally, as the parasites would be unable to hide.
Based on everything they found, the researchers conclude that (+)-SJ733 would make an ideal candidate for human drug trials. The compound is easy to make. In mice there is a large difference between the dose needed to treat malaria and the dose that is dangerous. The drug works quickly, helping the immune system clear the infection in as little as 2-4 days. And, while they were able to breed resistant strains of P. falciparum, they feel resistance is not a major concern. They base this on the fact that the course of treatment is very short (the shorter the treatment the better the compliance and the less chance for evolution by the parasites) and that there is a significant fitness cost for the parasites associated with becoming resistant. In fact, the resistant phenotype is quite similar to the phenotype of the treated susceptible parasites (higher intracellular sodium, swelling, and slow growth).
So what are my conclusions?
This is one of the most complicated papers I've tackled on this blog so far. Lots and lots of work went into this paper. I was actually a little surprised (and disappointed) that I didn't recognize any of the names on the author list. A few of my fellow graduate students at Clemson study/studied malaria parasites and worked with groups doing intelligent drug design. I have
I also liked the news article today. It was short and lacking in details, but I think that's understandable given how technical this paper was (even I was a bit thin on details for that very reason, there are lots of graphs and figures in the paper if you want more specifics). In spite of the article's short length it presented the main finding of the paper without any unnecessary drama and it had a link to the research paper!!!! So, I'll give World Science Report an A. I'd expect nothing less from a media outlet with such a name.
References
Jiménez-Díaz, María
Belén, et al. "(+)-SJ733, a clinical candidate for malaria that acts
through ATP4 to induce rapid host-mediated clearance of Plasmodium." Proceedings of the National Academy of Sciences (2014): 201414221.
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