Debopam Chakrabarti, Professor and head of Division Molecular Microbiology, talks about malaria and the efforts he and his colleagues are doing to find treatments.
Most of our viewers will have heard of malaria but may not have a real good idea of how people contract it, how prevalent it is. Can you tell me a little bit about malaria?
CHAKRABARTI: Sure. The malaria has been with us since the beginning of recorded history. If you go down the history, it was found in Egyptian mummies. Alexander the Great was reported to be of the case of malaria recorded during his invasion of India and the western part of India. Although it is debatable whether he died actually of malaria or not. In the medieval times Italy was endemic with malaria as a result popes and papal candidates died of malaria. It would be surprising to people that most of our early presidents got malaria because Foggy Bottom area was quite endemic for malaria. All the individuals on our dollar bills had malaria. I think the last president who got malaria was Theodore Roosevelt. Not in the United States, but because he was traveling he got it outside the country. Malaria is a vector-borne disease, which is the vector is the malaria. The mosquito, which is of anopheles type has a complex life cycle. The whole disease starts with the bite from an infected mosquito. During the bite, the mosquito injects the parasite into the bloodstream. It goes into the liver and then it flourishes there and it multiplies. After a few days, the new parasites in different forms are released into the bloodstream. Those are taken up by the RBC or red blood cells. And then the red blood cell cycle starts. So, every 48 hours, the parasite lyses the red blood cell and a bunch of new parasites are released into the bloodstream and they reinfect. This event causes the chill and fever, which is a typical symptom. There’s periodic episodes of fever and chill, which is every 48 hours because that’s what the life cycle within the RBC. Then certain signals dictates that the parasite differentiates or changes into a different shape called gametocytes. And those gametocytes are then taken up by mosquito during another blood meal, and then the mosquito cycle starts. In spite of all efforts to discover and control malaria, there are no effective vaccine yet. There are some promising candidates which have been reported recently but the drugs against malaria are becoming ineffective because of the emergence of resistance. As a result, about 420,000 people each year dies of malaria, mostly children in Africa. The cause of death is due to t malignant malaria or the cerebral malaria. The malaria parasite goes into the capillary and then occludes the capillary. And that causes the severe form of the malaria.
With that as background, sir, what are you and your colleagues doing and your colleagues here in this lab to try to come up and find some treatments or some therapies for malaria?
CHAKRABARTI: As I mentioned that the drugs which are effective against malaria are losing their efficacy because of the appearance of resistance. So, there is a need for new therapies or new drugs. What we are doing in the lab is to come up with new drug leads which can replace the existing drugs or can act as a partner to the existing drugs to alleviate the problem caused by drug resistance.
And if you could describe for our viewers how you’re going about looking at some of the microbes and then moving from, you know, what you’re looking here to something that might eventually lead to drug therapy. And I know it involves moldy Cheerios. Can explain how that works?
CHAKRABARTI: We started a collaboration with a research group at University of Oklahoma, which is led by Robert Cichewicz. He has created a huge collection of fungal specimens. As you know, that the fungi has been a good source, specifically, fungal metabolites have been a good source of drugs. For example, statin, penicillin, they all come from fungus. There are a huge number of fungi worldwide, but only five to 10 percent of those have been identified and explored. What Robert’s lab has done in Oklahoma, that he has used crowdsourcing to acquire samples from all over the United States. And he has isolated the fungi specimens from those soil samples. And in the process, he has now a collection of over 75,000 different fungal specimens. So, he grows them in media and during the course of his studies, he found by trial and error of different media component, that the breakfast cereal supports the growth of fungus the best and the growth is reproducible and which helps to maintain the program to discover the new the drug leads from fungal sources.
When their lab grows the fungus from the Cheerios, do they ship that to you or then what do you?
CHAKRABARTI: Oh, no.. After grpwing on solid phase cherrios, they extract the fungus to create an extract, the extraction can be done using various solvents. What we receive here is a plate with different sample extracts which are derived from different fungal specimens. Then we take those samples and we add to our malaria parasite culture, which we grow in the lab in human RBC samples, and then test for the activity. The activity is defined by inhibition of growth and when we see an inhibition by an extract sample, we report back to Robert and he then starts purifying that extract. The goal is to identify the compound present in the extract which is causing the inhibition of growth. This is a very involved process and it’s quite complex.
How many of those compounds has your lab identified at this point?
CHAKRABARTI: This is an NIH funded project. So far, we have screened over 5,000 extracts and there’s another 5,000 we are planning to screen, and from that we have about our initial hits identified. We only take the most potent ones, which inhibits growth over 90% compared to the control, and we have over 250 samples of those. Then we do a counter-screen. Counter-screen means that we test these active extracts against human liver cells. The idea is to identify extracts and compounds which are effective against malaria but is not going to have any effect on human cells, because we want the host to survive. Based on the selectivity index, preferably – acceptable one is 10 and ideally it should be 100 – that means it should be 100 times more effective on the parasite cells compared to the human cells. This is a multi-institution project. Another partner is the University of California, San Diego lab, Dr. Elizabeth Winzeler, and that fourth lab is in the University of Texas, San Antonio Kirsten Hanson. In the Winzeler lab, what they do is they’re testing in the liver stage growth. At the beginning I explained the lifecycle, where the parasite goes after it is released from the mosquito bite, is the liver. One of the main thrust in the malaria drug discovery is to identify compounds which are effective against multiple stages. The drugs against the red blood cell will cure the disease. But if you have a drug which is effective against the liver stage, that will be prophylactic, so that will be very useful for traveler and the military personnel who are in the malaria endemic region. There are two drugs which are used mainly for prophylaxis. One is doxycycline, the problem with that is you have to take every day and the other one is mefloquine that has some side effects and about 10% of individual has psychosomatic effects. Another drug malarone is also losing efficacy. So those are not ideal. We test for the liver stage activity in the Winzeler lab. Another thing we are doing in collaboration with the Winzeler lab is to identify the molecular target. So here is what we are doing, initially, we use a cell-based screen, which we call a phenotypic screen. There are two types of drug discovery. One is a target-based where you have a protein which is the cause of a disease. Although, in a true sense, the disease phenotype is more complex. A single molecule may be responsible predominantly, but there are other proteins which also play a role. So that’s a target-based approach. Which is a target-based rational drug discovery, the other one is cell-based screening, which is phenotypic screen, and you can also call it irrational drug discovery, jokingly. Because you do not know what you are looking for. There is an advantage to that. If you have a target-based discovery, you’re only targeting one protein or a handful of protein. But if you are doing the phenotypic screen your initial readout is the inhibition of growth. But then inside the cell, for example, in the case of malaria parasite in the blood stage, probably about 3,000 genes are involved to maintain the parasite growth in the blood stage. You’re essentially targeting 3,000 proteins. The goal of phenotypic screen here is to identify the compounds which are active, then define the target later. In collaboration with the Winzeler group we are defining the molecular target, because that’s what you need later on to fine-tune the compounds you have identified, you make analogues based on the molecular target.
How important is it, doctor, to have these multiple compounds that would work against multiple targets at different stages of the malaria cycle? How important is it to have all of these different options at some point for people with malaria?
CHAKRABARTI: It’s very important because our project goal is to define five targets each year. This is a five-year project, so that there will be 25 new targets. And in contrast, right now, the drugs against malaria only have a handful of targets. So essentially, we are going to expand the target landscape for malariaSo that’s why it’s going to be very significant.
If you could describe for our viewers what it is that you and your colleague are looking at.
CHAKRABARTI: We are going to demonstrate what we do to screen compounds. Essentially what the robot is doing is to deposit extracts in the plate, the extracts which we receive need to be diluted. So the goal is to make a serial dilutions and to find the most potent, that means that the extracts which are effective at the lowest concentration. You have to determine that. First we do the serial dilution in plates and then we add the malaria parasite-infected RBC, which we grow in the culture. Then they’re incubated for 72 hours in the incubator and after that, we add a lysis buffer which contains a dye. This dye, which is called SYBR green binds to the DNA and that will give a readout that means that will give a reading. The idea is in comparison to the control culture, we expect to see an inhibition in the treated culture, which will have less value or a lower reading because there will be less amount of cells as there is an inhibition of growth. Lower growth will lead to lower amount of DNA, and this dye binds to the DNA, so it will give a lower signal. So that’s what we are going to detect.
Is there anything I did not ask you that you would want to make sure that people who see this know about your work here in this lab?
CHAKRABARTI: Well, I think another aspect here is to understand the biology of the parasite. What I described so far is the drugs against malaria, which is an urgent need, but the parasite biology is very complex. Even the process of drug discovery is very complex, and that’s why drugs are so expensive. Most of the lead compounds which we would be identifying will not make it to the drug stage. Of the 25 or so which we expect to target and the lead compounds which we are going to identify, probably only one or two will pan out. Why they fail? For various reasons. The most of these compounds will fail because of their unwanted toxicity and also their pharmacokinetics that means the amount of the compound which will persist in the blood for time. Often as soon as the compounds goes into the bloodstream, various enzymes act on it and the compounds are degraded. They do not last long and that lead to the ineffective amount of compound persisting in the blood, which doesn’t cure the disease. We can get a lot of positive results in the animal model, but humans are not mice, so the actual challenge starts when the compounds get into the clinical trial. Another challenge with the malaria is the malaria is very species-specific. So the human malaria only affects human. But we have to test in a model system, we cannot test in human. So our model is the malaria model in rodents, in mice. Although mouse malaria is similar to human, but there are some differences and there are instances where the compounds which are effective in the rodent model are not effective in humans. There are different forms of parasites also, there’s a vivax malaria which is predominant in India and southeast Asia.. It has been reported that some of the compounds which are effective against falciparum malaria, which we work on, are not effective in the vivax malaria. There are a lot of challenges in malaria drug discovery. However, even the drugs which will fail in the clinical trial could be useful for as a probe. We call these chemical probe. So, we can use those molecules to probe different pathways to identify new targets and to understand a better biology of the parasite, which ultimately helps us to understand the parasite and develop improved therapies.
I know people will say, you know, why Cheerios?
CHAKRABARTI: I think the idea is because you need a carbohydrate source for growth, and also certain amount of. I think that Cheerios, when it’s manufactured, there’s a relatively good quality control and this consistency which helps the growth. I asked Dr. Cichewicz the same question, why? And that was his answer. I think the Cheerios has a consistency which probably helps to get the reproducible results, but that’s a solid source. Ultimately, when we are isolating the active component, we need to havethe culture grown in a larger volume. Then you move to the liquid culture, and he grows in bags for getting – harvesting larger amounts of secondary metabolites. These metabolic product are released into the media, and then you take the grown media and extract it from there. There’s another new discipline which has evolved from this science. It’s called synthetic biology. In synthetic biology – these compounds which are synthesized by the fungus enzymes are present on the genome. And because each of these metabolite is made in a multi-step process by the organism. And usually it has been seen that the genes which are responsible for synthesis is arranged in a cluster. So the idea in synthetic biology is to identify, what are the genes in the fungus is making these metabolites? So you can put the gene cluster, which is responsible for synthesizing the active components in a fungus in yeast. As yeast is easier to grow and the engineered yeast is going to make the same metabolites. The same concept has been used for the artemisinin. Artemisinin is now the drug of choice for malaria, and it is made in a Chinese plant which is difficult to grow and there is a lot of problem in the supply. It has been published that they identified the genes and the enzymes which are responsible for synthesis of artemisinin, they put that in yeast so that you can grow yeast and you make the compound. So that’s a synthetic biology.
Interview conducted by Ivanhoe Broadcast News.
END OF INTERVIEW
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