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UMiss Tekwani on Assaying Natural Products for Anti-Malarial Activity

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At A Glance

Name: Babu Tekwani

Position: Senior Scientist, National Center for Natural Products Research, University of Mississippi School of Pharmacy

Background: Senior Assistant Director, Central Drug Research Institute, Lucknow, India — 1984-2001; PhD, biochemistry, Lucknow University — 1984

Babu Tekwani supervises the anti-parasitic drug discovery program at the National Center for Natural Products Research at the University of Mississippi’s School of Pharmacy. He has spent the majority of his career studying the molecular basis of malaria and leishmania, two of the biggest infectious disease scourges in tropical regions of the world. His present research focuses on developing target-based assays for in vitro screening of new drug candidates against such infections. Last week, he discussed with Inside Bioassays the unique approach his laboratory is taking in this drug discovery effort.

How did you become interested in your current field of study?

I am a biochemist and molecular biologist by training. I did my PhD on molecular pathogenesis of cerebral infection by Acanthaamoeba culbertsoni, which is a free-living amoeba. This protozoan is responsible for causing a brain disease called primary amoebic meningo-encephalitis. Because I was working in this area, I became interested in protozoa. Subsequently, I realized that the most important protozoa are those causing the diseases of leishmania and malaria. So I then shifted my interest to tropical disease, particularly malaria and leishmania. Malaria affects 200 to 300 million people annually, and kills more than 2 million people annually, particularly in tropical regions — especially in Africa — and most of the people killed have been children under five years old. What I realized is that most of the pharmaceutical companies are not interested in this, because you don’t make money off of drugs against malaria or leishmania, or tropical diseases in general. Most pharmaceutical companies are not pursuing these types of programs. Since I was working in an academic center — the Central Drug Research Institute, which is a federally funded institute — their model is not to make any profit, but to work in disease areas that are of public importance. And tropical diseases are the kind where public awareness has to be built, and this is what these kinds of institutes do.

Tell me a little bit about your current organization, the National Center for Natural Products Research.

It is part of the School of Pharmacy at the University of Mississippi. They used to have one institute that was called the Research Institute of Pharmaceutical Sciences. They were working with a lot of natural products, as were a lot of people at the School of Pharmacy. With this model, they set up the center. I don’t know too much about the history, but the center is primarily involved in three areas. One is new drug discovery from natural products. Another is neutraceuticals, particularly dietary supplements, where they are primarily working on quality control — fingerprinting or analytical techniques — of dietary supplements. And another is the cultivation of pharmaceutically important plants. In the field of drug development, the center is dealing with four areas. One is anti-parasitic drugs — malaria and leishmania. We plan to enter into the area of trypanosomes very soon, as well, because they are also causing a lot of disease in South America and Africa. We plan to set up screening [techniques] for African trypanosomiasis and Chagas disease. Another area is anti-fungal drugs, and there’s a group working on immunomodulators, and one working on anti-cancer programs. My area is the anti-parasitic drug discovery.

Tell me about the metabolic pathway that you are studying related to Plasmodium infection.

The Plasmodium parasite grows inside the erythrocytes. Within these cells, it needs a lot of nutritional items that it has to obtain from the host. In the life cycle of the parasite, there are two hosts: the mosquito vector, which is responsible for transmission of the disease, and the human host. So one of the ways to develop drugs against this parasite is to look for some of the metabolic functions which are either unique in the parasite — unique in the sense that they are not present in the host — or maybe metabolic functions that are present in both the parasite and the host, but different in the parasite than in the host. In this area, the genome sequencing of one of the parasites, Plasmodium falciparum, was completed in October 2002. And one of the major benefits of the completion of this project is that with the help of the genome sequence, [scientists] have been able to make a preliminary draft of all the metabolic pathways in the malaria parasite. Once you have this draft, you have an idea of which pathways might be unique to the parasite or different in the parasite. One of the important things that has been discovered is that the parasite has a unique sub-cellular organelle that is known as the apicoplast. This is something like chloroplasts in plants. The presence of this is a distinct property of a group of parasites that is known as apicomplexan. In this group there is the malaria parasite, cryptosporidium, and toxoplasma. Some of the metabolic pathways that have been uncovered in these types of organisms are very similar to those found in plants and the lower bacteria. Similar pathways are not present in the host system. What we are doing in particular is trying to look at the functional importance of these different metabolic pathways in the parasite.

Are you focusing on one pathway in particular?

One of the pathways … is the thiamine, or vitamin B1, biosynthesis pathway. This pathway may be unique in the sense that the host system depends on thiamine — you have to have thiamine in your diet to fulfill the requirement. The mammalian host does not have the capability of biosynthesis. If you have a thiamin deficiency, or don’t have enough in your diet, you can develop some types of diseases. But what we discovered is that the malaria parasite may have the capacity to synthesize its own thiamine. Still, we know that thiamine will be important for the parasite, but since this is a very new pathway, it has to be determined whether it is really necessary for the parasite or not. One of the major points where the pathway may be a drug target has to be necessary for the survival of the parasite.

Have you begun developing drug screening assays against this pathway or any of the other pathways that have been uncovered?

Not exactly this pathway. When it comes to drug development for malaria, the most important assay that we have is that we have an in vitro culture system for the malaria parasite. We can culture the parasite using human red blood cells. This culture system is very common and has been used by many laboratories involved in malaria research. So we use this human RBC culture system for growing the parasite, and we can do this in 96-well microplates and can be used for screening compounds that might have anti-malarial activity.

What types of assays do you use in terms of reagents and instrumentation used?

For detecting anti-malarial activity, there are three ways to do that. There was initially a conventional method that many people used, which is the microscopic method. Once you cultured the parasites on the plates, you just take a drop of blood and put it on the slide and make a smear, and then try to observe the parasite under the microscope. But that was not a very comfortable method. Subsequently, some quantitative methods were developed, and there are two in this area. One is based on the incorporation of radioactive metabolic precursors, and on such precursor is known as H-hypoxanthine. One of the properties of the malaria parasite is that there are certain precursors for nucleic acid synthesis called purines. So the parasite depends on salvaging purines rather than synthesis of them. And hypocentine is one of the important precursors in this process. When the parasite grows in culture, RNA and DNA is synthesized, and the hypoxanthine is incorporated by the parasite, and you can quantitatively see how much has been incorporated. And if you add a test compound to the plate, you can see how much inhibition of this incorporation occurs. Another assay that we are using is a non-radiative spectrophotometric assay called malstat assay. This was developed by some researchers who discovered that the malaria parasite has a lactate dehydrogenase, which is very different in malaria parasite, and it can use one specific co-factor which is not used by humans. So based on this assay, you can do a spectrophotometric assay that you can read in a simple microplate reader or spectrophotometer. The quantity of the lactate dehydrogenase produced in the RBC’s is directly proportional to the growth of the parasite.

Are there any particular challenges in developing assays for screening drugs against infectious diseases or parasitic diseases in particular?

Everyone is moving toward high-throughput assays, and one of the important challenges in the field of malaria is that with the present status, it cannot be upgraded to a high-throughput system because you need to use the human RBC cultures, and when you culture the parasite using these cells, they are not in homogenous form. They tend to settle down. So the development of high-throughput assays based on this is not feasible.

Do you see ways to maybe increase the throughput in the future?

Yes, if we can get some kind of in vitro and in vivo correlation using the molecular target assays, those target assays may be more adaptable to high-throughput screening. But the only problem with the target-based assays is that they will only be able to identify a certain class of the compound. So you are likely to miss the compounds that are working through some other mechanism. What we are planning to do is use the target-based assays in primary screening, and the culture-based assays in secondary screening.

So with these secondary assays, you might be able to identify some off-target effects that you were not previously predicting?

That is true. One other thing I wanted to add is that when it comes to new anti-malarial drug discovery from natural products, the road that we follow is initially we get the crude extracts from different groups. One of the new programs that we are pursuing is the identification of anti-malarial drugs from marine sources. So these are the pre-natural product sources we are currently using. What we do is initially get the organic extracts of these natural sources from plants, microbial cultures, or marine sources. We screen the crude extract first, and then we extract the lead extract. There is a group of chemists working with us that fractionate these natural extracts, and then we get fractions and again test them for anti-parasitic activities, and tell them: OK, this is the fraction that has the highest anti-malarial activity. Then they go further and purify the active components and tell us: This is the active component of the natural product that is responsible for anti-malarial activity. This is the way we are able to identify new, natural products for anti-malarial activities. Besides testing these compounds for anti-malarial activity, we simultaneously test them for general cytotoxicity. This is important because we have to make sure — whether it’s an extract of a natural compound or it’s a synthetic compound — that it is showing good activity because it is acting on the parasite and not because it has general cytotoxicity. Besides in vitro assays, we have some small animal models for this.

Any promising leads?

One of the leads that we have from the School of Pharmacy is an alkaloid that has been isolated from sea sponge. This was isolated by one of the faculty in the department of pharmacognosy, Mark Hamann. He isolated this compound, and the lead compound shows very good anti-malarial activity in vitro, and also in animal models. This is presently being pursued by an agency in Geneva that is known as the Medicine for Malaria Venture. They have added some of our lead molecules to their portfolio, and we have funding from them. And one of the promising things that Mark’s group has recently discovered is that this compound is really produced by one of the microbes associated with the sea sponge. So that gives us confidence that sourcing of the compound in the future will not be difficult.

 

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