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“Forty years ago scientists thought that malaria could be eradicated, but drugs like chloroquine were used irresponsibly and this is one of the reasons why resistant strains of malaria develop,” says Dr Mandy Rousseau, a senior researcher in the CSIR's discovery chemistry research group. For scientists and practitioners in the medical field, the greatest challenge impacting treatment of malaria in the 21st century is the continued resistance of the malaria parasite to many of the trusted drugs that have been developed over time. The complex life cycle of Plasmodium - the parasite that causes malaria - and an incomplete understanding of the biology of this life cycle make it difficult for scientists to develop new strategies to control the disease.
Life cycle of the parasite (Nature, 2002,419, 495) |
Picture of the enzyme taken from a crystal structure |
Close up of the DHFR domain, with the active site a space
filled model in yellow and the co-factor NADPH bound. |
Malaria, often regarded as 'the poor man's disease', presents an overwhelming problem in the tropical regions and developing world. There are four Plasmodium species that cause malaria in humans, with Plasmodium falciparum, which is prevalent in Africa, responsible for the most fatalities.
The parasite is injected into the human bloodstream by an infected female Anopheles mosquito. Once in the bloodstream, there is only a brief opportunity for our immune system to react to the invading parasite before it enters the liver and is shielded from the host's immune response. In the liver it replicates and is released into the bloodstream in a form that invades red blood cells where it feeds on haemoglobin, multiplies and after 48 hours ruptures to release more parasites to repeat the cycle. In some forms of malaria, liver stages can lie dormant for many months, resulting in recurrence of the disease.
“One of the problems is that there has been misuse of readily available, cheap drugs. When malaria parasites are exposed to sub-therapeutic concentrations of a single drug, resistant strains are more likely to develop.” This is why artemisinin, a natural product found in the sweet wormwood, Artemisia annua and which displays potent activity against malaria, is specifically administered in combination with other drugs (artemisinin combined therapy, ACT) to treat complicated malaria. “The use of combination therapies significantly reduces the risk of the development of resistance to a particular drug,” she says.
“With the complex life cycle of the parasite there are many stages to target, including liver stages and several blood stages. Ideally a new drug should be able to act on all stages of the parasite life cycle in the host, but this is difficult to predict,” adds Rousseau who is working on the design of antifolates that inhibit dihydrofolate reductase (DHFR), an enzyme found in Plasmodium falciparum. DHFR and another enzyme present in the folate pathway, dihydropteroate synthase (DHPS), are validated targets that have been the focus of prophylaxis and treatment of malaria for the past 50 years. Unfortunately, recent resistance to the two drugs that target DHFR; cycloguanil (administered as a pro-drug, proguanil) and pyrimethamine has limited their clinical usefulness. This makes Rousseau's work all the more important.
Rousseau, with members of her team, has discovered a novel antifolate that shows good activity against both drug-sensitive and drug-resistant strains of malaria, and is non-toxic to mammalian cells at therapeutic levels. This work forms part of a provisional patent. They are also looking into making analogues of their 'hit' compound to explore structure-activity relationships and improve activity. “The benefits of these antifolates are that they are non-toxic and cheap to produce, which is an important consideration for the treatment of malaria in Africa, where 90% of the world's malaria cases are reported.”
Incorporating molecular modelling tools means Rousseau can achieve a near-accurate hit on her target DHFR. But she cautions, “One can use molecular modelling to see if a compound could potentially bind in the active site of the target enzyme. However, molecular modelling cannot tell you whether you have an active compound or not. One has to synthesise the compounds and submit them for biological screening.” Rousseau works in collaboration with the University of the Witwatersrand's Department of Pharmacy and Pharmacology, which carries out whole-cell screening against a drug-resistant strain of malaria, as well as with the University of Cape Town, which provides in vivo studies in mice and whole-cell screening against drug-sensitive strains.
Whole-cell screening is the act of infecting a sample of human blood with malaria and adding, for example, the compound Rousseau synthesised. “But you don't know if the compound is actually inhibiting the target enzyme unless the compound is screened in the specific biochemical enzyme assay.” Development of a biochemical enzyme assay involves cloning the genetic sequence of the protein into a vector such as E. coli, followed by expression, isolation and purification of the protein. As part of the project, the PfDHFR assay, which is unavailable in South Africa, is currently being developed at CSIR Biosciences.
Rousseau and her colleagues are in the last year of this three-year project. Her current work is a far cry from her original research focus on the development of fine chemicals when she joined the CSIR in 2000. She has found working on biological targets more interesting and relishes being part of a global force behind efforts to eradicate the malaria scourge.
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