BioWorld International Correspondent

LONDON - A better understanding of the genetic changes that allow the malaria parasite, Plasmodium falciparum, to become resistant to the malaria drug chloroquine could pave the way for a new generation of malaria drugs, researchers believe.

The new study demonstrated how point mutations in one of the parasite's genes, that encoding a protein called PfCRT, can influence whether Plasmodium falciparum is resistant to chloroquine or sensitive to it.

Stephen Ward, Walter Myers professor of parasitology and deputy director of the Liverpool School of Tropical Medicine in the UK, told BioWorld International: "Our work provides a significant advance in understanding the molecular basis of resistance to chloroquine. That should provide us with a platform on which we can rationally redesign novel chloroquine-like drugs that will be both cheap and safe, and effective against resistant parasites."

The work is reported in the Sept. 24, 2004, issue of Molecular Cell in a paper titled "Evidence for a Central Role for PfCRT in Conferring Plasmodium falciparum Resistance to Diverse Antimalarial Agents."

Chloroquine has been an ideal drug for the treatment of malaria because it is both cheap and safe - and was, until the 1960s, highly effective. But then the parasite began to show resistance to it. Recent increases in mortality from malaria have been blamed on the spread of resistance. Although other drugs exist, they are much more expensive and beyond the reach of many people in areas such as sub-Saharan Africa.

Earlier work by David Fidock and his colleagues at the Albert Einstein College of Medicine in the Bronx, N.Y., identified the parasite's gene, pfcrt, as one involved in conferring resistance to chloroquine. The most recent study, a collaboration between the New York and Liverpool teams, explored the function of the protein PfCRT further, and demonstrated how mutations in the gene affect how it works.

The team studied a range of parasites, each with resistance to one of several drugs, including chloroquine, another malaria drug called halofantrine and an antiviral drug called amantadine. The latter is used to treat influenza but also is known to be effective against chloroquine-resistant strains of P. falciparum even though its structure is unrelated to known antimalarials.

"We looked at the phenotypes of these strains of P. falciparum that were resistant to these drugs, and we found out, by comparison with genetically manipulated parasites, how we could make them sensitive again," Ward said. "When we looked at how the parasites had changed, we identified novel mutations in pfcrt that no one had identified before."

When P. falciparum infects a human host and invades the red blood cells, the parasites digest the hemoglobin within an organelle called the digestive vacuole. The result is a toxic byproduct called free heme. The parasite turns that into a non-toxic crystalline form, called malaria pigment, or hemozoin. Chloroquine and some other antimalarials form a complex with the free heme, preventing it from being inactivated by the parasite. The complex of drug and free heme also kills the parasite.

PfCRT is a transmembrane protein found on the membrane of the digestive vacuole. In wild-type parasites, Ward said, it appears that PfCRT acts as a molecular filter, preventing positively charged chloroquine from escaping from the vacuole. The "barrel" of the molecule has a positive charge.

Ward's team showed that, in parasites that are resistant to chloroquine, specific changes in amino acids resulting from mutations in the gene encoding PfCRT lead to loss of the positive charge from the transmembrane channel. As a result, drugs such as chloroquine can drift out, across the concentration gradient.

Ward added: "This finding is important because it gives us a mechanistic explanation for the observations we have. We now need to understand what are the characteristics that control the ability of this protein to allow the drug to egress through the mutated protein."

He and his colleagues are embarking on a study of a series of transfected pfcrt mutants. "We want to try to understand the relationship between the amino acid composition in the transporter, the physicochemical features of the pore of the transporter and the physicochemical features of a whole series of chloroquine analogues to try to produce the information required for rational redesign, based on structure-activity relationships," Ward said.

Interestingly, the results showed that mutations in pfcrt that increased the resistance of the parasite to chloroquine tended to reduce its resistance to halofantrine and amantadine, and vice versa. Ward said that is related to the site at which the drugs act: If the mutation opens the PfCRT channel, then it will tend to favor drugs that act outside the digestive vacuole, while mutations that allow the channel to remain closed tend to favor those drugs that act within the vacuole.

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