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Several mutations in a single gene make malaria resistant to chloroquine

Nearly a million children die each year of malaria, but the parasite became resistant to the cheapest drug. Now we know why.

By | November 17, 2000

LONDON Tom Wellems in the United States says it's taken him 15 years. David Warhurst in the UK says he's been studying the problem since 1963. Many others have been worrying at the issue at least as long. But now Wellems at the US National Institute for Allergy and Infectious Diseases (NIAID) and colleagues have pinned down the first of the serious drug resistances, chloroquine-resistant P. falciparum malaria, to a group of mutations in a single gene.

Wellems dubs the affected gene pfcrt, coding for a membrane protein — PFCRT — that sits in the wall of the parasite's food vacuole. The nomenclature means "P. falciparum chloroquine-resistance transporter", but exactly what the gene does, and how the mutations affect its action, is still up for research. Nevertheless it seems that its correlation with drug resistance is very close, in strains from all over the world.

David Warhurst, Professor of Protozoal Chemotherapy at the London School of Hygiene and Tropical Medicine, who has been privileged to work with Wellem's data for some months before publication, told BioMedcentral, "It seems clear that this is a very important result, and that these particular mutations may be the primary change that occurs in chloroquine resistance. The level of resistance they create is low, but they may open the gate to higher resistance by additional mutations. It doesn't say that explicitly in the paper, but having worked in this area for years, that's how it seems to me."

Chloroquine is still the cheapest and safest of the malaria drugs, but different degrees of resistance, from slight to total, have arisen throughout the tropics since its trumpeted introduction in the late 1940s. It still works in China, and in Central America and North Africa. In the 1950s, in combination with DDT against mosquitoes, it was expected to help eliminate malaria from the world — but resistance set in, first in South-East Asia in 1957, and then in South America in 1959. Moving from East to West, it covered tropical Africa between 1978 and 1985. Other drugs, such as mefloquine, halofantrine, pyramethamine and more recently artemisinin derivatives are far more expensive, and difficult for the poorest countries to afford. Yet malaria is said to kill 700,000 under-fives each year in Africa alone.

Wellems and co-workers made a false start three years ago, when his group reported another gene, dubbed cg2, was associated with resistance to chloroquine [see paper]. Wellems now says that was a mistake — but that the highly fragmented gene pfcrt lies in the same region, on the same chromosome, chromosome 7.

According to Wellems, "cg2 has now been ruled out" as the centre of chloroquine resistance. "We found cg2 by classical reverse genetics. At first it seemed to be the likely candidate gene. We'd mapped resistance to just 36 kb of chromosome 7, just 0.1% of genome. We'd removed 99.9% of the haystack, and sitting there was cg2, but it proved to be more difficult than we thought to pin down the resistance gene itself... The original software for P. falciparum sequencing, searching and identifying genes was based on other organisms, and it did not pick up pfcrt- although it is in the same region as cg2. We are still in the nascent phase for recognizing genes and their promoters from P. falciparum sequence information; pfcrt has 13 exons and 12 introns, and it was not showing up. No pfcrt exon has more than 100 amino acids, and the original software cut off below 100."

But haven't other genes on other chromosomes been related to drug resistant malaria in the past, BioMedcentral asked Warhurst? "There's been data on chromosome 5 since 1990, which looked as though it could pinpoint chloroquine resistance to the Pfmdr1 gene" Warhurst said, "but in some circumstances you can get all the changes you need there, but still get sensitivity to chloroquine. It looks as though chromosome 7 is the missing factor."

"There is more evidence this time than there was for cg2," said Warhurst, "cg2 was not clearly associated with the chloroquine resistance in South America, whereas pfcrt seems to fit the case everywhere."

"But I don't suppose this is the end of the story" he said. "Mefloquine resistance and artemisinin resistance, for example, seems to be mainly Pfmdr1."

So what might pfcrt gene do? Warhurst explained: "The protein coded by Pfmdr is believed to pump out drugs. Its structure is similiar to the transmembrane multidrug resistance (MDR) proteins that have been proven to pump drugs out of cancer cells. They get their energy from ATP breakdown. As for pfcrt, it seems also to make a transmembrane protein — as it also has membrane-soluble regions — and there is some similarity to TetA, which pumps tetracycline from E. coli using an intrinsic hydrogen ion gradient to supply the energy, a gradient produced by respiratory electron transport."

Malaria parasites feed on haemoglobin from the host, but that produces a waste product, hemin — iron in a porphyrin ring — which is toxic to the parasite. Normally the hemin polymerises into a form the parasite can eliminate. But chloroquine — and several other drugs including amodiaquine, quinine, mefloquine and halofantrine — complex with hemin and interrupt the polymerisation, leaving the toxic hemin and killing the parasite. Artemisinin, artemether and artesunate also interfere with the iron metabolism, releasing free radicals.

Careful optical work with fluorescent dyes by one of Wellems' team, Paul Robey, has shown that the pfcrt mutations increase the acidity of the food vacuole. According to Wellems, "we then have two hypotheses. Our first is that reducing the pH causes more efficient aggregation of hemin, thus raising the amount of chloroquine needed to have an effect. Our alternative idea is that the mutations reduce the flux of chloroquine across the membrane into the food vacuole."

But "whatever the details, the discovery of pfcrt changes the foundations for thinking about the whole process of chloroquine resistance" said Wellems. "There have been dozens of different theories, but now we have a specific molecule to focus on. It opens up new ways of thinking. Now we can think of a structurally specific action between a transporter and a drug."

This may lead chemists towards creating long-lived new drugs, according to Wellems. "If we can mimic the action of chloroquine with another drug that beats the pfcrt resistance mechanisms, it will have a long life" he believes. "Hemin is not going to mutate. Chloroquine came on scene in the late 1940s and was remarkably effective. It took only a single mutation to beat pyramethamine, but with chloroquine it turns out that multiple mutations were required in a single gene" — so resistance grew slowly.

However it is not clear how far this will really speed up drug development. It can take 15 years and up to $500 million to get from a research idea to a drug in the field. Wellems says that any modifications of chloroquine will have to be tested for side-effects, and so "this discovery may not reduce the cost of developing a substitute for chloroquine. Phase I, II & III trials, the packaging, dosing and distribution are going to cost as much as for any drug".

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