Australasian Science Magazine

Eggs from the African clawed frog have enabled scientists to determine how malaria parasites developed resistance to chloroquine.

By Tegan Dolstra

As the malaria parasite pits its all against the only treatment still standing, award-winning research has revealed the secret to reviving the most successful antimalarial drug in history.

More than one million people die from malaria every year, but this figure could be about to expand dramatically. The parasite responsible for 95% of fatal malaria cases has begun to develop resistance to artemisinin, the only undefeated treatment available. In the absence of a vaccine, the spread of artemisinin resistance through the parasite population will spell disaster.

A back-up drug is desperately needed, and former wonder drug chloroquine is an ideal candidate. Developed in the 1930s, this cheap, safe and effective drug was the first choice malaria treatment for three decades before the parasites caught up. Following the emergence and irrepressible spread of chloroquine-resistant parasites around the world, chloroquine is now ineffective in most cases.

We have recently uncovered the mechanism driving chloroquine resistance. This discovery has the potential to act as a basis for the design of new chloroquine-like drugs that bypass the resistance mechanism and resurrect the wonder drug to its former position as a frontline therapy.

Tiny Invaders
The agent that causes malaria is a unicellular parasite that is transmitted from person to person by female mosquitoes. While mother mozzie steals a nourishing blood meal from you to feed her offspring, she deposits malaria parasite stowaways from her salivary glands into your bloodstream.

These intruders quickly make their way to the haven of your liver cells, where they reproduce in the tens of thousands, undetected by your immune system. After a couple of weeks they burst back into your bloodstream, searching for a new home.

Red blood cells are the perfect refuge for the parasites because they are packed with a rich protein source – haemoglobin. Each parasite forces its way into a red blood cell and begins to break down haemoglobin into its component peptides and amino acids for its own use.

The digestion of haemoglobin occurs in the parasite’s acidic digestive vacuole. Here, a poisonous waste product of this breakdown, haem, is rendered harmless by crystallisation into an inert substance known as haemozoin.

This is where chloroquine enters the scene. The acidic environment of the digestive vacuole causes chloroquine to become trapped within it, accumulating to very high levels. Chloroquine can now carry out its mission of killing the parasite.

The widely accepted theory is that chloroquine binds to haem, interfering with the formation of haemozoin. The parasite, now defenceless against the toxic haem, is poisoned and dies.

Resistance Emerges
In its heyday, chloroquine was instrumental in eliminating malaria from 50 countries as part of an eradication attempt launched by the World Health Organisation in the 1950s. But the emergence of chloroquine-resistant parasites during this campaign was a disaster in the fight against malaria. Forty of these 50 countries are again affected by malaria, and the number of malaria deaths in some parts of Africa has more than doubled.

Chloroquine-resistant parasites first appeared in Colombia in the early 1960s. How did this microscopic parasite thwart such a powerful drug? The secret lies in the extent of chloroquine accumulation in the digestive vacuole: chloroquine-resistant parasites accumulate much less chloroquine than chloroquine-sensitive parasites.

The main protein responsible for chloroquine resistance was identified in 2000. Located on the membrane of the digestive vacuole, it was named the chloroquine resistance transporter (PfCRT) because of its similarities to other transport proteins.

One hypothesis was that the version of PfCRT harboured by chloroquine-resistant parasites is able to “leak” chloroquine out of the digestive vacuole, preventing the accumulation of the drug and its interference with haemozoin formation. However, for the next 10 years chloroquine transport via PfCRT could not be shown directly.

Frog Eggs 101
The main obstacle to measuring PfCRT activity directly is that the protein is hidden away on an internal membrane within the parasite. The answer to this problem is to express the protein on the outermost plasma membrane. We have recently been successful in expressing PfCRT on the plasma membrane using eggs from the African clawed frog.

Frog eggs are ideal for expressing PfCRT for two reasons. Measuring about 1.5 mm in diameter, they are large enough to inject with genetic material encoding mutant forms of PfCRT. Furthermore they do not contain haem or any other parasite transporters that may confound the direct measurement of chloroquine transport via PfCRT.

However, as 10 years of unsuccessful attempts attest, achieving expression of PfCRT on the egg plasma membrane was not straightforward. We first had to remove the parts of the coding sequence that cause PfCRT to be transported to an internal membrane.

Once PfCRT is positioned on the egg’s plasma membrane, direct measurements of its activity can be easily made. We begin by incubating eggs that have been injected with genetic material encoding PfCRT in an acidic solution (to mimic the parasite’s digestive vacuole) containing radioactive chloroquine. After 1–2 hours, the chloroquine that has been transported by PfCRT across the plasma membrane into the eggs is released by rupturing the eggs. The amount of radioactivity released is measured and compared with non-injected eggs that have undergone the same treatment.

Using this system, we have shown that the form of PfCRT found in chloroquine-resistant parasites is capable of transporting chloroquine out of the digestive vacuole, whereas the version of PfCRT found in chloroquine-sensitive parasites is not. This long-awaited finding provides the first direct evidence of the mechanism driving chloroquine resistance.

The new insights garnered from our findings have the potential to unlock the secret to reversing chloroquine resistance.

Old Drug, New Tricks
It took almost 20 years of intense selection pressure before resistance to chloroquine arose. This slow genesis is quite remarkable considering the drug’s successor didn’t last 5 years before it met resistance. Mass, uncontrolled usage – including the addition of chloroquine to table salt in Brazil in the 1950s and a plethora of counterfeit drugs on the black market – no doubt shortened the wonder drug’s reign.

What was the secret to chloroquine’s extraordinary longevity? The answer lies in its target, haemozoin formation, which is critical for the parasite’s survival. No parasite proteins are directly involved in this process, and haem itself is encoded by the human host.

This means that haemozoin formation is outside the genetic control of the parasite. Hence the genesis of resistance is not straightforward: the parasite could not mutate haem so it co-opted a protein of its own, PfCRT, to prevent chloroquine from making contact with haem.

The remarkably long life of chloroquine points to haemozoin formation as the malaria parasite’s Achilles’ heel, highlighting the need to look for ways to reinvent chloroquine. One such approach we are taking is the design of chloroquine-like drugs that bypass the PfCRT-mediated resistance mechanism.

This approach appears promising. Last month, a group at India’s Central Drug Research Institute modified the chemical structure of chloroquine to create a compound that appears to share chloroquine’s ability to inhibit haemozoin formation and is just as effective against chloroquine-resistant parasites as it is against chloroquine-sensitive strains.

Another tactic in the fight against malaria is the use of molecules that “undo” resistance to chloroquine. More than 40 such “resistance-reversers” have been described.

Along with collaborators at the University of Cape Town and the Australian National University’s Research School of Chemistry, we are using our frog egg system to test a broad range of resistance-reversers in the hope of identifying one that could be used in combination with chloroquine in humans.

Last year, by merging elements of both chloroquine and resistance-reversers, a research group in Portland synthesised a molecule that combines the lethal haemozoin-inhibiting properties of chloroquine with the ability to reverse chloroquine resistance. This compound can cure a mouse form of malaria, with no adverse side-effects.

We are currently collaborating with the designers of this new type of drug to determine whether it interacts with PfCRT. Further research into these hybrid molecules should prove exciting.

The normal function of PfCRT remains a mystery. This is of considerable interest because we know that parasites cannot survive without the protein. We are seeking to uncover the natural function of PfCRT in the hope that this discovery could lead to the development of a drug that abolishes this vital activity.

Meanwhile there is hope that chloroquine may return as the mainstay of antimalarial treatment via a much simpler route. Our research has shown that PfCRT can only transport chloroquine at a certain rate. This suggests that if the amount of chloroquine in the digestive vacuole is high enough, PfCRT will be overloaded and the drug will still accumulate to a sufficient level to kill the parasite.

In fact, in Guinea-Bissau, where chloroquine has always been prescribed at three times the regular dosage, the proportion of chloroquine-resistant parasites is exceptionally low. These findings raise an intriguing question: could millions of lives be saved simply by increasing the dosage?

The mechanism behind the malaria parasite’s resistance to one of the most successful drugs of all time has finally been revealed. Our research may bring about a renaissance in chloroquine therapy, restoring the former wonder drug to millions in need.