Erythrocyte G Protein as a Novel Target for Malarial Chemotherapy

Abstract

Background

Malaria remains a serious health problem because resistance develops to all currently used drugs when their parasite targets mutate. Novel antimalarial drug targets are urgently needed to reduce global morbidity and mortality. Our prior results suggested that inhibiting erythrocyte G _s signaling blocked invasion by the human malaria parasite Plasmodium falciparum.

Methods and Findings

We investigated the erythrocyte guanine nucleotide regulatory protein G _s as a novel antimalarial target. Erythrocyte “ghosts” loaded with a G _s peptide designed to block G _s interaction with its receptors, were blocked in β-adrenergic agonist-induced signaling. This finding directly demonstrates that erythrocyte G _s is functional and that propranolol, an antagonist of G protein–coupled β-adrenergic receptors, dampens G _s activity in erythrocytes. We subsequently used the ghost system to directly link inhibition of host G _s to parasite entry. In addition, we discovered that ghosts loaded with the peptide were inhibited in intracellular parasite maturation. Propranolol also inhibited blood-stage parasite growth, as did other β ₂-antagonists. β-blocker growth inhibition appeared to be due to delay in the terminal schizont stage. When used in combination with existing antimalarials in cell culture, propranolol reduced the 50% and 90% inhibitory concentrations for existing drugs against P. falciparum by 5- to 10-fold and was also effective in reducing drug dose in animal models of infection.

Conclusions

Together these data establish that, in addition to invasion, erythrocyte G protein signaling is needed for intracellular parasite proliferation and thus may present a novel antimalarial target. The results provide proof of the concept that erythrocyte G _s antagonism offers a novel strategy to fight infection and that it has potential to be used to develop combination therapies with existing antimalarials.

Abstract

Erythrocyte G protein signaling is needed for intracellular malarial parasite proliferation and thus may present a novel antimalarial target.

Abstract

Editors' Summary

Background.

New drugs for treatment of malaria are urgently needed, because the malaria parasite has evolved resistance against virtually all types of commonly used drugs. When a person is bitten by a malaria-infected mosquito, the parasite first infects the person's liver cells before going on to infect red blood cells, where the parasites multiply and develop into a parasite stage called a schizont. The red blood cells then burst and release more schizonts into the bloodstream; it is this “blood stage” of infection in humans that causes the symptoms of disease. Therefore efforts to develop new drugs against malaria often focus on this “blood stage” of infection. One strategy for developing new drugs is termed the “host-targeted” approach. This means that rather than trying to block processes occurring within the parasite itself, a drug can be developed which blocks processes within the person's red blood cells, and which would otherwise be needed for the parasite to complete its life cycle. It will be difficult for malaria parasites to evolve resistance to such a drug, because changes in a person's red blood cells occur much more slowly than in the parasites themselves.

Why Was This Study Done?

This research group has been studying a set of molecular processes within human red blood cells which seemed to be required for entry of malaria parasites into the cells. They wanted to get a better understanding of those processes and, specifically, to find out whether it would be possible to use particular molecules to block those processes, and by doing so to prevent malaria parasites from entering and multiplying within red blood cells. In particular, when the malaria parasites invade the red blood cell, they form membranes around the red blood cell, containing lipids and proteins “hijacked” from the red blood cell membrane. These researchers already knew that two particular proteins were hijacked in this way; the β ₂-adrenergic receptor (β ₂-AR) and heterotrimeric G protein (G _s). These two proteins act together to pass messages across the surface of the membrane to inside the cell. Small molecules could be used to block signaling through β ₂-AR and G _s, and therefore potentially to provide a new way of preventing malaria parasites from entering red blood cells and multiplying within them.

What Did the Researchers Do and Find?

Firstly, the researchers made red blood cell “ghosts” in which to study these molecular processes. This meant that they took fresh red blood cells from healthy human volunteers, burst them to remove half the contents and loaded them with markers and other cargoes before resealing the membranes of the cell. These resealed markers and cargoes allowed them to see what was happening inside the cells. Malaria parasites were able to invade these ghosts normally and multiply within them. When the researchers introduced a specific peptide (a molecule consisting of a short series of amino acids), they found that it blocked G _s signaling within the ghosts. This peptide also prevented malaria parasites from developing inside the ghosts. Therefore, they concluded that G _s signaling inside the red blood cell was important for the parasite life cycle. The researchers then examined a drug called propranolol which is already known to act on G _s signaling and which is commonly prescribed for high blood pressure. This drug also blocked development of malaria parasites inside the ghosts when used at a particular concentration. Finally, the researchers studied the effect of giving propranolol, along with other antimalarial drugs, to human malaria parasites in a culture dish and to mice injected with a malaria parasite that infects rodents. In these experiments, adding propranolol reduced the amount of other “parasite-targeted” drugs that were needed to effectively treat malarial infection in tissue culture and in mice.

What Do These Findings Mean?

Showing that the G _s signaling pathway is important for the malaria parasite's life cycle opens up new possibilities for drug development. Specifically, propranolol (which is already approved for treatment of high blood pressure and other conditions) might itself provide a new candidate therapy, either alone or in combination with existing drugs. These combinations would first, however, need to be tested in human clinical trials, perhaps by seeing whether they have antimalarial activity in people who have not responded to existing antimalarial drugs. Since it acts to lower blood pressure, which can already be low in some people with malaria, there are some concerns that propranolol might not be a suitable drug candidate for use, especially with existing antimalarial drugs that also reduce blood pressure. However, other molecules which block G _s signaling could be tested for activity against malaria should propranolol prove not to be an ideal drug candidate.

Additional Information.

Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030528.

• The World Health Organization publishes a minisite containing links to information about all aspects of malaria worldwide, including treatment, prevention, and current programs for malaria control

• Medicines for Malaria Venture is a collaboration between public and private organizations (including the pharmaceutical industry) that aims to fund and manage the development of new drugs for treatment and prevention of malaria

• Wikipedia entries for drug discovery and drug development (Wikipedia is an internet encyclopedia that anyone can edit)