Malaria: when an antimalarial drug is no longer fit for purpose
High fever, chills, cramps and severe anaemia are typical symptoms of malaria, even its mild form. According to World Health Organisation (WHO) estimates, around 660,000 people die from malaria every year. The disease is transmitted by female Anopheles mosquitoes infected by single-celled parasites of the genus Plasmodium. Malaria is difficult to treat or cure as it is caused by a eukaryotic organism that is biochemically more similar to humans than bacteria. Nevertheless, there are a number of drugs that can help prevent and treat malaria. One of them is atovaquone, which targets a respiratory chain protein in the plasmodia mitochondria and kills the parasites. However, mutations at a putative atovaquone-binding site are making more and more Plasmodium strains resistant to the drug. Prof. Dr. Carola Hunte and Dominic Birth from the Institute for Biochemistry at the University of Freiburg have shown how atovaquone binds to the protein and what happens at the molecular level in resistant plasmodia. Their research provides an impetus for structure-based drug design aimed at specifically improving the antimalarial.
History shows that it is possible to eliminate the disease. While the disease was still common worldwide a century ago, malaria is now largely restricted to tropical and subtropical regions in Africa and South-East Asia. According to WHO estimates, 3.3 billion people, or around half of the world population, are at risk of malaria. In 2012, there were an estimated 207 million cases of malaria that led to an estimated 600,000 deaths. Around 90% of all malaria deaths occurred in Africa, 77 percent in children under five. New cases of malaria have been reported in Greece every year since 2010. The disease is a persistent, global problem, with an emphasis on global. Insights into the underlying processes play a major role in the effort to make in-roads into the prevention and treatment of the disease as the mechanism of action of many antimalarial drugs is not yet known in detail. In the case of atovaquone, science is now one step further forward.
Atovaquone inhibits a protein in the respiratory chain
Atovaquone is an antiprotozoal drug mainly used for the prevention of protozoal infections when travelling, but it is also used for the treatment of malaria, toxoplasmosis and a form of pneumonia caused by the fungus Pneumocystis. “Atovaquone is effective against a broad range of single-celled pathogens,” says Prof. Dr. Carola Hunte from the Institute for Biochemistry and Molecular Biology at the University of Freiburg. Hunte and her team of researchers are investigating the mechanism and function of medically relevant proteins, particularly membrane proteins. Atovaquone interacts with this kind of enzyme complex in Plasmodium. Synthetic atovaquone fits perfectly into the three-dimensional pocket of the pathogen’s mitochondrial cytochrome bc1 complex, thus inhibiting the latter. “Atovaquone is similar to the natural substrate of cytochrome bc1 and is therefore able to bind in the same binding pocket as the natural substrate, thereby preventing the substrate from binding,” says Hunte explaining why atovaquone is a competitive inhibitor of the natural substrate.
The cytochrome bc1 complex is a key enzyme of the electron transport chain, generating energy in the form of ATP. It is the third of four protein complexes in the mitochondrial membrane that transfer electrons consecutively to oxygen. Ubiquinol, which is an essential component of the electron transport chain, binds in the centre of the cytochrome bc1 complex, transfers two electrons to the complex and is thus oxidised to ubiquinone. If atovaquone manages to bind to this particular site, ubiquinol is prevented from binding. The transfer of electrons is interrupted. Little is known about what happens next. Although inhibition of the cytochrome bc1 complex leads to the demise of the malaria parasite, the molecular details underlying its death are not yet understood in detail.
Ubiquinol is in a key position
It may be that the antiparasitic effect of the drug atovaquone is purely due to the fact that it disrupts the mitochondrial electron transport chain at the cytochrome bc1 complex, thereby preventing ATP synthesis. The cell is unable to generate the energy it needs to persist. However, it is possible that there is another reason for the drug’s effect. That said, the drug also prevents the regeneration of ubiquinone. After doing its work in the respiratory chain, ubiquinol becomes ubiquinone that enters the nucleic acid synthesis process where an essential pyrimidine biosynthesis enzyme is waiting for it. “We assume that the death of the parasite is caused by the inhibition of the cytochrome bc1 complex, which leads to the collapse of the mitochondrial membrane potential and disruption of pyrimidine biosynthesis,” says Hunte.
Many anti-cancer drugs target the biosynthesis of nucleotides, as cells require these pyrin and pyrimidine building blocks to copy the DNA before they divide into two new cells. As far as pyrimidine biosynthesis is concerned, mammals have a different pyrimidine synthesis pathway from that of protozoans, and this could be the reason why atovaquone specifically targets protozoan pyrimidine synthesis.
The drug atovaquone is prevented from exerting its deadly action when it cannot fit into the binding pocket of its target protein. And this happens more and more frequently. The structure and sequence analyses carried out by Hunte and her team have shown that point mutations in the binding region inhibit the enzyme. The most common mutation involves the exchange of an amino acid that leads to the elimination of the aromatic side chain in the enzyme’s catalytic centre, which normally stabilises the position of the drug. “The drug loses one of its most important docking sites and has a reduced affinity,” says Hunte. “Parasites with this particular mutation are resistant to atovaquone.”
X-ray crystallography brings it to light
Attempting to visualise the three-dimensional structure of the protein-drug complex is no small matter. Crystallisation of membrane proteins is rather difficult as they are bound to lipids and thus not soluble in water. Many research groups, including Prof. Hunte and her team, have been working long and hard to resolve the structure of the atovaquone-inhibited cytochrome bc1 complex. Hunte and her doctoral student Dominic Birth have now succeeded. “We used a special trick,” says Hunte. “We used recombinant antibodies produced in E. coli to crystallise this membrane protein, and they helped us to shed light on particularly challenging structures.” Rather than using the entire antibody, the researchers only used Fv fragments (variable fragment), which are the smallest antigen-binding elements of an antibody. “This helped us arrange the molecules in a relatively accurate three-dimensional grid,” says Hunte.
Hunte and her team used baker’s yeast (Saccharomyces cerevisiae) as their model organism because Plasmodium cytochrome bc1 is difficult to obtain in the quantity and purity required. Dr. Wei-Chun Kao, a post-doctoral researcher in Hunte’s team, carried out time-consuming sequence analyses and was able to show that the relevant amino acids in the catalytic centre of yeast corresponded to those of the parasite complex.
When they compared the cytochrome b sequences of parasites, humans and yeast, the team found “that the essential contact sites in the binding pocket with which the drug interacts are all identical”. The comparison of the sequences of these three species enables the position of amino acids in the Plasmodium cytochrome bc1 complex to be predicted. Specific sequence differences in the binding pocket of human cytochrome bc1 might potentially explain why atovaquone binds more weakly in humans.
Hunte now wants to pass the ball on to chemists who will be able to use this information for specifically modifying atovaquone. Knowing how the original molecule binds will help the researchers make useful suggestions on how to modify it in order to break the resistance of the parasites. “The pharmaceutical industry and generics producers now have the structure they need to develop new, more effective antimalarial drugs; and I hope they will be able to put this knowledge to good use,” says Hunte.
Prof. Dr. Carola Hunte
Institute for Biochemistry and Molecular Biology
University of Freiburg
Tel.: +49 (0)761 / 203 - 5279