parasitic diseases – Drug Discovery Opinion

Kinase Target for Toxoplasmosis, maybe Malaria

Crystal structure of inactivated form of CDPK1 from toxoplasma gondii - PDB ID 3KU2

Apicomplexan parasites such as Toxoplasma gondii and Plasmodium species can cause serious diseases in humans and domestic animals. Because the parasites are eukaryotes and share many metabolic pathways with their hosts, it has proved difficult to develop safe and effective treatments but researchers at Washington University School of Medicine in St. Louis have now identified an essential kinase in T. gondii which is unlike human kinases and more closely resembles those found in plants. In a study published in Nature, the team used conditional suppression to show that T. gondii calcium-dependent protein kinase 1 (TgCDPK1) is essential for survival of the parasite. The enzyme controls the ability of T. gondii parasites to secrete microneme proteins which allow the parasites to control their movement and move in and out of host cells.

It should be possible to exploit the differences between the parasite kinase and human kinases to develop potent and selective inhibitors of the parasite enzyme and the team have already identified compounds that block CDPK1 signalling without affecting human cells. Pyrazolopyrimidine-derived compounds such as 3-MB-PPI were found to specifically inhibit TgCDPK1 and disrupt the parasite’s life cycle at stages dependent on microneme secretion. The disruption was dependent on TgCDPK1 inhibition since parasites expressing a mutant kinase not sensitive to the inhibitors.

Calcium-dependent protein kinases have a kinase domain similar to that of calmodulin-dependent kinase, regulated by a calcium-binding domain in the C terminus. X-ray structures of TgCDPK1, published in Nature Structural and Molecular Biology, showed that, in the auto-inhibited (apo) form, the C-terminal activation domain resembles a calmodulin protein with an unexpected long helix in the N terminus that inhibits the kinase domain in the same manner as calmodulin-dependent kinase II. Calcium binding triggers reorganization of the C-terminal activation domain into a highly intricate fold, leading to its relocation around the base of the kinase domain to a site remote from the substrate binding site. This large conformational change constitutes a distinct mechanism in calcium signal-transduction pathways.

CDPK1 may play a similar role in Plasmodium species which cause malaria, but the researchers predict that it may be harder to selectively inhibit the Plasmodium enzymes.

Locking out or Locking in – It’s Not the Same Key for Malaria

Plasmodium parasites, responsible for malaria in humans, have a complex lifecycle that is dependent on mosquito and human hosts. In human blood, the merozoite stage of the parasite invades red blood cells (erythrocytes), growing and multiplying before rupturing the cell and escaping to infect other erythrocytes. It is this profound effect on erythrocytes that is responsible for the symptoms of malaria – fevers, chills and anaemia. Untreated, the disease can be fatal and drug resistance is an increasing problem. With up to half a billion people infected each year and nearly a million deaths, mostly in sub-Saharan Africa, there is an urgent need for new treatments.

Researchers at Harvard School of Public Health (HSPH) were attempting to identify the mechanism by which Plasmodium falciparum merozoites enter erthyrocytes, but instead found a parasite protein that is essential for escape from the cells. When the protein, P. falciparum calcium-dependent protein kinase (PfCDPK5), was suppressed the parasites were trapped in the host cell and unable to infect new cells. In further experiments the team showed that these merozoites were still able to invade erythrocytes if released from their host cell by other means, indicating separate mechanisms for invasion and egress from erythrocytes.

The findings reveal an essential step in the biology of P. falciparum and suggest a new, parasite-specific, drug target for fighting one of the world’s most common and dangerous infections. Whilst many scientists are looking for inhibitors of parasite egress and invasion of red blood cells, no anti-malarial drugs yet target these stages of the parasite lifecycle.

The study is published in Science.

Chagas Passes Down the Line

Several million people in Latin America are estimated to have Chagas disease, although most will not know that they are infected. The disease is caused by the protozoan Tryptanosoma cruzi and is transmitted by the bite of the blood-sucking beetles known as ‘kissing bugs’ because of their tendency to bite close to the mouth. Although the disease typically produces very mild symptoms in the early stages, chronic disease can lead to severe damage to the heart and GI tract and is often fatal. Severe disease and death occur many years after initial infection – even when the parasites are no longer present – and Brazilian scientists speculated that T. cruzi DNA might be retained in the body and that the resulting genomic alterations could explain the rejection of heart tissue by the host immune system. An unusual feature of virulent T. cruzi is that extra-nuclear mitochondrial DNA known as the kinetoplast (kDNA) accounts for 15-30% of the total cellular DNA. The kDNA is made up of thousands of minicircles which can insert into the genome of an infected individual. Depending on where the parasitic DNA is inserted, it can affect genes involved in cell communication, correct function of the immune system and even our sense of smell. The parasitic DNA can also be incorporated into the DNA of sperm and eggs and so can appear in the genome of children who have never themselves been in direct contact with the parasite.

Although it has been known for some time that the human genome contains significant amounts of viral DNA that have evolved with us over millions of years, the new research suggests that the transfer of mitochondrial DNA from T. cruzi to the human population may also be contributing to human genetic diversity. The parasite is especially well placed to fulfil this role since it infects millions of people, many of whom will migrate to other continents, infects many species of wild animals, and rarely kills its human victims below the age of forty.

The study is published in PLoS ONE.

Closantel May be Useful in Fighting River Blindness

Adult black fly with Onchocerca volvulus emerging from the insect’s antenna — Adult black fly with *Onchocerca volvulus* emerging from the insect’s antenna Photo: United States Department of Agriculture

Onchoceriasis – also known as river blindness – is the world’s second leading infectious cause of blindness. The disease is caused by the nematode, Onchocerca volvulus, and is transmitted to humans through the bite of a blackfly. Once inside the body, the female worm produces thousands of larval worms (microfilariae) which migrate to the skin and eyes. When the microfilariae die, they cause intense itching and a strong immune response that can destroy nearby tissue, leading eventually to blindness and disfiguring skin lesions. Control programmes have involved the use of larvicides to reduce blackfly populations and the use of ivermectin to treat infected people and limit the spread of disease. Ivermectin is most effective against the larval stage of the worm and is believed to kill the parasites by activating glutamate-gated chloride channels which are specific to invertebrates.

A team led by researchers at the Scripps Institute has now focused on a new way to kill the parasite. The protective outer cuticle of the worms is made of chitin and two classes of enzymes – chitin synthases and chitinases – are known to be critical for chitin formation and remodelling. One chitinase, OvCHT1, is expressed only in the infective third-stage larvae and is believed to be involved in development and host transmission. The team screened a small library of compounds for activity against OvCHT1 and found that closantel was able to inhibit the enzyme. When closantel was tested on cultured third-stage larvae, the compound prevented the larvae from moulting and developing into adult worms. Since the mechanism of action of closantel is completely different to that of ivermectin, it – or other chitinase inhibitors – could potentially be used to treat ivermectin-resistant worms. Closantel is a broad-spectrum anti-parasitic agent currently used in some countries in veterinary medicine.

The study is published in the Proceedings of the National Academy of Sciences.