Investigation of Plasmodium falciparum heat shock protein 20 as a novel target for antimalarial drug discovery

Abstract

Investigation of Plasmodium falciparum heat shock protein 20 (PfHsp20) as a novel target for antimalarial drug discovery:

Malaria is caused by a single-celled microorganism, a protozoan parasite of the genus Plasmodium, which is introduced into a person's blood in the form of sporozoites from the saliva of the female Anopheles mosquito. From there, the parasite travels to the liver where it matures and reproduces. The sporozoites remain in the liver for 7 to 10 days and are then released into the bloodstream as merozoites. These invade red blood cells and multiply until the cells burst, after which they invade more erythrocytes. The parasites evade the human immune system by constantly presenting different antigens at different stages of their life cycle, making vaccine development difficult. To date, more than 200 species of Plasmodium have been identified and described as parasitic to reptiles, birds, and mammals. P. falciparum is the cause of the most serious form of human malaria. In 2019, the WHO recorded 229 million cases of malaria worldwide and more than 400,000 deaths, with P. falciparum accounting for 99.7% of cases. If the disease is diagnosed early, it can potentially be treated with prescription drugs. The most common antimalarial drugs include chloroquine phosphate and artemisinin-based combination therapies (ACTs). However, malaria parasites have developed resistance to antimalarial drugs and there are high recrudescence rates (around 25%). The aim of this project is to search for a novel therapeutic target protein against the malaria parasite. The malaria parasite has a complex life cycle, passing from one host to another and being constantly exposed to sudden changes in growth conditions. To survive and proliferate the parasites need to develop multiple adaptation mechanisms and resistance responses. One of these mechanisms is to overexpress small heat-shock proteins. Small heat-shock proteins (sHsps) are among the most conserved proteins in organisms. It is known that sHsps are ATP-independent molecular chaperones, which stabilize partially unfolded target proteins that arise under stress conditions. The proteins refold into their native structure with the assistance of ATP-dependent chaperones. Small heat shock proteins play a crucial role in enhancing the survival of cells and protecting proteins from stress factors, so their roles are clinically significant. Structurally, sHsps are very dynamic and can form monomers, dimers, as well as ensembles of 24 to 40 multimeric complexes. Heat-shock protein 20 is a member of the small heat-shock protein family of molecular chaperones, with a subunit mass of 20 kDa. P. falciparum has three sHsps in its proteome. In this project, work was conducted on one of these, PfHsp20, which plays a crucial role in the survival of the parasite under conditions of thermal and oxidative stress. The parasite needs to survive in the cold-blooded invertebrate mosquito and in the warm-blooded vertebrate human host, requiring a thermal adaptation mechanism as well as an oxidate stress-coping mechanism, and both of these avenues offer options for finding novel drugs that could target malaria. This project highlights the key structural and functional characteristics of PfHsp20. A wide variety of biophysical, spectroscopic, and cell biological techniques were used. The major ones were CD spectroscopy, SEC-MALS, TEM, X-ray analysis, CLEM, palmitoylation assay, pull-down assay, and CellROX assay. We investigate the key properties of the protein and propose further steps that might be taken in order to devise a potentially effective anti-malarial drug. List of Abbreviations: CD=Circular dichroism, SEC-MALS=size exclusion chromatography multi-angle light scattering, TEM=transmission electron microscopy, CLEM=correlative light and electron microscopy Show more