Abstract:
With nearly half the world’s population at risk of malaria infection, 229 million new cases were recorded in 2019 with Sub-Saharan Africa accounting for most of these cases. In 2020, 384 000 malaria deaths were reported in the WHO African region. Plasmodium falciparum is the most virulent species responsible for over 90 % of all malaria infections. The P. falciparum life cycle occurs through multiple stages in humans and mosquitoes. Many advances have been made in the fight against malaria through vector control and treatment against infection by the parasite. However, resistance of P. falciparum to antimalarial medicines/treatment has hindered global efforts to control and eliminate malaria. A multidimensional approach to fighting malaria by targeting is essential protein machinery is needed. Heat shock proteins (Hsp) are molecular chaperones that are upregulated in response to stress and have long been projected as antimalarial drug targets. P. falciparum Hsp70-1 (PfHsp70-1; PF3D7_0818900) is an essential heat shock protein involved in the folding of newly synthesized and misfolded polypeptides and is also implicated in protein trafficking. PfHsp40 (PF3D7_1437900.1) is a type I Hsp40 co-chaperone of PfHsp70-1 that presents substrates to PfHsp70-1 and stimulates its ATPase activity, while PFF1010c (PF3D7_0620700.1) is a type IV Hsp40 found in the gametocytes that is yet to be characterized. The current study explored pyrimidine-quinoline hybrid (PQH1-4) compounds as possible inhibitors of PfHsp70-1 interaction with PfHsp40. Furthermore, this study sought to characterize the structure and function of PFF1010c. Notably, PFF1010c possess SVN residues in place of the HPD motif located in the so-called J domain of this Hsp40 co-chaperone. For this reason, PFF1010c is an atypical Hsp40 which may lack the capability to interact with PfHsp70-1. To characterise the role of PFF1010c, and consequently, its SVN motif, the SVN motif of PFF1010c was switched with the HPD motif of PfHsp40. The structure-function features of the mutants were characterized relative to the wild type Hsp40s. Bioinformatics based analysis was employed to predict the fold of the mutants relative to the wild type Hsp40s. Recombinant proteins were expressed in E. coli cells and purified using nickel affinity chromatography. Fluorescence spectroscopy was used to map out the tertiary structure of the Hsp40 proteins. It was noted that the HPD motif confers thermostability to the proteins. Possible interaction of PFF1010c with PfHsp70-1 was evaluated using
ATPase assay and SPR analysis. Furthermore, the same study was repeated for the motif switch mutants. PFF1010c was found to interact with PfHsp70-1, though the interaction would not be as conventional as the one between PfHsp40 and PfHsp70-1 because PFF1010c does not have the HPD motif. SPR analysis was also employed to investigate binding of the pyrimidine-quinoline hybrid compounds to PfHsp70-1. Compounds PQH1 and PQH4 were found to have high binding affinities for PfHsp70-1. The compounds also inhibited binding of PfHsp40 to PfHsp70-1. The study provides the first evidence that PfHsp70-1 interacts with a type IV Hsp40, where PfHsp70-1 and PFF1010c could be a druggable complex to reduce transmission of malaria from humans to mosquito. The pyrimidine-quinoline hybrid compounds would play a role in the proliferation of the malaria parasite by inhibiting the chaperone activities of PfHsp70-1 and PfHsp40.
