Identification of small molecule inhibitors targeting Plasmodium falciparum Hsp70-Hop pathway

Abstract

Malaria is caused by protozoan parasites of the Plasmodium genus. An estimated 405 000 million deaths due to malaria were reported in 2018. Plasmodium falciparum (P. falciparum) accounts for the deadliest form of the disease in Sub-Saharan Africa. P. falciparum has a complex life cycle spanning across both the mosquito vector and human hosts. During the lifecycle, the parasite is thus subjected to various physiological conditions. Therefore the parasite generally survives under stressful conditions and in response it upregulates its own molecular chaperones mainly the heat shock proteins (Hsps) in order to protect its protein constituents for survival. Heat shock protein 70 (Hsp70) together with heat shock protein 90 (Hsp90) are regarded as the most abundant cellular chaperones. Hsp70 and Hsp90 cooperate in order to exchange peptide substrates for folding and maturation. The cooperation of Hsp90 and Hsp70 is known to be facilitated by a co-chaperone, Hsp70-Hsp90 organizing protein (HOP) which serves as a module to facilitate interaction of the two chaperones. P. falciparum Hop (PfHop; PF3D7_1434300) brings together PfHsp70-1 and PfHsp90 into a functional complex. PfHop interacts with PfHsp70-1 (PF3D7_0818900) and PfHsp90 (PF3D7_0708400) through its tetracopeptide repeats (TPR) domains. PfHsp70-1 and PfHsp90 possess C-terminal EEVD and MEEVD motifs, respectively. The TPR1 and TPR2A of PfHop bind to the EEVD motif of PfHsp70-1 while TPR2B binds to the MEEVD motif of PfHsp90. It is thought that PfHsp70-1, PfHop and PfHsp90 form a functional complex that facilitates the folding of specific parasite proteins. Both PfHsp70-1 and PfHsp90 are essential molecules that could be specifically inhibited by potential antimalarial drugs. Therefore, this study evaluated the inhibition of the functional partnership between PfHop and PfHsp70-1 by colistin sulfate and pifithrin µ. In order to carry out in vitro assays, both PfHop and PfHsp70-1 were heterologously expressed in E. coli XL1 Blue cells and further purified by nickel affinity chromatography. Using ELISA, it was observed that the inhibition of the interaction between PfHsp70-1 and PfHop was more pronounced in the presence of colistin sulfate as compared to pifithrin µ. In addition, using surface plasmon resonance (SPR) assay, it was demonstrated that colistin sulfate and pifithrin µ directly bind to to PfHsp70-1 in nanomolar range. Furthermore, both colistin sulfate and pifithrin µ inhibited the PfHsp70-1 and PfHop interaction. Taken together, the findings from this study broadens the search for small molecule inhibitors of Hsp70 including repurposing some known antimicrobials such as colistin sulfate towards targeting malaria parasite.