Insertion of GGMP repeat residues of Plasmodium falciparum Hsp70-1 in the lid of DnaK adversely impacts client recognition.

Although Hsp70 is a conserved molecular chaperone, it exhibits some degree of functional specialisation across species. Features of Hsp70 regulating its functional specialisation remain to be fully established. We previously demonstrated that E. coli Hsp70 (DnaK) exhibits functional features that distinguishes it from PfHsp70-1, a canonical cytosolic Hsp70 of Plasmodium falciparum. One of the defining features of PfHsp70-1 is that it possesses GGMP repeat residues located in its C-terminal lid segment, while DnaK lacks this motif. Previously, we demonstrated that the insertion of GGMP repeat residues of PfHsp70-1 into E. coli DnaK abrogates the chaperone activity of DnaK. However, the role of the GGMP motif in regulating Hsp70 function remains to be fully understood. To explore the function of this motif, we expressed recombinant forms of wild type DnaK and its GGMP insertion motif, DnaK-G and systematically characterised the structure-function features of the two proteins using in silico analysis, biophysical approaches and an in cellulo complementation assay. Our findings demonstrated that the GGMP inserted in DnaK compromised various functional features such as nucleotide binding, allostery, substrate binding affinity and cellular proteome client selectivity. These findings thus, highlight the GGMP motif of Hsp70 as an important functional module.

Pharmacophore Model-Based Virtual Screening Workflow for Discovery of Inhibitors Targeting Plasmodium falciparum Hsp90.

Plasmodium falciparum causes the most lethal and widespread form of malaria. Eradication of malaria remains a priority due to the increasing number of cases of drug resistance. The heat shock protein 90 of P. falciparum (PfHsp90) is a validated drug target essential for parasite survival. Most PfHsp90 inhibitors bind at the ATP binding pocket found in its N-terminal domain, abolishing the chaperone's activities, which leads to parasite death. The challenge is that the NTD of PfHsp90 is highly conserved, and its disruption requires selective inhibitors that can act without causing off-target human Hsp90 activities. We endeavored to discover selective inhibitors of PfHsp90 using pharmacophore modeling, virtual screening protocols, induced fit docking (IFD), and cell-based and biochemical assays. The pharmacophore model (DHHRR), composed of one hydrogen bond donor, two hydrophobic groups, and two aromatic rings, was used to mine commercial databases for initial hits, which were rescored to 20 potential hits using IFD. Eight of these compounds displayed moderate to high activity toward P. falciparum NF54 (i.e., IC50s ranging from 6.0 to 0.14 µM) and averaged >10 in terms of selectivity indices toward CHO and HepG2 cells. Additionally, four compounds inhibited PfHsp90 with greater selectivity than a known inhibitor, harmine, and bound to PfHsp90 with weak to moderate affinity. Our findings support the use of a pharmacophore model to discover diverse chemical scaffolds such as FM2, FM6, F10, and F11 exhibiting anti-Plasmodium activities and serving as valuable new PfHsp90 inhibitors. Optimization of these hits may enable their development into potent leads for future antimalarial drugs.