Development of domain-specific probes of Plasmodium falciparum heat shock protein 70-1

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ABSTRACT In the malaria parasite Plasmodium falciparum , the essential chaperone Pf Hsp70-1 regulates proteostasis through protein folding, but its domain-specific functions remain poorly defined. The protein contains an N-terminal nucleotide-binding domain (NBD) and a C-terminal substrate-binding domain (SBD), where selective inhibitors are needed to probe domain-specific functions and advance Pf Hsp70-1 as an antimalarial drug target. Here, we identified small molecules that bind Pf Hsp70-1 using a high-throughput thermal shift screen, an unbiased approach that enables the discovery of ligands targeting any Pf Hsp70-1 domain. Molecules were prioritized by their affinity to Pf Hsp70-1 and anti- Plasmodium activity. These efforts led to the characterization of AMK3 and AMK4, which bind to Pf Hsp70-1 with low-micromolar affinity and exhibit selectivity (>25-fold) for Pf Hsp70-1 over the human homolog HSPA1A based on microscale thermophoresis. The Pf Hsp70-1 binding regions of AMK3 and AMK4 were isolated using a combination of proteomic and biochemical methods, which demonstrated that AMK3 binds to the NBD and competes with ATP binding. In contrast, AMK4 binds to the SBD and leads to disruption in peptide binding. A molecular dynamic-facilitated structure-activity relationship (SAR) screen was performed on AMK3, yielding a compound with retained anti- Plasmodium activity but reduced host cytotoxicity. This study identifies species-selective N-terminal and C-terminal inhibitors of Pf Hsp70-1, and suggests druggable binding pockets on the C-terminal domain that may be exploited for the disruption of essential protein-protein interactions. These domain-specific inhibitors are useful starting points for the development of probes to advance our knowledge of Pf Hsp70-1 functions during infection and as therapeutics to treat malaria.

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