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

Authors

Aaron M Keeler, Gaini Ibrasheva, Elizabeth G Boger, Yueqi Chen, Porter E Petruzziello, Erin A Schroeder, Christopher R Mansfield, Kayla Sylvester, Cameron D Fleming, Philip F Hughes, Timothy A J Haystead, Michael C Fitzgerald, Emily R Derbyshire

Published in

Antimicrobial agents and chemotherapy. Pages e0046626. Jul 02, 2026. Epub Jul 02, 2026.

Abstract

In the malaria parasite Plasmodium falciparum, the essential chaperone PfHsp70-1 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 PfHsp70-1 as an antimalarial drug target. Here, we identified small molecules that bind PfHsp70-1 using a high-throughput thermal shift screen, an unbiased approach that enables the discovery of ligands targeting any PfHsp70-1 domain. Molecules were prioritized by their affinity to PfHsp70-1 and anti-Plasmodium activity. These efforts led to the characterization of AMK3 and AMK4, which bind to PfHsp70-1 with low-micromolar affinity and exhibit selectivity (>25-fold) for PfHsp70-1 over the human homolog HSPA1A based on microscale thermophoresis. The PfHsp70-1 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 PfHsp70-1, 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 PfHsp70-1 functions during infection and as therapeutics to treat malaria.

PMID:
42390429
Bibliographic data and abstract were imported from PubMed on 02 Jul 2026.

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