AMK3 and AMK4 selectively target PfHsp70-1 domains, revealing new antimalarial drug pockets.

Malaria, caused by the parasite Plasmodium falciparum, remains a significant global health burden, with existing vaccines offering only modest and short-lived efficacy. This necessitates the urgent development of novel antimalarial drugs targeting essential parasite pathways. The Plasmodium falciparum heat shock protein 70-1 (PfHsp70-1) is a crucial molecular chaperone responsible for regulating proteostasis and protein folding, making it an attractive drug target. However, its domain-specific functions, particularly those of its N-terminal nucleotide-binding domain (NBD) and C-terminal substrate-binding domain (SBD), are poorly understood. This lack of specific inhibitors for each domain has hindered the full exploration of PfHsp70-1's therapeutic potential.

Researchers utilized a high-throughput thermal shift screen to identify small molecules that bind to PfHsp70-1, an unbiased approach to discover ligands for any domain. Molecules were prioritized based on their affinity to PfHsp70-1 and their anti-Plasmodium activity. This led to the characterization of AMK3 and AMK4. Their binding affinity and selectivity for PfHsp70-1 over the human homolog HSPA1A were assessed using microscale thermophoresis. Proteomic and biochemical methods were employed to precisely map the PfHsp70-1 binding regions of AMK3 and AMK4. Additionally, a molecular dynamics-facilitated structure-activity relationship (SAR) screen was conducted on AMK3 to identify derivatives with retained anti-Plasmodium activity but reduced host cytotoxicity.

Two small molecules, AMK3 and AMK4, were identified, demonstrating binding to PfHsp70-1 with low-micromolar affinity. Crucially, these compounds exhibited significant selectivity, showing >25-fold preference for PfHsp70-1 over its human counterpart, HSPA1A, based on microscale thermophoresis data. This species selectivity is vital for minimizing off-target effects in potential therapeutics. Detailed proteomic and biochemical analyses elucidated their distinct binding mechanisms:

AMK3 specifically binds to the NBD of PfHsp70-1, directly competing with ATP binding, thereby disrupting the energy-dependent chaperone cycle. In contrast, AMK4 targets the SBD, leading to a disruption in essential peptide binding, which is critical for substrate recognition and folding. Further SAR optimization of AMK3 successfully yielded a derivative that maintained potent anti-Plasmodium activity while significantly reducing host cytotoxicity, suggesting an improved therapeutic index. These findings highlight distinct druggable pockets on both the N-terminal and C-terminal domains of PfHsp70-1.