Peptidyl Protease Inhibitors Block ERM-BP Function and Suppress Entamoeba Encystation

Entamoeba encystation is a critical stage in the parasite's life cycle, contributing to its survival and transmission of disease. However, the precise mechanisms behind this transformation are not fully understood, making it an attractive target for new anti-amoebic treatments. ERM-BP, a transcription factor and key regulator of encystation, presents a promising therapeutic target.

To identify compounds that might inhibit ERM-BP activity, the study employed a target-based drug discovery approach, screening a library of 100 small molecules, including broad-spectrum nicotinamidase inhibitors and cysteine-reactive electrophiles, against ERM-BP using Protein Thermal Shift Assays (PTSA). Thirty-six compounds showed binding affinity to the wild-type ERM-BP, but further analysis of a mutant with a modified Cys-198 residue narrowed the focus to three compounds—PFMK (Z-Ala-Phe-FMK), LEK (Z-Leu-EK), and EBLL (Ethylbenzyl-Leu-Lys)—which exhibited Cys-198-dependent interactions.

These three peptidyl protease inhibitors were found to bind to ERM-BP-WT and inhibit the protein's DNA-binding activity in a dose-dependent manner. Additional cellular tests revealed that PFMK and EBLL significantly reduced trophozoite growth and encystation efficiency, resulting in irregular and structurally flawed cysts that were unable to excyst into trophozoites.

In contrast, LEK did not show significant effects on encystation efficiency or cyst morphology. These results suggest that PFMK and EBLL could serve as promising candidates for targeting ERM-BP and disrupting Entamoeba encystation, offering a potential framework for developing therapeutics aimed at controlling parasite differentiation and encystation.