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

Entamoeba encystation process is a crucial developmental process that support to parasite persistence and disease transmission, yet the molecular mechanisms governing this stage conversion remain poorly understood and represent an attractive opportunity for therapeutic intervention. The Encystation Regulatory Motif-Binding Protein (ERM-BP), a transcription factor and key regulator of encystation, therefore represents a promising target for anti-amoebic drug development to prevent disease transmission. In the present study, we applied a target-based drug discovery approach to screen small molecules that can block the activity of ERM-BP, with a particular focus on the Cys-198 residue of this protein, which is functionally important. A library comprising 100 compounds including broad-spectrum nicotinamidase inhibitors and cysteine-reactive electrophiles were screened against ERM-BP by Protein thermal shift assays (PTSA). Thirty-six compounds bound to wild-type ERM-BP while analysis using the C198A mutant narrowed the candidates to three compounds exhibiting Cys-198-dependent interactions. These three peptidyl protease inhibitors, PFMK (Z-Ala-Phe-FMK), LEK (Z-Leu-EK), and EBLL (Ethylbenzyl-Leu-Lys) binds to ERM-BP-WT and abolishes the DNA-binding activity of ERM-BP in a concentration-dependent way. Cellular evaluation further revealed distinct activity of PFMK, LEK and EBLL on trophozoite growth and PFMK and EBLL reduced encystation efficiency significantly, produced irregular, and structurally defective cysts, which fails to excyst to trophozoites, whereas LEK had no significant effects on encystation efficiency or cyst morphology. Altogether, these findings identify peptidyl protease inhibitors PFMK and EBLL as promising compounds to target ERM-BP and block the Entamoeba encystation, providing a framework for developing therapeutics targeting parasite differentiation and encystation.