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bioRxiv · 10.1101/2025.10.29.685357

PEG400 regulates Falcipain 2 activity through an unprecedented allosteric mechanism

Nath, B.·Chakraborty, S.·Biswas, S.
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Abstract

The malarial parasite Plasmodium falciparum cleaves host hemoglobin by cascade of proteolytic enzymes. The cysteine protease, Falcipain-2 (FP2) plays an essential role in the process and important for parasite survival, making it a potential drug target. However, similarities with host cysteine cathepsins hamper selective inhibition, thus necessitates detailed structural and functional characterizations of FP2. The present study uncovers a novel regulatory role of polyethylene glycol 400 (PEG400) on FP2 activity. PEG400 inhibits FP2 activity on small peptide substrate and azo-casein, while enhancing hemoglobin degradation, rendering a dual effect on FP2 catalysis. A mixed-type of inhibition has been observed for PEG400 against small peptide substrate of FP2, consistent with binding of PEG400 to catalytic cleft, confirmed by fluorescence quenching and docking studies. Unlike typical nonspecific PEG-protein interactions, PEG400 adopts a fit within catalytic region of FP2 and partially overlaps with leupeptin binding sites, albeit with lower affinity. Computational analysis further identifies a novel allosteric binding pocket of PEG400, supported by in-silico mutagenesis and molecular dynamics simulation. This pocket exhibits minimal conservation in human cathepsins, suggesting selective potential. In contrast to this inhibitory role, biochemical assay reveals that PEG400 promotes haemoglobin proteolysis. Spectroscopic analyse suggests PEG400 alter hemoglobin structural dynamics to favour proteolysis. ENM based normal mode analysis reveals upon haemoglobin binding, PEG400 restricts FP2 hinge-bending motion, improves FP2-hemoglobin proximity, and simultaneously PEG400 is dislodged from the active site, thereby promoting proteolysis. The combined experimental and computational findings reveal a novel mechanism of FP2 regulation, opening new therapeutic avenues.

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Nath, B., Chakraborty, S., Biswas, S.. 2025-10-29. PEG400 regulates Falcipain 2 activity through an unprecedented allosteric mechanism. https://doi.org/10.1101/2025.10.29.685357

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