Functional Characterization and Inhibition Analysis of a Glutathione Transferase from Cryptosporidium parvum: A Potential Target for Antiparasitic Drug Development.

Background/

Objectives

Cryptosporidiosis, caused by Cryptosporidium parvum, is a significant cause of diarrheal disease, particularly affecting young children and immunocompromised individuals. With current treatments offering limited efficacy, there is an urgent need for novel therapeutic targets.

Methods

In this study, we report the cloning, expression, and functional characterization of a glutathione transferase (GST) from C. parvum (CpGST).

Results

Biocomputing analysis revealed a single gene encoding a cytosolic enzyme with distinct structural features, compared to human cytosolic homologs. Structural modeling indicated a non-canonical thioredoxin fold and a truncated C-terminal domain, suggesting functional divergence. CpGST was expressed in Escherichia coli, and its enzymatic properties were characterized. Although the enzyme displayed a narrow substrate spectrum, it showed a distinct substrate preference, retaining catalytic activity toward the standard GST substrates 1-chloro-2,4-dinitrobenzene (CDNB) and cumene hydroperoxide (CuOOH). Steady-state kinetic analysis revealed limited affinity for both reduced glutathione (GSH) and CDNB. Inhibition analysis identified several polyphenols and synthetic curcumin analogues as potent inhibitors, with IC50 values in the low micromolar range. Kinetic analysis with the most potent inhibitor revealed a mixed-type inhibition mechanism.

Conclusions

These findings support the classification of CpGST as a structurally and functionally distinct member of the GST family, likely adapted to the parasite's physiology and metabolism. The enzyme's divergence from human GSTs, along with its favorable druggability profile, underscores its potential as a target for anti-cryptosporidial drug development, particularly in strategies aimed at disrupting stress response and detoxification pathways.