Publication: BIOLOGICAL EVALUATION OF SELECTED IN SILICO REPOSITIONED FDA-APPROVED DRUGS AS POTENTIAL INHIBITORS OF PORPHYROMONAS GINGIVALIS PEPTIDYLARGININE DEIMINASE
Date
2025
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Publisher
IMU University
Abstract
Rheumatoid arthritis (RA) is a chronic inflammatory condition which primarily affecting the joints and is characterized by dysregulated peptidylarginine deiminase (PAD) activity. PAD enzymes play a crucial role in catalysing the citrullination process and are implicated in RA pathogenesis by modifying RA-associated autoantigens. Meanwhile, Porphyromonas gingivalis (P. gingivalis), a key bacterium responsible for periodontitis, produces P. gingivalis peptidylarginine deiminase (PPAD). PPAD serves as a critical link between periodontitis and RA by generating autoantigens that trigger immune responses in RA patients. Given its role in autoimmunity, inhibiting PPAD activity presents a promising approach for managing RA. This study aims to evaluate the in vitro inhibitory effects of five FDA-approved compounds on PPAD and to identify key structural features responsible for protease inhibition. In this study, PPAD gene was cloned into pColdI plasmid and transformed into DH5α competent cells of Escherichia coli, then PPAD were overexpressed by the induction of isopropyl-β-D-1-thiogalactopyranoside (IPTG). The overexpressed cells then were sonicated to extract the PPAD enzyme (protein). The cells then pelleted because PPAD was in soluble form, then only the supernatant subjected for the citrulline colorimetric assay. In colorimetric assay, five FDA-approved compounds which had subjected to prior structure-based virtual screening, were evaluated in this study to confirm their inhibitory activity against the PPAD. Then the compounds that have the highest ability to reduce the citrullination activity, were subjected to IC50 study in order to assess the inhibitory effects of this top compound. The result indicated that PPAD gene was successfully inserted into the pColdI plasmid and transformed into DH5α competent cells. The DH5α-pColdI-PPAD cells were successfully overexpressed and subjected to sonication for PPAD enzyme extraction. The supernatant obtained after high-speed centrifugation was used for the citrulline colorimetric assay. In this assay, five drugs (Amifostine, 4-amino-3-hydroxybutyric acid, Eflornithine, Fludarabine, and Levetiracetam) were tested at 50 μM concentration against the PPAD enzyme. Among them, only Eflornithine exhibited the highest reduction in PPAD citrullination activity, showing a 16.3% decrease. Then further IC50 analysis revealed that Eflornithine could inhibit the PPAD citrullination activity by 50%, with an IC50 value of 108.6 μM. Although Eflornithine has demonstrated potential in reducing the PPAD enzyme activity, further validation is required before Eflornithine can be considered as appropriate treatment for RA.
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Keywords
Protein-Arginine Deiminases, Porphyromonas gingivalis, Arthritis, Rheumatoid, Drug Repositioning, Protein-Arginine Deiminase Type 4