Evaluation of Benzamidobenzoic Acids as Epigenetic Immune Regulators in Periodontitis

Objectives: The primary objective of this research was to develop novel adjunctive therapeutics for periodontal disease (PD) through epigenetic modulation of the immune response by small molecules.

Methods: In silico docking experiments were performed and consensus data was used to identify potential hit scaffolds. Bone-marrow-derived-macrophages (BMDMs) were isolated from 12-week-old C57BL/6 mice and pre-treated with selected hits. Pre-treated BMDMs were challenged with Aggregatibacter actinomycetemcomitans (A.a) lipopolysaccharide (LPS) for 24-hours and supernatant proteins were collected for ELISA analysis of IL-6 production. The benzamidobenzoic acid scaffold was chosen due to its baseline immunosuppressive capacity as well as ease of derivatization through modification of each of its three components. Several benzamidobenzoic acid derivatives were synthesized from economical starting materials. A quantitative structure/activity relationship (QSAR) model was developed to guide synthetic efforts. Compounds were tested for their ability to suppress the A.a LPS-induced production of IL-6 using ELISA, as well as their ability to modify methylation at H3K9 and H3K4 using immunofluorescence. Finally, twelve-week-old C57BL/6 mice (n=5/gp) were evaluated in a 5-day calvarial PD model using fixed A.a injections with and without immune modification using known and newly developed compounds.
Results: A custom derivative library and synthetic schema were developed for synthesis of previously unreported compounds. Several of these compounds cause a statistically significant reduction in IL-6 production in response to A.a LPS. A binary quantitative structure activity relationship was generated with accuracy in predicting both active and inactive compounds to guide current and future synthetic efforts. Immunosuppressive compounds caused a trend towards decreased bone loss in vivo following fixed A.a injections.
Conclusions: In silico docking coupled with phenotypic screens resulted in identification of new epigenetic modifying immunosuppressive benzamidobenzoic acids with potential to treat periodontal disease through localized immunosuppression and reduced bone loss.