Gingipain Inhibitors Penetrate and Inhibit Gingipains in Porphyromonas Gingivalis Biofilms

Objectives: Porphyromonas gingivalis (Pg), a keystone pathogen in the development of periodontal disease (PiD), is an assaccharolytic bacterium that relies on proteases called gingipains for its virulence and survival. Biofilm penetration of Pg directed treatments is critical for efficacy in PiD and other diseases associated with Pg including Alzheimer’s disease, atherosclerosis, and diabetes. A library of highly potent, oral, selective small-molecule gingipain inhibitors with brain penetration were developed and a lead molecule atuzaginstat (COR388) is currently being assessed in the GAIN Trial, a pivotal Phase 2/3 study in AD which includes a periodontal efficacy sub-study. Efficacy of atuzaginstat in PiD was demonstrated in a naturally occurring aged dog model, including a decrease in gingipain activity and bacterial burden in subgingival plaque biofilms and a reduction in periodontal pocket depths. Here, we further characterized the ability of gingipain inhibitors to penetrate in vitro surface attached biofilms.
Methods: Surface attached Pg biofilms were optimized on hydroxyapatite (HA) discs and analyzed for robustness and gingipain inhibitor penetration.
Results: Fluorescent viability stain and scanning electron microscopy methods confirmed robust biofilm formation. Growth kinetics showed that biofilm production was mature at 48 hours and measurable lysine-gingipain (Kgp) and arginine-gingipain (RgpB) activity was seen using substrate cleavage assays and Cy5 conjugated activity-based probes. Cultures demonstrated decreased potency of the broad-spectrum antibiotic amoxicillin relative to planktonic growth as expected for robust biofilms. Atuzaginstat and COR588, two potent Kgp inhibitors, exhibited significant time and concentration-dependent inhibition of lysine-gingipain activity within these biofilms compared to other potent gingipain inhibitors tested in this assay. These inhibitors maintained their lysine gingipain target selectivity within the biofilms.
Conclusions: Robust biofilms of Pg were grown on HA discs that can be used to characterize the biofilm penetration of gingipain inhibitors. Atuzaginstat and COR588 penetrate biofilms to inhibit lysine-gingipain at concentrations relevant for therapeutic efficacy in PiD.