Poster CC9 - Development of Complement 5a Receptor Antagonizing Self-Assembling Peptide Hydrogels

Introduction: Affecting approximately 8% of adults ages 30 and older, severe periodontitis is a disease of the subgingival space that remains difficult to treat and the cause of life-altering morbidities to the patient. In chronic periodontitis, the Complement 5a Receptor (C5aR) pathway is in a pathologically persistent active state. To address the chronic inflammation, a variety of antagonistic molecules including cyclic peptides have been proposed. Generally, these treatments have been compromised by their low bioavailability and limited transport to the subgingival space. To address the limitations of currently available treatments, we propose a lariat peptide that binds to C5aR. The self-assembling tail of the lariat peptide forms a hydrogel which can then be injected in the subgingival space, addressing the pharmacokinetic challenges of previous treatments.

Materials and

Methods: The peptide is designed against the transmembrane pocket of C5aR using the Generalized Kinematic Closure, PackRotamersMover, RotamerTrialsMover, and PeptideStub functions in Rosetta, parsed using the RosettaScripts interface. Sequences were selected based on their predicted energy of binding and their likelihood to adopt the conformation of the ligated posed. The peptide will be prepared in using Fmoc chemistry, rink-amide resin, and employ an on-resin Thiol-Michael acceptor click reaction to form the lariat structure. After verification of purity and identity using HPLC and MALDI-MS, respectively, the peptide will be solvated in a Phosphate Buffered Saline to form a hydrogel. The low strain stiffness of the hydrogel will be quantified using oscillatory rheometric analysis. To assess the antagonistic capability of the peptide, Human Embryonic Kidney 293T (HEK-293T) cells will be transfected to express Complement 5a Receptor. After stimulation with recombinant Complement 5a, the peptide will be introduced, and the cells incubated before staining with Flou-4 calcium binding dye. The magnitude of fluorescence will be quantified using a TECAN plate reader and calculated against the untreated control.

Results, Conclusions, and Discussions: We expect the peptide to perform similarly to previous candidates PMX53 and PMX205 in antagonizing the C5aR peptide. With the addition of the self-assembling tail, the peptide should form a hydrogel with a low strain storage modulus of 500-1000 Pa. When introduced against the Complement 5a Receptor expressing HEK-293 cells, we expect the presence of Complement 5a to induce a lower magnitude increase of calcium relative to the untreated control.

Acknowledgements (Optional): This study is supported by NIH NIDCR R01DE031812.