O26 - Impact of Peptoid Aromatic Residues on Pro-Inflammatory Cytokine Production

Introduction: : In the United States, more than 5 million people live with Alzheimer’s disease (AD), and this number is projected to reach nearly 14 million by 2050 [1]. Neuroinflammation has recently been identified as a driving force for the progression of AD [2]. Inflammatory signaling pathways are activated in AD partially due to the upregulation of the receptor for advanced glycation end-products (RAGE). RAGE ligands also upregulated in AD include Aβ fibrils or oligomers and S100 calcium-binding protein B (S100B) [3]. Additional inflammatory pathways activated due to this upregulated RAGE-ligand behavior will result in a positive feedback loop creating continuous inflammation in AD brain. New research aims to create therapeutics targeting this neuroinflammation using peptoids as anti-inflammatory agents. One such peptoid, JPT1, displays helicity via chiral monomers such that two aromatic groups are located on two faces of the helix mimicking the -sheet spacing of aggregated Aβ, a ligand of RAGE. In a previous study, JPT1 successfully reduced pro-inflammatory cytokine release induced by lipopolysaccharide (LPS) and exhibited nanomolar binding affinity for RAGE [3]. We hypothesize the number and placement of aromatic side chains on JPT1 is critical to its function. To test this hypothesis, additional peptoids were designed. JPT1C lacks aromatic side chains. JPT1FN presents two aromatic side chains with one aromatic present on each of two faces of the helix. JPT1FS presents two aromatic side chains on a single side of the helix. These peptoids were assessed for their ability to reduce LPS-induced pro-inflammatory cytokine release relative to JPT1.

Materials and

Methods: : In vitro immunoassay experiments are used to evaluate how peptoids with aromatic variations impact the manner in which immune cells respond in various inflammatory stimuli. Tohoku Hospital Pediatrics-1 (THP-1) monocytes are differentiated to macrophages in 400 nM phorbol 12-myristate 13 (PMA) for 72 h. To induce release of pro-inflammatory cytokines, THP-1 macrophages are then treated with 2 ng/mL LPS or 0.05 µg/mL S100B in the absence or presence of 50 µM JPT1, JPT1C, JPT1FN, JPT1FS, or a peptide with a sequence corresponding to JPT1 (peptide). Supernatant is collected after 72 h of treatment and centrifuged to void samples of cell debris. Concentrations of cytokines present in conditioned medium supernatant are measured via sandwich ELISAs (enzyme linked immunosorbent-assays) for each respective peptoid.

Results, Conclusions, and Discussions:: The presence of inflammatory cytokines was measured in conditioned media supernatants from THP-1 macrophages exposed to 2 ng/mL LPS or 0.05 µg/mL S100B. LPS and S100B both serve as chronic, low dose pro-inflammatory stimuli. Significant concentrations of cytokines were observed in positive control samples containing inflammatory stimulant only relative to non-stimulated negative controls (Figure 1). Additionally, the presence of 50 µM JPT1 significantly reduced cytokine levels, illustrated in Figures 1A and 1B for LPS treatment and S100B treatment, respectively, and measurement of IL-1β. In contrast, no significant difference in cytokine concentration was observed for cells receiving inflammatory stimulant and 50 µM JPT1C, JPT1FN, JPT1FS. The presence of a peptide in samples receiving S100B treatment resulted in significantly increased cytokine levels (Figure 1B).
Neuroinflammation is a major driving force of AD that intensifies neuronal damage. Peptoids are potential strong candidates for neuroinflammation treatment. Some benefits to peptoids include their small size, low toxicity, resistance to enzymatic degradation, and ability to cross the blood-brain barrier [2]. One such peptoid designed by our laboratory, JPT1, antagonizes the RAGE cell surface receptor [3]. This study explores the importance of the four aromatic residues and their placement in the peptoid design. While JPT1 continued to significantly decrease LPS- and S100B-induced cytokine release, a peptoid with no aromatic residues (JPT1C) failed to significantly alter cytokine release. Moreover, peptoids with only two aromatic residues, including one with one aromatic present on each of two faces of the helix (JPT1FN) and another with two aromatic side chains on a single side of the helix (JPT1FS) also did not significantly decrease inflammatory cytokine release. Overall, these results suggests that the presence and position of the aromatics is important to peptoid JPT1’s effectiveness. Future experimentation will evaluate the ability of peptoids to disrupt binding of LPS and S100B to RAGE. This work will demonstrate the promise for JPT1 as an AD therapeutic to target and reduce neuroinflammation.

Acknowledgements and/or References (Optional):: [1] “Alzheimer’s Facts | My BrainTM | Alzheimer’s Association,” My Brain. Accessed: Apr. 28, 2025. [Online]. Available: http://mybrain.alz.org/alzheimers-facts.asp
[2] M. T. Heneka et al., “Neuroinflammation in Alzheimer disease,” Nat Rev Immunol, vol. 25, no. 5, pp. 321–352, May 2025, doi: 10.1038/s41577-024-01104-7.
[3] M. L. Waugh, L. M. Wolf, J. P. Turner, L. N. Phillips, S. L. Servoss, and M. A. Moss, “Modulating the RAGE‐Induced Inflammatory Response: Peptoids as RAGE Antagonists,” ChemBioChem, vol. 24, no. 22, p. e202300503, Nov. 2023, doi: 10.1002/cbic.202300503.