(H-307) Peptoids as Potential Therapeutics Against RAGE-Associated Neuroinflammation in Alzheimer’s Disease

Introduction:: While the primary pathology of Alzheimer’s disease (AD) is defined by amyloid-β (Aβ) plaques and tau neurofibrillary tangles in the brain, chronic inflammation has emerged as an important factor in AD etiology. Recent studies have indicated that the receptor for advanced glycation end-products (RAGE), a key receptor of innate immune response, is upregulated in AD, and the RAGE ligand S100B is elevated in AD brain. Activation of RAGE by S100B can both upregulate RAGE expression and induce the release of inflammatory cytokines. Consequently, targeting RAGE to reduce chronic inflammation represents a novel therapeutic strategy for AD.

Peptoids are a novel, ideal class of potential AD therapeutics as they display resistance to proteolytic degradation by repositioning side-chains from the a-carbon to the amide nitrogen and exhibit facile blood-brain barrier permeability. We have previously shown that peptoids JPT1 and JPT1a, designed with strategic placement of aromatic side-chains to mimic RAGE ligands, demonstrate nanomolar binding affinity to RAGE. We hypothesize that these peptoids can attenuate S100B-induced activation of RAGE, thus reducing cytokine release associated with chronic neuroinflammation.


Materials and Methods:: To evaluate binding affinity between RAGE and RAGE antagonists, a solution phase equilibrium binding assay with modified ELISA was performed in the presence of 20 nM soluble RAGE and 0-2000 nM JPT1 or JPT1a. In addition, similar assay was performed with 0-2000 nM Azeliragon, previous RAGE antagonist clinical trial AD drug, in the presence of 20 nM soluble RAGE to compare its binding affinity to that between the peptoids and RAGE.

Effect of peptoids and Azeliragon on THP-1 macrophage cell viability was evaluated using an XTT cellular metabolic assay. THP-1 monocytes were incubated with phorbol 12-myristate 13-acetate (PMA) for three days to achieve macrophage differentiation. THP-1 macrophages were then treated with 0-50 μM of JPT1, JPT1a, or Azeliragon for three days. After 72-h treatment, cells were incubated with XTT reagent mixture for 4 h and absorbance was measured at 450 nm.

To assess the effects of JPT1 and JPT1a on RAGE function, THP-1 macrophages were exposed to chronic low-levels of S100B (0.05 mg/mL, 3 days) to confirm upregulation of cytokine release. Differentiated cells were also treated with S100B in the presence of 0-50 μM peptoids. To observe peptoid modulation of S100B-induced inflammation, cytokine analysis via multiplex assay was performed for cell culture supernatants.


Results, Conclusions, and Discussions:: Results and Discussions: When a solution phase equilibrium binding assay with modified ELISA was performed in the presence of 20 nM soluble RAGE and 0-1000 nM peptoid, both JPT1 and JPT1a exhibited nanomolar binding affinity for RAGE with K_d values of 78 ± 17 nM and 58 ± 12 nM, respectively. This binding affinity is higher than that between RAGE and Azeliragon (K_d = 239 ± 34 nM), a previous RAGE antagonist clinical trial AD drug.
Cell viability of human THP-1 macrophages incubated with 0-50 μM JPT1, JPT1a, or Azeliragon was evaluated via XTT assay. While 5-50 μM Azeliragon reduced the cell viability by more than 50%, cells treated with JPT1 and JPT1a were able to maintain 100% cell viability.


S100B-treated THP-1 macrophages elicited anticipated inflammatory response evidenced by a dose-dependent reduction in cytokine expression. When THP-1 macrophages were incubated with S100B in the presence of JPT1 or JPT1a, the peptoids significantly attenuated these S100B-induced pro-inflammatory responses (Figure 3). The cytokine-modulating effect was more pronounced for peptoid JPT1, which is comprised of chiral side chains to induce helical secondary structure.


Conclusions: We have demonstrated that the peptoids JPT1 and JPT1a are non-toxic to human macrophage culture and exhibit nanomolar binding affinity for RAGE. This binding affinity is higher than that between RAGE and Azeliragon, a previous RAGE antagonist clinical trial AD drug. In addition, peptoids JPT1 and JPT1a were able to significantly attenuate S100B-induced pro-inflammatory responses. Moreover, JPT1 demonstrates a more pronounced ability to augment inflammatory response, indicating that peptoid helical structure is advantageous. These results implicate peptoids as a potential therapeutic agent for not only AD but also for other inflammation-related illnesses. Because these peptoids also exhibit the ability to modulate Aβ aggregation, they may function as dual-target therapeutics for AD.






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