T7 - Evaluating Peptoids as Modulators of TLR4-Driven Inflammatory Signaling in Macrophages

Introduction: : Alzheimer’s disease (AD) is the most common form of dementia across the globe. With over six million Americans living with AD, the need for safe and efficacious therapeutics is evident. While amyloid plaques, comprised of aggregated amyloid-β protein (Aβ), are a defining characteristic of AD prognosis, increasing evidence suggests the role of chronic inflammation in AD pathogenesis. Numerous studies have linked the formation of Aβ plaques with inflammation in the AD brain, with some also denoting the additive impact this inflammation has on exacerbating Aβ pathology. Thus, targeting inflammation in AD may serve as an effective therapeutic strategy to diminish neurodegeneration and the progression of dementia. Peptoid-based therapeutics in the treatment of AD appear favorable given their low immunogenicity and toxicity as well as their ability to cross the blood-brain barrier. The peptoids examined throughout this study can function as antagonists for TLR4, a major immune receptor involved in chronic neuroinflammation in AD. The phenylalanine-like side groups are incorporated within the small-chained peptoids in these studies to achieve TLR4 ligand Aβ resemblance. Our peptoid JPT1, containing the sequence KIIFFIFF, is designed to mimic the hydrophobic and aromatic phenylalanine side groups that comprise the core KLVFF amino acid sequence of Aβ and extend the aromatic residues. Furthermore, this peptoid incorporates chiral side groups to facilitate a helical structure. This study aims to demonstrate that peptoid JPT1 will deter the production of pro-inflammatory cytokines and inhibit the binding of HMGB1 to TLR4 that is understood to propagate inflammation in AD.

Materials and

Methods: : THP-1 monocyte cells, a human acute monocytic leukemia cell line, were maintained in RPMI 1640 medium (American Type Culture Collection formulation) containing 10% FBS, penicillin (100 I.U./mL), and streptomycin (100 mg/mL). Cells were seeded on 6-well plates at 2.25E5 cells/well and differentiated into a macrophage phenotype using PMA at 200 nM for three days. Upon successful differentiation, cells were treated for three days with HMGB1 and/or peptoid. To identify an effective HMGB1 treatment concentration, ELISA was used to evaluate supernatant concentrations of two cytokines, IL-1β and TNF-α, known to be released following HMGB1 stimulation at varying concentrations. While HMGB1 concentrations lower than 10 µg/mL induced cytokine release, it was only at a treatment concentration of 10 µg/mL where significant amounts of both cytokines IL-1β and TNF-α were measured (Figure 1). In accordance with this finding, 10 µg/mL HMGB1 was determined to be an effective treatment concentration to simulate inflammatory conditions. To evaluate the cytokine-attenuation capabilities for JPT1, differentiated THP-1 cells were treated simultaneously with 50 µM peptoid and 10 µg/mL HMGB1. Cells treated with 10 µg/mL HMGB1 served as a positive control, and cells treated with vehicle (deionized water) served as a negative control. Following three days of treatment, supernatants were subsequently collected and stored at –80°C until cytokine analysis. ELISA was used to measure concentrations of IL-1β and TNF-α in cell culture supernatants. To determine whether helicity in the peptoid structure is important, JPT1a, which incorporates achiral side groups, was utilized as a functional control to JPT1.

Results, Conclusions, and Discussions:: A dose-dependent increase in cytokine release was observed from 2 to 10 µg/mL HMGB1. A robust response was observed following treatment with 10 µg/mL HMGB1, and this concentration was selected for future treatments. With the inclusion of peptoids, the results captured significant cytokine attenuation for both IL-1β and TNF-α in the presence of JPT1 compared to HMGB1 treatment alone (Figure 2). In addition, JPT1 significantly reduced pro-inflammatory cytokine production when compared to JPT1a. This difference in peptoid functionality demonstrates that incorporation of helicity into peptoid structure is fundamental for peptoid functionality in terms of cytokine attenuation. This helical structure places two aromatic groups on each of two faces of the helix to facilitate interaction with binding partners.

Previous research has indicated the involvement of TLR4 in perpetuating neuroinflammation in the AD brain. This work has demonstrated the potential of peptoid JPT1 in this therapeutic context. Future experimentation will probe the involvement of TLR4 in this therapeutic context, relative to other HMGB1 receptors, including the receptor for advanced glycation end products (RAGE) via antibody blocking experiments and binding measurements. These investigations will determine the receptor-specific binding and antagonist capabilities of our peptoids in response to HMGB1 treatment.

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