C-Terminal Amination of a Cationic Anti-Inflammatory Peptide Improves Bioavailability and Inhibitory Activity Against LPS-Induced Inflammation.

Lulu Zhang,Henan Guo,Yanfei Hou,Xubiao Wei,Matthew Koci,Dayong Si,Rijun Zhang,Baseer Ahmad

doi:10.3389/fimmu.2020.618312

Abstract

Lipopolysaccharide (LPS) has been implicated as a major cause of inflammation and an uncontrolled LPS response increases the risk of localized inflammation and sepsis. While some native peptides are helpful in the treatment of LPS-induced inflammation, the use of these peptides is limited due to their potential cytotoxicity and poor anti-inflammatory activity. Hybridization is an effective approach for overcoming this problem. In this study, a novel hybrid anti-inflammatory peptide that combines the active center of Cathelicidin 2 (CATH2) with thymopentin (TP5) was designed [CTP, CATH2 (1–13)-TP5]. CTP was found to have higher anti-inflammatory effects than its parental peptides through directly LPS neutralization. However, CTP scarcely inhibited the attachment of LPS to cell membranes or suppressed an established LPS-induced inflammation due to poor cellular uptake. The C-terminal amine modification of CTP (CTP-NH2) was then designed based on the hypothesis that C-terminal amidation can enhance the cell uptake by increasing the hydrophobicity of the peptide. Compared with CTP, CTP-NH2 showed enhanced anti-inflammatory activity and lower cytotoxicity. CTP-NH2 not only has strong LPS neutralizing activity, but also can significantly inhibit the LPS attachment and the intracellular inflammatory response. The intracellular anti-inflammatory effect of CTP-NH2 was associated with blocking of LPS binding to the Toll-like receptor 4-myeloid differentiation factor 2 complex and inhibiting the nuclear factor-kappa B pathway. In addition, the anti-inflammatory effect of CTP-NH2 was confirmed using a murine LPS-induced sepsis model. Collectively, these findings suggest that CTP-NH2 could be developed into a novel anti-inflammatory drug. This successful modification provides a design strategy to improve the cellular uptake and anti-inflammatory activity of peptide agents.

Highlights

Lipopolysaccharide (LPS), a major component of the cell wall of gram-negative bacteria, has been implicated as a major cause of inflammation [1, 2]
The cytotoxic activity of CTP and its parental peptides towards RAW264.7 macrophages was determined with Cell Counting Kit-8 (CCK-8) assays (Figure 1)
The results showed that LPS caused significant elevation of the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-a) (Figure 2A), IL-6 (Figure 2B), and IL-1b (Figure 2C) compared with untreated cells

Summary

INTRODUCTION

Lipopolysaccharide (LPS), a major component of the cell wall of gram-negative bacteria, has been implicated as a major cause of inflammation [1, 2]. Many native peptides have been reported to have certain inhibitory activities against LPS-induced inflammation, such as LL-37 [10,11,12,13], Cathelicidin 2 (CATH2) [14], and Thymopentin (TP5) [15, 16]. Effective strategy that can combine the advantages of different native peptides [25, 26], hybridization has been put forward to improve the anti-inflammatory activity and physiological stability and reduce the undesirable cytotoxic effects of these native peptides [25]. The new designed peptide, CTP, efficiently inhibited LPS-induced inflammation. We attempted to modify CTP to obtain a C-terminal amidated derivative peptide (CTP-NH2) to improve cellular uptake, intracellular distribution and antiinflammatory activity. Its anti-inflammatory effects were assessed through an LPS-induced murine model of sepsis

MATERIALS AND METHODS

RESULTS

DISCUSSION

CONCLUSION

ETHICS STATEMENT