Pentamidine Alleviates Inflammation and Lipopolysaccharide-Induced Sepsis by Inhibiting TLR4 Activation via Targeting MD2.

Siru Wu,Hongshuang Wang,Hongyuan Li,Cong Lin,Bo Zhang,Yushan Jin,Yibo Wang,Xiaohui Wang,Tianshu Zhang

doi:10.3389/fphar.2022.835081

Abstract

Toll-like receptor 4 (TLR4) is a pattern-recognition receptor (PRR) that can recognize lipopolysaccharides (LPS) and initiate the immune response, to protect the body from infection. However, excessive activation of TLR4 induced by LPS leads to substantial release of pro-inflammatory factors, which may bring a cytokine storm in the body and cause severe sepsis. Existing molecules specialized in sepsis therapy are either in clinical trials or show mediocre effects. In this study, pentamidine, an approved drug used in the treatment of trypanosomiasis, was identified as a TLR4 antagonist. Saturation transferred difference (STD)-NMR spectra indicated that pentamidine directly interacted with TLR4’s co-receptor myeloid differentiation protein 2 (MD2) in vitro. Cellular thermal shift assay (CETSA) showed that pentamidine binding decreased MD2 stability, which was supported by in silico simulations that pentamidine binding rendered most regions of MD2 more flexible. Pentamidine was found to inhibit the formation of the TLR4/MD2/MyD88 complex and the activation of the TLR4 signaling axes of NF-κB and MAPKs, therefore blocking LPS-induced TLR4 signaling downstream of the pro-inflammatory factors NO, TNF-α, and IL-1β. The bioisosteric replacement of the methylene group at the center 13′ site of pentamidine by the ether oxygen group significantly decreased its interactions with MD2 and abolished its TLR4 antagonist activity. Furthermore, pentamidine enhanced the survival rate of septic mice and exerted an anti-inflammatory effect on organs. All these data provide strong evidence that pentamidine may be an effective drug in alleviating inflammation and sepsis.

Highlights

During response to invading pathogens, macrophages play a key role in reacting to pathogens and infection by trigging inflammation, phagocytosis, and microbial activity (López-Collazo et al, 2014)
Since Toll-like receptor 4 (TLR4)/myeloid differentiation protein 2 (MD2) plays an important role in inflammatory responses, we first investigated the binding of pentamidine with MD2, which is responsible for ligand binding and the initiation of TLR4 signaling
Except for the active hydrogens whose signals were not shown in the spectrum, the remaining signals were from the hydrogens of the benzene ring (2′, 6′, 19′, 21′ and 3′, 5′, 18′, 22′ site) and carbon chain (11′, 15′, 12′, 14′, and 13’ site) of pentamidine, which indicated that pentamidine directly interacted with purified MD2 in vitro and that the binding sites of pentamidine with MD2 were nearly on the entire molecule (Figure 1B)

Summary

Introduction

During response to invading pathogens, macrophages play a key role in reacting to pathogens and infection by trigging inflammation, phagocytosis, and microbial activity (López-Collazo et al, 2014). Substantial invasion of pathogens leads to sepsis, which is the overwhelming activation and dysregulation of the immune response. Toll-like receptor 4 (TLR4) has been discovered to play a crucial role in activating an immune response (Raymond et al, 2017). Lipopolysaccharides (LPS) are a major component of the cell wall of Gram-negative bacteria and exhibit strong immunostimulatory activity by targeting the TLR4 signaling axis and triggering sepsis inside the body (Takeda and Akira, 2015). Targeting MD2 on macrophages to block the binding of LPS against inflammation has become a valuable strategy in the treatment of sepsis (Chen et al, 2018)

Methods

Results

Conclusion