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.