ACT001 Inhibits TLR4 Signaling by Targeting Co-Receptor MD2 and Attenuates Neuropathic Pain

Neuropathic pain is a common and challenging neurological disease, which renders an unmet need for safe and effective new therapies. Toll-like receptor 4 (TLR4) expressed on immune cells in the central nervous system arises as a novel target for treating neuropathic pain. In this study, ACT001, an o...

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Main Authors: Tianshu Zhang, Cong Lin, Siru Wu, Sha Jin, Xiaodong Li, Yinghua Peng, Xiaohui Wang
Format: Article
Language:English
Published: Frontiers Media S.A. 2022-06-01
Series:Frontiers in Immunology
Subjects:
Online Access:https://www.frontiersin.org/articles/10.3389/fimmu.2022.873054/full
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author Tianshu Zhang
Tianshu Zhang
Tianshu Zhang
Cong Lin
Cong Lin
Siru Wu
Siru Wu
Sha Jin
Xiaodong Li
Yinghua Peng
Xiaohui Wang
Xiaohui Wang
Xiaohui Wang
Xiaohui Wang
author_facet Tianshu Zhang
Tianshu Zhang
Tianshu Zhang
Cong Lin
Cong Lin
Siru Wu
Siru Wu
Sha Jin
Xiaodong Li
Yinghua Peng
Xiaohui Wang
Xiaohui Wang
Xiaohui Wang
Xiaohui Wang
author_sort Tianshu Zhang
collection DOAJ
description Neuropathic pain is a common and challenging neurological disease, which renders an unmet need for safe and effective new therapies. Toll-like receptor 4 (TLR4) expressed on immune cells in the central nervous system arises as a novel target for treating neuropathic pain. In this study, ACT001, an orphan drug currently in clinical trials for the treatment of glioblastoma, was identified as a TLR4 antagonist. In vitro quenching titrations of intrinsic protein fluorescence and saturation transfer difference (STD)-NMR showed the direct binding of ACT001 to TLR4 co-receptor MD2. Cellular thermal shift assay (CETSA) showed that ACT001 binding affected the MD2 stability, which implies that MD2 is the endogenous target of ACT001. In silico simulations showed that ACT001 binding decreased the percentage of hydrophobic area in the buried solvent-accessible surface areas (SASA) of MD2 and rendered most regions of MD2 to be more flexible, which is consistent with experimental data that ACT001 binding decreased MD2 stability. In keeping with targeting MD2, ACT001 was found to restrain the formation of TLR4/MD2/MyD88 complex and the activation of TLR4 signaling axes of NF-κB and MAPKs, therefore blocking LPS-induced TLR4 signaling downstream pro-inflammatory factors NO, IL-6, TNF-α, and IL-1β. Furthermore, systemic administration of ACT001 attenuated allodynia induced by peripheral nerve injury and activation of microglia and astrocyte in vivo. Given the well-established role of neuroinflammation in neuropathic pain, these data imply that ACT001 could be a potential drug candidate for the treatment of chronic neuropathic pain.
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spelling doaj.art-1ff7a98061a4460a96abd17f17f692e02022-12-22T02:31:33ZengFrontiers Media S.A.Frontiers in Immunology1664-32242022-06-011310.3389/fimmu.2022.873054873054ACT001 Inhibits TLR4 Signaling by Targeting Co-Receptor MD2 and Attenuates Neuropathic PainTianshu Zhang0Tianshu Zhang1Tianshu Zhang2Cong Lin3Cong Lin4Siru Wu5Siru Wu6Sha Jin7Xiaodong Li8Yinghua Peng9Xiaohui Wang10Xiaohui Wang11Xiaohui Wang12Xiaohui Wang13Laboratory of Chemical Biology, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, ChinaSchool of Applied Chemistry and Engineering, University of Science and Technology of China, Hefei, ChinaState Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing, ChinaLaboratory of Chemical Biology, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, ChinaSchool of Applied Chemistry and Engineering, University of Science and Technology of China, Hefei, ChinaLaboratory of Chemical Biology, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, ChinaSchool of Applied Chemistry and Engineering, University of Science and Technology of China, Hefei, ChinaState Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, ChinaBeijing Changping Huayou Hospital, Beijing, ChinaInstitute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, Changchun, ChinaLaboratory of Chemical Biology, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, ChinaSchool of Applied Chemistry and Engineering, University of Science and Technology of China, Hefei, ChinaState Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing, ChinaBeijing National Laboratory for Molecular Sciences, Beijing, ChinaNeuropathic pain is a common and challenging neurological disease, which renders an unmet need for safe and effective new therapies. Toll-like receptor 4 (TLR4) expressed on immune cells in the central nervous system arises as a novel target for treating neuropathic pain. In this study, ACT001, an orphan drug currently in clinical trials for the treatment of glioblastoma, was identified as a TLR4 antagonist. In vitro quenching titrations of intrinsic protein fluorescence and saturation transfer difference (STD)-NMR showed the direct binding of ACT001 to TLR4 co-receptor MD2. Cellular thermal shift assay (CETSA) showed that ACT001 binding affected the MD2 stability, which implies that MD2 is the endogenous target of ACT001. In silico simulations showed that ACT001 binding decreased the percentage of hydrophobic area in the buried solvent-accessible surface areas (SASA) of MD2 and rendered most regions of MD2 to be more flexible, which is consistent with experimental data that ACT001 binding decreased MD2 stability. In keeping with targeting MD2, ACT001 was found to restrain the formation of TLR4/MD2/MyD88 complex and the activation of TLR4 signaling axes of NF-κB and MAPKs, therefore blocking LPS-induced TLR4 signaling downstream pro-inflammatory factors NO, IL-6, TNF-α, and IL-1β. Furthermore, systemic administration of ACT001 attenuated allodynia induced by peripheral nerve injury and activation of microglia and astrocyte in vivo. Given the well-established role of neuroinflammation in neuropathic pain, these data imply that ACT001 could be a potential drug candidate for the treatment of chronic neuropathic pain.https://www.frontiersin.org/articles/10.3389/fimmu.2022.873054/fullneuropathic paintoll-like receptor 4myeloid differentiation protein 2chronic constriction injuryACT001
spellingShingle Tianshu Zhang
Tianshu Zhang
Tianshu Zhang
Cong Lin
Cong Lin
Siru Wu
Siru Wu
Sha Jin
Xiaodong Li
Yinghua Peng
Xiaohui Wang
Xiaohui Wang
Xiaohui Wang
Xiaohui Wang
ACT001 Inhibits TLR4 Signaling by Targeting Co-Receptor MD2 and Attenuates Neuropathic Pain
Frontiers in Immunology
neuropathic pain
toll-like receptor 4
myeloid differentiation protein 2
chronic constriction injury
ACT001
title ACT001 Inhibits TLR4 Signaling by Targeting Co-Receptor MD2 and Attenuates Neuropathic Pain
title_full ACT001 Inhibits TLR4 Signaling by Targeting Co-Receptor MD2 and Attenuates Neuropathic Pain
title_fullStr ACT001 Inhibits TLR4 Signaling by Targeting Co-Receptor MD2 and Attenuates Neuropathic Pain
title_full_unstemmed ACT001 Inhibits TLR4 Signaling by Targeting Co-Receptor MD2 and Attenuates Neuropathic Pain
title_short ACT001 Inhibits TLR4 Signaling by Targeting Co-Receptor MD2 and Attenuates Neuropathic Pain
title_sort act001 inhibits tlr4 signaling by targeting co receptor md2 and attenuates neuropathic pain
topic neuropathic pain
toll-like receptor 4
myeloid differentiation protein 2
chronic constriction injury
ACT001
url https://www.frontiersin.org/articles/10.3389/fimmu.2022.873054/full
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