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

Abstract

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.

ACT001 improved cardiovascular function in septic mice by inhibiting the production of proinflammatory cytokines and the expression of JAK-STAT signaling pathway

“…We found that ACT001 treatment reduced IL-6 expression on mRNA and protein levels in sepsis mice ( Figure 7 ). This is consistent with results from other disease models ( Patel et al, 2019 ; Zhang et al, 2022 ). It also decreased TNF-α in serum, suggesting ACT001 may ameliorate cytokine storm in sepsis mice.…”

MCL attenuates atherosclerosis by suppressing macrophage ferroptosis via targeting KEAP1/NRF2 interaction

“…Activation of TLR4/P65 pathway has been confirmed to promote the progression of AS by triggering inflammatory response. Previous studies have confirmed that MCL/ACT001 can inhibit the activation of TLR4/P65 pathway [ 54 , 57 ]. Our data also showed that MCL reduced serum levels of inflammatory factors.…”

Dissecting the chiral recognition of TLR4/MD2 with Neoseptin-3 enantiomers by molecular dynamics simulations

“…[5][6][7][8] Upon binding of agonistic ligands to MD2, it results in the formation of TLR4/MD2 dimerization, initiating the recruitment of the intracellular adaptor Toll/ Interleukin-1 receptor (TIR) domain and subsequently activating the downstream signaling pathway. 9 Dysregulation of the TLR4 signaling pathway can lead to numerous pathological conditions, including chronic inflammation, 10 asthma, 11 neuropathic pain, 12,13 etc. In addition, recent studies have indicated that psychoactive substances, including morphine, 14,15 cocaine, 16 and methamphetamine, 17 can activate TLR4/MD2 to enhance reward signaling, therefore contributing to drug addiction.…”