20 research outputs found
MOESM1 of Metabolomic investigation of regional brain tissue dysfunctions induced by global cerebral ischemia
No full textAdditional file 1: Figure S1. The 600-MHz 1H NMR NOESY spectra (δ 0.9-4.7, 5.3-9.4) of aqueous extracts from the hippocampus tissues of mice in female model group (A) and female sham group (B). The abbreviations of metabolites were denoted in Table S1. Figure S2. The PCA score plots, PLS-DA score plots and their corresponding validation plots derived from the 600 MHz 1H NMR spectra of cortex samples (A, A’, A’’) and hippocampus samples (B, B’, B’’) extracted from the mice in female sham group (■) and female model group (▲). The validation plots were obtained by using a permutation test that was randomly permuted for 350 times with the first component extracts. ▲ is for R2Y (cum), and ■ is for Q2 (cum). The vertical axis of validation plot represents the R2 and Q2 values, and the horizontal axis (A’’, B’’) represents the correlation coefficients. Figure S3. The PCA score plots, PLS-DA score plots and their corresponding validation plots derived from the 600 MHz 1H NMR spectra of cortex samples (A, A’, A’’) and hippocampus samples (B, B’, B’’) extracted from the mice in male sham group (■) and male model group (▲). The validation plots were obtained by using a permutation test that was randomly permuted for 350 times with the first component extracts. ▲ is for R2Y (cum), and ■ is for Q2 (cum). The vertical axis of validation plot represents the R2 and Q2 values, and the horizontal axis (A’’, B’’) represents the correlation coefficients. Of note, due to the poor scan qualities of original NMR spectra caused by the limited quantities of mouse hippocampus tissues, one hippocampus sample in the sham male group was excluded from the analysis. Figure S4. Influence of BCCAO on mitochondrial swelling. (A). The Ca2+ induced mitochondria swelling in the cerebral cortex tissues of mice in female/male model versus sham group. *P < 0.05, ***P < 0.005 versus sham group. (B). Representative swelling curves of mitochondria isolated from sham or BCCAO groups (Data are expressed as mean value of each group). Figure S5. The 600-MHz 1H NMR NOESY spectra of aqueous extracts with a total number of 11 samples from the hippocampus tissues of mice in sham groups. Figure S6. The 600-MHz 1H NMR NOESY spectra of aqueous extracts with a total number of 12 samples from the hippocampus tissues of mice in model groups. Table S1. NMR Resonance assignments of 26 aqueous metabolites extracted from regional brain tissues of experimental mice. Table S2. Quantitative comparisons of aqueous metabolites extracted from cerebral cortex and hippocampus tissues of female mice. Table S3. Quantitative comparisons of aqueous metabolites extracted from cerebral cortex and hippocampus tissues of male mic
Exploring Methamphetamine Nonenantioselectively Targeting Toll-like Receptor 4/Myeloid Differentiation Protein 2 by in Silico Simulations and Wet-Lab Techniques
No full textMethamphetamine (METH)
is one of the highly addictive nonopioid
psychostimulants, acting as a xenobiotic-associated molecular pattern
(XAMP) to target TLR4 and activate microglia. However, the molecule
recognition of METH by innate immune receptor TLR4/MD-2 is not well-understood.
METH exists in two enantiomeric forms, and it is unclear whether the
TLR4 innate immune recognition with METH is stereoselective. Herein,
molecular dynamics (MDs) simulations were performed to dissect the
recognition of (+)-METH and (−)-METH by TLR4/MD-2 at the atomic
level. Amphetamine (AMPH), which is an analogue of METH, was also
investigated for comparison. Computational simulations indicate that
METH binds into the interaction interface between MD-2 as well as
TLR4* that is from the adjacent copy of TLR4-MD-2, therefore stabilizing
the active heterotetramer (TLR4/MD-2)2 complex. The calculated
binding free energies and potential of mean force (PMF) values show
that (−)-METH and (+)-METH have similar TLR4/MD-2 binding affinity.
Further dynamics analyses of bindings with TLR4/MD-2 indicate that
(−)-METH and (+)-METH behave similarly. Unlike the stereoselective
neuron-stimulating activities of METH, no enantioselectivity was observed
for METH interacting with TLR4/MD-2 complex as well as activating
TLR4 signaling. Compared to METH, AMPH showed much weaker interactions
with TLR4/MD-2, indicating that the substituted methyl group is critical
in the molecular recognition of METH by TLR4/MD-2. In all, this study
provides molecular insight into the innate immune recognition of METH,
which demonstrates that METH could be nonenantioselectively sensed
by TLR4/MD-2
DataSheet9_Pentamidine Alleviates Inflammation and Lipopolysaccharide-Induced Sepsis by Inhibiting TLR4 Activation via Targeting MD2.ZIP
No full textToll-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.</p
DataSheet2_Pentamidine Alleviates Inflammation and Lipopolysaccharide-Induced Sepsis by Inhibiting TLR4 Activation via Targeting MD2.zip
No full textToll-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.</p
Image_1_ACT001 Inhibits TLR4 Signaling by Targeting Co-Receptor MD2 and Attenuates Neuropathic Pain.jpeg
No full textNeuropathic 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.</p
DataSheet7_Pentamidine Alleviates Inflammation and Lipopolysaccharide-Induced Sepsis by Inhibiting TLR4 Activation via Targeting MD2.ZIP
No full textToll-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.</p
DataSheet3_Pentamidine Alleviates Inflammation and Lipopolysaccharide-Induced Sepsis by Inhibiting TLR4 Activation via Targeting MD2.zip
No full textToll-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.</p
Image_2_ACT001 Inhibits TLR4 Signaling by Targeting Co-Receptor MD2 and Attenuates Neuropathic Pain.jpeg
No full textNeuropathic 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.</p
DataSheet5_Pentamidine Alleviates Inflammation and Lipopolysaccharide-Induced Sepsis by Inhibiting TLR4 Activation via Targeting MD2.ZIP
No full textToll-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.</p
DataSheet1_Pentamidine Alleviates Inflammation and Lipopolysaccharide-Induced Sepsis by Inhibiting TLR4 Activation via Targeting MD2.docx
No full textToll-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.</p
