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Characterization of Novel Src Family Kinase Inhibitors to Attenuate Microgliosis.

Manocha GD, Puig KL, Austin SA, Seyb K, Glicksman MA, Combs CK - PLoS ONE (2015)

Bottom Line: Cells were treated with the compounds to determine effects on active, phosphorylated levels of Src family kinases, Src and Lyn, as well as MAP kinases ERK, JNK and p38.Only one compound, LDDN-0003499, produced a dose dependent decrease in basal levels of active, phosphorylated Src and Lyn in the BV2 cells.This study identifies a novel small molecule Src family tyrosine kinase inhibitor with anti-inflammatory effects in response to Aβ stimulation of microglia.

View Article: PubMed Central - PubMed

Affiliation: Department of Basic Sciences, University of North Dakota, School of Medicine and Health Sciences, Grand Forks, ND, 58203, United States of America.

ABSTRACT
Microgliosis is a major hallmark of Alzheimer's disease (AD) brain pathology. Aβ peptide is hypothesized to act as a stimulus for microglia leading to activation of non-receptor tyrosine kinases and subsequent secretion of pro-inflammatory cytokines. Therefore, the signaling pathways mediating microglial activation may be important therapeutic targets of anti-inflammatory therapy for AD. Four novel compounds were chosen after high throughput screening kinase activity assays determined them as potential Lyn kinase inhibitors. Their kinase inhibitory and anti-inflammatory effect on Aβ-stimulated activation was assessed using the murine microglial cell line, BV2. Cells were treated with the compounds to determine effects on active, phosphorylated levels of Src family kinases, Src and Lyn, as well as MAP kinases ERK, JNK and p38. Only one compound, LDDN-0003499, produced a dose dependent decrease in basal levels of active, phosphorylated Src and Lyn in the BV2 cells. LDDN-0003499 treatment also attenuated the Aβ-stimulated increase in active, phosphorylated levels of Lyn/Src and TNFα and IL-6 secretion. This study identifies a novel small molecule Src family tyrosine kinase inhibitor with anti-inflammatory effects in response to Aβ stimulation of microglia. Further in vitro/in vivo characterization of LDDN-0003499 as well as structural modification may provide a new tool for attenuating microglial-mediated brain inflammatory conditions such as that occurring in AD.

No MeSH data available.


Related in: MedlinePlus

LDDN compounds were not toxic to microglial BV2 cells.Microglial BV2 cells were untreated (control), vehicle DMSO treated, or treated with 0.5nM, 5nM, 50nM, 0.5 μM, 5μM and 50 μM (A) LDDN-0003499, (B) LDDN-0075935, (C) LDDN-0125694, and (D) LDDN-0127164 for 24h. Following compound treatment, media were used to perform LDH release assay to determine cell viability. Three independent experiments with 8 replicates each were performed and absorbance values graphed and averaged ± SD.
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pone.0132604.g001: LDDN compounds were not toxic to microglial BV2 cells.Microglial BV2 cells were untreated (control), vehicle DMSO treated, or treated with 0.5nM, 5nM, 50nM, 0.5 μM, 5μM and 50 μM (A) LDDN-0003499, (B) LDDN-0075935, (C) LDDN-0125694, and (D) LDDN-0127164 for 24h. Following compound treatment, media were used to perform LDH release assay to determine cell viability. Three independent experiments with 8 replicates each were performed and absorbance values graphed and averaged ± SD.

Mentions: Although all four of the LDDN compounds had been characterized as possible Lyn inhibitory molecules from prior cell-free activity assays, their efficacy in cells had not yet been assessed. Therefore, before quantifying their ability to alter cellular kinase activities, we first determined whether the compounds exhibited any microglial toxicity. To perform this assessment we elected to use the common microglial cell line model, BV2 cells. They were treated with increasing concentrations of LDDN-0003499, LDDN-0075935, LDDN-0125694 and LDDN-0127164 from 0.5 nM up to 50 μM for 24h. The media were removed and used for LDH release assays to determine cell viability. Quantitation of LDH released from the media following compound treatment demonstrated that none of the compounds were toxic at any of the concentrations tested (Fig 1). For subsequent experiments assessing effects on tyrosine kinase inhibition, the concentration range of 0.5nM, 5nM, 50nM, 0.5μM, 5μM and 50μM was chosen for treatment of the BV2 cells.


Characterization of Novel Src Family Kinase Inhibitors to Attenuate Microgliosis.

Manocha GD, Puig KL, Austin SA, Seyb K, Glicksman MA, Combs CK - PLoS ONE (2015)

LDDN compounds were not toxic to microglial BV2 cells.Microglial BV2 cells were untreated (control), vehicle DMSO treated, or treated with 0.5nM, 5nM, 50nM, 0.5 μM, 5μM and 50 μM (A) LDDN-0003499, (B) LDDN-0075935, (C) LDDN-0125694, and (D) LDDN-0127164 for 24h. Following compound treatment, media were used to perform LDH release assay to determine cell viability. Three independent experiments with 8 replicates each were performed and absorbance values graphed and averaged ± SD.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4498792&req=5

pone.0132604.g001: LDDN compounds were not toxic to microglial BV2 cells.Microglial BV2 cells were untreated (control), vehicle DMSO treated, or treated with 0.5nM, 5nM, 50nM, 0.5 μM, 5μM and 50 μM (A) LDDN-0003499, (B) LDDN-0075935, (C) LDDN-0125694, and (D) LDDN-0127164 for 24h. Following compound treatment, media were used to perform LDH release assay to determine cell viability. Three independent experiments with 8 replicates each were performed and absorbance values graphed and averaged ± SD.
Mentions: Although all four of the LDDN compounds had been characterized as possible Lyn inhibitory molecules from prior cell-free activity assays, their efficacy in cells had not yet been assessed. Therefore, before quantifying their ability to alter cellular kinase activities, we first determined whether the compounds exhibited any microglial toxicity. To perform this assessment we elected to use the common microglial cell line model, BV2 cells. They were treated with increasing concentrations of LDDN-0003499, LDDN-0075935, LDDN-0125694 and LDDN-0127164 from 0.5 nM up to 50 μM for 24h. The media were removed and used for LDH release assays to determine cell viability. Quantitation of LDH released from the media following compound treatment demonstrated that none of the compounds were toxic at any of the concentrations tested (Fig 1). For subsequent experiments assessing effects on tyrosine kinase inhibition, the concentration range of 0.5nM, 5nM, 50nM, 0.5μM, 5μM and 50μM was chosen for treatment of the BV2 cells.

Bottom Line: Cells were treated with the compounds to determine effects on active, phosphorylated levels of Src family kinases, Src and Lyn, as well as MAP kinases ERK, JNK and p38.Only one compound, LDDN-0003499, produced a dose dependent decrease in basal levels of active, phosphorylated Src and Lyn in the BV2 cells.This study identifies a novel small molecule Src family tyrosine kinase inhibitor with anti-inflammatory effects in response to Aβ stimulation of microglia.

View Article: PubMed Central - PubMed

Affiliation: Department of Basic Sciences, University of North Dakota, School of Medicine and Health Sciences, Grand Forks, ND, 58203, United States of America.

ABSTRACT
Microgliosis is a major hallmark of Alzheimer's disease (AD) brain pathology. Aβ peptide is hypothesized to act as a stimulus for microglia leading to activation of non-receptor tyrosine kinases and subsequent secretion of pro-inflammatory cytokines. Therefore, the signaling pathways mediating microglial activation may be important therapeutic targets of anti-inflammatory therapy for AD. Four novel compounds were chosen after high throughput screening kinase activity assays determined them as potential Lyn kinase inhibitors. Their kinase inhibitory and anti-inflammatory effect on Aβ-stimulated activation was assessed using the murine microglial cell line, BV2. Cells were treated with the compounds to determine effects on active, phosphorylated levels of Src family kinases, Src and Lyn, as well as MAP kinases ERK, JNK and p38. Only one compound, LDDN-0003499, produced a dose dependent decrease in basal levels of active, phosphorylated Src and Lyn in the BV2 cells. LDDN-0003499 treatment also attenuated the Aβ-stimulated increase in active, phosphorylated levels of Lyn/Src and TNFα and IL-6 secretion. This study identifies a novel small molecule Src family tyrosine kinase inhibitor with anti-inflammatory effects in response to Aβ stimulation of microglia. Further in vitro/in vivo characterization of LDDN-0003499 as well as structural modification may provide a new tool for attenuating microglial-mediated brain inflammatory conditions such as that occurring in AD.

No MeSH data available.


Related in: MedlinePlus