Intracerebral hemorrhage (ICH) is devastating among
stroke types with high mortality. To date, not a single therapeutic intervention has been successful.
Cofilin plays a critical role in
inflammation and cell death. In the current study, we embarked on designing and synthesizing a first-in-class small-molecule inhibitor of
cofilin to target secondary complications of ICH, mainly
neuroinflammation. A series of compounds were synthesized, and two lead compounds SZ-3 and SK-1-32 were selected for further studies. Neuronal and microglial viabilities were assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide (MTT) assay using
neuroblastoma (SHSY-5Y) and human microglial (HMC-3) cell lines, respectively.
Lipopolysaccharide (LPS)-induced
inflammation in HMC-3 cells was used for neurotoxicity assay. Other assays include
nitric oxide (NO) by
Griess reagent,
cofilin inhibition by
F-actin depolymerization, migration by scratch
wound assay,
tumor necrosis factor (TNF-α) by
enzyme-linked
immunosorbent assay (ELISA),
protease-activated receptor-1 (PAR-1) by immunocytochemistry and Western blotting (WB), and
protein expression levels of several
proteins by WB. SK-1-32 increased neuronal/microglial survival, reduced NO, and prevented neurotoxicity. However, SZ-3 showed no effect on neuronal/microglial survival but prevented microglia from LPS-induced
inflammation by decreasing NO and preventing neurotoxicity. Therefore, we selected SZ-3 for further molecular studies, as it showed potent anti-inflammatory activities. SZ-3 decreased
cofilin severing activity, and its treatment of LPS-activated HMC-3 cells attenuated microglial activation and suppressed migration and proliferation. HMC-3 cells subjected to
thrombin, as an in vitro model for
hemorrhagic stroke, and treated with SZ-3 after 3 h showed significantly decreased NO and TNF-α, significantly increased
protein expression of phosphocofilin, and decreased PAR-1. In addition, SZ-3-treated SHSY-5Y showed a significant increase in cell viability by significantly reducing nuclear factor-κ B (NF-κB),
caspase-3, and high-temperature requirement (HtrA2). Together, our results support the novel idea of targeting
cofilin to counter
neuroinflammation during secondary injury following ICH.