Synthesis and Development of a Novel First-in-Class Cofilin Inhibitor for Neuroinflammation in Hemorrhagic Brain Injury

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, weembarked 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 neuro-blastoma (SHSY-5Y) and human microglial (HMC-3) cell lines, respectively.Lipopolysaccharide (LPS)-induced inflammation in HMC-3 cells was used forneurotoxicity assay. Other assays include nitric oxide (NO) by Griess reagent, cofilininhibition by F-actin depolymerization, migration by scratch wound assay, tumornecrosis factor (TNF-alpha) by enzyme-linked immunosorbent assay (ELISA), protease-activated receptor-1 (PAR-1) by immunocytochemistry and Western blotting (WB), and protein expression levels of several proteinsby WB. SK-1-32 increased neuronal/microglial survival, reduced NO, and prevented neurotoxicity. However, SZ-3 showed no effecton neuronal/microglial survival but prevented microglia from LPS-induced inflammation by decreasing NO and preventingneurotoxicity. Therefore, we selected SZ-3 for further molecular studies, as it showed potent anti-inflammatory activities. SZ-3decreased cofilin severing activity, and its treatment of LPS-activated HMC-3 cells attenuated microglial activation and suppressedmigration and proliferation. HMC-3 cells subjected to thrombin, as an in vitro model for hemorrhagic stroke, and treated with SZ-3after 3 h showed significantly decreased NO and TNF-alpha, significantly increased protein expression of phosphocofilin, and decreasedPAR-1. In addition, SZ-3-treated SHSY-5Y showed a significant increase in cell viability by significantly reducing nuclear factor-Kappa B(NF-Kappa B), 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.

Automated summary

This study designed and synthesized a cofilin small-molecule inhibitor for targeting secondary complications of ICH and found that the compound SZ-3 showed potent anti-neuroinflammation activity.