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Development of a cell death-based method for the screening of nuclear factor-kappaB inhibitors
Puneet Chopra1, Malini Bajpai, Sunanda G Dastidar
1Department of Pharmacology, New Drug Discovery Research, Ranbaxy Research Laboratories, Gurgaon, Haryana, India. p.chopra@ranbaxy.com
Abstract:
Nuclear factor kappa B (NF-kappaB) plays a significant role in immunity and inflammation and represents a first choice as pharmacological target for anti-inflammatory therapy. However, research in this field has been hampered by the fact that no convenient assay suitable for large-scale screening procedures is available. The present study provides a cell death-based assay method for screening of nuclear factor-kappaB inhibitors. In this study, we observed that four distinct pharmacologic inhibitors of NF-kappaB, pyrrolidine dithiocarbamate (PDTC), N-tosyl-L-lysyl chloromethyl ketone (TPCK), genistein and BAY11-7082, resulted in the cell death of murine macrophages, J774A.1. DNA-binding experiments showed that lethal doses were consistent with those required for NF-kappaB inhibition. DNA fragmentation analysis showed that cell death is apoptotic in nature. Further studies suggested that NF-kappaB inhibitors induced apoptosis is independent of the involvement of other markers of cell death such as caspases and p38 MAP (Mitogen activated protein) kinase. From this study, we conclude that NF-kappaB activation may represent an important survival mechanism in macrophages. This study also provides a new cell-based screening method, as any compound that will inhibit NF-kappaB activity will result in the death of macrophages.
Insights
Nuclear factor-kappaB (NF-kappaB) inhibitors induce apoptosis in macrophages, offering a novel cell death-based assay for screening anti-inflammatory drugs. This method identifies compounds inhibiting NF-kappaB by observing macrophage cell death.
Area of Science:
- Immunology
- Pharmacology
- Cell Biology
Background:
- Nuclear factor-kappaB (NF-kappaB) is crucial in immunity and inflammation, making it a key target for anti-inflammatory therapies.
- Current research is limited by the lack of suitable assays for large-scale screening of NF-kappaB inhibitors.
Purpose of the Study:
- To develop and validate a novel cell death-based assay for screening NF-kappaB inhibitors.
- To investigate the mechanism of cell death induced by NF-kappaB inhibitors in macrophages.
Main Methods:
- Utilized four NF-kappaB inhibitors (PDTC, TPCK, genistein, BAY11-7082) on murine macrophages (J774A.1).
- Assessed cell death, DNA binding, DNA fragmentation, and caspase/p38 MAP kinase activity.
- Correlated lethal doses with NF-kappaB inhibition levels.
Main Results:
- NF-kappaB inhibitors induced apoptosis in J774A.1 macrophages at doses consistent with NF-kappaB inhibition.
- The observed cell death was apoptotic and independent of caspases or p38 MAP kinase.
- NF-kappaB activation appears to be a survival mechanism in macrophages.
Conclusions:
- NF-kappaB inhibitors induce macrophage apoptosis, providing a new screening method for anti-inflammatory drug discovery.
- This assay enables identification of compounds that inhibit NF-kappaB activity through observable cell death.
