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A protocol for high-throughput screening of histone lysine demethylase 4 inhibitors using TR-FRET assay
Qiong Wu1, Wenwei Lin2, Zhen-Mei Li3
1Department of Surgery, St Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
Abstract:
Identification of diverse chemotypes of selective KDM4 inhibitors is important for exploring and validating the roles of KDM4s in the pathogenesis of human disease and for developing therapies. Here, we report a protocol for high-throughput screening of KDM4 inhibitors using TR-FRET demethylation functional assay. We describe this protocol for screen of KDM4B inhibitors, which can be modified to screen inhibitors of other JmjC-domain-containing KDMs. For complete details on the use and execution of this protocol, please refer to Singh et al. (2021).
Insights
We developed a high-throughput screening protocol to identify KDM4 inhibitors. This assay enables the discovery of novel therapeutic compounds targeting KDM4 proteins in human diseases.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- Lysine demethylases (KDMs) play crucial roles in human disease pathogenesis.
- KDM4 family proteins are implicated in various diseases, necessitating targeted inhibitors.
- Selective KDM4 inhibitors are vital for therapeutic development.
Purpose of the Study:
- To establish a high-throughput screening (HTS) protocol for identifying KDM4 inhibitors.
- To detail a protocol for screening KDM4B inhibitors using a TR-FRET assay.
- To provide a adaptable method for screening other JmjC-domain-containing KDMs.
Main Methods:
- Utilized a time-resolved fluorescence resonance energy transfer (TR-FRET) demethylation functional assay.
- Developed and optimized a protocol for HTS of KDM4 inhibitors.
- Applied the assay specifically for screening KDM4B inhibitors.
Main Results:
- Successfully established a robust TR-FRET based HTS protocol for KDM4 inhibitors.
- Demonstrated the protocol's applicability for KDM4B inhibitor screening.
- The protocol is adaptable for other JmjC-domain-containing KDMs.
Conclusions:
- The developed TR-FRET assay provides an efficient method for KDM4 inhibitor discovery.
- This protocol facilitates the exploration of KDM4 roles in disease.
- Enables the development of novel therapeutic strategies targeting KDM4s.