Homogeneous screening assay for human tankyrase.

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Tankyrase enzymes are key regulators of telomere homeostasis and Wnt signaling pathways.
• Dysregulation of tankyrase activity is implicated in various cancers, making it a significant drug target.
• Poly(ADP-ribosyl)ation, catalyzed by tankyrase, modifies protein interactions and downstream signaling.

Purpose of the Study:

• To optimize and apply a high-throughput screening (HTS) compatible, activity-based homogenous assay for identifying tankyrase inhibitors.
• To validate the assay's utility by screening a natural product library for novel tankyrase inhibitors.
• To assess the selectivity of identified inhibitors against related PARP enzymes.

Main Methods:

• Development of a fluorescence-based assay measuring substrate consumption via NAD+ conversion.
• Optimization of assay conditions for the auto-modification of a recombinant tankyrase 1 catalytic fragment.
• High-throughput screening of a natural product library and biochemical validation of hits.

Main Results:

• The optimized assay demonstrated robustness and suitability for HTS (Z'= 0.7).
• Flavone emerged as the most potent inhibitor with an IC(50) of 325 nM.
• Apigenin and isopropyl gallate showed micromolar potency and significant selectivity (>30-fold) for tankyrase over PARP1 and PARP2.

Conclusions:

• The developed homogenous assay is a cost-effective and efficient tool for discovering novel tankyrase inhibitors.
• The identification of flavone and other selective inhibitors provides a foundation for developing new therapeutic strategies targeting tankyrase.
• This assay facilitates the screening of diverse compound libraries for potential anti-cancer drug candidates.