Optimization and application of a fluorimetric assay for the identification of histone deacetylase inhibitors from plant origin

Abstract

Context: Histonedeacetylase inhibitors (HDACi) are the focus of enormous interest as a new class of anticancer drugs and discussed as novel treatment in cardiovascular diseases or memory enhancement. In the search for new active substances the structural diversity of secondary plant metabolites provides an indispensable resource. Several molecules from the plant kingdom have gained importance as anticancer drugs. Thus, a search for new therapeutic agents inhibiting histonedeacetylases (HDACs) is an important topic. To accelerate the isolation of potential candidates from plants bioassays well suited for screenings of extracts are an indispensable prerequisite.

Objective: In the presented study an enzymatic assay was modified, optimized and validated for the search for HDACi from plant origin.

Materials and methods: A fluorimetric assay was validated with respect to parameters such as temperature, incubation times, reproducibility, applicability of different enzyme sources and HDAC substrate. For the determination of the HDAC inhibitory potential of extracts the detailed study of the influence of different classes of primary and secondary metabolites probably interfering with the assay was most important.

Results: In the experimental design autofluorescent coumarins and tannins were identified to disrupt the assay. Possibilities to avoid disturbances are demonstrated and the applicability of the method in the bioactivity-guided search for new HDACi was proven on the example Leonuri herba (Leonurus cardiaca L.; Lamiaceae).

Conclusion: The optimization of the assay led to a highly efficient fluorimetric method to study plant extracts and fractions of medium/high polarity for HDAC inhibition. In the bioactivity-guided fractionation of extracts from Leonuri herba the applicability for the aimed purpose was clearly demonstrated.

Keywords::

• HDAC inhibition
• plant compounds
• Leonuri herba
• enzymatic assay

Acknowledgements

We are grateful to Prof. V. Dirsch, Department for Pharmacognosy, University of Vienna and Dr. D. Sorescu, M.D., Emory University Hospital Midtown, Atlanta, GA, for stimulative discussions. We thank Dr. R. Mazischek, Ph.D., Massachusetts General Hospital, Boston, MA, for provision of the HDAC substrate. We owe our thanks to Dr. V. Patel, Ph.D. and Prof. J. Clardy, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, for experimental suggestions and performed tests. We have to thank Associate Prof. Dr. H. Kaehlig, Department for Organic Chemistry, University of Vienna, for the NMR experiments and Dr. M. Zehl, Department of Pharmacognosy, University of Vienna, for the LC/MS experiments for structure elucidation of lavandulifolioside. We dedicate this work to em. O. Prof. Dr. Wolfgang Kubelka, Department of Pharmacognosy, University of Vienna, on the occasion of his 75th birthday.

Declaration of interest

The authors declare that they have no competing interests to report.