Discovery, characterization and SAR of gambogic acid as a potent apoptosis inducer by a HTS assay

doi:10.1016/j.bmc.2003.11.013

Bioorganic & Medicinal Chemistry

Volume 12, Issue 2, 15 January 2004, Pages 309-317

https://doi.org/10.1016/j.bmc.2003.11.013 Get rights and content

Abstract

Gambogic acid (2), a natural product isolated from the resin of Garcinia hurburyi tree, was discovered to be a potent apoptosis inducer using our cell- and caspase-based high-throughput screening assays. Gambogic acid was found to have an EC₅₀ of 0.78 μM in the caspase activation assay in T47D breast cancer cells. The apoptosis-inducing activity of gambogic acid was further characterized by a nuclear fragmentation assay and flow cytometry analysis in human breast tumor cells T47D. Gambogic acid was found to induce apoptosis independent of cell cycle, which is different from paclitaxel that arrests cells in the G2/M phase. To understand the structure–activity relationship (SAR) of gambogic acid, derivatives of 2 with modifications to different function groups were prepared. SAR studies of gambogic acid, as measured by the caspase activation assay, showed that the 9,10 carbon–carbon double bond of the α,β-unsaturated ketone is important for biological activity, while the 6-hydroxy and 30-carboxy group can tolerate a variety of modifications. The importance of the 9,10 carbon–carbon double bond was confirmed by the traditional growth inhibition assay. The high potency of 2 as an inducer of apoptosis, its novel mechanism of action, easy isolation and abundant supply, as well as the fact that it is amenable to chemical modification, makes gambogic acid an attractive molecule for the development of anticancer agents.

The discovery of gambogic acid as a potent inducer of apoptosis using a cell- and caspase-based HTS assay, as well as the characterization and SAR studies of gambogic acid as inducer of apoptosis, are reported.

Introduction

Programmed cell death, or apoptosis, plays important roles in normal cell development and tissue homeostasis.1, 2 Apoptosis can be activated by two major pathways: the extrinsic pathway, in which specific cell death receptors located on the cell surface membrane are activated by specific ligands; and the intrinsic pathway where mitochondria are primarily involved.³ Caspases play a crucial role in the execution of apoptosis with caspase 3 as the key enzyme in both the extrinsic and intrinsic pathways.4, 5, 6 It has been found that many cancer cells, while containing caspases, lack part of the molecular machinery that activate the caspase cascade, which results in a loss of the capacity to undergo apoptosis.⁷

Many current clinically used anticancer drugs are known to kill tumor cells through the induction of apoptosis. Therefore, the discovery and development of apoptosis inducers could lead to new anticancer agents.⁸ For example, it has been reported recently that retinoids can induce apoptosis and inhibit cell proliferation,9, 10 and several retinoids are currently being evaluated as preventive or therapeutic agents in clinical studies. In addition, compounds that kill tumor cells by inducing apoptosis may overcome tumor resistance to conventional anti-cancer agents. For example, trans-[PtCl₂(n,n-dimethylamine)(isopropylamine) has been found to have high cytotoxic activity for cisplatin-resistant cell lines and to induce apoptosis in these cells.¹¹ Several natural products also have been reported recently to induce apoptosis in cancer cells, including chloptosin from the culture broth of Streptomyces,¹² apoptolidin from Nocardiopsis sp,¹³ and a streptonigrin derivative from a Micromonospora strain.¹⁴

In our effort to discover and develop novel apoptosis inducers as potential anti-cancer drugs, we have created a cell-based high-throughput screening (HTS) assay for the identification of apoptosis inducers using a novel fluorescent caspase 3 substrate.¹⁵ The screening technology was designed for the discovery of compounds that activate caspase 3 through any apoptotic mechanism. Recently, we reported the discovery and SAR studies of substituted N-phenyl nicotinamides, exemplified by 6-methyl-N-(4-ethoxy-2-nitro-phenyl)-pyridine-3-carboxamide (1), as potent inducers of apoptosis using our HTS assay.¹⁶ Applying the same assay, we have identified gambogic acid (Scheme 1, Scheme 2, Scheme 5, Scheme 6, Chart 1) (Chart 1) as a novel apoptosis inducer from a commercial library.

Gambogic acid is a natural product isolated from the gamboge resin of Garcinia hanburyi tree in Southeast Asia. The resin is used as a traditional medicine as well as a color material for painting. The structure of gambogic acid was elucidated mainly by detailed NMR spectrum analysis¹⁷ and was confirmed recently by X-ray crystallographic analysis¹⁸ (Fig. 1). It contains a unique 4-oxatricyclo[4.3.1.0]decan-2-one ring system which is found in the natural products isolated from plants in the genus Garcinia.¹⁹ The first total synthesis of a compound containing this system, 1-O-methylforbesione (Chart 1), has been reported recently.²⁰ Gambogic acid and several related compounds have been reported previously to have cytotoxic activities.²¹ In addition, gaudichaudione A, a structurally related cytotoxic xanthone (Chart 1), has been reported recently to induce apoptosis in Jurkat cells through mitochondrial destabilization and caspase-3 activation.²² Herein, we report the discovery and characterization of gambogic acid as a potent apoptosis inducer in several cancer cell lines, as well as the SAR studies of gambogic acid as an inducer of apoptosis.

Section snippets

Chemistry

Gambogic acid (Scheme 1, Scheme 2, Scheme 5, Scheme 6, Chart 1) was isolated in overall yield of approximately 5% from the easily and widely available gamboge resin. It was purified by converting the crude extract from the gamboge resin into pyridine salt, followed by recrystallization.¹⁷ The structure of gambogic acid was confirmed by X-ray crystallographic analysis and is shown in Figure 1.¹⁸ There are many functional groups in the structure of Scheme 1, Scheme 2, Scheme 5, Scheme 6, Chart 1

Conclusion

In conclusion, gambogic acid, isolated from the resin of G. hurburyi tree, was identified as a potent apoptosis inducer through our cell-based high throughput screening assays. The ability of gambogic acid to induce apoptosis was further characterized in nuclear fragmentation and flow cytometry assays in T47D cells. The flow cytometry results indicate that gambogic acid does not induce apoptosis through cell cycle arrest. These data suggest that, similar to that of gaudichaudione A,²² a

General methods and materials

The ¹H NMR spectra were recorded at Varian 300 MHz. Chemical shifts are reported in ppm (δ) and J coupling constants are reported in Hz. Elemental analyses were performed by Numega Resonance Labs, Inc. (San Diego, CA, USA). Reagent grade solvents were used without further purification unless otherwise specified. Flash chromatography was performed on Merck silica gel 60 (230–400 mesh) using reagent grade solvents. Syto16 and N-(2-aminoethyl)biotinamide were obtained from Molecular Probes (Eugene,

Acknowledgements

This research was partially supported by a NIH SBIR Grant No. 1R43 CA91811-0. We would like to acknowledge the fine technical support given by Regina Brand and Ryan Yoshimura.

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Discovery, characterization and SAR of gambogic acid as a potent apoptosis inducer by a HTS assay

Abstract

Introduction

Section snippets

Chemistry

Conclusion

General methods and materials

Acknowledgements

Curr. Opin. Biotechnol.

A. Chem. Biol.

Cell

Bioorg. Med. Chem. Lett.

Bioconjugate Chem.

J. Chem. Crystallogr.

J. Org. Chem.

Tetrahedron Lett.

Curr. Med. Chem. Anticancer Agents

Carcinogenesis

Cancer Chemother. Pharmacol.

Annu. Rev. Cell Biol.

J. Clin. Oncol.

Curr. Med. Chem. AntiCancer Agents

J. Med. Chem.

J. Med. Chem.