Chung-Ming Sun

Andrographolide downregulates the v-Src and Bcr-Abl oncoproteins and induces Hsp90 cleavage in the ROS-dependent suppression of cancer malignancy

Andrographolide is a diterpenoid compound isolated from Andrographis paniculata that exhibits anticancer activity. We previously reported that andrographolide suppressed v-Src-mediated cellular transformation by promoting the degradation of Src. In the present study, we demonstrated the involvement of Hsp90 in the andrographolide-mediated inhibition of Src oncogenic activity. Using a proteomics approach, a cleavage fragment of Hsp90α was identified in andrographolide-treated cells. The concentration- and time-dependent induction of Hsp90 cleavage that accompanied the reduction in Src was validated in RK3E cells transformed with either v-Src or a human truncated c-Src variant and treated with andrographolide. In cancer cells, the induction of Hsp90 cleavage by andrographolide and its structural derivatives correlated well with decreased Src levels, the suppression of transformation, and the induction of apoptosis. Moreover, the andrographolide-induced Hsp90 cleavage, Src degradation, inhibition of transformation, and induction of apoptosis were abolished by a ROS inhibitor, N-acetyl-cysteine. Notably, Hsp90 cleavage, decreased levels of Bcr-Abl (another known Hsp90 client protein), and the induction of apoptosis were also observed in human K562 leukemia cells treated with andrographolide or its active derivatives. Together, we demonstrated a novel mechanism by which andrographolide suppressed cancer malignancy that involved inhibiting Hsp90 function and reducing the levels of Hsp90 client proteins. Our results broaden the molecular basis of andrographolide-mediated anticancer activity.

Design and Synthesis of New Biprivileged Molecular Scaffolds: Indolo-Fused Benzodiazepinyl/quinoxalinyl benzimidazoles

The present article describes the design and synthesis of new biprivileged molecular scaffolds with diverse structural features. Commercially available, simple heterocyclic building blocks such as 4-fluoro-3-nitrobenzoic acid, 2-chloro-3-nitrobenzoic acid, and indoline were utilized for the synthesis of the novel heterocycles. Pictet-Spengler-type condensation was used as a key step to construct tetracyclic indolo-benzodiazepines and indolo-quinoxalines linked with substituted benzimidazoles. Analysis of single crystals of representative compounds showed that these molecular skeletons have the potential to present various substituents with distinct three-dimensional orientations.

Multistep divergent synthesis of benzimidazole linked benzoxazole/benzothiazole via copper catalyzed domino annulation

An efficient, facile synthesis of structurally diverse benzimidazole integrated benzoxazole and benzothiazoles has been developed. In a multi-step synthetic sequence, 4-fluoro-3-nitrobenzoic acid was converted into benzimidazole bis-heterocycles, via the intermediacy of benzimidazole linked ortho-chloro amines. The amphiphilic reactivity of this intermediate was designed to achieve the title compounds by the reaction of various acid chlorides and isothiocyanates in a single step through the in situ formation of ortho-chloro anilides and thioureas under microwave irradiation. A versatile one pot domino annulation reaction was developed to involve the reaction of benzimidazole linked ortho-chloro amines with acid chlorides and isothiocyanates. The initial acylation and urea formation followed by copper catalyzed intramolecular C-O and C-S cross coupling reactions furnished the angularly oriented bis-heterocycles which bear a close resemblance to the streptomyces antibiotic UK-1.

Soluble polymer supported divergent synthesis of tetracyclic benzene-fused pyrazino/diazepino indoles: an advanced synthetic approach to bioactive scaffolds

The synthesis of indoline substituted nitrobenzene on a PEG support and its further elaboration to structurally diverse benzene-fused pyrazino/diazepino indoles is disclosed. A reagent based diversification approach coupled with Pictet-Spengler type condensation reactions furnished these fused polycyclic scaffolds. Microwave irradiation was used as a means of rate acceleration for soluble polymer-supported reactions. The efficiency of these fused heterocyclic molecules to inhibit the vascular endothelial growth factor receptor 3 (VEGFR-3) was examined in vitro using kinase receptor activation enzyme-linked immunosorbant assay (KIRA-ELISA). Based on the preliminary results obtained, a small set of potential drug candidates were identified as novel leads in this therapeutic area to be further explored as anti-metastatic agents.

Convergent Solution Phase Synthesis of Chimeric Oligonucleotides by a 2+2 and 3+3 Phosphoramidite Strategy

A chimeric oligonucleotide tetramer and hexamer were synthesized by the phosphoramidite approach using a 2+2 and 3+3 strategy, respectively. The concept of convergent synthesis provides an efficient route toward the synthesis of longer chimeric oligonucleotides, such as small interfering RNA oligonucleotides without the pollution of n – 1 or shorter failures. This methodology offers an efficient and economical way to scale-up the synthesis of high purity oligonucleotides for clinical trials and commercial uses.

Microwave‐Assisted Tandem Transformation on an Ionic‐Liquid Support: Efficient Synthesis of Pyrrolo/Pyridobenzimidazolones and Isoindolinone‐Fused Benzimidazoles

A tandem transformation that involves the formation of three bonds and two heterocyclic rings in a one-pot fashion through amino-alkylation of an ionic-liquid-immobilized diamine with keto acids followed by successive double intramolecular cyclizations to afford a tricyclic framework has been explored. This tandem cyclization has been utilized to develop a rapid and efficient method to synthesize various pyrrolo[1,2-a]benzimidazolones and pyrido[1,2-a]benzimidazolones on an ionic-liquid support by using focused microwave irradiation. The application of this tandem cyclization was further extended to the aromatic keto acids to provide isoindolinone-fused benzimidazoles, a structurally heterogeneous library with skeletal diversity. The outcome of the cascade reaction was confirmed by the X-ray crystallographic study of the product directly attached to the ionic-liquid support. Use of the ionic liquid as a soluble support facilitates purification by simple precipitation along with advantages like high loading capacity, homogeneous reaction conditions, and monitoring of the reaction progress by regular conventional spectroscopic methods, whereas application of microwave irradiation greatly accelerates the rate of the reactions.

Microwave-Assisted Synthesis of Tetracyclic 2,5-Diketopiperazines on a Soluble Polymer Support: A Structural Analogue of Tadalafil

Structural analogues of tadalafil that contain two diversity points have been synthesized from a soluble polymer support employing a Pictet–Spengler reaction using focussed microwave irradiation. Polymer-bound deprotected tryptophan reacts with various aldehydes to generate the tetrahydro-β-carbolines on the support. Subsequently immobilized tetrahydro-β-carboline underwent a highly efficient intramolecular N-heterocyclization in a traceless fashion from various in-situ generated α-alkyl and heteroalkyl amides in two steps to generate tetracyclic 2,5-diketopiperazines in high purity. All the compounds were isolated as cis and trans isomers with good yields.

Qualitative analysis of the fluorophosphonate-based chemical probes using the serine hydrolases from mouse liver and poly-3-hydroxybutyrate depolymerase (PhaZ) from Bacillus thuringiensis.

AbstractThe serine hydrolase family consists of more than 200 members and is one of the largest enzyme families in the human genome. Although up to 50xa0% of this family remains unannotated, there are increasing evidences that activities of certain serine hydrolases are associated with diseases like cancer neoplasia, invasiveness, etc. By now, several activity-based chemical probes have been developed and are applied to profile the global activity of serine hydrolases in diverse proteomes. In this study, two fluorophosphonate (FP)-based chemical probes were synthesized. Further examination of their abilities to label and pull down serine hydrolases was conducted. In addition, the poly-3-hydroxybutyrate depolymerase (PhaZ) from Bacillus thuringiensis was demonstrated as an appropriate standard serine hydrolase, which can be applied to measure the labeling ability and pull-down efficiency of FP-based probes. Furthermore, mass spectrometry (MS) was used to identify the serine residue that covalently bonded to the active probes. Finally, these FP-based probes were shown capable of establishing the serine hydrolase profiles in diverse mouse tissues; the serine hydrolases pulled down from mouse liver organ were further identified by MS. In summary, our study provides an adequate method to evaluate the reactivity of FP-based probes targeting serine hydrolases.n Figurexa0

Traceless synthesis of diketopiperazine fused tetrahydro-β-carbolines on soluble polymer support

The Pictet–Spengler reaction, using polyethylene glycol immobilized tryptophan ester with a variety of ketones, was achieved by refluxing condition in acidic chloroform. The linear as well as cyclic ketones were employed. All the ketones were reacted within 6–8xa0h to furnish soluble polymer-supported tetrahydro-β-carboline in good yields. Further expansion at N-terminus of tetrahydro-β-carbolines was achieved through a reaction with chloroacetyl chloride. Finally, the 2,5-diketopiperazine skeleton was constructed over a β-carboline by amination of the resulting N-chloroacetamides and subsequent intramolecular cyclization leading to cleavage of the polymer; constitutes a traceless synthesis of tetracyclic molecular architecture. Significantly, this strategy affords a straightforward and efficient approach for the construction of biological promising molecules with high purity and good yields.Graphical Abstract

Abstract LB-5: A proteomic approach to study molecular mechanisms of andrographolide-mediated degradation of Src oncoproteins.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnAndrographolide is a lactone diterpenoid compound isolated from Andrographis paniculata. Accumulating studies have demonstrated the anticancer activity of andrographolide. Andrographolide treatment could induce apoptosis of cancer cells and inhibit multiple malignant cancer phenotypes, such as aberrant proliferation, angiogenesis and metastasis. It is widely demonstrated that cancer cells require oncogene(s) for survival and maintenance of malignant phenotypes, a phenomenon called oncogene addiction. Thus, targeting oncoprotein(s) has been considered as an effective strategy for cancer therapy. Elevated protein expression and activity of Src oncoproteins have strong associations with carcinogenesis. Previous data from our laboratory have shown that andrographolide treatment led to degradation of Src proteins, suppression of Src-mediated cellular transformation and apoptosis of Src-transformed cells (J Biol Chem 2008; 283: 5023-33). To further reveal the molecular basis of andrographolide-mediated Src degradation, a proteomic approach was carried out to identify proteins specifically regulated by andrographolide. Proteomes from v-Src expressing RK3E cells treated with vehicle, andrographolide and NCTU-048 (an inactive andrographolide analog) were compared using two-dimensional electrophoresis and proteins with differential expression pattern in andrographolide-treated cells were further identified using mass spectrometry. Among the identified proteins, an N-terminal fragment of Hsp90 protein was selectively elevated (2.5-fold) in andrographolide-treated cells. Src oncoproteins are known client proteins of Hsp90 and their stability are modulated by Hsp90. We therefore investigated whether Hsp90 cleavage is a crucial event in andrographolide-mediated degradation of Src oncoproteins. Using Western blot analysis, we confirmed that Hsp90 cleavage was induced in various types of Src-transformed cells under andrographolide treatment in a concentration- and time-dependent manner. Notably, the Src-repressing activity of andrographolide and its derivatives correlated well with their ability to induce Hsp90 cleavage. Moreover, the amount of cleaved Hsp90 fragment induced by andrographolide and its derivatives positively correlated with the status of transformation suppression and apoptosis caused by these compounds. Notably, the andrographolide-induced Hsp90 cleavage, Src degradation, cellular transformation and induction of apoptosis were restored by ROS inhibitor, N-acetyl-cysteine (NAC). The molecular basis of andrographolide-induced biological effects via ROS is currently under investigation.nnCitation Format: Sheng-Hung Liu, Pei-Fen Chen, Chao-Hsiung Lin, Chung-Ming Sun, Chin-Wen Chi, Shu-Ling Fu. A proteomic approach to study molecular mechanisms of andrographolide-mediated degradation of Src oncoproteins. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-5. doi:10.1158/1538-7445.AM2013-LB-5