d-Amino Acid Position Influences the Anticancer Activity of Galaxamide Analogs: An Apoptotic Mechanism Study.

Galaxamide, an extract from Galaxaura filamentosa, is a cyclic pentapeptide containing five l-leucines. Due to the particular cyclic structure and the excellent anticancer activity, synthesis of Galaxamide and its analogs and their subsequent bio-applications have attracted great attention. In the present work, we synthesized six Galaxamide analogs by replacing one of the l-leucines with phenylalanine and varying the d-amino acid position. The anticancer effect of the synthesized Galaxamide analogs was tested against four in vitro human cancer cell lines, human hepatocellular cells (HepG2), human breast cancer cell (MCF-7), human breast adenocarcinoma cells (MDA-MB-435) and a human cervical carcinoma cell line (Hela). Results showed that Galaxamide analogs with different d-amino acid positions displayed distinct anticancer potential. The Galaxamide analog containing d-amino acid at position 5 (Analog-6) presented the strongest anticancer activity. The mechanism study revealed that Analog-6 could cause the early apoptosis of HepG2 cells by inhibiting their growth in the sub-G1 stage of the cell cycle and induce the chromatin condensation and fragmentation, which can be seen as 68% of HepG2 cells inhibited in the sub-G1 stage. Moreover, a mitochondria-mediated pathway was found to be involved in the apoptotic process of Analog-6 on HepG2 cells.

Design and Synthesis of Analogues of Marine Natural Product Galaxamide, an N-methylated Cyclic Pentapeptide, as Potential Anti-Tumor Agent in Vitro.

Herein, we report design and synthesis of novel 26 galaxamide analogues with N-methylated cyclo-pentapeptide, and their in vitro anti-tumor activity towards the panel of human tumor cell line, such as, A549, A549/DPP, HepG2 and SMMC-7721 using MTT assay. We have also investigated the effect of galaxamide and its representative analogues on growth, cell-cycle phases, and induction of apoptosis in SMMC-7721 cells in vitro. Reckon with the significance of conformational space and N-Me aminoacid (aa) comprising this compound template, we designed the analogues with modification in N-Me-aa position, change in aa configuration from l to d aa and substitute one Leu-aa to d/l Phe-aa residue with respective to the parent structure. The efficient solid phase parallel synthesis approach is employed for the linear pentapeptide residue containing N-Me aa, followed by solution phase macrocyclisation to afford target cyclo pentapeptide compounds. In the present study, all galaxamide analogues exhibited growth inhibition in A549, A549/DPP, SMMC-7721 and HepG2 cell lines. Compounds 6, 18, and 22 exhibited interesting activities towards all cell line tested, while Compounds 1, 4, 15, and 22 showed strong activity towards SMMC-7221 cell line in the range of 1-2 µg/mL IC50. Flow cytometry experiment revealed that galaxamide analogues namely Compounds 6, 18, and 22 induced concentration dependent SMMC-7721 cell apoptosis after 48 h. These compounds induced G0/G1 phase cell-cycle arrest and morphological changes indicating induction of apoptosis. Thus, findings of our study suggest that the galaxamide and its analogues 6, 18 and 22 exerted growth inhibitory effect on SMMC-7721 cells by arresting the cell cycle in the G0/G1 phase and inducing apoptosis. Compound 1 showed promising anti-tumor activity towards SMMC-7721 cancer cell line, which is 9 and 10 fold higher than galaxamide and reference DPP (cisplatin), respectively.