Screening enzyme-inhibitory activity in several ascidian species from Orkney Islands using protein tyrosine kinase (PTK) bioassay-guided fractionation

Protein tyrosine kinases (PTK) play a crucial role in cell growth, cell differentiation and proliferation. In vertebrates, they are considered as potential oncogenes in development and growth. Some invertebrates utilize PTK inhibitors as a protection against microbial colonialization. With particular emphasis on PTK inhibitory potential for novel anticancer agents, activity against the epidermal growth factor receptor (EGFR) tyrosine kinase has been tested in ascidians for the first time. Twelve ascidian species collected around the Orkney Islands north of Scotland (UK) were tested for their activity against the epidermal growth factor receptor using a protein tyrosine kinase assay (PTK-101 SIGMA). The crude extracts were partitioned according to their polarity (n-hexane, ethyl acetate, n-butanol and water) and tested for inhibitory properties, followed by bioassay-guided fractionation of the partitions using different chromatographic methods and the PTK-101 assay. Structure elucidation of purified and PTK-active fractions was performed by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS). Bioassay-guided fractionation led to the identification of several fractions enhancing or moderately reducing the enzyme activity. Strong inhibitory effects were detected in the ethyl acetate and the n-butanol fractions of the baked bean ascidian, Dendrodoa grossularia. NMR analysis indicated the presence of the guanidinostyrene derivative tubastrine. This is the first documentation of this metabolite in ascidians. Structure analysis of tubastrine in comparison to other known PTK inhibitors may enhance our understanding of the structure and effect of the compounds and may help in the development of efficient therapeutic agents. (c) 2005 Elsevier B.V All rights reserved.