Meridianins, a new family of protein kinase inhibitors isolated from the Ascidian Aplidium meridianum

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Abstract

Meridianins are brominated 3-(2-aminopyrimidine)-indoles which are purified from Aplidium meridianum, an Ascidian from the South Atlantic (South Georgia Islands). We here show that meridianins inhibit various protein kinases such as cyclin-dependent kinases, glycogen synthase kinase-3, cyclic nucleotide-dependent kinases and casein kinase 1. Meridianins prevent cell proliferation and induce apoptosis, a demonstration of their ability to enter cells and to interfere with the activity of kinases important for cell division and cell death. These results suggest that meridianins constitute a promising scaffold from which more potent and selective protein kinase inhibitors could be designed.

Introduction

The phosphorylation of proteins on serine, threonine and tyrosine residues by the ∼520 protein kinases encoded in the human genome constitutes one of the major mechanisms used by cells to regulate their metabolism and functions. The recent appreciation of the implication of abnormal protein phosphorylation in many human diseases has sparked considerable interest in the search for pharmacological inhibitors of kinases.1 In addition to their potential applications in many therapeutic indications, these pharmacological inhibitors also constitute molecular tools, in more basic research, to probe the functions of specific kinases, to synchronize cells, to modify their differentiation fate, to alter their metabolism, etc. New chemical inhibitors are constantly being described and characterized with respect of their selectivity, mechanism of action, cellular effects, potential medical use.2 In our laboratory we have focused our interest on a selection of kinases involved in cell cycle control and neuronal functions, namely the cyclin-dependent kinases (CDKs)3, 4 and glycogen synthase kinase-3 (GSK-3).5 Numerous pharmacological inhibitors of CDKs have been discovered, characterized and recently reviewed.6, 7, 8 In contrast, only very few GSK-3 inhibitors have been described.9

Marine organisms constitute a very promising and relatively poorly explored source of original bioactive molecules.10 Among a huge diversity of structures, indole alkaloids are frequently found in marine invertebrates.11, 12 In this article we report on the identification of meridianins, a family of 3-(2-aminopyrimidine)-indoles, as potent inhibitors of various protein kinases. Meridianins were initially isolated from Aplidium meridianum, an Ascidian collected in the South Atlantic (South Georgia Islands).13

Section snippets

Meridianins, a family of kinase inhibitors

The meridianins have been first isolated from the marine Ascidian Aplidium meridianum (Ascidiae, Polyclinidae family),14, 15 collected at a depth of 100 m in the vicinity of the South Georgia Islands, South Atlantic.13 These indoles substituted at C-3 with a 2-aminopyrimidine have now also been synthesized by two other groups.16, 17 While screening for new protein kinase inhibitors from natural sources, we discovered that meridianins (17) (Fig. 1) were potent inhibitors of several protein

Meridianins, structure–activity relationship

The very limited number of available meridianins precludes a detailed structure–activity relationship (SAR) study. However a few general features can be described. If we focus on CDK1 and CDK5, it is clear that a bromine substitution on position 7 of the indole (compare meridianins E and A or B) and a hydroxyl on position 4 (compare meridianins A and G, meridianins B and D) provide the best inhibitory activity. The removal of both bromine and hydroxyl substitutions (meridianin G) essentially

Cellular effects of meridianins

The effects of meridianins A–F on cellular viability were tested on cell lines from human and murine origin. The compounds induced cytotoxic effects, which were time-, dose- and cell line-dependent. For example, the effect of a 40 h treatment on human teratocarcinoma NT2 cells survival is depicted in Figure 2. Clearly, only meridianins B and E (assayed at doses up to 10 μM) had an effect on NT2 proliferating cells, suggesting that only most kinase-active meridianins may display

Natural products extraction and characterization

The green Ascidian Aplidium meridianum was collected by trawling at −100 m near the South Georgia Islands and stored at −20 °C until extraction. The frozen Ascidians were crushed and extracted three times with ethanol. The extract was dried under reduced pressure, and the yellow residue was flash-chromatographed on reverse phase Silica using a water/methanol gradient. Eluted fractions were chromatographed on Sephadex and further fractionated by HPLC.13

Kinase preparations and assays

CDK1/cyclin B, CDK2/cyclin A, CDK2/cyclin E,

Acknowledgements

We would like to thank Mrs. Alicia Rivelli, from the Institute of Oncology A. H. Roffo, for technical assistance with cell cultures and our colleagues for providing reagents: D. Alessi, M. Cobb, W. Harper, F. Hofmann, S. Lohmann, L. Pinna, H. Y. L. Tung, J. H. Wang. This research was supported by grants from the ‘Association pour la Recherche sur le Cancer’ (ARC5343) (L.M.) and the INSERM/CNRS ‘Molécules & Cibles Thérapeutiques’ Programme (L.M.).

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