Clavilactones, a novel class of tyrosine kinase inhibitors of fungal origin

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Abstract

Targeting of deregulated protein tyrosine kinases has been proposed as a new approach in the therapeutic intervention against pathological processes including proliferative disorders and cancer. Using a screening approach based on a comparative evaluation of antiproliferative effects in a panel of tumor cells with differential expression of protein tyrosine kinases, three benzoquinoid macrolidic fungal metabolites produced by Clitocybe clavipes, clavilactones A, B, and D (CA, CB, and CD) and two semisynthetic derivatives of these products, diacetyl-CA and dimethyl-CA, were identified as inhibitors of protein tyrosine kinases. Naturally occurring CA, CB, and CD showed inhibitory activity in kinase assays against the Ret/ptc1 and epidermal growth factor receptor (EGF-R) tyrosine kinases, while being less effective against the v-Abl tyrosine kinase and p34cdc2 serine/threonine kinase (ic50 2.8, 5.5, 81.3, and 128 μM respectively, for the most potent compound CD). CB was shown to be a non-competitive inhibitor of EGF-R with respect to ATP or poly(Glu6Ala3Tyr). CD also preferentially inhibited the growth of A431 cells, which overexpress a constitutively active EGF-R, as opposed to IGROV-1 and SKOV-3 cells, which express low levels of the receptor. Further, EGF-R was shown to be a target for clavilactones in A431 cells, since EGF-induced receptor autophosphorylation was inhibited in the presence of CB, CD, and diacetyl-CA. Both CD and diacetyl-CA displayed weak activity when administered daily (i.p.) to mice bearing ascitic A431 tumor. These findings indicate that clavilactones represent the prototypes of a new structural class of tyrosine kinase inhibitors deserving further investigation.

Section snippets

Clavilactones

The benzoquinoid macrolide structure of clavilactones under study is reported in Table 1. On the basis of the oxidation status of the aromatic ring, these molecules can be assigned to one of two groups, the first including the quinone derivatives CB and CD and the second the hydroquinones CA, dimethyl-CA, and diacetyl-CA. CA, CB, and CD were extracted from the C. clavipes culture medium as previously described 27, 28. Dimethyl-CA and diacetyl-CA are semisynthetic derivatives [28]. CA and CB

Antiproliferative activity of clavilactones towards tumor and oncogene-transformed cell lines

We focused on EGF-R and the Ret/ptc1 oncoprotein as possible target tyrosine kinases. To select inhibitory compounds, we adopted a cell and mechanism-based approach previously described 41, 42. As first step of the screening, we compared the antiproliferative activity profile of test compounds against a variety of cell lines characterized by differential expression of these two enzymes. The antiproliferative effects of clavilactones after 96-hr incubation of the three human cell lines A431,

Discussion

In an attempt to identify novel natural inhibitors of protein tyrosine kinases, we evaluated the three fungal metabolites, clavilactones CA, CB, and CD, and the two semisynthetic derivatives, diacetyl- and dimethyl-CA (structures in Table 1) 27, 28, by a cell and mechanism-based approach which previously allowed the identification of a new EGF-R inhibitor [41] and inhibitors of the Ret/ptc1 oncoprotein [42]. The comparison of the antiproliferative activity of these compounds against cell lines

Acknowledgements

We are grateful to Professor Lucio Merlini, Dr. Sergio Penco, and Dr. Giuseppe Cassinelli for helpful suggestions. NIH3T3PTC1 cells, NIH3T3H-RAS cells, and the anti-ret antibody were kindly provided by Dr. Marco A. Pierotti and NIH3T3HER1 cells by Dr. Elda Tagliabue. The expert technical assistance of Mr. Mario Azzini is acknowledged. This work was supported by the Ministero della Sanità, the Italy-USA Program on “Therapy of Tumors”, and the CNR (PF ACRO).

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