Received for publication September 27, 2004.
Revised December 19, 2004.
Accepted for publication December 20, 2004.

Selective inhibition of topoisomerase I and various steps of spliceosome assembly by diospyrin derivatives

Abstract

Pre-mRNA splicing is an essential step of the expression of most metazoan protein-coding genes, which is often regulated in a cell-type specific or developmental manner. Previously we have demonstrated that human DNA topoisomerase I, an extensively studied target for anticancer drugs, has also an intrinsic protein kinase activity which specifically phosphorylates proteins involved in splice sites selection. Accordingly, DNA topoisomerase I was recently shown to play a critical role in alternative splicing. Here we have exploited these novel properties of DNA topoisomerase I to develop entirely novel diospyrin derivatives targeting its protein kinase activity and thereby modulating pre-mRNA splicing. While some derivatives indeed inhibit kinase activity of topoisomerase I, they did not block reactions of topoisomerase I on DNA. However, these drugs interfere with camptothecin-dependent topoisomerase I-mediated DNA cleavage, implying that diospyrin derivatives mediate a conformational change of topoisomerase I. Importantly, in vitro splicing reactions revealed that diospyrin derivatives alter various steps of splicing. Some diospyrin derivatives inhibit either the first or the second catalytic step of splicing but not spliceosome assembly, while diospyrin itself prevents the formation of full spliceosome. Our data revealed for the first time that diospyrin derivatives are able to stall the dynamic assembly of the spliceosome and open the exciting possibility of using these derivatives to correct aberrant splicing in human genetic diseases.

Key words: Protein Kinases (other), Structure/function/mechanism, Topoisomerases