Furanocoumarins: Novel topoisomerase I inhibitors from Ruta graveolens L. Renuka Diwan, Nutan Malpathak * Department of Botany, University of Pune, Pune 411007, Maharashtra, India article info Article history: Received 13 February 2009 Revised 12 April 2009 Accepted 14 April 2009 Available online 17 April 2009 Keywords: Topoisomerase I inhibitors Furanocoumarins Ruta graveolens In vitro cultures abstract Topoisomerase I inhibitors from Ruta graveolens are reported for the first time. Potent topoisomerase I inhibitory activity from in vitro culture extracts R. graveolens were observed. Stabilization of DNA–topo- isomerase covalent complex was observed in all the tested extracts. The mechanism of topoisomerase inhibition was determined by preincubation studies. The irreversible topoisomerase I mediated relaxa- tion of plasmid in enzyme–substrate preincubation study, indicated that the observed inhibitory activity of extract constituents was not mediated through conformational changes in the DNA. Furthermore, the affinity of inhibitors with the enzyme was tested by enzyme–extract preincubation study. Increase in inhibition of topoisomerase activity and promotion of DNA–enzyme complex was observed after enzyme–extract preincubation. The activity could be assigned to furanocoumarins—psoralen, bergapten and xanthotoxin, identifying them as novel, potent topoisomerase I inhibitors. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction DNA topoisomerases are essential cellular enzymes required for cell proliferation and therefore, have recently emerged as impor- tant cellular targets for chemical intervention in the development of anti-cancer agents. Topoisomerases can be inhibited by two dis- tinct mechanisms, and are divided into two classes accordingly: class I and II. Class I inhibitors stabilize the enzyme–DNA-covalent complex and block the subsequent rejoining of DNA break, en- hance apoptosis through blocking the advancements of replication forks. Class II inhibitors prevent the enzyme and DNA from binding by interacting with either the topoisomerase enzyme or DNA (Ka- waik et al. 2007, Yonezawa et al. 2005). Topoisomerase inhibitors are very rare, the most widely studied and characterized inhibitors being camptothecin, a topo I poison, 3 and etoposide and doxorubicin, topo II poisons. 4–6 Although inhib- itors against topoisomerase I have been developed and applied clinically, their severe side effects remain a serious problem. Be- sides, there are fewer known inhibitors of DNA topoisomerase I. 7 The success of camptothecin and its analogues as anticancer agents has spurred a search for additional agents acting by inhibition of topoisomerase I; to date several new classes of inhibitors have been described, like nitidine, fagaronine, epiberberine 8,9 and cora- lyne. 10 In the development of new inhibitors, natural products from plant sources can be a valuable source of novel inhibitors and may also serve as a suitable lead for the production of semi- synthetic active agents. Ruta graveolens L. (Rutaceae) is a medicinal plant which has a long history of use as a domestic remedy. It is extensively used in homeopathic, ayurvedic and unani preparations. R. graveolens has been traditionally used in treatment of leucoderma, vitiligo, psoriasis, multiple sclerosis, cutaneous lymphomas, rheumatic arthritis. Recently its extracts were shown to have potent anti-can- cer activity. 11,12 Therefore R. graveolens was assessed for its topoi- somerase I inhibitory potential. Previously established culture lines, selected for their high furanocoumarin productivity, were also evaluated for their topoisomerase inhibitory activity. To determine the exact mechanism of topoisomerase inhibition the enzyme and DNA were preincubated prior to addition of ex- tract. Also to test the affinity of inhibitors with the enzyme, the en- zyme and extract were preincubated before addition of extract. Furanocoumarins (Illustration 1), the major active constituents of the extracts, were isolated and evaluated for their topoisomerase inhibitory potential. This is the first report on DNA topoisomerase I inhibitors from R. graveolens and its in vitro cultures leading to identification of furanocoumarins—psoralen, bergapten and xanthotoxin as novel topoisomerase I inhibitors. 0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2009.04.023 * Corresponding author. Tel.: +91 020 25601439; fax: +91 020 25690498. E-mail address: mpathak@unipune.ernet.in (N. Malpathak). Illustration 1. Bioorganic & Medicinal Chemistry 17 (2009) 7052–7055 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc