Diospyrin, A Bisnaphthoquinone: A Novel Inhibitor of Type I DNA Topoisomerase of Leishmania donovani SUTAPA RAY, 1 BANASRI HAZRA, BIDYOTTAM MITTRA, ADITI DAS, and HEMANTA K. MAJUMDER Molecular Parasitology Laboratory, Indian Institute of Chemical Biology, Calcutta 700 032, India (S.R., B.M., A.D., H.K.M.), and Department of Pharmacy, Jadavpur University, Calcutta 700 032, India (B.H.) Received March 2, 1998; Accepted August 31, 1998 This paper is available online at http://www.molpharm.org ABSTRACT Diospyrin is a plant product that has significant inhibitory effect on the growth of Leishmania donovani promastigotes. This compound inhibits the catalytic activity of DNA topoisomerase I of the parasite. Like camptothecin, it induces topoisomerase I mediated DNA cleavage in vitro. Treatment of DNA with dio- spyrin before addition of topoisomerase I has no effect. Prein- cubation of topoisomerase I with diospyrin before the addition of DNA in the relaxation reaction increases this inhibition. Our results suggest that this bis-naphthoquinone compound exerts its inhibitory effect by binding with the enzyme and stabilizing the topoisomerase I-DNA “cleavable complex.” Diospyrin is a specific inhibitor of the parasitic topoisomerase I. It does not inhibit type II topoisomerase of L. donovani and requires much higher concentrations to inhibit type I topoisomerase of calf thymus. The potent inhibitory effect of diospyrin on type I DNA topoisomerase from L. donovani can be exploited for rational drug design in human leishmaniasis. Leishmaniasis presents as a spectrum of diseases, ranging from benign cutaneous lesions through metastasizing muco- cutaneous forms to the often fatal visceralizing form (Walton, 1987). Current therapies are inadequate. The pentavalent antimonials sodium stibogluconate and meglumine anti- monate, the first line of drugs for visceral and cutaneous leishmaniasis, have variable efficacy and side effects (Thakur et al., 1988). The second line of drugs, amphotericin B and pentamidines, although used clinically, are often of limited efficacy and are very toxic (Iwu et al., 1994). Therefore, im- proved drug therapy of leishmanial infections is still desir- able and the need for new molecular targets on which to base future treatment strategies is clear and justified. Currently DNA topoisomerases have been recognized as po- tential chemotherapeutic targets for antitumor and antipara- sitic agents (Chakraborty and Majumder, 1988; Liu, 1989; Burri et al., 1996). DNA topoisomerases are ubiquitous en- zymes that control many vital cellular processes by making reversible DNA breaks, enabling a specific tyrosyl residue in the enzyme to covalently link to the phosphoryl group at the DNA break via a phosphodiester bond. They have been classi- fied into two types. The type I enzymes make a transient single stranded nick in absence of any high energy cofactor, whereas the type II enzymes make double-stranded breaks in the pres- ence of ATP, which allows supercoils to be removed from the circular DNAs. Both types of enzymes have been characterized in kinetoplastid hemoflagellated protozoan parasites (Riou, 1983; Chakraborty and Majumder, 1987; Melendy and Ray, 1987; Chakraborty and Majumder, 1991; Chakraborty et al., 1993). It has been suggested that topoisomerase I targeting agents may have broad spectrum antiprotozoal activity (Bodley et al., 1995). Our own studies have indicated that leishmanial DNA topoisomerases may well provide suitable targets for po- tential chemotherapy of antileishmanial drugs (Chakraborty and Majumder, 1988; Ray et al., 1996; Ray et al., 1997). Inhib- itors of DNA topoisomerases comprise a variety of structurally diverse compounds that interfere with the nicking-closing ac- tivities catalyzed by the enzymes. Clinically active antitumor drugs include inhibitors of topoisomerases, such as camptoth- ecin and three of its water soluble derivatives (Slichenmeyer et al., 1993), and inhibitors of topoisomerase II, such as acridines, anthracyclines, ellipticines, epipodophyllotoxins, and quino- lones, etc. (Liu, 1989). Some of these antitumor drugs (e.g., camptothecin, ellipticin, etoposides) also inhibit trypanosomal topoisomerases (Shapiro and Englund, 1990; Bodley and Sha- piro, 1995). In the present study, we describe the plant-derived bisnaphthoquinonoid compound diospyrin (Fig. 1). Diospyrin is an antitumor compound (Hazra et al., 1984) capable of This work was supported by grants from Department of Biotechnology, Government of India (BT/R&D/15/26/91 and BT/PRO/493/MED/09/096/96) to H.K.M. and University Grants Commission, India and International Founda- tion for Science, Sweden (Grant No. 1836/F-2) to B.H. 1 Current affiliation: Department of Pharmacology, University of Texas South Western Medical Center at Dallas, Dallas, TX 75235. ABBREVIATIONS: DMSO, dimethyl sulfoxide; SDS, sodium dodecyl sulfate; PIPES, piperazine-N,N'-bis [2-ethane sulfonic acid]; DTT, dithio- threitol, kDNA, kinetoplast DNA. 0026-895X/98/060994-06$3.00/0 Copyright © by The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 54:994 –999 (1998). 994 at ASPET Journals on October 13, 2017 molpharm.aspetjournals.org Downloaded from