Research Brief Leishmania donovani: Oral therapy with glycosyl 1,4-dihydropyridine analogue showing apoptosis like phenotypes targeting pteridine reductase 1 in intracellular amastigotes Jaspreet Kaur a , Nasib Singh b , Biswajit Kumar Singh c , Anuradha Dube b , Rama Pati Tripathi c , Prashant Singh d , Neeloo Singh a, * a Drug Target Discovery and Development Division, Central Drug Research Institute, Chattar Manzil Palace, P.O. Box No. 173, CSIR, Lucknow-226001, India b Division of Parasitology, Central Drug Research Institute, Chattar Manzil Palace, P.O. Box No. 173, CSIR, Lucknow-226001, India c Medicinal and Process Chemistry Division, Central Drug Research Institute, Chattar Manzil Palace, P.O. Box No. 173, CSIR, Lucknow-226001, India d Department of Chemistry, D.A.V. (P.G.) College, Dehradun-248001, India article info Article history: Received 31 August 2009 Received in revised form 19 February 2010 Accepted 23 February 2010 Available online 26 February 2010 Keywords: Leishmania donovani Dihydropyridine Antileishmanial activity Flow cytometry Cell cycle arrest abstract Glycosyl 1,4-dihydropyridine analogue (2,6-dimethyl-4-(3-O-benzyl-1,2-O-isopropylidene-b-L-threo pentofuranos-4-yl)-1-phenyl-1,4-dihydro-pyridine-3,5-dicarboxylic acid diethyl ester) synthesized in our laboratory, inhibited Leishmania donovani infection in vitro and in hamsters (Mesocricetus auratus) when administered orally. This analogue is nontoxic, cell-permeable and orally effective. This glycosyl dihydropyridine analogue functioned through arrest of cells in sub-G0/G1-phase, triggering mitochon- drial membrane depolarization-mediated programmed cell death of the intracellular amastigotes. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction The drugs recommended for the treatment of visceral leishman- iasis (VL), i.e., pentavalent antimonials, pentamidine, amphotericin B and its lipid formulations, paromomycin, allopurinol and miltefo- sine have limitations, including development of resistance, paren- teral administration, long courses of treatment, toxic side effects, and high costs (Murray et al., 2005; Sundar and Chatterjee, 2006). In order to generate an adequate armory of effective, safe and cheaper chemotherapeutic agents to treat VL, new and effec- tive drug targets are required to combat this disease. Enzymes, metabolites or proteins present in the parasite but absent from their mammalian host are considered as ideal targets for rational drug design. In the search for better therapeutics against leishman- iasis we focused on a validated drug target viz. folate biosynthetic pathway which is unique to the parasite (Bello et al., 1994). Pteri- dine reductase1 (PTR1) enzyme in Leishmania is essential for devel- opment of successful antifolate chemotherapy (Nichol et al., 1985). In search for specific inhibitors of PTR1 (Genbank Accession No. AY547305) we have previously reported phenyl 1,4-dihydropyri- dine ring as the lead structure showing impressive antileishmanial efficacy with an IC 50 of 18 lM in intracellular amastigotes (Kumar et al., 2008). Dihydropyridine analogues have exhibited important therapeutic and pharmacological properties as the integral back- bone of several calcium channel blockers (Kappe, 2000), antihyper- tensive agents (Atwal et al., 1991), a1a-antagonists (Kappe et al., 1997), and neuropeptide Y (NPY) antagonists (Poindexter et al., 2002). A broad range of biological effects including antiviral, anti- tumor, antibacterial and anti-inflammatory activities have been described for these compounds (Kappe, 1993). Amlodipine and lacidipine, conventional antihypertensive 1,4-dihydropyridine derivatives have been reported to be active against experimental VL and triggered programmed cell death (PCD) in promastigotes (Palit and Ali, 2008). The main objective of the present study was to evaluate the antileishmanial efficacy both in vitro and in vivo and their possible mode of killing mediated by glycosyl 1,4-dihy- dropyridine analogue (GDA) (2,6-dimethyl-4-(3-O-benzyl-1,2-O- isopropylidene-b-L-threo pentofuranos-4-yl)-1-phenyl-1,4-dihy- dro-pyridine-3,5-dicarboxylic acid diethyl ester) on intracellular amastigotes of Leishmania donovani clinical isolate. 0014-4894/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2010.02.011 * Corresponding author. Fax: +91 0522 2623405. E-mail address: neeloo888@yahoo.com (N. Singh). Experimental Parasitology 125 (2010) 310–314 Contents lists available at ScienceDirect Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr