B-Ring-Aryl Substituted Luotonin A Analogues with a New Binding Mode to the Topoisomerase 1-DNA Complex Show Enhanced Cytotoxic Activity Vı´ctor Gonza ´ lez-Ruiz 1,2 , Irene Pascua 3,4 , Tamara Ferna ´ ndez-Marcelo 3,4 , Pascual Ribelles 2,5 , Giulia Bianchini 2,5 , Vellaisamy Sridharan 5,6 , Pilar Iniesta 3,4 , M. Teresa Ramos 2,5 , Ana I. Olives 1,2 , M. Antonia Martı´n 1,2 , J. Carlos Mene ´ ndez 2,5 * 1 Seccio ´ n Departamental de Quı ´mica Analı ´tica, Facultad de Farmacia, Universidad Complutense, Madrid, Spain, 2 BIOHET (Biologically Relevant Heterocycles) group, Facultad de Farmacia, Universidad Complutense, Madrid, Spain, 3 Departamento de Bioquı ´mica y Biologı ´a Molecular II, Facultad de Farmacia, Universidad Complutense, Madrid, Spain, 4 Instituto de Investigacio ´ n Sanitaria del Hospital Clı ´nico San Carlos, Madrid, Spain, 5 Departamento de Quı ´mica Orga ´nica y Farmace ´utica, Facultad de Farmacia, Universidad Complutense, Madrid, Spain, 6 Department of Chemical and Biotechnology, SASTRA University, Thanjavur, India Abstract Topoisomerase 1 inhibition is an important strategy in targeted cancer chemotherapy. The drugs currently in use acting on this enzyme belong to the family of the camptothecins, and suffer severe limitations because of their low stability, which is associated with the hydrolysis of the d-lactone moiety in their E ring. Luotonin A is a natural camptothecin analogue that lacks this functional group and therefore shows a much-improved stability, but at the cost of a lower activity. Therefore, the development of luotonin A analogues with an increased potency is important for progress in this area. In the present paper, a small library of luotonin A analogues modified at their A and B rings was generated by cerium(IV) ammonium nitrate- catalyzed Friedla ¨nder reactions. All analogues showed an activity similar or higher than the natural luotonin A in terms of topoisomerase 1 inhibition and some compounds had an activity comparable to that of camptothecin. Furthermore, most compounds showed a better activity than luotonin A in cell cytotoxicity assays. In order to rationalize these results, the first docking studies of luotonin-topoisomerase 1-DNA ternary complexes were undertaken. Most compounds bound in a manner similar to luotonin A and to standard topoisomerase poisons such as topotecan but, interestingly, the two most promising analogues, bearing a 3,5-dimethylphenyl substituent at ring B, docked in a different orientation. This binding mode allows the hydrophobic moiety to be shielded from the aqueous environment by being buried between the deoxyribose belonging to the G(+1) guanine and Arg364 in the scissile strand and the surface of the protein and a hydrogen bond between the D-ring carbonyl and the basic amino acid. The discovery of this new binding mode and its associated higher inhibitory potency is a significant advance in the design of new topoisomerase 1 inhibitors. Citation: Gonza ´lez-Ruiz V, Pascua I, Ferna ´ndez-Marcelo T, Ribelles P, Bianchini G, et al. (2014) B-Ring-Aryl Substituted Luotonin A Analogues with a New Binding Mode to the Topoisomerase 1-DNA Complex Show Enhanced Cytotoxic Activity. PLoS ONE 9(5): e95998. doi:10.1371/journal.pone.0095998 Editor: A Ganesan, University of East Anglia, United Kingdom Received January 29, 2014; Accepted March 31, 2014; Published May 15, 2014 Copyright: ß 2014 Gonza ´lez-Ruiz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: MICINN (grant CTQ 2009-11312), www.micinn.es; Santander-UCM PR6/13-18859, www.ucm.es; Fundacio ´ n de Investigacio ´n Me ´ dica Mutua Madrilen ˜ a, www.fundacionmutua.es; MINECO (CTQ 2012-33272-BQU), www.mineco.gob.es. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: josecm@farm.ucm.es Introduction Cancer continues to be one of the leading causes of death worldwide. According to the latest data from the International Agency for Research on Cancer, in 2012 there were 14.1 million new cancer cases, 8.2 million cancer deaths and 32.6 million people living with cancer (within 5 years of diagnosis). Cancer is no longer a disease of the developed world, with 57% of newly diagnosed cases of cancer and 65% of deaths being associated with less developed regions [1]. Cancer therapy is still founded on the pillars of surgery, radiotherapy and chemotherapy, with immunotherapy having recently entered the stage as a fourth approach [2]. Nevertheless, the development of new anticancer drugs continues to be essential in the fight against the disease [3]. Topoisomerases are present in all living organisms and are crucial for relieving torsional tension in supercoiled DNA in the course of DNA replication, transcription and reparation [4]. Topoisomerases, and topoisomerase 1 in particular, are among the most relevant anticancer targets [5,6]. The camptothecins, specially irinotecan, topotecan and belote- can (Figure 1), are the main family of clinically relevant topoisomerase 1 inhibitors [7]. These compounds have a planar, pentacyclic core comprising a lactone functional group in ring E and containing a stereocenter at C-20, which must be in the S configuration for camptothecins to be active. Their pharmacologic target is the covalent topoisomerase 1-DNA binary complex, where they can bind non-covalently at the interphase formed between both macromolecules during the enzimatic catalytic cycle. This binding stabilizes the complex and retards its PLOS ONE | www.plosone.org 1 May 2014 | Volume 9 | Issue 5 | e95998