Differential inhibition of restriction enzyme cleavage by chromophore-modified analogues of the antitumour antibiotics mithramycin and chromomycin reveals structure–activity relationships Sylvia Mansilla a , Irene Garcia-Ferrer a , Carmen Me ´ ndez b , Jose ´ A. Salas b , Jose ´ Portugal a, * a Instituto de Biologia Molecular de Barcelona, CSIC, Parc Cientific de Barcelona, Baldiri Reixac, 10, E-08028 Barcelona, Spain b Departamento de Biologia Funcional-Instituto Universitario de Oncologia del Principado de Asturias, Universidad de Oviedo, E-33006 Oviedo, Spain 1. Introduction Mithramycin A (MTA) and chromomycin A 3 (CRO) are members of the aureolic acid family of anticancer antibiotics. MTA is produced by Streptomyces argillaceus (ATCC 12596), among other species, while CRO is produced by Streptomyces griseus subsp. griseus (ATCC 31053) [1]. These antibiotics are glycosylated aromatic polyketides, in which two different oligosaccharide chains are attached in the aromatic polyketide moiety (Fig. 1) [1,2]. MTA has been used in the treatment of Paget’s disease and advanced testicular carcinoma [3], while CRO has been used in Japan against advanced stomach cancers [4]. The aureolic acid family of molecules has gained renewed attention as therapeutic agents in both cancer and non-cancer related diseases [5–8]. MTA has been proposed for the treatment during antiangiogenic strategies against pancreatic cancer [9]. The activity of these antibiotics has been associated with their ability to bind to G/C- rich DNA tracts via the minor groove [10–14], and divalent cations such as Mg 2+ are essential requirement for the association with DNA at and above physiological pH [15,16]. As a consequence of the binding to DNA these antibiotics are able to inhibit transcription both in vivo and in vitro [17–20]. Sequence-selective binding of MTA and CRO to DNA is fundamentally achieved through direct hydrogen bonding between the 8-O-hydroxyl group in the antibiotic molecules and the 2-amino group of the guanine at GpC or GpG steps, while their respective trisaccharide moieties are essential for optimal binding [13,14,21,22]. The thermodynamic analysis of the MTA and CRO binding to DNA has shown that this is entropically driven [16,23], dominated by the hydrophobic transfer of the Mg 2+ -coordinated antibiotic dimers from solution to the DNA-binding site [23]. Hydrogen bonding also participates in the binding of the oligosaccharide chains along the minor groove [13,21,22,24]. Despite the high structural similarity between MTA and CRO (Fig. 1), the genetic organization of the biosynthesis gene clusters for both antitumour antibiotics is highly different [25]. These gene clusters have been studied in detail by gene sequencing, insertional inactivation and gene expression [1,11,25–29]. In fact, most of the biosynthetic intermediates in these pathways have been isolated and characterized, and some of them have shown enhanced antitumour activity [17,18,20,29]. Since MTA and CRO exhibit severe side-effects, we examined the possibility that some biosynthetically produced analogues (Fig. 1) may present lower toxicity and higher antitumour activity. Biochemical Pharmacology 79 (2010) 1418–1427 ARTICLE INFO Article history: Received 12 November 2009 Accepted 11 January 2010 Keywords: Mithramycin A Chromomycin A 3 Restriction enzymes Antitumour antibiotics Carcinoma cells ABSTRACT Differential cleavage at three restriction enzyme sites was used to determine the specific binding to DNA of the antitumour antibiotics mithramycin A (MTA), chromomycin A 3 (CRO) and six chromophore- modified analogues bearing shorter side chains attached at C-3, instead of the pentyl chain. All these antibiotics were obtained through combinatorial biosynthesis in the producer organisms. MTA, CRO and their six analogues showed differences in their capacity for inhibiting the rate of cleavage by restriction enzymes that recognize C/G-rich tracts. Changes in DNA melting temperature produced by these molecules were also analyzed, as well as their antiproliferative activities against a panel of colon, ovarian and prostate human carcinoma cell lines. Moreover, the cellular uptake of several analogues was examined to identify whether intracellular retention was related to cytotoxicity. These experimental approaches provided mutually consistent evidence of a seeming correlation between the strength of binding to DNA and the antiproliferative activity of the chromophore-modified molecules. Four of the analogues (mithramycin SK, mithramycin SDK, chromomycin SK and chromomycin SDK) showed promising biological profiles. ß 2010 Elsevier Inc. All rights reserved. * Corresponding author. Tel.: +34 93 403 4959; fax: +34 93 403 4979. E-mail address: jpmbmc@ibmb.csic.es (J. Portugal). Contents lists available at ScienceDirect Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm 0006-2952/$ – see front matter ß 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bcp.2010.01.005