Degradation of the morpholino ring in the crystal structure of cyanomorpholinodoxorubicin complexed with d(CGATCG)

Abstract

The cyanomorpholino analogue of antitumor anthracycline doxorubicin possesses an intense potency and differs from the parent compound in cross-resistance and other biological properties. The induction by cyanomorpholinodoxorubicin of both DNA cross-links and strand scission suggests an altered mode of action relative to doxorubicin with a different DNA-interacting capacity. We have co-crystallized 3′-(3-cyano-4-morpholinyl)-3′-desaminodoxorubicin (CMD) with the DNA hexamer d(CGATCG) and have determined the crystal structure at 0.16-nm resolution. The complex crystallizes in the space group P4 1 2 1 2 and is similar to the previously reported anthracycline/DNA structures, with the drug intercalated at the CpG step, forming hydrogen bonds with the guanine residue. The structure reveals that the morpholino moiety has undergone a major rearrangement with loss of the cyano group and opening of the morpholino ring. The compound actually bound to DNA in the complex resembles N-(2-hydroxyethyl)doxorubicin, which was previously identified as a hydrolysis product of CMD. No DNA alkylation has been observed. However, the structure shows that the active site of the morpholino ring, after dissociation of the cyano group, lies in the minor groove in proximity of the A/T base pair. This may indicate that a C/G base pair next to the intercalation site, with NH 2 group in the minor groove, is required for DNA alkylation.Keywords : anthracycline; drug/DNA complex ; anticancer drugs design ; X-ray structure ; DNA intercalation.Anthracycline antibiotics, of which daunomycin (daunorubicin) and adriamycin (doxorubicin) are the best known members, have been widely used as antitumor drugs for over two decades, and adriamycin has been one of the most successful chemotherapeutic agents in clinical use. Since their discovery, a number of studies have suggested that the cytotoxic action of anthracyclines is related to their ability to react with cellular DNA [1]. Though there is no simple relationship between DNAbinding affinity and antitumor activity, structural specificity of these drugs was clearly noted, and it also appeared that an intercalative DNA binding is necessary for drug cytotoxicity [2]. The binding of anthracyclines to DNA has been extensively studied by a variety of biochemical and biophysical methods [3]. Crystallographic data from several three-dimensional structures of DNA-anthracycline complexes have well characterized the molecular interaction between drug and DNA and have established specific sequences as preferred sites of drug intercalation [4Ϫ8].Despite intensive investigation, the mode of action of these drugs is still to be totally understood. Currently, the widely accepted view is that the mechanism of anthracycline cytotoxicity Correspondence to G. Ughetto, Istituto di Strutturistica Chimica 'Giordano Giacomello', C.N.R., Area della Ricerca di Roma, C.P. 10, I-00016 Monterotondo Stazione, Italy Fax: ϩ39 690 672630. E-mail: alex@isc.mlib.cnr.it Abbreviation. CMD, 3′-(3-cyano-4-morpholinyl)-3′-desaminodoxorubicin...

Release of the cyano moiety in the crystal structure of N‐cyanomethyl‐N‐(2‐methoxyethyl)‐daunomycin complexed with d(CGATCG)

“…Ettorre et al . [13] studied the X‐ray structure at 1.6 Å resolution of the crystal complex of the usual d(CGATCG) with an analogous compound of CMDa, the cyanomorpholino‐doxorubicin (CMoDx). The CMoDx‐d(CGATCG) and our complex are isomorphous.…”

“…Furthermore, experiments by fluorescence quenching method point out the high daunomycin binding affinities for duplexes containing GC base pairs as part of alternating purine‐pyrimidine sequence motifs [9]. At present, many crystal structures of drug‐DNA complexes describe the atomic interactions between anthracyclines and DNA [10–15].…”

“…We report these results and discuss them with a comparison of a cyano group release in other drug‐DNA complexes. A comparison of this crystal structure with those of daunomycin [10] (NDB entry code DDF020) or cyanomorpholino‐doxorubicin [13] (NDB entry code DDF078) d(CGATCG) complexes is reported. The effects of the groups attached at the N3′ position of the anthracyclines, on the DNA conformation, are detailed.…”

Anthracycline drug targeting: cytoplasmic versus nuclear – a fork in the road

“…65 In contrast to DOX, morpholinyl-, cyanomorpholinyl-and methoxymorpholinyl-doxorubicin convalently bind DNA though a mechanism by which the morpholino ring undergoes rearrangement/opening and is bound to DNA with a structure reported to resemble N-(2-hydroxyethyl)doxorubicin, based upon crystallographic analysis. 66 In the case of cyanomorpholinyldoxorubicin, the cyano group is released. 66 The interaction of this structure aligns the amino group in the minor groove as a requirement for DNA alkylation.…”

“…66 In the case of cyanomorpholinyldoxorubicin, the cyano group is released. 66 The interaction of this structure aligns the amino group in the minor groove as a requirement for DNA alkylation. The resulting cytotoxicity is not associated with topo II-mediated DNA cleavage but rather with enhanced topo I-mediated DNA cleavage 67 and DNA crosslinking.…”