Luzopeptin A

The luzopeptin-d(C-A-T-G) complex (1 drug/duplex) has been generated in aqueous solution and its structure characterized by a combined application of two-dimensional NMR experiments and molecular dynamics calculations. One equivalent of luzopeptin binds to the self-complementary tetranucleotide duplex with the 2-fold symmetry of the antitumor agent and the DNA oligomer retained on complex formation. We have assigned the exchangeable and nonexchangeable proton resonances of luzopeptin and the d(C-A-T-G) duplex in the complex and identified the intermolecular proton-proton NOEs that define the alignment of the antitumor agent at its binding site in duplex DNA. The analysis was greatly aided by a large number of intermolecular NOEs involving exchangeable protons on both the luzopeptin and the DNA in the complex. The molecular dynamics calculations were guided by 140 intramolecular nucleic acid distance constraints, 74 intramolecular luzopeptin distance constraints, and 96 intermolecular distance constraints between luzopeptin and the nucleic acid protons in the complex. The quinoline rings of luzopeptin bisintercalate at d(C-A).d(T-G) steps in the d(C-A-T-G) duplex and sandwich two Watson-Crick A.T base pairs between the bisintercalation site. The long axis of the quinoline rings are collinear with the long axis of the flanking Watson-Crick C1.G4 and A2.T3 base pairs such that the OCH3-6 group is directed toward the C1-A2 step and the OH-3 group is directed toward the T3-G4 step in the complex. The quinoline chromophore stacks with purines on both strands, with the quinoline A ring stacked on A2 and the quinoline B ring stacked on G4 in the complex. The C1.G4 and A2.T3 base pairs that flank the intercalation sites are parallel to each other with partial overlap of T3 and G4 in the T3-G4 step but no overlap of C1 and A2 in the C1-A2 step in the complex. The cyclic depsipeptide ring of luzopeptin is positioned in the minor groove of the d(C-A-T-G) duplex with the oligopeptide and oligonucleotide chains running antiparallel to each other. The cyclic depsipeptide backbone of luzopeptin exhibits cis peptide bonds at Pyr-Gly and Gly-Sar steps in the luzopeptin-d(C-A-T-G) complex in solution, in contrast to all trans peptide bonds for free luzopeptin in the crystalline state.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of luzopeptin on the mobility of DNA in polyacrylamide gels has been determined. Experiments on a mixture of DNA fragments of various lengths have shown that the drug does not form intermolecular cross-links. Gel analysis of complexes of the drug with short DNA fragments (15-35 base pairs) reveals a ladder of discrete bands in which each band appears to correspond to the addition of a further drug molecule. The results suggest that luzopeptin binds very strongly to DNA, occupying about four base pairs and displays little or no sequence selectivity. Luzopeptin renders certain adenine residues hyperreactive to diethylpyrocarbonate, these occur in different positions to those affected by echinomycin.

We have examined the binding of luzopeptin, an antitumor antibiotic, to five DNA fragments of varying base composition. The drug forms a tight, possibly covalent, complex with the DNA causing a reduction in mobility on nondenaturing polyacrylamide gels and some smearing of the bands consistent with intramolecular cross-linking of DNA duplexes. DNAase I and micrococcal nuclease footprinting experiments suggest that the drug binds best to regions containing alternating A and T residues, although no consensus di- or trinucleotide sequence emerges. Binding to other sites is not excluded and at moderate ligand concentrations the DNA is almost totally protected from enzyme attack. Ligand-induced enhancement of DNAase I cleavage is observed at both AT and GC-rich regions. The sequence selectivity and characteristics of luzopeptin binding are quite different from those of echinomycin, a bifunctional intercalator of related structure.