NMR studies of the anticancer drug pepleomycin and its complexes with DNA

Pepleomycin (PEP) is a metalloglycopeptide that has stronger anticancer activity and less pulmonary toxicity than bleomycin (BLM). PEP, like BLM, exerts its action by binding to and degrading DNA in the presence of oxygen and certain metals. Two cobalt-PEP ;species, HO2-Co(III)-PEP and its deglycosylated HO2-Co(III)-dPEP, have been studied by 2D-NMR and NOE refined structures were obtained. Two chiral conformers (form A or B) can exist for these molecules with either the beta-aminoalanine primary amine (A,NH2) or the mannose carbamoyl nitrogen (M,NH2) as the axial ligand. Analysis of our NOESY data shows convincingly that form A is the most probable conformer with the mannose carbamoyl M,NH, and the beta-aminoalanine primary amine A,NH2 as the axial ligands in CoPEP and CodPEP, respectively. The NOE crosspeaks resulting from the interactions between the N-terminus (i.e., the metal binding domain) and the C-terminus of CoPEP and CodPEP have similar patterns, suggesting that they both adopt compact structures with the bithiazole group folded back over the N-terminus. We have treated the self-complementary oligonucleotide d(CCrTACG)(2) with one equivalent of CoPEP or CodPEP and carefully examined these complexes - denoted CGTACG-CoPEP and CGTACG-CodPEP, respectively - by one- and two- dimensional H-1 NMR. Refined solution structures of CGTACG-CoPEP and CGTACG-CodPEP have been obtained by NOE restrained refinement procedures. Addition of CoPEP or CodPEP to d(CGTACG)(2) disrupts the two-fold symmetry of the DNA duplex, consistent with binding of the drugs to the DNA duplex. The binding mode of both CoPEP and CodPEP with respect to the DNA duplex is defined by approximately 60 intermolecular NOEs. The NOE data is consistent with a partial intercalative binding mode for both DNA-drug complexes with the drugs' metal-binding domain and peptide linker bound in the minor groove of the DNA close to residues G8 and T9, and the drug's bithiazole tail intercalated between the base pairs A4.T9 and T3.Al0. A structural comparison between CGTACG-CoPEP and CGTACG-CodPEP is made.