Μεταλλικά σύμπλοκα του αντικαρκινικού αντιβιοτικού Altromycin B: in vitro δραστικότητα, αλληλεπίδραση με φυσικό DNA και νουκλεοτίδια

Abstract

This thesis concerns the interaction of the anticancer antibiotic altromycin B with Pt(II), Pd(II), Cu(II) and Fe(III), the interaction of the formed complexes with double stranded calf thymus DNA, the interaction of the Pd(II)-altromycin B and Pt(II)-altromycin B complexes with nucleotides and the biological evaluation of all altromycin B metal complexes. At first, a complete assignment of the dichroic bands of altromycin B was proposed. Altromycin B metal complexes were studied using 1H-NMR, UV-Vis electronic absorption, circular dichroism and E.P.R. spectroscopy. Altromycin B coordinates to Pt(II) and Pd(II) ions via the N atom of the dimethylamino group to the C(10) bound dissacharide in 1:1 stoichiometry. The carbonyl C(12)=O and phenolic C(11)-OH oxygens of altromycin B are the coordination sites for Cu(II) and Fe(III) ions and a six-member chelate ring is formed. The proposed stoichiometries are 2:1 for the Cu(II)-altromycin B complex and 3:1 for the Fe(III)-altromycin B complex which are both octahedral. The binding constant for [Cu(II)(altroB)2] has an intrinsic value of (7.3±2.1)x1012M-2 whereas for [Fe(III)(altroB)3] is (2.4±0.9)x1019M-3. The interaction of altromycin B and its metal complexes with double stranded calf thymus DNA was studied using UV- thermal denaturation experiments, circular dichroism spectroscopy, temperature controlled spectrophotometric titrations and indirect thermodynamic studies. Altromycin B stabilizes the double helix by raising the Tm, by intercalation of its chromophore between the base pairs and interacting electrostatically via its sugar moieties with the edges of the DNA helix. Moreover, altromycin B induces a BaA structural transition of C.T. DNA. The effect on DNA stability and conformation depends on the metal ion. Pt(II) and Pd(II) complexes induce the BaA structural transition and stabilize the double helix similarly but they present lower final hyperchromicity due to premelting effect which was caused by intra- and interstrand crosslinking. On the contrary, Cu(II) and Fe(III) complexes do not cause such an effect although they also stabilize the double helix. The Cu(II) and Fe(III) complexes prevent the BaA transition by non specific electrostatic interactions with the phosphates, shielding the overall negative repulsions. Thus, a synergistic effect (negative or positive) of the metal ions to the altromycin B-C.T. DNA interaction is observed in all cases. The interaction of altromycin B and its metal complexes with C.T. DNA is energetically favored at the temperature range 18-37oC. The interaction of altromycin B and its Pt(II), Pd(II) and Cu(II) complexes is entropically driven whereas the DNA interaction of Fe(III)-altromycin B complex is enthalpically driven. Altromycin B interacts with 5’GMP, 5’AMP and 5’CMP by electrophilic attack of the opened epoxide ring to the N(7)G, N(1)/N(7)A and N(3)C and covalent binding. The [Pd(II)-altroB] complex dissociates in presence of the nucleotides, and various species of Pd(II)-nucleotide complexes, especially with 5’GMP, is formed. The [Pt(II)-altroB] complex dissociates too, but only one or two species of Pt(II)-nucleotide complexes are formed, and in the case of 5’AMP interaction the formation of a tertiary altroB-Pt(II)-5’AMP complex is proposed. 5’-TMP does not react as strongly as the other three nucleotides. These interactions were followed by 1H-NMR These complexes were studied in vitro against K562 leukemia sensitive and doxorubicin-resistant cells and GLC4 lung tumor cells, sensitive and doxorubicin-resistant. The activity of the complexes compared to the free drug is improved against resistant cells and is affected moderately against sensitive cells. Finally, a 20% of platinum added as altromycin B metal complex entered GLC4 cells.