Exploitable results
Molecular interactions of intoplicine, dual topo I and II inhibitor, with topos, plasmid DNA, in ternary cleavable complexes and in the reserved cleavable complexes were examined by means of SERS, CD Spectroscopy and by biochemical techniques. Intoplicine was found to be able to interact specifically with the topo II alone, but with topo I only when in the presence of DNA. It shows at least two modes of binding to the DNA: first, which was found to be dominant for its derivative - mist potent topo I inhibitor, and second - for the most potent topo II inhibitor. The possibility to form simultaneously these two types of complexes is suggested to be one of the main factors enabling the drug to be a dual topo I and topo II inhibitor. Direct interactions between the drug and topo II can play a critical role in the process of DNA recognition by the enzyme and topo II inhibition. The results suggest that the dual topo I and II inhibition, being critical for the antitumour activity of intoplicine, should not only be explained by effects induced at the DNA level, but by molecular interactions displayed by the drug in ternary and in reversed ternary complexes.
Differences of interactions of camptothecin (CPT) and its derivative CPT11, inhibitors of DNA topo I, with oligos derived from the sequences of the topo I-induced and CPT-enhanced cleavage sites were found by UV RR spectroscopy. CPT induces well-defined alterations of the oligos structure, whereas CPT11 interacts with oligos weaker and in the other manner as compared to CPT. Formation of the cleavable ternary complexes between CPT11, topo I and oligos turns over CPT11 to interact with oligos in the same fashion as was found for its parent compound CPT, and enhances this interaction as compared to the CPT-oligo complexes. The data present an evidence of molecular interactions of CPT11 with both partners (topo I and oligo) of the ternary cleavable complex.
Interactions of lactone, carboxylate, and J-type self-aggregated for of CPT with HSA and BSA, the principle mediators of transport and metabolism of medications, were studied by CD spectroscopy. HSA binding changes geometry of CPT covalent structure due to hydrophobic contacts of the chromophore within the protein interior and the carbonyl group of CPT interacts positively charged amino acid residues of HSA. Interaction with the HSA induces disaggregation of the aggregates of CPT. Analysis of HSA and BSA homology within the IIA and IIIA principle ligand-binding domains shows that the binding site for the CPT is located in the subdomain IIA of the HSA 3D-structure. Hydrophobic contacts with Leu203, Phe211, and Ala215 and electrostatic interactions with Lys199 and/or Arg222 of HSA may play a key role in stabilisation of the drug/protein complex.
An effect strongly influencing activity of some of the CPT drugs was found: formation of J-aggregates in a buffer solution. These aggregates were built up under certain dilution conditions of the stock DMSO solutions of 20(S)-CPT, 10, 11-CPT and SN38. They were formed by the stacking between quinoline rings of CPT chromophores with the inverse position of the nitrogen atoms. Self-aggregation prevents hydrolysis of the lactone ring at neutral pHs, thus preserving the drugs in a biologically active form. The aggregates were found to penetrate within the cells with much higher efficiency as compared with a monomeric drug. Cellular uptake correlated well with cytotoxic effects produced by the drug. In this manner, CPT's self-aggregation should be regarded as a favourable phenomenon producing species with more stable biologically active structure of the lactone ring and exhibiting enhanced cellular uptake levels relative to the monomeric forms of medications.
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