Abstract
Camptothecin (CPT) is a topoisomerase IB (TopIB) selective inhibitor whose derivatives are currently used in cancer therapy. TopIB cleaves DNA at any sequence, but in the presence of CPT the only stabilized protein–DNA covalent complex is the one having a thymine in position −1 with respect to the cleavage site. A metadynamics simulation of two TopIB–DNA–CPT ternary complexes differing for the presence of a thymine or a cytosine in position −1 indicates the occurrence of two different drug’s unbinding pathways. The free-energy difference between the bound state and the transition state is large when a thymine is present in position −1 and is strongly reduced in presence of a cytosine, in line with the different drug stabilization properties of the two systems. Such a difference is strictly related to the changes in the hydrogen bond network between the protein, the DNA and the drug in the two systems, indicating a direct role of the protein in determining the specificity of the cleavage site sequence stabilized by the CPT. Calculations carried out in presence of one compound of the indenoisoquinoline family (NSC314622) indicate a comparable energy difference between the bound and the transition state independently of the presence of a thymine or a cytosine in position −1, in line with the experimental results.
Highlights
Topoisomerase IB (TopIB) is an ubiquitous enzyme belonging to the class of DNA topoisomerases, a family of protein involved in the regulation of DNA topology that has a crucial role in removing DNA supercoiling occurring during replication and transcription [1,2]
In this work we present two metadynamics simulations of the ternary TopIB–DNA–CPT complex, following the CPT unbinding in two different situations, namely when in position À1 of the cleavage site a thymine or a cytosine is present
Our work shows that when a thymine is present in position À1 of the TopIB cleavage site (TG system) the hydrogen bond (H-bond) network between the protein and DNA forces CPT to leave its binding site crossing the DNA helix, an event that implies an high energetic cost, requiring the enlargement of bases at the cleavage site to permit the passage of the ethyl group on the CPT E-ring
Summary
Topoisomerase IB (TopIB) is an ubiquitous enzyme belonging to the class of DNA topoisomerases, a family of protein involved in the regulation of DNA topology that has a crucial role in removing DNA supercoiling occurring during replication and transcription [1,2]. TopIB catalytic cycle is conventionally divided in five steps: (i) DNA binding, (ii) DNA cleavage, (iii) DNA relaxation, (iv) DNA religation and (v) DNA unbinding. TopIB cleaves one strand of a double-stranded DNA through a nucleophilic attack operated by the Tyr723 residue resulting in a DNA-30-phosho-tyrosine covalent bond. After the energy equilibrium of supercoiled DNA is reached through modification of the DNA linking number, a second nucleophilic attack by the O50 DNA end restores an intact double strand and the enzyme is released. CPT does not bind to DNA or TopIB alone but it selectively interacts with the TopIB–DNA covalent complex, stabilizing it, intercalating between the base pairs in position À1 and +1 with respect to the cleavage site, slowing down the DNA relaxation and religation. The stabilization of the TopIB–DNA complex leads, in highly proliferating cell, to DNA damage as a consequence of a clash with the DNA replication machinery, eventually triggering the apoptotic pathways in cancer cells
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