Abstract
Amsacrine is an anilinoacridine derivative anticancer drug, used to treat a wide variety of malignancies. In cells, amsacrine poisons topoisomerase 2 by stabilizing DNA-drug-enzyme ternary complex. Presence of amsacrine increases the steady-state concentration of these ternary complexes which in turn hampers DNA replication and results in subsequent cell death. Due to reversible binding and rapid slip-out of amsacrine from DNA duplex, structural data is not available on amsacrine-DNA complexes. In the present work, we designed five oligonucleotide duplexes, differing in their minor groove widths and hydration pattern, and examined their binding with amsacrine using attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. Complexes of amsacrine with calf thymus DNA were also evaluated for a comparison. Our results demonstrate for the first time that amsacrine is not a simple intercalator; rather mixed type of DNA binding (intercalation and minor groove) takes place between amsacrine and DNA. Further, this binding is highly sensitive towards the geometries and hydration patterns of different minor grooves present in the DNA. This study shows that ligand binding to DNA could be very sensitive to DNA base composition and DNA groove structures. Results demonstrated here could have implication for understanding cytotoxic mechanism of aminoacridine based anticancer drugs and provide directions to modify these drugs for better efficacy and few side-effects.
Highlights
Topoisomerase enzymes are ubiquitous in nature because they play important task of regulation of superhelicity of DNA
Five dodecamer oligonucleotide duplexes having varied base sequence were designed: (AAAAAAAAAAAA)2, (ATATATATATAT)2, (TTAATTAATTAA)2, (AGAGCAACAGAG)2, and (AGAGACCAGAGA)2. These duplexes and Calf thymus DNA (ctDNA) were incubated with different molar ratios of amsacrine to obtain their complexes
Binding preference of amsacrine for (ATATATATATAT)2 and (AAAAAAAAAAAA)2 duplexes is apparent from these ATRFTIR spectroscopic results
Summary
In its course of action, topoisomerase 2 creates double strand breaks in opposite strands of DNA, passes an intact segment of DNA from this transient opening and reseals these openings in DNA This process, according to need of cell, either decatenates or changes topological number of DNA by 62 [4,5,6,7]. Topoisomerase 2 wrapped DNA fragments are short lived and generally termed as cleavable complex [8] Stabilization of these cleavable complexes by different topoisomerase 2 poisons including amsacrine though, results in their accumulation in the cell. These stabilized ternary complexes act as replication hurdles and stall the movement of replication machinery [9]. The critical phenomenon behind the poisoning of topoisomerase 2 by different antitumor agents is stabilization of the cleavable complexes [10]
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