Drug recognition and stabilisation of the parallel-stranded DNA quadruplex d(TTAGGGT)4 containing the human telomeric repeat

被引:169
作者
Gavathiotis, E
Heald, RA
Stevens, MFG
Searle, MS
机构
[1] Sch Chem, Nottingham NG7 2RD, England
[2] Sch Pharmaceut Sci, Nottingham NG7 2RD, England
关键词
quadruplex DNA; quinoacridinium cation; telomerase inhibition; NMR spectroscopy; molecular dynamics;
D O I
10.1016/j.jmb.2003.09.018
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The NMR structure of the parallel-stranded DNA quadruplex d(TTAGGGT)(4), containing the human telomeric repeat, has been determined in solution in complex with a fluorinated pentacyclic quino[4,3,2-kl]acri-2 dinium cation (RHPS4). RHPS4 has been identified as a potent inhibitor of telomerase at submicromolar levels (IC50 value of 0.33 (+/- 0.13) muM), exhibiting a wide differential between telomerase inhibition and acute cellular toxicity. All of the data point to RHPS4 exerting its chemotherapeutic potency through interaction with, and stabilisation of, four-stranded G-quadruplex structures. RHPS4 forms a dynamic interaction with d(TTAGGGT)(4), as evident from H-1 and F-19 linewidths, with fast exchange between binding sites induced at 318 K. Perturbations to DNA chemical shifts and 24 intermolecular nuclear Overhauser effects (NOEs) identify the 5'-ApG and 5'-GpT steps as the principle intercalation sites; a structural model has been refined using NOE-restrained molecular dynamics. The central G-tetrad core remains intact, with drug molecules stacking at the ends of the G-quadruplex. The partial positive charge on position 13-N of the acridine ring appears to act as a "pseudo" potassium ion and is positioned above the centre of the G-tetrad in the region of high negative charge density. In both ApG and GpT intercalation sites, the drug is seen to converge to the same orientation in which the pi-system of the drug overlaps primarily with two bases of each G-tetrad. The drug is held in place by stacking interactions with the G-tetrads; however, there is some evidence for a more dynamic, weakly stabilised A-tetrad that stacks partially on top of the drug at the 5'-end of the sequence. Together, the interactions of RHPS4 increase the t(m) of the quadruplex by similar to20 degreesC. There is no evidence for drug intercalation within the G-quadruplex; however, the structural model strongly supports end-stacking interactions with the terminal G-tetrads. (C) 2003 Elsevier Ltd. All rights reserved.
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页码:25 / 36
页数:12
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