Modulation of topoisomerase II alpha expression and chemosensitivity through targeted inhibition of NF-Y:DNA binding by a diamino p-anisyl-benzimidazole (Hx) polyamide

作者：Pett Luke; Kiakos Konstantinos; Satam Vijay; Patil Pravin; Laughlin Toth Sarah; Gregory Matthew; Bowerman Michael; Olson Kevin; Savagian Mia; Lee Megan; Lee Moses; Wilson W David; Hochhauser Daniel; Hartley John A^*

来源：Biochimica et Biophysica Acta-Gene Regulatory Mechanisms, 2017, 1860(5): 617-629.

DOI：10.1016/j.bbagrm.2016.10.005

• DNA-binding polyamides
• Sequence selectivity
• Transcription factor-DNA interactions
• Gene modulation
• NF-Y
• Topoisomerase II alpha (topo II alpha)
• Chemosensitisation

Background: Sequence specific polyamide HxIP 1, targeted to the inverted CCAAT Box 2 (ICB2) on the topoisomerase lice (topo lice) promoter can inhibit NF-Y binding, re-induce gene expression and increase sensitivity to etoposide. To enhance biological activity, diamino-containing derivatives (Hxl*P 2 and HxIP* 3) were synthesised incorporating an alkyl amino group at the N1-heterocyclic position of the imidazole/pyrrole.
Methods: DNase I footprinting was used to evaluate DNA binding of the diamino Hx-polyamides, and their ability to disrupt the NF-Y:ICB2 interaction assessed using EMSAs. Topo Ila mRNA (RT-PCR) and protein (Immunoblotting) levels were measured following 18 h polyamide treatment of confluent A549 cells.gamma H2AX was used as a marker for etoposide-induced DNA damage after pre-treatment with HxIP* 3 and cell viability was measured using Cell-Titer GlaD.
Results: Introduction of the N1-alkyl amino group reduced selectivity for the target sequence 5'-TACGAT-3' on the topo lice promoter, but increased DNA binding affinity. Confocal microscopy revealed both fluorescent diamino polyamides localised in the nucleus, yet Hxl*P 2 was unable to disrupt the NF-Y:ICB2 interaction and showed no effect against the downregulation of topo IIa. In contrast, inhibition of NF-Y binding by HxIP* 3 stimulated dose-dependent (0.1-2 mu M) re-induction of topo Ila and potentiated cytotoxicity of topo II poisons by enhancing DNA damage.
Conclusions: Polyamide functionalisation at the N1-position offers a design strategy to improve drug-like properties. Dicationic HxIP* 3 increased topo Hce expression and chemosensitivity to topo II-targeting agents. General significance: Pharmacological modulation of topo Ila expression has the potential to enhance cellular sensitivity to clinically-used anticancer therapeutics. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani.

• 出版日期2017-5