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

Highlights

• Polyamide N1 functionalisation offers a strategy to improve drug-like properties.

• Diamino Hx-polyamides contain an N1-alkyl amino group on the heterocyclic rings.

• Incorporating an additional cationic group enhances solubility and cellular uptake.

• Targeted inhibition of NF-Y:DNA binding by HxIP* re-stimulated topo IIα expression.

• Pharmacological modulation of topo IIα increases chemosensitivity to topo II poisons.

Abstract

Background

Sequence specific polyamide HxIP 1, targeted to the inverted CCAAT Box 2 (ICB2) on the topoisomerase IIα (topo IIα) promoter can inhibit NF-Y binding, re-induce gene expression and increase sensitivity to etoposide. To enhance biological activity, diamino-containing derivatives (HxI*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 IIα mRNA (RT-PCR) and protein (Immunoblotting) levels were measured following 18 h polyamide treatment of confluent A549 cells. γH2AX was used as a marker for etoposide-induced DNA damage after pre-treatment with HxIP* 3 and cell viability was measured using Cell-Titer Glo®.

Results

Introduction of the N1-alkyl amino group reduced selectivity for the target sequence 5′-TACGAT-3′ on the topo IIα promoter, but increased DNA binding affinity. Confocal microscopy revealed both fluorescent diamino polyamides localised in the nucleus, yet HxI*P 2 was unable to disrupt the NF-Y:ICB2 interaction and showed no effect against the downregulation of topo IIα. In contrast, inhibition of NF-Y binding by HxIP* 3 stimulated dose-dependent (0.1–2 μM) re-induction of topo IIα 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 IIα expression and chemosensitivity to topo II-targeting agents.

General significance

Pharmacological modulation of topo IIα 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.

Introduction

Gene expression is precisely regulated by the binding of the transcription machinery to specific DNA sequences. Dysregulation of transcriptional activity leading to aberrant gene expression is a fundamental driver of a diverse array of human diseases. Sequence selective (P) pyrrole-(I) imidazole polyamides are able to modulate gene expression through binding non-covalently to specific DNA sequences and disrupting the DNA interactions of transcription factors. These reversible DNA minor groove binders can arrange in a stacked, antiparallel 2:1 (ligand:DNA) orientation and afford programmable sequence recognition, governed by the side-by-side heterocyclic ring pairing rules [1], [2], [3], [4]. A P/P pairing degenerately targets A•T or T•A, whereas P/I recognises C•G and I/P preferentially binds to G•C. The reported anti-cancer biological activity of these cell permeable small molecules in both cellular and in vivo studies has highlighted the potential of therapeutic strategies that directly target transcription factor-DNA interfaces known to be implicated in certain malignant phenotypes [5], [6], [7], [8].

Nuclear Factor Y (NF-Y) is a heterotrimeric CCAAT-binding transcription factor involved in cell differentiation, proliferation and implicated in cancer progression [9], [10], [11], [12], [13]. NF-Y has been shown to bind to the promoter of the essential DNA processing enzyme, topoisomerase IIα (topo IIα) and regulates its transcription through interactions with the inverted CCAAT box (ICB) sequences located within the promoter [14], [15], [16]. Topo IIα plays a critical role in DNA metabolism, maintaining genomic stability [17], and is the target of clinically-used chemotherapeutic agents etoposide and doxorubicin [18], with low levels of topo IIα conferring cellular resistance to these anticancer agents [19], [20], [21], [22]. NF-Y acts as both an activator and repressor of topo IIα transcription with increased association of NF-Y to the promoter exerting a negative effect at confluence [15], [16]. The ICB2 has been identified as the crucial DNA regulatory element and its interaction with NF-Y (Fig. 1) mediates the confluence-induced downregulation of topo IIα and reduced chemosensitivity to topo II targeting therapeutics.

Chemical approaches that re-induce topo IIα expression have the potential to increase cellular sensitivity to topo II poisons and to this end, our group has used various DNA binding small molecules and polyamides to inhibit the repressive activity of NF-Y on the topo IIα promoter [16], [23], [24], [25], [26], [27]. Most recently, we reported the synthesis and biological activity of a novel polyamide incorporating the p-anisylbenzimidazole (Hx) DNA recognition element [28], [29]. Designed to enhance polyamide-DNA binding and cellular uptake, the Hx moiety exhibits intrinsic fluorescence upon binding DNA enabling the direct visualisation of polyamide nuclear localisation. Hx-polyamide HxIP (1, Fig. 2A) designed to target the 5′-flanking sequence 5′-TACGAT-3′ of the ICB2 (Fig. 1), binds to DNA with high affinity and sequence selectivity, and disrupts the NF-Y:ICB2 interaction resulting in the upregulation of topo IIα expression at confluence. HxIP pre-treatment enhanced etoposide-induced DNA damage, providing further evidence that sequence specific polyamides can re-sensitise confluence-arrested cancer cells to topo IIα poisons [28].

The development of HxIP provides a new framework for the design of fluorescent sequence selective DNA binding molecules, distinct in configuration from the prototypical hairpin polyamide, yet capable of efficient nuclear localisation and in vitro gene regulation. In parallel, we have continued to explore an alternative strategy for further optimisation of polyamide physicochemical and DNA binding properties, through the introduction of an additional alkyl amino group at the N1 position of the heterocyclic rings [30], [31], [32], [33]. DNA binding studies by our groups revealed the small diamino polyamide containing an orthogonal positioned propyl amino group, f-IP*I (*denotes modified heterocycle) to have greater binding affinity than its monoamino counterpart and analogous sequence selectivity. Importantly, the inclusion of an extra amino group, which is cationic at physiological pH, also increases water solubility and may afford greater polyamide nuclear uptake. We aim to exploit this potential combination of improved DNA binding and solubility properties to engineer a potentially more potent generation of dicationic polyamides.

This study presents the DNA binding and biological activities of the diamino Hx-polyamides HxI*P 2 and HxIP* 3, which incorporate the N1-alkyl amino group modification (Fig. 2A). Polyamides 2 and 3 are functionalised derivatives of HxIP 1 and like their monoamino predecessor are designed to target the ICB2 5′-flanking sequence 5′-TACGAT-3′ on the topo IIα promoter (Fig. 2B) and disrupt NF-Y binding, inducing topo IIα expression at confluence. Comparison with the monoamino HxIP 1 will reveal the effect the inclusion of an N1-alkyl amino group in the Hx-framework has on DNA binding and the feasibility of using functionalisation of the N1 position to enhance polyamide cellular uptake and biological activity. Additionally, we shall assess the potential chemosensitising effects of the diamino generation of polyamide inhibitors of NF-Y:DNA binding and the effectiveness of pharmacological modulation of topo IIα expression as a strategy for overcoming the drug resistance exhibited by confluence-arrested cells.