Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms
Modulation of topoisomerase IIα expression and chemosensitivity through targeted inhibition of NF-Y:DNA binding by a diamino p-anisyl-benzimidazole (Hx) polyamide☆
Graphical abstract
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.
Section snippets
Synthesis of polyamides
Details of the synthesis and characterisation of diamino Hx-polyamides 2 and 3 are provided in the Supplemental materials and methods (Scheme S1).
Thermal denaturation studies
Thermal denaturation (ΔTM) studies were performed using a Cary Bio 100 spectrophotometer UV–Vis instrument (Palo Alto, CA) as previously described by Chavda et al. [29]. DNA oligomers were purchased from Operon and the sequences are provided in the Supplemental materials. Experiments for diamino Hx-polyamides 2 and 3 were performed at a concentration
DNA binding affinity and sequence selectivity of diamino Hx-polyamides 2 and 3
The DNA binding properties of the diamino Hx-polyamides 2 and 3 were investigated to assess the effect of introducing an alkyl amino group at the heterocyclic N1 position. Thermal denaturation analysis probed the binding affinity and selectivity of polyamides 2 and 3 by measuring their ability to stabilise duplex DNA. The Hx recognition element behaves similarly to two consecutive pyrrole units and as a result, polyamides 1, 2 and 3 can degenerately bind sequences 5′-ATCGAT-3′ and 5′-TACGAT-3′,
Discussion
DNA binding polyamides with programmed sequence recognition are able to chemically control transcription and their gene regulatory activities have been confirmed in various biological contexts, targeting a range of transcription factors such as nuclear hormone receptors [6], [8], hypoxia-inducible factor 1 (HIF-1) [38], nuclear factor κβ (NF-κβ) [7] and c-Myc [39]. Nevertheless, design strategies that further enhance polyamide activity are still required in order to realise the therapeutic
Conclusion
Polyamide functionalisation via the N1 position of the pyrrole and imidazole rings in the Hx-framework presents a promising approach to improving the drug-like properties of these small molecules. The introduction of an additional cationic alkyl amino functionality enhances water solubility and facilitates the nuclear uptake of the diamino polyamides 2 and 3. However, the N1-alkyl amino group had a detrimental effect on DNA sequence selectivity, and binding affinity for the target sequence
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Acknowledgements
This work was supported by a program grant from Cancer Research UK (C2259/A16569 to J.A.H and D.H), a Medical Research Council Doctoral Training Grant awarded to UCL (L.P) and a grant from the National Science Foundation (CHE 0809162 to M.L and W.D.W). The biosensor-SPR work was supported by the National Institutes of Health for W.D.W (GM 111749).
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2020, European Journal of Medicinal ChemistryCitation Excerpt :The interaction between NF–Y and ICB2 is linked to the confluence-induced downregulation of topo-IIα and the consequential resistance to topo-IIα targeting drugs. In 2017, Pett et al. [43] designed and synthesized pyrrole-polyamides, incorporating the p-anisylbenzimidazole (Hx) DNA recognition element, to target the 5′-flanking sequence 5′-TACGAT-3′ of the ICB2, and disrupt the interaction between NF–Y and ICB2. Compound 19a was shown to display a dose-dependent inhibition of NF–Y binding to the ICB2 at doses ≥3 μM using an electrophoretic mobility shift assay (EMSA).
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This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani.
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Current address: University of Vienna, Research Platform “Translational Cancer Therapy Research”, Waehringer Str. 42, A-1090 Vienna, Austria.