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

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 18h 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.

Bioactive pyrrole-based compounds with target selectivity

“…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).…”

Benzimidazole Scaffold as Anticancer Agent: Synthetic Approaches and Structure–Activity Relationship

“…Nayak et al reported compounds 597c and 597p which exhibited significant anti‐proliferative activity against MCF‐7 cell lines and induced mitochondrial mediated (intrinsic apoptotic pathway) apoptosis. Pett et al reported diamino polyamide 598a–c with an alkyl amino group at the N1 position of the imidazole/pyrrole ring and assayed for modulation of topo IIα and inhibition of NF‐Y binding. Chojnacka et al synthesized benzimidazole and benzotriazole derivatives containing (Scheme ) tetrazole moiety through N ‐alkylation of 5‐aryltetrazole with 4,5,6,7‐tetrabromo‐1‐(3‐chloropropyl)‐1 H ‐benzimidazole and 4,5,6,7‐tetrabromo‐2‐(3‐chloropropyl)‐2 H ‐benzotriazole in which compounds 601 and 603 showed most potent anticancer results against CCRF‐CEM and MCF‐7 cell lines.…”

“…Nayak et al[118] reported compounds 597c and 597p which exhibited significant anti-proliferative activity against MCF-7 cell lines and induced mitochondrial mediated (intrinsic apoptotic pathway) apoptosis. Pett et al[119] reported diamino polyamide 598a-c with an alkyl amino group at the N1 position of the imidazole/pyrrole ring and assayed for modulation of topo IIa and inhibition of NF-Y binding.Scheme 64. Synthesis of benzimidazole derivatives linked to five membered heterocyclic ring.…”

DNA‐Binding Properties of New Fluorescent AzaHx Amides: Methoxypyridylazabenzimidazolepyrroleimidazole/pyrrole

“…In a recent study, an extra positive charge was added to Hx‐IP at either P or I. These molecules were demonstrated to have not only higher binding affinity but also improved cell uptake in NIH3T3 mouse fibroblast cells and A549 cancer cells …”

“…These molecules have demonstrated to not only have higher binding affinity but also improved cell uptake in NIH3T3 mouse fibroblast cells and A549 cancer cells. [13]…”