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1.
Bioorg Med Chem Lett ; 47: 128158, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34058343

RESUMO

Five X-HxIP (Hx-amides) 6a-e, in which the N-terminus p-anisyl moiety is modified, were designed and synthesised with the purpose of optimising DNA binding, improving cellular uptake/nuclear penetration, and enhancing the modulation of the topoisomerase IIα (TOP2A) gene expression. The modifications include a fluorophenyl group and other heterocycles bearing different molecular shapes, size, and polarity. Like their parent compound HxIP 3, all five X-HxIP analogues bind preferentially to their cognate sequence 5'-TACGAT-3', which is found embedded on the 5' flank of the inverted CCAAT box-2 (ICB2) site in the TOP2A gene promoter, and inhibit protein complex binding. Interestingly, the 4-pyridyl analog 6a exhibits greater binding affinity for the target DNA sequence and abolishes the protein:ICB2 interaction in vitro, at a lower concentration, compared to the prototypical compound HxIP 3. Analogues 6b-e, display improved DNA sequence specificity, but reduced binding affinity for the cognate sequence, relative to the unmodified HxIP 3, with polyamides 6b and 6e being the most sequence selective. However, unlike 3 and 6b, 6a was unable to enter cells, access the nucleus and thereby affect TOP2A gene expression in confluent human lung cancer cells. These results show that while DNA binding affinity and sequence selectivity are important, consideration of cellular uptake and concentration in the nucleus are critical when exerting biological activity is the desired outcome. By characterising the DNA binding, cellular uptake and gene regulatory properties of these small molecules, we can elucidate the determinants of the elicited biological activity, which can be impacted by even small structural modifications in the polyamide molecular design.


Assuntos
Amidas/farmacologia , DNA Topoisomerases Tipo II/genética , DNA de Neoplasias/efeitos dos fármacos , Proteínas de Ligação a Poli-ADP-Ribose/genética , Amidas/síntese química , Amidas/química , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular Tumoral , DNA Topoisomerases Tipo II/metabolismo , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Relação Estrutura-Atividade
2.
Chembiochem ; 19(18): 1979-1987, 2018 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-29974647

RESUMO

DNA minor groove binding polyamides have been extensively developed to control abnormal gene expression. The establishment of novel, inherently fluorescent 2-(p-anisyl)benzimidazole (Hx) amides has provided an alternative path for studying DNA binding in cells by direct observation of cell localization. Because of the 2:1 antiparallel stacking homodimer binding mode of these molecules to DNA, modification of Hx amides to 2-(p-anisyl)-4-azabenzimidazole (AzaHx) amides has successfully extended the DNA-recognition repertoire from central CG [recognized by Hx-I (I=N-methylimidazole)] to central GC [recognized by AzaHx-P (P=N-methylpyrrole)] recognition. For potential targeting of two consecutive GG bases, modification of the AzaHx moiety to 2- and 3-pyridyl-aza-benzimidazole (Pyr-AzaHx) moieties was explored. The newly designed molecules are also small-sized, fluorescent amides with the Pyr-AzaHx moiety connected to two conventional five-membered heterocycles. Complementary biophysical methods were performed to investigate the DNA-binding properties of these molecules. The results showed that neither 3-Pyr-AzaHx nor 2-Pyr-AzaHx was able to mimic I-I=N-methylimidazole-N-methylimidazole to target GG dinucleotides specifically. Rather, 3-Pyr-AzaHx was found to function like AzaHx, f-I (f=formamide), or P-I as an antiparallel stacked dimer. 3-Pyr-AzaHx-PI (2) binds 5'-ACGCGT'-3' with improved binding affinity and high sequence specificity in comparison to its parent molecule AzaHx-PI (1). However, 2-Pyr-AzaHx is detrimental to DNA binding because of an unfavorable steric clash upon stacking in the minor groove.


Assuntos
Benzimidazóis/química , DNA/química , Corantes Fluorescentes/química , Nylons/química , Pirróis/química , Sequência de Bases , Benzimidazóis/metabolismo , Sítios de Ligação , Dicroísmo Circular , DNA/metabolismo , Corantes Fluorescentes/metabolismo , Conformação de Ácido Nucleico , Nylons/metabolismo , Pirróis/metabolismo , Ressonância de Plasmônio de Superfície
3.
Bioorg Med Chem Lett ; 25(17): 3681-5, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26122210

RESUMO

The design, synthesis, and DNA binding properties of azaHx-PI or p-anisyl-4-aza-benzimidazole-pyrrole-imidazole (5) are described. AzaHx, 2-(p-anisyl)-4-aza-benzimidazole-5-carboxamide, is a novel, fluorescent DNA recognition element, derived from Hoechst 33258 to recognize G·C base pairs. Supported by theoretical data, the results from DNase I footprinting, CD, ΔT(M), and SPR studies provided evidence that an azaHx/IP pairing, formed from antiparallel stacking of two azaHx-PI molecules in a side-by-side manner in the minor groove, selectively recognized a C-G doublet. AzaHx-PI was found to target 5'-ACGCGT-3', the Mlu1 Cell Cycle Box (MCB) promoter sequence with specificity and significant affinity (K(eq) 4.0±0.2×10(7) M(-1)).


Assuntos
Benzimidazóis/química , DNA/metabolismo , Corantes Fluorescentes/química , Nylons/química , Pirróis/química , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Pareamento de Bases , Benzimidazóis/síntese química , Benzimidazóis/metabolismo , Sítios de Ligação , Técnicas de Química Sintética , Dicroísmo Circular , DNA/química , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease I/química , Desenho de Fármacos , Corantes Fluorescentes/metabolismo , Nylons/síntese química , Regiões Promotoras Genéticas , Pirróis/síntese química , Pirróis/metabolismo
4.
Bioorg Med Chem ; 21(20): 6162-70, 2013 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-23769166

RESUMO

The synthesis together with biophysical and biological evaluation of a series of tetra-substituted naphthalene diimide (ND) compounds, are presented. These compounds are positional isomers of a recently-described series of quadruplex-binding ND derivatives, in which the two N-methyl-piperidine-alkyl side-chains have now been interchanged with the positions of side-chains bearing a range of end-groups. Molecular dynamics simulations of a pair of positional isomers are in accord with the quadruplex stabilization and biological data for these compounds. Analysis of structure-activity data indicates that for compounds where the side-chains are not of equivalent length then the positional isomers described here tend to have improved cell proliferation potency and in some instances, superior quadruplex stabilization ability.


Assuntos
Quadruplex G , Imidas/química , Imidas/farmacologia , Naftalenos/química , Naftalenos/farmacologia , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Imidas/síntese química , Isomerismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Estrutura Molecular , Naftalenos/síntese química , Relação Estrutura-Atividade
5.
Biochim Biophys Acta Gene Regul Mech ; 1860(5): 617-629, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-27750031

RESUMO

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 (0.1-2µM) 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.


Assuntos
Antígenos de Neoplasias/biossíntese , Fator de Ligação a CCAAT/metabolismo , Dano ao DNA , DNA Topoisomerases Tipo II/biossíntese , Proteínas de Ligação a DNA/biossíntese , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Nylons/farmacologia , Regiões Promotoras Genéticas , Células A549 , Animais , Antígenos de Neoplasias/genética , Fator de Ligação a CCAAT/genética , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/genética , Etoposídeo/efeitos adversos , Etoposídeo/farmacologia , Regulação Enzimológica da Expressão Gênica/genética , Camundongos , Células NIH 3T3 , Nylons/química
6.
Curr Top Med Chem ; 15(14): 1293-322, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25866278

RESUMO

DNA interactive agents have been used in the clinical setting for the treatment of cancer since the beginning of modern-era chemotherapy. Despite a shift of focus towards molecular targeted therapy, DNA remains a critical macromolecular target for anti-cancer intervention and the next generation of agents must conform to the optimum combination of increased therapeutic activity and reduced off-target toxicity. We evaluate the potential of non-covalent DNA binding small molecules as "gene-control" agents, exploiting inherent or engineered sequence selectivity, to target critical genomic sequences. In addition we review examples of natural products and synthetic derivatives that exert their activity through sequence specific DNA-covalent modification.


Assuntos
Sítios de Ligação/genética , Produtos Biológicos/farmacologia , DNA de Neoplasias/efeitos dos fármacos , DNA de Neoplasias/genética , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/genética , Sequência de Bases , Sítios de Ligação/efeitos dos fármacos , Produtos Biológicos/síntese química , Produtos Biológicos/química , Humanos , Especificidade por Substrato
7.
Chem Biol ; 22(7): 862-75, 2015 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-26119998

RESUMO

Synthetic pyrrole (P)-imidazole (I) containing polyamides can target predetermined DNA sequences and modulate gene expression by interfering with transcription factor binding. We have previously shown that rationally designed polyamides targeting the inverted CCAAT box 2 (ICB2) of the topoisomerase IIα (topo IIα) promoter can inhibit binding of transcription factor NF-Y, re-inducing expression of the enzyme in confluent cells. Here, the A/T recognizing fluorophore, p-anisylbenzimidazolecarboxamido (Hx) was incorporated into the hybrid polyamide HxIP, which fluoresces upon binding to DNA, providing an intrinsic probe to monitor cellular uptake. HxIP targets the 5'-TACGAT-3' sequence of the 5' flank of ICB2 with high affinity and sequence specificity, eliciting an ICB2-selective inhibition/displacement of NF-Y. HxIP is readily taken up by NIH3T3 and A549 cells, and detected in the nucleus within minutes. Exposure to the polyamide at confluence resulted in a dose-dependent upregulation of topo IIα expression and enhanced formation of etoposide-induced DNA strand breaks.


Assuntos
Sondas de DNA/farmacologia , Corantes Fluorescentes/farmacologia , Nylons/farmacologia , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Sondas de DNA/genética , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Expressão Gênica/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Camundongos , Células NIH 3T3 , Sinais de Localização Nuclear/efeitos dos fármacos , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Pirróis/farmacologia
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