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1.
Chemistry ; 29(39): e202301181, 2023 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-37115041

RESUMO

The structural determinants of the interaction of the G-quadruplex (G4) motif found in precursor miRNA 149 (rG4) with the acridine orange derivative C8 , a G4 ligand stabilizer possessing anticancer activity, and the protein nucleolin (overexpressed in cancer cells) were investigated by Nuclear Magnetic Resonance (NMR) spectroscopy. For the rG4/C8 complex, the results revealed a strong stabilizing interaction between the aromatic core and the iodinated ring of the C8 ligand with the rG4 structure. The NMR study revealed also different interaction patterns between nucleolin and rG4 and nucleolin and rG4/C8 complex. In the absence of the ligand, rG4 establishes interactions with polar residues of the protein while for the rG4/C8 complex, these contacts are mainly established with amino acids that have hydrophobic side chains. However, nucleolin chemical shift perturbation studies in the presence of rG4 or rG4/C8 reveal the same location between domains 1 and 2 of the protein, which suggests that the rG4 and rG4/C8 complex bind in this region. This puzzling structural study opens a new framework to study rG4/ligand/nucleolin complexes that might impact the biogenesis of miRNA 149.


Assuntos
Quadruplex G , MicroRNAs , Humanos , Ligantes , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Carcinogênese , Nucleolina
2.
Int J Mol Sci ; 23(21)2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-36361696

RESUMO

MST1R (RON) is a receptor of the MET tyrosine kinase receptor family involved in several cancers such as pancreas, breast, ovary, colon, and stomach. Some studies have shown that overexpression of MST1R increases the migratory and invasive properties of cancer cells. The promoter region of the oncogene MST1R is enriched in guanine residues that can potentially form G-quadruplexes (G4s), as it was observed in other oncogenic promoters such as KRAS and c-MYC. There is abundant literature that links the presence of G4s in promoter regions of oncogenes to diverse gene regulation processes that are not well understood. In this work, we have studied the reverse and forward sequence of MST1R promoter region using the G4Hunter software and performed biophysical studies to characterize the best scored sequences.


Assuntos
Quadruplex G , Regiões Promotoras Genéticas , Guanina/química , Receptores Proteína Tirosina Quinases/metabolismo , Proto-Oncogenes
3.
J Pharm Biomed Anal ; 215: 114757, 2022 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-35462282

RESUMO

Let-7e precursor microRNA has the potential to adopt a G-quadruplex (rG4) structure and recently, its roles in oncology have been the focus of much attention, as it is now known that let-7e pre-miRNA is frequently dysregulated in cancers. Therefore, it is crucial to unveil and fully characterize its ability to adopt a rG4 structure, which could be stabilized or destabilized by small molecules and proteins such as nucleolin, a protein that is deeply associated with miRNA biogenesis. Herein, by combining a set of different methods such as circular dichroism (CD), nuclear magnetic resonance (NMR), UV spectroscopy (thermal difference spectra (TDS) and isothermal difference spectra (IDS)) and polyacrylamide gel electrophoresis (PAGE), we demonstrate the formation of the rG4 structure found in let-7e pre-miRNA sequence in the presence of K+ (5'-GGGCUGAGGUAGGAGG-3'). The ability of eight small molecules (or ligands) to bind to and stabilize this rG4 structure was also fully assessed. The dissociation constants for each RNA G-quadruplex/ligand complex, determined by surface plasmon resonance (SPR), ranged in the 10-6 to 10-9 M range. Lastly, the binding of the rG4 structure to nucleolin in the presence and absence of ligands was evaluated via CD, SPR, PAGE and confocal microscopy. The small molecules 360 A and PDS demonstrated attractive properties to targetthe rG4 structure of let-7e pre-miRNA and control its biology. Our findings also highlighted that the interaction of TMPyP4 with the G-quadruplex of let-7e precursor miRNA could block the formation of the complex between the rG4 and nucleolin. Overall, this study introduces an approach to target the rG4 found in let-7e pre-miRNA which opens up a new opportunity to control the microRNA biogenesis.


Assuntos
Quadruplex G , MicroRNAs , Ligantes , MicroRNAs/metabolismo , Fosfoproteínas , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Nucleolina
4.
Trends Cell Biol ; 32(7): 561-564, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35410819

RESUMO

Nucleolin protein is involved in a plethora of cellular pathways across the nucleolus, nucleus, and cytoplasm. The association of its RNA-binding domain (RBD) and its RGG (arginine-glycine-glycine-rich) domain allows it to interact with G-quadruplex structures in nucleic acids. We highlight evidence that the nucleolin/G-quadruplex partnership is of extensive relevance to neurodegenerative disease, cancer, and viral infections.


Assuntos
Quadruplex G , Fosfoproteínas , Proteínas de Ligação a RNA , Humanos , Neoplasias , Doenças Neurodegenerativas , Fosfoproteínas/metabolismo , Ligação Proteica , Proteínas de Ligação a RNA/metabolismo , Viroses , Nucleolina
5.
ACS Omega ; 6(49): 34092-34106, 2021 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-34926957

RESUMO

Recent studies have proven that the genetic landscape of pancreatic cancer is dominated by the KRAS oncogene. Its transcription is controlled by a G-rich motif (called 32R) located immediately upstream of the TSS. 32R may fold into a G-quadruplex (G4) in equilibrium between two G4 conformers: G9T (T M = 61.2 °C) and G25T (T M = 54.7 °C). We found that both G4s bind to hnRNPA1 and its proteolytic fragment UP1, promoting several contacts with the RRM protein domains. 1D NMR analysis of DNA imino protons shows that, upon binding to UP1, G25T is readily unfolded at both 5' and 3' tetrads, while G9T is only partially unfolded. The impact of hnRNPA1 on KRAS expression was determined by comparing Panc-1 cells with two Panc-1 knockout cell lines in which hnRNPA1 was deleted by the CRISPR/Cas9 technology. The results showed that the expression of KRAS is inhibited in the knockout cell lines, indicating that hnRNPA1 is essential for the transcription of KRAS. In addition, the knockout cell lines, compared to normal Panc-1 cells, show a dramatic decrease in cell growth and capacity of colony formation. Pull-down and Western blot experiments indicate that conformer G25T is a better platform than conformer G9T for the assembly of the transcription preinitiation complex with PARP1, Ku70, MAZ, and hnRNPA1. Together, our data prove that hnRNPA1, being a key transcription factor for the activation of KRAS, can be a new therapeutic target for the rational design of anticancer strategies.

6.
Pharmaceuticals (Basel) ; 14(8)2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34451866

RESUMO

Progress in the design of G-quadruplex (G4) binding ligands relies on the availability of approaches that assess the binding mode and nature of the interactions between G4 forming sequences and their putative ligands. The experimental approaches used to characterize G4/ligand interactions can be categorized into structure-based methods (circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography), affinity and apparent affinity-based methods (surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and mass spectrometry (MS)), and high-throughput methods (fluorescence resonance energy transfer (FRET)-melting, G4-fluorescent intercalator displacement assay (G4-FID), affinity chromatography and microarrays. Each method has unique advantages and drawbacks, which makes it essential to select the ideal strategies for the biological question being addressed. The structural- and affinity and apparent affinity-based methods are in several cases complex and/or time-consuming and can be combined with fast and cheap high-throughput approaches to improve the design and development of new potential G4 ligands. In recent years, the joint use of these techniques permitted the discovery of a huge number of G4 ligands investigated for diagnostic and therapeutic purposes. Overall, this review article highlights in detail the most commonly used approaches to characterize the G4/ligand interactions, as well as the applications and types of information that can be obtained from the use of each technique.

7.
Nucleic Acids Res ; 49(16): 9548-9559, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34379785

RESUMO

Genomic sequences susceptible to form G-quadruplexes (G4s) are always flanked by other nucleotides, but G4 formation in vitro is generally studied with short synthetic DNA or RNA oligonucleotides, for which bases adjacent to the G4 core are often omitted. Herein, we systematically studied the effects of flanking nucleotides on structural polymorphism of 371 different oligodeoxynucleotides that adopt intramolecular G4 structures. We found out that the addition of nucleotides favors the formation of a parallel fold, defined as the 'flanking effect' in this work. This 'flanking effect' was more pronounced when nucleotides were added at the 5'-end, and depended on loop arrangement. NMR experiments and molecular dynamics simulations revealed that flanking sequences at the 5'-end abolish a strong syn-specific hydrogen bond commonly found in non-parallel conformations, thus favoring a parallel topology. These analyses pave a new way for more accurate prediction of DNA G4 folding in a physiological context.


Assuntos
Quadruplex G , Nucleotídeos/genética , Oligonucleotídeos/genética , Polimorfismo Genético/genética , Dicroísmo Circular , DNA/genética , DNA/ultraestrutura , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Nucleotídeos/química , Oligonucleotídeos/química , RNA/genética , RNA/ultraestrutura
8.
Nanomedicine ; 36: 102429, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34174419

RESUMO

Nanoparticles offer targeted delivery of drugs with minimal toxicity to surrounding healthy tissue and have great potential in the management of human papillomavirus (HPV)-related diseases. We synthesized lipid-modified AS1411 aptamers capable of forming nanoaggregates in solution containing Mg2+. The nanoaggregates presented suitable properties for pharmaceutical applications such as small size (100 nm), negative charge, and drug release. The nanoaggregates were loaded with acridine orange derivative C8 for its specific delivery into cervical cancer cell lines and HPV-positive tissue biopsies. This improved inhibition of HeLa proliferation and cell uptake without significantly affecting healthy cells. Finally, the nanoaggregates were incorporated in a gel formulation with promising tissue retention properties aiming at developing a local delivery strategy of the nanoaggregates in the female genital tract. Collectively, these findings suggest that the nanoformulation protocol has great potential for the delivery of both anticancer and antiviral agents, becoming a novel modality for cervical cancer management.


Assuntos
Antineoplásicos , Antivirais , Aptâmeros de Nucleotídeos , Proliferação de Células/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Oligodesoxirribonucleotídeos , Neoplasias do Colo do Útero/tratamento farmacológico , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Antivirais/química , Antivirais/farmacocinética , Antivirais/farmacologia , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/farmacocinética , Aptâmeros de Nucleotídeos/farmacologia , Feminino , Células HeLa , Humanos , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/farmacocinética , Oligodesoxirribonucleotídeos/farmacologia , Neoplasias do Colo do Útero/metabolismo
9.
Nucleic Acid Ther ; 31(1): 68-81, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33121376

RESUMO

Herein, we report, for the first time, the screening of several ligands in terms of their ability to bind and stabilize G-quadruplexes (G4) found in seven human Papillomavirus (HPV) genomes. Using a variety of biophysical assays, HPV G-quadruplexes were shown to possess a high degree of structural polymorphism upon ligand binding, which may have an impact on transcription, replication, and viral protein production. A sequence found in high-risk HPV16 genotype folds into multiple non-canonical DNA structures; it was converted into a major G4 conformation upon interaction with a well-characterized highly selective G4 ligand, PhenDC3, which may have an impact on the viral infection. Likewise, HPV57 and 58, which fold into multiple G4 structures, were found to form single stable complexes in the presence of two other G4 ligands, C8 and pyridostatin, respectively. In addition, one of the selected compounds, the acridine derivative C8, demonstrated a significant antiviral effect in HPV18-infected organotypic raft cultures. Altogether, these results indicate that targeting HPV G4s may be an alternative route for the development of novel antiviral therapies.


Assuntos
Quadruplex G/efeitos dos fármacos , Papillomavirus Humano 16/genética , Papillomavirus Humano 18/genética , Viroses/tratamento farmacológico , Aminoquinolinas/farmacologia , Complemento C8/genética , Complemento C8/farmacologia , Proteínas de Ligação a DNA/genética , Genoma Viral/efeitos dos fármacos , Genoma Viral/genética , Genótipo , Papillomavirus Humano 16/efeitos dos fármacos , Papillomavirus Humano 16/patogenicidade , Papillomavirus Humano 16/ultraestrutura , Papillomavirus Humano 18/efeitos dos fármacos , Papillomavirus Humano 18/ultraestrutura , Humanos , Ligantes , Terapia de Alvo Molecular , Conformação de Ácido Nucleico/efeitos dos fármacos , Ácidos Picolínicos/farmacologia , Viroses/genética , Viroses/patologia
10.
Nucleic Acids Res ; 48(16): 9336-9345, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32432667

RESUMO

KRAS is one of the most mutated oncogenes and still considered an undruggable target. An alternative strategy would consist in targeting its gene rather than the protein, specifically the formation of G-quadruplexes (G4) in its promoter. G4 are secondary structures implicated in biological processes, which can be formed among G-rich DNA (or RNA) sequences. Here we have studied the major conformations of the commonly known KRAS 32R, or simply 32R, a 32 residue sequence within the KRAS Nuclease Hypersensitive Element (NHE) region. We have determined the structure of the two major stable conformers that 32R can adopt and which display slow equilibrium (>ms) with each other. By using different biophysical methods, we found that the nucleotides G9, G25, G28 and G32 are particularly implicated in the exchange between these two conformations. We also showed that a triad at the 3' end further stabilizes one of the G4 conformations, while the second conformer remains more flexible and less stable.


Assuntos
DNA/genética , Quadruplex G , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Sítios de Ligação/genética , Dicroísmo Circular , Humanos , Terapia de Alvo Molecular , Neoplasias/genética , Neoplasias/terapia , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores
11.
Bioorg Chem ; 100: 103920, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32413624

RESUMO

The G-quadruplex (G4)-forming sequence within the AS1411 derivatives with alternative nucleobases and backbones can improve the chemical and biological properties of AS1411. Zn(II) phthalocyanine (ZnPc) derivatives have potential as high-affinity G4 ligands because they have similar size and shape to the G-quartets. The interactions of four Zn(II) phthalocyanines with the G4 AS1411 aptamer and its derivatives were determined by biophysical techniques, molecular docking and gel electrophoresis. Cell viability assay was carried out to evaluate the antiproliferative effects of Zn(II) phthalocyanines and complexes. CD experiments showed structural changes after addition of ZnPc 4, consistent with multiple binding modes and conformations shown by NMR and gel electrophoresis. CD melting confirmed that ZnPc 2 and ZnPc 4, both containing eight positive charges, are able to stabilize the AT11 G4 structure (ΔTm > 30 °C and 18.5 °C, respectively). Molecular docking studies of ZnPc 3 and ZnPc 4 suggested a preferential binding to the 3'- and 5'-end, respectively, of the AT11 G4. ZnPc 3 and its AT11 and AT11-L0 complexes revealed pronounced cytotoxic effect against cervical cancer cells and no cytotoxicity to normal human cells. Zn(II) phthalocyanines provide the basis for the development of effective therapeutic agents as G4 ligands.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Indóis/química , Indóis/farmacologia , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/farmacologia , Compostos Organometálicos/química , Compostos Organometálicos/farmacologia , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Quadruplex G , Células HeLa , Humanos , Isoindóis , Simulação de Acoplamento Molecular , Neoplasias/tratamento farmacológico , Compostos de Zinco
12.
Trends Mol Med ; 26(9): 848-861, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32467069

RESUMO

The clinical applicability of G-quadruplexes (G4s) as anticancer drugs is currently being evaluated. Several G4 ligands and aptamers are undergoing clinical trials following the notable examples of quarfloxin and AS1411, respectively. In this review, we summarize the latest achievements and breakthroughs in the use of G4 nucleic acids as both therapeutic tools ('friends', as healing anticancer drugs) and targets ('foes', within the harmful cancer cell), particularly using aptamers and quadruplex-targeted ligands, respectively. We explore the recent research on synthetic G4 ligands toward the discovery of anticancer therapeutics and their mechanism of action. Additionally, we highlight recent advances in chemical and structural biology that enable the design of specific G4 aptamers to be used as novel anticancer agents.


Assuntos
Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Quadruplex G/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Animais , Aptâmeros de Nucleotídeos/farmacologia , Aptâmeros de Nucleotídeos/uso terapêutico , Humanos , Ligantes , Ácidos Nucleicos/farmacologia , Oligodesoxirribonucleotídeos/farmacologia , Oligodesoxirribonucleotídeos/uso terapêutico
13.
Eur J Pharm Sci ; 149: 105337, 2020 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-32311457

RESUMO

The oncogene KRAS is involved in the pathogenesis of many tumors such as pancreatic, lung and colorectal cancers, thereby representing a relevant target for the treatment of these diseases. The KRAS P1 promoter contains a nuclease hypersensitive, guanine-rich sequence able to fold into a G-quadruplex motif (G4). The stabilization of this G4 structure by small molecules is emerging as a feasible approach to downregulate KRAS expression. Here, a set of novel stabilizing molecules was identified through a virtual screening campaign on the NMR structure of the 22-mer KRAS G4. The most promising hits were then submitted to structure-activity relationships studies which allowed improving their binding affinity and selectivity over double helix DNA and different G4 topologies. The best derivative (19) underwent fluorescence titration experiments and further computational studies to disclose its binding mechanism to KRAS G4. Finally, biological assays showed that this compound is capable to reduce the viability of colorectal cancer cells in which mutated KRAS acts as a driver oncogene. Thus, 19 might represent the prototype of a new class of drugs for the treatment of tumors that, expressing mutated forms of KRAS, are refractory to current therapeutic regimens.

14.
J Biomol Struct Dyn ; 38(8): 2276-2286, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31204609

RESUMO

Using a molecular dynamics approach, the study of the interaction between six different known ligands and a predicted pre-miRNA 149 RNA G-quadruplex (rG4) structure is reported. The stabilization of rG4 structures formed within the pre-miRNA stem-loop regions using small ligands is an attractive anticancer strategy. Particularly, miRNA-149 is upregulated in a variety of cancers such as prostate cancer and is therefore a potential target for drug development. The results show that ligands C8 and PhenDC3 interact with the rG4 structure via stacking interactions with the end G-quartets. Ligands [16]phenN2, [32]phen2N4 and pyridostatin on the other hand bind the loops/groove interface of the rG4 being H-bonding and electrostatic interactions the driving force of the interaction. The C8 precursor, C8-NH2, emphasizes the structural nuances of the rG4 short loops as the lack of a large terminal aromatic moiety produced a mixed stacking-groove binding mode. Overall, this study may help the design of specific ligands for pre-miRNA rG4 towards anticancer therapeutics development.Communicated by Ramaswamy H. Sarma.


Assuntos
Quadruplex G , MicroRNAs , Humanos , Ligantes , Masculino , MicroRNAs/genética , Simulação de Dinâmica Molecular , Eletricidade Estática
15.
Biochim Biophys Acta Gen Subj ; 1864(1): 129423, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31476357

RESUMO

BACKGROUND: We previously reported the identification of the aapA1/IsoA1 locus as part of a new family of toxin-antitoxin (TA) systems in the human pathogen Helicobacter pylori. AapA1 belongs to type I TA bacterial toxins, and both its mechanism of action towards the membrane and toxicity features are still unclear. METHODS: The biochemical characterization of the AapA1 toxic peptide was carried out using plasmid-borne expression and mutational approaches to follow its toxicity and localization. Biophysical properties of the AapA1 interaction with lipid membranes were studied by solution and solid-state NMR spectroscopy, plasmon waveguide resonance (PWR) and molecular modeling. RESULTS: We show that despite a low hydrophobic index, this toxin has a nanomolar affinity to the prokaryotic membrane. NMR spectroscopy reveals that the AapA1 toxin is structurally organized into three distinct domains: a positively charged disordered N-terminal domain (D), a single α-helix (H), and a basic C-terminal domain (R). The R domain interacts and destabilizes the membrane, while the H domain adopts a transmembrane conformation. These results were confirmed by alanine scanning of the minimal sequence required for toxicity. CONCLUSION: Our results have shown that specific amino acid residues along the H domain, as well as the R domain, are essential for the toxicity of the AapA1 toxin. GENERAL SIGNIFICANCE: Untangling and understanding the mechanism of action of small membrane-targeting toxins are difficult, but nevertheless contributes to a promising search and development of new antimicrobial drugs.


Assuntos
Proteínas de Bactérias/química , Toxinas Bacterianas/química , Membrana Celular/química , Infecções por Helicobacter/genética , Helicobacter pylori/química , Alanina/química , Alanina/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/ultraestrutura , Toxinas Bacterianas/genética , Membrana Celular/genética , Membrana Celular/ultraestrutura , Infecções por Helicobacter/microbiologia , Helicobacter pylori/patogenicidade , Humanos , Espectroscopia de Ressonância Magnética , Conformação Proteica em alfa-Hélice/genética , Domínios Proteicos/genética , Ressonância de Plasmônio de Superfície , Sistemas Toxina-Antitoxina/genética
16.
Int J Pharm ; 568: 118511, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31301466

RESUMO

Nucleic acid aptamers can specifically bind to target molecules on the cell membrane that mediate their entrance into the cells. Their small size, high binding affinity, specificity, good biocompatibility, stability and low immunogenicity make them ideal drug delivery systems for cancer therapy. These biopharmaceuticals have potential for the delivery of anticancer compounds to diseased tissues, increasing their effectiveness while mitigating the off-target toxicity towards healthy cells. Herein, we have studied two quadruplex-forming DNA sequences derived from the nucleolin-targeted aptamer AS1411 as supramolecular carriers for the cancer-selective delivery of acridine orange derivatives (C3, C5 and C8) in cervical cancer cells. The devised delivery strategy relied on the non-covalent association of the acridine derivatives and the G-quadruplex (G4) structures. This association is done with a high binding strength, as suggested by the obtained KD values in the 10-6-10-7 M range, leading to the thermal stabilization of the G4 structures, particularly for C8. The stability of the resulting supramolecular conjugates was evaluated in fetal bovine serum, which proved their resistance against serum nucleases up to 48 h. Previous studies showed that the tested acridine orange derivatives were cytotoxic towards cervical cancer cells (HeLa) and non-malignant cells. However, when conjugated to AS1411 derivatives, the cytotoxicity of the free ligands towards non-malignant cells was restrained. Furthermore, conjugated C3 showed an enhanced cytotoxicity against HeLa cancer cells. Confocal microscopy indicated that both G4 sequences appear to colocalize with nucleolin, suggesting their ability to recognize and bind nucleolin on the cell surface. Additionally, the results confirmed the internalization of these delivery systems into HeLa cancer cells and their sustained cell trafficking, although being able to dissociate intracellularly to deliver C8 to the nucleoli. Overall, we showed that AS1411-derived G4s can be used as a potential cancer drug delivery system for cervical cancer.


Assuntos
Laranja de Acridina/química , Aptâmeros de Nucleotídeos/química , Sistemas de Liberação de Medicamentos , Quadruplex G , Oligodesoxirribonucleotídeos/química , Laranja de Acridina/administração & dosagem , Laranja de Acridina/análogos & derivados , Aptâmeros de Nucleotídeos/administração & dosagem , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Ligantes , Oligodesoxirribonucleotídeos/administração & dosagem , Neoplasias do Colo do Útero/metabolismo
17.
Sci Rep ; 9(1): 7945, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138870

RESUMO

AS1411 is a G-rich DNA oligonucleotide that functions as an aptamer of the protein nucleolin, found at high levels on the surface of cancer cells but not on the surface of normal cells. Herein, we have studied AS1411 as a supramolecular carrier for the delivery of an acridine-based G-quadruplex ligand, C8, to HeLa cancer cells. Two AS1411 derivatives, LNA-AS1411 and U-AS1411, were also tested, in an attempt to compare AS1411 pharmacological properties. The results showed that AS1411-C8 complexation was made with great binding strength and that it lowered the ligand's cytotoxicity towards non-malignant cells. This effect was suggested to be due to a decreased internalization of the complexed versus free C8 as shown by flow cytometry. The AS1411 derivatives, despite forming a stable complex with C8, lacked the necessary tumour-selective behaviour. The binding of C8 to AS1411 G-quadruplex structure did not negatively affect the recognition of nucleolin by the aptamer. The AS1411-C8 repressed c-MYC expression at the transcriptional level, possibly due to C8 ability to stabilize the c-MYC promoter G-quadruplexes. Overall, this study demonstrates the usefulness of AS1411 as a supramolecular carrier of the G-quadruplex binder C8 and the potential of using its tumour-selective properties for the delivery of ligands for cancer therapy.


Assuntos
Antineoplásicos/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , Quadruplex G , Regulação Neoplásica da Expressão Gênica , Oligodesoxirribonucleotídeos/farmacologia , Fosfoproteínas/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas de Ligação a RNA/genética , Células A549 , Antineoplásicos/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Técnicas de Transferência de Genes , Células HeLa , Humanos , Ligantes , Oligodesoxirribonucleotídeos/metabolismo , Especificidade de Órgãos , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica , Nucleolina
18.
J Am Chem Soc ; 141(6): 2516-2525, 2019 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-30715863

RESUMO

A number of foldamer backbones have been described as useful mimics of protein secondary structure elements, enabling for example the design of synthetic oligomers with the ability to engage specific protein surfaces. Synthetic folded backbones can also be used to create artificial proteins in which a folded peptide segment (e.g., an α-helix, a loop) is replaced by its unnatural counterpart, with the expectation that the resulting molecule would maintain its ability to fold while manifesting new exploitable features. The similarities in screw sense, pitch, and polarity between peptide α-helices and oligourea 2.5-helices suggest that a tertiary structure could be retained when swapping the two backbones in a protein sequence. In the present work, we move a step toward the creation of such composite proteins by replacing the 10-residue long original α-helical segment in the Cys2His2 zinc finger 3 of transcription factor Egr1 (also known as Zif268) by an oligourea sequence bearing two appropriately spaced imidazole side chains for zinc coordination. We show by spectroscopic techniques and mass spectrometry analysis under native conditions that the ability of the peptide/oligourea hybrid to coordinate the zinc ion is not affected by the foldamer replacement. Moreover, detailed NMR analysis provides evidence that the engineered zinc finger motif adopts a folded structure in which the native ß-sheet arrangement of the peptide region and global arrangement of DNA-binding side chains are preserved. Titration in the presence of the Egr1 target DNA sequence supports binding to GC bases as reported for the wild-type zinc finger.


Assuntos
Desenho de Fármacos , Proteína 1 de Resposta de Crescimento Precoce/química , Dedos de Zinco , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Simulação de Dinâmica Molecular , Conformação Proteica em alfa-Hélice , Domínios Proteicos
19.
Org Biomol Chem ; 16(15): 2776-2786, 2018 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-29611599

RESUMO

Targeting quadruplex DNA structures with small molecules is a promising strategy for anti-cancer drug design. Four phenanthroline polyazamacrocycles were studied for their binding affinity, thermal stabilization, inhibitory effect on the activity of helicase towards human telomeric 22AG and oncogene promoter c-MYC G-quadruplexes (G4s), and their ability to inhibit Taq polymerase-mediated DNA extension. The fluorescence resonance energy transfer (FRET) melting assay indicates that the melting temperature increases (ΔTm values) of c-MYC and 22AG G4s are 17.2 and 20.3 °C, respectively, for the ligand [32]phen2N4 followed by [16]phenN4 (11.3 and 15.0 °C, for c-MYC and 22AG, respectively). Competitive FRET assays show that [32]phen2N4 and [16]phenN4 exhibit G4 selectivity over duplex DNA. Different G4s were compared; no considerable selectivity of the ligands for a specific G4 was found. Circular dichroism (CD) confirms the formation of G4 structures and the melting experiments show that [16]phenN4 and [32]phen2N4 are the most stabilizing ligands with a ΔTm of 19.3 °C and 15.1 °C, respectively, at 5 molar equivalents for the c-MYC G4. The fluorescent intercalator displacement (FID) assay also demonstrates that ligand [32]phen2N4 furnishes very low DC50 values (0.87-1.24 µM), indicating high stabilization of c-MYC and 22AG G4s. These results suggest that the hexyl chain in these compounds plays an important role in regulating the stabilization of these G4s. Binding constants, determined by fluorescence titrations, indicate a moderate ligand-G4 binding with KSV between 105 and 106 M-1 in which [16]phenN4 has a slightly higher apparent binding constant for telomeric 22AG G4 than that for the c-MYC G4. The ligand's ability to inhibit Taq polymerase confirms the biological activity of [16]phenN4 and [32]phen2N4 against the c-MYC G4. In addition, ligands [32]phen2N4 and [16]phenN4 affect the unwinding activity of Pif1 in the presence of DNA systems harboring c-MYC and telomeric G4 motifs.


Assuntos
Antineoplásicos/síntese química , Compostos Aza/síntese química , DNA/química , Quadruplex G , Compostos Macrocíclicos/síntese química , Fenantrolinas/síntese química , Antineoplásicos/farmacologia , Compostos Aza/farmacologia , Sobrevivência Celular/efeitos dos fármacos , DNA Helicases/química , Desenho de Fármacos , Genes myc , Células HeLa , Humanos , Ligantes , Compostos Macrocíclicos/farmacologia , Fenantrolinas/farmacologia , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/química , Relação Estrutura-Atividade , Taq Polimerase/química , Taq Polimerase/genética , Telômero/química , Termodinâmica
20.
Biochimie ; 144: 144-152, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29129745

RESUMO

KRAS is often found mutated in lethal cancers and should be an important target for anticancer drug development. However, no effective inhibitor has been reported so far, prompting the scientific community to describe the RAS proteins as nearly "undruggable". Recent approaches developed to modulate KRAS protein expression comprises the targeting of G-quadruplex (G4) structures formed within the nuclease hypersensitive element of KRAS promoter region, by designing small and specific ligands to stabilize the tertiary fold and reduce gene expression. In this work, we report in vitro and in silico studies of novel acridine orange (AO) derivatives (C3-C8), developed as G4 stabilizing agents. The results show that the ligands bind with high affinity and stabilize KRAS22-RT G4 with modest specificity over duplex DNA. The most promising ligand C8 stabilizes the structure by ≈ 40 °C. Molecular docking using NMR-derived distance restraints reveal atomic details about the ligand structural features in the interaction with KRAS22-RT G4. In vitro studies with HeLa cells show that the ligands are cytotoxic with IC50 values between 0.9 µM and 5.7 µM. Moreover, the ligands tend to localize in the nucleus as shown by confocal fluorescence microscopy. Overall, these results show that the reported AO ligands display favourable properties as G4 ligands and this study provides structural detail for the development of lead KRAS G4 ligands.


Assuntos
Laranja de Acridina/química , Laranja de Acridina/farmacologia , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacologia , Quadruplex G/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/genética , Laranja de Acridina/metabolismo , Transporte Biológico , Proliferação de Células/efeitos dos fármacos , Corantes Fluorescentes/metabolismo , Células HeLa , Humanos , Espaço Intracelular/metabolismo , Ligantes
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