RESUMO
The interactions of intermolecular G-quadruplex RNA and small molecules have been investigated by computational studies. Various anthraquinone, bisbenzimidazole, and carbazole-benzimidazole based ligands have shown a distinct preference to G-quadruplex structures as opposed to the corresponding duplex forms of DNA that were docked with telomeric G-quadruplex RNA. The comparative binding study of such ligands with G-quadruplex (G4) RNA showed higher binding affinities toward carbazole-benzimidazole ligands than those of the anthraquinone and bisbenzimidazole based ligands. A molecular dynamics simulation study was used to examine quadruplex-ligand interactions. Analysis of the binding free energy indicated the formation of the thermodynamically favorable RNA-ligand complex. The formation of several H-bonding interactions and the change of the solvent accessible surface area (SASA) also support the effective binding of the carbazole-benzimidazole ligands with G4 RNA structures. Thus, the library screening approach has assisted in getting a structure-activity relationship for the selected small molecules toward the G-quadruplex RNA binding, which can be applied in the targeting of G-quadruplex RNA medicated anticancer therapeutics.
Assuntos
Quadruplex G , Simulação de Dinâmica Molecular , Ligantes , Simulação de Acoplamento Molecular , RNA , TelômeroRESUMO
Pyrrole-imidazole (Py-Im) polyamides are synthetic molecules that can be rationally designed to target specific DNA sequences to both disrupt and recruit transcriptional machinery. While in vitro binding has been extensively studied, in vivo effects are often difficult to predict using current models of DNA binding. Determining the impact of genomic architecture and the local chromatin landscape on polyamide-DNA sequence specificity remains an unresolved question that impedes their effective deployment in vivo. In this report we identified polyamide-DNA interaction sites across the entire genome, by covalently crosslinking and capturing these events in the nuclei of human LNCaP cells. This technique confirms the ability of two eight ring hairpin-polyamides, with similar architectures but differing at a single ring position (Py to Im), to retain in vitro specificities and display distinct genome-wide binding profiles.
Assuntos
Proteínas de Ligação a DNA/genética , Genoma Humano/efeitos dos fármacos , Conformação de Ácido Nucleico/efeitos dos fármacos , Nylons/farmacologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Humanos , Imidazóis/farmacologia , Pirróis/farmacologiaRESUMO
We have designed and synthesized anthraquinone containing compounds which have oligopyrrole side chains of varying lengths. These compounds stabilized the G-quadruplex DNA formed in the promoter regions of c-MYC oncogenes selectively over the duplex DNA. These observations were recorded using UV-vis spectroscopic titrations, fluorescence measurements and circular dichroism (CD) spectral titrations. The potency of the compounds to stabilize the G4 DNA has been shown from the thermal denaturation experiments. The compound interacts with c-MYC G-quadruplex DNA through stacking mode as obtained from ethidium bromide displacement assay, cyclic voltammetric titration, and docking experiments. Molecular modeling studies suggested that the stacking of the anthraquinone moiety over the G-tetrad of the G4 structures are responsible for the stability of such quadruplex secondary structure. Furthermore, polymerase stop assay also supported the formation of stable G4 structures in the presence of the above-mentioned compounds. The compounds have shown selective cancer cell (HeLa and HEK293T) cytotoxicity over normal cells (NIH3T3 and HDFa) under in vitro conditions as determined from MTT based cell viability assay. Apoptosis was found to be the mechanistic pathway underlying the cancer cell cytotoxicity as obtained from Annexin V-FITC and PI dual staining assay which was further substantiated by nuclear morphological changes as observed by AO/EB dual staining assay. Cellular morphological changes, as well as nuclear condensation and fragmentation upon treatment with these compounds, were observed under bright field and confocal microscopy.
Assuntos
Antracenos/química , Dimerização , Distamicinas/química , Distamicinas/farmacologia , Quadruplex G/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-myc/genética , Antineoplásicos/química , Antineoplásicos/farmacologia , DNA/química , DNA/genética , Desenho de Fármacos , Modelos MolecularesRESUMO
Phosphorylation of the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) governs stage-specific interactions with different cellular machines. The CTD consists of Y1S2P3T4S5P6S7 heptad repeats and sequential phosphorylations of Ser7, Ser5 and Ser2 occur universally at Pol II-transcribed genes. Phosphorylation of Thr4, however, appears to selectively modulate transcription of specific classes of genes. Here, we identify ten new Thr4 kinases from different kinase structural groups. Irreversible chemical inhibition of the most active Thr4 kinase, Hrr25, reveals a novel role for this kinase in transcription termination of specific class of noncoding snoRNA genes. Genome-wide profiles of Hrr25 reveal a selective enrichment at 3' regions of noncoding genes that display termination defects. Importantly, phospho-Thr4 marks placed by Hrr25 are recognized by Rtt103, a key component of the termination machinery. Our results suggest that these uncommon CTD kinases place phospho-Thr4 marks to regulate expression of targeted genes.
Assuntos
Proteínas Quinases/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/fisiologia , Sequência de Aminoácidos , Caseína Quinase I/metabolismo , Fosforilação , Filogenia , Domínios Proteicos , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Treonina/metabolismo , Transcrição GênicaRESUMO
An iron-responsive optical probe has been developed for simultaneous sensing of both ferritin and apoferritin proteins at pH 7.4 in water. The compound showed an exclusive response (turn-off signal) towards ferritin among a wide range of proteins even at nanomolar concentration. In contrast, apoferritin dissociates the preformed iron complex and revives the green colored fluorescence of the native probe (turn-on signal). Subsequently, various parameters associated with the serum iron level are evaluated, which are beneficial for clinical diagnosis of many iron-related diseases, including anemia. Estimation of iron was achieved in a wide range of edible plant materials as well as pharmaceutical formulations. Subsequently, different kinds of natural water samples were screened for quantification of soluble iron contents. In addition to traditional spectroscopic tools, dye-coated paper strips were developed as an alternative strategy for onsite 'instrument-free' detection of iron. Highly specific bioimaging of Fe3+ was achieved in cervical cancer cells (HeLa).
Assuntos
Apoferritinas/análise , Ferritinas/análise , Corantes Fluorescentes/química , Ferro/sangue , Água Doce/análise , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Ferro/análise , Microscopia Confocal , Teoria Quântica , Espectrofotometria , Raios UltravioletaRESUMO
Recent studies support the idea that G-quadruplex structures in the promoter regions of oncogenes and telomere DNA can serve as potential therapeutic targets in the treatment of cancer. Accordingly, several different types of organic small molecules that stabilize G-quadruplex structures and inhibit telomerase activity have been discerned. Here, we describe the binding of benzimidazole-carbazole ligands to G-quadruplex structures formed in G-rich DNA sequences containing the promoter regions of human c-MYC, c-KIT1, c-KIT2, VEGF and BCL2 proto-oncogenes. The fluorescence spectroscopic data indicate that benzimidazole-carbazole ligands bind and stabilize the G-quadruplexes in the promoter region of oncogenes. The molecular docking studies provide insights into the mode and extent of binding of this class of ligands to the G-quadruplexes formed in oncogene promoters. The high stability of these G-quadruplex structures was validated by thermal denaturation and telomerase-catalyzed extension of the 3' end. Notably, benzimidazole-carbazole ligands suppress the expression of oncogenes in cancer cells in a dose-dependent manner. We anticipate that benzimidazole-carbazole ligands, by virtue of their ability to stabilize G-quadruplex structures in the promoter regions of oncogenes, might reduce the risk of cancer through the loss of function in the proteins encoded by these genes.
Assuntos
Antineoplásicos/química , Benzimidazóis/metabolismo , Carbazóis/metabolismo , Quadruplex G/efeitos dos fármacos , Proteínas Oncogênicas/antagonistas & inibidores , Oncogenes/genética , Regiões Promotoras Genéticas/genética , Telômero/genética , Antineoplásicos/metabolismo , Benzimidazóis/farmacologia , Carbazóis/farmacologia , Humanos , Ligantes , Simulação de Acoplamento Molecular , Telômero/químicaRESUMO
The release of paused RNA polymerase II into productive elongation is highly regulated, especially at genes that affect human development and disease. To exert control over this rate-limiting step, we designed sequence-specific synthetic transcription elongation factors (Syn-TEFs). These molecules are composed of programmable DNA-binding ligands flexibly tethered to a small molecule that engages the transcription elongation machinery. By limiting activity to targeted loci, Syn-TEFs convert constituent modules from broad-spectrum inhibitors of transcription into gene-specific stimulators. Here we present Syn-TEF1, a molecule that actively enables transcription across repressive GAA repeats that silence frataxin expression in Friedreich's ataxia, a terminal neurodegenerative disease with no effective therapy. The modular design of Syn-TEF1 defines a general framework for developing a class of molecules that license transcription elongation at targeted genomic loci.
Assuntos
Cromatina/metabolismo , Ataxia de Friedreich/genética , Proteínas de Ligação ao Ferro/genética , Ativação Transcricional , Fatores de Elongação da Transcrição/síntese química , Fatores de Elongação da Transcrição/metabolismo , Inativação Gênica , Humanos , RNA Polimerase II/metabolismo , Transcrição Gênica , FrataxinaRESUMO
An easily synthesized fluorescein-based luminescent dye has been utilized for the dual-mode detection of histamine at nanomolar concentrations at pHâ 7.0 in water. The specific response to histamine was achieved by imidazole-catalyzed 'imine formation' reaction. The protocol was subsequently applied for the estimation of histamine in complex biological milieu such as human blood serum and urine samples. Furthermore, the dose-dependent cellular uptake of histamine and de novo synthesis (by thapsigargin treatment) was visualized in RAW 264.7, a mouse macrophage cell line. We have also developed portable paper strips for rapid, on-site detection of histamine without involving costly instruments.
Assuntos
Histamina/análise , Espectrometria de Fluorescência , Animais , Linhagem Celular , Corantes Fluorescentes/química , Histamina/sangue , Histamina/urina , Humanos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Microscopia Confocal , Teoria Quântica , Células RAW 264.7 , Tapsigargina/farmacologiaRESUMO
Bombyx mori (B. mori) is important due to its major role in the silk production. Though DNA binding ligands often influence gene expression, no attempt has been made to exploit their use in sericulture. The telomeric heterochromatin of B. mori is enriched with 5'-TTAGG-3' sequences. These sequences were also found to be present in several genes in the euchromatic regions. We examined three synthetic oligopyrrole carboxamides that target 5'-TTAGG-3' sequences in controlling the gene expression in B. mori. The ligands did not show any defect or feeding difference in the larval stage, crucial for silk production. The ligands caused silencing of various isoforms of the broad-complex transcription factor and cuticle proteins which resulted in late pupal developmental defects. Furthermore, treatment with such drugs resulted in statistically enhanced cocoon weight, shell weight, and silk yield. This study shows for the first time use of oligopyrrole carboxamide drugs in controlling gene expression in B. mori and their long term use in enhancing silk production.
Assuntos
Bombyx/genética , Técnicas de Silenciamento de Genes/métodos , Inativação Gênica , Seda/genética , Aminopiridinas/química , Animais , Bombyx/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Pirróis/química , Seda/biossíntese , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Metal based salen complexes have been considered as an important scaffold toward targeting of DNA structures. In the present work, we have synthesized nickel(II) and palladium(II) salen and salphen complexes by using readily available fluorescein as the backbone to provide an extended aromatic surface. The metal complexes exhibit affinity toward the human telomeric G-quadruplex DNA with promising inhibition of telomerase activity. This has been ascertained by their efficiency in the long term cell proliferation assay which showed significant cancer cell toxicity in the presence of the metal complexes. Confocal microscopy showed cellular internalization followed by localization in the nucleus and mitochondria. Considerable population at the sub-G1 phase of the cell cycle showed cell death via apoptotic pathway.
Assuntos
DNA/química , Fluoresceína/química , Quadruplex G/efeitos dos fármacos , Níquel/química , Compostos Organometálicos/farmacologia , Paládio/química , Telomerase/antagonistas & inibidores , Células A549 , Apoptose/efeitos dos fármacos , DNA/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Etilenodiaminas/química , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Compostos Organometálicos/química , Compostos Organometálicos/metabolismo , Fenilenodiaminas/químicaRESUMO
DNA targeting by various metal complexes is a key strategy toward the restriction of cancer cell proliferation. Toward this end, we designed and synthesized novel salen-based Ni(II) and Pd(II) metal complexes with positively charged flanking side chains comprising N-methylpyrrole carboxamides of varying lengths. The compounds showed high specificity toward G-quadruplex DNA over duplex DNA. Sufficient inhibition of the telomerase activity was observed, which was ascertained by the prominent restriction of cancer cell proliferation in the long-term cell viability and telomerase inhibition assays. The compounds exhibited selective cancer cell death following an apoptotic pathway. Analysis of the binding mode showed partial stacking of the salen moiety over the G-tetrads and association of the pendant oligopyrrole carboxamide units with the grooves. The conjugation of the tetrad-binding metal salen core with groove-oriented flexible oligopyrrole moieties resulted in the high selectivity and stabilization of the human G-quadruplex DNA structures.
Assuntos
Antineoplásicos/farmacologia , DNA de Neoplasias/efeitos dos fármacos , DNA de Neoplasias/genética , Inibidores Enzimáticos/farmacologia , Quadruplex G/efeitos dos fármacos , Compostos Organometálicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Morte Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Etilenodiaminas/química , Etilenodiaminas/farmacologia , Humanos , Modelos Moleculares , Estrutura Molecular , Níquel/farmacologia , Compostos Organometálicos/síntese química , Compostos Organometálicos/química , Paládio/química , Paládio/farmacologia , Pirróis/química , Pirróis/farmacologia , Relação Estrutura-Atividade , Telomerase/antagonistas & inibidores , Telomerase/metabolismoRESUMO
Minor groove binding distamycin like moieties were conjugated with core salens and the corresponding Fe(iii) and Co(ii) complexes were synthesized. Herein, we have shown efficient DNA minor groove binding specificities along with excellent DNA cleavage capacities with metallosalen conjugates. The metal complexes showed toxicity toward various cancer cells over normal cells with high specificity. Interestingly, the Co(ii) complexes exhibited greater activity than the Fe(iii) complexes in accordance with the stronger affinity of the former in the biophysical studies. Active DNA damage, and prominent nuclear condensation along with the release of cytochrome-c from the mitochondria unanimously showed that the metal complexes followed apoptotic pathways to induce cell death.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Cobalto/farmacologia , Complexos de Coordenação/farmacologia , Dano ao DNA , DNA/efeitos dos fármacos , Distamicinas/farmacologia , Compostos Férricos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Sobrevivência Celular/efeitos dos fármacos , Quelantes/farmacologia , Complexos de Coordenação/síntese química , DNA/biossíntese , Clivagem do DNA , Etilenodiaminas/farmacologia , Compostos Férricos/síntese química , Células HEK293 , Células HeLa , Humanos , Mitocôndrias/metabolismoRESUMO
The binding of ligand 5,10,15,20-tetra(N-methyl-4-pyridyl)porphine (TMPyP4) with telomeric and genomic G-quadruplex DNA has been extensively studied. However, a comparative study of interactions of TMPyP4 with different conformations of human telomeric G-quadruplex DNA, namely, parallel propeller-type (PP), antiparallel basket-type (AB), and mixed hybrid-type (MH) G-quadruplex DNA, has not been done. We considered all the possible binding sites in each of the G-quadruplex DNA structures and docked TMPyP4 to each one of them. The resultant most potent sites for binding were analyzed from the mean binding free energy of the complexes. Molecular dynamics simulations were then carried out, and analysis of the binding free energy of the TMPyP4-G-quadruplex complex showed that the binding of TMPyP4 with parallel propeller-type G-quadruplex DNA is preferred over the other two G-quadruplex DNA conformations. The results obtained from the change in solvent excluded surface area (SESA) and solvent accessible surface area (SASA) also support the more pronounced binding of the ligand with the parallel propeller-type G-quadruplex DNA.
Assuntos
DNA/química , Quadruplex G , Porfirinas/química , Telômero/química , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular , TermodinâmicaRESUMO
Cancer has always been a dreadful disease and continues to attract extensive research investigations. Various targets have been identified to restrain cancer. Among these DNA happens to be the most explored one. A wide variety of small molecules, often referred to as 'ligands', has been synthesized to target numerous structural features of DNA. The sole purpose of such molecular design has been to interfere with the transcriptional machinery in order to drive the cancer cell toward apoptosis. The mode of action of the DNA targeting ligands focuses either on the sequence-specificity by groove binding and strand cleavage, or by identifying the morphologically distinct higher order structures like that of the G-quadruplex DNA. However, in spite of the extensive research, only a tiny fraction of the molecules have been able to reach clinical trials and only a handful are used in chemotherapy. This review attempts to record the journey of the DNA binding small molecules from its inception to cancer therapy via various modifications at the molecular level. Nevertheless, factors like limited bioavailability, severe toxicities, unfavorable pharmacokinetics etc. still prove to be the major impediments in the field which warrant considerable scope for further research investigations.