RESUMO
Telomeres are known to prevent chromosome ends from being recognized as DNA double-strand breaks. Conversely, many DNA damage response proteins, including ATM, are thought to participate to telomere maintenance. However, the precise roles of ATM at telomeres remain unclear due to its multiple functions in cell checkpoints and apoptosis. To gain more insights into the role of ATM in telomere maintenance, we determined the effects of the G-quadruplex ligand 360A in various cell lines lacking functional ATM. We showed, by using Fluorescence in situ hybridization (FISH) and Chromosome Orientation-FISH using telomere PNA probes, that 360A induced specific telomere aberrations occurring during or after replication, mainly consisting in sister telomere fusions and also recombinations that involved preferentially the lagging strand telomeres. We demonstrate that ATM reduced telomere instability independently of apoptosis induction. Our results suggest thus that ATM has a direct role in preventing inappropriate DNA repair at telomeres, which could be related to its possible participation to the formation of protected structures at telomeres.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Reparo do DNA , Proteínas de Ligação a DNA/fisiologia , Quadruplex G/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/fisiologia , Piridinas/toxicidade , Quinolinas/toxicidade , Telômero/química , Proteínas Supressoras de Tumor/fisiologia , Apoptose , Proteínas Mutadas de Ataxia Telangiectasia , Ciclo Celular , Proteínas de Ciclo Celular/antagonistas & inibidores , Aberrações Cromossômicas , Dano ao DNA , Proteínas de Ligação a DNA/antagonistas & inibidores , Células HeLa , Humanos , Ligantes , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Transdução de Sinais , Troca de Cromátide Irmã/efeitos dos fármacos , Telômero/efeitos dos fármacos , Proteínas Supressoras de Tumor/antagonistas & inibidoresRESUMO
The G-overhangs of telomeres are thought to adopt particular conformations, such as T-loops or G-quadruplexes. It has been suggested that G-quadruplex structures could be stabilized by specific ligands in a new approach to cancer treatment consisting in inhibition of telomerase, an enzyme involved in telomere maintenance and cell immortality. Although the formation of G-quadruplexes was demonstrated in vitro many years ago, it has not been definitively demonstrated in living human cells. We therefore investigated the chromosomal binding of a tritiated G-quadruplex ligand, 3H-360A (2,6-N,N'-methyl-quinolinio-3-yl)-pyridine dicarboxamide [methyl-3H]. We verified the in vitro selectivity of 3H-360A for G-quadruplex structures by equilibrium dialysis. We then showed by binding experiments with human genomic DNA that 3H-360A has a very potent selectivity toward G-quadruplex structures of the telomeric 3'-overhang. Finally, we performed autoradiography of metaphase spreads from cells cultured with 3H-360A. We found that 3H-360A was preferentially bound to chromosome terminal regions of both human normal (peripheral blood lymphocytes) and tumor cells (T98G and CEM1301). In conclusion, our results provide evidence that a specific G-quadruplex ligand interacts with the terminal ends of human chromosomes. They support the hypothesis that G-quadruplex ligands induce and/or stabilize G-quadruplex structures at telomeres of human cells.
Assuntos
Cromossomos Humanos/química , DNA/metabolismo , Piridinas/metabolismo , Quinolinas/metabolismo , Telômero/química , Sítios de Ligação , Linhagem Celular Tumoral , Células Cultivadas , Cromossomos Humanos/metabolismo , DNA/química , Quadruplex G , Guanina/química , Humanos , Ligantes , Linfócitos/ultraestrutura , Metáfase , Piridinas/química , Quinolinas/química , Telômero/metabolismoRESUMO
Telomerase represents a relevant target for cancer therapy. Molecules able to stabilize the G-quadruplex (G4), a structure adopted by the 3'-overhang of telomeres, are thought to inhibit telomerase by blocking its access to telomeres. We investigated the cellular effects of four new 2,6-pyridine-dicarboxamide derivatives displaying strong selectivity for G4 structures and strong inhibition of telomerase in in vitro assays. These compounds inhibited cell proliferation at very low concentrations and then induced a massive apoptosis within a few days in a dose-dependent manner in cultures of three telomerase-positive glioma cell lines, T98G, CB193 and U118-MG. They had also antiproliferative effects in SAOS-2, a cell line in which telomere maintenance involves an alternative lengthening of telomeres (ALT) mechanism. We show that apoptosis was preceded by multiple alterations of the cell cycle: activation of S-phase checkpoints, dramatic increase of metaphase duration and cytokinesis defects. These effects were not associated with telomere shortening, but they were directly related to telomere instability involving telomere end fusion and anaphase bridge formation. Pyridine-based G-quadruplex ligands are therefore promising agents for the treatment of various tumors including malignant gliomas.
Assuntos
Apoptose/fisiologia , Instabilidade Genômica/fisiologia , Telômero/genética , Telômero/metabolismo , Ciclo Celular , Divisão Celular/genética , Divisão Celular/imunologia , Glioma/tratamento farmacológico , Humanos , Ligantes , Piridinas/farmacologia , Telomerase/antagonistas & inibidoresRESUMO
A total of 11 structures of the (pp60)src SH2 domain with non-peptidic inhibitors based on the same two closely related inhibitor scaffolds were determined using X-ray crystallography. Surprisingly, the inhibitors that have an IC(50) value between 4 and 2700 nM bind in three different binding modes. Structure comparisons show that the inhibitors aim to maximize the interaction between the hydrophobic substituent and the hydrophobic pY+3 pocket. This is achieved either by an alternative binding mode of the phenyl phosphate or by including water molecules that mediate the interaction between the inhibitor scaffold and a rigid surface of the SH2 domain. The combination of the rigid pY+3 pocket and the rigid protein surface to which the scaffolds bind results in severe distance and angular restraints for putative scaffolds and their substituents. The X-ray data suggest that these restraints seem to be compensated in our system by including water molecules, thereby increasing the flexibility of the system.
Assuntos
Inibidores Enzimáticos/química , Proteínas Proto-Oncogênicas pp60(c-src)/química , Domínios de Homologia de src , Compostos de Bifenilo/química , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Relação Estrutura-AtividadeRESUMO
Results from a novel approach which uses protein crystallography for the screening of a low affinity inhibitor fragment library are analyzed by comparing the X-ray structures with bound fragments to the structures with the corresponding full length inhibitors. The screen for new phospho-tyrosine mimics binding to the SH2 domain of (pp60)src was initiated because of the limited cell penetration of phosphates. Fragments in our library typically had between 6 and 30 atoms and included compounds which had either millimolar activity in a Biacore assay or were suggested by the ab initio design program LUDI but had no measurable affinity. All identified fragments were located in the phospho-tyrosine pocket. The most promising fragments were successfully used to replace the phospho-tyrosine and resulted in novel nonpeptidic high affinity inhibitors. The significant diversity of successful fragments is reflected in the high flexibility of the phospho-tyrosine pocket. Comparison of the X-ray structures shows that the presence of the H-bond acceptors and not their relative position within the pharmacophore are essential for fragment binding and/or high affinity binding of full length inhibitors. The X-ray data show that the fragments are recognized by forming a complex H-bond network within the phospho-tyrosine pocket of SH2. No fragment structure was found in which this H-bond network was incomplete, and any uncompensated H-bond within the H-bond network leads to a significant decrease in the affinity of full length inhibitors. No correlation between affinity and fragment binding was found for these polar fragments and hence affinity-based screening would have overlooked some interesting starting points for inhibitor design. In contrast, we were unable to identify electron density for hydrophobic fragments, confirming that hydrophobic interactions are important for inhibitor affinity but of minor importance for ligand recognition. Our results suggest that a screening approach using protein crystallography is particularly useful to identify universal fragments for the conserved hydrophilic recognition sites found in target families such as SH2 domains, phosphatases, kinases, proteases, and esterases.
Assuntos
Inibidores Enzimáticos/química , Proteínas Proto-Oncogênicas pp60(c-src)/antagonistas & inibidores , Domínios de Homologia de src , Sítios de Ligação , Simulação por Computador , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Ligação Proteica , Proteínas Proto-Oncogênicas pp60(c-src)/químicaRESUMO
(pp60)Src is a protein involved in signal transduction and is mainly expressed in neurones, platelets, and osteoclasts. Its precise biological role was recently discovered with the KO experiments by Soriano that gave rise to no other apparent phenotype than osteopetrosis, a disease resulting in excedent bone formation. The SH2 domain of the Src family specifically recognizes a sequence of tetrapeptide featuring a phosphotyrosine and a lipophilic aminoacid at the +1 and +3 positions. Recently we engaged in the search for SH2 ligands via modular peptidomimicry of this tetrapetide. This gave rise to several families of nanomolar inhibitors; the best one incorporated a caprolactam scaffold, a biphenyl moiety, and a phosphotyrosine. However, these inhibitors still incorporated the phosphate group that confers good binding affinity to the protein. Phosphates have undesirable features for drug candidates, namely, high rate of hydrolysis of the phosphate group by phosphatases and high charge content precluding cell penetration. Therefore, while searching for optimal non-peptide ligands for Src SH2, we looked for phosphate replacements. For this, we have designed an SAR by fragment crystallography approach. The start of this work resulted from two experimental observations. First, the fact that phenyl phosphate itself displayed detectable binding affinity for Src SH2 permitted us to perform a screening of small aromatic compounds as phenyl phosphate surrogates. Second, the obtention of large Src SH2 crystals displaying a channel large enough for soaking purposes allowed structure determination of over 40 of these small aromatic compounds bound in the phosphotyrosine binding pocket. This search and the way it gave rise to low nanomolar range Src SH2 inhibitors devoid of phosphate groups will be the subject of the present paper.
Assuntos
Inibidores Enzimáticos/química , Proteínas Proto-Oncogênicas pp60(c-src)/antagonistas & inibidores , Domínios de Homologia de src , Cristalografia por Raios X , Desenho de Fármacos , Modelos Moleculares , Organofosfatos/química , Ligação Proteica , Proteínas Proto-Oncogênicas pp60(c-src)/química , Relação Estrutura-AtividadeRESUMO
Starting from known Src SH2 inhibitors incorporating five-membered heterocycles or benzamide scaffolds, we prepared tetrasubstituted imidazole compounds able to interact with the pY, pY+1 and pY+3 binding sites of the Src SH2 protein. The synthesis and biological data are presented.
Assuntos
Imidazóis/metabolismo , Domínios de Homologia de src , Imidazóis/química , LigantesRESUMO
Various small fragments bearing phosphate, phosphonate or phosphonic acid moieties have been prepared through parallel synthesis and their binding potencies evaluated on the Src SH2 protein using a BIAcore assay. This provided us insight into the requirement of the Src SH2 pTyr binding pocket and some promising small ligands have been characterised.
Assuntos
Fosfotirosina/metabolismo , Domínios de Homologia de src , Sítios de Ligação , LigantesRESUMO
A parallel G-quadruplex structure was recently identified in the NHE III(1) element of the c-myc gene promoter that functioned as a transcriptional repressor. Different series of telomeric G-quadruplex interacting ligands reported to block telomerase activity were evaluated in a new PCR stop assay on the c-myc quadruplex (Pu22myc). Results indicated that the cationic porphyrin TMPyP4 previously described to stabilize c-myc quadruplex and to cause transcription inhibition efficiently inhibited the assay but with a narrow selectivity when parallel experiments were performed with an oligonucleotide (Pu22mu) containing mutations in the guanine repeat which is unable to form a quadruplex. Other ligands presented potent inhibitory properties with IC(50) in the submicromolar range. 307A, a new 2,6-pyridin-dicarboxamide derivative was found to present the highest selectivity as compared to Pu22mu oligonucleotide (>90-fold). Comparison with telomeric G-quadruplex using TRAP-G4 and PCR stop assays also indicated that ligands 307A, telomestatin, and TMPyP4 are equipotent against both c-myc and telomeric sequences while other ligands displayed some partial selectivity (2- to 6-fold) towards one of these sequences. This work provides evidence that G-quadruplex ligands reported as telomerase inhibitors efficiently stabilized c-myc promoter intramolecular quadruplex and may also potentially be used to inhibit c-myc gene transcription in tumor cells.
Assuntos
Proteínas de Ligação a DNA/química , Reação em Cadeia da Polimerase/métodos , Compostos de Quinolínio/química , Telomerase/química , Fatores de Transcrição/química , Triazinas/química , Sítios de Ligação , Ativação Enzimática , Inibidores Enzimáticos/química , Estabilidade Enzimática , Ligantes , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , Telomerase/antagonistas & inibidoresRESUMO
Optimization of the hydrophobic moiety of caprolactam/thiazepinone based compounds led to the identification of potent Src SH2 binders in two different series incorporating a phosphotyrosine group (RU 81843) or a phosphobenzoic group (RU 79181). The X-ray co-structures with the Src SH2 domain revealed different binding modes for RU 81843 and RU 79181, and an excellent fit between RU81843 and the Src SH2 protein thus explaining its high potency (9 nM, 15-fold more potent than pYEEI reference peptide).
Assuntos
Benzoatos/metabolismo , Caprolactama/metabolismo , Compostos Organofosforados/metabolismo , Fosfotirosina/metabolismo , Domínios de Homologia de src , Benzoatos/química , Caprolactama/análogos & derivados , Caprolactama/química , Estrutura Molecular , Compostos Organofosforados/química , Fosfotirosina/análogos & derivados , Fosfotirosina/química , Relação Estrutura-Atividade , Difração de Raios XRESUMO
The Src-SH2 domain has been determined to play a key role in many signaling pathways, especially in osteoclast-mediated bone resorption. Therefore, non-peptidic small molecules, mimicking the natural pYEEI peptide ligand, have been designed, to inhibit SH2-mediated protein-protein interactions and provide therapeutic treatment of certain diseases such as osteoporosis. However it has been shown in vitro that phosphopeptidic ligands of the SH2 domain are able to increase Src kinase activity by disrupting the intramolecular interactions between the Tyr(521)-phosphorylated C-terminal tail and the SH2 domain, thereby inducing a change from a "closed" inactive to an "open" active conformation of Src. Thus it was not clear whether non-peptidic ligands would limit their action to the inhibition of the signaling cascade by interfering with the intermolecular SH2 binding, or would activate the enzyme as do phosphopeptides. To address this question we have investigated the effects of a series of both peptidic and non-peptidic ligands of the SH2 domain on Src kinase activation, both in vitro in an ELISA based assay and in vivo using csk and src double transformed Schizosaccharomyces pombe. We found that, in the peptide series, the extent of c-Src activation is directly correlated to the respective binding affinity for Src-SH2. By contrast such correlation is not valid for non-peptidic ligands, some high-affinity SH2 binders showing no detectable Src activation in vivo. These results have significant implications for the design of SH2 binders, as they allow a way to inhibit Src-SH2-mediated signal transduction in target cells, without activating Src in non-target cells, thereby reducing the possibility of side effects.
Assuntos
Inibidores Enzimáticos/metabolismo , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Tirosina/metabolismo , Animais , Proteína Tirosina Quinase CSK , Ativação Enzimática , Inibidores Enzimáticos/química , Ensaio de Imunoadsorção Enzimática , Humanos , Ligantes , Peptídeos/química , Peptídeos/metabolismo , Fosforilação , Proteínas Tirosina Quinases/genética , Ratos , Schizosaccharomyces/genética , Transformação Genética , Domínios de Homologia de src , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
The enzyme telomerase is involved in the replication of telomeres, specialized structures that cap and protect the ends of chromosomes. Its activity is required for maintenance of telomeres and for unlimited lifespan, a hallmark of cancer cells. Telomerase is overexpressed in the vast majority of human cancer cells and therefore represents an attractive target for therapy. Several approaches have been developed to inhibit this enzyme through the targeting of its RNA or catalytic components as well as its DNA substrate, the single-stranded 3'-telomeric overhang. Telomerase inhibitors are chemically diverse and include modified oligonucleotides as well as small diffusable molecules, both natural and synthetic. This review presents an update of recent investigations pertaining to these agents and discusses their biological properties in the context of the initial paradigm that the exposure of cancer cells to these agents should lead to progressive telomere shortening followed by a delayed growth arrest response.