Targeted inhibition of Snail family zinc finger transcription factors by oligonucleotide-Co(III) Schiff base conjugate.
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ABSTRACT:
SUBMITTER: Harney AS
PROVIDER: S-EPMC2728951 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Snail family proteins are core EMT (epithelial-mesenchymal transition) regulatory factors that play essential roles in both development and disease processes and have been associated with metastasis in carcinomas. Snail factors are required for the formation of neural crest stem cells in most vertebrate embryos, as well as for the migratory invasive behavior of these cells. Snail factors have recently been linked to the formation of cancer stem cells, and expression of Snail proteins may be associated with tumor recurrence and resistance to chemotherapy and radiotherapy. We report that Co(III)-Ebox is a potent inhibitor of Snail-mediated transcriptional repression in breast cancer cells and in the neural crest of Xenopus. We further show that the activity of Co(III)-Ebox can be modulated by temperature, increasing the utility of this conjugate as a Snail inhibitor in model organisms. We exploit this feature to further delineate the requirements for Snail function during neural crest development, showing that in addition to the roles that Snail factors play in neural crest precursor formation and neural crest EMT/migration, inhibition of Snail function after the onset of neural crest migration leads to a loss of neural crest derived melanocytes. Co(III)-Ebox-mediated inhibition therefore provides a powerful tool for analysing the function of these core EMT factors with unparalleled temporal resolution. Moreover, the potency of Co(III)-Ebox as a Snail inhibitor in breast cancer cells suggests its potential as a therapeutic inhibitor of tumor progression and metastasis.
| S-EPMC3290632 | biostudies-literature
Project description:Transcription factors are key regulators in both normal and pathological cell processes. Affecting the activity of these proteins is a promising strategy for understanding gene regulation and developing effective therapeutics. Co(III) Schiff base complexes ([Co(acacen)(L)2](+) where L=labile axial ligands) have been shown to be potent inhibitors of a number of zinc metalloproteins including Cys2His2 zinc finger transcription factors. Inhibition by [Co(acacen)(L)2](+) of the target protein is believed to occur through a dissociative exchange of the labile axial ligands for histidine (His) residues essential for function. Here, we report a series of spectroscopic investigations with model peptides of zinc fingers that elucidate the interaction between [Co(acacen)(L)2](+) complexes and zinc finger transcription factors. Observed changes in NMR chemical shifts and 2D (1)H-(1)H NOESY NMR spectra demonstrate the preference of [Co(acacen)(L)2](+) complexes to coordinate His residues over other amino acids. The conformation of [Co(acacen)(L)2](+) upon His coordination was characterized by (1)H NMR spectroscopy, near-UV CD, and electronic absorption. These studies reveal that the resulting His-coordinated [Co(acacen)(L)2](+) complex possesses an octahedral structure. The effects of [Co(acacen)(L)2](+) complexes on the zinc-finger structure were assessed by the degree of hydrogen bonding (probed by 2D NMR spectroscopy) and secondary-structure profiles measured by far-UV CD. These structural studies demonstrate the ability of [Co(acacen)(L)2](+) complexes to disrupt the ??? structure of zinc fingers, resulting in primarily random-coil conformations. A mechanism is described wherein [Co(acacen)(L)2](+) complexes inhibit zinc finger transcription factor activity through selectively coordinating His residues in the zinc finger by dissociative ligand exchange and disrupting the ??? structural motif required for gene regulation.
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Project description:The transition from a non-invasive to an invasive phenotype is an essential step in tumor metastasis. The Snail family of transcription factors (TFs) is known to play a significant role in this transition. These TFs are zinc fingers that bind to the CAGGTG Ebox consensus sequence. Co(III) -Ebox is a cobalt(III) complex attached to an Ebox oligonucleotide that confers specificity towards Snail TFs. Co(III) -Ebox has been shown to inhibit Snail-mediated embryonic neural crest development in Xenopus laevis, but its efficacy in inhibiting Snail-induced cancer cell invasiveness has not been explored. Here, we describe the efficacy of Co(III) -Ebox in inhibiting the invasive aspects of heregulin-?1(HRG)-treated breast cancer cells. Co(III) -Ebox was found to inhibit the capacity of Snail to repress target genes after HRG induction. Snail inhibition by Co(III) -Ebox reduced the invasive propensity of cells in 2D and 3D, thereby demonstrating promise in inhibiting metastasis.
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Project description:We describe the use of Co(III) Schiff base-DNA conjugates, a versatile class of research tools that target C2H2 transcription factors, to inhibit the Hedgehog (Hh) pathway. In developing mammalian embryos, Hh signaling is critical for the formation and development of many tissues and organs. Inappropriate activation of the Hedgehog (Hh) pathway has been implicated in a variety of cancers including medulloblastomas and basal cell carcinomas. It is well-known that Hh regulates the activity of the Gli family of C2H2 zinc finger transcription factors in mammals. In Drosophila the function of the Gli proteins is performed by a single transcription factor with an identical DNA binding consensus sequence, Cubitus Interruptus (Ci). We have demonstrated previously that conjugation of a specific 17 base-pair oligonucleotide to a Co(III) Schiff base complex results in a targeted inhibitor of the Snail family C2H2 zinc finger transcription factors. Modification of the oligonucleotide sequence in the Co(III) Schiff base-DNA conjugate to that of Ci's consensus sequence (Co(III)-Ci) generates an equally selective inhibitor of Ci. Co(III)-Ci irreversibly binds the Ci zinc finger domain and prevents it from binding DNA in vitro. In a Ci responsive tissue culture reporter gene assay, Co(III)-Ci reduces the transcriptional activity of Ci in a concentration dependent manner. In addition, injection of wild-type Drosophila embryos with Co(III)-Ci phenocopies a Ci loss of function phenotype, demonstrating effectiveness in vivo. This study provides evidence that Co(III) Schiff base-DNA conjugates are a versatile class of specific and potent tools for studying zinc finger domain proteins and have potential applications as customizable anticancer therapeutics.
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Project description:Four cobalt(iii) complexes of the general formula [Co(Schiff base)(L)2]+, where L is ammonia (NH3) or 3-fluorobenzylamine (3F-BnNH2), were synthesized. The complexes were characterized by NMR spectroscopy, mass spectrometry, and X-ray crystallography. Their electrochemical properties, ligand substitution mechanisms, and ligand exchange rates in aqueous buffer were investigated. These physical properties were correlated to the cellular uptake and anticancer activities of the complexes. The complexes undergo sequential, dissociative ligand substitution, with the exchange rates depending heavily on the axial ligands. Eyring analyses revealed that the relative ligand exchange rates were largely impacted by differences in the entropy, rather than enthalpy, of activation for the complexes. Performing the substitution reactions in the presence of ascorbate led to a change in the reaction profile and kinetics, but no change in the final product. The cytotoxic activity of the complexes correlates with both the ligand exchange rate and reduction potential, with the more easily reduced and rapidly substituted complexes showing higher toxicity. These relationships may be valuable for the rational design of Co(iii) complexes as anticancer or antiviral prodrugs.
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Project description:Cobalt(III) Schiff base complexes have been used as potent inhibitors of protein function through the coordination to histidine residues essential for activity. The kinetics and thermodynamics of the binding mechanism of Co(acacen)(NH(3))(2)Cl [Co(acacen); where H(2)acacen is bis(acetylacetone)ethylenediimine] enzyme inhibition has been examined through the inactivation of matrix metalloproteinase 2 (MMP-2) protease activity. Co(acacen) is an irreversible inhibitor that exhibits time- and concentration-dependent inactivation of MMP-2. Co(acacen) inhibition of MMP-2 is temperature-dependent, with the inactivation increasing with temperature. Examination of the formation of the transition state for the MMP-2/Co(acacen) complex was determined to have a positive entropy component indicative of greater disorder in the MMP-2/Co(acacen) complex than in the reactants. With further insight into the mechanism of Co(acacen) complexes, Co(III) Schiff base complex protein inactivators can be designed to include features regulating activity and protein specificity. This approach is widely applicable to protein targets that have been identified to have clinical significance, including matrix metalloproteinases. The mechanistic information elucidated here further emphasizes the versatility and utility of Co(III) Schiff base complexes as customizable protein inhibitors.
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Project description:Cobalt(III) Schiff base complexes ([Co(acacen)(L)2](+), where L = NH3) inhibit histidine-containing proteins through dissociative exchange of the labile axial ligands (L). This work investigates axial ligand exchange dynamics of [Co(acacen)(L)2](+) complexes toward the development of protein inhibitors that are activated by external triggers such as light irradiation. We sought to investigate ligand exchange dynamics to design a Co(III) complex that is substitutionally inert under normal physiological conditions for selective activation. Fluorescent imidazoles (C3Im) were prepared as axial ligands in [Co(acacen)(L)2](+) to produce complexes (CoC3Im) that could report on ligand exchange and, thus, complex stability. These fluorescent imidazole reporters guided the design of a new dinuclear Co(III) Schiff base complex containing bridging diimidazole ligands, which exhibits enhanced stability to ligand exchange with competing imidazoles and to hydrolysis within a biologically relevant pH range. These studies inform the design of biocompatible Co(III) Schiff base complexes that can be selectively activated for protein inhibition with spatial and temporal specificity.
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