RESUMO
Recent studies have identified and characterized a novel putative transcriptional repressor site in a 5' untranslated region of the Aγ-globin gene that interacts with the Ly-1 antibody reactive clone (LYAR) protein. LYAR binds the 5'-GGTTAT-3' site of the Aγ-globin gene, and this molecular interaction causes repression of gene transcription. In ß-thalassemia patients, a polymorphism has been demonstrated (the rs368698783 G>A polymorphism) within the 5'-GGTTAT-3' LYAR-binding site of the Aγ-globin gene. The major results gathered from surface plasmon resonance based biospecific interaction analysis (SPR-BIA) studies (using crude nuclear extracts, LYAR-enriched lysates, and recombinant LYAR) support the concept that the rs368698783 G>A polymorphism of the Aγ-globin gene attenuates the efficiency of LYAR binding to the LYAR-binding site. This conclusion was fully confirmed by a molecular docking analysis. This might lead to a very important difference in erythroid cells from ß-thalassemia patients in respect to basal and induced levels of production of fetal hemoglobin. The novelty of the reported SPR-BIA method is that it allows the characterization and validation of the altered binding of a key nuclear factor (LYAR) to mutated LYAR-binding sites. These results, in addition to theoretical implications, should be considered of interest in applied pharmacology studies as a basis for the screening of drugs able to inhibit LYAR-DNA interactions. This might lead to the identification of molecules facilitating induced increase of γ-globin gene expression and fetal hemoglobin production in erythroid cells, which is associated with possible reduction of the clinical severity of the ß-thalassemia phenotype. Graphical abstract.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Mutação , Proteínas Nucleares/metabolismo , Polimorfismo Genético , Ressonância de Plasmônio de Superfície/métodos , Talassemia beta/genética , gama-Globinas/genética , Sítios de Ligação , Células HEK293 , Humanos , Células K562 , Simulação de Acoplamento Molecular , Ligação Proteica , gama-Globinas/metabolismoRESUMO
Overcoming the silencing of the fetal γ-globin gene has been a long-standing goal in the treatment of sickle cell disease (SCD). The major transcriptional enhancer of the ß-globin locus, called the locus control region (LCR), dynamically interacts with the developmental stage-appropriate ß-type globin genes via chromatin looping, a process requiring the protein Ldb1. In adult erythroid cells, the LCR can be redirected from the adult ß- to the fetal γ-globin promoter by tethering Ldb1 to the human γ-globin promoter with custom-designed zinc finger (ZF) proteins (ZF-Ldb1), leading to reactivation of γ-globin gene expression. To compare this approach to pharmacologic reactivation of fetal hemoglobin (HbF), hematopoietic cells from patients with SCD were treated with a lentivirus expressing the ZF-Ldb1 or with chemical HbF inducers. The HbF increase in cells treated with ZF-Ldb1 was more than double that observed with decitabine and pomalidomide; butyrate had an intermediate effect whereas tranylcypromine and hydroxyurea showed relatively low HbF reactivation. ZF-Ldb1 showed comparatively little toxicity, and reduced sickle hemoglobin (HbS) synthesis as well as sickling of SCD erythroid cells under hypoxic conditions. The efficacy and low cytotoxicity of lentiviral-mediated ZF-Ldb1 gene transfer compared with the drug regimens support its therapeutic potential for the treatment of SCD.
Assuntos
Anemia Falciforme/metabolismo , Cromatina/metabolismo , Hemoglobina Fetal/metabolismo , Adulto , Antígenos CD34/metabolismo , Proteínas de Ligação a DNA , Células Eritroides/metabolismo , Hemoglobina Falciforme , Humanos , Proteínas com Domínio LIM , Fatores de Transcrição , Dedos de ZincoRESUMO
The work describes a new low-impact synthetic route to Pt(ii)-carboxylate complexes, a class of compounds provided with established anticancer activity. The process is based on the ligand substitution on [PtCO3(Me2SO-S)2] (), a new synthon that can be easily prepared in water with high yield, is stable as a solid, and is reactive in solution where all its ligands can be easily replaced. It reacts with acidic O-donors releasing CO2 as the only side-product, whose development also supplies a driving force toward the products. The substitution of carbonate led to new Pt-DMSO carboxylate complexes , while the total substitution of the ligands of complex gave new Pt-phosphino carboxylates in high yields. The X-ray crystal structures of complexes [Pt(d(-)-quinate-O,O')(Me2SO-S)2] (), [Pt(salicylate)(Me2SO-S)2] () and [Pt(salicylate)(PPh3)2] () were determined. The tests of the antiproliferative activity of complexes on two human tumoral cell lines, A2780 (cisplatin-sensitive) and SKOV-3 (cisplatin-resistant), showed that the PTA (PTA = 1,3,5-triaza-7-phosphaadamantane) complexes were the most active on both cell lines.
Assuntos
Antineoplásicos/síntese química , Carbonatos/química , Ácidos Carboxílicos/síntese química , Dimetil Sulfóxido/química , Compostos Organoplatínicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Carbonatos/síntese química , Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Técnicas de Química Sintética , Cristalografia por Raios X , Dimetil Sulfóxido/síntese química , Humanos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Compostos Organoplatínicos/química , Compostos Organoplatínicos/farmacologiaRESUMO
In the treatment of hemoglobinopathies, amending altered hemoglobins and/or globins produced in excess is an important part of therapeutic strategies and the selective inhibition of globin production may be clinically beneficial. Therefore the development of drug-based methods for the selective inhibition of globin accumulation is required. In this study, we employed peptide nucleic acids (PNAs) to alter globin gene expression. The main conclusion of the present study was that PNAs designed to target adult murine ß-globin mRNA inhibit hemoglobin accumulation and erythroid differentiation of murine erythroleukemia (MEL) cells with high efficiency and fair selectivity. No major effects were observed on cell proliferation. Our study supports the concept that PNAs may be used to target mRNAs that, similar to globin mRNAs, are expressed at very high levels in differentiating erythroid cells. Our data suggest that PNAs inhibit the excess production of globins involved in the pathophysiology of hemoglobinopathies.