RESUMO
The binding of nuclear factor Y (NF-Y) to inverted CCAAT boxes (ICBs) within the promoter region of DNA topoisomerase IIα results in control of cell differentiation and cell cycle progression. Thus, NF-Y inhibitory small molecules could be employed to inhibit the replication of cancer cells. A library of pyrrolobenzodiazepine (PBD) C8-conjugates consisting of one PBD unit attached to tri-heterocyclic polyamide fragments was designed and synthesized. The DNA-binding affinity and sequence selectivity of each compound were evaluated in DNA thermal denaturation and DNase I footprinting assays, and the ability to inhibit binding of NF-Y to ICB1 and ICB2 was studied using an electrophoretic mobility shift assay (EMSA). 3a was found to be a potent inhibitor of NF-Y binding, exhibiting a 10-fold selectivity for an ICB2 site compared to an ICB1-containing sequence, and showing low nanomolar cytotoxicity toward human tumor cell lines. Molecular modeling and computational studies have provided details of the covalent attachment process that leads to formation of the PBD-DNA adduct, and have allowed the preference of 3a for ICB2 to be rationalized.
Assuntos
Benzodiazepinas/química , DNA/metabolismo , Nylons/química , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , DNA/química , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Camundongos , Modelos Moleculares , Células NIH 3T3 , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
A series of novel DNA-interactive C8-linked pyrrolobenzodiazepine (PBD)-heterocycle polyamide conjugates has been synthesised to explore structure/sequence-selectivity relationships. One conjugate (2d) has a greater selectivity and DNA binding affinity for inverted CCAAT sequences within the Topoisomerase IIα promoter than the known C8-bis-pyrrole PBD conjugate GWL-78 (1b).
Assuntos
Amidas/química , Benzodiazepinas/química , Regiões Promotoras Genéticas , Pirróis/química , Amidas/síntese química , Amidas/metabolismo , Antígenos de Neoplasias/genética , Sequência de Bases , Benzodiazepinas/síntese química , Benzodiazepinas/metabolismo , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/genética , Modelos Moleculares , Regiões Promotoras Genéticas/genética , Ligação Proteica , Pirróis/síntese química , Pirróis/metabolismo , Relação Estrutura-AtividadeRESUMO
1. Manipulation of micro opioid receptor expression either by chronic morphine treatment or by deletion of the gene encoding micro opioid receptors leads to changes in adenosine receptor expression. Chronic administration of the opioid receptor antagonist naltrexone leads to upregulation of micro receptor binding in the brain. 2. To investigate if there are any compensatory alterations in adenosine systems in the brains of chronic naltrexone-treated mice, we carried out quantitative autoradiographic mapping of A(1) and A(2A) adenosine receptors in the brains of mice treated for 1 week with naltrexone (8 mg(-1) kg(-1) day(-1)), administered subcutaneously via osmotic minipump. 3. Adjacent coronal brain sections were cut from chronic saline- and naltrexone-treated mice for the determination of binding of [(3)H] D-Ala(2)-MePhe(4)-Gly-ol(5) enkephalin ([(3)H] DAMGO), [(3)H]1,3-dipropyl-8-cyclopentylxanthine ([(3)H] DPCPX) or [(3)H] 2-[p-(2-carbonylethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine ([(3)H] CGS21680) to micro, A(1) and A(2A) receptors, respectively. 4. A significant increase in micro and A(1) receptor binding was detected in chronic naltrexone-treated brains. The changes in micro receptors were significant in several regions, but changes in A(1) were relatively smaller but showed significant upregulation collectively. No significant change in A(2A) receptor binding was detected in chronic naltrexone-treated brains. 5. The results show that blockade of opioid receptors causes upregulation of A(1) receptors, but not A(2A) receptors, by as yet undefined mechanisms.
