RESUMO
The p53 tumor suppressor protein is regulated by its interaction with HDM2, which serves as a ubiquitin ligase (E3) to target p53 for degradation. We have identified a family of small molecules (HLI98) that inhibits HDM2's E3 activity. These compounds show some specificity for HDM2 in vitro, although at higher concentrations effects on unrelated RING and HECT domain E3s are detectable, which could be due, at least in part, to effects on E2-ubiquitin thiol-ester levels. In cells, the compounds allow the stabilization of p53 and HDM2 and activation of p53-dependent transcription and apoptosis, although other p53-independent toxicity was also observed.
Assuntos
Inibidores Enzimáticos/farmacologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Quinase 4 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Quinases Ciclina-Dependentes/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte , Inibidores Enzimáticos/química , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Flavinas/química , Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Estrutura Molecular , Ubiquitina-Proteína Ligases Nedd4 , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas/antagonistas & inibidores , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2 , Transfecção , Proteína Supressora de Tumor p53/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The HIV-1 nucleocapsid (NC) protein is a small, basic protein containing two retroviral zinc fingers. It is a highly active nucleic acid chaperone; because of this activity, it plays a crucial role in virus replication as a cofactor during reverse transcription, and is probably important in other steps of the replication cycle as well. We previously reported that NC binds with high-affinity to the repeating sequence d(TG)n. We have now analyzed the interaction between NC and d(TG)4 in considerable detail, using surface plasmon resonance (SPR), tryptophan fluorescence quenching (TFQ), fluorescence anisotropy (FA), isothermal titration calorimetry (ITC) and electrospray ionization Fourier transform mass spectrometry (ESI-FTMS). Our results show that the interactions between these two molecules are surprisngly complex: while the K(d) for binding of a single d(TG)4 molecule to NC is only approximately 5 nM in 150 mM NaCl, a single NC molecule is capable of interacting with more than one d(TG)4 molecule, and conversely, more than one NC molecule can bind to a single d(TG)4 molecule. The strengths of these additional binding reactions are quantitated. The implications of this multivalency for the functions of NC in virus replication are discussed.
Assuntos
Proteínas do Capsídeo/química , Produtos do Gene gag/química , Oligodesoxirribonucleotídeos/química , Proteínas Virais/química , Sítios de Ligação , Ligação Competitiva , Calorimetria , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Fluorescência , Polarização de Fluorescência , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Mutação , Espectrometria de Massas por Ionização por Electrospray , Ressonância de Plasmônio de Superfície , Triptofano/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência HumanaRESUMO
The human immunodeficiency virus (HIV) epidemic is an important medical problem. Although combination drug regimens have produced dramatic decreases in viral load, current therapies do not provide a cure for HIV infection. We have used structure-based design and combinatorial medicinal chemistry to identify potent and selective HIV-1 reverse transcriptase (RT) inhibitors that may work by a mechanism distinct from that of current HIV drugs. The most potent of these compounds (compound 4, 2-naphthalenesulfonic acid, 4-hydroxy-7-[[[[5-hydroxy-6-[(4-cinnamylphenyl)azo]-7-sulfo-2-naphthalenyl]amino]carbonyl]amino]-3-[(4-cinnamylphenyl)azo], disodium salt) has an IC(50) of 90 nM for inhibition of polymerase chain extension, a K(d) of 40 nM for inhibition of DNA-RT binding, and an IC(50) of 25-100 nM for inhibition of RNaseH cleavage. The parent compound (1) was as effective against 10 nucleoside and non-nucleoside resistant HIV-1 RT mutants as it was against the wild-type enzyme. Compound 4 inhibited HIV-1 RT and murine leukemia virus (MLV) RT, but it did not inhibit T(4) DNA polymerase, T(7) DNA polymerase, or the Klenow fragment at concentrations up to 200 nM. Finally, compound 4 protected cells from HIV-1 infection at a concentration more than 40 times lower than the concentration at which it caused cellular toxicity.