RESUMO
Keap1 binds to the Nrf2 transcription factor to promote its degradation, resulting in the loss of gene products that protect against oxidative stress. While cell-active small molecules have been identified that modify cysteines in Keap1 and effect the Nrf2 dependent pathway, few act through a non-covalent mechanism. We have identified and characterized several small molecule compounds that specifically bind to the Keap1 Kelch-DC domain as measured by NMR, native mass spectrometry and X-ray crystallography. One compound upregulates Nrf2 response genes measured by a luciferase cell reporter assay. The non-covalent inhibition strategy presents a reasonable course of action to avoid toxic side-effects due to non-specific cysteine modification.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas de Transporte , Cristalografia por Raios X , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteína 1 Associada a ECH Semelhante a Kelch , Fator 2 Relacionado a NF-E2/química , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-Atividade , TermodinâmicaRESUMO
We report the use of a fragment-based lead discovery method, Tethering with extenders, to discover a pyridinone fragment that binds in an adaptive site of the protein PDK1. With subsequent medicinal chemistry, this led to the discovery of a potent and highly selective inhibitor of PDK1, which binds in the 'DFG-out' conformation.
Assuntos
Desenho de Fármacos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Cristalografia por Raios X , Descoberta de Drogas , Inibidores Enzimáticos/química , Concentração Inibidora 50 , Modelos Biológicos , Estrutura Molecular , Piridonas/química , Piridonas/farmacologia , Piruvato Desidrogenase Quinase de Transferência de Acetil , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-AtividadeRESUMO
Therapeutic antibodies directed against the type 1 insulin-like growth factor receptor (IGF-1R) have recently gained significant momentum in the clinic because of preliminary data generated in human patients with cancer. These antibodies inhibit ligand-mediated activation of IGF-1R and the resulting down-stream signaling cascade. Here we generated a panel of antibodies against IGF-1R and screened them for their ability to block the binding of both IGF-1 and IGF-2 at escalating ligand concentrations (>1 microm) to investigate allosteric versus competitive blocking mechanisms. Four distinct inhibitory classes were found as follows: 1) allosteric IGF-1 blockers, 2) allosteric IGF-2 blockers, 3) allosteric IGF-1 and IGF-2 blockers, and 4) competitive IGF-1 and IGF-2 blockers. The epitopes of representative antibodies from each of these classes were mapped using a purified IGF-1R library containing 64 mutations. Most of these antibodies bound overlapping surfaces on the cysteine-rich repeat and L2 domains. One class of allosteric IGF-1 and IGF-2 blocker was identified that bound a separate epitope on the outer surface of the FnIII-1 domain. Using various biophysical techniques, we show that the dual IGF blockers inhibit ligand binding using a spectrum of mechanisms ranging from highly allosteric to purely competitive. Binding of IGF-1 or the inhibitory antibodies was associated with conformational changes in IGF-1R, linked to the ordering of dynamic or unstructured regions of the receptor. These results suggest IGF-1R uses disorder/order within its polypeptide sequence to regulate its activity. Interestingly, the activity of representative allosteric and competitive inhibitors on H322M tumor cell growth in vitro was reflective of their individual ligand-blocking properties. Many of the antibodies in the clinic likely adopt one of the inhibitory mechanisms described here, and the outcome of future clinical studies may reveal whether a particular inhibitory mechanism leads to optimal clinical efficacy.
Assuntos
Epitopos/química , Receptores de Somatomedina/química , Sítio Alostérico , Animais , Células CHO , Varredura Diferencial de Calorimetria , Cricetinae , Cricetulus , Mapeamento de Epitopos , Humanos , Fator de Crescimento Insulin-Like II/química , Cinética , Ligantes , Conformação Molecular , Receptor IGF Tipo 1/metabolismoRESUMO
The control of myelination by oligodendrocytes in the CNS is poorly understood. Here we show that LINGO-1 is an important negative regulator of this critical process. LINGO-1 is expressed in oligodendrocytes. Attenuation of its function by dominant-negative LINGO-1, LINGO-1 RNA-mediated interference (RNAi) or soluble human LINGO-1 (LINGO-1-Fc) leads to differentiation and increased myelination competence. Attenuation of LINGO-1 results in downregulation of RhoA activity, which has been implicated in oligodendrocyte differentiation. Conversely, overexpression of LINGO-1 leads to activation of RhoA and inhibition of oligodendrocyte differentiation and myelination. Treatment of oligodendrocyte and neuron cocultures with LINGO-1-Fc resulted in highly developed myelinated axons that have internodes and well-defined nodes of Ranvier. The contribution of LINGO-1 to myelination was verified in vivo through the analysis of LINGO-1 knockout mice. The ability to recapitulate CNS myelination in vitro using LINGO-1 antagonists and the in vivo effects seen in the LINGO-1 knockout indicate that LINGO-1 signaling may be critical for CNS myelination.