RESUMO
Modulation of protein-protein interactions (PPIs) is becoming increasingly important in drug discovery and chemical biology. While a few years ago this 'target class' was deemed to be largely undruggable an impressing number of publications and success stories now show that targeting PPIs with small, drug-like molecules indeed is a feasible approach. Here, we summarize the current state of small-molecule inhibition and stabilization of PPIs and review the active molecules from a structural and medicinal chemistry angle, especially focusing on the key examples of iNOS, LFA-1 and 14-3-3.
Assuntos
Proteínas 14-3-3/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Humanos , Ligação Proteica/efeitos dos fármacos , Mapas de Interação de Proteínas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/químicaRESUMO
One-third of all human cancers harbor somatic RAS mutations. This leads to aberrant activation of downstream signaling pathways involving the RAF kinases. Current ATP-competitive RAF inhibitors are active in cancers with somatic RAF mutations, such as BRAF(V600) mutant melanomas. However, they paradoxically promote the growth of RAS mutant tumors, partly due to the complex interplay between different homo- and heterodimers of A-RAF, B-RAF, and C-RAF. Based on pathway analysis and structure-guided compound identification, we describe the natural product cotylenin-A (CN-A) as stabilizer of the physical interaction of C-RAF with 14-3-3 proteins. CN-A binds to inhibitory 14-3-3 interaction sites of C-RAF, pSer233, and pSer259, but not to the activating interaction site, pSer621. While CN-A alone is inactive in RAS mutant cancer models, combined treatment with CN-A and an anti-EGFR antibody synergistically suppresses tumor growth in vitro and in vivo. This defines a novel pharmacologic strategy for treatment of RAS mutant cancers.
Assuntos
Proteínas 14-3-3/metabolismo , Produtos Biológicos/uso terapêutico , Diterpenos/uso terapêutico , Neoplasias/tratamento farmacológico , Quinases raf/metabolismo , Animais , Produtos Biológicos/farmacologia , Linhagem Celular Tumoral , Diterpenos/farmacologia , Humanos , Camundongos , Camundongos SCID , Modelos Moleculares , Neoplasias/genética , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-raf/metabolismo , Quinases raf/genética , Proteínas ras/genéticaRESUMO
Chemically induced dimerization is an important tool in chemical biology for the analysis of protein function in cells. Here we report the use of the natural product fusicoccin (FC) to induce dimerization of 14-3-3-fused target proteins with proteins tagged to the C terminus (CT) of the H(+)-ATPase PMA2. To prevent nonproductive or detrimental interactions of the 14-3-3 proteins and CT fusions with endogenous cell proteins, their interaction surface was engineered to facilitate FC-induced dimerization exclusively between the introduced protein constructs. Live-cell imaging documented the reversible FC-induced translocation of 14-3-3 and CT to different cell compartments depending on localization sequences fused to their dimerization partner protein. The functionality of this system was demonstrated by the FC-induced importation of the NF-κB-CT into the nucleus. In HeLa cells, FC-mediated dimerization of the NF-κB-CT with a constitutively nuclear-localized 14-3-3 protein led to an NF-κB-specific cellular response by inducing IL-8 secretion.
Assuntos
Glicosídeos/farmacologia , NF-kappa B/química , Multimerização Proteica/efeitos dos fármacos , Transdução de Sinais , Proteínas 14-3-3/química , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Sítios de Ligação , Far-Western Blotting , Núcleo Celular/metabolismo , Glicosídeos/química , Células HEK293 , Células HeLa , Humanos , Interleucina-8/metabolismo , Cinética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Modelos Moleculares , Estrutura Molecular , NF-kappa B/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Fatores de TempoRESUMO
The 14-3-3 proteins are a class of eukaryotic acidic adapter proteins, with seven isoforms in humans. 14-3-3 proteins mediate their biological function by binding to target proteins and influencing their activity. They are involved in pivotal pathways in the cell such as signal transduction, gene expression, enzyme activation, cell division and apoptosis. The Yes-associated protein (YAP) is a WW-domain protein that exists in two transcript variants of 48 and 54 kDa in humans. By transducing signals from the cytoplasm to the nucleus, YAP is important for transcriptional regulation. In both variants, interaction with 14-3-3 proteins after phosphorylation of Ser127 is important for nucleocytoplasmic trafficking, via which the localization of YAP is controlled. In this study, 14-3-3σ has been cloned, purified and crystallized in complex with a phosphopeptide from the YAP 14-3-3-binding domain, which led to a crystal that diffracted to 1.15 A resolution. The crystals belonged to space group C222(1), with unit-cell parameters a=82.3, b=112.1, c=62.9 A.