RESUMO
We report first non-covalent and exclusively extracellular inhibitors of 14-3-3 protein-protein interactions identified by virtual screening. Optimization by crystal structure analysis and in vitro binding assays yielded compounds capable of disrupting the interaction of 14-3-3σ with aminopeptidase N in a cellular assay.
Assuntos
Proteínas 14-3-3/antagonistas & inibidores , Ensaios de Triagem em Larga Escala , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Dose-Resposta a Droga , Fibroblastos/química , Fibroblastos/citologia , Humanos , Pulmão/química , Pulmão/citologia , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismo , Modelos Moleculares , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Regulação para Cima/genéticaRESUMO
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
Dendrimers have several unique properties that make them attractive scaffolds for use in biomedical applications. To date, multivalent and multimodal dendritic structures have been synthesized predominantly by statistical modification of peripheral groups. However, the potential application of such probes in patients demands well-defined and monodisperse materials that have unique structures. Current progress in the field of chemical biology, in particular chemoselective ligation methods, renders this challenge possible. In this Minireview, we outline the different available synthetic strategies, some applications that already make use of this new generation of multivalent and multimodal architectures, and the challenges for future developments.
Assuntos
Materiais Biocompatíveis/síntese química , Engenharia Biomédica/métodos , Dendrímeros/síntese química , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Engenharia Biomédica/tendências , Dendrímeros/química , Dendrímeros/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Sistemas de Liberação de Medicamentos/tendências , Relação Estrutura-AtividadeRESUMO
Protein switches use the binding energy gained upon recognition of ligands to modulate the conformation and binding properties of protein segments. We explored whether the programmable nucleic acid mediated recognition might be used to design or mimic constraints that limit the conformational freedom of peptide segments. The aim was to design nucleic acid-peptide conjugates in which the peptide portion of the conjugate would change the affinity for a protein target upon hybridization. This approach was used to control the affinity of a PNA-phosphopeptide conjugate for the signal transduction protein Src kinase, which binds the cognate phosphopeptides in a linear conformation. Peptide-nucleic acid arms were attached to known peptide binders. The chimeric molecules were studied in three modes: 1) as single strands, 2) constrained by intermolecular hybridization (duplex formation) and 3) constrained by intramolecular hybridization (hairpin formation). Of note, duplexes that were designed to accommodate bulged peptide structures (for example, in hairpins or bulges) had lower binding affinities than duplexes in which the peptide was allowed to adopt a more relaxed conformation. Greater than 90-fold differences in binding affinities were observed. It was, thus, feasible to make use of DNA hybridization to reversibly switch from no to almost complete inhibition of Src-SH2-peptide binding, and vice versa. A series of DNA and PNA-based hybridization experiments revealed the importance of charges and conformational effects. Nucleic acid mediated switching was extended to the use of RNA; this enabled a regulation of the enzymatic activity of the Src kinase. The proof-of-principle results demonstrate for the first time that PNA-peptide chimeras can transduce changes of the concentration of a given RNA molecule to changes of the activity of a signal transduction enzyme.
Assuntos
DNA/metabolismo , Proteínas Quinases/metabolismo , RNA/metabolismo , Sequência de Aminoácidos , Sequência de Bases , DNA/genética , Ativação Enzimática , Hibridização de Ácido Nucleico , Ácidos Nucleicos Peptídicos/genética , Ácidos Nucleicos Peptídicos/metabolismo , Proteínas Quinases/química , RNA/genética , Transdução de Sinais , Quinases da Família src/metabolismoRESUMO
Oligonucleotide-peptide conjugates have frequently been used to control the localisation of the conjugate molecule. For example, the oligonucleotide segment has allowed spatially addressed immobilization of peptides and proteins on DNA-arrays via hybridisation while the peptide part has most frequently been used to confer transfer of oligonucleotide cargo into live cells. The regulation of functional properties such as the affinity of these bioconjugates for protein targets has rarely been addressed. This review article describes the current developments in the application of smart oligonucleotide-peptide hybrids. The mutual recognition between nucleic acid segments is used to constrain the structure or control the distance between peptide and protein segments. Application of these new type of oligonucleotide-peptide hybrids allowed not only the regulation of binding affinity of peptide ligands but also control of enzymatic and optical activity of proteins.
Assuntos
Ácidos Nucleicos/metabolismo , Peptídeos/metabolismo , Proteínas/metabolismo , Sequência de Bases , Metabolismo dos Carboidratos , Ligantes , Ácidos Nucleicos/genética , Proteínas/químicaRESUMO
The rules that govern the formation of DNA duplex structures are well known. On one hand, this process is used in the design of probe molecules that report the presence of target nucleic acids by responding to changes of structure and reactivity. On the other hand, molecules may be developed that transduce changes of nucleic acid structure to changes of peptide structure, and vice versa. Applications in the fields of bioanalytical chemistry and synthetic biology are discussed.
Assuntos
Sondas de Ácido Nucleico/química , Ácidos Nucleicos Peptídicos/química , Ativação Enzimática , Hibridização de Ácido Nucleico , Análise de Sequência de DNA , Domínios de Homologia de src , Quinases da Família src/metabolismoAssuntos
Antineoplásicos/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Netropsina/análogos & derivados , Netropsina/farmacologia , Proteína Supressora de Tumor p53/genética , Regulação para Cima/efeitos dos fármacos , Actinobacteria/química , Furanos , Estrutura Molecular , Netropsina/química , Netropsina/isolamento & purificaçãoRESUMO
The ability to accurately quantify specific nucleic acid molecules in complex biomolecule solutions in real time is important in diagnostic and basic research. Here we describe a DNA-PNA (peptide nucleic acid) hybridization assay that allows sensitive quantification of specific nucleic acids in solution and concomitant detection of select single base mutations in resulting DNA-PNA duplexes. The technique employs so-called FIT (forced intercalation) probes in which one base is replaced by a thiazole orange (TO) dye molecule. If a DNA molecule that is complementary to the FIT-PNA molecule (except at the site of the dye) hybridizes to the probe, the TO dye exhibits intense fluorescence because stacking in the duplexes enforces a coplanar arrangement even in the excited state. However, a base mismatch at either position immediately adjacent to the TO dye dramatically decreases fluorescence, presumably because the TO dye has room to undergo torsional motions that lead to rapid depletion of the excited state. Of note, we found that the use of d-ornithine rather than aminoethylglycine as the PNA backbone increases the intensity of fluorescence emitted by matched probe-target duplexes while specificity of fluorescence signaling under nonstringent conditions is also increased. The usefulness of the ornithine-containing FIT probes was demonstrated in the real-time PCR analysis providing a linear measurement range over at least seven orders of magnitude. The analysis of two important single nucleotide polymorphisms (SNPs) in the CFTR gene confirmed the ability of FIT probes to facilitate unambiguous SNP calls for genomic DNA by quantitative PCR.