RESUMO
Modulation of protein-protein interactions (PPIs) by small molecules has emerged as a valuable approach in drug discovery. Compared to direct inhibition, PPI stabilization is vastly underexplored but has strong advantages, including the ability to gain selectivity by targeting an interface formed only upon association of proteins. Here, we present the application of a site-directed screening technique based on disulfide trapping (tethering) to select for fragments that enhance the affinity between protein partners. We target the phosphorylation-dependent interaction between the hub protein 14-3-3σ and a peptide derived from Estrogen Receptor α (ERα), an important breast cancer target that is negatively regulated by 14-3-3σ. We identify orthosteric stabilizers that increase 14-3-3/ERα affinity up to 40-fold and propose the mechanism of stabilization based on X-ray crystal structures. These fragments already display partial selectivity toward ERα-like motifs over other representative 14-3-3 clients. This first of its kind study illustrates the potential of the tethering approach to overcome the hurdles in systematic PPI stabilizer discovery.
Assuntos
Proteínas 14-3-3/química , Neoplasias da Mama/química , Descoberta de Drogas , Receptor alfa de Estrogênio/química , Proteínas 14-3-3/metabolismo , Neoplasias da Mama/metabolismo , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Modelos Moleculares , Fosforilação , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Estabilidade Proteica/efeitos dos fármacosRESUMO
Proteins typically interact with multiple binding partners, and often different parts of their surfaces are employed to establish these protein-protein interactions (PPIs). Members of the class of 14-3-3 adapter proteins bind to several hundred other proteins in the cell. Multiple small molecules for the modulation of 14-3-3 PPIs have been disclosed; however, they all target the conserved phosphopeptide binding channel, so that selectivity is difficult to achieve. Here we report on the discovery of two individual secondary binding sites that have been identified by combining nuclear magnetic resonance-based fragment screening and X-ray crystallography. The two pockets that these fragments occupy are part of at least three physiologically relevant and structurally characterized 14-3-3 PPI interfaces, including those with serotonin N-acetyltransferase and plant transcription factor FT. In addition, the high degree of conservation of the two sites implies their relevance for 14-3-3 PPIs. This first identification of secondary sites on 14-3-3 proteins bound by small molecule ligands might facilitate the development of new chemical tool compounds for more selective PPI modulation.
Assuntos
Proteínas 14-3-3/metabolismo , Biomarcadores Tumorais/metabolismo , Exorribonucleases/metabolismo , Modelos Moleculares , Fatores de Transcrição/metabolismo , Proteínas 14-3-3/química , Proteínas 14-3-3/genética , Aciltransferases , Sequência de Aminoácidos , Sítios de Ligação , Biomarcadores Tumorais/química , Biomarcadores Tumorais/genética , Sequência Conservada , Cristalografia por Raios X , Exorribonucleases/química , Exorribonucleases/genética , Deleção de Genes , Humanos , Cinética , Ligantes , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Biblioteca de Peptídeos , Fosforilação , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Binding of tyrosine kinase inhibitors such as imatinib was shown to induce a novel open-inhibited conformation of BCR-ABL, in which Tyr245 is exposed and prone to phosphorylation. To evaluate whether this leads to priming of the kinase in cellular systems, we probed activation of downstream signaling as a result of Tyr245 phosphorylation in a series of cellular washout experiments. While a spike in Tyr245 phosphorylation was observed both in overexpression and endogenous settings, no induction of downstream signaling was detected, showing that the priming hypothesis is not relevant for the therapeutic situation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Transdução de Sinais , Tirosina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/química , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Fosforilação , Fator de Transcrição STAT5/metabolismo , Tirosina/químicaRESUMO
Successful treatment of chronic myelogenous leukemia is based on inhibitors binding to the ATP site of the deregulated breakpoint cluster region (Bcr)-Abelson tyrosine kinase (Abl) fusion protein. Recently, a new type of allosteric inhibitors targeting the Abl myristoyl pocket was shown in preclinical studies to overcome ATP-site inhibitor resistance arising in some patients. Using NMR and small-angle X-ray scattering, we have analyzed the solution conformations of apo Abelson tyrosine kinase (c-Abl) and c-Abl complexes with ATP-site and allosteric inhibitors. Binding of the ATP-site inhibitor imatinib leads to an unexpected open conformation of the multidomain SH3-SH2-kinase c-Abl core, whose relevance is confirmed by cellular assays on Bcr-Abl. The combination of imatinib with the allosteric inhibitor GNF-5 restores the closed, inactivated state. Our data provide detailed insights on the poorly understood combined effect of the two inhibitor types, which is able to overcome drug resistance.
Assuntos
Benzamidas/química , Proteínas de Fusão bcr-abl/química , Modelos Moleculares , Complexos Multiproteicos/química , Piperazinas/química , Conformação Proteica , Proteínas Proto-Oncogênicas c-abl/química , Proteínas Proto-Oncogênicas c-abl/metabolismo , Pirimidinas/química , Regulação Alostérica , Benzamidas/metabolismo , Isótopos de Carbono/análise , Escherichia coli , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Mesilato de Imatinib , Estrutura Molecular , Isótopos de Nitrogênio/análise , Ressonância Magnética Nuclear Biomolecular , Piperazinas/metabolismo , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Pirimidinas/metabolismo , Espalhamento a Baixo ÂnguloRESUMO
For a wide range of proteins of high interest, the major obstacle for NMR studies is the lack of an affordable eukaryotic expression system for isotope labeling. Here, a simple and affordable protocol is presented to produce uniform labeled proteins in the most prevalent eukaryotic expression system for structural biology, namely Spodoptera frugiperda insect cells. Incorporation levels of 80% can be achieved for (15)N and (13)C with yields comparable to expression in full media. For (2)H,(15)N and (2)H,(13)C,(15)N labeling, incorporation is only slightly lower with 75 and 73%, respectively, and yields are typically twofold reduced. The media were optimized for isotope incorporation, reproducibility, simplicity and cost. High isotope incorporation levels for all labeling patterns are achieved by using labeled algal amino acid extracts and exploiting well-known biochemical pathways. The final formulation consists of just five commercially available components, at costs 12-fold lower than labeling media from vendors. The approach was applied to several cytosolic and secreted target proteins.
Assuntos
Proteínas de Insetos/química , Marcação por Isótopo/métodos , Spodoptera/metabolismo , Aminoácidos/química , Animais , Carboidratos/química , Isótopos de Carbono/química , Isótopos de Nitrogênio/química , Ressonância Magnética Nuclear Biomolecular/métodos , Reprodutibilidade dos Testes , Spodoptera/citologiaRESUMO
Drug discovery is a complex process, and a variety of technologies contribute to its success. Biophysical methods have gained widespread attention within the last decade, and in particular NMR spectroscopy as the most versatile biophysical method has seen numerous applications and significant impact to drug discovery. Here we summarize the potential of NMR to support drug discovery, and highlight a number of recent applications.
Assuntos
Descoberta de Drogas/tendências , Ressonância Magnética Nuclear Biomolecular , Animais , Biofísica , HumanosRESUMO
Drug discovery is a complex process, and a variety of technologies contribute to its success. Biophysical methods have gained widespread attention within the last decade, and in particular NMR spectroscopy as the most versatile biophysical method has seen numerous applications and significant impact to drug discovery. Here we summarize the potential of NMR to support drug discovery, and highlight a number of recent applications.
RESUMO
Misfolding of the natively α-helical prion protein into a ß-sheet rich isoform is related to various human diseases such as Creutzfeldt-Jakob disease and Gerstmann-Sträussler-Scheinker syndrome. In humans, the disease phenotype is modified by a methionine/valine polymorphism at codon 129 of the prion protein gene. Using a combination of hydrogen/deuterium exchange coupled to NMR spectroscopy, hydroxyl radical probing detected by mass spectrometry, and site-directed mutagenesis, we demonstrate that stop mutants of the human prion protein have a conserved amyloid core. The 129 residue is deeply buried in the amyloid core structure, and its mutation strongly impacts aggregation. Taken together the data support a critical role of the polymorphic residue 129 of the human prion protein in aggregation and disease.
Assuntos
Amiloide/genética , Códon de Terminação/genética , Mutação/genética , Polimorfismo Genético , Príons/genética , Sequência de Aminoácidos , Centrifugação , Humanos , Metionina/genética , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Príons/química , Estrutura Quaternária de Proteína , SolventesRESUMO
Misfolding intermediates play a key role in defining aberrant protein aggregation and amyloid formation in more than 15 different human diseases. However, their experimental characterization is challenging due to the transient nature and conformational heterogeneity of the involved states. Here, we demonstrate that direct carbon-detected NMR experiments allow observation, assignment, and structural analysis of molten globule amyloid intermediates that are severely broadened by conformational exchange. The method is used to characterize the structure and dynamics of partially unfolded intermediates of the 99-residue protein beta-2-microglobulin, which is the major component of insoluble aggregates occurring in dialysis-related amyloidosis. Comparison of the conformational properties of the molten globule-like intermediates with levels of deuterium incorporation into amyloid fibrils of beta-2-microglobulin revealed a close relationship between the conformational properties of the metastable intermediates and the beta-sheet-rich insoluble aggregates of beta-2-microglobulin.
Assuntos
Amiloide/química , Desnaturação Proteica , Dobramento de Proteína , Microglobulina beta-2/química , Amiloidose , Medição da Troca de Deutério , Humanos , Espectroscopia de Ressonância Magnética/métodos , Conformação Proteica , Estabilidade Proteica , SolubilidadeRESUMO
In modern kinase drug discovery, allosteric inhibitors have become a focus of attention due to their potential selectivity, but such compounds are difficult to identify. Here we describe an NMR-based competition assay using 19F-containing reporter molecules, which allows for rapid identification and discrimination between ATP-competitive and allosteric kinase inhibitors. We illustrate the principle of such a dual-site competition assay with the example of catalytic and allosteric ABL1 kinase inhibitors. The assay can also be used to identify and characterize mixed binding modes of well-known drugs, as shown for crizotinib and fingolimod.
RESUMO
Protein targets of contemporary research are often membrane proteins, multiprotein complexes, secreted proteins, or other proteins of human origin. These are difficult to express in the standard expression host used for most nuclear magnetic resonance (NMR) studies, Escherichia coli. Insect cells represent an attractive alternative, since they have become a well-established expression system and simple solutions have been developed for generation of viruses to efficiently introduce the target protein DNA into cells. Insect cells enable production of a larger fraction of the human proteome in a properly folded way than bacteria, as insect cells have a very similar set of cytosolic chaperones and a closely related secretory pathway. Here, the limited and defined glycosylation pattern that insect cells produce is an advantage for structural biology studies. For these reasons, insect cells have been established as the most widely used eukaryotic expression host for crystallographic studies. In the past decade, significant advancements have enabled amino acid type-specific as well as uniform isotope labeling of proteins in insect cells, turning them into an attractive expression host for NMR studies.
Assuntos
Proteínas de Insetos/metabolismo , Insetos/metabolismo , Marcação por Isótopo , Animais , Baculoviridae/genética , Insetos/citologiaRESUMO
Amyloid fibrils are the pathological hallmark of a large variety of neurodegenerative disorders. The structural characterization of amyloid fibrils, however, is challenging due to their non-crystalline, heterogeneous, and often dynamic nature. Thus, the structure of amyloid fibrils of many proteins is still unknown. We here show that the structure calculation program CS-Rosetta can be used to obtain insight into the core structure of amyloid fibrils. Driven by experimental solid-state NMR chemical shifts and taking into account the polymeric nature of fibrils CS-Rosetta allows modeling of the core of amyloid fibrils. Application to the Y145X stop mutant of the human prion protein reveals a left-handed ß-helix.
Assuntos
Amiloide/química , Príons/química , Amiloide/genética , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Mutação Puntual , Príons/genética , Estrutura Secundária de Proteína , SoftwareRESUMO
Prion diseases are caused by the aggregation of the native alpha-helical prion protein PrP(C) into its pathological beta-sheet-rich isoform PrP(Sc). In current models of PrP(Sc), helix1 is assumed to be preferentially converted into beta-sheet during aggregation of PrP(C). This was supported by the NMR structure of PrP(C) since, in contrast to the isolated helix1, helix2 and helix3 are connected by a small loop and are additionally stabilized by an interhelical disulfide bond. However, helix1 is extremely hydrophilic and has a high helix propensity. This prompted us to investigate the role of helix1 in prion aggregation using humPrP(23-159) including helix1 (144-156) compared with the C-terminal-truncated isoform humPrP(23-144) corresponding to the pathological human stop mutations Q160Stop and Y145Stop, respectively. Most unexpectedly, humPrP(23-159) aggregated significantly faster compared with the truncated fragment humPrP(23-144), clearly demonstrating that helix1 is involved in the aggregation process. However, helix1 is not resistant to digestion with proteinase K in fibrillar humPrP(23-159), suggesting that helix1 is not converted to beta-sheet. This is confirmed by Fourier transformation infrared spectroscopy since there is almost no difference in beta-sheet content of humPrP(23-159) fibrils compared with humPrP(23-144). In conclusion, we provide strong direct evidence that in contrast to earlier assumptions helix1 is not converted into beta-sheet during aggregation of PrP(C) to PrP(Sc).