RESUMO
Fragment-based drug design consists of identifying low-molecular weight compounds that weakly bind to a target macromolecule and will then be modified or linked to yield potent inhibitors. The specificity of these low-complexity and low-affinity molecules has rarely been discussed in the literature. To address this question, NMR spectroscopy was used to investigate the interactions of 150 fragments with five proteins: three proteins from the Bcl-2 family (Bcl-x(L), Bcl-w, and Mcl-1), human peroxiredoxin 5, for which very few ligands have been reported, and human serum albumin, which is known to bind a large number of ligands. Our results show that the fragments are rather versatile binders and able to identify binding hot spots in very different targets. Despite the different hit rates observed related to the druggability of the proteins, two scaffolds appear as preferred binders for all proteins. Low specificity was observed between homologous proteins or unrelated poorly druggable proteins, while higher specificity could be achieved with highly druggable targets.
Assuntos
Desenho de Fármacos , Ligantes , Peroxirredoxinas/química , Preparações Farmacêuticas/química , Proteínas Proto-Oncogênicas c-bcl-2/química , Albumina Sérica/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética , Ligação Proteica , Solubilidade , Relação Estrutura-AtividadeRESUMO
The search for protein ligands is a crucial step in the inhibitor design process. Fragment screening represents an interesting method to rapidly find lead molecules, as it enables the exploration of a larger portion of the chemical space with a smaller number of compounds as compared to screening based on drug-sized molecules. Moreover, fragment screening usually leads to hit molecules that form few but optimal interactions with the target, thus displaying high ligand efficiencies. Here we report the screening of a homemade library composed of 200 highly diverse fragments against the human Peroxiredoxin 5 protein. Peroxiredoxins compose a family of peroxidases that share the ability to reduce peroxides through a conserved cysteine. The three-dimensional structures of these enzymes ubiquitously found throughout evolution have been extensively studied, however, their biological functions are still not well understood and to date few inhibitors have been discovered against these enzymes. Six fragments from the library were shown to bind to the Peroxiredoxin 5 active site and ligand-induced chemical shift changes were used to drive the docking of these small molecules into the protein structure. The orientation of the fragments in the binding pocket was confirmed by the study of fragment homologues, highlighting the role of hydroxyl functions that hang the ligands to the Peroxiredoxin 5 protein. Among the hit fragments, the small catechol molecule was shown to significantly inhibit Peroxiredoxin 5 activity in a thioredoxin peroxidase assay. This study reports novel data about the ligand-Peroxiredoxin interactions that will help considerably the development of potential Peroxiredoxin inhibitors.
Assuntos
Peroxirredoxinas/química , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X/métodos , Cisteína/química , Humanos , Cinética , Ligantes , Espectroscopia de Ressonância Magnética/métodos , Modelos Químicos , Modelos Moleculares , Peroxidases/química , Ligação Proteica , Conformação ProteicaRESUMO
Fragment-based drug design consists of screening low-molecular-weight compounds in order to identify low-affinity ligands that are then modified or linked to yield potent inhibitors. The method thus attempts to build bioactive molecules in a modular way and relies on the hypothesis that the fragment binding mode will be conserved upon elaboration of the active molecule. If the inverse process is considered, do the fragments resulting from the deconstruction of high-affinity inhibitors recapitulate their binding mode in the large molecule? Few studies deal with this issue. Here, we report the analysis of 22 fragments resulting from the dissection of 9 inhibitors of the antiapoptotic protein Bcl-x(L). To determine if the fragments retained affinity toward the protein and identify their binding site, ligand-observed and protein-observed NMR experiments were used. The analysis of the fragments behavior illustrates the complexity of low-affinity protein-ligand interactions involved in the fragment-based construction of bioactive molecules.
Assuntos
Compostos Orgânicos/química , Compostos Orgânicos/farmacologia , Proteína bcl-X/antagonistas & inibidores , Sítios de Ligação , Ligação Competitiva , Desenho de Fármacos , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Peso Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Relação Estrutura-Atividade , Proteína bcl-X/química , Proteína bcl-X/genéticaRESUMO
The binding of phosphorylated peptides to the receptor plays a major role in many basic cellular processes in a variety of pathological states. Human beta-TrCP is a key component of a recently characterized E3 ubiquitin ligase complex that regulates protein degradation through the ubiquitin-dependent proteasome pathway. Docking studies were carried out to explore the structural requirements for the beta-TrCP substrates. Docking studies were performed on the bound conformation of the phosphorylated peptides determined by NMR, whereas the beta-TrCP structure was derived by X-ray from Protein Data Bank. After the docking calculation, during which the peptides were conformationally restrained, the complex presented herein was analyzed in terms of ligand-protein interactions and properties of contacting surfaces. The structural requirements for phosphorylated substrates in interaction with beta-TrCP were explored and compared with experimental data from TRNOESY and STD NMR results. The analysis revealed that the bend of the peptide structures, which is indispensable for beta-TrCP recognition, aligns two charged phosphate groups and a central hydrophobic group in a favorable arrangement that leads to the burial of the peptide surface in the binding cleft upon complexation. Through docking simulations, we have identified different specific binding pockets of beta-TrCP according to the ligand in interaction. These data should be valuable in the rational design of a ligand to be used in therapeutic approaches.
Assuntos
Proteínas Ligases SKP Culina F-Box/química , Sequência de Aminoácidos , Domínio Catalítico , Simulação por Computador , Sequência Consenso , Cristalografia por Raios X , Humanos , Técnicas In Vitro , Ligantes , Substâncias Macromoleculares/química , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Fosforilação , Proteínas Ligases SKP Culina F-Box/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , beta Catenina/química , beta Catenina/genética , beta Catenina/metabolismoRESUMO
ATF4 plays a crucial role in the cellular response to stress. The E3 ubiquitin ligase, SCF beta-TrCP protein responsible for ATF4 degradation by the proteasome, binds to ATF4 through a DpSGXXXpS phosphorylation motif, which is similar but not identical to the DpSGXXpS motif found in most other substrates of beta-TrCP. NMR studies were performed on the free and bound forms of a peptide derived from this ATF4 motif that enabled the elucidation of the conformation of the ligand complexed to the beta-TrCP protein and its binding mode. Saturation transfer difference (STD) NMR allowed the study of competition for binding to beta-TrCP, between the phosphorylation motifs of ATF4 and beta-catenin, to characterize the ATF4 binding epitope. Docking protocols were performed using the crystal structure of the beta-catenin-beta-TrCP complex as a template and NMR results of the ATF4-beta-TrCP complex. In agreement with the STD results, in order to bind to beta-TrCP, the ATF4 DpSGIXXpSXE motif required the association of two negatively charged areas, in addition to the hydrophobic interaction in the beta-TrCP central channel. Docking studies showed that the ATF4 DpSGIXXpSXE motif fits the binding pocket of beta-TrCP through an S-turning conformation. The distance between the two phosphate groups is 17.8 A, which matched the corresponding distance 17.1 A for the other extended DpSGXXpS motif in the beta-TrCP receptor model. This study identifies the residues of the beta-TrCP receptor involved in ligand recognition. Using a new concept of STD competition experiment, we show that ATF4 competes and inhibits binding of beta-catenin to beta-TrCP.
Assuntos
Fator 4 Ativador da Transcrição/química , Espectroscopia de Ressonância Magnética/métodos , Peptídeos/química , beta Catenina/química , Proteínas Contendo Repetições de beta-Transducina/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Peptídeos/síntese química , Peptídeos/metabolismo , Fosforilação , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , beta Catenina/metabolismo , Proteínas Contendo Repetições de beta-Transducina/genética , Proteínas Contendo Repetições de beta-Transducina/metabolismoRESUMO
ATF4 plays a crucial role in the cellular response to stress and the F-box protein beta-TrCP, the receptor component of the SCF E3 ubiquitin ligase responsible for ATF4 degradation by the proteasome, binds to ATF4, and controls its stability. Association between the two proteins depends on ATF4 phosphorylation of serine residues 219 and 224 present in the context of DpSGXXXpS, which is similar but not identical to the DpSGXXpS motif found in most other substrates of beta-TrCP. We used NMR spectroscopy to analyze the structure of the 23P-ATF4 peptide. The 3D structure of the ligand was determined on the basis of NOESY restraints that provide an hairpin loop structure. In contrast, no ordered structure was observed in the NMR experiments for the nonphosphorylated 23-ATF4 in solution. This structural study provides information, which could be used to study the beta-TrCP receptor-ligand interaction in docking procedure. Docking studies showed that the binding epitope of the ligand, is represented by the DpSGIXXpSXE motif. 23P-ATF4 peptide fits the binding pocket of protein beta-TrCP very well, considering that the DpSGIXXpSXE motif adopts an S-turning conformation contrary to the extended DpSGXXpS motif in the other known beta-TrCP ligands.
Assuntos
Fator 4 Ativador da Transcrição/química , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Ligação de Hidrogênio , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosforilação , Conformação Proteica , Proteínas Contendo Repetições de beta-Transducina/química , Proteínas Contendo Repetições de beta-Transducina/metabolismoRESUMO
The IkappaB-alpha protein, inhibitor of the transcription factor nuclear factor-kappaB (NF-kappaB), is a cellular substrate of beta-transducin repeat containing protein (beta-TrCP). beta-TrCP is the F-box protein component of an Skp1/Cul1/F-box (SCF)-type ubiquitin ligase complex. beta-TrCP targets the protein IkappaB-alpha for ubiquitination, followed by proteasome degradation. The SCF-beta-TrCP complex specifically recognizes an IkappaB-alpha peptide containing the DpSGXXpS motif in a phosphorylation-dependent manner. A fragment comprising 24 amino acids residues for the phosphorylated peptide at the two sites Ser32 and Ser36 and thus termed 24P-IkappaBalpha (P-IkappaBalpha21-44) was characterized conformationally by NMR spectroscopy and molecular dynamics simulation. In the free states, 24P-IkappaBalpha exhibits mainly a random coil conformation, although the presence of a nascent bend was detected between residues 30 and 36, flanked by two N- and C-terminal disordered regions. The bound conformation of the phosphorylated IkappaB-alpha peptide was obtained using transfer nuclear Overhauser effect spectroscopy (TRNOESY) experiments. To further elucidate the basis of the beta-TrCP interaction, a complex between 24P-IkappaBalpha peptide and beta-TrCP protein was studied using saturation transfer difference (STD) NMR experiments. The conformation of 24P-IkappaBalpha bound to beta-TrCP presents a bend corresponding to the 31DpSGLDpS36 motif and on both sides N- and C-terminal turn regions (Lys22-Asp31 and Met37-Glu43). The bound structure of the phosphorylated peptide suggests that these domains are crucial for the interaction of the peptide with its receptor showing the protons identified by STD NMR as exposed in close proximity to the beta-TrCP surface.
Assuntos
Proteínas I-kappa B/química , NF-kappa B/antagonistas & inibidores , Sequência de Aminoácidos , Sítios de Ligação , Dicroísmo Circular , Mapeamento de Epitopos , HIV-1/metabolismo , Proteínas do Vírus da Imunodeficiência Humana , Humanos , Modelos Moleculares , Inibidor de NF-kappaB alfa , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Proteínas Virais Reguladoras e Acessórias/metabolismo , Proteínas Contendo Repetições de beta-Transducina/química , Proteínas Contendo Repetições de beta-Transducina/fisiologiaRESUMO
Reestablishment of digestive continuity after radical surgery for severe esophagojejunostomic leakage is a major challenge associated with high morbidity and uncertain feasibility. In this case report we describe a conservative double exclusion technique involving cervical stapling and loop jejunostomy. This technique greatly simplifies subsequent reconstruction and is suitable in most cases.