RESUMO
The estrogen receptor (ER) is the number one target for the treatment of endocrine responsive breast cancer and remains a highly attractive target for new drug development. Despite considerable efforts to understand the role of ER post-translational modifications (PTMs), the complexity of these modifications and their impact, at the molecular level, are poorly understood. Using a chemical biology approach, fundamentally rooted in an efficient protein semisynthesis of tyrosine phosphorylated ER constructs, the complex role of the ER tyrosine phosphorylation is addressed here for the first time on a molecular level. The semisynthetic approach allows for the site-specific introduction of PTMs as well as biophysical probes. A combination of biophysical techniques, including NMR, with molecular dynamics studies reveals the role of the phosphorylation of the clinically relevant tyrosine 537 (Y537) in ERα and the analogous tyrosine (Y488) in ERß. Phosphorylation has important effects on the dynamics of the ER Helix 12, which is centrally involved in receptor activity regulation, and on its interplay with ligand and cofactor binding, but with differential regulatory effects of the analogous PTMs on the two ER subtypes. Combined, the results bring forward a novel molecular model of a phosphorylation-induced subtype specific ER modulatory mechanism, alternative to the widely accepted ligand-induced activation mechanism.
Assuntos
Coativadores de Receptor Nuclear/metabolismo , Receptores de Estrogênio/metabolismo , Sítios de Ligação , Modelos Moleculares , Coativadores de Receptor Nuclear/química , Fosforilação , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Receptores de Estrogênio/químicaRESUMO
The human nuclear factor related to kappa-B-binding protein (NFRKB) is a 1299-residue protein that is a component of the metazoan INO80 complex involved in chromatin remodeling, transcription regulation, DNA replication and DNA repair. Although full length NFRKB is predicted to be around 65% disordered, comparative sequence analysis identified several potentially structured sections in the N-terminal region of the protein. These regions were targeted for crystallographic studies, and the structure of one of these regions spanning residues 370-495 was determined using the JCSG high-throughput structure determination pipeline. The structure reveals a novel, mostly helical domain reminiscent of the winged-helix fold typically involved in DNA binding. However, further analysis shows that this domain does not bind DNA, suggesting it may belong to a small group of winged-helix domains involved in protein-protein interactions.
Assuntos
Proteínas de Ligação a DNA/química , Fatores de Transcrição Winged-Helix/química , Sequência de Aminoácidos , Proteínas de Bactérias/química , Cristalografia por Raios X , Proteínas Culina/química , DNA/metabolismo , Humanos , Dados de Sequência Molecular , Ligação Proteica , Desnaturação Proteica , Mapas de Interação de Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Repressoras/química , Saccharomyces cerevisiae/química , Alinhamento de Sequência , Homologia Estrutural de Proteína , TemperaturaRESUMO
A library of small tetrahydroisoquinoline ligands, previously identified via structure- and chemistry-based hierarchical organization of library scaffolds in tree-like arrangements, has been generated as novel estrogen receptor agonistic fragments via traditional medicinal chemistry exploration. The approach described has allowed for the rapid evaluation of a structure-activity relationship of the ligands concerning estrogen receptor affinity and estrogen receptor ß subtype selectivity. The structural biological insights obtained from the fragments aid the understanding of larger analogues and constitute attractive starting points for further optimization.
Assuntos
Desenho de Fármacos , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Tetra-Hidroisoquinolinas/química , Tetra-Hidroisoquinolinas/metabolismo , Receptor alfa de Estrogênio/agonistas , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor alfa de Estrogênio/química , Receptor beta de Estrogênio/agonistas , Receptor beta de Estrogênio/antagonistas & inibidores , Receptor beta de Estrogênio/química , Ligantes , Modelos Moleculares , Estrutura Terciária de Proteína , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Especificidade por Substrato , Tetra-Hidroisoquinolinas/farmacologiaRESUMO
An efficient and rapid on-bead screening method was established to identify non-natural peptides that target the Androgen Receptor-cofactor interaction. Binding of the Androgen Receptor ligand binding domain to peptide sequences displayed on beads in a One-Bead-One-Compound format could be screened using fluorescence microscopy. The method was applied to generate and screen both a focussed and a random peptide library. Resynthesis of the peptide hits allowed for the verification of the affinity of the selected peptides for the Androgen Receptor in a competitive fluorescence polarization assay. For both libraries strong Androgen Receptor binding peptides were found, both with non-natural and natural amino acids. The peptides identified with natural amino acids showed great similarity in terms of preferred amino acid sequence with peptides previously isolated from biological screens, thus validating the screening approach. The non-natural peptides featured important novel chemical transformations on the relevant hydrophobic amino acid positions interacting with the Androgen Receptor. This screening approach expands the molecular diversity of peptide inhibitors for nuclear receptors.
Assuntos
Peptídeos/química , Receptores Androgênicos/química , Sequência de Aminoácidos , Polarização de Fluorescência , Microscopia de Fluorescência , Dados de Sequência MolecularAssuntos
Receptor alfa de Estrogênio/química , Receptor beta de Estrogênio/química , Ligantes , Sequência de Aminoácidos , Cristalografia por Raios X , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/química , Fosforilação , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Miniprotein phage display screening yields structured peptides with high affinity for the Estrogen Receptor (ER). Hits from apamin phage libraries feature a LXXLL motif specifically placed on the predefined miniprotein helical segment. The apamin scaffold also allows optimization of flanking amino acids to ensure an optimal ER binding affinity.
Assuntos
Peptídeos/química , Receptores de Estrogênio/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Dissulfetos/química , Dados de Sequência Molecular , Biblioteca de Peptídeos , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Estrogênio/metabolismoRESUMO
Insertion of 3 to 4 mutations, based on in silico modelling, in a diverse set of natural miniproteins generates potent androgen receptor (AR) binders and a clear insight into the structure-activity relationship of such coactivator mimics concerning helix length.
Assuntos
Androgênios , Proteínas de Ligação a DNA/química , Desenho de Fármacos , Peptídeos/química , Peptídeos/metabolismo , Receptores Androgênicos/metabolismo , Fatores de Transcrição/química , Sequência de Aminoácidos/genética , Apamina/química , Apamina/genética , Ligação Competitiva , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Bases de Dados de Proteínas , Humanos , Modelos Moleculares , Mimetismo Molecular , Dados de Sequência Molecular , Mutação/genética , Peptídeos/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Estrutura Secundária de Proteína , Venenos de Escorpião/química , Venenos de Escorpião/genética , Relação Estrutura-Atividade , Toxinas Biológicas/química , Toxinas Biológicas/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The structure- and chemistry-based hierarchical organization of library scaffolds in tree-like arrangements provides a valid, intuitive means to map and navigate chemical space. We demonstrate that scaffold trees built using bioactivity as the key selection criterion for structural simplification during tree construction allow efficient and intuitive mapping, visualization and navigation of the chemical space defined by a given library, which in turn allows correlation of this chemical space with the investigated bioactivity and further compound design. Brachiation along the branches of such trees from structurally complex to simple scaffolds with retained yet varying bioactivity is feasible at high frequency for the five major pharmaceutically relevant target classes and allows for the identification of new inhibitor types for a given target. We provide proof of principle by identifying new active scaffolds for 5-lipoxygenase and the estrogen receptor ERalpha.