RESUMO
Despite decades of research in the epidermal growth factor receptor (EGFR) signalling field, and many targeted anti-cancer drugs that have been tested clinically, the success rate for these agents in the clinic is low, particularly in terms of the improvement of overall survival. Intratumoral heterogeneity is proposed as a major mechanism underlying treatment failure of these molecule-targeted agents. Here we highlight the application of fluorescence lifetime microscopy (FLIM)-based biosensing to demonstrate intratumoral heterogeneity of EGFR activity. For sensing EGFR activity in cells, we used a genetically encoded CrkII-based biosensor which undergoes conformational changes upon tyrosine-221 phosphorylation by EGFR. We transfected this biosensor into EGFR-positive tumour cells using targeted lipopolyplexes bearing EGFR-binding peptides at their surfaces. In a murine model of basal-like breast cancer, we demonstrated a significant degree of intratumoral heterogeneity in EGFR activity, as well as the pharmacodynamic effect of a radionuclide-labeled EGFR inhibitor in situ. Furthermore, a significant correlation between high EGFR activity in tumour cells and macrophage-tumour cell proximity was found to in part account for the intratumoral heterogeneity in EGFR activity observed. The same effect of macrophage infiltrate on EGFR activation was also seen in a colorectal cancer xenograft. In contrast, a non-small cell lung cancer xenograft expressing a constitutively active EGFR conformational mutant exhibited macrophage proximity-independent EGFR activity. Our study validates the use of this methodology to monitor therapeutic response in terms of EGFR activity. In addition, we found iNOS gene induction in macrophages that are cultured in tumour cell-conditioned media as well as an iNOS activity-dependent increase in EGFR activity in tumour cells. These findings point towards an immune microenvironment-mediated regulation that gives rise to the observed intratumoral heterogeneity of EGFR signalling activity in tumour cells in vivo.
Assuntos
Técnicas Biossensoriais/métodos , Neoplasias da Mama , Receptores ErbB/metabolismo , Neoplasias Mamárias Experimentais , Proteínas de Neoplasias/metabolismo , Transfecção/métodos , Microambiente Tumoral , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Receptores ErbB/genética , Feminino , Fluorescência , Humanos , Lipossomos , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Proteínas de Neoplasias/genética , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismoRESUMO
A novel route for the synthesis of cyclic peptides constrained by an aliphatic bridge between two C(alpha)sites, using a triply orthogonal protecting group strategy, is described. The synthesis of the orthogonally protected bis-amino acid 1, via an enantioselective route utilizing the Schöllkopf and Evans methodologies, is first described. This is then incorporated into a short, alanine-rich peptide 13, using a novel triply orthogonal protecting group strategy to couple first one, then the other, amino acid moiety in such a way that an aliphatic bridge is formed between the i and i + 4 positions. Unexpectedly, the resulting constrained peptide does not adopt a helical conformation: instead, it is shown by CD at low temperature to adopt a left-handed type II beta-turn conformation in aqueous media and a right-handed type I beta-turn conformation in TFE.
Assuntos
Aminoácidos/síntese química , Peptídeos Cíclicos/síntese química , Aminoácidos/química , Dicroísmo Circular , Biossíntese Peptídica , Peptídeos Cíclicos/química , Estrutura Secundária de ProteínaRESUMO
The factors that determine the binding of a chromophore between the base pairs in DNA intercalation complexes are dissected. The electrostatic potential in the intercalation plane is calculated using an accurate ab initio based distributed multipole electrostatic model for a range of intercalation sites, involving different sequences of base pairs and relative twist angles. There will be a significant electrostatic contribution to the binding energy for chromophores with a predominantly positive electrostatic potential, but this varies significantly with sequence, and somewhat with twist angle. The usefulness of these potential maps for understanding the binding of intercalators is explored by calculating the electrostatic binding energy for 9-aminoacridine, ethidium, and daunomycin in a variety of model binding sites. The electrostatic forces play a major role in the positioning of an intercalating 9-aminoacridine and a significant stabilizing role in the binding of ethidium in its sterically constrained position, but the intercalation of daunomycin is determined by the side-chain binding. Sequence preferences are likely to be determined by a complex and subtle mixture of effects, with electrostatics being just one component. The electrostatic binding energy is also unlikely to be a major determinant of the twist angle, as its variation with angle is modest for most intercalation sites. Overall, the electrostatic potential maps give guidance on how positively charged chromophores can be chemically adapted by heteroatomic substitution to optimise their binding.