RESUMO
The formation of new types of sensitive conductive surfaces for the detection and transduction of cell-extracellular matrix recognition events in a real time, label-free manner is of great interest in the field of biomedical research. To study molecularly defined cell functions, biologically inspired materials that mimic the nanoscale order of extracellular matrix protein fibers and yield suitable electrical charge transfer characteristics are highly desired. Our strategy to achieve this goal is based on the spatial self-organization of patches of cell-adhesive molecules onto a gold-nanoparticle-patterned indium tin oxide electrode. Fibroblast adhesion response to selective ligands for integrins α5ß1 and αvß3, which are both relevant in cancer progression, is investigated by simultaneous electrochemical impedance spectroscopy and optical microscopy. Adhesive cells on α5ß1-selective nanopatterns showed enhanced membrane dynamics and tighter binding, compared with cells on αvß3-selective nanopatterns. The surface of the electrode exhibits high sensitivity to small changes in surface properties, because of the constitution of specific cell-surface interactions. Moreover, such sensitivity enables differentiation between cell types. This is exemplified by analyzing distinct features in the electrochemical readout of MCF-7 breast cancer cells versus MCF-10A mammary epithelial cells, when subjected to individual adhesive nanopatches.
Assuntos
Técnicas Eletroquímicas , Ouro/química , Nanopartículas Metálicas/química , Imagem Óptica , Compostos de Estanho/química , Animais , Adesão Celular , Células Cultivadas , Humanos , Integrina alfa5beta1/antagonistas & inibidores , Integrina alfa5beta1/metabolismo , Integrina alfaVbeta3/antagonistas & inibidores , Integrina alfaVbeta3/metabolismo , Ligantes , Células MCF-7 , Microeletrodos , Tamanho da Partícula , Ratos , Propriedades de SuperfícieRESUMO
The interaction of specific surface receptors of the integrin family with different extracellular matrix-based ligands is of utmost importance for the cellular adhesion process. A ligand consists of an integrin-binding group, here cyclic RGDfX, a spacer molecule that lifts the integrin-binding group from the surface and a surface anchoring group. c(-RGDfX-) peptides are bound to gold nanoparticle structured surfaces via polyproline, polyethylene glycol or aminohexanoic acid containing spacers of different lengths. Although keeping the integrin-binding c(-RGDfX-) peptides constant for all compounds, changes of the ligand's spacer chemistry and length reveal significant differences in cell adhesion activation and focal adhesion formation. Polyproline-based peptides demonstrate improved cell adhesion kinetics and focal adhesion formation compared with common aminohexanoic acid or polyethylene glycol spacers. Binding activity can additionally be improved by applying ligands with two head groups, inducing a multimeric effect. This study gives insights into spacer-based differences in integrin-driven cell adhesion processes and remarkably highlights the polyproline-based spacers as suitable ligand-presenting templates for surface functionalization.
RESUMO
Peptides have the specificity and size required to target the protein-protein interactions involved in many diseases. Some cyclic peptides have been utilised as scaffolds for peptide drugs because of their stability; however, other cyclic peptide scaffolds remain to be explored. θ-Defensins are cyclic peptides from mammals; they are characterised by a cyclic cystine ladder motif and have low haemolytic and cytotoxic activity. Here we demonstrate the potential of the cyclic cystine ladder as a scaffold for peptide drug design by introducing the integrin-binding Arg-Gly-Asp (RGD) motif into the θ-defensin RTD-1. The most active analogue had an IC50 of 18 nM for the αv ß3 integrin as well as high serum stability, thus demonstrating that a desired bioactivity can be imparted to the cyclic cystine ladder. This study highlights how θ-defensins can provide a stable and conformationally restrained scaffold for bioactive epitopes in a ß-strand or turn conformation. Furthermore, the symmetry of the cyclic cystine ladder presents the opportunity to design peptides with dual bioactive epitopes to increase activity and specificity.
Assuntos
Cistina/química , Defensinas/metabolismo , Integrina alfaVbeta3/metabolismo , Oligopeptídeos/química , Animais , Defensinas/síntese química , Defensinas/química , Desenho de Fármacos , Integrina alfaVbeta3/química , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
We present a click chemistry-based molecular toolkit for the biofunctionalization of materials to selectively control integrin-mediated cell adhesion. To this end, α5ß1-selective RGD peptidomimetics were covalently immobilized on Ti-based materials, and the capacity to promote the selective binding of α5ß1 was evaluated using a solid-phase integrin binding assay. This functionalization strategy yielded surfaces with a nine-fold increased affinity for α5ß1, in comparison to control samples, and total selectivity against the binding of the closely related integrin αvß3. Moreover, our methodology allowed the screening of several phosphonic acid containing anchoring units to find the best spacer-anchor moiety required for establishing an efficient binding to titanium and to promote selective integrin binding. The integrin subtype specificity of these biofunctionalized surfaces was further examined in vitro by inducing selective adhesion of genetically modified fibroblasts, which express exclusively the α5ß1 integrin. The versatility of our molecular toolkit was proven by shifting the cellular specificity of the materials from α5ß1- to αvß3-expressing fibroblasts by using an αvß3-selective peptidomimetic as coating molecule. The results shown here represent the first functionalization of Ti-based materials with α5ß1- or αvß3-selective peptidomimetics that allow an unprecedented control to discriminate between α5ß1- and αvß3-mediated adhesions. The role of these two integrins in different biological events is still a matter of debate and is frequently discussed in literature. Thus, such bioactive titanium surfaces will be of great relevance for the study of integrin-mediated cell adhesion and the development of new biomaterials targeting specific cell types.
Assuntos
Integrina alfa5beta1/química , Integrina alfaVbeta3/química , Oligopeptídeos/química , Peptidomiméticos/química , Titânio , Animais , Materiais Biocompatíveis , Adesão Celular , Química Click , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Integrina alfa5beta1/metabolismo , Integrina alfaVbeta3/metabolismo , Peptidomiméticos/farmacologia , Ligação ProteicaRESUMO
The selective targeting of the αvß3 integrin subtype without affecting the structurally closely related receptor α5ß1 is crucial for understanding the details of their biological and pathological functions and thus of great relevance for diagnostic and therapeutic approaches in cancer treatment. Here, we present the synthesis of highly active RGD peptidomimetics for the αvß3 integrin with remarkable selectivity against α5ß1. Incorporation of a methoxypyridine building block into a ligand scaffold and variation of different functional moieties led to αvß3-antagonistic activities in the low nanomolar or even subnanomolar range. Furthermore, docking studies were performed to give insights into the binding modes of the novel compounds. The presented library comprises powerful ligands for specific addressing and blocking of the αvß3 integrin subtype, thereby representing privileged tools for integrin-based personalized medicine.
Assuntos
Integrina alfa5beta1/antagonistas & inibidores , Integrina alfaVbeta3/antagonistas & inibidores , Peptidomiméticos/síntese química , Integrina alfa5beta1/química , Integrina alfaVbeta3/química , Ligantes , Simulação de Acoplamento Molecular , Oligopeptídeos/metabolismo , Peptidomiméticos/farmacologia , Relação Estrutura-AtividadeRESUMO
Integrins are the major class of cell adhesion proteins. Their interaction with different ligands of the extracellular matrix is diverse. To get more insight into these interactions, artificial ligands endowed with a well-defined activity/selectivity profile are necessary. Herein, we present a library of cyclic pentapeptides, based on our previously reported peptide motif c(-phg-isoDGR-X-), in which high activity toward fibronectin binding integrins α5ß1 and αvß6 and not on vitronectin binding integrins αvß3 and αvß5 has been achieved by changing the flanking amino acids. The structure of the most promising candidates has been determined using a combined approach of NMR, distance geometry, and molecular dynamics simulations, and docking studies have been further used to elucidate the peptide-integrin interactions at the molecular level. The peptides' binding affinity has been characterized by enzyme linked immunosorbent assay experiments, and the results have been verified by cell adhesion experiments on specifically functionalized surfaces.
Assuntos
Aminoácidos/química , Antígenos de Neoplasias/metabolismo , Fibronectinas/metabolismo , Integrina alfa5beta1/metabolismo , Integrinas/metabolismo , Oligopeptídeos/química , Peptídeos Cíclicos/química , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Fibrinogênio/metabolismo , Ouro/química , Proteínas de Ligação a TGF-beta Latente/metabolismo , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Nanopartículas , Oligopeptídeos/síntese química , Oligopeptídeos/farmacologia , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/farmacologia , Ligação Proteica , Ratos , Estereoisomerismo , Relação Estrutura-Atividade , Vitronectina/metabolismoRESUMO
Integrin heterodimeric cell adhesion and signaling receptors bind ligands of the extracellular matrix and relay signals bidirectionally across cell membranes. Thereby, integrins adopt multiple conformational and functional states that control ligand binding affinity and linkage to cytosolic/cytoskeletal proteins. Here, we designed an integrin chimera encompassing the strongly dimerizing transmembrane domain (TMD) of glycophorin A (GpA) in the context of the otherwise unaltered integrin αvß3. We hypothesized that this chimera should have a low basal affinity to soluble ligand but should be force-activatable. By cellular expression of this chimera, we found a decreased integrin affinity to a soluble peptide ligand and inhibited intracellular signaling. However, under external forces applied by an atomic force microscope or by a spinning disc device causing shear forces, the mutant caused stronger cell adhesion than the wild-type integrin. Our results demonstrate that the signaling- and migration-incapable integrin αvß3-TMD mutant TMD-GpA shows the characteristics of a primed integrin state, which is of low basal affinity in the absence of forces, but may form strong bonds in the presence of forces. Thus, TMD-GpA may mimic a force-activatable signaling intermediate.
Assuntos
Glicoforinas/metabolismo , Integrina alfaVbeta3/metabolismo , Adesão Celular , Linhagem Celular , Glicoforinas/genética , Humanos , Integrina alfaVbeta3/genética , Fenômenos Mecânicos , Microscopia de Força Atômica , Modelos Biológicos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Intervention in integrin-mediated cell adhesion and integrin signaling pathways is an ongoing area of research in medicinal chemistry and drug development. One key element in integrin-ligand interaction is the coordination of the bivalent cation at the metal ion-dependent adhesion site (MIDAS) by a carboxylic acid function, a consistent feature of all integrin ligands. With the exception of the recently discovered hydroxamic acids, all bioisosteric attempts to replace the carboxylic acid of integrin ligands failed. We report that phosphinates as well as monomethyl phosphonates represent excellent isosters, when introduced into integrin antagonists for the platelet integrin αIIbß3. The novel inhibitors exhibit in vitro and ex vivo activities in the low nanomolar range. Steric and charge requirements of the MIDAS region were unraveled, thus paving the way for an in silico prediction of ligand activity and in turn the rational design of the next generation of integrin antagonists.