RESUMEN
The extra domain B splice variant (EDB) of human fibronectin selectively expressed in the tumor vasculature is an attractive target for cancer imaging and therapy. Here, we describe the generation and characterization of EDB-specific optical imaging probes. By screening combinatorial cystine-knot miniprotein libraries with phage display technology we discover exquisitely EDB-specific ligands that share a distinctive motif. Probes with a binding constant in the picomolar range are generated by chemical oligomerization of selected ligands and fluorophore conjugation. We show by fluorescence imaging that the probes stain EDB in tissue sections derived from human U-87 MG glioblastoma xenografts in mice. Moreover, we demonstrate selective accumulation and retention of intravenously administered probes in the tumor tissue of mice with U-87 MG glioblastoma xenografts by in vivo and ex vivo fluorescence imaging. These data warrants further pursuit of the selected cystine-knot miniproteins for in vivo imaging applications.
Asunto(s)
Miniproteínas Nodales de Cistina/metabolismo , Fibronectinas/metabolismo , Glioblastoma/irrigación sanguínea , Proteínas Recombinantes/metabolismo , Secuencias de Aminoácidos , Animales , Sitios de Unión , Línea Celular Tumoral , Miniproteínas Nodales de Cistina/química , Miniproteínas Nodales de Cistina/genética , Miniproteínas Nodales de Cistina/uso terapéutico , Fibronectinas/genética , Colorantes Fluorescentes/química , Colorantes Fluorescentes/uso terapéutico , Glioblastoma/diagnóstico por imagen , Glioblastoma/metabolismo , Humanos , Ratones , Ratones Desnudos , Imagen Óptica , Biblioteca de Péptidos , Ingeniería de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Resonancia por Plasmón de Superficie , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The cystine-knot miniproteins present in tomato fruit (TCMPs) have been shown to exert anti-angiogenic effects by inhibiting endothelial cell migration and to display resistance to gastrointestinal proteolytic attack. To better define the pharmacological potential of TCMPs, their oral bioavailability and their resistance to industrial processing must be assessed. To explore the intestinal transport of TCMPs we used the differentiated Caco-2 cells model. After 24h incubation, 37.73±9.34% of TCMPs crossed the epithelium, without altering the integrity of the cell layer. To assess the effects of the industrial processing on the biochemical features and the biological activity of TCMPs, we developed a method for purifying the proteins from tomato paste. The tomato-paste purified TCMPs retained the resistance to gastrointestinal digestion and the inhibitory activity towards endothelial cell migration. Our previous and present results collectively demonstrate that TCMPs possess interesting features for drug development.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Miniproteínas Nodales de Cistina/uso terapéutico , Absorción Intestinal/efectos de los fármacos , Solanum lycopersicum/química , Miniproteínas Nodales de Cistina/administración & dosificación , Manipulación de Alimentos , HumanosRESUMEN
Peptides with the cystine-knot architecture, often termed knottins, are promising scaffolds for biomolecular engineering. These unique molecules combine diverse bioactivities with excellent structural, thermal, and proteolytical stability. Being different in the composition and structure of their amino acid backbone, knottins share the same core element, namely cystine knot, which is built by six cysteine residues forming three disulfides upon oxidative folding. This motif ensures a notably rigid framework that highly tolerates both rational and combinatorial changes in the primary structure. Being accessible through recombinant production and total chemical synthesis, cystine-knot miniproteins can be endowed with novel bioactivities by variation of surface-exposed loops and incorporation of non-natural elements within their non-conserved regions towards the generation of tailor-made peptidic compounds. In this chapter the topology of cystine-knot peptides, their synthesis and applications for diagnostics and therapy is discussed.
Asunto(s)
Miniproteínas Nodales de Cistina/uso terapéutico , Diseño de Fármacos , Neoplasias/tratamiento farmacológico , Fragmentos de Péptidos/uso terapéutico , Ingeniería de Proteínas/métodos , Animales , Miniproteínas Nodales de Cistina/síntesis química , Humanos , Neoplasias/diagnósticoRESUMEN
Cystine-knot miniproteins, also known as knottins, constitute a large family of structurally related peptides with diverse amino acid sequences and biological functions. Knottins have emerged as attractive candidates for drug development as they potentially fill a niche between small molecules and protein biologics, offering drug-like properties and the ability to bind to clinical targets with high affinity and selectivity. Due to their extremely high stability and unique structural features, knottins also demonstrate promise in addressing challenging drug development goals, including the potential for oral delivery and the ability to access intracellular drug targets. Several naturally-occurring knottins have recently received approval for treating chronic pain and irritable bowel syndrome, while others are under development for tumor imaging applications. To expand beyond nature's repertoire, rational and combinatorial protein engineering methods are generating tumor-targeting knottins for use as cancer diagnostics and therapeutics.
Asunto(s)
Antineoplásicos/uso terapéutico , Miniproteínas Nodales de Cistina/uso terapéutico , Neoplasias/tratamiento farmacológico , Radiofármacos , Animales , Antineoplásicos/metabolismo , Biomarcadores de Tumor/metabolismo , Dolor Crónico/tratamiento farmacológico , Ciclotidas/uso terapéutico , Miniproteínas Nodales de Cistina/metabolismo , Humanos , Síndrome del Colon Irritable/tratamiento farmacológico , Imagen Molecular/métodos , Neoplasias/diagnóstico , Neoplasias/metabolismo , Ingeniería de Proteínas , Radiofármacos/metabolismoRESUMEN
Cystine knot miniproteins define a class of peptides in the size range of approximately 28-35 amino acid residues that often combine high chemical and biological stability with high potency and selectivity. They share a common structural motif that is defined by three intramolecular disulfide bonds that gives rise to a very stable scaffold. Members of this family cover a broad spectrum of natural bioactivities ranging from antimicrobial and antiviral activities to selective blockage or activation of ion channels, cell surface receptors and extracelluar proteases. In recent years, the spectrum of natural bioactivities of this class of miniproteins was further expanded by application of protein design and directed evolution technologies. Miniproteins have been developed that inhibit platelet aggregation, block asthma-related proteases, act as growth factor mimics or address human tumor targets. Recent reports on miniproteins binding to cancer specific targets indicate that these biomolecules due to their particularly high in vivo stability, high target affinity, good tissue distribution, and fast body clearance are very promising agents that can be endowed with important beneficial features for imaging and therapeutic applications. With the first cystine-knot miniprotein already marketed as an analgesic, more candidates can be expected to find their way into the clinic for diagnostic and therapeutic applications over next years.
Asunto(s)
Miniproteínas Nodales de Cistina , Descubrimiento de Drogas , Ingeniería de Proteínas , Administración Oral , Secuencia de Aminoácidos , Animales , Fenómenos Biofísicos , Miniproteínas Nodales de Cistina/síntesis química , Miniproteínas Nodales de Cistina/genética , Miniproteínas Nodales de Cistina/uso terapéutico , Estabilidad de Medicamentos , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Neoplasias/diagnóstico , Neoplasias/tratamiento farmacológico , Inhibidores de Agregación Plaquetaria/síntesis química , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/farmacología , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Conformación Proteica , Estabilidad Proteica , Técnicas de Síntesis en Fase SólidaRESUMEN
PURPOSE: Cystine knot peptides (knottins) 2.5D and 2.5F were recently engineered to bind integrin receptors with high affinity and specificity. These receptors are overexpressed on the surface of a variety of malignant human tumor cells and tumor neovasculature. In this study, 2.5D and 2.5F were labeled with a therapeutic radionuclide, (177)Lu, and the resulting radiopeptides were then evaluated as potential radiotherapeutic agents in a murine model of human glioma xenografts. METHODS: Knottins 2.5D and 2.5F were synthesized using solid phase peptide synthesis, folded in vitro, and site-specifically coupled with 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) at their N terminus for (177)Lu radiolabeling. The stability of the radiopeptides (177)Lu-DOTA-2.5D and (177)Lu-DOTA-2.5F was tested in both phosphate-buffered saline (PBS) and mouse serum. Cell uptake assays of the radiolabeled peptides were performed in U87MG integrin-expressing human glioma cells. The biodistribution studies of both (177)Lu-DOTA-2.5D and (177)Lu-DOTA-2.5F were examined in U87MG tumor-bearing athymic nu/nu mice. Radiation absorbed doses for the major tissues of a human adult male were calculated based on the mouse biodistribution results. RESULTS: DOTA-2.5D and DOTA-2.5F were labeled with (177)Lu at over 55% efficiency. High radiochemical purity for both radiocomplexes (> 95%) could be achieved after high performance liquid chromatography (HPLC) purification. Both radiopeptides were stable in PBS and mouse serum. Compared to (177)Lu-DOTA-2.5D (0.39 and 0.26 %ID/g at 2 and 24 h, respectively), (177)Lu-DOTA-2.5F showed much higher tumor uptake (2.16 and 0.78 %ID/g at 2 and 24 h, respectively). It also displayed higher tumor to blood ratios than that of (177)Lu-DOTA-2.5D (31.8 vs 18.7 at 24 h and 52.6 vs 20.6 at 72 h). Calculation of radiodosimetry for (177)Lu-DOTA-2.5D and (177)Lu-DOTA-2.5F suggested that tumor and kidney were tissues with the highest radiation absorbed doses. Moreover, (177)Lu-DOTA-2.5F had a higher tumor to kidney radiation absorbed dose ratio than that of (177)Lu-DOTA-2.5D. CONCLUSION: Cystine knot peptides can be successfully radiolabeled with (177)Lu for potential therapeutic applications. Knottin 2.5F labeled with (177)Lu exhibits favorable distribution in murine U87MG xenograft model; thus, it is a promising agent for radionuclide therapy of integrin-positive tumors.