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1.
J Biol Chem ; 295(9): 2866-2884, 2020 02 28.
Artículo en Inglés | MEDLINE | ID: mdl-31959628

RESUMEN

Aberrant Ras signaling drives 30% of cancers, and inhibition of the Rho family small GTPase signaling has been shown to combat Ras-driven cancers. Here, we present the discovery of a 16-mer cyclic peptide that binds to Cdc42 with nanomolar affinity. Affinity maturation of this sequence has produced a panel of derived candidates with increased affinity and modulated specificity for other closely-related small GTPases. The structure of the tightest binding peptide was solved by NMR, and its binding site on Cdc42 was determined. Addition of a cell-penetrating sequence allowed the peptides to access the cell interior and engage with their target(s), modulating signaling pathways. In Ras-driven cancer cell models, the peptides have an inhibitory effect on proliferation and show suppression of both invasion and motility. As such, they represent promising candidates for Rho-family small GTPase inhibitors and therapeutics targeting Ras-driven cancers. Our data add to the growing literature demonstrating that peptides are establishing their place in the biologics arm of drug discovery.


Asunto(s)
Descubrimiento de Drogas , Péptidos Cíclicos/farmacología , Transducción de Señal/efectos de los fármacos , Proteína de Unión al GTP cdc42/antagonistas & inhibidores , Proteínas ras/metabolismo , Sitios de Unión , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Péptidos de Penetración Celular , GTP Fosfohidrolasas/antagonistas & inhibidores , Humanos , Estructura Molecular , Invasividad Neoplásica/prevención & control , Neoplasias/tratamiento farmacológico , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Proteína de Unión al GTP cdc42/metabolismo
2.
Am J Pathol ; 180(6): 2576-89, 2012 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-22521648

RESUMEN

In vivo phage display selection is a powerful strategy for directly identifying agents that target the vasculature of normal or diseased tissues in living animals. We describe here a new in vivo biopanning strategy in which a human phage single-chain antibody (scFv) library was injected into high-fat diet-fed ApoE(-/-) mice. Extracellular and internalized phage scFvs were selectively recovered from atherosclerotic vascular endothelium and subjacent tissues. After three successive biopanning rounds, a panel of six clones with distinct gene sequences was isolated. Four scFvs produced and purified in soluble form were shown to interact in vitro with a rabbit atheromatous protein extract by time-resolved fluorescence resonance energy transfer and to target the endothelial cell surface and inflamed intima-related regions of rabbit and human tissue sections ex vivo. These new scFvs selected in a mouse model recognized both rabbit and human tissue, underlying the interspecies similarities of the recognized epitopes. By combining immunoprecipitation and mass spectrometry, one of the selected scFvs was shown to recognize carbonic anhydrase II, an up-regulated enzyme involved in resorption of ectopic calcification. These results show that in vivo biopanning selection in hypercholesterolemic animals makes it possible to identify both scFvs homing to atherosclerotic endothelial and subendothelial tissues, and lesion-associated biomarkers. Such scFvs offer promising opportunities in the field of molecular targeting for the treatment of atherosclerosis.


Asunto(s)
Aterosclerosis/metabolismo , Anticuerpos de Cadena Única/farmacocinética , Animales , Aorta Torácica/metabolismo , Apolipoproteínas E/deficiencia , Biomarcadores/metabolismo , Anhidrasa Carbónica II/metabolismo , Enfermedad de la Arteria Coronaria/metabolismo , Grasas de la Dieta/administración & dosificación , Endotelio Vascular/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Hipercolesterolemia/metabolismo , Masculino , Ratones , Ratones Noqueados , Biblioteca de Péptidos , Placa Aterosclerótica/metabolismo , Unión Proteica , Conejos , Anticuerpos de Cadena Única/aislamiento & purificación
3.
Int J Mol Sci ; 13(6): 6902-6923, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22837671

RESUMEN

Atherosclerosis is a chronic, progressive inflammatory disease that may develop into vulnerable lesions leading to thrombosis. To interrogate the molecular components involved in this process, single-chain variable fragments (scFvs) from a semi-synthetic human antibody library were selected on the lesions induced in a rabbit model of atherosclerosis after two rounds of in vivo phage display. Homing Phage-scFvs were isolated from (1) the injured endothelium, (2) the underlying lesional tissue and (3) the cells within the intima. Clones selected on the basis of their redundancy or the presence of key amino acids, as determined by comparing the distribution between the native and the selected libraries, were produced in soluble form, and seven scFvs were shown to specifically target the endothelial cell surface and inflamed intima-related regions of rabbit tissue sections by immunohistology approaches. The staining patterns differed depending on the scFv compartment of origin. This study demonstrates that large-scale scFv binding assays can be replaced by a sequence-based selection of best clones, paving the way for easier use of antibody libraries in in vivo biopanning experiments. Future investigations will be aimed at characterizing the scFv/target couples by mass spectrometry to set the stage for more accurate diagnostic of atherosclerosis and development of therapeutic strategies.


Asunto(s)
Aterosclerosis/fisiopatología , Aterosclerosis/terapia , Anticuerpos de Cadena Única/química , Secuencia de Aminoácidos , Animales , Aorta Abdominal/patología , Células Espumosas/metabolismo , Humanos , Inmunohistoquímica , Inflamación/patología , Imagen por Resonancia Magnética , Masculino , Espectrometría de Masas , Ratones , Datos de Secuencia Molecular , Biblioteca de Péptidos , Conejos , Homología de Secuencia de Aminoácido , Túnica Íntima/patología
4.
NMR Biomed ; 24(4): 413-24, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21192086

RESUMEN

The noninvasive imaging of atherosclerotic plaques at an early stage of atherogenesis remains a major challenge for the evaluation of the pathologic state of patients at high risk of acute coronary syndromes. Recent studies have emphasized the importance of platelet-endothelial cell interactions in atherosclerosis-prone arteries at early stages, and the prominent role of P-selectin in the initial loose contact between platelets and diseased vessel walls. A specific MR contrast agent was developed here for the targeting, with high affinity, of P-selectin expressed in large amounts on activated platelets and endothelial cells. For this purpose, PEGylated dextran/iron oxide nanoparticles [PEG, poly(ethylene glycol)], named versatile ultrasmall superparamagnetic iron oxide (VUSPIO) particles, labeled with rhodamine were coupled to an anti-human P-selectin antibody (VH10). Flow cytometry and microscopy experiments on human activated platelets were highly correlated with MRI (performed at 4.7 and 0.2 T), with a 50% signal decrease in T(2) and T(1) values corresponding to the strong labeling of activated vs resting platelets. The number of 1000 VH10-VUSPIO nanoparticles attained per activated platelet appeared to be optimal for the detection of hypo- and hyper-signals in the platelet pellet on T(2) - and T(1) -weighted MRI. Furthermore, in vivo imaging of atherosclerotic plaques in ApoE mice at 4.7 T showed a spatial resolution adapted to the imaging of intimal thickening and a hypo-signal at 4.7 T, as a result of the accumulation of VH10-VUSPIO nanoparticles in the plaque. Our work provides support for the further assessment of the use of VH10-VUSPIO nanoparticles as a promising imaging modality able to identify the early stages of atherosclerosis with regard to the pertinence of both the target and the antibody-conjugated contrast agent used.


Asunto(s)
Aterosclerosis/sangre , Aterosclerosis/diagnóstico , Plaquetas/metabolismo , Imagen por Resonancia Magnética/métodos , Selectina-P/sangre , Activación Plaquetaria , Animales , Anticuerpos/metabolismo , Aorta/efectos de los fármacos , Aorta/metabolismo , Aorta/patología , Apolipoproteínas E/deficiencia , Plaquetas/efectos de los fármacos , Dextranos/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/patología , Citometría de Flujo , Humanos , Nanopartículas de Magnetita , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Activación Plaquetaria/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Receptores de Trombina/metabolismo , Trombina/farmacología
5.
FASEB J ; 23(2): 575-85, 2009 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18957574

RESUMEN

Identification of short, structured peptides able to mimic potently protein-protein interfaces remains a challenge in drug discovery. We report here the use of a naive cyclic peptide phage display library to identify peptide ligands able to recognize and mimic IgG1-Fc functions with Fc gammaRI. Selection by competing off binders to Fc gammaRI with IgG1 allowed the isolation of a family of peptides sharing the common consensus sequence TX(2)CXXthetaPXLLGCPhiXE (theta represents a hydrophobic residue, Phi is usually an acidic residue, and X is any residue) and able to inhibit IgG1 binding to Fc gammaRI. In soluble form, these peptides antagonize superoxide generation mediated by IgG1. In complexed form, they trigger phagocytosis and a superoxide burst. Unlike IgG, these peptides are strictly Fc gammaRI-specific among the Fc gammaRs. Molecular modeling studies suggest that these peptides can adopt 2 distinct and complementary conformers, each able to mimic the discontinuous interface contacts constituted by the Cgamma2-A and -B chains of Fc for Fc gammaRI. In addition, by covalent homodimerization, we engineered a synthetic bivalent 37-mer peptide that retains the ability to trigger effector functions. We demonstrate here that it is feasible to maintain IgG-Fc function within a small structured peptide. These peptides represent a new format for modulation of effector functions.


Asunto(s)
Materiales Biomiméticos/química , Epítopos/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Péptidos Cíclicos/química , Péptidos Cíclicos/inmunología , Receptores de IgG/inmunología , Secuencia de Aminoácidos , Materiales Biomiméticos/metabolismo , Materiales Biomiméticos/farmacología , Células Cultivadas , Bases de Datos de Proteínas , Humanos , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/metabolismo , Datos de Secuencia Molecular , Péptidos Cíclicos/metabolismo , Péptidos Cíclicos/farmacología , Fagocitosis/efectos de los fármacos , Unión Proteica , Multimerización de Proteína , Receptores de IgG/metabolismo , Superóxidos/metabolismo
6.
Peptides ; 26(11): 2302-13, 2005 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-15893405

RESUMEN

We identified a large number of peptide mimotopes of the adrenocorticotropic hormone (ACTH) and the alpha-melanocyte stimulating hormone (alpha-MSH) to analyze better the structure-function relationships of these hormones with the human MC1 receptor (hMC1R). We have investigated the use of phage-display technology to isolate specific peptides of this receptor by using three monoclonal anti-ACTH antibodies (mAbs). A library of 10(8) phage-peptides displaying randomized decapeptides was constructed and used to select phage-peptides that bind to mAbs. Forty-five phage-peptides have been isolated and from their amino acid sequences, we have identified two consensus sequences, EXFRWGKPA and WGXPVGKP, corresponding to the regions 5-13 and 9-16 of ACTH, respectively. A biological assay on cells expressing the hMC1-R was developed to determine the capacity of phage-peptides to stimulate the receptor. Only two phage-peptides showed detectable activity. Thirty-one peptides were synthesized to analyze their biological effect. We identified two weak agonists, EC50=16 and 11 microM, two strong agonists, EC50=25 and 14 nM and a partial antagonist, IC50=36 microM. This work confirmed the modulator agonist role of the regions 11-12 of alpha-MSH and ACTH, and the importance of the methionine residue at position 4 for the stimulation of the hMC1-R. We also identified analogues of the regions 8-17 of ACTH that exhibited a weak activator effect, and of one analogue of the N-terminal regions 1-9 of ACTH and alpha-MSH having a partial antagonist effect. These results may be useful in the development of potential agonists or antagonists of the hMC1R.


Asunto(s)
Oligopéptidos/farmacología , Biblioteca de Péptidos , Receptor de Melanocortina Tipo 1/agonistas , Receptor de Melanocortina Tipo 1/antagonistas & inhibidores , alfa-MSH/farmacología , Línea Celular , Relación Dosis-Respuesta a Droga , Humanos , Oligopéptidos/genética , alfa-MSH/genética
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