RESUMEN
PDE4 inhibitors have the potential to alleviate the symptoms and underlying inflammation associated with dry eye. Disclosed herein is the development of a novel series of water-soluble PDE4 inhibitors. Our studies led to the discovery of coumarin 18, which is effective in a rabbit model of dry eye and a tear secretion test in rats.
Asunto(s)
4-Aminopiridina/análogos & derivados , Antiinflamatorios/química , Cumarinas/química , Inhibidores de Fosfodiesterasa 4 , Agua/química , 4-Aminopiridina/síntesis química , 4-Aminopiridina/química , 4-Aminopiridina/uso terapéutico , Animales , Antiinflamatorios/síntesis química , Antiinflamatorios/uso terapéutico , Sitios de Unión , Simulación por Computador , Cumarinas/síntesis química , Cumarinas/uso terapéutico , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Modelos Animales de Enfermedad , Síndromes de Ojo Seco/tratamiento farmacológico , Conejos , RatasRESUMEN
Immobilized metal affinity chromatography (IMAC) has rapidly become one of the most widespread affinity purification techniques employed in recombinant protein expression. However, the high purity demands of certain applications are occasionally unattainable through a single IMAC separation. GNC92H2scFv is a cocaine-binding single-chain antibody fragment that is unstable during long-term storage in aqueous solution. To circumvent this problem, a reversed-phase HPLC separation was performed following IMAC purification of GNC92H2scFv from Escherichia coli cell culture supernatant. The resulting HPLC effluent was then freeze-dried to afford a salt-free lyophilizate amenable to long-term storage with minimal loss in binding activity. HPLC purification also effectively removed an 80-kDa protein contaminant that co-eluted with the IMAC-purified protein. Of special importance for in vivo applications of recombinantly expressed protein therapeutics, an HPLC purification step afforded a 1000-fold reduction in lipopolysaccharide (LPS) endotoxin contamination in the final GNC92H2scFv product.
Asunto(s)
Cocaína/metabolismo , Fragmentos de Inmunoglobulinas/aislamiento & purificación , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Ensayo de Inmunoadsorción Enzimática , Fragmentos de Inmunoglobulinas/metabolismo , Lipopolisacáridos/aislamiento & purificaciónRESUMEN
Cocaine is a highly addictive drug, and despite intensive efforts, effective therapies for cocaine craving and addiction remain elusive. In recent years, we and others have reported advances in anti-cocaine immunopharmacotherapy based on specific antibodies capable of sequestering the drug before it reaches the brain. In an effort to obtain high affinity therapeutic anti-cocaine antibodies, either whole IgGs or other antibody constructs, fluorescence spectroscopic techniques could provide a means of assisting selection and engineering strategies. We report the synthesis of a series of cocaine-fluorophore conjugates (GNC-F1, GNC-F2, GNC-I) and the functional evaluation of these compounds against single-chain Fv antibodies obtained via crystallographic analysis/engineering and against commercially available anti-cocaine monoclonal antibodies with a wide range of cocaine-binding affinities. From these studies, we determined that the GNC-F2 fluorophore reproduced affinity constants obtained using [(3)H]-labeled cocaine. We anticipate that the readily synthesized and nonradioactive GNC-F2 will find use both as a tool for bioimaging and in the high-throughput selection and engineering of potential therapeutic antibodies against cocaine.
Asunto(s)
Anticuerpos Monoclonales/química , Cocaína/análogos & derivados , Colorantes Fluorescentes/síntesis química , Inmunoconjugados/química , Fragmentos de Inmunoglobulinas/química , Animales , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Cocaína/química , Cocaína/inmunología , Humanos , Inmunoconjugados/inmunología , Fragmentos de Inmunoglobulinas/inmunología , Inmunoglobulina G/química , Inmunoglobulina G/inmunología , Cinética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Biblioteca de Péptidos , Ingeniería de Proteínas , Espectrometría de FluorescenciaRESUMEN
Overexpression of the cell-surface glycosphingolipid G(M3) is associated with a number of different cancers, including those of the skin, colon, breast, and lung. Antibodies against the G(M3) epitope have potential application as therapeutic agents in the treatment of these cancers. We describe the chemoenzymatic synthesis of two G(M3)-derived reagents and their use in the panning of a phage-displayed human single-chain Fv (scFv) antibody library derived from the blood of cancer patients. Three scFv-phage clones, GM3A6, GM3A8, and GM3A15, were selected for recombinant expression and were characterized using BIAcore and flow cytometry. BIAcore measurements using the purified, soluble scFvs yielded dissociation constants (K(d)) ranging from 4.2 x 10(-7) to 2.1 x 10(-5) M. Flow cytometry was used to evaluate the ability of each scFv to discriminate between normal human cells (human dermal fibroblast, HDFa), melanoma cells (HMV-1, M21, and C-8161), and breast cancer cells (BCM-1, BCM-2, and BMS). GM3A6 displayed cross-reactivity with normal cells, as well as tumor cells, and GM3A15 possessed little or no binding activity toward any of the cell lines tested. However, GM3A8 bound to five of the six tumor cell lines and showed no measurable reactivity against the HDFa cells. Hence, we have demonstrated that a synthetic G(M3) panning reagent can be used to isolate a fully human scFv that is highly specific for native G(M3) on the surface of tumor cells. The result is a significant step toward effective immunotherapies for the treatment of cancer.