RESUMEN
Humans and higher primates are unique in that they lack uricase, the enzyme capable of oxidizing uric acid. As a consequence of this enzyme deficiency, humans have high serum uric acid levels. In some people, uric acid levels rise above the solubility limit resulting in crystallization in joints, acute inflammation in response to those crystals causes severe pain; a condition known as gout. Treatment for severe gout includes injection of non-human uricase to reduce serum uric acid levels. Krystexxa® is a hyper-PEGylated pig-baboon chimeric uricase indicated for chronic refractory gout that induces an immunogenic response in 91% of treated patients, including infusion reactions (26%) and anaphylaxis (6.5%). These properties limit its use and effectiveness. An innovative approach has been used to develop a therapeutic uricase with improved properties such as: soluble expression, neutral pH solubility, high E. coli expression level, thermal stability, and excellent activity. More than 200 diverse uricase sequences were aligned to guide protein engineering and reduce putative sequence liabilities. A single uricase lead candidate was identified, which showed low potential for immunogenicity in >200 human donor samples selected to represent diverse HLA haplotypes. Cysteines were engineered into the lead sequence for site specific PEGylation and studies demonstrated >95% PEGylation efficiency. PEGylated uricase retains enzymatic activity in vitro at neutral pH, in human serum and in vivo (rats and canines) and has an extended half-life. In canines, an 85% reduction in serum uric acid levels was observed with a single subcutaneous injection. This PEGylated, non-immunogenic uricase has the potential to provide meaningful benefits to patients with gout.
Asunto(s)
Gota/tratamiento farmacológico , Urato Oxidasa/uso terapéutico , Animales , Rastreo Diferencial de Calorimetría , Perros , Escherichia coli/metabolismo , Semivida , Humanos , Concentración de Iones de Hidrógeno , Cinética , Papio , Polietilenglicoles/química , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/uso terapéutico , Especificidad por Sustrato , Porcinos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Urato Oxidasa/efectos adversos , Urato Oxidasa/inmunologíaRESUMEN
Tn3 proteins are a novel class of binding molecules based on the third fibronectin type III domain of human tenascin C. Target-specific Tn3 proteins are selected from combinatorial libraries in which three surface-exposed loops have been diversified. Here, the cocrystallization of two different Tn3 proteins in complex with CD40L, a therapeutic target for immunological disease, is reported. These crystal structures are the first to be reported of Tn3 proteins and will help to reveal how these engineered molecules achieve specific recognition of a cognate target.
Asunto(s)
Ligando de CD40/química , Fibronectinas/química , Péptidos/química , Secuencia de Aminoácidos , Sitios de Unión , Ligando de CD40/genética , Ligando de CD40/aislamiento & purificación , Cristalografía por Rayos X , Escherichia coli/genética , Fibronectinas/genética , Expresión Génica , Humanos , Datos de Secuencia Molecular , Biblioteca de Péptidos , Péptidos/genética , Péptidos/aislamiento & purificación , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Alineación de SecuenciaRESUMEN
The formation of protein complexes between phosphorylated R-Smads and Smad4 is a central event in the TGF-beta signaling pathway. We have determined the crystal structure of two R-Smad/Smad4 complexes, Smad3/Smad4 to 2.5 angstroms, and Smad2/Smad4 to 2.7 angstroms. Both complexes are heterotrimers, comprising two phosphorylated R-Smad subunits and one Smad4 subunit, a finding that was corroborated by isothermal titration calorimetry and mutational studies. Preferential formation of the R-Smad/Smad4 heterotrimer over the R-Smad homotrimer is largely enthalpy driven, contributed by the unique presence of strong electrostatic interactions within the heterotrimeric interfaces. The study supports a common mechanism of Smad protein assembly in TGF-beta superfamily signaling.