RESUMO
Chemical synthesis in combination with precision polymer modification allows the systematic exploration of the effect of protein properties, such as charge and hydrodynamic radius, on potency using defined, homogeneous conjugates. A series of polymer-modified synthetic erythropoiesis proteins were constructed that had a polypeptide chain similar to the amino acid sequence of human erythropoietin but differed significantly in the number and type of attached polymers. The analogs differed in charge from +5 to -26 at neutral pH and varied in molecular weight from 30 to 54 kDa. All were active in an in vitro cell proliferation assay. However, in vivo potency was found to be strongly dependent on overall charge and size. The trends observed in this study may serve as starting points for the construction of more potent synthetic EPO analogs in the future.
Assuntos
Eritropoese/fisiologia , Polímeros/síntese química , Proteínas/síntese química , Sequência de Aminoácidos , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Eritropoetina/síntese química , Eritropoetina/metabolismo , Eritropoetina/fisiologia , Humanos , Macaca fascicularis , Camundongos , Dados de Sequência Molecular , Polímeros/metabolismo , Polímeros/farmacologia , Proteínas/metabolismo , Proteínas/fisiologia , RatosRESUMO
We report the design and total chemical synthesis of "synthetic erythropoiesis protein" (SEP), a 51-kilodalton protein-polymer construct consisting of a 166-amino-acid polypeptide chain and two covalently attached, branched, and monodisperse polymer moieties that are negatively charged. The ability to control the chemistry allowed us to synthesize a macromolecule of precisely defined covalent structure. SEP was homogeneous as shown by high-resolution analytical techniques, with a mass of 50,825 +/-10 daltons by electrospray mass spectrometry, and with a pI of 5.0. In cell and animal assays for erythropoiesis, SEP displayed potent biological activity and had significantly prolonged duration of action in vivo. These chemical methods are a powerful tool in the rational design of protein constructs with potential therapeutic applications.
