RESUMO
Thymidylyltransferases (thymidine diphospho pyrophosphorylases) are nucleotidylyltransferases that play key roles in the biosynthesis of carbohydrate components within bacterial cell walls and in the biosynthesis of glycosylated natural products. They catalyze the formation of sugar nucleotides concomitant with the release of pyrophosphate. Protein engineering of thymidylyltransferases has been an approach for the production of a variety of non-physiological sugar nucleotides. In this work, we have explored chemical approaches towards modifying the activity of the thymidylyltransferase (Cps2L) cloned from S. pneumoniae, through the use of chemically synthesized 'activated' nucleoside triphosphates with enhanced leaving groups, or by switching the metal ion co-factor specificity. Within a series of phosphonate-containing nucleoside triphosphate analogues, thymidylyltransferase activity is enhanced based on the acidity of the leaving group and a Brønsted-type analysis indicated that leaving group departure is rate limiting. We have also determined IC50 values for a series of bisphosphonates as inhibitors of thymidylyltransferases. No correlation between the acidity of the inhibitors (pKa) and the magnitude of enzyme inhibition was found.
Assuntos
Difosfonatos/química , Difosfonatos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Nucleosídeo-Trifosfatase/química , Nucleosídeo-Trifosfatase/metabolismo , Timidina/química , Sítios de Ligação , Ativação Enzimática/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Nucleosídeo-Trifosfatase/genética , Engenharia de Proteínas , Streptococcus pneumoniae/enzimologia , Streptococcus pneumoniae/genética , Especificidade por Substrato , Timidina/genéticaRESUMO
OBJECTIVE: The study objectives were to assess the efficacy of N,O carboxymethyl chitosan film in reducing postsurgical adhesion in a rabbit cardiac injury model and to confirm the efficacy of N,O carboxymethyl chitosan gel and film in reducing postsurgical adhesion formation in a pig cardiac injury model. METHODS: (1) Rabbit cardiac injury model: Cardiac injury was generated by abrading the anterior surface of the heart and desiccation with oxygen. N,O carboxymethyl chitosan solution and film were administered to the injured surface. (2) Pig cardiac injury model: Cardiac injury was generated as described above. N,O carboxymethyl chitosan solution and gel (or film) were administered to the injured surface. The severity and area of adhesion between the heart and the sternum were evaluated at 14 days postcardiac surgery. RESULTS: (1) Rabbits treated with N,O carboxymethyl chitosan film plus solution showed significantly reduced severity and area of adhesion formation. (2) Both N,O carboxymethyl chitosan gel plus solution and N,O carboxymethyl chitosan film plus solution significantly reduced adhesion formation in the pig model. CONCLUSIONS: Application of N,O carboxymethyl chitosan products significantly reduces severity of postsurgical adhesion formation after cardiac surgery in the rabbit and pig models. N,O carboxymethyl chitosan products may act as a biophysical barrier.
