Tesis doctoral de Daniel Fernandez Fleischhauer
Hydrolases are enzymes catalyzing the breakdown of the amide or peptide bond, and are therefore called proteases or peptidases as well. In the human genome, proteases made up about 2% of the genome, or about 600 gene products. There are six major groups of peptidases according to the catalytic residue. In our work we focused on the m14 family of peptidases, also called metallocarboxypeptidases (cps) because of their catalytic activity hinges on the zinc ion present in the active site of the enzyme. In the human genome there are identified at least 26 genes encoding for cps. M14 peptidases in the gastrointestinal tract are the main metalloproteases responsible of the liberation of free aminoacids from the protein content of the diet. In other compartments of the body, cps may perform specialized and tightly controlled tasks such as neuropeptide, cytokine and hormone maturation. In some instances an imbalance in their activity leads to disease states in man. Increasing evidence shows carboxypeptidase involvement in acute pancreatitis, diabetes, inflammation, fibrinolysis and cancer. Although some aspects have become clearer, much of their activity remain poorly understood. Besides, carboxypeptidases are interesting targets for drug development, and therefore we pursued a multidisciplinary approach to identify and characterize novel small molecular weight compounds able to interfere carboxypeptidase activity. in this work we combined modern computational tools, in vitro screening, molecular modelling and x-ray crystallography to obtain new chemical entities useful as scaffolds for drug design. Based on a bioinformatics tools, the optimal docking area method, we identified protein-protein and protein-ligand binding sites over the surface of m14 peptidases. This knowledge was employed to find out a new class of small molecular weight inhibitors which exploit the differential binding provided by hydrophobic patches. a further class of inhibitors was identified from in silico screening of collections of compounds. In vitro analysis revealed that the leads were potent inhibitors against the target proteases with interesting features like an oxadiazole zinc-chelating moiety. Compounds from the organic chemistry department were also screened, and unexpectedly, afforded some good inhibitors with unprecedented atomic bonding. One further class involved inhibitors that attach covalently to the target enzyme. In this case the structure of the complex obtained at high resolution by x-ray crystallography allowed the structure-guided design of new generation of compounds. The catalytic mechanism of m14 peptidases was also revisited based on our crystallographic and computational analysis of a new cpb crystal form at high resolution. Overall, our study provided new lead small molecular weight inhibitors which can be the foundation for further developments in the design of drugs and bioimaging or diagnostic agents targeted to physiologically-relevant metallocarboxypeptidases.
Datos académicos de la tesis doctoral «Discovery and characterization of small molecular weight metallocarboxypeptidase inhibitors.«
- Título de la tesis: Discovery and characterization of small molecular weight metallocarboxypeptidase inhibitors.
- Autor: Daniel Fernandez Fleischhauer
- Universidad: Autónoma de barcelona
- Fecha de lectura de la tesis: 14/12/2009
Dirección y tribunal
- Director de la tesis
- Josep Vendrell Roca
- Tribunal
- Presidente del tribunal: m. victí²ria Nogués bara
- ignacio Fita rodriguez (vocal)
- (vocal)
- (vocal)