Discovery and characterizatopn of small molecular weight metallocarboxypeptidase inhibitors

Descripció del recurs: el 02 de novembre de 2010Las hidrolasas son enzimas que catalizan la ruptura del enlace amida o peptódico, y por lo tanto son denominadas también proteasas o peptidasas. Las proteasas constituyen cerca del 2 % del genoma humano, lo que representa unos 600 productos génicos. De acuerdo con el residuo catalóticamente activo, existen seis grandes grupos de peptidasas. En este trabajo nos centraremos en la familia M14 de peptidasas, también denominadas metalocarboxipeptidasas (CPs) debido a que su actividad catalótica reside en el ion zinc presente en el sitio activo de la enzima. En el genoma humano, se han identificado al menos 26 genes que codifican carboxipeptidasas. Las peptidasas de la familia M14 que actúan en el tracto gastrointestinal son las principales metaloproteasas responsables de la obtención de aminoácidos libres de la proteína de la dieta. En otros compartimientos corporales, las CPs pueden llevar a cabo tareas especializadas y altamente reguladas como ser la maduración de neuropéptidos, citokinas y hormonas peptódicas. En algunos casos, una actividad catalótica fuera de control puede conducir a enfermedades. Cada vez existe una mayor evidencia experimental que demuestra la actividad carboxipeptidasa en procesos como la pancreatitis aguda, la diabetes, la inflamación, la fibrinólisis y el cáncer. A pesar de ciertos avances en algunos aspectos, la actividad específica de las CPs es pobremente conocida. Además, las carboxipeptidasas son blancos terapéuticos interesantes para el desarrollo de fármacos, y por lo tanto se ha decidido emplear una aproximación multi-disciplinaria para la identificación y caracterización de nuevas moléculas de bajo peso molecular capaces de interferir la actividad carboxipeptidasa. Así, en este trabajo se han combinado modernas herramientas computacionales, screening in vitro, modelado molecular y cristalografía de rayos X con el fin de obtener nuevas entidades quφmicas como base para el desarrollo de fármacos. Con base en herramientas computacionales, aplicando el método de Optimal Docking Areaö, se han caracterizado sitios de unión proteína-proteína y proteína-ligando en la superficie de las peptidasas de la familia M14. A partir de aquí, se identificó una nueva clase de compuestos químicos capaces de explotar las diferencias existentes entre enzimas de la familia por unión a regiones hidrofóbicas. Otros inhibidores fueron identificados mediante un screening in silico de grandes colecciones de compuestos. Ensayos in vitro demostraron que los compuestos líderes inhibieron de manera potente a las carboxipeptidasas blanco con otras características interesantes como la posibilidad de coordinación del ion zinc catalítico por intermedio de un anillo oxadiazol. A través de una colaboración con el Departamento de Química Orgánica se obtuvieron y caracterizaron nuevos compuestos químicos con conectividades atómicas novedosas que, inesperadamente, demostraron ser potentes inhibidores de carboxipeptidasas. Una clase adicional de molécula de bajo peso molecular caracterizada corresponde a inhibidores que se unen covalentemente al enzima blanco. En este caso, se logró obtener la estructura tridimensional del complejo a resolución atómica mediante cristalografφa de rayos X, lo que ha permitido el dise±o basado en la estructura de una nueva generación de compuestos. Basados en otros datos de cristalografía de rayos X y análisis computacional, se ha revisado y ampliado el mecanismo de acción catalítica de las peptidasas de la familia M14 a partir de una nueva forma cristalina de CPB a alta resolución. En conjunto, nuestro trabajo ha permitido la obtención de nuevas moléculas líderes de bajo peso molecular que podrían servir como base para futuros desarrollos en el diseño de fármacos y agentes de diagnóstico o imaginería dirigidos a metalocarboxipeptidasas fisiológicamente activas.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 obtained 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

Direct interaction between a human digestive protease and the mucoadhesive poly(acrylic acid)

Carboxypeptidase A1 has been the subject of extensive research in the last 30 y and is one of the most widely studied zinc metalloenzymes. However, the three-dimensional structure of the human form of the enzyme is not yet available. This report describes the three-dimensional structure of human carboxypeptidase A1 (hCPA1) derived from crystals that belong to the tetragonal space group P43212 and diffract to 1.6 Å resolution. A description of the ternary complex hCPA1-Zn2+-poly(acrylic acid) is included as a model of the interaction of mucoadhesive polymers with proteases in the gastrointestinal tract. The direct mode of interaction between poly(acrylic acid) and the active site of the target protease was confirmed by in vitro inhibition assays. The structure was further analyzed in silico through the optimal docking-area method. The characterization of binding sites on the surface of hCPA1 and a comparison with other available carboxypeptidase structures provided further insights into the formation of multiprotein complexes and the activation mechanisms of carboxypeptidase zymogens. The high-resolution structure of hCPA1 provides an excellent template for the modelling of physiologically relevant carboxypeptidases and could also contribute to the design of specific agents for biomedical purposes

Direct interaction between a human digestive protease and the mucoadhesive poly(acrylic acid)

Carboxypeptidase A1 has been the subject of extensive research in the last 30 y and is one of the most widely studied zinc metalloenzymes. However, the three-dimensional structure of the human form of the enzyme is not yet available. This report describes the three-dimensional structure of human carboxypeptidase A1 (hCPA1) derived from crystals that belong to the tetragonal space group P43212 and diffract to 1.6 Å resolution. A description of the ternary complex hCPA1-Zn2+-poly(acrylic acid) is included as a model of the interaction of mucoadhesive polymers with proteases in the gastrointestinal tract. The direct mode of interaction between poly(acrylic acid) and the active site of the target protease was confirmed by in vitro inhibition assays. The structure was further analyzed in silico through the optimal docking-area method. The characterization of binding sites on the surface of hCPA1 and a comparison with other available carboxypeptidase structures provided further insights into the formation of multiprotein complexes and the activation mechanisms of carboxypeptidase zymogens. The high-resolution structure of hCPA1 provides an excellent template for the modelling of physiologically relevant carboxypeptidases and could also contribute to the design of specific agents for biomedical purposes

Structural and functional analysis of the complex between citrate and the zinc peptidase carboxypeptidase A.

A high-resolution carboxypeptidase-Zn(2+)-citrate complex was studied by X-ray diffraction and enzyme kinetics for the first time. The citrate molecule acts as a competitive inhibitor of this benchmark zinc-dependent peptidase, chelating the catalytic zinc ion in the active site of the enzyme and inducing a conformational change such that carboxypeptidase adopts the conformation expected to occur by substrate binding. Citrate adopts an extended conformation with half of the molecule facing the zinc ion, while the other half is docked in the S1' hydrophobic specificity pocket of the enzyme, in contrast with the binding mode expected for a substrate like phenylalanine or a peptidomimetic inhibitor like benzylsuccinic acid. Combined structural and enzymatic analysis describes the characteristics of the binding of this ligand that, acting against physiologically relevant zinc-dependent proteases, may serve as a general model in the design of new drug-protecting molecules for the oral delivery of drugs of peptide origin