46 research outputs found
Affinity chromatography as tool for characterization of drug binding to immobilized human serum albumin
BIORECOGNITION STUDIES: MULTIMETHODOLOGICAL APPROACH
Molecular recognition phenomena play crucial roles in a number of biological processes. Thus the elucidation of these processes deserves the understanding of the basic forces that determine the involved protein-protein or small molecule-protein interactions. This aim determines a growing need of methodologies for studying any specific biomolecular event as well as the structural elements that drive the process. In order to determine the equilibrium and/or kinetic constants for binding, these techniques must factor the concentrations of free probe and target molecules, as well as of the corresponding complex. Many methodologies are well suited to get information on the complex formation as well as on the funzionalizing high structures. However a multimethodological approach is often essential to give a deeper insight in the understanding of the recognition process, taking advantage of the peculiarity of each methodology.
Here we report the use of biochromatography, circular dichroism, and optical biosensors in key activities in drug discovery and developments, e.g. identification of protein function modulators, and early determination of ADME parameters. In particular, applications of the different methodologies will be presented for determining the distribution parameters of anticancer and antiviral drugs, studying the drug binding to target proteins, and getting information on the association of functional proteins into homo and hetero-oligomers (1-4). As far as the distribution parameters determination is concerned, the binding of drugs to albumins from different species have been characterized in terms of bound fraction, affinity constant, binding area localization, and stereochemistry of the drug-protein complex. The relevance of target discovery and validation will be then addressed by applying optical biosensor technique and circular dichroism spectroscopy to anticancer drugs. Finally, the characterization of the molecular interactions between herpes simplex virus (HSV) cellular receptors and glycoproteins showing receptor-binding activity will be reported. The use of different methodologies resulted of great relevance for understanding the glycoprotein-mediated interference to infection and then to get information on the mechanism involved in the virus entry.
1. S. Cimitan, M.T. Lindgren, C. Bertucci, U.H. Danielson
J.Med.Chem., 2005, 48:7282-9.
2. T. Gianni, A. Piccoli, C. Bertucci, G. Campadelli-Fiume. J Virol., 2006, 80:2216-24.
3. G.A. Ascoli, E. Domenici, C. Bertucci, Chirality, 2006, 18:667-79.
4. B. Sanavio, A. Piccoli, T. Gianni, C. Bertucci, BBA-PRO, 2007, in press
Structural characterization of recombinant therapeutic proteins by circular dichroism.
Most of the protein therapeutics are now produced by recombinant DNA technology. The advantages of recombinant proteins are related to their higher specificity and to their safety as exposure to animal or human diseases. However, several problems are still present in development of recombinant proteins as therapeutics, such as low bioavailability, short serum half-life, and immune response. Their successful application hinges on the protein stereochemical stability, and on the folding and the tendency to aggregate induced by purification steps and storage. All these aspects determine the failure of many potential protein therapies, and limitations in the development of the formulation. The application of multiple analytical techniques is important in order to obtain a detailed product profile and to understand how manufacturing can influence product structure and activity. Surely the protein conformation is a key aspect to be assessed, because a specific conformation is often essential for the biological function of the protein. Thus, there is a growing need to perform structural studies under the conditions in which the proteins operate, and to monitor the structural changes of the protein. Circular dichroism has been increasingly recognised as a valuable and reliable technique to get this information. In particular, examples will be here reported on the use of circular dichroism spectroscopy in the structural characterization of free and formulated recombinant proteins, looking at the prediction of the secondary structure, propensity to conformational changes, stability, and tendency to aggregate
Circular dichroism in drug discovery and development: an abridged review.
Chirality plays a fundamental role in determining the pharmacodynamic and pharmacokinetic properties of drugs, and contributes significantly to our understanding of the mechanisms that lie behind biorecognition phenomena. Circular dichroism spectroscopy is the technique of choice for determining the stereochemistry of chiral drugs and proteins, and for monitoring and characterizing molecular recognition phenomena in solution. The role of chirality in our understanding of recognition phenomena at the molecular level is discussed here via several selected systems of interest in the drug discovery and development area. The examples were selected in order to underline the utility of circular dichroism in emerging studies of protein-protein interactions in biological context. In particular, the following aspects are discussed here: the relationship between stereochemistry and pharmacological activity--stereochemical characterization of new leads and drugs; stereoselective binding of leads and drugs to target proteins--the binding of drugs to serum albumins; conformational transitions of peptides and proteins of physiological relevance, and the stereochemical characterization of therapeutic peptides