FOCUS-2D method

For peptides larger than approx 80-100 amino acids, 2D methods become increasingly cumbersome, and larger proteins are assigned using 3D or fourdimensional (4D) methods that require the protein to be uniformly enriched with 13C, 15N, or both. These techniques will not be described here because we rather focus on peptide NMR, but the reader is referred to excellent articles on this topic (69-72). 

This method was applied to the design of a tripeptoid library. Experimental work on that library had been described by Zuckermann et al. who had shown that a few members of the library had high affinities for a 1-adrenergic or p-opiate receptors. The results of that published work were used as a retrospective test case to evaluate the effectiveness of Focus-2D. 

To identify potentially active compounds in the virtual library, FOCUS-2D employs stochastic optimization methods such as SA (228, 229) and (jA (230-232). The latter algorithm was used for targeted pentapeptide library design as follows. Initially, a population of 100 peptides is randomly generated and encoded by use of topological indices or amino acid-dependent physicochemical descriptors. The fitness of each peptide is evaluated by its biological activity predicted from a precon-structed QSAR equation (see below). Two par-... 

First Dimension Optimization After the second-dimension separation has been developed, the first-dimension flow rate is determined. This includes selecting a first-dimension column diameter to work at the flow rate selected. We illustrate the selection process with an application that addresses a column method for proteins that functions as a replacement for planar 2D gel electrophoresis (2DGE) within a narrow molecular weight and p/range. In the planar experiment, isoelectric focusing is performed in the first dimension and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS/PAGE) in the second dimension. 

The examples described in this chapter are meant to illustrate the actual application of NMR for the study of protein-ligand interactions in the pharmaceutical industry. In our group, the focus has been mainly on protein observe techniques, as we have been fortunate enough to have a dedicated laboratory for providing both unlabeled and labeled proteins in large quantities. At present, two-dimensional HSQC spectra offer the highest content of information which is not accessible that rapidly by other methods. 2D NMR spectroscopy provides... 

Decornez et al. used a generalized kinase model and a combination of 2D (fingerprint based similarity) and 3D methods (docking) to develop a kinase family focused library (15). The authors used 2800 kinase inhibitors compounds as a reference for a 2D search of their in-house database of 260 compounds... 

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