Springer protocols feed by Pharmacology/Toxicology 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Virtual High-Throughput Ligand Screening
In Structural Genomics projects, virtual high-throughput ligand screening can be utilized to provide important functional details for newly determined protein structures. Using a variety of publicly available software tools, it is possible to computationally model, predict, and evaluate how different ligands interact with a given protein. At the Center for Structural Genomics of Infectious Diseases (CSGID) a series of protein analysis, docking and molecular dynamics software is scripted into a single hierarchical pipeline allowing for an exhaustive investigation of protein–ligand interactions. The ability to conduct ...
Source: Springer protocols feed by Pharmacology/Toxicology - March 5, 2014 Category: Drugs & Pharmacology Source Type: news
Structure Determination, Refinement, and Validation
It can be a tremendous advantage to have the X-ray crystal structure of a protein that is targeted for drug discovery. Due to recent advances in methods, software and hardware, crystallographic structure determination no longer requires a specialist in the method, but rather it has become a technique that can be readily applied to many research problems. The high-throughput approaches developed and used by structural genomics projects can be adapted and used to aid drug discovery efforts. It should be emphasized, however, that one cannot blindly accept the results of automated approaches and that it is essential to careful...
Source: Springer protocols feed by Pharmacology/Toxicology - March 5, 2014 Category: Drugs & Pharmacology Source Type: news
Data Collection for Crystallographic Structure Determination
Diffraction data measurement is the final experimental step of crystal structure analysis; all subsequent stages are computational. Good-quality data, optimized for a particular application, make the structure solution and refinement easier and enhance the accuracy of the final models. This chapter describes the principles of the rotation method of data collection and discusses various scenarios that are useful for different types of applications, such as anomalous phasing, molecular replacement, ligand identification, etc. Some typical problems encountered in practice are also discussed. (Source: Springer protocols feed b...
Source: Springer protocols feed by Pharmacology/Toxicology - March 5, 2014 Category: Drugs & Pharmacology Source Type: news
Salvage or Recovery of Failed Targets by Mutagenesis to Reduce Surface Entropy
The success of macromolecular crystallization depends on the protein’s ability to form specific, cohesive intermolecular interactions that serve as crystal contacts. In the cases where the protein lacks surface patches conducive to such interactions, crystallization may not occur. However, it is possible to enhance the likelihood of crystallization by engineering such patches through site-directed mutagenesis, targeting specifically residues with high side chain entropy and replacing them with small amino acids (i.e., surface entropy reduction, SER). This method has proven successful in hundreds of crystallographic a...
Source: Springer protocols feed by Pharmacology/Toxicology - March 5, 2014 Category: Drugs & Pharmacology Source Type: news
Salvage of Failed Protein Targets by Reductive Alkylation
The growth of diffraction-quality single crystals is of primary importance in protein X-ray crystallography. Chemical modification of proteins can alter their surface properties and crystallization behavior. The Midwest Center for Structural Genomics (MCSG) has previously reported how reductive methylation of lysine residues in proteins can improve crystallization of unique proteins that initially failed to produce diffraction-quality crystals. Recently, this approach has been expanded to include ethylation and isopropylation in the MCSG protein crystallization pipeline. Applying standard methods, 180 unique proteins were ...
Source: Springer protocols feed by Pharmacology/Toxicology - March 5, 2014 Category: Drugs & Pharmacology Source Type: news
Salvage or Recovery of Failed Targets by In Situ Proteolysis
In situ proteolysis is the method of proactively adding tiny amounts of nonspecific proteases to aid in the crystallization of proteins and protein macromolecular complexes. The simplicity of the procedure and high recovery rate make it a method of first choice for recalcitrant targets. An improved and updated in situ proteolysis protocol used in high-throughput structural biology platforms is described. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - March 5, 2014 Category: Drugs & Pharmacology Source Type: news
Screening Proteins for NMR Suitability
NMR spectroscopy is a valuable tool in structural genomics. Identification of protein samples that are amenable to structure determination by NMR spectroscopy requires efficient screening. The preparation of multiple samples in parallel and screening by NMR is described. The method described is applicable to large structural genomics projects but can easily be scaled down for application to small structural biology projects. All the equipment used is commonly found in any NMR structural biology laboratory. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - March 5, 2014 Category: Drugs & Pharmacology Source Type: news
High-Throughput Crystallization Screening
Protein structure determination by X-ray crystallography is dependent on obtaining a single protein crystal suitable for diffraction data collection. Due to this requirement, protein crystallization represents a key step in protein structure determination. The conditions for protein crystallization have to be determined empirically for each protein, making this step also a bottleneck in the structure determination process. Typical protein crystallization practice involves parallel setup and monitoring of a considerable number of individual protein crystallization experiments (also called crystallization trials). In these t...
Source: Springer protocols feed by Pharmacology/Toxicology - March 5, 2014 Category: Drugs & Pharmacology Source Type: news
Oxidative Refolding from Inclusion Bodies
This protocol describes the growth and purification of bacterial inclusion body proteins with an option to selenomethionine label the targeted protein through feedback inhibition of methionine biosynthesis in common (non-auxotrophic) strains of E. coli. The method includes solubilization of inclusion body proteins by chemical denaturation and disulfide reduction, renaturation of the solubilized material through rapid dilution by pulsed injection into refolding buffer containing arginine and a mixture of oxidized and reduced glutathione, recovery of the recombinant protein using a stirred cell concentrator, and removal of t...
Source: Springer protocols feed by Pharmacology/Toxicology - March 5, 2014 Category: Drugs & Pharmacology Source Type: news
Parallel Protein Purification
Structural genomics has increased demands on protein production laboratories to supply large numbers of different proteins in high quantities and qualities in shorter times. Extensive experience with many different technologies and strategies developed and implemented by structural genomics groups have helped identify an optimal approach to purification of a large number of widely different proteins. This standardized protocol can be applied to many samples and can be used in parallel protein purification. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - March 5, 2014 Category: Drugs & Pharmacology Source Type: news
Enzyme Kinetics of Oxidative Metabolism: Cytochromes P45
The cytochrome P450 enzymes (CYPs) are the most important enzymes in the oxidative metabolism of hydrophobic drugs and other foreign compounds (xenobiotics). The versatility of these enzymes results in some unusual kinetic properties, stemming from the simultaneous interaction of multiple substrates with the CYP active site. Often, the CYPs display kinetics that deviate from standard hyperbolic saturation or inhibition kinetics. Non-Michaelis–Menten or “atypical” saturation kinetics include sigmoidal, biphasic, and substrate inhibition kinetics (see
Chapter 3
...
Source: Springer protocols feed by Pharmacology/Toxicology - February 17, 2014 Category: Drugs & Pharmacology Source Type: news
Enzyme Kinetics in Drug Metabolism: Fundamentals and Applications
Enzymes are protein catalysts that lower the energy barrier for a reaction and speed the rate of a chemical change. The kinetics of reactions catalyzed by enzymes, as well as several mechanisms underlying the kinetics, have been comprehensively studied and written in textbooks (1, 2). The importance of quantitative evaluation of enzymatic processes has been recognized in many fields of study, including biochemistry, molecular biology, and pharmaceutical sciences to name a few. In pharmaceutical sciences, the applications of enzyme kinetics range from hit finding efforts for new chemical entities on a pharmacological target...
Source: Springer protocols feed by Pharmacology/Toxicology - February 14, 2014 Category: Drugs & Pharmacology Source Type: news
Fundamentals of Enzyme Kinetics
This chapter provides a general introduction to the kinetics of enzyme-catalyzed reactions, with a focus on drug-metabolizing enzymes. A prerequisite to understanding enzyme kinetics is having a clear grasp of the meanings of “enzyme” and “catalysis.” Catalysts are reagents that can increase the rate of a chemical reaction without being consumed in the reaction. Enzymes are proteins that form a subset of catalysts. These concepts are further explored below. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - February 14, 2014 Category: Drugs & Pharmacology Source Type: news
Different Enzyme Kinetic Models
As described in Chapter
2
, a large number of enzymatic reactions can be adequately described by Michaelis–Menten kinetics. The Michaelis–Menten equation represents a rectangular hyperbola, with a y-asymptote at the V
max value. In many cases, more complex kinetic models are required to explain the observed data. Atypical kinetic profiles are believed to arise from the simultaneous binding of multiple molecules within the active site of the enzyme (Tracy and Hummel, Drug Metab Rev 36:231–242, 2004). Several cytochromes P450 have large a...
Source: Springer protocols feed by Pharmacology/Toxicology - February 14, 2014 Category: Drugs & Pharmacology Source Type: news
Reversible Mechanisms of Enzyme Inhibition and Resulting Clinical Significance
Inhibition of a drug-metabolizing enzyme by the reversible interaction of a drug with the enzyme, thus decreasing the metabolism of another drug, is a major cause of clinically significant drug–drug interactions. This chapter defines the four reversible mechanisms of inhibition exhibited by drugs: competitive, noncompetitive, uncompetitive, and mixed competitive/noncompetitive. An in vitro procedure to determine the potential of a drug to be a reversible inhibitor is also provided. Finally, a number of examples of clinically significant drug–drug interactions resulting from reversible inhibition are described. ...
Source: Springer protocols feed by Pharmacology/Toxicology - February 14, 2014 Category: Drugs & Pharmacology Source Type: news
