Induced circular dichroism as a tool to investigate the binding of drugs to carrier proteins: Classic approaches and new trends.

Induced circular dichroism (ICD) is a spectroscopic phenomenon that provides versatile and useful methods for characterizing the structural and dynamic properties of the binding of drugs to target proteins. The understanding of biorecognition processes at the molecular level is essential to discover and validate new pharmacological targets, and to design and develop new potent and selective drugs. The present article reviews the main applications of ICD to drug binding studies on serum carrier proteins, going from the classic approaches for the derivation of drug binding parameters and the identification of binding sites, to an overview of the emerging trends for the characterization of binding modes by means of quantum chemical (QC) techniques. The advantages and limits of the ICD methods for the determination of binding parameters are critically reviewed; the capability to investigate the binding interactions of drugs and metabolites to their target proteins is also underlined, as well as the possibility of characterizing the binding sites to obtain a complete picture of the binding mechanism and dynamics. The new applications of ICD methods to identify stereoselective binding modes of drug/protein complexes are then reviewed with relevant examples. The combined application of experimental ICD spectroscopy and QC calculations is shown to identify qualitatively the bound conformations of ligands to target proteins even in the absence of a detailed structure of the binding sites, either obtained from experimental X-ray crystallography and NMR measurements or from computational models of the complex.

Physical characterization and formulation development of a recombinant pneumolysoid protein-based pneumococcal vaccine.

Streptococcus pneumoniae is a major cause of death in children worldwide. There are more than 90 known pneumococcus serotypes that vary by geographical location. Pneumolysin is a protein toxin produced by virtually all invasive strains of S. pneumoniae and is considered an important virulence factor. Pneumolysin is immunogenic and has the potential to be a new vaccine antigen offering broad serotype-independent coverage. To develop a stable vaccine formulation, the conformational stability of a recombinant pneumolysin mutant (pneumolysoid L460D) was characterized by various techniques. Three data visualization diagrams were constructed to summarize the biophysical data of the L460D pneumolysoid; the protein is most stable in solution at pH 6-7, and loses conformational integrity above 48°C. Excipient screening assays were performed and sugars such as trehalose and sucrose stabilized the pneumolysin mutant with respect to improving thermal transition temperatures and minimizing aggregation. In addition, the protein antigen showed efficient binding to aluminum hydroxide adjuvant. The conformational stability of the L460D pneumolysoid on the surface of alhydrogel adjuvant was little affected by adsorption, either with or without excipients. These studies provide important preformulation characterization information useful for the development of a stable pneumolysin mutant-based vaccine.

Recent theoretical and experimental advances in the electronic circular dichroisms of planar chiral cyclophanes.

The chiroptical properties, such as electronic and vibrational circular dichroism and optical rotation, of planar chiral cyclophanes have attracted much attention in recent years. Although the chemistry of cyclophanes has been extensively explored for more than 60 years, the studies on chiral cyclophanes are rather limited. Experimentally, the use of chiral stationary phases in HPLC becomes more popular and facilitates the enantiomer separation of chiral cyclophanes of interest. Almost all chiral cyclophanes can be readily separated, in analytical and preparative scales, most typically on a Daicel OD type column, which is based on cellulose tris(3,5-dimethylphenylcarbamate). The CD spectra of chiral cyclophanes are unique in their fairly large, significantly coupled Cotton effects observed in all the ¹B (b), ¹L (a), and ¹L (b) band regions. Theoretically, the time-dependent density functional theory, or TD-DFT, method becomes a cost-efficient, yet accurate, theoretical method to reproduce the electronic circular dichroisms and the absorption spectra of a variety of cyclophanes. The direct comparison of the experimental CD spectra with the theoretical ones readily leads to the unambiguous assignment of the absolute configuration of cyclophanes. In addition, the analysis of configuration interaction and molecular orbitals allows detailed interpretation of the electronic transitions and Cotton effects in the UV and CD spectra. Through the study of the CD spectra of chiral cyclophanes as model systems, the effects of intra- and intermolecular interactions on the chiroptical properties of molecules can be explored, and the results thus obtained are valuable in comprehensively elucidating the structure-chiroptical property relationship. In this review the recent progress in experimental and theoretical investigations of the electronic CD spectra of chiral cyclophanes is discussed.

Vibrational circular dichroism spectroscopy of chiral molecules.

In this chapter, new developments and main applications of vibrational circular dichroism (VCD) spectroscopy reported in the last 5 years are described. This includes the determinations of absolute configurations of chiral molecules, understanding solvent effects and modeling solvent-solute explicit hydrogen bonding networks using induced solvent chirality, studies of transition metal complexes and their peculiar and enormous intensity enhancements in VCD spectra, investigations of conformational preference of chiral ligands bound to gold nano particles, and two new advances in applying matrix isolation VCD spectroscopy to flexible, multi-conformational chiral molecules and complexes, and in development of femtosecond laser based VCD instruments for transient VCD monitoring. A brief review of the experimental techniques and theoretical methods is also given. The purpose of this chapter is to provide an up-to-date perspective on the capability of VCD to solve significant problems about chiral molecules in solution, in thin film states, or on surfaces.

Protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases.

Circular dichroism (CD) spectroscopy has been a valuable method for the analysis of protein secondary structures for many years. With the advent of synchrotron radiation circular dichroism (SRCD) and improvements in instrumentation for conventional CD, lower wavelength data are obtainable and the information content of the spectra increased. In addition, new computation and bioinformatics methods have been developed and new reference databases have been created, which greatly improve and facilitate the analyses of CD spectra. This article discusses recent developments in the analysis of protein secondary structures, including features of the DICHROWEB analysis webserver.

New synchrotron radiation circular dichroism end-station on DISCO beamline at SOLEIL synchrotron for biomolecular analysis.

The novel Synchrotron Radiation Circular Dichroism (SRCD) technique is becoming a new tool of investigation for the molecular structures of biomolecules, like proteins, carbohydrates or others bio-materials. Here, we describe the characteristics of a new experimental end-station for circular dichroism studies, in construction on DISCO beamline at SOLEIL synchrotron (Saint-Aubin, France). This experimental end-station will be an open facility for the community of researchers in structural biology. In order to show the kind of information accessible with this type of technique, we give an example: the conformational study of the galactose mutarotase from Escherichia coli, an enzyme involved in the galactose metabolism. This study was made using an operational SRCD station available at SRS (Daresbury Laboratory, UK).