In the exploratory stage of traditional drug discovery, two steps are taken to evaluate promising drug candidates. The first step is to determine whether the new chemical entity (NCE) interacts with an active site of a biopolymer of therapeutic interest. The second step is to determine whether the new chemical entity interferes with a cell-based biological process. The first approach is generally evaluated with an in vitro assay involving an isolated biopolymer, generally a protein, deoxyribonucleic acid (DNA) model, or ribonucleic acid (RNA) model, while the second approachis generally evaluated with an in vivo cell-based assay. In vitro screening of new drug candidates in low and high throughput formats with mass spectrometry (MS) as the preferred detector is the subject of this review. The mass spectrometric techniques currently in vogue for drug screening are principally electrospray ionization (ESI) MS, and to a much lesser extent, matrix-assisted laser-desorption/ionization (MALDI) MS.

A general goal of in vitro screening is to study the noncovalent interactions between a biopolymer and drug candidate, and to identify those components

Integrated Strategies for Drug Discovery Using Mass Spectrometry, Edited by Mike S. Lee © 2005 John Wiley & Sons, Ltd.

that selectively bind to the active site of the biopolymer. In many applications, the drug candidates consist of a library of known chemical entities, which can be analyzed individually or as mixtures. Likewise, the biopolymers used in these studies are generally fully characterized and can be used either as single components or as mixtures together with the drug candidates for studying the biopolymer-drug noncovalent interactions. The components that react with the active site may inhibit or activate the biological activity of the biopolymer. Nevertheless, the identification of compounds that bind specifically to a biopolymer does not certify activity in a biological system. Activity can only be demonstrated with an in vivo biological assay.

Three mass spectrometric approaches have been taken to study bio-polymer-drug noncovalent complexes produced under native conditions. One approach is to study the biopolymer-drug complex directly in the gas phase by ESI-MS [1-7] and to assay the complex with tandem MS (MS/MS) techniques. The second approach uses condensed-phase separation techniques to resolve the biopolymer-drug complex from unreacted drug or biopolymer, followed by the analysis of the drug by MS techniques [8]. In the first approach, the ESI-MS analysis is conducted under less sensitive native conditions, while in the second approach the complex can be analyzed by ESI-MS under more sensitive denaturing conditions, and in special cases, even by MALDI-MS. The third approach is the study of screened compounds that were noncova-lently bound to an active surface or even covalently bound to a bead surface by releasing and analyzing the small molecules with MALDI-MS techniques.

In this review, the in vitro drug-screening method is illustrated with the direct gas-phase ESI-MS analysis method for peptide-drug, protein-drug, RNA-drug, and DNA-drug complexes. The indirect drug-screening method, with separation techniques coupled with ESI-MS, is illustrated principally for protein-drug complexes using the ancillary techniques of affinity chro-matography, ultrafiltration, ultracentrifugation, gel permeation chromatogra-phy (GPC) spin-columns, GPC reverse-phase high-performance liquid chromatographic (HPLC) and capillary electrophoretic methods. MALDI-MS methods for solid-phase samples are illustrated for samples bound to affinity probe tips and for the analysis of sorted combinatorial chemistry beads. Figure 2.1 summarizes schematically all the strategies used today for screening noncovalent biopolymer-ligand complexes with mass-spectrometric detection for exploratory and early drug discovery.

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