BIOANALYSIS IN DRUG DISCOVERY: AN INTRODUCTION

For several years, bioanalytical techniques and in particular mass spectrometry (MS), have found several applications in the different stages of drug development, including identification of new compounds (organic, inorganic and metallo-organic compounds), their structure elucidation and confirmation, identification of drugs and their metabolites in body-fluids, and assessment of compound purity. With the advent of electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) techniques, the use of MS has also extended to all the discovery stages and in particular: 1) target identification and characterization, 2) screening and hit evaluation, 3) lead identification, and optimization. Drug target identification attempts to identify new targets, normally proteins, whose modulation might inhibit or reverse disease progression. Not too long ago, scientists searched for new targets employing a long and costly process of trial and error. Today, the collective contribution of “omics” approaches allow for the much more rapid and precise discovery of those genes and/or proteins involved in the etiology of certain diseases. In particular, proteomics represents a promising tool for drug target discovery, and this thank to the development of bioanalytical approaches for protein separation and identification/characterization leading to the analysis of protein expression on a broad scale and in complex mixtures. Examples of proteomics application in drug target identification and validation will be given during the seminar. Regarding screening and hit evaluation, ESI-MS has been widely recognized as a suitable technique in the measurement of the effects of compounds on the biological activity of a target molecule (function-based screening). The MS approach (mainly LC-ESI-MS) permits to quantitate analytes by selected ion monitoring (SIM) or multiple reaction monitoring (MRM) and to evaluate the inhibition of a target enzyme at a single compound concentration (% inhibition) or by serial concentrations to obtain IC50. MS is also useful to determine the affinities of compounds for target macromolecules such as proteins, RNA, DNA (affinity-based screening). The screening is based on a MS approach able to study non-covalent complexes between the target and ligands, which can be characterized either by direct detection of the complexes by MS (gas-phase drug screening) or by detection of bound compound after the compound is dissociated from the complex (condensed-phase drug screening). Several condensed-phase ESI-MS approaches have been up to now set-up and applied, among them: frontal affinity chromatography, affinity ultrafiltration, pulsed ultrafiltration, GPC-spin column.