Current Topics in Medicinal Chemistry - Volume 2, Issue 1, 2002

Off-line miniaturized “nano-spray” formats for electrospray ionization mass spectrometry (ESI-MS) enable the routine identification of femtomole quantities of protein or peptide. Even greater strides have been achieved using on-line miniaturized ESI-MS methods, such as nanobore LC-MS and CE-MS. On-line methods enable greater sensitivity (sub-attomole limit of detection), dynamic range, and throughput. In either off- or on-line methods for protein analysis, samples are typically isolated and digested enzymatically, with MS analysis of the peptide fragments, yielding 5-50 percent sequence coverage, in a “bottom-up” approach. Obtaining biologically relevant (structure / function) information (such as the localization of regions of error or post-transnational modifications) often demands 100 percent sequence coverage and this may be obtained by analyzing intact proteins by MS with a “top-down”methodology. Proteome wide success with top-down methods will require the development of novel miniaturized approaches for sample preparation along with new tools for bioinformatics. As these miniaturized formats continue to power proteomics applications, they will undoubtedly pollinate “cross-over” applications in LC-MS ranging from drug discovery to development. An example of metabolite identification using an order of magnitude less sample than usually required, with a concurrent order of magnitude increase in signal, illustrates the potential of miniaturized formats in lead characterization activities.

Electrospray ionization (ESI) and matrix-assisted laser desorption / ionization (MALDI) mass spectrometric methods useful for early discovery drug screening are reviewed. All methods described involve studies of non-covalent complexes between biopolymer receptors and small molecule ligands formed in the condensed phase. The complexes can be sprayed intact directly into the gas phase by ESI-MS using gentle experimental conditions. Gas phase screening applications are illustrated for drug ligand candidates non-covalently interacting with peptides, proteins, RNA, and DNA. In the condensed phase, the complexes can be also isolated, denatured and analyzed by ESI-MS to identify the small molecule ligands. Condensed phase drug screening exam-ples are illustrated for the ESI-MS ancillary techniques of affinity chromatography, ultrafiltration, ultracentri-fugation, gel permeation chromatography (GPC), reverse phase-high performance liquid chromatography (RP-HPLC) and capillary electrophoretic methods. Solid phase drug screening using MALDI-MS is illustrated for small molecule ligands bound to MALDI affinity probe tips and to beads. Since ESI and MALDI principally produce molecular ions, high throughput screening is achieved by analyzing mass indexed mixtures.

Biofluid NMR spectroscopy is a powerful tool providing a comprehensive metabolic profile of the low molecular weight components in biofluids that reflect concentrations and fluxes of endogenous metabolites involved in key intermediary cellular pathways, thereby giving an indication of an organisms physiological or pathophysiological status [1]. The interaction of pharmacological agents with cells and tissues can also be monitored using recently developed high resolution magic-angle spinning (HRMAS) NMR spectroscopic technology for biological matrices [1]. However, recent developments in both spectrometer and software technology has resulted in improved capacity for sample handling, leading to a rapid growth in the size of toxicological spectral databases, and increased the complexity of the biological spectral data generated. Thus more emphasis has been placed on the need to develop improved automated procedures for data processing and interpretation. By harnessing chemometric tools for analysis of complex spectral data, the toxicological consequences of xenobiotic exposure can be evaluated efficiently on line. Automation of spectral processing procedures and the construction of mathematically based ‘expert systems’ for the prediction of drug-induced toxicity founded on 1H NMR spectral profiles have now been achieved. Chemometric analysis of biological NMR spectra has provided the main analytical platform for metabonomic analysis, providing a systems approach to evaluating pathophysiological or genetic influences on the metabolic status of an organism [1]. This technology is currently being given high-priority in the pharmaceutical industry with respect to development of efficient high throughput toxicity screening systems for lead candidate selection. In this article, we review the recent developments in metabonomics and consider their application in toxicological screening, disease diagnosis and functional genomics.

LC / MS / MS based bioanalysis using atmospheric pressure ionization (API)-style interfaces has now been applied for over a decade. This technology, which initially found application for clinical bioanalysis, is now firmly established as the primary bioanalytical tool for ADME studies related to drug discovery and lead optimization (LO). This review focuses on recent advances in LC / MS / MS based bioanalysis in support of drug discovery and LO.The initial part of the article reviews the principal components of LC / MS / MS bioanalysis: sample preparation, chromatography, ionization and mass analysis. In each section, factors affecting high throughput bioanalysis are addressed. Because of the importance of on-line column switching methods to discovery bioanalysis, the section on sample preparation is divided into off-line and on-line approaches. In addition, the discussion of chromatography is limited to reversed phase liquid chromatography with emphasis given to the trend towards high-flow gradient elution techniques.The latter part of the review focuses on considerations for experimental design. In this section, pooling methods such as cassette dosing are discussed along with more highly integrated strategies linking bioanalysis with protocol generation and sample collection. The article concludes by briefly reviewing factors, which affect bioanalytical precision and accuracy, such as ion suppression, analyte stability and metabolite interference.

The past years have seen only the beginning of our understanding of metabolic processes and the importance of these processes to the development of safe and effective medicines. The trend to bring more detailed information into earlier stages of drug discovery will continue to drive improvements in technology and in experimental and analytical procedures for the study of biotransformation of drugs. The challenges are significant, but so is the promise of the contributions that can be made by biotransformation studies.

Organ perfusion techniques bridge the methodological gap between in vivo studies on the one hand and in vitro studies on the other. In drug candidate selection and subsequent development the differences between these systems should be considered carefully in study design, as one approach may be more suitable than the other depending on the question(s) being asked and, in particular, how the data will be used.This article is not concerned with the mechanics, the surgery or composition of perfusates as there are numerous reviews / books covering these aspects. Instead, using perfused gut, liver, lung, kidney and brain as examples, the emphasis is on the usefulness (or otherwise) of the data generated with respect to drug absorption, metabolism, pharmacokinetics (PK) and the factors which affect these parameters.Perfusion systems are not difficult to set up but do require ‘high maintenance’ for routine use. For this reason they have been used sparingly by the pharmaceutical industry mainly for problem solving or mechanistic studies. The latter part of this article shows how simultaneous dosing of numerous compounds followed by multiple - component analysis using LC / MS / MS has proved to be an effective way to improve the throughput of absorption, pharmacokinetics and metabolism screening ex vivo.

The field of pharmaceutical profiling in drug discovery is described. The pharmaceutical properties of drug candidates determine how much of the drug safely reaches the therapeutic target. Drug candidates often fail in discovery and development due to inadequate properties, resulting in lost opportunities and resources for developing new drugs. Pharmaceutical profiling assays have been developed and implemented to measure the properties of large numbers of drug candidates starting at the earliest stages of discovery. This information is used for informed decisions in drug candidate selection and synthetic optimization. A holistic process of parallel activity and property optimization has emerged in drug discovery. The assays, strategies, and data management associated with pharmaceutical profiling are discussed.

The rapid growth of mass spectrometry (MS)-based computer software applications has been fueled by the unprecedented need to capture and analyze MS data and provide the information necessary for decision-making. Shorter timelines and a significantly greater number of samples has resulted in a tremendous focus on streamlined approaches that provide scientists, managers, and executives the capability to readily obtain, or even request, the necessary information that leads to accelerated product development. The generation of analytical data using roboticized high-throughput hard-ware has produced a bottleneck since data can be generated faster than it can be analyzed. New techniques including MS / MS and accurate mass experiments are feasible only using computers to capture and manage the enormous amounts of data necessary to perform the experiments. Whatever the nature of the experiments conducted, the MS analysis strategy is to extract the appropriate information required for decision-making in as facile a manner as possible. We will review here a survey of the creation of commercial and laboratory specific reference databases and associated searching algorithms and also recent efforts to introduce advanced processing and analysis algorithms to the hands of the masses, specifically as an aid to structure elucidation.