Miniaturized Formats
for Efficient Mass Spectrometry-Based Proteomics and TherapeuticDevelopment Pp-1-12
Gary A. Valaskovic and Neil L. Kelleher
Early Discovery Drug Screening Using Mass
Spectrometry Pp-13-33
Marshall M. Siegel
Metabonomic Applications in Toxicity
Screening and Disease Diagnosis
Pp-35-51
John P. Shockcor and Elaine Holmes
Recent Advances in use of LC/MS/MS for
Quantitative High-Throughput Bioanalytical Support of Drug Discovery Pp-53-66
Bradley L. Ackermann, Michael J. Berna and Anthony T. Murphy
The Emergence and Application of Technological
Advances in Biotransformation Studies
Pp-67-76
Carmen L. Fernández-Metzler and Richard C. King
Organ Perfusion and Mass Spectrometry: A
Timely Merger for Drug Development Pp-77-86
C. Gerald Curtis, Ben Chien, David Bar-Or and Kumar Ramu
Multivariate Pharmaceutical Profiling for
Drug Discovery Pp-87-98
Edward H. Kerns and Li Di
Applications of Computer Software for the
Interpretation and Management of Mass Spectrometry Data in Pharmaceutical
Science Pp-99-107
Antony Williams
[Back to top] Miniaturized
Formats for Efficient Mass Spectrometry-Based Proteomics and
Therapeutic Development
Gary A. Valaskovic and Neil L.
Kelleher
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% 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% 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.
[Back to top] Early
Discovery Drug Screening Using Mass Spectrometry
Marshall M. Siegel
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.
[Back to top] Metabonomic
Applications in Toxicity Screening and Disease Diagnosis
John P. Shockcor and Elaine
Holmes
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.
[Back to top] Recent Advances in use of LC/MS/MS for
Quantitative High-Throughput Bioanalytical Support of Drug
Discovery
Bradley L. Ackermann,
Michael J. Berna and Anthony T. Murphy
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.
[Back to top]
The Emergence and Application of
Technological Advances in Biotransformation Studies
Carmen L. Fernández-Metzler and Richard C. King
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.
[Back to top] Organ
Perfusion and Mass Spectrometry: A Timely Merger for Drug Development
C. Gerald Curtis, Ben
Chien, David Bar-Or and Kumar Ramu
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.
[Back
to top] Multivariate
Pharmaceutical Profiling for Drug Discovery
Edward H. Kerns and Li Di
The field of pharmaceutical
profiling in drug discovery is described. The pharmaceutical properties of drug
candidates determine how much of the drug safelyreaches 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.
[Back
to top] Applications of Computer
Software for the Interpretation and Management of Mass Spectrometry Data in Pharmaceutical Science
Antony Williams
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 nalysis algorithms to the hands of the masses,
specifically as an aid to structure elucidation.