Testing for drug-human serum albumin binding using fluorescent probes and other methods

ABSTRACT

Introduction: Drug plasma protein binding remains highly relevant to research and drug development, making the assessment and profiling of compound affinity to plasma proteins essential to drug discovery efforts. Although there are a number of fully-characterized methods, they lack the throughput to handle large numbers of compounds. As the evaluation of adsorption, distribution, metabolism, and excretion is addressed earlier in the drug development timeline, the need for higher-throughput methods has grown.

Areas Covered: This review will highlight recent developments on methods for profiling drug plasma binding, with an emphasis on fluorescent probes and emerging high-throughput methodologies.

Expert Opinion: There have been a number of high-throughput assays developed in recent years to meet the scaled up demands for compound profiling. Ultimately, the selection of assay technology relies on a number of factors, such as capabilities of the laboratory and the breadth and amount of data required. Fluorescent probe displacement assays are highly flexible and amenable to high-throughput screening, easily scaling up to handle large compound libraries. Recent developments in fluorescence technologies, such as homogenous time-resolved fluorescence and probes utilizing the aggregation-induced emission effect, have improved the sensitivity of these assays. Other technologies, such as microscale thermophoresis and quantitative structure-activity relationship modeling, are gaining popularity as alternative techniques for drug plasma protein binding characterization.     

Introduction to micro-analytical systems: bioanalytical and pharmaceutical applications.

This review presents a brief overview of recent developments in miniaturization of analytical instruments utilizing microfabrication technology. The concept 'Micro-Total Analysis Systems micro-TAS)', also termed 'Lab-on-a-chip', and the latest progresses in the development of microfabricated separation devices and on-chip detection techniques are discussed. Applications of micro-analytical methods to bioanalytical and pharmaceutical studies are also described, including chemical reactions, assays, and analytical separations of biomolecules in micro-scale.     

Current in vitro kinase assay technologies: the quest for a universal format

The rapidly growing interest in kinases as drug targets has prompted the development of many kinase assay technologies. These technologies can be grouped into three categories: radiometric assays, phospho-antibody-dependent fluorescence/luminescence assays, and phospho-antibody-independent fluorescence/luminescence assays. This article will review some of the major kinase assay technologies on the market, with particular emphasis on the newest systems. We will describe the physical principles, the practical advantages and drawbacks, and the potential applications of these technologies in kinase drug discovery. Most of these technologies are suitable for HTS, but only a few can be utilized for kinetic and mechanistic studies. Significant progress towards development of generic assays, free of radioisotopes and custom reagents such as phospho-specific antibodies, has been made in recent years. However, due to various limitations of each format, none of these generic assay technologies can yet claim to be truly universal. Several factors, including the intended applications, cost, timeline, expertise, familiarity, and comfort level, should be considered prior to pursuing a particular kinase assay technology.     

High-throughput techniques for compound characterization and purification

A new paradigm in drug discovery is the synthesis of structurally diverse collections of compounds, so-called libraries, followed by high-throughput biological screening. High-throughput characterization and purification techniques are required to provide high-quality compounds and reliable biological data, which has led to the development of faster methods, system automation and parallel approaches. This review summarizes recent advances in support of analytical characterization and preparative purification technologies. Notably, mass spectrometry (MS) and supercritical fluid chromatography (SFC) are among the areas where new developments have had a major impact on defining these high-throughput applications.     

Highly sensitive fluorescence detection technology currently available for HTS

Homogeneous fluorescence methods are providing an important tool for HTS technologies. A wide range of different techniques have been established on the market, with read-outs ranging from total fluorescence intensity to statistical analysis of fluorescence fluctuations for biochemical assays or fluorescence imaging techniques for cellular systems. Each method has its own advantages and limitations, which have to be accounted for when designing a specific assay. Here, recently developed fluorescence techniques and some of their applications, with a particular focus on sensitivity, are summarized and their principles are presented.     

Recent advances in inkjet dispensing technologies: applications in drug discovery

Introduction: Inkjet dispensing technology is a promising fabrication methodology widely applied in drug discovery. The automated programmable characteristics and high-throughput efficiency makes this approach potentially very useful in miniaturizing the design patterns for assays and drug screening. Various custom-made inkjet dispensing systems as well as specialized bio-ink and substrates have been developed and applied to fulfill the increasing demands of basic drug discovery studies. The incorporation of other modern technologies has further exploited the potential of inkjet dispensing technology in drug discovery and development. Areas covered: This paper reviews and discusses the recent developments and practical applications of inkjet dispensing technology in several areas of drug discovery and development including fundamental assays of cells and proteins, microarrays, biosensors, tissue engineering, basic biological and pharmaceutical studies. Expert opinion: Progression in a number of areas of research including biomaterials, inkjet mechanical systems and modern analytical techniques as well as the exploration and accumulation of profound biological knowledge has enabled different inkjet dispensing technologies to be developed and adapted for high-throughput pattern fabrication and miniaturization. This in turn presents a great opportunity to propel inkjet dispensing technology into drug discovery.     

Cell-impedance-based label-free technology for the identification of new drugs

Introduction: Drug discovery has progressed from relatively simple binding or activity screening assays to high-throughput screening of sophisticated compound libraries with emphasis on miniaturization and automation. The development of functional assays has enhanced the success rate in discovering novel drug molecules. Many technologies, originally based on radioactive labeling, have sequentially been replaced by methods based on fluorescence labeling. Recently, the focus has switched to label-free technologies in cell-based screening assays.

Areas covered: Label-free, cell-impedance-based methods comprise of different technologies including surface plasmon resonance, mass spectrometry and biosensors applied for screening of anticancer drugs, G protein-coupled receptors, receptor tyrosine kinase and virus inhibitors, drug and nanoparticle cytotoxicity. Many of the developed methods have been used for high-throughput screening in cell lines. Cell viability and morphological damage prediction have been monitored in three-dimensional spheroid human HT-29 carcinoma cells and whole Schistosomula larvae.

Expert opinion: Progress in label-free, cell-impedance-based technologies has facilitated drug screening and may enhance the discovery of potential novel drug molecules through, and improve target molecule identification in, alternative signal pathways. The variety of technologies to measure cellular responses through label-free cell-impedance based approaches all support future drug development and should provide excellent assets for finding better medicines.     

The A,B, Cs of G-protein-coupled receptor pharmacology in assay development for HTS

G-protein-coupled receptors represent one of the most important areas of research in the pharmaceutical industry, being one of the largest druggable gene families. Recognising this fact, manufacturers have developed a huge variety of homogeneous assay technologies that facilitate the quantification of receptor ligand binding events and their downstream signalling cascades. However, while early emphasis was placed on the most sensitive, high-throughput and cost-effective screening technologies to enable identification of the most lead matter for further development, in recent years emphasis has shifted to a focus on maximising the identification of compounds that are new and developing assays that are more biologically/pharmacologically relevant. Therefore, this review provides an overview of the binding and functional techniques available for high-throughput screening, with particular attention on how assay application and configuration can be maximised to ensure their successful identification of relevant chemical matter and thereby optimising project success.     

Informatics and modeling challenges in fragment-based drug discovery

This review summarizes recent developments in fragment-based drug-discovery methods with an emphasis on informatics and modeling requirements. Fragment-based methods have become established as a powerful approach in structure-based lead discovery. A number of successful projects have been announced recently, where fragments have had a central role in hit generation and lead optimization, leading to candidates being considered for clinical trials. Despite these successes, there are still many opportunities for new development, such as improving the structural diversity of fragment libraries, strategies for fragment evolution, and methods for predicting fragment binding modes.     

Rapid methods for deoxynivalenol and other trichothecenes

Method development for deoxynivalenol (DON) and other trichothecenes in recent years was driven by the analytical necessities arising from its widespread (and increasing) occurrence in foods and feeds. This has resulted in the establishment of guideline levels for animal feed, tolerable daily intake (TDI) levels for humans, and most importantly, in the prospect of low-tolerance levels for these toxins in foods in the near future. In order to ensure reliable determination of the toxin content at the tolerance levels, routine analytical methods must have detection limits of less than the tolerance level. This paper intends to give an overview of current analytical developments of rapid testing for deoxynivalenol and other trichothecene mycotoxins, with a special focus on antibody-based techniques. This includes high-throughput instrumental analysis for the laboratory environment, as well as rapid visual tests for on-site testing. The applicability of rapid tests within an integrated detection system for mycotoxins in foods is discussed.     

Parallel confocal detection of single biomolecules using diffractive optics and integrated detector units

The past few years we have witnessed a tremendous surge of interest in so-called array-based miniaturised analytical systems due to their value as extremely powerful tools for high-throughput sequence analysis, drug discovery and development, and diagnostic tests in medicine (see articles in Issue 1). Terminologies that have been used to describe these array-based bioscience systems include (but are not limited to): DNA-chip, microarrays, microchip, biochip, DNA-microarrays and genome chip. Potential technological benefits of introducing these miniaturised analytical systems include improved accuracy, multiplexing, lower sample and reagent consumption, disposability, and decreased analysis times, just to mention a few examples. Among the many alternative principles of detection-analysis (e.g.chemiluminescence, electroluminescence and conductivity), fluorescence-based techniques are widely used, examples being fluorescence resonance energy transfer, fluorescence quenching, fluorescence polarisation, time-resolved fluorescence, and fluorescence fluctuation spectroscopy (see articles in Issue 11). Time-dependent fluctuations of fluorescent biomolecules with different molecular properties, like molecular weight, translational and rotational diffusion time, colour and lifetime, potentially provide all the kinetic and thermodynamic information required in analysing complex interactions. In this mini-review article, we present recent extensions aimed to implement parallel laser excitation and parallel fluorescence detection that can lead to even further increase in throughput in miniaturised array-based analytical systems. We also report on developments and characterisations of multiplexing extension that allow multifocal laser excitation together with matched parallel fluorescence detection for parallel confocal dynamical fluorescence fluctuation studies at the single biomolecule level.     

药材中微量元素测定方法的研究进展

微量元素包含人体必需与可能必需的,随着现代分析技术的不断发展,进行微量元素分析和测定的方法也层出不穷。查阅近几年国内外相关文献,通过对目前的药材中微量元素含量测定方法的研究进展进行综述,包括原子吸收分光光度法、电感耦合等离子体质谱法、荧光光谱法、激光诱导击穿光谱法等,对比分析了各种微量元素含量测定方法的优缺点,以期为提升元素检测标准提供借鉴,不断提高药材质量标准。     

Biomarkers: unrealized potential in sports doping analysis

The fight against doping in sport using analytical chemistry is a mature area with a history of approximately 100 years in horse racing and at least 40 years in human sport. Over that period, the techniques used and the breadth of coverage have developed significantly. These improvements in the testing methods have been matched by the increased sophistication of the methods, drugs and therapies available to the cheat and, as a result, testing has been a reactive process constantly adapting to meet new threats. Following the inception of the World Anti-Doping Agency, research into the methods and technologies available for human doping control have received coordinated funding on an international basis. The area of biomarker research has been a major beneficiary of this funding. The aim of this article is to review recent developments in the application of biomarkers to doping control and to assess the impact this could make in the future.     

Recent advances in inkjet dispensing technologies: applications in drug discovery

Introduction: Inkjet dispensing technology is a promising fabrication methodology widely applied in drug discovery. The automated programmable characteristics and high-throughput efficiency makes this approach potentially very useful in miniaturizing the design patterns for assays and drug screening. Various custom-made inkjet dispensing systems as well as specialized bio-ink and substrates have been developed and applied to fulfill the increasing demands of basic drug discovery studies. The incorporation of other modern technologies has further exploited the potential of inkjet dispensing technology in drug discovery and development.

Areas covered: This paper reviews and discusses the recent developments and practical applications of inkjet dispensing technology in several areas of drug discovery and development including fundamental assays of cells and proteins, microarrays, biosensors, tissue engineering, basic biological and pharmaceutical studies.

Expert opinion: Progression in a number of areas of research including biomaterials, inkjet mechanical systems and modern analytical techniques as well as the exploration and accumulation of profound biological knowledge has enabled different inkjet dispensing technologies to be developed and adapted for high-throughput pattern fabrication and miniaturization. This in turn presents a great opportunity to propel inkjet dispensing technology into drug discovery.     

Annual banned‐substance review: the Prohibited List 2008—analytical approaches in human sports drug testing

The list of prohibited substances and methods of doping issued by the World Anti‐Doping Agency is updated and modified annually based on most recent developments and scientific data. Compounds and methods are maintained, added, or removed from the list, or they are placed in so‐called monitoring programmes that have been established to obtain reliable data on the prevalence of particular substances and methods in‐ and/or out‐of‐competition. Consequently, doping control laboratories continuously update, modify and optimize existing screening and confirmation assays to ensure utmost comprehensiveness in detecting the prohibited and monitored substances as well as chemically and pharmacologically related analogs. The annual banned‐substance review for human sports drug testing critically summarizes recent innovations in analytical approaches supporting the detection of established and newly outlawed substances and methods of doping. Literature from January 2007 through September 2008 as indexed in Medline and Web of Science was screened and articles on detection methods for substances and methods of doping in humans were compiled according to the 2008 Prohibited List of the World Anti‐Doping Agency. Few new approaches were presented for individual doping agents and the majority of reports demonstrated new options for increasing the comprehensiveness of existing doping control assays. In addition, new techniques in separation and/or ionization of analytes complementary to commonly used procedures were described, which, so far, did not meet all requirements of sports drug testing. Copyright © 2009 John Wiley & Sons, Ltd.     

Intact protein analysis in the biopharmaceutical field

In recent years, a growing number of biopharmaceutical proteins have been produced and are already available, or will be soon available, in the market. These molecules are more complex to analyze than conventional low molecular weight drugs, and thus need powerful analytical approaches for the entire development and delivery process. This review summarizes the analytical techniques available for intact protein determination and the main development steps in which they are applicable. A strong emphasis has been put on separation techniques, liquid chromatography and electrophoretic techniques, but mass spectrometry and spectroscopic approaches are also mentioned. Overall, we highlight how several analytical strategies are necessary to obtain global information.     

Microcoil nuclear magnetic resonance spectroscopy

In comparison with most analytical chemistry techniques, nuclear magnetic resonance has an intrinsically low sensitivity, and many potential applications are therefore precluded by the limited available quantity of certain types of sample. In recent years, there has been a trend, both commercial and academic, towards miniaturization of the receiver coil in order to increase the mass sensitivity of NMR measurements. These small coils have also proved very useful in coupling NMR detection with commonly used microseparation techniques. A further development enabled by small detectors is parallel data acquisition from many samples simultaneously, made possible by incorporating multiple receiver coils into a single NMR probehead. This review article summarizes recent developments and applications of “microcoil” NMR spectroscopy.     

Overview of the metallometabolomic methodology for metal-based drug metabolism

Metallometabolomics is an emerging field integrating the research technologies related to the comprehensive analysis of metabolites of a metallodrug in a biologically relevant sample, requiring high-throughput and targeted analyses of the transformation, speciation, localization and structural characteristics of the metallometabolites. This review discusses the concept of metallometabolomics with a focus on analytical techniques and methods, particularly the hyphenated approaches that combine high resolution separation techniques (liquid chromatography or capillary electrophoresis) with highly sensitive detection methods such as mass spectrometry (elemental (ICP) or molecular (ESI)) or nuclear analytical methods (X-ray fluorescence/absorption/emission/diffraction and nuclear magnetic resonance). The application of these advanced analytical technologies in the speciation analysis, identity determination and structural elucidation of metallometabolites will be selectively outlined, along with their advantages and limitations.     

High-throughput analytical techniques for reaction optimization.

This review describes recent developments in approaches to high-throughput reaction optimization, as well as the associated analytical techniques. The studies discussed include the use of UV-visible, IR-thermographic and mass spectrometric methods for application in catalyst development, process optimization and materials science. Other methods of potential use in a high-throughput format are also discussed.     

Recent instrumental progress in mass spectrometry: advancing resolution, accuracy, and speed of drug detection

Fast and unequivocal drug detection is of considerable importance in numerous fields of analytical chemistry, and today mass spectrometry-based approaches are often the method of choice due to their sensitive and specific nature. Mass spectrometry is in constant flux with innovations and thus supports the development of new, complementary assays for rapid determination of drugs and toxins as well as their metabolic products in clinical, forensic, and doping control laboratories. Examples of such innovations that have greatly aided the worldwide bioanalytical efforts are the modern improvements in ion trap, for example, Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometer--Orbitrap, and time-of-flight (TOF) mass analyzers when coupled with sensitive ionization techniques such as electrospray ionization. In this perspective, the utility of state-of-the-art mass spectrometers and recent instrumental developments such as new and/or improved hybrid analyzers are discussed and selected applications presented.