Abstract
Laboratory Automation and High-Throughput Chemistry
Application of Combinatorial Chemistry Science on Modern Drug Discovery
The application of combinatorial chemistry science has revolutionized high-throughput screening (HTS) paradigms, chemical lead optimization, library purification, and postpurification sample handling, as well as in vitro and in vivo drug metabolism and pharmacokinetic assays. Although no longer in the spotlight and heralded as the savior of the drug industry, combinatorial chemistry science is more prevalent and widespread than ever before. C. W. Lindsley et al. summarize the current status of the field in a perspectives article (J. Comb. Chem.
The Synergy between Combinatorial Chemistry and High-throughput Screening
D. J. Diller highlights in a review some of the strengths of large library combinatorial chemistry as a means of generating molecules for lead discovery, such as providing robust structure–activity relationships around each hit series. The challenges and concepts emerging from traditional high-throughput screening and fragment-based drug design, how these methods influence the design of large combinatorial libraries, and the interpretation of the ensuing high-throughput screening data also are discussed (Curr. Opin. Drug Discov. Dev.
Recent Trends in Library Design: Rational Design Revisited
Diversity has historically played a critical role in the design of combinatorial libraries, screening sets, and corporate collections for lead discovery. Large library design dominated the field of lead discovery in the 1990s, with design methods ranging from arbitrary and property-based reagent selection to product-based approaches. In recent years, there has been a trend toward smaller library size as new technologies greatly increased the volume of information concerning desired targets. Concurrently, computing grids have facilitated the development of structure-based tools that are able to screen hundreds of thousands of molecules. Smaller combinatorial and focused parallel libraries have replaced the unfocused large libraries in the drug design paradigm of the 21st century. Although diversity continues to play a role in lead discovery, D. M. Schnur discusses how the focus of current library design methods has shifted to scaffold design and bio-isostere searching, with a greatly needed emphasis on synthetic feasibility (Curr. Opin. Drug Discov. Dev.
High-Throughput Analytics
Application of LC/MS and Related Techniques to High-Throughput Drug Discovery
The broad implementation of common platform technologies, such as liquid chromatography and mass spectrometry (LC/MS), is a key factor in achieving increased throughput, sensitivity, and data quality for pharmaceutical discovery. A. Espada et al. review the current status and future advances for the applications of LC/MS and related techniques to high-throughput drug discovery (Drug Discov. Today
Microfluidic Chip Technology and Microreactor Technology
A Modular Flow Reactor for Performing Curtius Rearrangements as a Continuous Flow Process
The use of a mesofluidic flow reactor is described by S. V. Ley et al. for performing Curtius rearrangement reactions of carboxylic acids in the presence of diphenylphosphoryl azide and trapping of the intermediate isocyanates with various nucleophiles (Org. Biomol. Chem.
Azide Monoliths as Convenient Flow Reactors for Efficient Curtius Rearrangement Reactions
The preparation and use of an azide-containing monolithic reactor is described by S. V. Ley et al. for use in a flow chemistry device and in particular for conducting Curtius rearrangement reactions via acid chloride inputs (Org. Biomol. Chem.
Transition Metal-Catalyzed Carbon–Carbon Bond Formation Suzuki, Heck, and Sonogashira Reactions Using Microwave and Microtechnology
The fast growing field of microwave and microreactor technology has a significant impact on scaling-up when combined with microwave irradiation for a wide variety of transition metal-catalyzed reactions. The combination of reactor design with immobilization techniques is very important for the flow process, allowing maximal interaction between reagents and a catalyst with no clogging problem. V. S. Parmar et al. review the scale-up possibilities for a variety of transition metal-catalyzed C–C bond-forming reactions applying microwave heating and microtechnology (Org. Process Res. Dev.
Bioautomation and Screening
Which Aspects of HTS are Empirically Correlated with Downstream Success?
In a recent article by M. Glick et al., experience at Novartis with respect to factors influencing the success of HTS campaigns is discussed. An inherent measure of HTS quality can be defined by certain assay factors, the number of hits and their biological potencies. However, such measures of quality do not always correlate with the advancement of hits to the later stages of drug discovery. Furthermore, for many target classes, such as kinases, it is easy to identify hits, but, as a result of selectivity, intellectual property, and other issues, the projects do not result in lead series. In this article, HTS success is defined as the fraction of HTS campaigns that advance into the later stages of drug discovery, and the major influencing factors are examined. Interestingly, screening compounds in individual wells or in mixtures did not have a major impact on the HTS success and, equally interesting, there is no difference in the progression rates of biochemical and cell-based assays. Particular target types, assay technologies, structure–activity relationships, and powder availability have a much greater impact on success rates (Curr. Opin. Drug Discov. Dev.
Prioritizing Hits from Phenotypic High-Content Screens
Advances in the field of high-content screening (HCS) have provided researchers with a powerful new screening tool to observe treatment effects on multiple experimental parameters. Although extremely useful, HCS has resulted in the collection of large data sets of increased complexity that require intensive analysis. Recently, approaches have been developed to analyze multiparametric HCS data more completely, and when used in conjunction with RNA interference, target-based biochemistry, and structural analysis, these approaches have begun to unlock the potential of this screening format in aiding drug discovery. J. J. Sutherland et al. illustrate in a review how the combination of these technologies are used to successfully drive the drug discovery process (Curr. Opin. Drug Discov. Dev.
New Strategies in Drug Discovery for GPCRs: High-Throughput Detection of Cellular ERK Phosphorylation
G-protein coupled receptors (GPCRs) are a large family of receptors for a wide range of stimulants, including hormones, neurotransmitters, and taste and olfactory chemicals. Compared to other receptor families, the GPCRs have been a rich source of drugs. At several levels, however, current screening technologies of drug development for GPCRs are lacking. Firstly, responses from many GPCRs are not easily measured in large screening programs by current techniques. Secondly, there are few options for detecting agonists of orphan GPCRs. Thirdly, it is now clear that the signaling from GPCRs is more complex than once thought, and the measurement of Ca2+ and cAMP can account for only a fraction of the biological information emanating from an activated GPCR. Studies of the discrete and sometimes separable activation of the Ras/Raf/Mek/ERK cascade by many GPCRs are likely to offer development of new agonists and antagonists, contribute to new pharmacologies from receptors, and raise the potential for novel drug candidates in this important area of biology. Downstream activation of the ERK pathway, with or without transactivation of growth factor receptors, has not been measurable by high-throughput methodologies. Michael F. Crouch and Ron I. W. Osmond present recent advances and applications for screening of GPCRs and other receptor species through the rapid measurement of protein phosphorylation events, such as ERK phosphorylation, as new readouts for drug discovery (Comb. Chem. High Throughput Screen.
Label-Free Cell-Based Assays for GPCR Screening
With recent advances in instrumentation and understanding of cellular mechanisms for signals measured, biosensor-centered label-free cell-assay technologies have become a very active area for GPCR screening. Y. Fang et al. review the principles and potential of current label-free cell-assay technologies in GPCR drug discovery (Comb. Chem. High Throughput Screen.
State-Selective Binding Peptides for Heterotrimeric G-Protein Subunits: Novel Tools for Investigating G-Protein Signaling Dynamics
Heterotrimeric G-proteins, comprising Gα, Gβ, and Gγ subunits, are molecular switches that regulate numerous signaling pathways involved in cellular physiology. This is achieved by the adoption of two principal states: an inactive state in which guanosine diphosphate-bound Gα is complexed with the Gβγ dimer, and an active state in which guanosine triphosphate-bound Gα is freed of its Gβγ binding partner. Recently, several screening approaches have been used to identify peptide sequences capable of interacting with Gα (and free Gβγ) in nucleotide-dependent fashions. These peptides have demonstrated applications in direct modulation of the nucleotide cycle, assessing the structural basis for aspects of Gα and Gβγ signaling, and serving as biosensor tools in assays for Gα activation, including high-throughput drug screening. D. P. Siderovski et al. review some of the methods used for such discoveries and discuss the insights that can be gleaned from application of these identified peptides (Comb. Chem. High Throughput Screen.
A High-Throughput Fluorescence Polarization Assay for Inhibitors of the GoLoco Motif/G-Alpha Interaction
The GoLoco motif is a short Gα-binding polypeptide sequence. It is often found in proteins that regulate cell surface receptor signaling as well as in proteins that regulate mitotic spindle orientation and force generation during cell division. A. Simeonov et al. describe a high-throughput fluorescence polarization assay using fluorophore-labeled GoLoco motif peptides for identifying inhibitors of the GoLoco motif interaction with a G-protein alpha subunit. The assay exhibits considerable stability over time and is tolerant to DMSO up to 5%. The assay is miniaturized to a 4 μL final volume. In a fully automated run, the Sigma-Aldrich LOPAC1280 collection is screened three times with every library compound being tested over a range of concentrations. Excellent assay performance is found (Comb. Chem. High Throughput Screen.
G-Protein βγ Subunits as Targets for Small Molecule Therapeutic Development
In drug discovery in the field of GPCRs, most interest has been in the development of selective GPCR agonists and antagonists that activate or inhibit specific GPCRs. Some recent thinking has focused on the idea that some pathologies are the result of the actions of an array of GPCRs, suggesting that targeting single receptors may have limited efficacy. Therefore, targeting pathways common to multiple GPCRs that control critical pathways involved in disease has potential therapeutic relevance. G-protein βγ subunits released from some GPCRs upon receptor activation regulate a variety of downstream pathways to control various aspects of mammalian physiology. From cell-based and animal models, there is evidence that excess Gβγ signaling can be detrimental and blocking Gβγ signaling has positive effects in a number of pathological models. Gβγ regulates downstream pathways through modulation of enzymes that produce cellular second messengers, or through regulation of ion channels by direct protein–protein interactions. Thus, blocking Gβγ functions requires development of small molecule agents that disrupt Gβγ protein interactions with downstream partners. A. V. Smrcka et al. discuss evidence that small molecule targeting Gβγ could be of therapeutic value. The concept of disruption of protein–protein interactions by targeting a hot spot on Gβγ is delineated, and the biochemical and virtual screening strategies for identification of small molecules that selectively target Gβγ functions are outlined (Comb. Chem. High Throughput Screen.
Functional Selectivity in GPCR Modulator Screening
In high-throughput screening systems, a single concentration of a new compound is tested in a biological system to detect direct effects (agonists) or effects on other ligands (antagonists). In this latter case, the chemical context of the assay is defined by a balance of maximal sensitivity and maximal window to observe effect. For allosteric modulators, there are other factors that should be considered in high-throughput screening environments. T. Kenakin gives an overview of functional selectivity in GPCR modulator screening (Comb. Chem. High Throughput Screen.
Immunoassays: Biological Tools for High-Throughput Screening and Characterization of Combinatorial Libraries
In the field of proteomics, there is an urgent need for affinity-catcher molecules to implement effective and high-throughput methods for analyzing the human proteome or parts of it. Antibodies have an essential role in this endeavor, and selection, isolation, and characterization of specific antibodies represent key issues to meet success. Alternatively, it is expected that new, well-characterized affinity reagents generated in rapid and cost-effective manners also will be used to facilitate the deciphering of the function, location, and interactions of the high number of encoded protein products. Combinatorial approaches combined with high-throughput screening technologies have become essential for the generation and identification of robust affinity reagents from biological combinatorial libraries, and the lead discovery of active molecules in large chemical libraries. Phage and yeast provide the means for engineering a multitude of antibodylike molecules against any desired antigen. The construction of peptide libraries is commonly used for the identification and characterization of ligand-receptor specific interactions, and the search for novel ligands for protein purification. Selection and characterization of leads is a most relevant task. Immunological assays, in microtiter plates, biosensors, or microarrays, are highly valuable biological tools for the iterative screening of combinatorial ligand libraries for tailored specificities, and improved affinities. M. A. Taipa states that enzyme-linked immunosorbent assays are frequently the method of choice in a large number of screening strategies for both biological and chemical libraries (Comb. Chem. High Throughput Screen.