Abstract
Laboratory Automation and High-Throughput Chemistry
Blood–Brain Barrier Permeability Considerations for CNS-Targeted Compound Library Design
A further refinement of the concept of drug-likeness is required for compound libraries intended for central nervous system (CNS) targets to account for the limitations imposed by blood–brain barrier permeability. Stephen A. Hitchcock describes criteria and processes that can be applied in the de novo design and assembly of libraries to increase the odds of compounds residing within CNS-accessible chemical space. A number of published examples where CNS activity and/or penetration characteristics have been a factor in library design are discussed (Curr. Opin. Chem. Biol.
Isocyanide-Based Multicomponent Reactions in Drug Discovery
I. Akritopoulou-Zanze describes recent advances in the application of isocyanide-based multicomponent reactions (IMCRs) in drug discovery and summarizes the various chemotypes used to probe biological targets. In the past couple of years, IMCR-derived ligands have been used to develop agents against infectious diseases and to interfere with protein–protein interactions. Additionally, they were active against a variety of targets such as enzymes, G protein-coupled receptors (GPCRs), and ion channels. The rational for the chemical biologist to apply such diversity generating chemistries is also discussed (Curr. Opin. Chem. Biol.
Kerstin Thurow, Ph.D.
Hilmar Weinmann, Ph.D.
Chemical Synthesis in Nanosized Cavities
M. Patek and M. Drew review methods of incavity synthesis with the main focus on molecularly imprinted polymers and self-assembled capsules. Discussed are examples that highlight recent progress and outline key challenges for the further development of in-cavity synthesis. Emphasis is placed on potential applications in drug discovery and combinatorial technologies (Curr. Opin. Chem. Biol.
Coverage and Bias in Chemical Library Design
The design of chemical libraries directed to target classes is an activity that requires the availability of ligand pharmacological data and/or protein structural data. On the basis of the knowledge derived from these data, chemical libraries directed mainly to GPRS, kinases, proteases, and nuclear receptors have been assembled. However, current design strategies widely overlook assessing the potential ability of the compounds contained in a focused library to provide uniform ample coverage of the protein family they intend to target. J. Mestres et al. discuss the use of in silico target profiling methods as a means to estimate the actual scope of chemical libraries to probe entire protein families and illustrate its applicability in optimizing the composition of compound sets to achieve maximum coverage of the family with minimum bias to particular targets (Curr. Opin. Chem. Biol.
New Directions in Library Design and Analysis
The high costs associated with HTS coupled with the limited coverage and bias of current screening collections is such that diversity analysis continues to be an important criterion in lead generation. Whereas early approaches to diversity analysis were based on traditional descriptors such as two-dimensional fingerprints, a recent emphasis has been on assessing scaffold coverage to ensure that a variety of different chemotypes are represented. V. J. Gillet discusses that it is widely recognized that designs should aim to achieve balance in the number of different properties, and that multiobjective optimization provides an effective way of achieving such designs (Curr. Opin. Chem. Biol.
Structure-Directed Combinatorial Library Design
In recent years, pharmaceutical companies have used structure-based drug design and combinatorial library design techniques to speed up their drug discovery efforts. Both approaches are routinely used in the lead discovery and lead optimization stages of the drug discovery process. Fragment-based drug design, a new powerful tool in the drug design toolbox, is also gaining acceptance across the pharmaceutical industry. J. Zhongxiang Zhou focuses in his review on the interplay between these three design techniques and recent developments in computational methodologies that enhance their integration. Examples of successful synergistic applications of these three techniques are described, and an outlook on future directions in this field is given (Curr. Opin. Chem. Biol.
Library Synthesis Using 5,6,7,8-Tetrahydro-1,6-naphthyridines as Scaffolds
The chemistry of 5,6,7,8-tetrahydro-1,6-naphthyridine scaffolds, synthesized by intramolecular cobalt-catalyzed [2 + 2 + 2] cyclizations, is exploited for library synthesis by J. K. Snyder et al. Urea, amide, and sulphonamide formations are used in the synthesis of a 101-membered library. Screening of the library for antituberculosis activity reveals three lead compounds (J Combinat. Chem.,
Maximizing Efficiency in the Production of Compound Libraries
Efficiency is one of the most important criteria in departments responsible for the production of compounds in a library format. M. Koppitz reports how this key factor has been implemented in the initial design of an automated medicinal chemistry department, and how it has improved processes and workflows and optimized constantly (J. Combinat. Chem.,
Reaction Technology and Liquid Delivery Technologies
Drop on Demand in a Microfluidic Chip
Lab-on-a-chip systems exist in various forms and are used for a lot of purposes. To achieve desired aims with such devices, several components in a miniaturized form have to be established on it, such as channels, mixers, and pumps. This article demonstrates the integration of a drop-on-demand system into a microfluidic chip. Drop-on-demand technology based on a piezoelement is well known as a dispensing technology. A pressure impulse, caused by energizing the piezoelement, is induced in a liquid filled reservoir and a droplet is expelled through a very small nozzle. This technology is adapted from ink-jet printing and allows the generation of reproducible droplets in a range of 50–500 pL. To integrate this principle into a small lab-on-a-chip system, much must be considered. This article describes the major concerns of the construction, the system behavior, and the testing of a prototype. For realization, several calculations and experiments with different designs are made. With the help of measurement equipment and a high speed camera, some results are presented. In contrast to usual drop-on-demand dispensing procedures, there is a liquid–liquid connection instead of dispensing droplets through the air. The droplets are expelled directly from one liquid into the other. The droplets are induced into a channel where a flow can be applied. This design and the opportunities of drop-on-demand releasing enables a kind of digital microfluidics. Precise and high reproducible amounts of liquids can be controlled and made to flow through a chip. Many available video examples underline the opportunities. The entire process of dispensing from liquid directly to another liquid on a lab-on-a-chip system is called: in-chip-drop on demand. The system is a prototype, but suggestions are made about how the commercial adoption can be made easier by using mass produced audio electronics as a control system (J. Micromech. Microeng.,
High-Throughput Analytics
Parallel SFC/MS-MUX Screening to Assess Enantiomeric Purity
Enantiomeric excess (EE) is evaluated by L. Zeng et al. for two internally synthesized compound libraries using a HT automated, intelligent four-channel parallel supercritical fluid chromatography/MS system equipped with a multiplexed ion source interface (SFC/MS-MUX). The two libraries contain compounds spanning a wide range of enantiomeric ratios with structurally diverse chemical scaffolds and stereogenic centers. The system analyzes each sample simultaneously against four chiral columns using up to six organic modifiers. The presented methods demonstrate the value and utility of HT EE determinations to support drug discovery and development programs (Chirality
Easy Ambient Sonic-Spray Ionization Mass Spectrometry Combined with Thin-Layer Chromatography
The analysis of samples directly from their natural matrixes without further preparation, and under atmospheric pressure and room temperature, is one of the most convenient advances in MS. M. N. Eberlin and colleagues from ThoMSon Mass Spectrometry Laboratory (State University of Campinas, Campinas SP, Brazil) describe the advantages of a combination of thin-layer chromatography (TLC) with easy ambient sonic-spray ionization MS (EASI-MS) (Anal. Chem.
TLC is one of the least expensive, simplest, and most easily performed chromatographic methods, and therefore, a very widespread method. EASI-MS uses no voltages, no electrical discharges, no UV or laser beams, and no high temperatures, which make it an excellent detection device for TLC. The coupling provides fast, selective, and sensitive on-spot TLC detection and characterization operating in MS as well as in MS/MS mode. As examples, mixtures of semi-polar nitrogenated compounds, pharmaceutical drugs, and vegetable oils are tested. In general, the received mass spectra are clean and intense, due to the suppression of background ions. With on-spot TLC-EASI-MS/MS, the anticipated fragments of the drug samples are obtained. Reaction monitoring as a straightforward and advantageous application is illustrated for a Ritter-type reaction. Furthermore, an oil analysis via TLC-EASI-MS proves the ability to typify oil spots after TLC separation. The simplicity of the EASI source, concerning construction and installation, enables the exploration of further interesting applications and abilities.
Subambient Pressure Ionization with Nanoelectrospray Source and Interface for Improved Sensitivity in Mass Spectrometry
Electrospray ionization (ESI) has become one of the most widely applied ionization techniques for MS, but the ultimate achievable level of performance is still limited due to the ion losses during transmission from atmospheric pressure to low-pressure regions of the mass analyzer. R. D. Smith and co-workers from Biological Sciences Division (Pacific Northwest National Laboratory, Washington, USA) have designed a nanoelectrospray ionization MS source and interface that enables ion production and transmission in a subambient pressure environment to diminish these limitations (Anal. Chem.
Bioautomation and Screening
Automated On-Chip Rapid Microscopy, Phenotyping, and Sorting of Caenorhabditis elegans
Forward genetics, such as screens of mutagenized populations, and reverse genetics, such as the analysis of RNA interference lines, are classical and modern methods, respectively, to exploit the potential of genetic model organisms. Both approaches are very powerful investigative tools, but both of them depend on large sample sizes. To circumvent this problem, the automation of the different processes in the evaluation and sorting of mutants would be a crucial step forward.
Chung and colleagues present a set-up for automating a model organism, C. elegans. The core of this integrated and automated microsystem is a specific microfluidic chip with on-chip valves. The flow of the worms is observed with a microscope and the cellular or subcellular phenotypes are analyzed by a special closed-loop software, which also controls sorting. The complete process proceeds in four steps. First, a single worm enters the chip. Second, the worm is positioned in the detection zone. Third, it is cooled and imaged, and while phenotyping, a sorting decision is made. In the last step, the worm exits and is sorted to its destination.
According to the presented results, the system works with relatively high velocity and accuracy. It has been tested in three different realistic genetic screening scenarios, and the rate of false evaluation and sorting was below 5% at a speed of 150–900 worms/h depending on experiment. Also, due to the noninvasive analysis, all of the worms survive the procedure and can be characterized in further detail, which is another advantage of this system.
The automation operates independently from human input. Furthermore, it is very flexible in the integration of microscopes, cameras, and further screening tools. This also allows the application of this system to other small model organisms, such as drosophila and zebrafish embryos. Therefore, this automation approach may solve many problems in large-scale genetic experiments of some model organisms, and lead to substantial progress in the fields of developmental biology and functional genomics (Nat. Meth.
High-Content Assays in Oncology Drug Discovery: Opportunities and Challenges
High-content screening (HCS), a process that combines fluorescence microscopic imaging and automated image analysis, has had a significant impact on drug discovery since its introduction in the mid-1990s. The application of HCS within pharmaceutical drug discovery has become widespread, notably within oncology drug discovery. The trends, challenges, and considerations for HCS that affect the successful and pragmatic implementation of this process in drug discovery are outlined by E. H. Mouchet and P. B. Simpson (IDrugs
Massively Parallel Screening of the Receptorome
The National Institute of Mental Health (NIMH) Psychoactive Drug Screening Program (PDSP) is a resource that provides free screening of novel compounds to academic investigators. This program differs from other public-sector screening programs in that compounds are screened against a large panel of transmembrane receptors, channels, and transporters, a selection that currently includes a large portion of the whole neuroreceptorome. N. H. Jensen and B. L. Roth discuss in their review the research areas that can profit from this resource, exemplified by recent findings. The first area is the identification of side effects of medications. Examples include the identification of the histamine H1 receptor as being responsible for weight gain under antipsychotic treatment and the association of 5-HT2B receptor agonism with cardiac valvulopathy, which led to the removal of several medications. A second area is the identification of mechanisms of actions of medications and natural products. Examples include findings of how the kappa opioid receptor is the pharmacological target of the potent hallucinogen salvinorin A, that ephedrine and related compounds are not acting through direct sympathomimetic action, the identification of a strong dopaminergic action of WAY-100635, a compound that had been used as a selective 5-HT1A antagonist, and the discovery that the metabolite desmethylclozapine activates M1 muscarinic receptors, an activity that might contribute to the clinical efficacy of the antipsychotic drug clozapine (Combinat. Chem. High Throughput Screen.,
Affinity Selection-Mass Spectrometry and its Emerging Application to the High Throughput Screening of G Protein-Coupled Receptors
Advances in combinatorial chemistry and genomics have inspired the development of novel affinity selection-based screening techniques that rely on MS to identify compounds that preferentially bind to a protein target. Of the many affinity selection-MS techniques so far documented, only a few solution-based implementations that separate target-ligand complexes away from unbound ligands persist today as routine HTS platforms. Because affinity selection-MS techniques do not rely on radioactive or fluorescent reporters or enzyme activities, they can complement traditional biochemical and cell-based screening assays and enable scientists to screen targets that may not be easily amenable to other methods. In addition, by using MS for ligand detection, these techniques enable HTS of massive library collections of pooled compound mixtures, vastly increasing the chemical space that a target can encounter during screening. Of all drug targets, G protein-coupled receptors yield the highest percentage of therapeutically effective drugs. C. E. Whitehurst and D. A. Annis present the emerging application of affinity selection-MS to the HTS of G protein-coupled receptors. They also review how affinity selection-MS can be used as an analytical tool to guide receptor purification, and further used after screening to characterize target–ligand binding interactions (Combinat. Chem. High Throughput Screen.,
Antibodies Against G Protein-Coupled Receptors: Novel Uses in Screening and Drug Development
Modern biomedical research has used very specific and efficient antibodies in the diagnosis of diseases, localization of gene products, and the rapid screening of targets for drug discovery and testing. In addition, the introduction of antibodies with fluorescent or enzymatic tags has significantly contributed to advances in imaging and microarray technology, which are revolutionizing disease research and the search for effective therapeutics. More recently, antibodies have been used in the isolation of dimeric G protein-coupled receptor (GPCR) complexes. L. A. Devi et al. discuss, in a review, antibodies as powerful research tools for studying GPCRs, and their potential to be developed as drugs (Combinat. Chem. High Throughput Screen.,
Applications of High-Throughput Microsomal Stability Assay in Drug Discovery
HT in vitro microsomal stability assays are widely used in drug discovery as an indicator for in vivo stability, which affects pharmacokinetics. This is based on in-depth research involving a limited number of model drug-like compounds that are cleared predominantly by cytochrome P450 metabolism. However, drug discovery compounds are often not drug-like, are assessed with HT assays, and have many potential uncharacterized in vivo clearance mechanisms. Therefore, it is important to determine the correlation between HT in vitro microsomal stability data and abbreviated discovery in vivo pharmacokinetics study data for a set of drug discovery compounds to have evidence for how the in vitro assay can be reliably applied by discovery teams for making critical decisions. L. Di et al. discuss the relationship between in vitro single time point HT microsomal stability and in vivo clearance from abbreviated drug discovery pharmacokinetics studies using 306 real world drug discovery compounds. The results demonstrate that in vitro Phase I microsomal stability half-life is significantly correlated to in vivo clearance. This demonstrates that HT microsomal stability data are very effective in identifying compounds with significant clearance liabilities in vivo. For compounds with high in vitro rat microsomal stability, no significant differentiation is observed between high and low clearance compounds. This is likely due to other clearance pathways, in addition to cytochrome P450 metabolism that enhances in vivo clearance (Combinat. Chem. High Throughput Screen.,
How Large Does a Compound Screening Collection Need To Be?
Chemical libraries are often produced with focus on a biological target or group of related targets rather than simply being constructed in a combinatorial fashion. A screening collection compiled from such libraries will contain multiple analogs of a number of discrete series of compounds. The question arises as to how many analogs are necessary to represent each series to ensure that an active series will be identified. Based on a simple probabilistic argument and supported by in-house screening data, M. J. Lipkin et al. give guidelines for the number of compounds necessary to achieve a “hit” or series of hits at various levels of certainty. Obtaining more than one hit from the same series is useful, because this enables early acquisition of structure–activity relationship (SAR) and confirms a hit is not a singleton. The authors show that screening collections composed of only small numbers of analogs of each series are suboptimal for SAR acquisition. Based on these studies, a minimum series size of about 200 compounds is recommended. This gives a high probability of confirmatory SAR (i.e., at least two hits from the same series). More substantial early SAR (at least five hits from the same series) can be gained by using series of about 650 compounds each according to the authors (Combinat. Chem. High Throughput Screen.,
Molecular Diversity in the Context of Leadlikeness: Compound Properties that Enable Effective Biochemical Screening
Molecular diversity is of vital importance in drug screening in general, and for the discovery and development of new pharmacophores in particular. Biochemical screening is a powerful tool for pharmacophore development given an understanding of the properties of a good lead compound operating in the biochemical environment. The properties of leadlikeness have evolved to accommodate the artificial conditions of a biochemical assay. Accordingly, the properties of leadlikeness that are suited for screening at protein targets biochemically are different and complementary to the properties of drug-likeness used to guide the selection of good compounds studied biologically in cellular studies and animal models. The benefits of leadlikeness in the biochemical screening arena, including fragment-based screening and cocrystallization studies, are described by G. M. Rishton, and recommendations are forwarded for the generation of leadlike molecular diversity. Chemically stable low molecular weight minimalist compounds (or fragments) with dense heteroatom substitution and variable conformational constraint are promoted as conceptually superior compounds for biochemical screening (Curr. Opin. Chem. Biol.
Computational Analysis of Ligand Relationships within Target Families
Computational tools for the large-scale analysis and prediction of ligand–target interactions and the identification of small molecules having different selectivity profiles within target protein families complement research in chemical genetics and chemogenomics. For computational analysis and design, such tasks require a departure from the traditional focus on single targets, hit identification, and lead optimization. Recently, studies have been reported that profile compounds in silico against arrays of targets or systematically map ligand–target space. J. Bajorath describes that to identify small molecular probes that are suitable for chemical genetics applications, molecular diversity needs to be viewed in a way that partly differs from principles guiding conventional library design (Curr. Opin. Chem. Biol.
Dissimilarity-Based Approaches to Compound Acquisition
The concept of molecular diversity has been integrated in drug discovery efforts for many years. Applications of molecular diversity have been used to identify compounds for screening and to select compounds to augment proprietary collections. These early efforts were crude and suffered from a number of faults, but their evolution has, over the years, led to an improvement in the computational procedures used to identify new commercial compounds for acquisition. Although not much has recently been written about modern methods for augmenting compound collections, this activity is still a very relevant and important task for those involved with the development of compound collections. M. Lajiness and I. Watson focus in their review on the process and software used to identify compounds deemed worthy of acquisition (Curr. Opin. Chem. Biol.
A Cellular Conformation-Based Screen for Androgen Receptor Antagonists
The androgen receptor (AR), a member of the steroid nuclear receptor family of transcription factors, regulates a wide range of physiological processes. Androgen signaling is also associated with numerous human diseases, including prostate cancer. All current antiandrogen therapies reduce ligand access to AR, whether by competitive antagonism or inhibition of androgen production, but are limited by acquired resistance and serious side effects. Thus, novel antiandrogens that target events after ligand binding could have important therapeutic value. Jeremy O. Jones and Marc I. Diamond report a HT assay that exploits fluorescence resonance energy transfer (FRET) to measure ligand-induced conformation change in AR. They directly compare this assay to a transcription-based assay in a screen of FDA-approved compounds and natural products. The FRET-based screen identifies compounds with previously unrecognized antiandrogen activities, with equivalent sensitivity and superior specificity compared with a reporter-based screen. This approach could, therefore, potentially improve the identification of small molecule AR inhibitors (ACS Chem. Biol.