High‐Throughput Approaches in Carbohydrate‐Active Enzymology: Glycosidase and Glycosyl Transferase Inhibitors,Evolution, and Discovery

Carbohydrates are attached and removed in living systems through the action of carbohydrate‐active enzymes such as glycosyl transferases and glycoside hydrolases. The molecules resulting from these enzymes have many important roles in organisms, such as cellular communication, structural support, and energy metabolism. In general, each carbohydrate transformation requires a separate catalyst, and so these enzyme families are extremely diverse. To make this diversity manageable, high‐throughput approaches look at many enzymes at once. Similarly, high‐throughput approaches can be a powerful way of finding inhibitors that can be used to tune the reactivity of these enzymes, either in an industrial, a laboratory, or a medicinal setting. In this review, we provide an overview of how these enzymes and inhibitors can be sought using techniques such as high‐throughput natural product and combinatorial library screening, phage and mRNA display of (glyco)peptides, fluorescence‐activated cell sorting, and metagenomics.     

A Direct Fluorescent Activity Assay for Glycosyltransferases Enables Convenient High‐Throughput Screening: Application to O‐GlcNAc Transferase

Glycosyltransferases carry out important cellular functions in species ranging from bacteria to humans. Despite their essential roles in biology, simple and robust activity assays that can be easily applied to high‐throughput screening for inhibitors of these enzymes have been challenging to develop. Herein, we report a bead‐based strategy to measure the group‐transfer activity of glycosyltransferases sensitively using simple fluorescence measurements, without the need for coupled enzymes or secondary reactions. We validate the performance and accuracy of the assay using O‐GlcNAc transferase (OGT) as a model system through detailed Michaelis–Menten kinetic analysis of various substrates and inhibitors. Optimization of this assay and application to high‐throughput screening enabled screening for inhibitors of OGT, leading to a novel inhibitory scaffold. We believe this assay will prove valuable not only for the study of OGT, but also more widely as a general approach for the screening of glycosyltransferases and other group‐transfer enzymes.     

Exploring the Fundamental Structures of Life: Non‐Targeted,Chemical Analysis of Single Cells and Subcellular Structures

Cells are a basic functional and structural unit of living organisms. Both unicellular communities and multicellular species produce an astonishing chemical diversity, enabling a wide range of divergent functions, yet each cell shares numerous aspects that are common to all living organisms. While there are many approaches for studying this chemical diversity, only a few are non‐targeted and capable of analyzing hundreds of different chemicals at cellular resolution. Here, we review the non‐targeted approaches used to perform comprehensive chemical analyses, provide chemical imaging information, or obtain high‐throughput single‐cell profiling data. Single‐cell measurement capabilities are rapidly increasing in terms of throughput, limits of detection, and completeness of the chemical analyses; these improvements enable their application to understand ever more complex physiological phenomena, such as learning, memory, and behavior.     

On‐Demand Production of Flow‐Reactor Cartridges by 3D Printing of Thermostable Enzymes

The compartmentalization of chemical reactions is an essential principle of life that provides a major source of innovation for the development of novel approaches in biocatalysis. To implement spatially controlled biotransformations, rapid manufacturing methods are needed for the production of biocatalysts that can be applied in flow systems. Whereas three‐dimensional (3D) printing techniques offer high‐throughput manufacturing capability, they are usually not compatible with the delicate nature of enzymes, which call for physiological processing parameters. We herein demonstrate the utility of thermostable enzymes in the generation of biocatalytic agarose‐based inks for a simple temperature‐controlled 3D printing process. As examples we utilized an esterase and an alcohol dehydrogenase from thermophilic organisms as well as a decarboxylase that was thermostabilized by directed protein evolution. We used the resulting 3D‐printed parts for a continuous, two‐step sequential biotransformation in a fluidic setup.     

Pseudilins: Halogenated,Allosteric Inhibitors of the Non‐Mevalonate Pathway Enzyme IspD

The enzymes of the non‐mevalonate pathway for isoprenoid biosynthesis have been identified as attractive targets with novel modes of action for the development of herbicides for crop protection and agents against infectious diseases. This pathway is present in many pathogenic organisms and plants, but absent in mammals. By using high‐throughput screening, we identified highly halogenated marine natural products, the pseudilins, to be inhibitors of the third enzyme, IspD, in the pathway. Their activity against the IspD enzymes from Arabidopsis thaliana and Plasmodium vivax was determined in photometric and NMR‐based assays. Cocrystal structures revealed that pseudilins bind to an allosteric pocket by using both divalent metal ion coordination and halogen bonding. The allosteric mode of action for preventing cosubstrate (CTP) binding at the active site was elucidated. Pseudilins show herbicidal activity in plant assays and antiplasmodial activity in cell‐based assays.     

Applications of Activity‐Based Protein Profiling (ABPP) and Bioimaging in Drug Discovery

Activity‐based protein profiling (ABPP) and bioimaging have been developed in recent years as powerful technologies in drug discovery. Specifically, both approaches can be applied in critical steps of drug development, such as therapy target discovery, high‐throughput drug screening and target identification of bioactive molecules. We have been focused on the development of various strategies that enable simultaneous activity‐based protein profiling and bioimaging studies, thus facilitating an understanding of drug actions and potential toxicities. In this Minireview, we summarize these novel strategies and applications, with the aim of promoting these technologies in drug discovery.     

Nucleic Acid‐Barcoding Technologies: Converting DNA Sequencing into a Broad‐Spectrum Molecular Counter

The emergence of high‐throughput DNA sequencing technologies sparked a revolution in the field of genomics that has rippled into many branches of the life and physical sciences. The remarkable sensitivity, specificity, throughput, and multiplexing capacity that are inherent to parallel DNA sequencing have since motivated its use as a broad‐spectrum molecular counter. A key aspect of extrapolating DNA sequencing to non‐traditional applications is the need to append nucleic‐acid barcodes to entities of interest. In this review, we describe the chemical and biochemical approaches that have enabled nucleic‐acid barcoding of proteinaceous and non‐proteinaceous materials and provide examples of downstream technologies that have been made possible by DNA‐encoded molecules. As commercially available high‐throughput sequencers were first released less than 15 years ago, we believe related applications will continue to mature and close by proposing new frontiers to support this assertion.     

Combinatorial Methods,Automated Synthesis and High‐Throughput Screening in Polymer Research: Past and Present

Combinatorial techniques, parallel experimentation and high‐throughput methods represent a very promising approach in order to speed up the preparation and investigation of new polymeric materials: a large variety of parameters can be screened simultaneously resulting in new structure/property relationships. The field of polymer research seems to be perfectly suited for parallel and combinatorial methods due to the fact that many parameters can be varied during synthesis, processing, blending as well as compounding. In addition, numerous important parameters have to be investigated, such as molecular weight, polydispersity, viscosity, hardness, stiffness and other application‐specific properties. A number of corresponding high‐throughput techniques have been developed in the last few years and their introduction into the commercial market further boosted the development. These combinatorial approaches can reduce the time‐to‐market for new polymeric materials drastically compared to traditional approaches and allow a much more detailed understanding of polymers from the macroscopic to the nanoscopic scale. Here we provide an overview of the present status of combinatorial and parallel polymer synthesis and high‐throughput screening.

     

A Quadruplex‐Based,Label‐Free,and Real‐Time Fluorescence Assay for RNase H Activity and Inhibition

We demonstrate a unique quadruplex‐based fluorescence assay for sensitive, facile, real‐time, and label‐free detection of RNase H activity and inhibition by using a G‐quadruplex formation strategy. In our approach, a RNA–DNA substrate was prepared, with the DNA strand designed as a quadruplex‐forming oligomer. Upon cleavage of the RNA strand by RNase H, the released G‐rich DNA strand folds into a quadruplex in the presence of monovalent ions and interacts with a specific G‐quadruplex binder, N‐methyl mesoporphyrin IX (NMM); this gives a dramatic increase in fluorescence and serves as a reporter of the reaction. This novel assay is simple in design, fast in operation, and is more convenient and promising than other methods. It takes less than 30 min to finish and the detection limit is much better or at least comparable to previous reports. No sophisticated experimental techniques or chemical modification for either RNA or DNA are required. The assay can be accomplished by using a common spectrophotometer and obviates possible interference with the kinetic behavior of the catalysts. Our approach offers an ideal system for high‐throughput screening of enzyme inhibitors and demonstrates that the structure of the G‐quadruplex can be used as a functional tool in specific fields in the future.     

In‐depth insight into the methods of plasma protein‐drug interaction studies: Comparison of capillary electrophoresis‐frontal analysis,isothermal titration calorimetry,circular dichroism and equilibrium dialysis

Plasma protein‐drug binding assays are routinely performed during the early stages of drug discovery and development, which creates demand for an automated high‐throughput screening assay to increase laboratory efficiency. A comprehensive comparison of the four methods typically used for determining the binding parameters is presented in this study with respect to the above demand. Capillary electrophoresis‐frontal analysis, isothermal titration calorimetry, circular dichroism and equilibrium dialysis were used to study the affinity of human serum albumin for diclofenac and lidocaine. These model drugs were chosen due to their different physico‐chemical properties and different binding sites on the albumin molecule, also resulting in different binding strength. The binding parameters estimated under the conditions as similar as possible were comparable among all these approaches as well as to the literature values. Besides this, the comparison of the results and especially other considerations demonstrated the benefits and drawbacks of the selected methods, with capillary electrophoresis‐frontal analysis being the best candidate for such studies.     

Fullerene‐sp2‐Iminosugar Balls as Multimodal Ligands for Lectins and Glycosidases: A Mechanistic Hypothesis for the Inhibitory Multivalent Effect

Concerted functioning of lectins and carbohydrate‐processing enzymes, mainly glycosidases, is essential in maintaining life. It was commonly assumed that the mechanisms by which each class of protein recognizes their cognate sugar partners are intrinsically different: multivalency is a characteristic feature of carbohydrate–lectin interactions, whereas glycosidases bind to their substrates or substrate‐analogue inhibitors in monovalent form. Recent observations on the glycosidase inhibitory potential of multivalent glycomimetics have questioned this paradigm and led to postulate an inhibitory multivalent effect. Here the mechanisms at the origin of this phenomenon have been investigated. A D ‐gluco‐configured sp²‐iminosugar glycomimetic motif, namely 1‐amino‐5N,6O‐oxomethylydenenojirimycin (1N‐ONJ), behaving, simultaneously, as a ligand of peanut agglutinin (PNA) lectin and as an inhibitor of several glycosidases, has been identified. Both the 1N‐ONJ–lectin‐ and 1N‐ONJ–glycosidase‐recognition processes have been found to be sensitive to multivalency, which has been exploited in the design of a lectin–glycosidase competitive assay to explore the implication of catalytic and non‐glycone sites in enzyme binding. A set of isotropic dodecavalent C₆₀‐fullerene–sp²‐iminosugar balls incorporating matching or mismatching motifs towards several glycosidases (inhitopes) was synthesized for that purpose, thereby preventing differences in binding modes arising from orientational preferences. The data supports that: 1) multivalency allows modulating the affinity and selectivity of a given inhitope towards glycosidases; 2) multivalent presentation can switch on the inhibitory capacity for some inhitope–glycosidase pairs, and 3) interactions of the multivalent inhibitors with non‐glycone sites is critical for glycosidase recognition. The ensemble of results point to a shift in the binding mode on going from monovalent to multivalent systems: in the first case a typical ′′key–lock′′ model involving, essentially, the high‐affinity active site can be assumed, whereas in the second, a lectin‐like behavior implying low‐affinity non‐glycone sites probably operates. The differences in responsiveness to multivalency for different glycosidases can then be rationalized in terms of the structure and accessibility of the corresponding carbohydrate‐binding regions.     

Three‐Dimensional Arrays Using GlycoPEG Tags: Glycan Synthesis,Purification and Immobilisation

Glycan arrays have become the premier tool for rapidly establishing the binding or substrate specificities of lectins and carbohydrate‐processing enzymes. New approaches for accelerating carbohydrate synthesis to address the enormous complexity of natural glycan structures are necessary. Moreover, optimising glycan immobilisation is key for the development of selective, sensitive and reproducible array‐based assays. We present a tag‐based approach that accelerates the preparation of glycan arrays on all levels by improving the synthesis, the purification and immobilisation of oligosaccharides. Glycan primers were chemically attached to bifunctional polyethyleneglycol (PEG) tags, extended enzymatically with the help of recombinant glycosyltransferases and finally purified by ultrafiltration. When printed directly onto activated glass slides, these glycoPEG tags afforded arrays with exceptionally high sensitivity, low background and excellent spot morphology. Likewise, the conjugation of glycoPEG tags to latex nanoparticles yielded multivalent scaffolds for carbohydrate‐binding assays with very low non‐specific binding.     

A High‐Throughput Assay for Arylamine Halogenation Based on a Peroxidase‐Mediated Quinone–Amine Coupling with Applications in the Screening of Enzymatic Halogenations

Arylhalides are important building blocks in many fine chemicals, pharmaceuticals and agrochemicals, and there has been increasing interest in the development of more “green” halogenation methods based on enzyme catalysis. However, the screening and development of new enzymes for biohalogenation has been hampered by a lack of high‐throughput screening methods. Described herein is the development of a colorimetric assay for detecting both chemical and enzymatic arylamine halogenation reactions in an aqueous environment. The assay is based on the unique UV/Vis spectrum created by the formation of an ortho‐benzoquinone‐amine adduct, which is produced by the peroxidase‐catalysed benzoquinone generation, followed by Michael addition of either a halogenated or non‐halogenated arylamine. This assay is sensitive, rapid and amenable to high‐throughput screening platforms. We have also shown this assay to be easily coupled to a flavin‐dependent halogenase, which currently lacks any convenient colorimetric assay, in a “one‐pot” workflow.     

Glycomimetics Targeting Glycosyltransferases: Synthetic,Computational and Structural Studies of Less‐Polar Conjugates

The Leloir donors are nucleotide sugars essential for a variety of glycosyltransferases (GTs) involved in the transfer of a carbohydrate to an acceptor substrate, typically a protein or an oligosaccharide. A series of less‐polar nucleotide sugar analogues derived from uridine have been prepared by replacing one phosphate unit with an alkyl chain. The methodology is based on the radical hydrophosphonylation of alkenes, which allows coupling of allyl glycosyl compounds with a phosphate unit suitable for conjugation to uridine. Two of these compounds, the GalNAc and galactose derivatives, were further tested on a model GT, such as GalNAc‐T2 (an important GT widely distributed in human tissues), to probe that both compounds bound in the medium–high micromolar range. The crystal structure of GalNAc‐T2 with the galactose derivative traps the enzyme in an inactive form; this suggests that compounds only containing the β‐phosphate could be efficient ligands for the enzyme. Computational studies with GalNAc‐T2 corroborate these findings and provide further insights into the mechanism of the catalytic cycle of this family of enzymes.     

High‐throughput droplet‐based microfluidics for directed evolution of enzymes

Natural enzymes have evolved over millions of years to allow for their effective operation within specific environments. However, it is significant to note that despite their wide structural and chemical diversity, relatively few natural enzymes have been successfully applied to industrial processes. To address this limitation, directed evolution (DE) (a method that mimics the process of natural selection to evolve proteins toward a user‐defined goal) coupled with droplet‐based microfluidics allows the detailed analysis of millions of enzyme variants on ultra‐short timescales, and thus the design of novel enzymes with bespoke properties. In this review, we aim at presenting the development of DE over the last years and highlighting the most important advancements in droplet‐based microfluidics, made in this context towards the high‐throughput demands of enzyme optimization. Specifically, an overview of the range of microfluidic unit operations available for the construction of DE platforms is provided, focusing on their suitability and benefits for cell‐based assays, as in the case of directed evolution experimentations.     

Microfluidic chips for mass spectrometry‐based proteomics

Microfluidic devices coupled to mass spectrometers have emerged as excellent tools for solving the complex analytical challenges associated with the field of proteomics. Current proteome identification procedures are accomplished through a series of steps that require many hours of labor‐intensive work. Microfluidics can play an important role in proteomic sample preparation steps prior to mass spectral identification such as sample cleanup, digestion, and separations due to its ability to handle small sample quantities with the potential for high‐throughput parallel analysis. To utilize microfluidic devices for proteomic analysis, an efficient interface between the microchip and the mass spectrometer is required. This tutorial provides an overview of the technologies and applications of microfluidic chips coupled to mass spectrometry for proteome analysis. Various approaches for combining microfluidic devices with electrospray ionization (ESI) and matrix‐assisted laser desorption/ionization (MALDI) are summarized and applications of chip‐based separations and digestion technologies to proteomic analysis are presented. Copyright © 2009 John Wiley & Sons, Ltd.     

Total Synthesis of Dansylated Park's Nucleotide for High‐Throughput MraY Assays

The membrane protein translocase I (MraY) is a key enzyme in bacterial peptidoglycan biosynthesis. It is therefore frequently discussed as a target for the development of novel antibiotics. The screening of compound libraries for the identification of MraY inhibitors is enabled by an established fluorescence‐based MraY assay. However, this assay requires a dansylated derivative of the bacterial biosynthetic intermediate Park's nucleotide as the MraY substrate. Isolation of Park's nucleotide from bacteria and subsequent dansylation only furnishes limited amounts of this substrate, thus hampering the high‐throughput screening for MraY inhibitors. Accordingly, the efficient provision of dansylated Park's nucleotide is a major bottleneck in the exploration of this promising drug target. In this work, we present the first total synthesis of dansylated Park's nucleotide, affording an unprecedented amount of the target compound for high‐throughput MraY assays.     

Data‐Driven Materials Exploration for Li‐Ion Conductive Ceramics by Exhaustive and Informatics‐Aided Computations

Interest in all‐solid‐state Li‐ion batteries (LIBs) using non‐flammable Li‐conducting ceramics as solid electrolytes has increased, as safe and robust batteries are urgently desired as power sources for (hybrid) electric vehicles. However, the low Li‐ion conductivities of ceramics have hindered all‐solid‐state LIB commercialization; many researchers have attempted to develop fast Li‐ion conductors. We introduce two efficient high‐throughput computational approaches for materials exploration: (i) exhaustive search and (ii) informatics‐aided prediction. For demonstration, ～400 Li‐ and Zn‐containing oxide (Li?Zn?X?O) compounds of varied crystal structures are extracted from Materials Project datasets. We calculate the migration energies for Li‐ion conduction and the phase stabilities (decomposition energies) of these materials by simulation and apply Bayesian optimization to determine the material with the highest ionic conductivity. The results show much greater efficiency than a random search algorithm.