Estimating protein-ligand binding affinity using high-throughput screening by NMR

Many of today's drug discovery programs use high-throughput screening methods that rely on quick evaluations of protein activity to rank potential chemical leads. By monitoring biologically relevant protein-ligand interactions, NMR can provide a means to validate these discovery leads and to optimize the drug discovery process. NMR-based screens typically use a change in chemical shift or line width to detect a protein-ligand interaction. However, the relatively low throughput of current NMR screens and their high demand on sample requirements generally makes it impractical to collect complete binding curves to measure the affinity for each compound in a large and diverse chemical library. As a result, NMR ligand screens are typically limited to identifying candidates that bind to a protein and do not give any estimate of the binding affinity. To address this issue, a methodology has been developed to rank binding affinities for ligands based on NMR screens that use 1D (1)H NMR line-broadening experiments. This method was demonstrated by using it to estimate the dissociation equilibrium constants for twelve ligands with the protein human serum albumin (HSA). The results were found to give good agreement with previous affinities that have been reported for these same ligands with HSA.     

Chip-based high-throughput screening of herbal medicines

At present, high-throughput screening (HTS) programs in drug discovery rely mainly on compound libraries from combinational chemistry. Similarly, natural flora has been used as a prominent origin for new and potent herbal drugs. Herbal medicines have been used worldwide for thousands of years to cure many diseases. As such, herbal secondary metabolites show a remarkable structural diversity that supplements chemically synthesized compound analogs in drug discovery screening. Unfortunately, there is often a considerable deterioration in the quality of herbal drugs in such screening programs as there are time-consuming manual processes involved in the isolation of active ingredients from the highly complex mixtures of herbal plant products. The quality and quantity of herbal samples are critical for the success of HTS programs. In the recent past, there have been substantial improvements in HTS due to the miniaturization and integration of microchip (e.g., Herbochip(?), DNA chip, protein chip, cell chip, etc.)-based technologies so as to design herbal drugs that compete with synthetic drug analogs. Here we will review various technologies used for HTS of herbal medicines. Finally, we will summarize our efforts to develop a novel chip-based HTS assay to explore the antioxidant and radioprotective properties of herbal plants.     

Bipolar electrochemistry for high-throughput corrosion screening

It is demonstrated that bipolar electrochemistry can be used for high-throughput corrosion testing covering a wide potential range in one single experiment and that this, combined with rapid image analysis, constitutes a simple and convenient way to screen the corrosion behaviour of conducting materials and corrosion protective coatings. Stainless steel samples (SS304), acting as bipolar electrodes, were immersed in sulphuric and hydrochloric acid and exposed to an electric field to establish a potential gradient along the surface. In this way, the same steel sample was exposed to a wide range of cathodic and anodic conditions, ranging from potentials yielding hydrogen evolution to potentials well into the transpassive region. This wireless approach enables rapid simultaneous comparison of numerous samples, and also provides the opportunity to perform experiments on samples that are of a complex shape, or which otherwise are difficult to employ in standard electrochemical corrosion tests.     

Automated high-throughput nanoliter-scale protein crystallization screening

A highly efficient method is developed for automated high-throughput screening of nanoliter-scale protein crystallization. The system integrates liquid dispensing, crystallization and detection. The automated liquid dispensing system handles nanoliters of protein and various combinations of precipitants in parallel to access diverse regions of the phase diagram. A new detection scheme, native fluorescence, with complementary visible-light detection is employed for monitoring the progress of crystallization. This detection mode can distinguish protein crystals from inorganic crystals in a nondestructive manner. A gas-permeable membrane covering the microwells simplifies evaporation rate control and probes extended conditions in the phase diagram. The system was successfully demonstrated for the screening of lysozyme crystallization under 81 different conditions.     

Frequency-swept HMQC sequences for high-throughput NMR analysis

We describe here new versions of the DEPT phase-encoded HMQC experiment that offer robust performance and improved sensitivity. The new sequences rely on frequency-swept proton and carbon pulses to minimize signal losses from miscalibrated pulses while providing 'J compensation' to optimize the signal strength over a range of heteronuclear coupling constants. By including both proton and carbon-swept pulses, the new sequences also offer an additional signal gain of roughly 10% over well-calibrated hard-pulse experiments. The new sequences also demonstrate that one can construct a sequence that incorporates both 90 degrees and 180 degrees frequency-swept pulses. Although individual pulses in the sequence cause severe phase roll, the phase roll can be eliminated by the proper choice of pulse lengths and sweep directions.     

高通量药物筛选现代检测技术研究进展

高通量药物筛选是发现创新药物的重要技术途径.高通量筛选结果必须通过适当的检测方法才能反映出来,检测技术是实现高通量药物筛选的基础.本文综述了近年来有关光学分析、色谱分析、热分析、电化学分析、质谱、核磁共振等现代检测技术在高通量药物筛选研究中的进展.     

Frequency-swept HSQC sequences for high-throughput NMR analysis

This article describes new versions of the DEPT phase-edited heteronuclear single quantum correlation (HSQC) pulse sequence with sensitivity enhancement. The sequences incorporate frequency-swept carbon and proton pulses. The new experiments are inherently robust, well-suited for a high-throughput setting in which sample-to-sample variations may be ignored. The observed signal has the obvious benefit of sensitivity enhancement resulting from the preservation of two magnetization transfer pathways. The two pathways are maintained even in the version of the sequence in which all pulses are frequency-swept. There is an additional signal gain of roughly 10% that derives from the use of both proton and carbon frequency-swept pulses. Furthermore, the sequences use J compensation to provide optimal signal over a range of heteronuclear coupling constants. We demonstrate that the new sequences offer good sensitivity and perform well even when the NMR probe is deliberately mistuned.     

Design and prioritization of plates for high-throughput screening.

A general algorithm for the prioritization and selection of plates for high-throughput screening is presented. The method uses a simulated annealing algorithm to search through the space of plate combinations for the one that maximizes some user-defined objective function. The algorithm is robust and convergent, and permits the simultaneous optimization of multiple design objectives, including molecular diversity, similarity to known actives, predicted activity or binding affinity, and many others. It is shown that the arrangement of compounds among the plates may have important consequences on the ability to design a well-targeted and cost-effective experiment. To that end, two simple and effective schemes for the construction of homogeneous and heterogeneous plates are outlined, using a novel similarity sorting algorithm based on one-dimensional nonlinear mapping.     

NMR and in silico screening

NMR-based screening and virtual, or in silico, screening can be highly complementary and synergistic. NMR-based screening is a rapid and reliable method for validating hits that come from in silico screens. In addition, ligand-binding data derived from NMR-based screens can focus and direct subsequent in silico screening. We will first give a short overview of existing NMR and in silico screening methods, discuss the drawbacks associated with each, and finally present applications that highlight the combination of the two technologies.     

Gas chromatographic high-throughput screening techniques in catalysis

Discovering highly efficient catalysts is of great scientific and economical interest. Advances in high-throughput assays in combination with sophisticated analytical techniques have increased the rapidity with which catalysts can be identified and optimized. Understanding how kinetics in the mechanism of catalysis is controlled by structural parameters is essential for a directed design of catalysts. To identify such rate-controlling elementary steps and to develop and refine models, comprehensive experimental kinetic data of a broad variety of substrates are necessary. In the present article concepts of high-throughput screening techniques in catalysis using gas chromatography are reviewed in a survey covering the period from 1998 to 2007. To cover also the origins of concepts and groundbreaking experiments in this research area milestones going back to 1950 are also reviewed. The first part of the review will focus on off-line gas chromatographic analysis, the second part on on-line gas chromatographic analysis covering sequential, parallelized and high-throughput multiplexing gas chromatography. The third part presents recent advances in the integration of chemical transformation and analysis in gas chromatography. The present review article describes the state-of-the-art, scope and limitations, and applications of these different high-throughput screening approaches.     

Large-scale plasmonic microarrays for label-free high-throughput screening

Microarrays allowing simultaneous analysis of thousands of parameters can significantly accelerate screening of large libraries of pharmaceutical compounds and biomolecular interactions. For large-scale studies on diverse biomedical samples, reliable, label-free, and high-content microarrays are needed. In this work, using large-area plasmonic nanohole arrays, we demonstrate for the first time a large-scale label-free microarray technology with over one million sensors on a single microscope slide. A dual-color filter imaging method is introduced to dramatically increase the accuracy, reliability, and signal-to-noise ratio of the sensors in a highly multiplexed manner. We used our technology to quantitatively measure protein-protein interactions. Our platform, which is highly compatible with the current microarray scanning systems can enable a powerful screening technology and facilitate diagnosis and treatment of diseases.     

Spontaneous membrane-translocating peptides by orthogonal high-throughput screening

Combinatorial peptide chemistry and orthogonal high-throughput screening were used to select peptides that spontaneously translocate across synthetic lipid bilayer membranes without permeabilization. A conserved sequence motif was identified that contains several cationic residues in conserved positions in an otherwise hydrophobic sequence. This 9-residue motif rapidly translocates across synthetic multibilayer vesicles and into cells while carrying a large polar dye as a "cargo" moiety. The extraordinary ability of this family of peptides to spontaneously translocate across bilayers without an energy source of any kind is distinctly different from the behavior of the well-known, highly cationic cell-penetrating peptides, such as the HIV tat peptide, which do not translocate across synthetic bilayers, and enter cells mostly by active endocytosis. Peptides that translocate spontaneously across membranes have the potential to transform the field of drug design by enabling the delivery of otherwise membrane-impermeant polar drugs into cells and tissues. Here we describe the chemical tools needed to rapidly identify spontaneous membrane translocating peptides.     

Strategic pooling of compounds for high-throughput screening.

Bringing new medicines to the market depends on the rapid discovery of new and effective drugs, often initiated through the biological testing of many thousands of compounds in high-throughput screening (HTS). Mixing compounds together into pools for screening is one way to accelerate this process and reduce costs. This paper contains both theoretical and experimental data which suggest that careful selection of compounds to be pooled together is necessary in order to reduce the risk of reactivity between compounds within the pools.     

Fluorescence assays for high-throughput screening of protein kinases

Protein kinases comprise one of the most important group of targets for drug discovery research today. Methods to identify novel kinase inhibitors by high-throughput screening have evolved rapidly in recent years. An important aspect is the availability of fluorescent probes that can be applied in a homogeneous, or mix-and-measure, assay format. Here, we illustrate the application of fluorescence read-out technologies for kinase targets in light of our own experiences in assay development and high-throughput screening.     

Setup for high-throughput impedance screening of gas-sensing materials

This paper reports on the setup for a high-throughput impedance measurement system that allows rapid screening of the electrical and dielectrical properties of solid-state sample libraries in variable atmospheres and temperatures. Using multielectrode arrays, most time-consuming steps in the workflow are parallelized. In addition, an approach for automated data evaluation of impedance spectra is presented. For reasons of verification of robust measuring results and reproducibility, screening results of a sample library composed of doped indium(III) oxide as a resistive-type gas-sensing material are discussed on the basis of the determined sensitivities focusing temperature and testing gas gradients.     

Optical chemical biosensors for high-throughput screening of drugs

Optical biosensors have been commercially available since the early 1990s, and have been used extensively in many areas of research in the life sciences. Optical biosensors developed for drug analysis generally exploit the high selectivity of the antigen-antibody and drug-protein interaction. Optical biosensors can be made based on optical diffraction or electro-chemiluminescence. High-throughput screening, (HTS) which includes automated preparation of a large number of samples and then screening of their properties in multi-well plates, improves the efficiency of research in many scientific areas, e.g., catalyst screening, food processing, chemical synthesis, drug discovery, absorption, distribution, metabolism, and excretion and toxicological and cell based screening. The three most common detection techniques used in HTS are UV-VIS absorbance, fluorescence and luminescence. In this review, we summarize some recent trends and developments in the construction of optical chemical biosensors used in high-throughput screening of drugs. Also, we have included environmental, biological and other medical applications of biosensors.     

Synthesis and high-throughput screening of N-acetyl-beta-hexosaminidase inhibitor libraries targeting osteoarthritis

C1 Nitrogen iminocyclitols are potent inhibitors of N-acetyl-beta-hexosaminidases. Given hexosaminidases' important roles in osteoarthritis, we developed two straightforward and efficient syntheses of C1 nitrogen iminocyclitols from two readily available starting materials, D-mannosamine hydrochloride and the microbial oxidation product of fructose. A diversity-oriented synthetic strategy was then performed by coupling these core structures with various aldehydes, carboxylic acids, and alkynes to generate three separate libraries. High-throughput screening of the generated libraries with human N-acetyl-beta-hexosaminidases produced only moderate inhibitory activities. However, the synthetic approach and screening strategy for these compounds will be applied to develop new potent inhibitors of human N-acetyl-beta-hexosaminidases, particularly when combined with the structural information of these enzymes.     

Assay development and high-throughput screening of caspases in microfluidic format

Caspase proteases are familiar targets in drug discovery. A common format for screening to identify caspase inhibitors employs fluorogenic or colorimetric tetra-peptide substrates in 96, 384, or 1536 -well microtiter plates. The primary motivation for increasing the number of wells per plate is to reduce the reagent cost per test and increase the throughput of HTS operations. There are significant challenges, however, to moving into or beyond the 1536-well format, such as submicroliter liquid handling, liquid evaporation, increased surface area-to-volume ratios, and the potential for artifacts and interference from small air-borne particles such as lint. Therefore, HTS scientists remain keenly interested in technologies that offer alternatives to the ever-shrinking microtiter plate well. Microfluidic assay technology represents an attractive option that, in theory, consumes only subnanoliter volumes of reagents per test. We have successfully employed a microfluidic assay technology in fluorogenic screening assays for several caspase isoforms utilizing the Caliper Technologies Labchip platform. Caspase-3 is used as a representative case to describe microfluidic assay development and initial high-throughput screening results. In addition, microfluidic screening and plate-based screening are compared in terms of reagent consumption, data quality, and ease of operation.