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.     

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.     

MALDI-target integrated platform for affinity-captured protein digestion

To address immunocapture of proteins in large cohorts of clinical samples high throughput sample processing is required. Here a method using the proteomic sample platform, ISET (integrated selective enrichment target) that integrates highly specific immunoaffinity capture of protein biomarker, digestion and sample cleanup with a direct interface to mass spectrometry is presented. The robustness of the on-ISET protein digestion protocol was validated by MALDI MS analysis of model proteins, ranging from 40 fmol to 1 pmol per nanovial. On-ISET digestion and MALDI MS/MS analysis of immunoaffinity captured disease-associated biomarker PSA (prostate specific antigen) from human seminal plasma are presented.     

Fluorine-NMR competition binding experiments for high-throughput screening of large compound mixtures

High-throughput ligand-based NMR screening with competition binding experiments is extended to (19)F detection. Fluorine is a favorable nucleus for these experiments because of the significant contribution of the Chemical Shift Anisotropy (CSA) to the (19)F transverse relaxation of the ligand signal when bound to a macromolecular target. A low to moderate affinity ligand containing a fluorine atom is used as a reference molecule for the detection and characterization of new ligands. Titration NMR experiments with the selected reference compound are performed for finding the optimal set-up conditions for HTS and for deriving the binding constants of the identified NMR hits. Rapid HTS of large chemical mixtures and plant or fungi extracts against the receptor of interest is possible due to the high sensitivity of the (19)F nucleus and the absence of overlap with the signals of the mixtures to be screened. Finally, a novel approach for HTS using a reference molecule in combination with a control molecule is presented.     

Polymorphism - integrated approach from high-throughput screening to crystallization optimization

Crystal structure (polymorphism) as well as crystal shape (morphology) and size have a huge practical and commercial impact on active substances all the way from research to manufacture of the final product. For an optimal development process, it is important to have an integrated approach to these issues ranging from a systematic polymorphism screening to a controlled scale-up of the crystallization process. The polymorphism program has to be tailored according to the development stage. Particularly suitable for an early development stage is a high-throughput polymorphism screening, which is the basis for a more thorough investigation if the product proceeds further in development. Such a comprehensive polymorphism investigation involves further crystallization experiments and extensive physicochemical characterization of the various forms. In this article the high-throughput polymorphism screening method that we have developed is described. Using carbamazepine as an example, the power of this high-throughput polymorphism screening system is demonstrated. Not only were all published forms found, but also new forms were identified. In the second part of the article, important considerations for crystallization optimization are discussed, again using the example of carbamazepine. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Impedimetric immunosensor with on-chip integrated electrodes for high-throughput screening of liver fibrosis markers

This paper deals with an immunoassay based on microelectromechanical system (MEMS) technology with integrated electrochemical cells on a chip and locally copolymerized semi-insulating membrane of poly o-phenylenediamine (PPD) with antibodies. The hepatic fibrosis marker lamin (LN) and its antibody anti-lamin (α-LN) are selected to illustrate the functionality of the sensor. MEMS technology endows this sensor with the features of a small size, a high reproducibility, a low cost, and the potential integration with IC circuitry for portable applications. The sensor is characterized by impedance spectroscopy and calibrated by cyclic voltammetry. The active-site density is found to be directly related to the copolymerization cycles and antibody concentration. The results show that controlling the current in the range from −16 to −13 μA at 0.8 V provides the optimized semi-insulation characteristic that is most suitable for all detections. The impedance analysis reveals that a low-frequency measurement for concentration-dependent impedance is applicable for such a sensor. The evaporation of the electrolyte is found to be responsible for the impedance changes at a very high frequency. The sensitivity of the immunosensor to LN is 0.6208%/(ng/mL) with a linear detection concentration range between 0 and 90 ng/mL. The text was submitted by the authors in English.     

An integrated optofluidic platform for Raman-activated cell sorting

We report on integrated optofluidic Raman-activated cell sorting (RACS) platforms that combine multichannel microfluidic devices and laser tweezers Raman spectroscopy (LTRS) for delivery, identification, and simultaneous sorting of individual cells. The system allows label-free cell identification based on Raman spectroscopy and automated continuous cell sorting. Two optofluidic designs using hydrodynamic focusing and pinch-flow fractionation are evaluated based on their sorting design and flow velocity effect on the laser trapping efficiency at different laser power levels. A proof-of-principle demonstration of the integrated optofluidic LTRS system for the identification and sorting of two leukemia cell lines is presented. This functional prototype lays the foundation for the development of a label-free cell sorting platform based on intrinsic Raman markers for automated sampling and sorting of a large number of individual cells in solution.     

An integrated bioanalytical method development and validation approach: case studies

We proposed an integrated bioanalytical method development and validation approach: (1) method screening based on analyte's physicochemical properties and metabolism information to determine the most appropriate extraction/analysis conditions; (2) preliminary stability evaluation using both quality control and incurred samples to establish sample collection, storage and processing conditions; (3) mock validation to examine method accuracy and precision and incurred sample reproducibility; and (4) method validation to confirm the results obtained during method development. This integrated approach was applied to the determination of compound I in rat plasma and compound II in rat and dog plasma. The effectiveness of the approach was demonstrated by the superior quality of three method validations: (1) a zero run failure rate; (2) >93% of quality control results within 10% of nominal values; and (3) 99% incurred sample within 9.2% of the original values. In addition, rat and dog plasma methods for compound II were successfully applied to analyze more than 900 plasma samples obtained from Investigational New Drug (IND) toxicology studies in rats and dogs with near perfect results: (1) a zero run failure rate; (2) excellent accuracy and precision for standards and quality controls; and (3) 98% incurred samples within 15% of the original values.     

Chemical protein synthesis-assisted high-throughput screening strategies for d-peptides in drug discovery

_D-peptides are recognized as a new class of synthetic chemical drugs and they possess many interesting advantages such as high enzymatic stability,improved oral bioavailability,as well as high binding affinity and specificity.Recently,_D-peptide drugs have been attracting increasing attention in both academic and industrial researches over recent years.One _D-peptide etelcalcetide has even entered the market that targets the calcium(Ca²⁺)-sensing recepto...     

An empirical process for the design of high-throughput screening deck filters

A process for objective identification and filtering of undesirable compounds that contribute to high-throughput screening (HTS) deck promiscuity is described. Two methods of mapping hit promiscuity have been developed linking SMARTS-based structural queries with historical primary HTS data. The first compares an expected assay hit rate to actual hit rates. The second examines the propensity of an individual compound to hit multiple assays. Statistical evaluation of the data indicates a correlation between the resultant functional group filters and compound promiscuity. These data corroborate a number of commonly applied filters as well as producing some unexpected results. Application of these models to HTS collection triage reduced the number of in-house compounds considered for screening by 12%. The implications of these findings are further discussed in the context of the HTS screening set and combinatorial library design as well as compound acquisition.     

高通量、自动化蛋白质结晶筛选技术以及相关仪器的研究进展

基于X射线晶体学的蛋白质结构解析主要依赖于大规模结晶条件筛选获得的高衍射分辨率的蛋白质晶体。近年来,自动化、高通量的液体操控技术和相关仪器的快速发展为蛋白质结晶筛选提供了高效、可靠的研究手段,显著推动了蛋白质结构生物学的研究。文章综述了蛋白质结晶筛选的自动化液体处理技术的发展,包括移液器、注射泵、同步纳升定量吸取注射、喷墨打印、超声喷射以及微流控等技术。文章详细介绍了各技术所对应的典型商品化仪器及其在蛋白质结晶筛选中的应用。此外,文章还介绍了集成多孔板的储存和操控、编码扫描、环境控制和软件管理等诸多功能的一体化液体操纵平台。     

High throughput screening platform for new inhibitors of protein biosynthesis

The search for new antibiotics is an important task, which is of interest for both basic research and health care practices. It is essential to elucidate the mechanism of antimicrobial action during the screening for antimicrobial activity and at the same time be able to test thousands of compounds. A robotic screening system for potential antibiotics developed at the Department of Chemistry at Moscow State University has been described that enables the immediate identification of those that inhibit protein biosynthesis.     

VSDMIP: virtual screening data management on an integrated platform

A novel software (VSDMIP) for the virtual screening (VS) of chemical libraries integrated within a MySQL relational database is presented. Two main features make VSDMIP clearly distinguishable from other existing computational tools: (i) its database, which stores not only ligand information but also the results from every step in the VS process, and (ii) its modular and pluggable architecture, which allows customization of the VS stages (such as the programs used for conformer generation or docking), through the definition of a detailed workflow employing user-configurable XML files. VSDMIP, therefore, facilitates the storage and retrieval of VS results, easily adapts to the specific requirements of each method and tool used in the experiments, and allows the comparison of different VS methodologies. To validate the usefulness of VSDMIP as an automated tool for carrying out VS several experiments were run on six protein targets (acetylcholinesterase, cyclin-dependent kinase 2, coagulation factor Xa, estrogen receptor alpha, p38 MAP kinase, and neuraminidase) using nine binary (actives/inactive) test sets. The performance of several VS configurations was evaluated by means of enrichment factors and receiver operating characteristic plots. ángel R. Ortiz deceased on May 5, 2008.     

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.     

An integrated microfluidic device for the high-throughput screening of microalgal cell culture conditions that induce high growth rate and lipid content

This study describes the development of a microfluidic device for the high-throughput screening of culture conditions, such as the optimum sodium acetate concentration for promoting rapid growth and high lipid accumulation of Chlamydomonas reinhardtii. An analysis of the microalgal growth on the microfluidic device revealed an optimum sodium acetate concentration of 5.72 g L^?1. The lipid content, determined by the 4,4-Difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY® 505/515) staining method, increased with the sodium acetate concentration. The results were found to be statistically reproducible with respect to cell growth and lipid production. Other nutrient conditions, including the nitrogen and phosphorus concentrations, can also be optimized on the same microfluidic platform. The microfluidic device performance results agreed well with the results obtained from the flask-scale experiments, validating that the culture conditions were scalable. Finally, we, for the first time, established a method for the absolute quantification of the microalgal lipid content in the picoliter culture volumes by comparing the on-chip and off-chip data. In conclusion, we successfully demonstrated the high-throughput screening of sodium acetate concentrations that induced high growth rates and high lipid contents in C. reinhardtii cells on the microfluidic device.

Hybrid gravitational field-flow fractionation using immunofunctionalized walls for integrated bioanalytical devices

In this work, the biospecific recognition antigen–antibody reaction was implemented in gravitational field-flow fractionation (GrFFF), a flow-assisted separation technique for micron-sized particles, in order to realize a hybrid immunomodulated GrFFF system in which two different principles are combined to achieve highly versatile fractionation. Micron-sized polystyrene beads coated with horseradish peroxidase (HRP) were used as a model sample, and anti-HRP antibodies were immobilized on the accumulation wall of the GrFFF channel. Ultrasensitive chemiluminescence imaging was employed to visualize the beads during elution and to optimize experimental conditions. The same principle was then applied to real biological samples composed by Yersinia enterocolitica and Escherichia coli cells. Results show the possibility to modify the elution of selected sample components and even to retain them into the channel. The hybrid immunomodulated GrFFF system is a step towards the development of a module that could be integrated in a lab-on-a-chip-based point-of-care testing device which includes sample pre-analytical cleanup and analysis.     

Nucleoside optimization for RNAi: a high-throughput platform

The RNA induced silencing complex (RISC) contains at its core the endonuclease Argonaute (Ago) that allows for guide strand (GS)-mediated sequence-specific cleavage of the target mRNA. Functionalization of the sugar/phosphodiester backbone of the GS, which is in direct contact with Ago, presents a logical opportunity to affect RISC's activity. A systematic evaluation of modified nucleosides requires the synthesis of phosphoramidites corresponding to all four canonical bases (A, U, C, and G) and their sequential evaluation at each position along the 21-nucleotide-long GS. With the use of a platform approach, the sequential replacement of canonical bases with inosine greatly simplifies the problem and defines a new activity baseline toward which the corresponding sugar-modified inosines are compared. This approach was validated using 2'-O-benzyl modification, which demonstrated that positions 5, 8, 15, and 19 can accommodate this large group. Application of this high-throughput methodology now allows for hypothesis-driven rational design of highly potent, immunologically silent and stable siRNAs suitable for therapeutic applications.     

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.