Fluorescence correlation spectroscopy: lead discovery by miniaturized HTS

Auer, Manfred; Moore, Keith J. M.; Meyer‐Almes, Franz‐Josef; Guenther, Rolf; Pope, Andrew J.; Stoeckli, K.

doi:10.1016/s1359-6446(98)01240-9

Cited by 97 publications

(64 citation statements)

References 9 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The binding of a small ligand to a macromolecule will significantly reduce the diffusion rate of the ligand. This idea provides the basis for measurements of noncovalent ligand-macromolecule interactions by pulsed field gradient NMR [4 -11] and fluorescence correlation spectroscopy [12][13][14]. In a similar fashion, fluorescence polarization measurements rely on changes of the rotational diffusion coefficient upon complex formation [15].…”

mentioning

confidence: 99%

Diffusion measurements by electrospray mass spectrometry for studying solution-phase noncovalent interactions

Clark

Konermann

2003

J. Am. Soc. Mass Spectrom.

View full text Add to dashboard Cite

This study describes a novel approach for monitoring noncovalent interactions in solution by electrospray mass spectrometry (ESI-MS). The technique is based on measurements of analyte diffusion in solution. Diffusion coefficients of a target macromolecule and a potential low molecular weight binding partner are determined by measuring the spread of an initially sharp boundary between two solutions of different concentration in a laminar flow tube (Taylor dispersion), as described in Rapid Commun. Mass Spectrom. 2002Spectrom. , 16, 1454Spectrom. -1462. In the absence of noncovalent interactions, the measured ESI-MS dispersion profiles are expected to show a gradual transition for the macromolecule and a steep transition for the low molecular weight compound. However, if the two analytes form a noncovalent complex in solution the dispersion profiles of the two species will be very similar, since the translational diffusion of the small compound is determined by the slow Brownian motion of the macromolecule. In contrast to conventional ESI-MS-based techniques for studying noncovalent complexes, this approach does not rely on the preservation of solution-phase interactions in the gas phase. On the contrary, "harsh" conditions at the ion source are required to disrupt any potential gasphase interactions between the two species, such that their dispersion profiles can be monitored separately. The viability of this technique is demonstrated in studies on noncovalent heme-protein interactions in myoglobin. Tight noncovalent binding is observed in solutions of pH 10, both in the absence and in the presence of 30% acetonitrile. In contrast, a significant disruption of the noncovalent interactions is seen at an acetonitrile content of 50%. Under these conditions, the diffusion coefficient of heme in the presence of myoglobin is only slightly lower than that of heme in a protein-free solution. A breakdown of the noncovalent interactions is also observed in aqueous solution of pH 2.4, where myoglobin is known to adopt an acid-unfolded conformation. (J Am Soc Mass Spectrom 2003, 14, 430 -441)

show abstract

mentioning

confidence: 99%

Diffusion measurements by electrospray mass spectrometry for studying solution-phase noncovalent interactions

Clark

Konermann

2003

J. Am. Soc. Mass Spectrom.

View full text Add to dashboard Cite

show abstract

“…[14] Biochemical assays can be heterogeneous or homogenous, heterogeneous assays requiring multiple steps, compared to simpler homogeneous assays that are simply 'mix and read' type systems. Detection techniques in HTS are predominantly fluorescence and luminescence based techniques, including standard fluorescence and luminescence assays as well as extensions of these such as fluorescence resonance energy transfer (FRET) [25], fluorescence polarization (FP) [26], homogeneous time resolved fluorescence (HTRF) [27] and fluorescence correlation spectroscopy (FCS) [28]. Assays are developed to detect specific binding processes or to probe particular pathways, networks or phenotypes.…”

Section: Current High Throughput Screening Techniques: Could Vibratiomentioning

confidence: 99%

Vibrational spectroscopy as a tool for studying drug-cell interaction: Could high throughput vibrational spectroscopic screening improve drug development?

Jamieson

Byrne²

2017

Vibrational Spectroscopy

View full text Add to dashboard Cite

. (2017). Vibrational spectroscopy as a tool for studying drug-cell interaction: could high throughput vibrational spectroscopic screening improve drug development? Vibrational Spectroscopy, vol. 91, July, pp. 16-30. doi.org/10.1016/ j.vibspec.2016 Vibrational spectroscopy as a tool for studying drug-cell interaction: could high throughput vibrational spectroscopic screening improve drug development? AbstractVibrational spectroscopy is currently widely explored as a tool in biomedical applications. An area at the forefront of this field is the use of vibrational spectroscopy for disease diagnosis, ultimately aiming towards spectral pathology. However, while this field shows promising results, moving this technique into the clinic faces the challenges of widespread clinical trials and legislative approval. While spectral pathology has received a lot of attention, there are many other biomedical applications of vibrational spectroscopy, which could potentially be translated to applications with greater ease. A particularly promising application is the use of vibrational spectroscopic techniques to study the interaction of drugs with cells. Many studies have demonstrated the ability to detect biochemical changes in cells in response to drug application, using both infrared and Raman spectroscopy. This has shown potential for use in high throughput screening (HTS) applications, for screening of efficacy and mode of action of potential drug candidates, to speed up the drug discovery process. HTS is still a relatively new and growing area of research and, therefore, there is more potential for new techniques to move into and shape this field. Vibrational spectroscopic techniques come with many benefits over the techniques used currently in HTS, primarily based on fluorescence assays to detect specific binding interactions or phenotypes. They are label free, and an infrared or Raman spectrum provides a wealth of biochemical information, and therefore could reveal not only information about a specific interaction, but about how the overall biochemistry of a cell changes in response to application of a drug candidate. Therefore, drug mode of action could be elucidated. This review will investigate the potential for vibrational spectroscopy, particularly FTIR and Raman spectroscopy, to benefit the field of HTS and improve the drug development process. In addition to FTIR and Raman spectroscopy, surface enhanced Raman spectroscopy (SERS), coherent anti-Stokes Raman spectroscopy (CARS) and stimulated Raman spectroscopy (SRS), will be investigated as an alternative tool in the HTS process.

show abstract

“…Therefore, such spiking experiments are an integral part of validating the miniaturized assay prior to primary screening. Both oxytocin and vasopressin were randomly placed in 16 nanoplate. Both agonists were added at 1 µM, the primary screening concentration, which is significantly higher than their EC 50 values.…”

Section: Miniaturization Of An Agonist Assaymentioning

confidence: 99%

Miniaturization of Whole Live Cell-Based GPCR Assays Using Microdispensing and Detection Systems

Kornienko¹,

Lacson²,

Kunapuli³

et al. 2004

SLAS Discovery

View full text Add to dashboard Cite

Cell-based β-lactamase reporter gene assays designed to measure the functional responses of G-protein-coupled receptors (GPCRs) were miniaturized to less than 2 µL total assay volume in a 3456-well microplate. Studies were done to evaluate both receptor agonists and antagonists. The pharmacology of agonists and antagonists for target GPCRs originally developed in a 96-well format was recapitulated in a 3456-well microplate format without compromising data quality or EC 50 /IC 50 precision. These assays were employed in high-throughput screening campaigns, allowing the testing of more than 150,000 compounds in 8 h. The instrumentation used and practical aspects of the assay development are discussed. (Journal of Biomolecular

show abstract

Fluorescence correlation spectroscopy: lead discovery by miniaturized HTS

Cited by 97 publications

References 9 publications

Diffusion measurements by electrospray mass spectrometry for studying solution-phase noncovalent interactions

Diffusion measurements by electrospray mass spectrometry for studying solution-phase noncovalent interactions

Vibrational spectroscopy as a tool for studying drug-cell interaction: Could high throughput vibrational spectroscopic screening improve drug development?

Miniaturization of Whole Live Cell-Based GPCR Assays Using Microdispensing and Detection Systems

Contact Info

Product

Resources

About