High-throughput screening: advances in assay technologies

Sittampalam, G. Sitta; Kahl, Steven D.; Janzen, William P.

doi:10.1016/s1367-5931(97)80078-6

Cited by 135 publications

(90 citation statements)

References 64 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…In high-throughput enzyme inhibition assays, more than a million compounds are usually tested for their ability to inactivate the target enzyme. Often the process involves two consecutive screens [1,2]. The first screen identifies compounds that inhibit the enzymatic activity and is usually performed at a single compound concentration.…”

Section: B S T R a C Tmentioning

confidence: 99%

A fluorescence quenching assay to discriminate between specific and nonspecific inhibitors of dengue virus protease

Bodenreider

Beer

Keller

et al. 2009

Analytical Biochemistry

View full text Add to dashboard Cite

In drug discovery, the occurrence of false positives is a major hurdle in the search for lead compounds that can be developed into drugs. A small-molecular-weight compound that inhibits dengue virus protease at low micromolar levels was identified in a screening campaign. Binding to the enzyme was confirmed by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR). However, a structure-activity relationship study that ensued did not yield more potent leads. To further characterize the parental compound and its analogues, we developed a high-speed, low-cost, quantitative fluorescence quenching assay. We observed that specific analogues quenched dengue protease fluorescence and showed variation in IC (50) values. In contrast, nonspecifically binding compounds did not quench its fluorescence and showed similar IC(50) values with steep dose-response curves. We validated the assay using single Trp-to-Ala protease mutants and the competitive protease inhibitor aprotinin. Specific compounds detected in the binding assay were further analyzed by competitive ITC, NMR, and surface plasmon resonance, and the assay's utility in comparison with these biophysical methods is discussed. The sensitivity of this assay makes it highly useful for hit finding and validation in drug discovery. Furthermore, the technique can be readily adapted for studying other protein-ligand interactions. b s t r a c tIn drug discovery, the occurrence of false positives is a major hurdle in the search for lead compounds that can be developed into drugs. A small-molecular-weight compound that inhibits dengue virus protease at low micromolar levels was identified in a screening campaign. Binding to the enzyme was confirmed by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR). However, a structure-activity relationship study that ensued did not yield more potent leads. To further characterize the parental compound and its analogues, we developed a high-speed, low-cost, quantitative fluorescence quenching assay. We observed that specific analogues quenched dengue protease fluorescence and showed variation in IC 50 values. In contrast, nonspecifically binding compounds did not quench its fluorescence and showed similar IC 50 values with steep dose-response curves. We validated the assay using single Trp-to-Ala protease mutants and the competitive protease inhibitor aprotinin. Specific compounds detected in the binding assay were further analyzed by competitive ITC, NMR, and surface plasmon resonance, and the assay's utility in comparison with these biophysical methods is discussed. The sensitivity of this assay makes it highly useful for hit finding and validation in drug discovery. Furthermore, the technique can be readily adapted for studying other proteinligand interactions. Ó 2009 Elsevier Inc. All rights reserved.High-throughput screening (HTS) 2 is a major method in drug discovery for new small lead molecules. In high-throughput enzyme inhibition assays, more than a million compounds are usua...

show abstract

Section: B S T R a C Tmentioning

confidence: 99%

A fluorescence quenching assay to discriminate between specific and nonspecific inhibitors of dengue virus protease

Bodenreider

Beer

Keller

et al. 2009

Analytical Biochemistry

View full text Add to dashboard Cite

show abstract

“…However, miniaturisation of assays leads to a series of key challenges, reviewed by a range of authors late in the 20th century (Hertzberg and Pope 2000, Sittampalam et al 1997, Silverman et al 1998, Lemmo et al 1998 such as (i) assay methods and detection, (ii) liquid handling and robotics and (iii) process flow and information management. It is the second challenge for which inkjet printing was identified as a potentially important tool (Blanchard et al 1996, Lemmo et al 1997, Sittampalam et al 1997, Burbaum et al 1997, Lemmo et al 1998, Oldenburg et al 1998, Schena et al 1998, Rose 1999, Dunn and Feygin 2000, Bellavance et al 2000, Taylor et al 2002.…”

Section: High Throughput 'System Discovery' Techniquesmentioning

confidence: 99%

“…It is the second challenge for which inkjet printing was identified as a potentially important tool (Blanchard et al 1996, Lemmo et al 1997, Sittampalam et al 1997, Burbaum et al 1997, Lemmo et al 1998, Oldenburg et al 1998, Schena et al 1998, Rose 1999, Dunn and Feygin 2000, Bellavance et al 2000, Taylor et al 2002. The key features of inkjet that lend themselves to the liquid handling challenges of micro-array technologies are (i) noncontact deposition with a significant stand-off distance, ensuring that the size of the well is no longer limited by the size of the dosing nozzle, (ii) repeatability and accuracy of inkjet-deposited drops once their formation is optimised, (iii) accurate control of both individual drop volumes and total volumes by waveform and print signal controls, (iv) low reservoir volume requirements and (v) minimal space requirements for the system.…”

Section: High Throughput 'System Discovery' Techniquesmentioning

confidence: 99%

Inkjet printing for pharmaceutics – A review of research and manufacturing

Daly

Harrington

Martin

et al. 2015

International Journal of Pharmaceutics

271

185

View full text Add to dashboard Cite

Abstract:Global regulatory, manufacturing and consumer trends are driving a need for change in current pharmaceutical sector business models, with a specific focus on the inherently expensive research costs, high-risk capital-intensive scale-up and the traditional centralised batch manufacturing paradigm. New technologies, such as inkjet printing, are being explored to radically transform pharmaceutical production processing and the end-to-end supply chain. This review provides a brief summary of inkjet printing technologies and their current applications in manufacturing before examining the business context driving the exploration of inkjet printing in the pharmaceutical sector. We then examine the trends reported in the literature for pharmaceutical printing, followed by the scientific considerations and challenges facing the adoption of this technology. We demonstrate that research activities are highly diverse, targeting a broad range of pharmaceutical types and printing systems. To mitigate this complexity we show that by categorising findings in terms of targeted business models and Active Pharmaceutical Ingredient (API) chemistry we have a more coherent approach to comparing research findings and can drive efficient translation of a chosen drug to inkjet manufacturing.

show abstract

“…In addition to SPA assays,¯uores-cence-based assays using FRET (Stryer, 1978) or uorescence polarization (FP) have been the most common solution to this problem (Silverman et al, 1998;Sittampalam et al, 1997). FRET is a process that occurs when a donor¯uorescent molecule has an emission wavelength that overlaps the absorption wavelength of an acceptor.…”

Section: Simplified`mix and Read' Homogeneous Assaysmentioning

confidence: 99%