Biomedical subjects

Philip Gribbon

Publications and source records attributed to Philip Gribbon.

7 recordsLinked to original sources

PubMed2006

The application of fluorescence lifetime readouts in high-throughput screening.

Measurement of fluorescence lifetime is a well-established technique, which has recently been introduced into the portfolio of assay formats used in high-throughput screening (HTS). This investigation establishes appropriate conditions for using lifetime measurements to reduce the impact of compound interference effects during large-scale HTS of corporate screening files. Experimental data on mixtures of standard fluorophores and interfering compounds (from 5 HTS campaigns) have been combined with a theoretical model to identify the minimum data quality required, defined by the photon count in the peak channel, for discrimination of biological activity. Single-component fluorophore lifetimes can be recovered with an error of 1%, with a peak photon count of 10(2), but the same accuracy with a 2-component decay requires a peak photon count of 10(3). When a 3rd component is introduced, the minimum peak count increases to 10(4). The influence of scattered light on lifetime determination was investigated using an emulsion (diameters 25-675 nm). The measured decays of interfering compounds, identified as autofluorescent, show that the vast majority have a very short lifetime that can readily be resolved from the reporter fluorophore, using appropriate data-fitting methods.

Drug Evaluation, Preclinical↗

PubMed2006

A novel method for analyzing [Ca2+] flux kinetics in high-throughput screening.

Driven by multiparameter fluorescence readouts and the analysis of kinetic responses from biological assay systems, the amount and complexity of high-throughput screening data are constantly increasing. As a consequence, the reduction of data to a simple number, reflecting a percentage activity/inhibition, is no longer an adequate approach because valuable additional information, for example, about compound-or process-induced artifacts, is lost. Time series data such as the transient calcium flux observed after activation of Gq-coupled G protein-coupled receptors (GPCRs), are especially challenging with respect to quantity of data; typically, responses are followed for several minutes. Based on measurements taken on the fluorometric imaging plate reader, the authors have introduced a mathematical model to describe the time traces of cellular calcium fluxes mediated by the activation of GPCRs. The model describes the time series using 13 parameters, reducing the amount of data by 90% while guiding the detection of compound-induced artifacts as well as the selection of compounds for further characterization.

Animals↗

PubMed2006

Analysing the output from primary screening.

From a perspective of process knowledge and enhancement, the analysis of the results of biological screening should not be limited to the outcome of specific projects, but additionally encompass a process centric view. Summarising outcomes across multiple projects is a powerful tool to gain a greater understanding of biological screening that will also enable optimisation of the strategy for specific projects or target classes. We have analysed a set of 73,651 compounds with reproducible (confirmed) results from 63 high-throughput screening (HTS) campaigns to reveal the underlying trends in the population of active compounds. We have focused on the overall physico-chemical profile of compound populations derived from biological screening since the in vivo activity of drug molecules is the result of physico-chemical and structural properties of the compound.

Automation↗

PubMed2005

NanoStore: a concept for logistical improvements of compound handling in high-throughput screening.

Small molecule screening, the systematic encounter of biology space with chemical space, has provoked the emergence of a whole industry that recreates itself by constant iterative improvements to this process. The authors describe an approach to tackle the problem for one of the most time-consuming steps in the execution of a screening campaign, namely, the reformatting of high-throughput screening test compounds from master plates to daughter assay plates used in the execution of the screen. Through an engineered storage procedure, they prepare plates ahead of the screening process with the respective compounds in a ready-to-use format. They show the biological inertness of the method and how it facilitates efficient recovery of compound activity. This uncoupling of normally interconnected processes provides time and compound savings, avoids repeated freeze-thaw cycles of compound solutions, and removes the problems associated with the DMSO sensitivity of certain assays types.

Automation↗

PubMed2005

Evaluating real-life high-throughput screening data.

High-throughput screening (HTS) is the result of a concerted effort of chemistry, biology, information technology, and engineering. Many factors beyond the biology of the assay influence the quality and outcome of the screening process, yet data analysis and quality control are often focused on the analysis of a limited set of control wells and the calculated values derived from these wells. Taking into account the large number of variables and the amount of data generated, multiple views of the screening data are necessary to guarantee quality and validity of HTS results. This article does not aim to give an exhaustive outlook on HTS data analysis but tries to illustrate the shortfalls of a reductionist approach focused on control wells and give examples for further analysis.

Biological Assay↗

PubMed2004

Experiences in implementing uHTS--cutting edge technology meets the real world.

Driven by growing corporate compound files, the demands of target biology, and attempts to cut cost, the number of solutions to HTS has spiralled. In quick succession new assay technologies and screening platforms are appearing on the market, with the promise of screening faster than ever in low volume high density formats whilst providing high quality data. Within this world of rapid change, Pfizer has applied cutting edge technology to HTS by introducing screening in 1 microl formats utilising single molecule detection technology. Instead of resource intensive in-house development, Pfizer entered into a collaboration with Evotec OAI / Evotec Technologies and introduced their Mark-II EVOscreen platform. In this article we will outline the benefits of the approach taken at Pfizer, Sandwich, and introduce the Mark-II EVOscreen platform, illustrating the potential but also possible pitfalls of HTS miniaturisation.

Cloning, Molecular↗

PubMed2003

Fluorescence readouts in HTS: no gain without pain?

Fluorescence-based detection technologies are frequently applied in biological testing, due to their unique advantages in setting up homogeneous, sensitive assays in miniaturized formats. However, the wide application of these readouts has highlighted challenges in reagent design and problems with interference from biological reagents and compounds. Here, we summarize the current application of fluorescence-based detection methodologies, focusing on the problems faced by assay developers and on solutions to reduce false positive and negative results in fluorescence-based HTS.

Calmodulin-Binding Proteins↗