Christine Williams

cAMP detection methods in HTS: selecting the best from the rest

The number of technologies that enable high-throughput functional screening of G-protein-coupled receptors has expanded markedly over the past 5 years. Consequently, choosing the most appropriate technology can be a daunting task, particularly for Gi- or Gs-coupled receptors. The most common systems for cyclic AMP detection are reviewed, highlighting the practical and theoretical aspects that are important in their application to high-throughput screening. Current technologies can do the job, but it is likely that the future may require development of technologies that provide even greater biological information.

Patch clamping by numbers.

Abstract Many ion channels are recognized as amenable targets for a range of disease states and conditions. However, the process of discovering drugs is highly influenced by the chemical doability, the biological confidence in rationale of the approach and the ‘screenability’. To date, the absence of informative high throughput technologies for ion channel screening has resulted in ion channels remaining a largely unexplored class of drug targets. This, however, is about to change – a large increase in the number of data points per day should be achieved by the introduction of automated ‘high throughput’ patch clamp machines.

Measuring Intracellular Calcium Fluxes in High Throughput Mode

The measurement of intracellular calcium fluxes in real time is widely applied within the pharmaceutical industry to measure the activation of G-protein coupled receptors (GPCRhyp;s), either for pharmacological characterisation or to screen for new surrogate ligands. Initially restricted to G(q) coupled GPCRs, the introduction of promiscuous and chimeric G-proteins has further widened the application of these assays. The development of new calcium sensitive dyes and assays has provided sensitive, homogeneous assays which can be readily applied to high throughput screening (HTS). In this paper we describe the full automation of this assay type using a fluorometric imaging plate reader (FLIPR ) integrated into a Beckman/Sagian system to establish a simple robotic system that is well suited for the current medium throughput screening in this area of lead discovery. Using a recently completed HTS we discuss important determinants for FLIPR based screening, highlight some limitations of the current approach, and look at the requirements for future automated systems capable of keeping up with expanding compound files.

Insights into GPCR pharmacology from the measurement of changes in intracellular cyclic AMP; advantages and pitfalls of differing methodologies

It is clear that the G protein‐coupled receptor family play a key role in the pharmaceutical industry, with a significant proportion of approved drugs targeting this protein class. While our growing understanding of the complexity of G protein‐coupled receptor pharmacology is playing a key role in the future success of these endeavours, with allosteric mechanisms now well integrated into the industrial community and G protein‐independent signalling mechanisms establishing themselves as novel phenomenon to be exploited, it is still possible to underestimate the complexity of G protein signal transduction mechanisms and the impact that inappropriate study of these mechanisms can have on data interpretation. In this manuscript we review different approaches to measuring the cAMP signal transduction pathway, with particular emphasis on key parameters influencing the data quality and biological relevance.

GPCR Signaling: Understanding the Pathway to Successful Drug Discovery

Modulators of G protein-coupled receptors (GPCRs) form a key area for the pharmaceutical industry, representing approximately 27% of all Food and Drug Administration (FDA)-approved drugs. Consequently, there are a wide variety of in vitro plate-based screening technologies that enable the measurement of compound affinity, potency, and efficacy for almost every type of GPCR. However, to maximize success it is prudent to ensure that (i) the most suitable assay formats are identified, (ii) they are configured optimally to detect the desired compound activity, and (iii) that they form a basis for predicting clinical effects. To achieve this, an understanding of the pathways and mechanisms of receptor activation relevant to the disease mechanism, as well as the benefits and/or limitations of the specific techniques, is key.

Development and Automation of a 384-Well Cell Fusion Assay to Identify Inhibitors of CCR5/CD4-Mediated HIV Virus Entry:

This article describes the automation of an in vitro cell-based fusion assay for the identification of novel inhibitors of receptor mediated HIV-1 entry. The assay utilises two stable cell lines: one expressing CD4, CCR5 and an LTR-promoter/β-galactosidase reporter construct, and the other expressing gp160 and tat. Accumulation of β-galactosidase can only occur following fusion of these two cell lines via the gp160 and receptor mediators, as this event facilitates the transfer of the tat transcription factor between the two cell types. Although similar cell fusion systems have been described previously, they have not met the requirements for HTS due to complexity, throughput and reagent cost. The assay described in this article provides significant advantage, as (a) no transfection/infection events are required prior to the assay, reducing the potential for variability, (b) cells are mixed in solution, enhancing fusion efficiency compared to adherent cells, (c) miniaturisation to low volume enables screening in 384-well plates; and (d) online cell dispensing facilitates automated screening. This assay has been employed to screen ~650,000 compounds in a singleton format. The data demonstrate that the assay is robust, with a Z′ consistently above 0.6, which compares favourably with less complex biochemical assays.

Improving the design and analysis of high-throughput screening technology comparison experiments using statistical modeling.

Contemporary small-molecule drug discovery frequently involves the screening of large compound files as a core activity. Subsequently cost, speed, and safety become critical issues. In order to meet this need, numerous technologies have been developed to allowmix andmeasure approaches, facilitate miniaturization, and to increase speed and tominimize the use of potentially hazardous reagents such as radioactive materials. However, despite the on-paper advantages of these new technologies, risks can remain undefined. For example, the question of whether the novel method will facilitate identification of active chemical series in a way that is comparable with conventional methods arises. In order to address this question, we have taken the approach of carrying out experiments to directly compare the output of high-throughput screens using a given novel approach and a traditionalmethod. The concordance between the screening methods can then be determined via comparison of the numbers and structures of the active molecules identified. This article describes the approach taken in our laboratory to minimize variability in such experiments and shows data that exemplifies the general result of lower than expected concordance. Statistical modeling was subsequently used to facilitate this interpretation. The model used distribution function to generate a real-activity frequency relationship with added normal random error and occasional outliers to represent assay variability. Hence, the effect of assay parameters such as the threshold, the number of real actives, and the number of outliers and the standard deviation could readily be explored. The model was found to describe the data reasonably and moreoverwas found to be of great utility when it came to planning further optimal experiments. A key conclusion from the model was that concordance between screening methods could appear poor even when one approach is compared with itself. This occurs simply because the result is a function of assay threshold, standard deviation and the true compound activity. In response to this finding we have adopted alternative experimental designs that more reliably measure the concordance between screening methods.

Plate-based diversity subset screening: an efficient paradigm for high throughput screening of a large screening file

The screening files of many large companies, including Pfizer, have grown considerably due to internal chemistry efforts, company mergers and acquisitions, external contracted synthesis, or compound purchase schemes. In order to screen the targets of interest in a cost-effective fashion, we devised an easy-to-assemble, plate-based diversity subset (PBDS) that represents almost the entire computed chemical space of the screening file whilst comprising only a fraction of the plates in the collection. In order to create this file, we developed new design principles for the quality assessment of screening plates: the Rule of 40 (Ro40) and a plate selection process that insured excellent coverage of both library chemistry and legacy chemistry space. This paper describes the rationale, design, construction, and performance of the PBDS, that has evolved into the standard paradigm for singleton (one compound per well) high-throughput screening in Pfizer since its introduction in 2006.

Plate-based diversity subset screening generation 2: An improved paradigm for high throughput screening of large compound files

High-throughput screening (HTS) is an effective method for lead and probe discovery that is widely used in industry and academia to identify novel chemical matter and to initiate the drug discovery process. However, HTS can be time consuming and costly and the use of subsets as an efficient alternative to screening entire compound collections has been investigated. Subsets may be selected on the basis of chemical diversity, molecular properties, biological activity diversity or biological target focus. Previously, we described a novel form of subset screening: plate-based diversity subset (PBDS) screening, in which the screening subset is constructed by plate selection (rather than individual compound cherry-picking), using algorithms that select for compound quality and chemical diversity on a plate basis. In this paper, we describe a second-generation approach to the construction of an updated subset: PBDS2, using both plate and individual compound selection, that has an improved coverage of the chemical space of the screening file, whilst only selecting the same number of plates for screening. We describe the validation of PBDS2 and its successful use in hit and lead discovery. PBDS2 screening became the default mode of singleton (one compound per well) HTS for lead discovery in Pfizer.