Assay development is an adaptation of the benchtop assay to render it automation-friendly, and miniaturizable. In our facility assay development has a higher likelihood of success and timely delivery with the active participation of the sponsoring scientists. Familiarity with the actual biological system contributes greatly to the success of the project. During this dynamic period, experiments will be planned and performed collaboratively. Deviations from initial plans are to be expected therefoire, maintaining flexibility is important.
The goals of assay development include (but are not limited to):
- Rendering the assay rautomation-friendly
- Assay miniaturization to 384 or 1536 well plate format to reduce reagent cost and decrease overall screening campaign cost and timelines
- Assay stability (positive and negative controls, overall S/N ratio, time and temperature dependency)
- Robustness to incubation with compounds including decision on compound concentration and sensitivity to DMSO concentrations, time and length of exposure
- Data analysis parameters
Upon the successful completion of assay development a final HTS protocol will be recommended during a milestone meeting that will authorize a Standard Operating Procedure (SOP) to facilitate the performance and reproducibility of the screening campaign.
High Throughput Screening (HTS)
HTS is a popular approach in drug discovery in which a relatively large number of chemical perturbants can be tested against a target or several targets.
HTS brings together expertise in liquid handling and robotic automation and multi-mode plate readers to perform automated biological reactions over a relatively short space of time. Benchtop assays are scaled down through miniaturization to 384-well or 1536-well formats for significant savings in reagent costs and dramatic increases in throughput.
A screen typically produces a number of ‘hits’ – inhibitors or activators depending upon the target of interest and the goal of the screener. The number of hits produced usually varies from 0.1%-2% of compounds screened, and highly depends upon the assay and the compounds in the screening set. The resulting hits can then be interrogated in much finer detail through secondary hit validation and selection of potent modulators for further study. An in depth familiarity with the mechanism of the screening assay will facilitate filtering of false positive hits and minimizing false negatives that could be missed in the primary screen.
Ultimately, a portfolio of biologically relevant compounds will be assembled following a successful HTS campaign. This method promotes a more structured and focused approach to later stage screening.
The HTS should be tailored to the system under investigation. Assays of greater complexity, multiple steps, and prolonged incubations will be less amenable to screening of larger groups of compounds. In such cases, focused libraries with structures and activity closely associated with a target class may be selected. Bioactive molecules such as known drugs or natural products may also be used a source of enriched activity in a smaller screening set.
High Content Screening (HCS)
HCS is a defined subset of assay technologies that employ automated microscopy and imaging platforms as the detection step in the small molecule or gene knock-down screen. The term “high content” relates to the multiple data readouts associated with each compound treatment. As microscopy images are analyzed with various software packages, numerous attributes are quantified on a per cell basis, and generate multivariate profiles associated with each phenotype.
Throughput of HCS is frequently much lower than HTS, as the acquisition time of the automated microscope is significantly longer than plate-reader based HTS. Further, extensive computational power must be applied to transform image-based readouts into meaningful numerical data. Phenotypic screens using HCS can be performed using live or fixed cells, and attention should be devoted to preserving the relevant phenotype when setting up the screening assay.
HCS is frequently used as a primary assay when a defined target of interest is not known, or a cell-free option is elusive. HCS is many times used as a secondary assay following an HTS campaign against a defined molecular target, where hit compounds can be rapidly triaged for toxicity or a relevant biological response in a cellular system. Automated microscopy based screens can also be adapted for whole organism readouts in systems such as zebrafish (not currently oferred here).
Cheminformatics and Virtual Screening
Computational approaches are widely used in pharmaceutical research to enhance efficiency of the drug discovery and development pipeline. Computer-aided drug discovery (CADD) exploits state-of-the-art computational technologies to speed up the drug development process, compared to traditional drug screening. Such in-silico methods are fast and automatic, and feature low costs while maintaining high success rates. Both ligand-based and structure-based drug design approaches are powerful technologies that can be applied to virtual screening for lead identification and optimization. Traditional drug screening is a random approach, whereas the in-silico approach has the capacity to intellectually build an effective virtual small-molecule library for screening, based on known structures and/or known potent ligands. Thus, CADD helps scientists focus on the most promising compounds, so as to minimize the synthetic, in vitro and in vivo testing efforts..
The HTS unit uses a number of software packages to analyze the screening data.
- Genedata Screener – The HTS unit uses this package to analyze primary screen data as well as multi-compound dose-response curve fitting and multi-assay profiling of compounds for structure-activity studies. This is a commercial license based package and is only available at workstations in the unit. Normalized data and curve-fits are easily exported in CSV/Excel/PDF formats.
- MetaExpress – High content analysis software package from molecular devices. Standard modules for major phenotypic readouts and also custom protocols. Several commercial network-seat licenses are available in the unit.
- CellProfiler – Open source high content analysis package from the Broad Institute. This package may be used to set up additional analysis pipelines not supported in commercial packages.
- BIOVIA Pipeline Pilot – Commercial software package that's automates the scientific analysis of data, enabling users across the enterprise to rapidly explore, visualize and report research results.
- BIOVIA Discovery Studio & Schrodinger Suite - Software solution for scientific computational chemistry; contains all the tools necessary for lead discovery and optimization, modeling biologics, antibodies and proteins and cheminformatics
- Collaborative Drug Discovery (CDD) – Web-based registry of compound data and screening results.
The HTS unit maintains and tracks the library of ~100,000 screening compounds from a physical and an informatics perspective. Accurate and precise distribution of compounds to the assay system are vital elements in the screening process. Compound Management uses highly sophisticatedb instrumentation such as acoustic dispensing (Labcyte Echo) and an integrated robotics system for plate replication . Compounds from our screening collections can be used or a screener can bring a set of molecules of interest which we can register and then deploy according to the experimental design.