Screening Strategies in Drug Discovery – Multimedia

In order to identify host genes required for M. tuberculosis infection and persistence, we developed a phenotypic cell-based assay in both murine and human cells and adapted it for high throughput and high content screening purposes. Knock-down efficiencies above 80% were achieved in “hard-to-Transfect” macrophage cells. Validation of the assay performed with control siRNAs will be discussed.

The COP9 signalosome (CSN) is involved in cell cycle regulation, possesses kinase activity and is therefore an interesting therapeutic target for anti-tumor drugs. Known inhibitors of the kinase activity exhibit low binding constants. Using them a 3D similarity screening for our in-house database is performed. The superposition algorithm enables the explicit consideration of conformers reflecting the structural flexibility.

The multimode microplate reader PHERAstar from BMG combines the rapid plate reading necessary for HTS with enhanced performance and sensitivity needed to read small liquid volumes along with the flexibility to tackle the most demanding assays. The performance and features of the PHERAstar are presented in this poster with commercially available HTS kits such as HTRF®, AlphaScreen™, and PolarScreen™.

In order to facilitate high throughput screening of AD drug candidates, we have developed a new SensoLyte™ 520 b-secretase assay kit using a fluorescence resonance energy transfer (FRET) peptide, HiLyte Fluor™ 488-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys(QXL™ 520)-OH. The longer excitation and emission wavelengths of HiLyte Fluor™488 minimize the interference from autofluorescence and absorbance of test compounds.

At Southern Research Institute we routinely conduct HTS campaigns using infectious agents at the BSL-2 (Biosafety Level) and BSL-3 level. Here we discuss how we have successfully screened over one million compounds in anti-viral (Severe Acute Respiratory Syndrome, Influenza, Highly Pathogenic Avian Influenza) and anti-microbial (Staphylococcus aureus, Escherichia coli, Mycobacterium tuberculosis) screens in BSL-2 and BSL-3 containment.

Today’s high-throughput screening facilities face increasing demands to generate more information from their existing compound libraries. In this poster, we demonstrate the combination of several Promega cell-based assays (CellTiter-Blue®, Apo-One® and Caspase-Glo® 3/7) multiplexed in both low-volume 384 and 1536-well plate formats. The BMG LABTECH PHERAstar microplate reader is used to record both luminescence and fluorescence, depending on the multiplex combination.

Image-based high-content screening assays, demand solutions for image segmentation and cellular compartment encoding to track critical events – for example those presented by GFPreporters within cell cycle tracking and GPCR translocation assays. We have designed nuclear and cytoplasm discriminator CyTRAK™ probes - spectrally compatible with all variants of GFPreporters offering new solutions in cytometry.

To facilitate high throughput screening of renin inhibitors, we have developed new renin assay kits utilizing FRET peptide substrates. The SensoLyte™ 520 Renin Assay Kit contains FRET peptide labeled with the quencher QXL™520 and the fluorophore 5-FAM. These newly developed assays were validated with several known renin inhibitors. Assay with QXL™ 520/5-FAM substrate was approximately 40 fold more sensitive than an existing assay which uses EDANS/DABCYL FRET substrate.

The main success of QSAR predictive technology is mainly related to the modelling of the single activity/property endpoint in chemical space. Unfortunately an optimisation of the targeted endpoint may result in losing other desired properties, for example, toxicity is increased or solubility reduced. Because of this, attempts have been made in the last few years to model chemical space against multiple endpoints.

Assay-ready plates are becoming key to maximizing throughput for many HTS departments. These plates contain test compounds prepared from library stocks ahead of time at the correct concentrations. DMSO is routinely used for compound dissolution, but its presence at concentrations above 1% can markedly affect assay performance. Thus, accurate nanolitre quantities of DMSO-based stock solution are required to keep concentrations low when preparing assay-ready plates.