Fragment-based drug discovery is an emerging technique that involves screening low molecular weight molecules and subsequent structure-guided medicinal chemistry optimization to turn the weakly active fragment hits into drug candidates. The use of high throughput fragment screening (HTFS) has been successfully demonstrated by Evotec using single-molecule confocal Fluorescence Correlation Spectroscopy (FCS) and its variant detection techniques to ensure high data precision, reproducibility and pharmacological sensitivity on the EVOscreen™. The use of biochemical assays for fragment screening, is the only technique which allows the primary screen to determine if a fragment has an effect on the normal function of the protein or not.
The EVOscreen™ HTFS platform features nanolitre liquid handling technology and confocal fluorescence reading devices that enable single molecule detection and analysis to generate assay readouts based on physical properties such as diffusion, rotation or specific fluorophore brightness and lifetime. There are multiple benefits associated with this over conventional fluorescence screening:
- The statistically most robust assay readout is selected from a portfolio of options; robust readouts allow the HTS scientist to focus on the pharmacological sensitivity of the assay that is typically associated with low enzyme concentration and low levels of substrate conversion or complex formation in catalytic and binding assay setups, respectively.
- Fluorescent readout multiplexing can frequently be applied
- Compound induced fluorescence artifacts can be detected and corrected if the fluorescent properties of the compound are sufficiently different from that of the assay fluorophore
- Due to the high throughput (100,000 data points per day) and level of miniaturisation of these well established HTS techniques multiple fragment concentrations can be assessed in the initial screen and 1,000s of fragments are tested per day with low consumption of reagents
- Full dose response curves are either obtained at the outset for every fragment tested or subsequently in a second round of screening
- Fragment screening experiments can be conducted in presence and absence of ligands and modulators to tailor the assay conditions for particular target protein pockets and sites. Fragment sets can be enriched and screened with little extra effort if desired
Together these provide high quality information allowing the best fragments to be selected for subsequent optimization. For fragment screens it has been found to be advantageous to conduct the screening free of DMSO, a solvent commonly used for dissolution of screening compounds and which at high concentration may interfere with enzyme function. This was achieved by the application of NanoStore™ which enables DMSO free screening through the use of a pharmaceutical compatible excipient (2-hydroxypropyl-_-cyclodextrin) to aid compound solubilisation.