Biophysics: for HTS hit validation, chemical lead optimization, and beyond.

There are many challenges to the drug discovery process, including the complexity of the target, its interactions, and how these factors play a role in causing the disease. Traditionally, biophysics has been used for hit validation and chemical lead optimization. With its increased throughput and sensitivity, biophysics is now being applied earlier in this process to empower target characterization and hit finding.

Applications of Biophysics in High-Throughput Screening Hit Validation.

For approximately a decade, biophysical methods have been used to validate positive hits selected from high-throughput screening (HTS) campaigns with the goal to verify binding interactions using label-free assays. By applying label-free readouts, screen artifacts created by compound interference and fluorescence are discovered, enabling further characterization of the hits for their target specificity and selectivity. The use of several biophysical methods to extract this type of high-content information is required to prevent the promotion of false positives to the next level of hit validation and to select the best candidates for further chemical optimization.

Base pair mismatch recognition using plasmon resonant particle labels.

We demonstrate the use of silver plasmon resonant particles (PRPs), as reporter labels, in a microarray-based DNA hybridization assay in which we screen for a known polymorphic site in the breast cancer gene BRCA1. PRPs (40-100 nm in diameter) image as diffraction-limited points of colored light in a standard microscope equipped with dark-field illumination, and can be individually identified and discriminated against background scatter. Rather than overall intensity, the number of PRPs counted in a CCD image by a software algorithm serves as the signal in these assays.