The rockefeller university

Medium-Throughput Protein–Ligand Binding Analysis

The NanoTemper Dianthus is a 384-well microplate reader designed for medium-throughput analysis of protein–ligand interactions. It uses specialized microplates and fluorescence-based measurements to detect changes in a labeled target protein following ligand or compound binding.

The Dianthus can provide two complementary readouts:

• Temperature-induced fluorescence intensity changes, which can report changes in the stability or fluorescence behavior of a labeled protein upon ligand binding.
• Spectral shifts, which can provide an additional measure of changes in the fluorescent environment of the labeled target.

Applications in Drug Discovery

At the Fisher Drug Discovery Resource Center, the Dianthus is particularly useful for fragment-based screening and for assay development.

For fragment screening, purified labeled target protein can be exposed to compound libraries and analyzed for changes associated with binding. The 384-well format provides substantially greater throughput than many conventional biophysical techniques, making the Dianthus useful for screening larger numbers of compounds.

The instrument can also be used during assay development, particularly when developing direct binding assays in which ligand or compound binding to a purified target can be measured without relying on a downstream functional response.

Advantages

The principal requirement is that the target protein must be fluorescently labeled. In return, the Dianthus offers several important advantages:

• 384-well format for medium-throughput screening
• Direct measurement of protein–ligand interactions
• Two independent fluorescence-based readouts—temperature-induced intensity changes and spectral shifts
• Useful for both fragment screening and development of binding assays
• Requires relatively small quantities of purified target protein compared with many traditional biophysical approaches

The Dianthus therefore occupies a useful position between low-throughput biophysical characterization and high-throughput biochemical screening, providing a practical approach for identifying and characterizing compounds that interact directly with a purified target protein.