Kinetic analysis of small drug-like molecules binding to protein targets is a crucial aspect of drug discovery. Small molecules present several unique challenges for interaction analysis. Most obvious is their size, which is typically less than 500 daltons, resulting in small binding signals and low signal to noise. They also exhibit fast association and dissociation rates with proteins, the accurate measurement of which can be challenging. In addition, small molecules are generally less soluble in standard aqueous buffers than proteins and require the addition of organic co-solvents such as DMSO.
To combat the low signal to noise seen in small molecule experiments, two pathways have been pursued simultaneously. New super streptavidin (SSA) coated biosensors with increased binding capacity for biotinylated capture molecules increase signal three- to eightfold depending on the molecular system. In addition, higher sensitivity spectrometers allow the newest Octet instruments (Octet RED and Octet RED384) to have an eight- to tenfold reduction in noise. Together, the two approaches have enabled the analysis of binding kinetics of small molecules as small as 157 daltons.
The use of cosolvents at levels that are compatible with most proteins (1–5% DMSO) are easily tolerated in BLI-based analysis, but should be kept consistent throughout the assay. In Figure 2, kinetic analyses of three different known inhibitors for carbonic anhydrase are shown. Biotinylated carbonic anhydrase was immobilized onto the SSA biosensor surface. The analysis for each inhibitor was performed in duplicate at five different concentrations in 5% DMSO. Kinetic constants determined for these inhibitors agree well with literature values.