Biosensor-based small molecule fragment screening with biolayer interferometry

Abstract

Biosensor-based fragment screening is a valuable tool in the drug discovery process. This method is advantageous over many biochemical methods because primary hits can be distinguished from non-specific or non-ideal interactions by examining binding profiles and responses, resulting in reduced false-positive rates. Biolayer interferometry (BLI), a technique that measures changes in an interference pattern generated from visible light reflected from an optical layer and a biolayer containing proteins of interest, is a relatively new method for monitoring small molecule interactions. The BLI format is based on a disposable sensor that is immersed in 96-well or 384-well plates. BLI has been validated for small molecule detection and fragment screening with model systems and well-characterized targets where affinity constants and binding profiles are generally similar to those obtained with surface plasmon resonsance (SPR). Screens with challenging targets involved in protein-protein interactions including BCL-2, JNK1, and eIF4E were performed with a fragment library of 6,500 compounds, and hit rates were compared for these targets. For eIF4E, a protein containing a PPI site and a nucleotide binding site, results from a BLI fragment screen were compared to results obtained in biochemical HTS screens. Overlapping hits were observed for the PPI site, and hits unique to the BLI screen were identified. Hit assessments with SPR and BLI are described.

Characterizing Antibodies

“…Although there is little published data systematically comparing the performances of these varied approaches (ELISA, optical methods, ITC, etc. ), the few available studies suggest that the affinity constant estimations are similar across platforms (Myszka et al 2003;Wartchow et al 2011), whereas another study comparing ELISA with SPR concluded that they are not only consistent but also complementary (Heinrich et al 2010). This latter study is interesting because the ELISA distinguished two distinct binding populations in solution (high-and low-affinity constants), but the affinity constants obtained via SPR were dependent on whether binding occurred in solution or at the surface of the biosensor: A higher-affinity constant was detected under solution binding conditions and a lower one when the antibody was bound to the surface or the Biacore chip.…”

EFN-4/Ephrin functions in LAD-2/L1CAM-mediated axon guidance in Caenorhabditis elegans

“…To further evaluate the binding of EFN-4 to LAD-2, we used biolayer interferometry (BLI), an optical biosensing technique similar to surface plasmon resonance that can measure the association and disassociation of biomolecules (Abdiche et al, 2008;Wartchow et al, 2011;Wilson et al, 2010). In BLI assays, we immobilized EFN-4::Fc or the Fc control onto an anti-human Fc capture probe, after which we immersed the probe into conditioned tissue culture medium isolated from HEK293T cells transiently expressing the LAD-2 extracellular domain fused to alkaline phosphatase (LAD-2::AP).…”

Metal Impurities Cause False Positives in High-Throughput Screening Campaigns

“…ForteBio and Biacore biosensor-based binding assays provided similar results and were consistent with these findings. 23 The measured IC 50 and K D values were in the low micromolar range, making these low molecular weight series attractive starting points for development into a lead series (compounds 1.1, 2.1, 3.1, and 4.1; Table 1). …”