Applications
Biochemical Applications >> Small Molecule & Fragment-based Screening
Fragment-based and small molecule screening often employ biophysical techniques such as X-ray crystallography, NMR, surface plasmon resonance and calorimetry to screen libraries for direct binding leads. These technologies have limited throughput, require specialized users and often consume large quantities of the target protein. BIND® provides an efficient lead discovery solution by offering label-free detection of direct binding in a high throughput 96-, 384- or 1536-well format, reducing target consumption and identifying the desired binding characteristics for a wide range of affinities from 10-9 to 10-3M.

Application Features
  • Rapid Assay Development
  • Plug-and-play Chemistry
  • Low to Ultra-high Throughput Formats
  • Affinity, Specificity & Stoichiometry Determination
  • Complementary pre-screening tool to SPR
Label-Free Direct Binding Assays
BIND label-free detection provides an assay format where a target is probed under more native conditions. Identification of specific binding sites not detectable by assays requiring labels or competitive substrates expands the diversity of identified leads. Proteins, peptides, fragments and small molecules are just a few examples of BIND direct binding assay capabilities. SRU offers biosensors with a number of different surface chemistries for flexibility in immobilizing assay targets.
Small Molecule Binding
BIND is highly robust and enables screening of even small molecules in high throughput formats. BIND is able to detect weak binding interactions and compatible with high concentrations of compounds. The direct binding assays provide information on affinity, stoichiometry and specificity of binding as well as mechanisms of action. This information is key during hit identification and characterization.

Kinase Direct Binding Assay

BIND Biosensors has a variety of available surface chemistry allowing for immobilization of any target. The BIND assay shown in Figure 1 depicts a direct binding assay of AMP Kinase (AMPK) and AMP. Immobilization of the target on the biosensor surface causes a shift in BIND peak wavelength value. This allows for real time monitoring of target immobilization as well as calculation of the amount of target immobilized on the biosensor. This information can then be used in examining the stoichiometry of binding.

A 130 kDa version of AMP Kinase (AMPK) was immobilized on a 384-well biosensor over a 30 minute period using 15-20µL of a 100ug/mL solution (Figure 1A). Approximately 200ng of protein was immobilized on the biosensor surface of each well with excellent uniformity across the target containing wells. A less concentrated protein solution could have been employed for a slightly longer incubation time. Following a quick rinse for removal of the unbound AMPK, the sensor is ready to use immediately for ligand binding tests.

An AMPK surface (8.4nm immobilization shift) was titrated for direct binding of adenosine monophosphate (AMP; MW = 365 Da), a known AMPK ligand (Figure 1B). The titration maximum binding PWV shift signal (26pm) is coincidental with calculations for maximum expected signal based upon the molecular masses (protein & ligand) and amount of AMPK immobilized, suggesting fully functional protein at 1:1 binding stoichiometry. Stoichiometry (AMP:AMPK) = (0.026/8.4)*(130000/365) =1.1 mol/mol.

Additionally, no signal was observed on the AMPK surface with GMP nor was signal observed where AMP was added to a surface without AMPK. The fitted titration of AMP on AMPK provides a Kd in line with literature reports. SRU BIND is capable of detecting non-specific interactions often found with aggregating compounds. AMP kinase displayed no aggregation signal.
Figure 1 – A). A 384-well GA3 BIND Biosensor was coated with AMPK using a 15-20mL of a 100µg/mL solution for 30 minutes and the kinetics of immobilization monitored on a BIND Reader. B). Various concentrations of AMP were added to biosensors either with immobilized AMPK or an uncoated reference biosensor and direct binding monitored on a BIND Reader. Standard curve fitting equation for 1:1 stoichiometry applied using Grafit application.

Small Molecule Protease Inhibition

BIND direct binding assays can be performed in a variety of well-based formats from 16-well BIND cartridge biosensors to 1536-well microplate biosensors. Figure 2 is a small molecule inhibition assay using the 16-well cartridge system which is ideal for use in assay development or with low sample numbers.
Figure 2 - 16-well BIND Biosensor Cartridges were coated with a bacterial protease (target) and incubated with various concentrations of a small molecule inhibitor. Peak wavelengths (PWV) of samples were detected on the BIND Cartridge Reader once a minute for 20 minutes.
Fragment-based Screening
BIND direct binding assays provide a powerful fragment-based screening and profiling platform. These assays can detect the binding of low molecular weight compounds to fragments with 100x the molecular weight of the compounds.

ATPase Fragment Screening

A 26kDa, ATP-ase domain target was attached to a BIND biosensor and used to detect the direct binding of 500 small molecular weight compounds (Figure 3). The compounds were in 2% DMSO and had affinities ranging from 1nM to 1mM. All positive and negative controls were identified. The hits identified in this assay correlated strongly with those identified using lower throughput biophysical methods such NMR and X-ray crystallography.

Figure 3 - Binding of a fragment library with average molecular weights <300 Da and affinities ranging from 1nM to approximately 1mM, to BIND Biosensors coated with a 26 kDa ATPase. Data courtesy of Astex Therapeutics, UK.
High Throughput Small Molecule Screening
The NR binding assay in which a biotinylated cofactor peptide was immobilized to streptavidin biosensors and assessed for small molecule modulation of orphan NR binding was used to screen a 10,000 compound library. 32 specific, titratable agonists were identified, several with super-stoichiometric binding (Figure 4).
Figure 4 - A 10K compound screen was performed using cofactor peptide-coated BIND Biosensors. Identified hits were confirmed for concentration-dependent activity (data not shown). Data courtesy of GSK.