Assay design for small molecule screening

Before implementing the screening assay as a routine method, run small-scale pilot experiments to establish that the assay is robust. Evaluate the pilot runs in the same way as you intend to evaluate the full screen, but pay particular attention to the following points:

Small molecule interactions are often characterized by rapid binding and dissociation and regeneration can be omitted
Regeneration performance: is regeneration satisfactory over a range of different analytes? Check the consistency of response levels from control samples repeated throughout the run. (It is not sufficient just to test the regeneration performance with known controls: the purpose of the pilot experiments is to identify potential problems in the spectrum of unknown compounds that are tested in a screen.)
Binding to reference surface: are the relative responses on the reference surface consistently low? If not, you may need to revise the way the reference surface is treated or use a different sensor chip type.
Precipitation: many small organic molecules are sparingly soluble in aqueous buffer, and microprecipitates can form even in the presence of DMSO, particularly if the sample rack temperature is low. Precipitation is usually seen as marked irregularities in the sensorgrams, often accompanied by carry-over problems. Increase the rack tray temperature or the DMSO concentration in both samples and running buffer if precipitation is a problem. (Remember to adjust the DMSO concentrations in solvent correction solutions accordingly.)
Carry-over problems: significant responses in the carry-over control injection can indicate “sticky” compounds that remain to some extent in the flow system after the cycle is completed. Problems of this kind can affect the interpretation of subsequent cycles. Include wash injections of buffer or up to 50% DMSO to wash the compound out of the system if carry-over is a general problem. (High concentrations of DMSO can damage immobilized ligands). Compounds known or expected to carry over to the next cycle can be placed at the end of the sample series so that the consequences of carry-over for the assay as a whole are minimized.

Sample preparation for small molecule screening

Sample solubility

Many small molecules will require the use of organic solvents. Solvent correction is not required for compounds dissolved in aqueous solutions, providing that DMSO is not used in the running buffer during the assay, but it is still important to prepare samples and running buffer in the same way to match refractive indices as closely as possible.

For screening small organic compounds that require DMSO to maintain solubility, the running buffer should contain the same DMSO concentration as the samples. DMSO contributes considerably to the bulk refractive index of the sample (approximately 1200 RU per % DMSO), and accurate matching of samples and running buffer is important for reliable measurement of the low response levels obtained from small molecules. 2% DMSO is the recommended “standard” concentration, but up to 10% DMSO may be used in running buffer and samples if required for a particular compound.

Be careful when diluting stock solutions. Reducing the DMSO concentration may result in sample aggregation or precipitation. Carry-over disturbances during the run may indicate a problem with sample precipitation.

Matching buffer composition

Differences in bulk refractive index between running buffer and samples, arising for example from slight differences in salt or DMSO concentration, can lead to responses that may be confused with binding of analyte to the surface. This effect is largely eliminated by subtraction of the response from the reference spot with no attached ligand. However, the level of response expected for lead compounds is frequently low (less than 100 RU and often as low as 10-20 RU) and solvent effects can result in errors in reference subtraction of the same order of magnitude.

For this reason it is important to match the composition of running buffer and samples as closely as possible, particularly with respect to salt and DMSO (which are the major contributors to the bulk refractive index). A recommended procedure to facilitate buffer matching is to prepare a concentrated buffer stock solution containing all additives except DMSO, and to use the same solution for preparing both running buffer and sample dilutions. Suggested protocols for sample and buffer preparation are available.

A common cause of variations in bulk refractive index is variable evaporation of samples that are left uncovered for any length of time. Always use correct caps to close vials and foil to cover microplates to minimize evaporation. Do not degas running buffer excessively after the buffer has been used for sample dilution, since evaporation under vacuum can introduce discrepancies between running buffer and samples.

Sample dilution

Prepare all solutions containing DMSO fresh each day.

However you prepare your solutions, it is essential that solvent correction solutions, samples and running buffer are all prepared from the same stock solutions using as far as possible the same dilution procedures. Any differences in preparation routines will introduce potential errors in the solvent correction procedure.

Read more: Buffer and sample preparation for direct binding assay in DMSO using Biacore

Points to watch

Do not store DMSO in polystyrene vessels. Make sure that all plastic equipment used in handling the solutions is resistant to DMSO. Use Teflon or nylon membranes to filter DMSO solutions: do not use cellulose acetate membranes.
Certain plastics may release small quantities of contaminants into DMSO that have been observed to interfere with binding assays in Biacore. Deterioration of the plastic on contact with DMSO is not always apparent: running test analyses with negative controls (buffer containing DMSO) is recommended for all assays.
DMSO from different suppliers may vary in quality, and may sometimes contain contaminants which interfere with the assays. Good results have been obtained at Biacore using DMSO from Riedel de Haën (analytical reagent grade, maximum 0.03% H₂O).
Prepare all solutions carefully. Keep all vials and microplates covered with correct caps and foil respectively to avoid evaporation of samples.
Beware of running duplicate samples from the same well at widely separated intervals during the run, evaporation from the well once the foil covering the microplate has been pierced by the needle can lead to significant changes in concentration during the course of a run. Running duplicates in consecutive cycles is recommended.

Lab protocols

Lab procedure Buffer and sample preparation for Biacore direct binding assay in 2% or 5% DMSO, resp., is available for download on www.cytivalifesciences.com/solutions/protein-research/Interaction-analysis-with-Biacore-surface-plasmon-resonance-SPR/Get-started-with-surface-plasmon-resonance-SPR-interaction-analysis

Special considerations for small molecule screening

Small molecule screening assays are based on two main strategies, along with some variations on these formats:

Direct binding assay
The direct binding assay format is the most straightforward approach to screening, and is recommended as the method of choice for most purposes. Ligand covalently immobilized to chip surface or ligand captured by an immobilized capturing molecule
Read more: Direct binding assay
Surface competition assay
Single macromolecule analyte competition or enhancement molecule to amplify response of competing analyte
Read more: Surface competition assay

Preparing the active surface for small molecule screening

Ligand capture versus covalent immobilization

The choice of immobilization or capture is based largely on the characteristics of the targets.

For the most straightforward direct binding assays, the target molecule is immobilized covalently as the ligand on the sensor chip surface, and is regenerated (if necessary) after each sample injection.

An alternative approach to attaching the target molecule is to use ligand capture, where a capturing molecule is covalently immobilized on the surface and the ligand is captured through a high affinity interaction. Regeneration is generally simpler with a capture approach, since both target and analyte are removed in the regeneration step. The capturing approach can address the problem that some target molecules may lose activity when they are covalently immobilized on the surface or when they are exposed to regeneration conditions. However, capture assays involve an extra step in every cycle (injection of ligand), and interpretation of the results may be more difficult if ligand dissociates from the capturing molecule during the course of the assay cycle.

Immobilization levels

Low molecular weight analytes give intrinsically low response levels since the mass of analyte that binds to the surface is small. Immobilize enough target molecule to give an analyte binding capacity of at least 30-50 RU.

Buffers for small molecule screening

Buffers

All buffers used in Biacore, both as running buffer and for sample and reagent preparation, should be filtered through a 0.22 µm filter and degassed. Use Teflon or nylon filters for buffers containing DMSO: do not use cellulose acetate filters.

Phosphate-buffered saline (PBS) is recommended as a buffer for most routine assays. Many different “standard” recipes are available for PBS, and any of these should be acceptable. Use ionic strength at or close to physiological levels (150 mM monovalent ions) to reduce non-specific binding of compounds to the sensor surface. For work with kinases as target molecules, Tris-based buffers are recommended instead of PBS.

When DMSO is included in the buffer, the pH of stock buffer solutions will need to be adjusted to compensate for the effect of DMSO on the pH in the final working buffer (addition of DMSO raises the pH slightly).

Buffer compatibility

It is important that all solutions used (running buffer, sample solutions, regeneration and wash solutions) are compatible with each other and do not precipitate on mixing.

When using PBS as a running buffer, bear in mind that phosphate buffers can form precipitates with additives such as calcium, or on contact with some regeneration solutions such as high concentrations of MgCl₂. If MgCl₂ is used for regeneration, do not exceed 3M MgCl2 for regeneration and 10 mM phosphate for running buffer. Higher concentrations of either will cause precipitation at the injection interface between regeneration solution and running buffer.

Control samples in screening experiments

Use one or more known binders as positive controls and buffer injections as negative controls. If you intend to evaluate the screen by ranking multiple levels of binding strength, include different controls that allow you to place the ranking boundaries at corresponding positions in different plots. Control samples are prepared in exactly the same way as other samples.

Positive controls in screening assays may serve a dual purpose, for checking the activity of the sensor surface throughout the run (suggested frequency every 20 sample cycles) and as reference points for ranking boundaries. Only one positive control per spot is used for target activity adjustment.

Control samples also provide information for run-to-run comparisons when multiple runs are collated. Details of control samples that are repeated each time the assay is run (to provide a check on run-to-run variability of the assay) are suitably specified in the method. The default setting is to specify one positive and one negative control with variables defined in the method.

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