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Sensitive drug discovery

1st April 2013


Two types of light scattering technologies, static light scattering (SLS) and dynamic light scattering (DLS), have been applied extensively to biomolecular and compound characterisation, often playing complementary roles to each other.

SLS measures the time-average intensity of scattered light from the molecules in solution. The intensity is proportional to the concentration and molecular weight (MW) of the molecule in the solution.

SLS, especially multi-angle light scattering (MALS) detectors, are widely used in measuring absolute MW of proteins and other polymers independent of the shape of the molecules, avoiding the need for column calibration. SLS also has been used to determine the second viral coefficient, A2, a thermodynamic parameter that is associated with the conditions for protein crystallisation.

In addition, SLS can be employed whenever MW change is of significance in the compound screening process. Measurements can be carried out either in a stand-alone batch mode or, more often, in conjunction with a size exclusion chromatography (SEC, also known as FPLC) system. Consequently, the automation of MALS or MW measurements has generally followed the automation pathway of chromatography technology.

Today’s chromatography systems are commonly available with sophisticated software and integrated autosamplers capable of managing and loading hundreds of samples from industry standard microplates. Automated MALS or ‘absolute MW’ measurements are thus achieved by coupling readily available MALS technology to any modern SEC system.

Automated DLS, on the other hand, has lagged behind the more rapid implementation of automated MALS systems. This lag in implementation is largely due to the application of DLS as an ‘off-line’ batch format technique, playing a complementary role with SEC and thereby remaining independent of the SEC technology pathway. DLS contrasts with MALS in its fundamental measurement too, analysing the rapid time intensity fluctuations in the scattered light (which are due to the Brownian motion of the molecules in solution), rather than the time average intensity of scattered light.
The rate of the time intensity changes is directly related to the hydrodynamic size and size distribution of the molecules. DLS size distributions provide information about the homogeneity of the molecules, often measuring trace amounts of large particles (aggregates) not detected by other means.

Early batch DLS technologies became widely adopted in protein crystallisation, therapeutic drug development, and other areas because of the value in detecting aggregation quickly and easily. Early automated DLS technologies such as titration devices, Linbro plate readers, and autosamplers did not become widely adopted. Now a novel DLS plate reader recently commercialised by Wyatt Technology Corporation overcomes the limitations of prior attempts to automate DLS, requiring as little as 50 microlitre sample volumes, utilising disposable microplates (96 or 384 well formats) common to the drug discovery process, and detecting chemical aggregates at compound concentrations in the range of 3 to 10°M.

Drug discovery groups continue to be placed under significant pressure to improve productivity, by obtaining more information more quickly. Laboratory automation and integrated information management systems alleviate productivity pressure, automating virtually every screening technique.

Yet even a high level of automation does not guarantee a more fruitful compound development or discovery process. Drug discovery projects are often fruitless because of false-positives encountered during the screening of compound libraries.

False-positives exhibit confusing and contradictory data that are profoundly difficult to interpret, due to a shortage of accepted and definitive explanations for their behaviour.
Several known common mechanisms explain many but not all false-positives, including: compounds that inhibit multiple proteins within a protein family, or that covalently bind with the functional group of the target protein (leading to irreversible inhibition); and experimental artefacts arising from the detection methods comprising screening assays.

Recently McGovern et al discovered another mechanism underlying false-positives, referred to as compound or chemical aggregation. These so called ‘aggregate-based inhibitors’ aggregate under bioassay buffer conditions, and then non-specifically inhibit receptors and enzymes, producing a false-positive result.

Aggregate-based inhibitors share several characteristic features or modes of behaviour that can be exploited for the purposes of their detection. These troublesome inhibitors inhibit multiple enzymes in a time-dependent manner; exhibit an inhibitory effect that is sensitive to the presence of detergent and to enzyme concentration; and, perhaps most importantly, form large particles readily detectable by optical detection technologies including DLS.

The discovery of aggregate-based inhibitors coupled with automated DLS technology represents a new tool for drug discovery. New, critical information about a chemical compound can be determined quickly, typically seconds to minutes per well, without perturbation of the sample. The detection of compound aggregation can be applied during the various stages of drug discovery, whether in the preliminary formulation of a compound or as a secondary-screening diagnostic tool.

In a typical compound screening experiment, compounds are first dissolved in DMSO and then diluted and transferred to an assay mixture to make a range of desired compound concentration, typically 3 to 10 µM or higher, with DMSO concentration of around one per cent.

DLS can detect compound aggregates under these same conditions due to the large light scattering signal produced by the large aggregates. Large particles scatter exponentially more light than small particles. Though a typical compound molecule may have a MW less than 1000 (typically less than 0.5 nanometres in radius), the compound aggregate may reach particle sizes of tens to hundreds of nanometres in radius. Aggregates this large can be detected at concentrations well below the micromolar range of the compound concentration.

Drug discovery productivity is enhanced both by detecting these aggregate forming compounds early in the drug discovery process, as well as by incorporating an automated DLS technology for detecting chemical aggregation rather than batch DLS systems.

The DLS Plate Reader (Fig. 1) improves productivity over batch DLS by an order of magnitude, as shown in Table 1. Working with industry standard microplates enables samples to be more readily prepared and loaded by incorporating automated liquid handling systems, usually already available on site. Once the microplate is loaded into the DLS Plate Reader, personnel can initiate hands-free software controlled measurements, and spend their time on more vital project activities including data analysis and interpretation. Conventional batch DLS systems require personnel to stand by the instrument to remove the cuvette and to load samples.

Summary

DLS technologies are slowly but surely evolving as critical automated tools in drug discovery. Compared to other techniques and tools, automated dynamic light scattering is a non-invasive, reproducible and sensitive method for quick and easy detection of aggregate-based inhibitors in a wide variety of solvents and additives. u

Dr Bingyi Yao, Bob Collins and Dr Michelle Chen are with Wyatt Technology Corporation. For more information visit www.wyatt.com





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