Today’s high-tech, high-investment, and high-risk pharmaceutical industry has thus created a hugely competitive, complex environment in which a careful balance is needed between harnessing innovative technologies to develop better, safer drugs and making a profitable return on increased R&D spending. In theory, the wealth of new disease data and the abundance of early-stage assay “hits” from high-throughput screening (HTS) programs should equate to more drugs reaching the market. In practice, however, the pharmaceutical industry is still failing to turn potential into profit, and only a small number of promising compounds will make it successfully through development. The failure rate is high and, with the average cost of drug development hitting the $350 million to $500 million mark, companies need to significantly improve the efficiency of their discovery and development processes to stay in the game.

One of the present bottlenecks is in knowing which candidates to pursue from the vast numbers of HTS hits. This would save both time and cost, and it would help facilitate the progress of high-quality candidates to clinical trials. The ideal solution is the use of automated in vitro analytical techniques that can characterize drug target relationships and enable predictive preclinical analyses on, for example, how a potential drug may be absorbed, metabolized, distributed, and excreted from the body. This capability would give researchers a much better platform from which to make informed decisions on drug lead selection.

A novel solution
Surface plasmon resonance (SPR) is beginning to have a growing and widespread impact on drug discovery and development, having already been proven for a decade in basic and applied life sciences for analyzing biomolecular interactions. In early drug discovery upstream of HTS, SPR technology is enabling proteomics applications for drug target identification, validation, and optimization, as well as high-throughput assay development for HTS. The technology determines the binding specificity between two molecules, assesses how much of a given molecule is present and active, and quantitatively defines both the kinetics and affinity of binding. This real-time analytical capability can be widely applied to investigate the properties of binding between combinations of low and high molecular weight biomolecules, including DNA, protein, antibodies, lipids, and whole eukaryotic cells. In addition, this biomolecular analysis is carried out without the need for fluorescent tags or radioactive markers, and often without the need for prior purification. (See box, “To serve and detect”.)

In proteomics research, numerous receptors have predicted biological function but no known ligands. Such receptors are commonly referred to as orphans. Ligand fishing or deorphaning is the process by which these receptors are screened against a multitude of compounds or cell/tissue extracts to identify putative ligands for the receptors. This process can also be applied to novel proteins that have no known binding partners. During the drug target identification process, the binding partner of a receptor or a protein must be found in order to determine possible ways to challenge malfunction and address the pathological pathway (1, 2).

The ability of SPR-based instrumentation systems in proteomics to detect specific binding interactions in complex matrices allows materials such as crude cell and tissue homogenates to be directly screened as potential sources of ligands for orphan receptors. In proteomics research, the technology can be classed as unique because it allows the concentration of bound ligand to be determined together with simultaneous monitoring of the binding affinity, kinetics, and specificity, all in one system. SPR technology can also be integrated with mass spectrometry, which enables protein detection, separation, and kinetics analysis to be linked with mass determination. Combined, these technologies provide a powerful system for the elucidation of protein structure and function (3, 4).

Because SPR technology can detect and quantify functionally active molecules, it serves as a monitor and information provider for tracing targeted components during purification development and optimization. When selecting suitable gene expression systems, the functional integrity of the purified recombinant and the quantity of active protein produced can be assessed. The information provided not only enables faster analysis compared with conventional techniques, it also assists in optimizing purification to increase product yield (5, 6).

A significant advantage of SPR technology is its ability to show real-time interactions without altering the natural kinetic characteristics of the interacting partners by labeling. Analytical systems based on SPR are therefore routinely used to develop and troubleshoot molecular interaction-based high-throughput screens, including ELISA, scintillation proximity assay, and fluorescent-based systems. For example, the real-time kinetic analysis of recombinant receptor–ligand interactions enables the selection of the most suitable binding partners. The chosen interaction can then be further characterized to evaluate the effect of labeling. The optimum assay conditions can then be determined, ready for the HTS process. In HTS, in which hundreds of thousands of compounds are tested, the resulting validated and robust assays can help reduce false positive/negative rates, thereby increasing efficiency and data quality (7, 8).

Downstream of HTS, new high-performance systems are being developed that will offer specific applications in drug lead optimization and preclinical analysis, including compound interaction studies and certain absorption, distribution, metabolism, and excretion (ADME) evaluations. In addition, kinetic characterization of HTS hits will provide detailed insight into the action of lead compounds. Such information is available because SPR technology can be used to directly detect and quantify, without labels, the kinetic binding characteristics of low molecular weight compounds to therapeutic target proteins. Coupled with the ability to test compounds against a series of targets in a single assay, this capability can significantly enhance the data available for hit selection and improve the quality of compounds prioritized. SPR technology is also being used to develop the next generation of instrumentation systems for this area of application (9–11).

Initial applications of the system in ADME analysis will help to determine the extent to which drug compounds bind to plasma proteins, providing an indication of the drug candidate’s distribution and bioavailability. Researchers will be able to highlight potential problems, such as undesirable binding properties, earlier in the discovery process. The aim of these systems is to reduce the cost and time of specific tests, such as human serum albumin and a-1-acid glycoprotein binding, while at the same time providing quantitative data at an earlier stage in the drug discovery process to improve drug lead selection. (See box, “On the pharm”.)

What is SPR?

Figure 1. SPR-based biosensors. The sensor chip provides a gold-coated surface through which minute changes in biomolecule concentration, due to molecular binding, are detected in real time. These changes lead to changes in the reflected light that are captured by the optical detection unit and converted to a resonance signal. (Courtesy of Biacore International AB.)

Figure 2. SPR sensorgram. As molecules bind to and dissociate from the sensor chip surface, the resulting changes in the resonance signal create a sensorgram. The shape and amplitude of the measurements can then be used to determine overall concentration and the kinetics of the interaction. (Courtesy of Biacore International AB.)

SPR biosensors visualize the progress of biomolecular binding through time by defining the change in mass concentration that occurs on a sensor chip surface during the binding and dissociation process. The chips are composed of a glass surface coated with a thin layer of gold that provides the physical conditions necessary for the SPR reaction (Figure 1). Light is shone on the reverse side of this chip, propagating an electron charge density wave phenomenon that arises at the surface of the metallic film. This takes the form of an evanescent wave that extends beyond the sensor surface and detects mass changes on the surface. Light does not actually enter the sample at any time; thus, the quenching or absorbance problems characteristic of spectrophotometric and fluorescent techniques are eliminated.

The chip is constructed by covering the gold surface with a layer of dextran on which one of the interacting molecules under study (e.g., a cell-surface receptor) is immobilized. A microfluidic system then injects the analyte solution containing the other interacting molecule (e.g., receptor ligands or druglike compounds) over the sensor surface. Binding is quantitatively assessed; and once the analyte solution has completed its pass over the sensor surface, the dissociation rate is also measured (Figure 2). Flow conditions are controlled by using a liquid-handling system comprising precision pumps and an integrated fluidic cartridge that forms the flow cells on the chip surface and directs the flow of liquid over the surface.

Future developments
SPR technology is also setting the foundation for a new generation of biosensor chip applications that, although a few years from market, are expected to dramatically increase the technology’s drug discovery throughput capabilities. At present, SPR biosensors can monitor up to approximately 100 biological evaluations per day. The new technology—known as SPR array chip technology—aims to increase this number 1000-fold to 100,000 biological evaluations per day.

Conclusion
If pharmaceutical companies are to increase the drugs they bring to market, it is critical for them to maximize the value of their development portfolios by using resources only for projects with real potential. Successful management of the research-to-development interface will require better quality decision making in the selection of entrants to the development process.

SPR technology has already proved its value in life science research and is now being used in drug discovery processes upstream of HTS. The technology rapidly provides accurate, high-quality data that can enable crucial decisions to be made early in drug selection processes. New systems that will specifically enhance the selection of drug leads from HTS, coupled with the potential of the new array technology, mean SPR biosensors are paving the way for smarter drug discovery and development.

References

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Julian Abery is vice president and head of the pharmaceutical and biotechnology business unit of Biacore International AB (Neuchâtel, Switzerland). Send your comments or questions regarding this article to mdd@acs.org or the Editorial Office by fax at 202-776-8166 or by post at 1155 16th Street, NW; Washington, DC 20036.