Several approaches are pursued to increase the throughput. One approach is to organize a large number of flow cells in an array format on a chip. Several companies are engaged in developing protein biochip systems.64 BIAcore (Uppsala, Sweden) has recently launched its SPR-based array technology and signed the first commercial deal. Applied Biosystems (Foster City, CA, USA) together with HTS Biosystems (Hopkinton, MA, USA) have developed the 8500 Affinity Chip Analyzer, based on grating-coupled surface plasmon resonance. Grating coupling is based on a fine grating on the chip surface, which provides coupling and allows imaging of the entire surface at once.64 BIAcore has now acquired all assets from HTS Biosystems including the FLEX Chip System. Another approach to increase throughput is based on the use of colloidal gold or silver nanoparticles. Englebienne and co-workers65 used colloidal gold nanoparticles sensitized with the binding protein. Gold nanoparticles coated with protein are able to directly report ligand association and dissociation by shifts in the SPR band of gold (localized surface plasmon resonance, LSPR). Protein-protein interactions as well as protein-small molecule interactions could be monitored. The same throughput has been calculated to be about 3000 samples per day using a clinical chemistry analyzer. The use of triangular silver nanoparticles in LSPR has been demonstrated with biotin-streptavidin as a model system66 and more recently applied by Haes and colleagues67,68 to monitor amyloid b-derived diffusible ligands.
Small molecule microarrays (SMMs) are an expanding field, where small synthetic or semisynthetic molecules are immobilized on a variety of surfaces.69,70 Arrays are used to probe small molecule-ligand interactions or protein function profiling. The protein-binding partner can either be fluorescently labeled or detected with a labeled antibody to the binding protein. Scientists at Graffinity Pharmaceuticals (Heidelberg, Germany) have developed a SPR-based fragment-screening platform.71 The microarrays in this case consist of gold-coated micro-structured glass plates. The whole array surface is then coated with a self-assembled monolayer on which chemical compounds are immobilized. A special plasmon reader has been developed to read the >9000-compound-rich sensor slides. A fragment-based affinity fingerprint is generated with what is called 'RAISE' (rapid affinity instructed structure evolution). The technology has been validated by the successful identification of fragments that bind to factor VIIa.72
Other label-free optical readout technologies are pursued in high-throughput screening. Gauglitz and colleagues are using reflectometric interference spectroscopy (RIfS).73 Part of white light is reflected at the interface of a thin layer and another part is reflected at the second interface. The two reflected beams can either be superimposed or form an interference pattern. This pattern can be constructive, where waves add in phase, or destructive, producing smaller peaks than either wave alone. The pattern depends on incidence, wavelengths, and optical density of the layer (given by refractive index and physical thickness of the layer). The properties of the interference pattern are sensitive to changes in or at the layer, e.g., by binding of proteins and ligands. For direct optical detection of binding events, simultaneous imaging with a CCD camera can be applied. Assays can be run in 96- or 384-well formats. A high-throughput assay for the identification of thrombin inhibitors has been developed on the basis of RIfS.74 In this case plates were coated with a SiO2 layer onto which a dextran layer was added. A known thrombin inhibitor was covalently attached to the dextran layer, which enabled the detection of thrombin binding and inhibition by small molecules.
A new system called BIND (biomolecular interaction detection system) has been developed by scientists at SRU Biosystems (Woburn, MA, USA).75 It is based on a new class of optical biosensors, so-called guided-mode resonant filters. They are made from a few homogeneous dielectric layers combined with a grating. This layout exhibits a narrow reflection spectral band due to the grating. The system works such that the incoming light is trapped in the waveguide via evanescent coupling. However coupling becomes leaky due to the grating layer and energy is coupled out of the waveguide into radiation modes. This light interferes destructively with the incoming light, similar to resonance conditions. Outside this resonance the light does not couple and is transmitted or reflected. Since coupling is highly sensitive to the wavelength of light and the angle of incidence, sharp resonant peaks can be observed in the reflected light when these parameters are changed. By applying biomolecules or cells to the surface of the sensor, the resonant coupling of light is modified and so the reflected and transmitted output is tuned. SRU has developed sensors in 96- and 384-well formats. A readout instrument based on a two-dimensional CCD camera determines the peak wavelength values of reflected light at resonant reflection conditions. Binding of biochemical material to the sensor shifts the peak wavelength values to greater wavelengths.75 As examples for the application of the technology, protein A-IgG interaction and human serum albumin as well as carbonic anhydrase II binding to small molecule drugs were selected. In the case of interaction with small molecules, the protein partner is immobilized on the sensor chip. Equilibrium dissociation constants determined with the BIND system correlate well with published data. The system also allows cellular assays to be performed and can determine cell density, cell death, and detachment. The IC50 determined for vinblastine on CHO cells is similar to values determined for other cell lines with other methods. In addition, when the surface of the chip is coated with antibodies directed toward cell surface molecules, the interaction of whole cells with the antibodies could be quantified. This application might be of interest to identify inhibitors of protein-cell or cell-cell interactions. The BIND system offers a sensitive, label-free assay technology with a broad range of applications.
3.27.6 Turbidometric Assays: Isomerases
Turbidometric assays make use of fine suspensions, which are formed during material precipitation such as protein denaturation, either by measuring light scattering (nephelometry) or by determining absorbed light spectro-photometrically. The development of high-throughput assays for certain enzymes has proven difficult. Protein disulfide isomerases represent a protein family with multiple biological functions and with potential implications in disease processes.76-78 Smith and colleagues79 have recently described the successful adaptation of a protein disulfide isomerase assay to high-throughput mode using a turbidometric assay. The assay makes use of the catalytic reduction of insulin by the isomerase and the subsequent aggregation of insulin chains in the presence of dithiothreitol. The turbidity can be monitored spectrophotometrically at 650 nm. A signal to background ratio of 3-6 was obtained and the assay was generally linear for 30 min. An important step that enabled a high-throughput mode was the successful conversion of the kinetic assay into an endpoint assay by the addition of H2O2, which acts as an oxidant and immediately depletes the reductant dithiothreitol. The assay showed good performance with Z0 values above 0.7 in 384-well plates. At least 100 plates could be screened per day making this assay a true high-throughput assay to search for protein disulfide isomerase modulators. The methodology developed should also have broader applications in transforming kinetic turbidimetric assays into endpoint screening assays.
Optical assays play an important role in hit/lead finding and high-throughput screening. ELISA is highly sensitive, robust, and reproducible and in most cases inexpensive. A major disadvantage is inhomogeneity, but a broad range of antibodies and luminescent readout technologies make ELISAs attractive. Assays where colored products are formed represent ideal homogeneous formats. Oxidoreductases, glyoxalases, caspases, and phosphatases form a large group of drug targets that can be assayed directly or indirectly by color changes of components of the assay reaction. Radioactive assays still play an important role in drug discovery. Filtration assays and more recently SPA beads and FlashPlates are important for a broad range of targets: kinases, GPCRs, fatty acid synthases, DNA polymerases, primases, helicases, and CYPs among others. Radioactive assays are easy to set up and robust. A major disadvantage remains, i.e., the risk involved in handling radioactive materials. Optical biosensors represent a more recent technology, which finds increasing applications in drug discovery. Optical biosensors make use of the behavior of light when it hits metal films in a prism. The behavior of light is altered when the metal film is coated with biomolecules. Optical sensors are therefore label-free technologies that develop from low- to high-throughput applications and become highly valuable in all phases of drug discovery and development.
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Bernhard Schnurr is currently Vice President Assay Development & Profiling at Discovery Partners International AG in Switzerland. He joined Discovery Partners International AG in 2001. Previously, Dr Schnurr was Head of HTS Factory at Discovery Technology Ltd, which merged in 2001 with Discovery Partners International AG. From 1996 to 1997 he was Project and Team Leader for Analytics and HTS at Burecco AG. He joined the Tumor Biology Center in Freiburg i. Br., Germany in 1994 as a Team leader for Assay Development and HTS (Institute for Molecular Medicine and Natural Compound Research) until 1996. In 1993 Dr Schnurr obtained his PhD in Biology from the University of Freiburg i. Br., Germany. He performed his thesis at Goedecke AG, Freiburg i. Br. in Germany.
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