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"SPA, scintillation proximity assay, a technology developed by Amersham.

"SPA, scintillation proximity assay, a technology developed by Amersham.

be reduced down to nanoliter volumes and can even detect single molecule interactions (23,24) (see also www.evotec.com). Another method, homogeneous time-resolved fluorescence, is used for kinases, receptors, proteases, and other targets. As an example, in a protease assay one label (e.g., europium cryptate) is attached to one part of the protein substate, an allophy-cocyanin label to the other side. Excitation at 337 nm leads to an energy transfer from the cryptate to the allophycocyanin, which causes emission of a delayed fluorescence signal at 620 nm. Protease cleavage leads to a larger distance between the two labels, which results in a lower signal. This method is of particular interest in natural product screening because the interference with autofluorescence and quenching in such screening samples is limited (23,24). A similar concept has been developed for isotopic assays.

For most methods, standard plate readers are being used, although digital imaging will be the readout of choice for very large throughput (19). Other technologies currently being developed are using microfluidic devices (see, for example, www.orchid.com,www.aclara.com) (25,26).

A significant number of the potential drug targets are tested in insect or mammalian cell cultures, which are also run in 96-, 384-, 1536-plate or lower volume formats (27-29). An example is the screening for modulators of G protein-coupled receptors, the most important class of drug targets. If the interaction of a natural ligand causes an increase in the intracellular cyclic adenosine monophosphate (cAMP) concentration, such a receptor can be overexpressed in a mammalian cell line and the increase in cAMP quantified by the activity of a reporter gene linked to a cAMP-responsive element (CRE). If coupling of the G protein-coupled receptor is not via the cAMP pathway but through increase of intracellular calcium, dyes are used for quantification of calcium (27,29). Other targets are screened by quantification, e.g., by enzyme-linked immunosorbent assay (ELISA), of the expression of endogenous proteins in the selected cell culture after compound application.

For natural product screening, particularly for extracts, cellular screening systems are often difficult to use because of the presence of cytotoxic material in the extracts or other nonactive material, which interferes with the assay (e.g., by signal quenching). In most assays such undesired effects lead to false-negative results; i.e., if any biological activity was present in the extract, it is not discovered because of side effects.

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