In Vitro Assays

The availability of large chemical collections and numerous protein targets for herbicides necessitates the development of in vitro assays that are amenable to miniaturization and HTS.  Many enzymes with well-defined functions can be assayed through direct measurement of the product, or by linking the activity to a colorimetric or fluorimetric assay detectable on a spectrophotometer.  Combining this approach with HTS using microtiter plates (Figure 3) allows massive numbers of compounds and targets to be screened simultaneously.  However, this strategy is limited if the enzyme product is unknown or difficult to visualize.  The availability of high-throughput mass spectrometry will now allow these assays, and possibly those with several enzymes and/or substrates, to be conducted efficiently.

Figure 3.  A microtiter plate containing lesser duckweed (Lemna minor) plants showing growth inhibitors (circled wells).  

Assays to screen potential herbicides that interfere with protein-protein interactions are also in development.  The yeast two-hybrid system, and more recently a reverse two-hybrid assay, can detect compounds that interrupt signal transduction pathways or other cellular functions that were previously impossible to assay.