'Target-Based' Discovery and Screening

The recent explosion in molecular biology technologies has provided some extremely powerful tools for herbicide discovery.  For example, whole genome sequencing of Arabidopsis and other model plants has identified many genes whose proteins could represent potentially new herbicide target sites.  There are about 28,000 genes in Arabidopsis, of which about 20% are annotated as enzymes.  As noted above, only a few of these enzymes are current targets for commercial herbicides.  Thus, a logical approach would be to pick one or more of these unknown enzymes and try to develop chemical inhibitors as herbicides.  

This approach is a very simple example of the ‘rational design’ or ‘targeted’ strategies now being explored, in which the emphasis is first placed on the biochemical target, and secondly on the chemical itself.  However, such a strategy requires that several assumptions must be met before embarking on a discovery program.  Among them are:

1. Will inhibition of the target enzyme result in sufficient growth inhibition to provide acceptable levels of weed control?  Practically speaking, 100% inhibition of a target enzyme is almost never achievable under field conditions.  Abell (1996) suggested that an enzyme should be a good target if 60% to 80% inhibition leads to a severe growth phenotype (i.e., severe growth inhibition).
2. Can the enzyme be obtained in sufficient quantities and in the correct catalytic conformation to conduct in vitro assays in a HTS format?  This requirement assumes that the gene and/or the cDNA has been cloned and is available, can be properly expressed in a eukaryotic expression system, and that the enzyme can be purified while retaining its activity.
3. Can the enzyme’s activity be measured in an in vitro assay that is compatible with highly-mechanized, HTS systems?  Catalytic activities of most enzymes can often be linked to colorimetric or fluorescent reactions to create relatively simple assays.  However, assaying the inhibition of an enzyme’s regulatory site or protein-protein interaction sites is much more difficult, and introduces another level of complexity.
4. Is information on the enzyme active site (and regulatory sites) known in molecular detail?  These are perhaps the most important criteria, since all the chemical modeling methods discussed above require this level of detail in order to predict binding and activity of novel inhibitors and their structural analogs.  Such information is obtained from analysis of the enzyme’s crystal structure.

If these assumptions are met, detailed work on identifying potential herbicide inhibitors can be conducted rapidly using HTS screening systems.  Also, strategies like comparing proteinsequences or active site information between monocots and dicots may identify potential enzyme targets for selective herbicides.