The Genetic Approach
If the molecule is active in a model system for which appropriate tools are available, genetic strategies can be used to isolate drug resistant mutant organisms. The mutations causing resistance often define targets because they disrupt critical side-chain drug interactions.
In the case of an antagonist, target-site mutations may present themselves with dominant inheritance patterns, agonist target-site mutations often behave recessively, and if a target is dosage sensitive, semi-dominant or haplo-insufficient behavior can be observed for both agonists and antagonists. Since other resistance mechanisms are possible, such as defects in pro-drug activation or defects in the pathways that may be needed for bioavailability of a given molecule under normal circumstances, careful analysis of mutant phenotypes, inheritance patterns, in vivo drug levels and metabolism are used to assess which mutations isolated are most likely to uncover target proteins.
Unlike the biochemical approached outlined below, the genetic approach does not require knowledge of structure-activity relationships for a given molecule, which simplifies its application. With sequencing technologies evolving at their current rates, whole genome resequencing is likely to be affordable to most biologists relatively soon. This will greatly accelerate the genetic identification of targets by removing time consuming map-based cloning steps from the process.
However, as appealing as the genetic approach is, it is clear from systematic analyses of herbicides in plants, that resistance mutations may not be isolated for every molecule analyzed (given a finite, but large population size). Like all methods the genetic approach is not guaranteed to work all of the time. Additionally, because of the rarity of target-site mutations, the genetic approach usually requires drug-induced phenotypes that are coupled to easily scored traits such as growth. In spite of these significant limitations, the genetic approach is a simple, inexpensive and general approach to target identification.
The Genetic Approach