Target-directed evolution of plant biosynthesis

Naprogenix is developing a biotechnology platform, based on evolutionary concepts, which re-invents plant drug discovery. Conventional drug discovery requires chemical synthesis of compounds followed by their screening for interactions with specific target proteins. In Naprogenix’ technology, the target protein is expressed in plant cells so that metabolites which interact with this protein confer a survival advantage under a specific selection procedure. A large mutant population of these cells, in which genes are activated randomly, is then exposed to the selection procedure. This "evolves" the plant genome toward the biosynthesis of metabolites that engage the target protein. Commonly about half of those mutant plant cells that survive are overproducing known or novel metabolites with the desired activity on the target. This technology therefore mimics conventional drug discovery, but substitutes plant biosynthesis for chemical synthesis, and target-directed selection for screening. In this way we re-direct plant metabolism toward the medicinal activity that we want the plant to produce. It accesses the whole genomic capacity of the plant to synthesize active metabolites to our specification.

Applications of the technology
Biosynthetic production of natural products: The technology provides a way of increasing the efficiency of production of drugs in plant cells. This is important when a plant metabolite is too complex to be chemically synthesized, and is present in low concentrations in the plant. In this case, its development as a drug is often abandoned because separation and purification will be uneconomic. Naprogenix’ technology can generate mutant plant cells that are biosynthesizing greatly increased amounts of the active natural product. These cells can be used as a novel production system, which can be patented because it is based on a unique mutation in the plant cell. As an example, we applied the technology to a Lobelia species which contains small amounts of a metabolite that could be a lead for Parkinson’s Disease, but which cannot be synthesized chemically. Many of the mutant Lobelia cells that survived the target-directed selection procedure showed increased activity at the target protein, together with greatly increased yields of the known active metabolite. These cells can now be used as biosynthetic production systems for this potential drug.

Biosynthetic discovery of active lead compounds: The technology can identify active compounds in plants that would not be discovered by conventional methods, and can even cause the plant cell to make "new" compounds that are not naturally found in the plant. Once again this is important when an active plant metabolite is too complex for chemical synthesis. Complexity makes it difficult to use a plant metabolite as a “lead” for the chemical synthesis of active compounds that can be patented. However, complexity is no barrier for biosynthesis in the plant cell. By switching on plant genes at random, our technology can activate biosynthetic pathways that do not normally exist in the plant, leading to new active compounds which may be more suitable as drugs than the original metabolite. This enables us to utilize previous research on medicinal plants that would otherwise be a dead end. In the example above, many of the Lobelia mutants which survived selection showed increased activity on the target protein which could not be explained by the major known active metabolite. These mutants contained 20 other active metabolites, about one third of which were novel, and are now under investigation as potential drugs.

Biosynthetic optimization of plant extracts: In many countries, traditional medicines are plant extracts containing a mixture of therapeutically valuable metabolites together with potentially toxic, unwanted metabolites. It is very difficult to separate all these chemically to improve medicinal value, but Naprogenix’ technology can use cells of the medicinal plant to biosynthesize "optimized" mixtures. This is achieved by expressing more than one target in the plant cell, so that desirable activity at one target promotes cell survival, and undesirable activity at a different target promotes cell death. The selection of mutants now results in survival of plant cells which produce extracts with better medicinal properties. We are using this technology to modify the phytoestrogens produced by licorice root cells, so that extracts will be safer and more effective treatment for menopausal symptoms. Instead of arduously separating all the good and bad ingredients, we simply tell the plant cell to synthesize the activity we want, and not to synthesize that which we do not want.

Conclusion: target-directed biosynthesis is a very powerful "green chemistry" in which mankind achieves control over plant secondary metabolism. Instead of simply using metabolites that the plant has evolved for its own survival, this technology re-directs the plant to produce metabolites that are more valuable for human use.