Selection-driven plant metabolites for treatment of CNS diseases

DESCRIPTION provided by applicant This project aims to demonstrate that the evolution of plant biosynthetic pathways can be accelerated and driven to favor the synthesis of ligands which interact with a specific human target protein This is achieved by subjecting mutant plant cells to selection pressures favoring the survival of mutants with the phenotype of interest As an example this approach will be used to optimize pharmacological activity in Lobelia cardinalis which inhibits the human dopamine transporter hDAT putatively by its ability to synthesize the complex alkaloid lobinaline A stable transgenic line of L cardinalis plant cells expressing the hDAT has been established These cells show increased sensitivity to toxins transported into the cell by the hDAT including the neurotoxin MPP A large genetically diverse population of gain of function mutants expressing the hDAT has now been generated and selected on medium containing uM MPP which kills the vast majority of the transgenic mutants However individual mutants that are over producing inhibitors of the hDAT have a survival advantage so that the MPP resistant population is greatly andquot enrichedandquot in clones with this bioactivity Preliminary GC MS analysis of individual MPP resistant mutants with increased hDAT inhibitory activity indicates that many of these are overproducing lobinaline but the rest are generating other metabolites some of which are not detectable in the wild type plant Phase II is designed to demonstrate that this biotechnology can be used to a generate novel natural products with a specific valuable pharmacology b provide a biosynthetic production system for these compounds in mutant plants The process should be of particular value when a plant derived lead compound such as lobinaline is too complex for chemical synthesis The first specific aim is to analyze the remaining MPP resistant population to determine those individuals in which lobinaline content cannot explain increased hDAT inhibitory activity Separation assay guided preparative HPLC and tentative identification GC MS of active compounds will be followed by pharmacological evaluation in vitro in comparison with lobinaline and a synthetic inhibitor of the hDAT The most active compounds will then be tested for functional effects on the DAT in rat brain in vivo using electrochemistry In parallel studies the mutant clonal cultures which are overproducing active metabolites to the greatest extent will be regenerated to intact mutant plants and extracts analyzed to establish whether the pharmacological chemical phenotype is retained The ultimate aim is to commercialize the technology as a platform for discovering and producing novel plant derived natural products targeted on specific human CNS proteins PUBLIC HEALTH RELEVANCE Plants are a major source of existing medicines but the pharmaceutical industry has now almost abandoned this source in favor of synthetic chemicals One reason is that plant metabolites are often too complex for synthetic methods to be used for their modification or production The technology being developed by Naprogenix uses the plants own biosynthetic capacity to modify the active chemicals contained in the plant It is also capable of increasing the amounts of the active chemicals that are present The andquot directed evolutionandquot of a plant species using this technology is capable of generating novel compounds that may be of value in many CNS diseases for example the targets in this project are important in drug dependence and neurodegenerative disease