Mutant transgenic plant cells as a novel source of drugs

Abstract of administrative supplement The original objective was to use a novel plant biotechnologytarget directed evolutionto produce novel inhibitors of the human dopamine transporterDATas leads for medications for alcohol use disorderThis approach evolves plant biosynthesis toward a specific target by a combination of mutagenesis and selectionLobelia cardinalis was used as the plant species because it contains lobinalinea previously uninvestigated inhibitor of the DAT that also has partial agonist activity at nicotinic receptorsBrown et alThese proteins are molecular targets in alcohol and nicotine use disordersand lobinaline inhibits alcohol consumption in rodents with no evidence of abuse potentialLobinaline would be an excellent lead compound for pharmacotherapy of alcohol and nicotine use disorders except that it is a highly complexinsoluble alkaloid with no chemical synthesisHowevertarget directed evolution of Lcardinalis mutants should generate lobinaline derivatives with greater potency and or solubilityas well as other metabolites with activity on the DATThis was shown to be true using a combination of GC MS and pharmacological analysesBrown et alHoweverto definitively identify the active metabolites in mutant plant cell cultures the applicants had to develop novel analytical methodologyThis has identified at leastnovel metabolites that are markedly overproduced in those mutants that contain increased inhibitory activity on the DATTwo of these are already known to be activeeither against the DAT or against MPPneurotoxicityand four metabolites are derivatives of lobinalineThese lobinaline metabolites are N oxidesand derivatives with new double bonds in the ring structureThe lobinaline N oxides have been tested in vitro and in vivo with exciting resultsTheir pharmacology in vitro differs only modestly from lobinalinebut they have an importantdruggableadvantage in increased aqueous solubilityHoweverwhen tested in vivo by microperfusion in rat brain the lobinaline N oxides have clear initial inhibitory effects on rate of dopamine clearanceas does lobinalinebut this is rapidly converted to an increase in dopamine clearance capacityThispartial antagonisteffect of these metabolites on the DAT is unlike lobinalineor indeed any other known DAT inhibitorIt is theoretically of considerable value in drug dependencebut this has not been tested in intact animalsand this was not included in the original applicationOne specific aim in this supplementary proposal is to test the lobinaline N oxides in animal models of alcohol and nicotine use disorderand to test theother novel metabolites for activity in vitroIn additionthe mutant transgenic cultures that contain novel active metabolites are a major resourceestimated cost to date is $K cloneand for sufficient metabolites to be obtained for testing they must continue to be maintained and sub culturedIf this supplemental application is successful the applicants intend to use the data for another NIH application to continue development of these novel plant metabolites as treatments for alcohol use disorder Project narrative The company is developing a technology that directs mutant cells of a plant species to produce drugs with activity at a specific targetThis proposal applies the technology to plant drugs that are of potential value in drug dependence and alcoholismUnder the original award we have found several plant metabolites with the required activity and the supplemental application is to test these for their ability to reduce drug dependence