The objective is further development of a biotechnology that evolves biosynthesis in a plant species toward bioactive metabolites with a specific molecular target. In wild-type plants, bioactive metabolites have generally evolved by mutation and natural selection over millennia [1], whereas here, target-directed evolution can change the active metabolite profile in mutant plant cells within months. Proof of concept has been obtained in Lobelia cardinalis, which contains the complex alkaloid lobinaline. This is a novel inhibitor of the dopamine transporter (DAT) [2], a molecular target in Parkinson's Disease and drug dependence [3,4 ]. First, hairy root cultures of this species were transformed to express the human DAT. This made these transgenic (hDAT) plant cells highly susceptible to toxicity induced by the neurotoxin MPP+, which is accumulated intracellularly by activity of the DAT. Gain of function mutants of these transgenic cells were then generated in a concentration of MPP+ that is lethal to non-mutants. This selection procedure favors survival of mutants that overproduce metabolites that inhibit the DAT. As a result, more than half the >100 MPP+-resistant mutants were significantly overproducing DAT inhibitory activity relative to controls. In the majority of these mutants, enhanced DAT inhibition could be ascribed to lobinaline, or other known active metabolites, but 25 mutants contain unknown DAT inhibitors. In these mutants there are 9 novel HPLC peaks that are not observed in wild-type, and these all contain DAT inhibitory activity. The first specific aim is to separate sufficient quantities of these metabolites for chemical identification and pharmacological analysis (DAT inhibition in vitro and in vivo). This will establish whether novel DAT inhibitory metabolites with therapeutic potential exist in these mutants. Many of the remaining MPP+-resistant population (mutants that do not overproduce DAT inhibitors) appear to overproduce metabolites that inhibit the intracellular mechanism of MPP+. Extracts from these mutants protect the dopaminergic cell line SH-SY5Y against MPP+, and so may contain novel neuroprotective metabolites. The second specific aim is to separate and analyze these also, using the same approach based on separation and analysis of novel HPLC peaks, so that their potential therapeutic value can be ascertained. Successful completion of these aims will establish proof of application for the technology as a platform for plant drug discovery. The same approach can also be used to generate overproducing mutants to become biosynthetic production systems, and as a means of optimizing biosynthesis of therapeutic metabolites in medicinal plants. In addition, although this proposal uses the human DAT and L. cardinalis as examples, the technology can be applied to many other targets and plant species. If adopted by the global pharmaceutical industry this could be a transformative technology. The applicants intend to pursue this in phase III by seeking partnerships with major pharmaceutical and/or biotechnology companies. Thus, the main purpose of the proposal is technology development, to be achieved by demonstrating that this approach is capable of discovering novel active metabolites with therapeutic potential in mutant transgenic plant cells.
Public Health Relevance Statement: Project narrative The company is developing a technology which enables us to tell mutant cells of a plant species what kind of drug we want them to make. This proposal applies the technology to drugs that may be of value in Parkinson's Disease or drug dependence. The main objective is to show that the technology works, so that it can be adopted by the industry as an alternative to chemical synthesis as a source of new drugs.
Project Terms: 1-Methyl-4-phenylpyridinium; abstracting; Adopted; Alkaloids; Anabolism; aqueous; base; Biotechnology; Carbon; Cell Line; Cells; chemical synthesis; Chemicals; commercialization; Complex; cytotoxicity; Development; Directed Molecular Evolution; dopamine transporter; Dopaminergic Cell; Drug Addiction; drug discovery; Drug Industry; drug production; Evaluation; Freeze Drying; gain of function; gain of function mutation; High Pressure Liquid Chromatography; Human; In Vitro; in vitro activity; in vivo; Industry; inhibitor/antagonist; Lobelia; Mass Fragmentography; Mass Spectrum Analysis; Medicinal Plants; Methanol; Methodology; Molecular Target; mutant; Mutation; N-Methylaspartate; Natural Selections; Neurons; Neurotoxins; novel; novel therapeutics; Nuclear Magnetic Resonance; Parkinson Disease; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Plant Roots; Plants; Population; Procedures; Production; Proteins; Protons; Resistance; Resolution; Source; Spectrometry; Spectrum Analysis; Structure; Synapses; System; Technology; technology development; Testing; Therapeutic; therapeutic target; Toxic effect; Toxin; Traditional Medicine; Transgenic Organisms; Transgenic Plants; uptake; Work
