Human nuclear receptor (HNR) ligands have potential value as pharmacotherapies or chemopreventive agents for many hormone-responsive cancers. The primary aim of this STTR is to transfer an "academic" plant genomics technology into drug discovery, targeting HNR ligands. Estrogen receptor (ER) ligands in plants will be used for proof of application, because these have clear commercial and therapeutic value in their own right. In phase1 transgenic Arabidopsis thaliana seedlings expressing a human ERalpha/GFP construct were used to screen extracts from a native plant library for ER ligands. 9 previously uninvestigated aquatic species were identified, including species with agonist or antagonist activity at ERs in mammalian cell-based screens. In phase 2, representatives of these species will be investigated further, using assay-guided HPLC fractionation to isolate active compounds, which will be tested on breast cancer cells in vitro and in vivo. However, the major aim in phase 2 is to apply the novel plant genomics technology to optimize the production of ER ligands in two plant species. In this technology, transgenic hairy root cultures of a plant species are generated, expressing either the ERalpha/GFP construct, or a novel ERbeta/GFP construct. These cultures are then subjected to activation tagging mutagenesis (ATM), which induces random gain-of-function mutations in the genome. Now, any individual mutant in which the production of ER ligands is increased, should be identifiable by bright green fluorescence. This rapidly screens the "genomic capability" of the species to generate ligands for either ERalpha or ERbeta. In phase 1, proof of concept for this approach was achieved in a mutant population of transgenic A thaliana seedlings expressing ERalpha/GFP, and in phase 2 we propose initially to apply this approach to hairy root cultures of Glycine max (soybean - known to contain high levels of ER ligands) and Cacalia plantaginea (a native plantain with high levels of ERalpha/beta agonist activity). Positive cultures will be characterized by their GFP expression, and on the activity of culture extracts in conventional screens for ER activity. Attempts will also be made to regenerate intact mutant plants with the same over-producing phenotype, and active fractions from mutant cultures or plants evaluated on breast cancer cells as above. The approach can be used with any HNR target, and the major aim is to show that the technology can be transferred to anti-cancer natural product discovery. The commercialization plan is aimed at licensing the technology, or partnering with major companies to apply the technology to targets specified by the partner.
The technology transfer in this STTR promises to rapidly evaluate plant species for appropriate therapeutic activity at human nuclear receptors. The example chosen is for plant compounds with activity at the two types of human estrogen receptors (ERalpha and beta) which have different therapeutic implications, particularly for breast cancer. This is the most common form of cancer in women and is the major cause of death for women under 60 in the USA. Many breast cancers are (initially at least) "hormone responsive" in that drugs which inhibit ERs markedly slow the growth of the cancer cells, and this is a very active research area in the pharmaceutical industry. Plants are a known rich source of ligands for the ER, but it has proven difficult to exploit this source because conventional plant drug discovery is often time-consuming and expensive. This technology promises to change this by applying genomics approaches to plant drug discovery in this area.