Eukaryotic translation factor inhibitors as selective translation factor probes and anticancer therapeutics Project Summary The goal of this project is to establish anticancer activities for eukaryotic translation factor inhibitors (ETFIs) possessing the scaffold of the myxobacterial natural product, gephyronic acid. Gephyronic acid inhibits eukaryotic initiation factor 2 a (eIF2a)-phosphorylation through a potential direct interaction with eIF2a with antiproliferative activities reported at low nanomolar concentrations. Access to microbial ETFIs remains limited due to difficult culturing conditions and low production titers from the cultivable native producing myxobacteria. This absence of accessibility has significantly impeded investigation of microbial ETFIs for both, translation factor and cancer cell specificities and efficacies. We propose two distinct approaches to provide access to such ETFIs as well as generate novel ETFI new chemical entities (NCEs): we intend to (1) optimize production of gephyronic acid and ETFI NCEs from the native hosts Archangium violaceum and Archangium gephyra and (2) develop heterologous production platforms in hosts such as Myxococcus xanthus, Streptomyces albus, and Escherichia coli. We will optimize the metabolic and physiological conditions of A. violaceum and A. gephyra for the production of gephyronic acid for application in in breast cancer xenografted zebrafish assays conducted in collaboration with Dr. Kristie Willett, also in the UM School of Pharmacy, as well as tumor specificity provided by the NCI-60 Human Tumor Cell Lines Screen. Because myxobacteria perceive and react to surrounding quorum sensing networks of neighboring organisms, exogenous supplementation of ubiquitous, fungal quorum sensing molecules will be evaluated as inducers of ETFI production in an attempt to increase the production titers of both native hosts. Analogous ETFI production capabilities will be investigated in A. violaceum and A. gephyra using precursor directed biosynthesis experiments with supplementation of methionine analogs for incorporation via the rare embedded a-methyltransferase domains replete throughout the gephyronic acid biosynthetic pathway. Also, RecET/Redab methodology will be employed to construct gephyronic acid biosynthetic pathway expression plasmids for the development of heterologous ETFI production platforms in M. xanthus, S. albus, and E. coli hosts. Precursor directed biosynthesis experiments as done in the native hosts will also be conducted in developed heterologous hosts. The development of optimized, tunable ETFI production platforms would drastically increase access to ETFIs and ETFI NCEs and facilitate their application as translation factor probes as well as anticancer therapeutics. Established ETFI probes and therapeutics would directly supplement the National Cancer Institute?s efforts to improve cancer prevention, detection, diagnosis, and survivorship.
Eukaryotic translation factor inhibitors as selective translation factor probes and anticancer therapeutics Project Narrative Through the development of native and heterologous gephyronic acid production platforms, we will increase access to eukaryotic translation factor inhibitor scaffolds and investigate their application as translation factor probes and anticancer therapeutics primarily in breast cancer xenografted zebrafish assays.
