Nannocystin reagents will be used to interrogate the role of the protein elongation factor 1A in apoptosis of cancer cells. Nannocystins are small molecule natural products isolated from myxobacteria that potently inhibit cancer cell proliferation and trigger apoptosis at an early time point. Nannocystin is a hybrid molecule consisting of an upper tripeptide domain and a lower polyketide domain. Nannocystin binds the protein eukaryotic elongation factor 1? (EEF1A), however it is presently unclear how binding to this protein causes apoptosis. The related molecule didemnin B also binds EEF1A, inhibiting EEF1A-mediated elongation at micromolar concentrations but triggering apoptosis at nanomolar concentrations. Elongation factors are vital for protein synthesis, but additional cellular roles have recently been found. In particular, EEF1A was found to inhibit p53's activity as a tumor suppressor, and cancer cells under stress increase EEF1A production. It is hypothesized that nannocystin disrupts EEF1A-p53 binding, which releases p53 to activate the apoptosis pathway, leading to cell death. Nannocystin is thought to bind EEF1A at the same site as didemnin B. However, there are no cocrystal structures of EEF1A-nannocystin, and a recent structure-activity relationship study challenged this putative binding model. Details of the binding site are critical for rational development of new anticancer therapeutics related to nannocystin. To investigate apoptosis in cancer cells, we propose studies in which nannocystin and molecular probe reagents will be prepared by total synthesis, an approach that requires efficient and convergent chemical synthesis. We will also employ a tandem reaction between a simple alkene and alkyne to access the unique polyketide domain. This proposal has three specific aims: (1) to use Ru coupling and Co-promoted isomerization in the total synthesis of nannocystin, for which we have made significant progress toward both the tripeptide and polyketide fragments; (2) to synthesize two bifunctional photoaffinity reagents to elucidate the site of binding between nannocystin and EEF1A, incorporating a novel tripeptide to introduce the photoaffinity probe, designed to covalently bond to the protein at the two extreme ends of the binding site; and (3) to determine if the p53 pathway is involved in nannocystin-triggered apoptosis and identify the downstream effectors of p53 activation.
The total synthesis of the eukaryotic elongation factor 1? (EEF1A)-binding small-molecule nannocystin will enable the preparation of nannocystin reagents to study apoptosis signaling in cancer cells. EEF1A, though important for protein synthesis, also has noncanonical roles, such as binding to and regulating p53. It is hypothesized that nannocystin binds to EEF1A, disrupts p53 binding, and triggers apoptosis.
