The control of translation (mRNA-encoded protein synthesis) is crucial for cell proliferation and differentiation which is manifested in the fact that protein synthesis is rapidly adjusted in response to environmental stimuli including diet, stress and infections. More than 10 eukaryotic translation initiation factors (IFs) are known to be involved in assembly of the 80S ribosome-RNA complex enabling interaction of the initiator Met-tRNA with the start codon of the mRNA. Involvement of such a large number of proteins in the process suggests an important role for protein-protein interactions in translation initiation. The main mechanism used by most cellular mRNAs, named cap dependent translation, requires cap binding complex eIF4F. The eIF4F complex composed of the cap binding subunit eIF4E, the RNA helicase eIF4A, and the scaffolding protein eIF4G. The ATP-dependent RNA helicase eIF4A unwinds secondary structure in the 5'untranslated region (UTR) of mRNAs in preparation for scanning for the start codon. This unwinding process enables translation of mRNAs with long, structured 5'UTRs. Long, structured 5'UTRs are a common feature of growth-regulatory genes (e.g. those encoding for oncoproteins) as well as of some viral RNAs. eIF4A alone shows low unwinding and ATPase activity. This activity is stimulated when eIF4A is part of a multiprotein complex including eIF4G, eIF4E, eIF4B and eIF4H. The interaction between eIF4A and eIF4G presents an important human drug target since inhibition of this interaction would primarily decrease translation of growth-regulatory genes, e.g. oncogenes, and viral RNA utilizing the human translation machinery. Therefore, we expect these inhibitors to exhibit anti-tumor or anti- viral activity. High Throughput Screening (HTS) allows the rapid analysis of thousands of compounds. Thus, it has become a key tool in modern drug discovery.
Our specific aims for this application are: to develop a functional assay based on the ATPase activity of eIF4A;to develop a secondary screen based on a binding assay and to evaluate the assays and perform a pilot screen. The primary screen for HTS will be based on the ATPase activity of eIF4A. We will utilize known assays for the detection of the ATP hydrolysis products ADP and inorganic phosphate and optimize them for the use in high throughput screening. In a secondary screen false positives will be eliminated and the activity of identified hits specified. We will develop an assay suitable for use as a secondary screen based on directly monitoring the binding between eIF4G/eIF4A/RNA using a fluorescence polarization (FP) or scintillation proximity assays (SPA). Possible candidates for drug development and their interaction with eIF4A and eIF4G will be characterized using NMR.
The biosynthesis of proteins depends on complex machinery including the ribosome and numerous protein factors. The identification of the small molecule inhibitors proposed in this study will specifically modulate the synthesis of oncoproteins and protein from RNA viruses. The proposed study will allow us to obtain leads for anti-cancer and anti-viral drug design.
