Translational Initiation Factor 2 Inhibitors Development of a High-Throughput Scintillation Proximity Assay for the Identification of C-Domain

ABSTRACTTranslational initiation factor 2 (IF2) is the largest of the 3 factors required for translation initiation in prokaryotes andhas been shown to be essential in Escherichia coli . It stimulates the binding of fMet-tRNA fMet to the 30S ribosomal sub-unit in the presence of GTP. The selectivity is achieved through specific recognition of the tRNA fMet blocked -aminogroup.IF2iscomposedof3structuraldomains:N-domain,whosefunctionisnotknown;G-domain,whichcontainstheGTP/GDP binding site and the GTPase catalytic center; and C-domain, which recognizes and binds fMet-tRNA fMet .Itsactivity is strictly bacteria specific and highly conserved among prokaryotes. So far, antibiotics targeting IF2 functionarenotknown,andthismakesitanidealtargetfornewdrugswithmechanismsofresistancenotyetdeveloped.Afewas-says have been developed in the past, which allow the detection of IF2 activity either directly or indirectly. In both in-stances,theassaysarebasedonradioactivedetectionanddonotallowforhighthroughputbecauseoftheneedforsepa-ration or solvent extraction steps. The authors describe a novel biochemical assay for IF2 that exploits the molecularrecognitionoffMet-tRNA