Inhibition of Eukaryotic Ribosomal Function by the Sesquiterpenoid Antibiotic Fusarenon‐X

1 Detailed studies have been carried out on the sesquiterpenoid antibiotic fusarenon‐X to determine its precise mode of action as an inhibitor of eukaryotic protein synthesis. 2 In reticulocyte cell‐free systems low concentrations of fusarenon‐X (2 μg/ml) cause extensive ‘run off’ of polyribosomes and parallel studies on time courses of protein synthesis indicate that the drug inhibits initiation. As the concentration of fusarenon‐X is increased, progressively less ‘run off’ is observed but this effect is coupled with a progressive rise in the level of inhibition of protein synthesis. Inhibition becomes complete at fusarenon‐X concentrations approaching 100 μg/ml and under these conditions polyribosomes are stabilized. 3 The effects of the drug were studied on the puromycin‐induced release of nascent peptides from sucrose‐washed polyribosomes, an assay for peptidyl transferase activity. Fusarenon‐X did not inhibit the reaction when present at a concentration of 8 μg/ml but did so progressively as the drug concentration was increased above this level. 4 An assay system has been developed whereby initiation complexes, involving ribosomes, [ 35 S]Met‐tRNA f and poly(A,U,G), can be formed in reticulocyte lysates. This system is sensitive to low levels of puromycin (10 μg/ml) and can be used to assay the formation of the ‘first’ peptide bond in protein synthesis. All concentrations of fusarenon‐X tested inhibited the puromycin reaction in this system, the degree of inhibition increasing progressively with increasing concentrations of fusarenon‐X. 5 It is concluded that the ribosomal target site for fusarenon‐X is the peptidyl transferase catalytic centre but that the ability of the drug to bind to this centre on polyribosomes (carrying long nascent polypeptide chains) is critically dependent upon the concentration of fusarenon‐X employed.