Contribution of chain termini to the conformational stability and biological activity of onconase

Onconase, a member of the RNase A superfamily, is a potent antitumor agent which is undergoing phase III clinical trials as an antitumor drug. We have recently shown that onconase is an unusually stable protein. Furthermore, the protein is resistant to the action of proteases, which could influence its use as a drug, prolonging its biological life, and leading to its renal toxicity. Our investigation focused on the contribution of chain termini t o onconase conformational stability and biological activities. We used diff erential scanning calorimetry, isothermal unfolding experiments, limited pr oteolysis, and catalytic and antitumor activity determinations to investiga te the effect of the elimination of the two blocks at the chain termini, th e N-terminal cyclized glutamine and the C-terminal disulfide bridge between the terminal Cys104 and Cys87. The determination of the thermodynamic para meters of the protein led to the conclusion that the two blocks at onconase chain termini are responsible for the unusual stability of the protein. Mo reover, the reduced stability of the onconase mutants does not influence gr eatly their catalytic and antitumor activities. Thus, our data would sugges t that an onconase-based drug, with a decreased toxicity, could be obtained through the use of less stable onconase variants.