title: Disruption of shape-complementarity markers to create cytotoxic variants of ribonuclease A.
abstract: Onconase (ONC), an amphibian member of the bovine pancreatic ribonuclease A (RNase A) superfamily, is in phase III clinical trials as a treatment for malignant mesothelioma. RNase A is a far more efficient catalyst of RNA cleavage than ONC but is not cytotoxic. The innate ability of ONC to evade the cytosolic ribonuclease inhibitor protein (RI) is likely to be a primary reason for its cytotoxicity. In contrast, the non-covalent interaction between RNase A and RI is one of the strongest known, with the RI.RNase A complex having a K(d) value in the femtomolar range. Here, we report on the use of the fast atomic density evaluation (FADE) algorithm to identify regions in the molecular interface of the RI.RNase A complex that exhibit a high degree of geometric complementarity. Guided by these "knobs" and "holes", we designed variants of RNase A that evade RI. The D38R/R39D/N67R/G88R substitution increased the K(d) value of the pRI.RNase A complex by 20 x 10(6)-fold (to 1.4 microM) with little change to catalytic activity or conformational stability. This and two related variants of RNase A were more toxic to human cancer cells than was ONC. Notably, these cytotoxic variants exerted their toxic activity on cancer cells selectively, and more selectively than did ONC. Substitutions that further diminish affinity for RI (which has a cytosolic concentration of 4 microM) are unlikely to produce a substantial increase in cytotoxic activity. These results demonstrate the utility of the FADE algorithm in the examination of protein-protein interfaces and represent a landmark towards the goal of developing chemotherapeutics based on mammalian ribonucleases.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Rutkoski
authors.forename: Erin L
authors.initials: EL
authors.lastname: Kurten
authors.forename: Julie C
authors.initials: JC
authors.lastname: Mitchell
authors.forename: Ronald T
authors.initials: RT
authors.lastname: Raines
mesh_terms: D000465:Algorithms; D019943:Amino Acid Substitution; D000818:Animals; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects*; D019295:Computational Biology; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity*
chemical_list: D012259:Ribonuclease, Pancreatic