ABSTRACT
Onconase is an amphibian protein that is now in Phase III clinical trials as a cancer chemotherapeutic. Human pancreatic ribonuclease (RNase 1) is homologous to Onconase but is not cytotoxic. Here, ERDD RNase 1, which is the L86E/N88R/G89D/R91D variant of RNase 1, is shown to have conformational stability and ribonucleolytic activity similar to that of the wild-type enzyme but > 10(3)-fold less affinity for the endogenous cytosolic ribonuclease inhibitor protein. Most significantly, ERDD RNase 1 is toxic to human leukemia cells. The addition of a non-native disulfide bond to ERDD RNase 1 not only increases the conformational stability of the enzyme but also increases its cytotoxicity such that its IC(50) value is only 8-fold greater than that of Onconase. Thus, only a few amino acid substitutions are necessary to make a human protein toxic to human cancer cells. This finding has significant implications for human cancer chemotherapy.
Subject(s)
Antineoplastic Agents/pharmacology , Ribonuclease, Pancreatic/chemistry , Ribonuclease, Pancreatic/pharmacology , Ribonuclease, Pancreatic/toxicity , Ribonucleases/pharmacology , Amino Acid Sequence , Amino Acids/chemistry , Cell Division , Cysteine/chemistry , DNA, Complementary/metabolism , Disulfides , Dose-Response Relationship, Drug , Electrophoresis, Agar Gel , Humans , Inhibitory Concentration 50 , K562 Cells , Kinetics , Leukemia/drug therapy , Models, Biological , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Conformation , Ribonuclease, Pancreatic/metabolism , Spectrometry, Fluorescence , Temperature , Tumor Cells, CulturedABSTRACT
Onconase((R)) (ONC) is a homolog of ribonuclease A (RNase A) that has unusually high conformational stability and is toxic to human cancer cells in vitro and in vivo. ONC and its amphibian homologs have a C-terminal disulfide bond, which is absent in RNase A. Replacing this cystine with a pair of alanine residues greatly decreases the conformational stability of ONC. In addition, the C87A/C104A variant is 10-fold less toxic to human leukemia cells. These data indicate that the synapomorphic disulfide bond of ONC is an important determinant of its cytotoxicity.
