Ricin-Mechanism, Transition State and Inhibitor Design

DESCRIPTION (provided by applicant): Ricin immunoconjugates are in clinical trials as anticancer agents. However their use leads to vascular leak syndrome, an unacceptable side effect. It is proposed to avoid this side effect by the design and production of powerful inhibitors that bind tightly to unwanted ricin, inactive it and prevent it from damaging normal tissue. The transition state structure of ricin A-chain is known and is being used to design powerful transition state analogue inhibitors. Mimics of the transition state will be chemically synthesized and made into stem-loop RNA or RNA/DNA hybrids containing mimics of the ricin A- chain transition state. In addition to the transition state structure, inhibitor design will be guided by x-ray crystallography and NMR. Covalently closed circular RNA, DNA and RNA/DNA hybrids containing chemically stable elements of transition state features are being developed. A second goal of this research is to develop new methods to detect ricin catalytic activity. These detection methods are important for measuring the amount of ricin-linked immunochemotherapy agents in the blood of patients undergoing ricin therapy. It is the catalytic activity of ricin that is toxic to humans. Methods that detect ricin catalytic activity would also be useful to detect ricin in case it is used as a bioterrorism agent. The broader significance of this work is to provide new catalytic insights into enzymes that process RNA. Methods developed to solve the transition state structure of ricin will be used to investigate the transition state structures of tRNA deaminase (TadA) and mismatched double-stranded RNA adenylate deaminase (ADAR-1) and to design transition state analogues for these enzymes. In summary, the results of these studies are proposed to provide;1) sensitive methods for the detection of ricin activity, 2) powerful transition state analogue inhibitors of ricin with long biological lifetimes, 3) transition state structures and powerful transition state analogue inhibitors for TadA and ADAR-1. Crystallization and high-resolution NMR studies of inhibitors with the target proteins has potential for additional mechanistic insights into this developing area of nucleic acid enzymology. Ricin is a powerful toxin obtained from castor beans. It is being used in clinical trials for cancer therapy. When it becomes too active, it begins to damage normal tissue as well as the cancer. This research proposes a way to make an antidote to reduce damage to normal tissue. Ricin is also a bioterrorism threat. A similar antidote would be useful to prevent damage to populations exposed to ricin during a terrorism episode.