Friday, September 14, 2018 - 10:30am
PhD Exam Seminar
ABSTRACT IRE1 and RNase L are a unique set of proteins found in the kinome, which possess both a kinase and an endoribonuclease domain. Although both IRE1 and RNase L share a similar structural domain architecture, they are involved in different cellular pathways. Cells endure cellular distress when their homeostasis is perturbed, which interferes with the proper folding and assembly of proteins in the endoplasmic reticulum (ER) resulting in the accumulation of misfolded proteins in the ER that further leads to ER stress. However, the unfolded protein response (UPR) system is able to detect and resolve ER stress, and further re-establish ER and cellular homeostasis through the activation of three protein-signaling pathways. In my Ph.D. studies I focused on investigating how small molecules can regulate the most ancient member of the ER stress transducers, IRE1. Cells also endure distress during viral infection, which leads to the accumulation of viral single stranded RNA in the cells, which are then translated into viral proteins through the host ribosomal machinery. However, the interferon induced antiviral pathways activate proteins that are involved in the degradation of viral RNA, inhibit viral protein synthesis and replication therefore lending to an antiviral state within the cell. RNase L is a highly regulated protein that is activated during viral infection and serves to help mount an innate immune response against viruses by cleaving viral and cellular ssRNA thereby leading to inhibition of viral replication and protein synthesis. In my Ph.D. studies I focused on investigating how small molecules can regulate RNase L ribonuclease activity. In the first chapter, I crystallized the structure of the IRE1 enzyme bound to three hydroxy-aryl-aldehyde (HAA) small molecule analogues that inhibit IRE1’s ribonuclease activity. These three structures depicted that the HAA analogues engaged a preformed pocked in the ribonuclease active site of IRE1 supported by an essential covalent yet reversible Schiff base interaction between the lysine 907 and the aldehyde moiety of the inhibitor. Further mutational studies, modeling, SAR, and enzymological characterization were performed in collaboration to provide insight and validate the HAA inhibitor mechanism of action. These structures are the first structural view of any inhibitor engaging the ribonuclease active site of IRE1, which has far reaching implications since ribonuclease active sites in general have made poor drug targets, with no other ribonuclease inhibitor of promising therapeutic value been described to date. In the second chapter, I discovered two potent polyphenol compounds from the OICR kinase library small molecule screen that selectively inhibit RNase L’s ribonuclease activity over IRE1. I performed further SAR and enzymological characterization to determine the potential binding mode and mechanism of action of these small molecules. In collaboration, further in cell studies were performed with these inhibitors that revealed that they exert their effects by specifically inhibiting the RNase L enzyme thereby providing a proof-of-principle concept that of RNase L can be a viable therapeutic target.
Dr. Frank Sicheri
Department of Molecular Genetics