Structural and functional characterization of dual kinase-endoribonucleases, IRE1 and RNase L

IRE1 and RNase L are a unique set of protein kinases found in the human kinome. They are unique in that both possess a protein kinase domain fused to an endoribonuclease domain. Although IRE1 and RNase L share similar structural domain architectures, they are involved in regulating different cellular pathways. IRE1 is involved in the unfolded protein response (UPR) signaling pathway while RNase L is involved in the interferon antiviral response signaling pathway. In the first data chapter, I describe purified and crystallized IRE1 bound to three hydroxy-aryl-aldehyde (HAA) small molecule analogues that inhibit IRE1’s ribonuclease activity but not its protein kinase activity. The IRE1-HAA structures revealed that the HAA analogues engaged a preformed pocket in the ribonuclease active site of IRE1. Inhibitors binding to the pocket is 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 enzymology characterizations were performed to validate the binding mode and mechanism of action of the HAA inhibitors. These IRE1-HAA structures provided the first view of an inhibitor engaging the ribonuclease active site of IRE1. In the second data chapter, I discovered two potent polyphenol compounds that potently and selectively inhibited RNase L’s ribonuclease activity relative to its closest relative IRE1. I performed further SAR and enzymology characterization to gain insight into the binding mode and mechanism of action of these small molecules. Additionally, in collaboration with the Silverman and Weiss labs, we performed in cell studies that demonstrated that our newly discovered inhibitors are functional against RNase L enzyme and superior to the only other RNase L inhibitor characterized to date, namely sunitinib. These polyphenol compounds represent the first small molecules to inhibit RNase L’s ribonuclease activity by binding specifically to the enzyme- RNA substrate complex.Ph.D.2019-11-19 00:00:0