Further investigation into FCR is warranted to promote evidence-based care for this significant cancer survivorship concern.

Endoplasmic reticulum (ER) stress activates the unfolded protein response and its dysfunction is linked to multiple diseases. The stress transducer IRE1α is a transmembrane kinase endoribonuclease (RNase) that cleaves mRNA substrates to re-establish ER homeostasis. Aromatic ring systems containing hydroxy-aldehyde moieties, termed hydroxy aryl aldehydes (HAA), selectively inhibit IRE1α RNase and thus represent a novel chemical series for therapeutic development. We solved crystal structures of murine IRE1α in complex with three HAA inhibitors. HAA inhibitors engage a shallow pocket at the RNase active site through pi-stacking interactions with His910 and Phe889, an essential Schiff base with Lys907 and a H-bond with Tyr892. Structure activity studies and mutational analysis of contact residues define the optimal chemical space of inhibitors and validate the inhibitor binding site. These studies lay the foundation for understanding both the biochemical and cellular functions of IRE1α using small molecule inhibitors and suggest new avenues for inhibitor design.

Summary RNase L is an ankyrin repeat domain containing dual endoribonuclease-pseudokinase that is activated by unusual 2′,5′-oligoadenylate (2-5A) second messengers and which impedes viral infections in higher vertebrates. Despite its importance in interferon regulated antiviral innate immunity, relatively little is known about its precise mechanism of action. Here, we present a functional characterization of 2.5 Å and 3.25 Å X-ray crystal and small angle x-ray scattering structures of RNase L bound to a natural 2-5A activator with and without ADP or the non-hydrolysable ATP mimetic AMP-PNP. These studies reveal how recognition of 2-5A through interactions with the ankyrin repeat domain and the pseudokinase domain together with nucleotide binding, impose a rigid intertwined dimer configuration that is essential for RNase catalytic and anti-viral functions. The involvement of the pseudokinase domain of RNase L in 2-5A sensing, nucleotide binding, dimerization, and ribonuclease functions highlights the evolutionary adaptability of the eukaryotic protein kinase fold.