Recent studies show that the duration and amplitude of UPR activation powerfully affects both cell function and fate7,8,29,30
. Indeed, many cell-degenerative diseases such as diabetes mellitus feature increased ER stress and UPR activation in affected cells3,31,32
. These same markers are evident in a wide range of solid and hematopoietic malignancies33
. To properly ascertain the role of the UPR in these disease contexts will require development of tool compounds that target critical nodes in the UPR in both positive and negative directions. The master UPR regulator IRE1α, which controls cell fate under ER stress, offers two enzymatic targets that could be modulated with small molecules. In this work, we exploited the unusual mechanistic relationship between these two catalytic domains to inhibit the RNase from a distance by inhibiting the kinase.
Starting with known pharmacophores that stabilize an inactive conformation in other protein kinases, we optimized a type II inhibitor lead to produce 3. Despite inhibiting IRE1α kinase autophosphorylation similarly to the type I inhibitor APY29, 3 inhibits XBP1 mRNA splicing, even during ER stress. Consistent with competition studies, footprinting experiments strongly suggest that 3 and APY29 bind to the same ATP-binding pocket. However, these same footprinting experiments indicate that these inhibitors cause divergent effects on the activation loop of IRE1α, and support a model in which 3 and APY29 promote distinct, mutually exclusive, movements of the DFG-motif contained within the activation loop.
The aforementioned experiments, combined with modeling studies, lead to a parsimonious model of IRE1α modulation by kinase inhibitors () that posits that the protein can adopt either a canonical DFG-in or a DFG-out conformation, as is seen with other kinases under the influence of types I and II inhibitors, respectively. However, while for other kinases these two distinct modes of inhibition stereotypically shut down kinase function, for the multi-domain kinase, IRE1α, the two inhibition modes have opposite and divergent results on the attached RNase activity. To our knowledge, this ability to modulate a second catalytic activity in a multi-domain kinase in two different directions with distinct classes of ATP-competitive inhibitors has not been reported to date. We expect that this ability may be extended to many of the other known multi-domain kinases.
Intriguingly, opposite effects on oligomeric state were found using the two compounds: while type I inhibitors increase the dimeric and possibly oligomeric state of IRE1α and the catalytic activity of the RNase, type II inhibitors decrease both in tandem. Given previous reports of a direct mechanistic relationship between the degree of order and RNase activity in IRE1 proteins16
, we speculate that the inactive conformation that 3
stabilizes in IRE1α promotes the monomeric state.
It is of course conceivable that a different, previously unidentified active site conformation is adopted in the presence of 3
; to fully resolve this particular point in the future will require atomic level co-crystal structures. Regardless, the particular kinase active site conformation stabilized by 3
has the unique and novel property of preserving the mechanistic coupling between the kinase and the RNase in IRE1α, allowing full inhibition of both activities in concert. We propose that this represents a new alternative to aldehyde-based covalent inhibitors of the RNase such as STF-083010 (or another recently reported compound called 4µ8C34
), which leave kinase autophosphorylation and oligomerization intact. In contrast to the action of direct RNase inhibitors, any biological signaling through the kinase that is dependent on phosphorylation of non-autonomous substrates or kinase-mediated scaffolding should be simultaneously quenched with type II kinase inhibitors.
In summary, the ability to now inhibit the effector RNase domain of IRE1α with type II kinase inhibitors complements our previous ability to activate the RNase with type I inhibitors, independent of upstream ER stress, establishing opposite directions of control over this master UPR regulator. Thus, type II kinase inhibitors of IRE1α will expand on a toolkit that includes chemical-genetic systems to test and validate the UPR’s role in ER stress-related diseases. While 3 is not completely selective for IRE1α over other protein kinases, this compound serves as a starting point for the generation of more potent and selective inhibitors that may eventually be developed into disease-modifying drugs for ER stress-related disorders. Moreover, the ability to toggle the IRE1α RNase on and off through its kinase domain may serve as a precedent for pharmacologically targeting the many other kinase-coupled enzymes present in eukaryotes.