Targeting noncatalytic cysteine residues with irreversible acrylamide-based inhibitors is a powerful approach for enhancing pharmacological potency and selectivity. Nevertheless, concerns about off-target modification motivate the development of reversible cysteine-targeting strategies. Here we show that electron-deficient olefins, including acrylamides, can be tuned to react with cysteine thiols in a rapidly reversible manner. Installation of a nitrile group increased the olefins’ intrinsic reactivity, yet paradoxically eliminated the formation of irreversible adducts. Incorporation of these electrophiles into a noncovalent kinase recognition scaffold produced slowly dissociating, covalent inhibitors of the p90 ribosomal protein S6 kinase, RSK. A cocrystal structure revealed specific noncovalent interactions that stabilize the complex by positioning the electrophilic carbon near the targeted cysteine. Disruption of these interactions by protein unfolding or proteolysis promoted instantaneous cleavage of the covalent bond. Our results establish a chemistry-based framework for engineering sustained covalent inhibition without accumulating permanently modified proteins and peptides.

Deficiencies in the protein folding capacity of the endoplasmic reticulum (ER) in all eucaryotic cells lead to ER stress and triggers the unfolded protein response (UPR)1–3. ER stress is sensed by Ire1, a transmembrane kinase/endoribonuclease, which initiates the non-conventional splicing of the mRNA encoding a key transcription activator, Hac1 in yeast or XBP-1 in metazoans. In the absence of ER stress, ribosomes are stalled on unspliced HAC1 mRNA. The translational control is imposed by a base pairing interaction between the HAC1 intron and the HAC1 5′ untranslated region (5′UTR)4. After excision of the intron, tRNA ligase joins the severed exons5,6, lifting the translational block and allowing synthesis of Hac1 from the spliced HAC1 mRNA to ensue4. Hac1 in turn drives the UPR gene expression program comprising 7–8% of the yeast genome7 to counteract ER stress. We show here that upon activation, Ire1 molecules cluster in the ER membrane into discrete foci of higher-order oligomers, to which unspliced HAC1 mRNA is recruited by means of a conserved bipartite targeting element contained in the 3′ untranslated region (3′UTR). Disruption of either Ire1 clustering or of HAC1 mRNA recruitment impairs UPR signaling. The HAC1 3′UTR element is sufficient to target other mRNAs to Ire1 foci, as long as their translation is repressed. Translational repression afforded by the intron fulfills this requirement for HAC1 mRNA. Recruitment of mRNA to signaling centers provides a new paradigm for the control of eukaryotic gene expression.

Background Diabetes mellitus (DM) is a major risk factor for cardiovascular mortality by increasing endothelial cell (EC) dysfunction and subsequently accelerating atherosclerosis. Extracellular-signal regulated kinase 5 (ERK5) is activated by steady laminar flow and regulates EC function by increasing eNOS expression and inhibiting EC inflammation. However, the role and regulatory mechanisms of ERK5 in EC dysfunction and atherosclerosis are poorly understood. Here, we report the critical role of the p90 ribosomal S6 kinase (p90RSK)/ERK5 complex in EC dysfunction in DM and atherosclerosis. Methods and Results Inducible EC-specific ERK5 knockout (ERK5-EKO) mice showed increased leukocyte rolling and impaired vessel reactivity. To examine the role of endothelial ERK5 in atherosclerosis, we used inducible ERK5-EKO-LDLR−/− mice and observed increased plaque formation. When activated, p90RSK associated with ERK5, and this association inhibited ERK5 transcriptional activity and up-regulated VCAM-1 expression. In addition, p90RSK directly phosphorylated ERK5 S496 and reduced eNOS expression. p90RSK activity was increased in diabetic mouse vessels, and FMK-MEA, a specific p90RSK inhibitor, ameliorated EC-leukocyte recruitment and diminished vascular reactivity in DM mice. Interestingly, in ERK-EKO mice, increased leukocyte rolling and impaired vessel reactivity were resistant to the beneficial effects of FMK-MEA, suggesting a critical role for endothelial ERK5 in mediating the salutary effects of FMK-MEA on endothelial dysfunction. FMK-MEA also inhibited atherosclerosis formation in ApoE−/− mice. Conclusions Our study highlights the importance of the p90RSK/ERK5 module as a critical mediator of EC dysfunction in DM and atherosclerosis formation, thus revealing a potential new target for therapeutic intervention.

Fragment-based ligand design and covalent targeting of noncatalytic cysteines have been employed to develop potent and selective kinase inhibitors. Here, we combine these approaches, starting with a panel of low-molecular weight, heteroaryl-susbstituted cyanoacrylamides, which we have previously shown to form reversible covalent bonds with cysteine thiols. Using this strategy, we identified electrophilic fragments with sufficient ligand efficiency and selectivity to serve as starting points for the first reported inhibitors of the MSK1 C-terminal kinase domain. Guided by x-ray co-crystal structures, indazole fragment 1 was elaborated to afford 12 (RMM-46), a reversible covalent inhibitor that exhibits high ligand efficiency and selectivity for MSK/RSK-family kinases. At nanomolar concentrations, 12 blocked activation of cellular MSK and RSK, as well as downstream phosphorylation of the critical transcription factor, CREB.