Background: JAK JH2s (pseudokinase domains) mediate important regulatory functions; it is unclear whether TYK2 JH2 binds ATP and possesses enzymatic activity. Results: TYK2 JH2 binds ATP, but is catalytically inactive; ATP stabilizes JH2 and modulates TYK2 activity. Conclusion: ATP binding to JH2 is functionally important; the rigid activation loop probably hinders substrate phosphorylation. Significance: The TYK2 JH2 domain can be targeted with ATP-competitive compounds for therapeutics.

Alzheimer's disease (AD) is the most common form of dementia in the elderly and its prevalence is set to increase rapidly in coming decades. However, there are as yet no available drugs that can halt or even stabilize disease progression. One of the main pathological features of AD is the presence in the brain of senile plaques mainly composed of aggregated β amyloid (Aβ), a derivative of the longer amyloid precursor protein (APP). The amyloid hypothesis proposes that the accumulation of Aβ within neural tissue is the initial event that triggers the disease. Here we review research efforts that have attempted to inhibit the generation of the Aβ peptide through modulation of the activity of the proteolytic secretases that act on APP and discuss whether this is a viable therapeutic strategy for treating AD.

A large number of natural products have been advocated as anticancer agents. Many of these compounds contain functional groups characterized by chemical reactivity. It is not clear whether distinct mechanisms of action can be attributed to such compounds. We used a chemical library screening approach to demonstrate that a substantial fraction (~20%) of cytotoxic synthetic compounds containing Michael acceptor groups inhibit proteasome substrate processing and induce a cellular response characteristic of proteasome inhibition. Biochemical and structural analyses showed binding to and inhibition of proteasome-associated cysteine deubiquitinases, in particular ubiquitin specific peptidase 14 (USP14). The results suggested that compounds bind to a crevice close to the USP14 active site with modest affinity, followed by covalent binding. A subset of compounds was identified where cell death induction was closely associated with proteasome inhibition and that showed significant antineoplastic activity in a zebrafish embryo model. These findings suggest that proteasome inhibition is a relatively common mode of action by cytotoxic compounds containing Michael acceptor groups and help to explain previous reports on the antineoplastic effects of natural products containing such functional groups.

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The non-genotoxic nature of proteasome inhibition makes it an attractive therapeutic option for the treatment of pediatric malignancies. We recently described the small molecule VLX1570 as an inhibitor of proteasome deubiquitinase (DUB) activity that induces proteotoxic stress and apoptosis in cancer cells. Here we show that acute lymphoblastic leukemia (ALL) cells are highly sensitive to treatment with VLX1570, resulting in the accumulation of polyubiquitinated proteasome substrates and loss of cell viability. VLX1570 treatment increased the levels of a number of proteins, including the chaperone HSP70B’, the oxidative stress marker heme oxygenase-1 (HO-1) and the cell cycle regulator p21Cip1. Unexpectedly, polyubiquitin accumulation was found to be uncoupled from ER stress in ALL cells. Thus, increased phosphorylation of eIF2α occurred only at supra-pharmacological VLX1570 concentrations and did not correlate with polyubiquitin accumulation. Total cellular protein synthesis was found to decrease in the absence of eIF2α phosphorylation. Furthermore, ISRIB (Integrated Stress Response inhibitor) did not overcome the inhibition of protein synthesis. We finally show that VLX1570 can be combined with L-asparaginase for additive or synergistic antiproliferative effects on ALL cells. We conclude that ALL cells are highly sensitive to the proteasome DUB inhibitor VLX1570 suggesting a novel therapeutic option for this disease.