Abstract
JAK1 depletion or downregulation was previously reported to account for coronavirus inhibition. Here, we found that AG1024, an IR (insulin receptor) and IGF-1R (insulin-like growth factor 1 receptor) inhibitor, diminishes JAK1 protein levels and exerts anti-coronaviral activities with EC50 values of 5.2 ± 0.3 μM against transmissible gastroenteritis coronavirus (TGEV) and 4.3 ± 0.3 μM against human flu coronavirus OC43. However, although the IR and IGF-1R signaling pathways are activated by insulin or IGF-1 in swine testis cells, they are not triggered upon TGEV infection. AG1024, therefore, inhibits coronaviral replication and downregulates JAK1 protein levels independently of IR and IGF-1R. Moreover, JAK1 proteolysis caused by AG1024 was found through activation of upstream Ndfip1/2 and its effector NEDD4-like E3 ligase Itch. In addition, ouabain, which was reported to mediate JAK1 proteolysis causing anti-coronaviral activity by activation of Ndfip1/2 and NEDD4 E3 ligase, additively inhibited anti-coronaviral activity and JAK1 diminishment in combination with AG1024. This study provides novel insights into the pharmacological effects of AG1024 and Itch E3 ligase mediated JAK1 proteolysis and identified Ndfip1/2 as a cognate effector for JAK1 proteolysis via the diversified E3 ligases NEDD4 and NEDD4-like Itch. These findings are expected to provide valued information for the future development of anti-viral agents.
Highlights
The Janus kinase (JAK) family comprises four tyrosine kinases, TYK2, Janus kinase 1 (JAK1), JAK2, and JAK3, which share significant structural homology and initiate signaling transduction by associating with cytokine and hormone receptors [1,2]
During our previous screening of inhibitors for the signaling pathways involved the anti-coronaviral activity of ouabain [8,23], we discovered that the cardenolide ouabain suppressed coronaviral replication via augmenting a Na+ /K+ -ATPase-dependent PI3K_PDK1 axis signaling, and that this effect could be antagonized by LY294002, a PI3K inhibitor [23]
We discovered that ouabain suppressed coronaviral replication by downregulating JAK1 mediated by Ndfip1/2 and NEDD4 E3 ligase via Na+ /K+ -ATPaseindependent proteolysis and that tyrphostin AG1024 but not picropodophyllin (PPP) significantly inhibited transmissible gastroenteritis coronavirus (TGEV) replication by ~84% at a concentration of 10 μM, and enhanced the anti-TGEV activity of ouabain (Table 1)
Summary
The Janus kinase (JAK) family comprises four tyrosine kinases, TYK2, JAK1, JAK2, and JAK3, which share significant structural homology and initiate signaling transduction by associating with cytokine and hormone receptors [1,2]. JAK1 degradation is associated with the regulation of its coupled biological functions, but only a few ubiquitination proteases and phosphatases, as well as the Ndfip1/2 activated proteolysis ligase NEDD4 (Neural Precursor Cell Expressed Developmentally DownRegulated Protein 4; NEDD4 E3 Ubiquitin Protein Ligase), are involved in JAK1 degradation [5,6,7]. We discovered that JAK1 depletion or downregulation can account for coronavirus inhibition and that cardenolides down-regulate JAK1 in a Na+ /K+ -ATPaseindependent manner by activation of NEDD4 family-interacting protein 1/2 (Ndfip1/2). Activation of Ndfip1/2 by other means may constitute a novel route for JAK1 down-regulation. 4.0/).
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