Interleukin-10 and Small Molecule SHIP1 Allosteric Regulators Trigger Anti-Inflammatory Effects Through SHIP1/STAT3 Complexes

Abstract

28The anti-inflammatory actions of interleukin-10 (IL10) are thought to be mediated primarily by the STAT3 29 transcription factor, but pro-inflammatory cytokines such as interleukin-6 (IL6) also act through STAT3. 30We now report that IL10, but not IL6 signaling, induces formation of a complex between STAT3 and the 31 inositol polyphosphate-5-phosphatase SHIP1 in macrophages. Both SHIP1 and STAT3 translocate to the 32 nucleus in macrophages. Remarkably, sesquiterpenes of the Pelorol family we previously described as 33 allosteric activators of SHIP1 phosphatase activity, could induce SHIP1/STAT3 complex formation in cells, 34 and mimic the anti-inflammatory action of IL10 in a mouse model of colitis. Using crystallography and 35 docking studies we identified a drug-binding pocket in SHIP1. Our studies reveal new mechanisms of 36 action for both STAT3 and SHIP1, and provide a rationale for use of allosteric SHIP1-activating compounds 37 which mimic the beneficial anti-inflammatory actions of IL10. 38 39 1999) much like an IL10 -/mouse (Kuhn et al., 1993, Zigmond et al., 2014. However, STAT3 becomes 63 tyrosine phosphorylated and activated by many stimuli including the pro-inflammatory cytokine 64 interleukin-6 (IL6) (Garbers et al., 2015), so STAT3 activation must differ downstream of IL10 and IL6 65 signaling in order to mediate their opposing actions. 66The SHIP1 phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase is a cytoplasmic protein expressed 67 predominantly in hematopoietic cells (Hibbs et al., 2018, Fernandes, 2013 #1400, Huber et al., 1999 68 Krystal, 2000, Pauls and Marshall, 2017). In response to extracellular signals, SHIP1 can be recruited to 69 the cell membrane and one of its actions can be to turn off phosphoinositide 3-kinase (PI3K) signaling 70 (Brown et al., 2010) by dephosphorylating the PI3K product PIP3 into PI(3,4)P2 (Fernandes et al., 2013, 71 Huber et al., 1999, Krystal, 2000, Pauls and Marshall, 2017. We have shown that SHIP1 phosphatase 72 activity is allosterically activated by its product PI(3,4)P2 and that small molecules of the pelorol family 73 (ZPR-MN100 and ZPR-151) also allosterically enhance SHIP1 phosphatase activity (Meimetis et al., 2012, 74 Ong et al., 2007. These data suggest that stimulating SHIP1 phosphatase activity with small molecule 75 SHIP1 activators could be used to treat inflammatory diseases caused by inappropriately sustained PI3K 76 production of PI(3,4)P2. 77However, in addition to its enzymatic function in hydrolyzing PIP3, SHIP1 can also act as a docking protein 78 for assembly of signaling complexes (Pauls and Marshall, 2017). We previously showed that IL10R 79 signaling requires SHIP1 to inhibit TNFα translation (Chan et al., 2012) but whether SHIP1 and STAT3 80 worked independently or together was not determined. We now report that a SHIP1 protein containing 81 point mutations, which inactivates its phosphatase activity, could still mediate the anti-inflammatory 82 action of IL10, and that SHIP1 and STAT3 associate with each other in respo...

Synthetic Studies on the Indane SHIP1 Agonist AQX-1125.

“…Recently Mui and co-workers demonstrated that AQX-1125 is ineffective in treating inflammation in IL-10 knockout mice, while more potent SHIP1 agonists are effective in this model. 31 This report also showed that AQX-1125 only binds to SHIP1 weakly, with SHIP1 perhaps not being the primary cellular target of the molecule. These results may explain the lack of activity of AQX-1125 on OPM2 cells.…”

Targeting SHIP1 and SHIP2 in Cancer

“…Mui and co-workers have reported the identification of the site on the C2 domain of SHIP1 where PI(3,4)P 2 and SHIP1 agonists bind to accelerate the phosphatase reaction [ 148 ]. The identification of this site should accelerate the development of SHIP1 agonists, which also may exhibit antitumor properties [ 145 ].…”

“…One of the drawbacks of the terpenoid scaffold was the generally poor water solubility of the pelorol analogs. This was addressed by adding basic nitrogens to the scaffold with the synthesis of 16 and 17, which significantly increased water solubility and oral bioavailability [ 148 , 178 , 180 ]. AQX-435 was recently shown to significantly reduce tumor volume in murine models using a panel of mice bearing TMD8 or DLBCL PDX tumors [ 180 ].…”

“…Unfortunately, AQX-1125 did not show significant efficacy to continue development as a treatment for interstitial cystitis (IC) and bladder pain syndrome (BPS), and its development has been discontinued. More recent studies have questioned whether AQX-1125 actually binds to SHIP1, indicating the molecule may be acting through other mechanisms [ 148 ].…”

“…A summary of the most utilized SHIP1 agonists is shown below in Table 2 . The most well developed and characterized SHIP1 agonist has been AQX-1125 18, but the recent disclosure that AQX-1125 only weakly binds to SHIP1 [ 148 ] will likely result in a reinvigoration of research in this field, as the poor clinical results of AQX-1125 may be explained by the lack of binding. Further studies on the pelorol agonists ZPR-151 16 and AQX-435 17 would seem to be a logical starting point for this area.…”