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  • The potential off target activity of against


    The potential off-target activity of against other ATP-dependent enzymes, such as kinases, was also investigated. Encouragingly, there was no significant inhibition of ATP binding to 97 human kinases, when was evaluated at 10μM within a DiscoveRx scanEDGE® kinome screen (, ). All together, these data represent, to the best of our knowledge, the first selective UBA5 inhibitor developed to date, and support the noncompetitive nature by which mediates its activity. Finally, the activity of was evaluated within a cellular environment by measuring the proliferation of leukotriene receptor antagonists in which high UBA5 protein expression levels were detected ( and ). Previous work demonstrated that knock-down of UFM1 protein labeling machinery increased pancreatic cell susceptibility to apoptosis under ER stress, and genetic silencing of UBA5 led to decreased breast cancer proliferation. In line with these observations, suppressed the proliferation of Sk-Luci6 cancer cells that express high levels of UBA5 (at concentrations above 50μM) while no effect was observed on A549 cancer cells or MRC9 lung fibroblasts (up to 200μM of ), which express significantly lower levels of UBA5 ( and ). The detailed intracellular mechanism by which elicits its anti-proliferative effect is currently being determined. In conclusion, we have developed a selective UBA5 inhibitor using a structure-based design approach. Inhibitor incorporates an adenosine scaffold appended to a zinc(II)cyclen complex, and exhibits single digit micromolar activity against UBA5. Notably, is selective for UBA5 over other E1 enzymes as well as a panel of 97 human kinases. In culture, demonstrated selective anti-proliferative effects on cells expressing higher levels of UBA5. Compound might serve as a useful biological probe for selective inhibition of intracellular UBA5-mediated UFMylation, and may be used as a valuable tool for future Ub/Ubl research. Acknowledgements This work was supported by grants from the Canadian Cancer Society Research Innovation Grant 701486 to P.T.G., the National Institutes of Health Grant R01 GM081776 to H.L. and the Natural Sciences and Engineering Research Council of Canada to S.R.D. The authors thank Dr. Leda Raptis from Queen’s University (Kingston, ON, Canada) for her generous donation of all cell lines used for this manuscript. The authors also acknowledge DiscoveRx for the kinome screen. Finally, the authors wish to acknowledge Dr. Sirano Dhe-Paganon for his support and guidance in the writing of this manuscript.
    Introduction Bone remodeling is the dynamic process to maintain the integrity of skeletal system in which old/damaged bone tissues are resorbed by osteoclasts and new bone tissues are synthesized by osteoblasts (Raggatt & Partridge, 2010). An imbalance between bone formation and bone resorption activities leads to skeletal abnormalities such as osteoporosis (Lerner, 2004). Osteoporosis is a skeletal disorder featured with loss of bone mass, strength, degradation of bone micro-architecture and elevated risk of fractures (Kanis, 1994). Most drugs/therapies to treat osteoporosis are antiresorptive agents that target at osteoclasts. Current researches provide further molecular insights into the communication between osteoblasts and osteoclasts, and the orchestrating signaling network, which offers novel molecular targets against osteoporosis (Rachner, Khosla, & Hofbauer, 2011). Phosvitin (PV) is a highly phosphorylated protein from egg yolk (Byrne et al., 1984). Phosvitin and its derived phosvitin hydrolysate (PVH) were reported to exert various physiological activities such as anti-inflammatory activity (Hu et al., 2013, Xu et al., 2012, Young et al., 2011), antioxidant activities (Katayama et al., 2006, Xu et al., 2007) and antimicrobial activities (Ma et al., 2013, Wang et al., 2011). Previously, phosvitin was applied to induce biomineralization in dental study (Ito et al., 2011, Onuma, 2005). Phosvitin hydrolysate in animal diet was reported to increase calcium absorption, calcium to ash ratios, bone mineral density and bone mineral content in femurs and tibias of Sprague Dawley (SD) rats (Choi, Jung, Choi, Kim, & Ha, 2005; Zhong et al., 2016). In a study with mouse calvarial organ culture, phosvitin promoted bone formation activities by upregulating collagen synthesis, calcium deposition, and several biomarkers of bone formation (Liu et al., 2013). It was proposed that phosvitin mirrored the role of ascorbic acid in physiological conditions and the activity of phosvitin was related to its antioxidant activity or reducing ability (Li et al., 2014, Liu et al., 2013). This was confirmed by the facts that both ascorbic acid treated and phosvitin treated osteoblastic cells produced similar expression levels of osteogenic gene markers, collagen type I, osteocalcin (OCN), runt-related transcription factor 2 (RUNX2), and bone morphogenetic protein-2 (BMP-2; Liu, Li, Geng, Huang, & Ma, 2017). This hypothesis was further supported by other studies that oxidative stress and chronic inflammation were involved in development of osteoporosis by directly/indirectly regulating the balance between osteoclasts and osteoblasts (Manolagas & Parfitt, 2010); while functional foods with antioxidant/antiinflammation activities, like green tea, grapefruit pulp and citrus extract were reported to increase bone mass or prevent bone loss in vivo (Mandadi et al., 2009, Shen et al., 2009). Inflammation plays a critical role in many chronic diseases. It was reported that the elevation of inflammation status is related to bone loss in arthritis and osteoporosis (Di Benedetto, Gigante, Colucci, & Grano, 2013). Tumor necrosis factor alpha (TNF-α) is an inflammatory cytokine that was previously believed to inhibit osteoblast differentiation (Abbas et al., 2003, Gilbert et al., 2002). However, depending on the concentration, cell type and duration of treatment, TNF-α could also induce osteogenic differentiation by upregulating expression of gene markers for osteoblast differentiation (Huang et al., 2011, Lu et al., 2012). Osteoblasts are not only responsible for forming new bone tissue but also for expressing inflammatory chemokines including interleukin (IL)-8, growth-regulated oncogene-alpha (GRO-α), monocyte chemoattractant protein-1 (MCP-1), regulated on activation, normal T cell expressed and secreted (RANTES), macrophage inflammatory proteins-1 (MIP-1) alpha and MIP-1 beta under stimulation of inflammatory cytokines (Lisignoli et al., 2002, Zhu et al., 1994). These chemokines play an important role in bone remodeling by recruiting osteoclast progenitors and stimulating osteoclastogenesis (Graves, Jiang, & Valente, 1999).