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  • EGTA In conclusion we designed novel

    2020-07-02

    In conclusion, we designed novel E1 inhibitors based on the three-dimensional structure of E1 in complex with ubiquitin. Following an enzymatic assay evaluating synthetic compounds , , and , we identified compound as a novel E1 inhibitor. Comparing the inhibitory activity of compound with and , we found that the benzoyl group in compound is important for E1 inhibition. A competition assay using additional ubiquitin suggested that E1 inhibition by compound is reversible. In a cellular assay, the reversible E1 inhibitor caused an accumulation of p53 and inhibited the growth of MCF-7 cells. We believe that the reversible E1 inhibitor presented here will provide the basis for new tools to investigate the biology of E1 and uncover new strategies for cancer treatment.
    Introduction Glucocorticoids are steroid hormones exhibiting effects to the immune system, wound healing, metabolism such as gluconeogenesis and lipolysis, fetal development, and CNS maturation in the human body [1]. Circulating cortisol, the predominant glucocorticoid produced by the human adrenal glands, is regulated by the hypothalamic–pituitary–adrenal (HPA) feedback system. But intracellular levels of glucocorticoid concentrations are mediated by 11β-hydroxysteroid dehydrogenases (11β-HSDs). 11β-HSDs are enzymes involved in the bidirectional conversion of cellular glucocorticoids (Fig. 1). While 11β-HSD type 1 (11β-HSD1) is a ubiquitous NADPH-dependent oxoreductase in vivo and regenerates glucocorticoid receptor-active cortisol from cortisone, 11β-HSD type 2 (11β-HSD2) is a NAD-specific dehydrogenase which inactivates cortisol to receptor-inactive cortisone, thereby providing mineralocorticoid specificity of EGTA in aldosterone target tissues. It has significant expression in mineralocorticoid target tissues including the distal nephron, colon and sweat and salivary glands. 11β-HSD1, as a predominant 11β-reductase in most intact cells, has its highest expression in the liver, adipose tissue and skeletal muscle [2]. First identified in 1953, Amelung and colleagues found conversion of cortisone to cortisol in rat homogenates of various tissues in vitro [3], revealing the enzyme in charge of conversion was 11β-HSD1. Although being bidirectional in in vitro tissue homogenates and in purified form from mouse liver [4], 11β-HSD1 acts as a predominant oxoreductase in vivo and in most intact cells, thereby functionally amplifying glucocorticoid action [5,6]. 11β-HSD1 belongs to the superfamily of the short-chain dehydrogenases/reductases (SDR) [7] and, interestingly enough, has been shown to participate as a carbonyl reducing enzyme in the phase I biotransformation system of pharmacologically and toxicologically relevant xenobiotics [8,9]. The enzyme is membrane-bound and was successfully purified in an active state from human liver [10]. 11β-HSD1 is dependent on NADP(H), located to the inner leaflet of the endoplasmic reticulum (ER) and interacts with hexose-6-phosphate dehydrogenase (H6PDH) which generates a favorable NADPH/NADP+ ratio in the ER lumen for 11β-HSD1 reductase activity [[11], [12], [13]]. The enzyme is widely expressed with its highest levels in liver [14,15], but also occurs in adipose tissue [16], vasculature [17], ovary [18], testis [15,19], brain [14,20,21], uterus [22], immune and inflammatory cells [23], skeletal muscle [24] and heart [25]. 11β-HSD1 regulation is known to be highly tissue-specific and driven by two distinct promoters [26]. Physiologically, 11β-HSD1 seems to play important roles in the development of the metabolic syndrome as well as at the interface of inflammation and obesity [27].
    The role of 11β-HSD1 in the metabolic syndrome Based on a series of findings, dysregulation of cortisol action has been presumed as a central feature of the metabolic syndrome [28]. But there are conflicting observations regarding the link between 11β-HSD1 expression and activity with the development of the metabolic syndrome in humans (Fig. 2). The metabolic syndrome is defined as a cluster of metabolic disorders, including hypertension, glucose intolerance, dyslipidemia and obesity [29,30]. To test the hypothesis that 11β-HSD1 contributes to obesity and its metabolic complications via its cortisol regenerating activity, a number of respective studies have been performed in animal models and in humans.