Inhibition of human and rat 11β-hydroxysteroid dehydrogenase type 1 by 18β-glycyrrhetinic acid derivatives

Abstract

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) plays an important role in regulating the cortisol availability to bind to corticosteroid receptors within specific tissue. Recent advances in understanding the molecular mechanisms of metabolic syndrome indicate that elevation of cortisol levels within specific tissues through the action of 11β-HSD1 could contribute to the pathogenesis of this disease. Therefore, selective inhibitors of 11β-HSD1 have been investigated as potential treatments for metabolic diseases, such as diabetes mellitus type 2 or obesity. Here we report the discovery and synthesis of some 18β-glycyrrhetinic acid (18β-GA) derivatives (2–5) and their inhibitory activities against rat hepatic11β-HSD1 and rat renal 11β-HSD2. Once the selectivity over the rat type 2 enzyme was established, these compounds’ ability to inhibit human 11β-HSD1 was also evaluated using both radioimmunoassay (RIA) and homogeneous time resolved fluorescence (HTRF) methods. The 11-modified 18β-GA derivatives 2 and 3 with apparent selectivity for rat 11β-HSD1 showed a high percentage inhibition for human microsomal 11β-HSD1 at 10 μM and exhibited IC₅₀ values of 400 and 1100 nM, respectively. The side chain modified 18β-GA derivatives 4 and 5, although showing selectivity for rat 11β-HSD1 inhibited human microsomal 11β-HSD1 with IC₅₀ values in the low micromolar range.

Introduction

Metabolic syndrome is a condition characterized by a cluster of metabolic disorders including insulin resistance, glucose intolerance, visceral obesity, hypertension and dyslipidemia, which are widely recognized as high risk factors for cardiovascular disease [1]. The rapidly increasing prevalence of the metabolic syndrome and obesity has focused the need for novel treatments, thus attracting intense interest from pharmaceutical research laboratories across academia and industry. Recent clinical evidence and animal genetic studies have indicated that glucocorticoid action in many aspects is associated with obesity and insulin resistance, the two underlying causes of metabolic syndrome [2], [3], [4]. It is well known that the glucocorticoid action on target tissues depends on both circulating hormone level and intracellular pre-receptor metabolism, which is mediated by 11β-hydroxysteroid dehydrogenases (11β-HSDs) [5].

11β-HSDs are members of the short-chain dehydrogenase/reductase (SDR) family and are microsomal enzymes catalysing the inter-conversion of active glucocorticoids and their 11-keto counterparts in specific tissues [6]. Currently, two isozymes of 11β-HSD have been characterized in human at the molecular level. The 11β-HSD1 isoform, highly expressed in liver, adipose tissue and central nervous system, converts cortisone to the active glucocorticoid cortisol and therefore locally amplifies glucocorticoid receptor activation [7]. The 11β-HSD1 knock out mouse resists stress-induced hyperglycaemia and has decreased cholesterol and triglyceride levels [8]. Transgenic mice over-expressing 11β-HSD1 selectively in adipose tissue exhibited insulin-resistant diabetes, hyperlipidemia and visceral obesity [9]. Clinical studies with a non-selective 11β-HSD inhibitor suggested that inhibition leads to improvement of insulin sensitivity in healthy individuals [10]. Therefore, the discovery of 11β-HSD1 inhibitors could provide potentially new approaches as a treatment for metabolic diseases, such as diabetes mellitus type 2 and obesity [11].

The 11β-HSD2 isoform is primarily found in mineralocorticoid target tissues such as the kidney and colon. The main function of 11β-HSD2 is inactivating physiological glucocorticoid cortisol to inactive cortisone in specific tissues, thereby preventing glucocorticoid occupation of mineralocorticoid receptor (MR) and allowing regulation of the receptor by aldosterone [12]. Impaired 11β-HSD2 activity leads to cortisol-induced MR activation with hypernatremia and hypokalemia causing hypertension [13], [14]. To avoid the possible side effects of inhibiting 11β-HSD, the selectivity over 11β-HSD2 was regarded as an important criterion in searching for novel potent 11β-HSD1 inhibitors for potential clinical use.

18β-Glycyrrhetinic acid (18β-GA, 1), a principal active ingredient of liquorice root, and its hemisuccinate derivative carbenoxolone (CBX) are potent non-selective inhibitors of 11β-HSD with IC₅₀ values in the nanomolar range (Fig. 1) [15], [16]. Despite their restricted use because of non-selective inhibition, the use of CBX in a clinical study resulted in increased hepatic insulin sensitivity and decreased glucose production [10]. The topical application of 18β-GA in healthy women could reduce the thickness of subcutaneous thigh fat possibly through the blocking of 11β-HSD1 [17].

Our previous work on the discovery of novel inhibitors of the 11β-HSDs from natural products showed that 18β-GA analogues are potent inhibitors of rat microsomal 11β-HSDs. While most of these compounds exhibit no selectivity for rat hepatic 11β-HSD1 and some are in favour of rat renal 11β-HSD2, a few 18β-GA derivatives have been identified with selectivity for 11β-HSD1 [18], [19], [20]. These 18β-GA analogues (2–5) were further studied for their inhibitory activities against 11β-HSD1 from human hepatic microsomes using a radioimmunoassay (RIA) and a homogeneous time resolved fluorescence (HTRF) assay and here we report the results from both these assays (Fig. 2).

Since a number of crystal structures of human 11β-HSD1 have been solved and published in the Protein Data Bank [21], [30], we have performed docking studies with selected 18β-GA analogues into the crystal structure of human 11β-HSD1. The results revealed how the molecules may interact with the substrate and cofactor binding sites of the enzyme and have been used to guide further design of potent 11β-HSD1 inhibitors.