Small molecule receptors involved in the metabolic syndrome

Abstract

This thesis is concerned with small molecule receptors in the development of the metabolic syndrome. This thesis focuses on the most common small molecule receptor classes, the nuclear receptors and the G-protein coupled receptors (GPCRs). Chapter 1 is concerned with the role of the nuclear receptor Retinoid-related Orphan Receptor γ (RORγ) in mature brown adipocytes. RORγ is known to be involved in adipogenesis in white adipose tissue, while its role in brown adipose tissue has not been investigated, yet. Since RORγ is known to be involved in the regulation of the circadian rhythm, we investigated its circadian expression in murine brown adipose tissue, both on the mRNA, as well as protein level. RORγ expression was found to be subject to a circadian rhythm, with its expression being lower during the light phase. RORγ expression was found to increase during the differentiation of both primary and immortalized brown adipocytes. The role of RORγ in differentiated immortalized brown cells was investigated using different viral approaches. To this end, different RORγ constructs were generated, a construct overexpressing endogenous RORγ, as well as a constitutively active (VP16) and inactive (dominant negative, ΔH12) RORγ mutant. For in vitro studies, lentiviral vectors of these constructs were generated and used to overexpress RORγ in infected differentiated immortalized brown adipocytes as well as in pre-adipocytes in order to generate a polyclonal stable cell line overexpressing the constructs. It was found that that RORγ overexpression increases the expression of RevErbα, while it leads to a decrease in UCP-1 expression. This thesis therefore provides the first evidence of a regulation of UCP-1 by RORγ in BAT. In Chapter 2, the ligand binding of G-protein coupled receptor 55 (GPR55), a GPCR involved in obesity, was investigated using synthetic analogs of the endogenous ligand arachidonoyl-sn-glycero-3-phosphoinositol (AGPI). The arachidonic acid chain of AGPI was modified by replacing the terminal double bond with either a vicinal tri-chloro or di-fluoro motif. These halogen modifications increase the molecule’s flexibility compared to AGPI, while still allowing it to preserve the structure of a Z-double bond in the fatty acid chain. The ability of the analogs to activate GPR55 was measured in β-arrestin recruitment assays and compared to the endogenous ligand AGPI. Dose-dependent activation of GPR55 was found for the di-fluorinated analogs, which exhibited potencies comparable to AGPI. Our results show that di-fluoro motifs can be used to replace Z-double bonds without loss of biological activity. In Chapter 3, the physiochemical properties of t-butyl-isosteres were evaluated in various assays. The t-butyl group is a frequently used substituent in drugs. Since the incorporation of a t-butyl group often introduces undesired characteristics to the molecule, surrogates for this group are highly sought after. Here, analogs of bosentan and vercirnon were synthesized, in which the t-butyl group was replaced by CF3, SF5, cyclopropyl-CF3, and bicycle[1.1.1]pentane groups. The physiochemical properties of those analogs were investigated and compared amongst each other for both drugs. Many pharmacologically important parameters, such as solubility and permeability, were improved by the introduction of the isosteres. At the same time all synthesized compounds remained biologically active, demonstrating that these substituents are suitable replacements for a t-butyl group and should be considered in future drug discovery efforts. In Chapter 4, the endogenous agonist of the δ-opioid receptor, Leu-Enkephalin, was modified using oxetanyl dipeptides as replacements for amide bonds in all possible positions. The resulting analogs showed increased metabolic stability in human serum and two analogs retained an affinity towards the receptor comparable to the parent molecule. This establishes the proof of concept, that oxetanes can be suitable substituents for amide bonds in biological settings, while at the same time endowing the molecule with improved pharmacokinetic parameters.