The nuclear receptor NR superfamily of ligand regulated tran
The nuclear receptor (NR) superfamily of ligand regulated transcription factors has proven to be a rich source of targets for the development of therapeutics for a wide range of human diseases. The NR3B subfamily known as the estrogen-related receptor (ERRα[NR3B1], ERRβ[NR3B2] and ERRγ[NR3B3]) regulate several physiological processes, including mitochondrial function, glucose and lipid metabolism, and muscle fiber type determination. The ERR’s are constitutively active orphan nuclear receptors, and while ERRα and ERRβ are more ubiquitously expressed,, , ERRγ is more restricted to metabolically active and highly vascularized tissues such as heart, kidney, ONC201 and skeletal muscle., ERRγ mice fail to thrive shortly after birth due to abnormal heart and spinal cord development, but haploinsufficient ERRγ mice are viable and phenotypically normal in the absence of stress. ERRγ mice exhibit decreased exercise capacity and muscle mitochondrial function compared to their WT littermates. In mice, muscle-specific forced expression of ERRγ increased oxygen consumption, treadmill endurance, mitochondrial function and these animals were resistant to diet-induced weight gain. Interestingly, repression of ERRγ expression in db/db mice ameliorated hyperglycemia via inhibition of hepatic gluconeogenesis. Synthetic modulators of ERRγ including agonists, antagonists or inverse agonists may hold utility in the treatment of a myriad of human disorders, including obesity and type-2 diabetes, cardiovascular disease and muscle atrophy. The functions of ERRγ using genetically engineered mice has provided valuable insight into the role of ERRγ in the context of overexpression or depletion of the receptor; however, pharmacological modulation of ERRγ using selective small molecule chemical probes would complement and validate these data in a more translational context. Although endogenous ligands for the ER’s have shown no activity at ERRs, some synthetic ligands have demonstrated activity towards both ER’s and ERRs. Diethylstilbesterol, a synthetic ER agonist, has been demonstrated to function as an inverse agonist for all three ERRs. 4-hydroxytamoxifen, a synthetic ER antagonist, functions as an inverse agonist of ERRβ and ERRγ, but displays no activity at ERRα., GSK5182 functions as a dual ERRγ /ERα inverse agonist., A few ERR selective ligands have also been identified: XCT790 is an ERRα selective inverse agonist and GSK4716 an ERRβ/γ selective agonist. Given the receptors specific tissue distribution and important physiological role, the identification of ERRγ-selective small molecules would be valuable as chemical probes and pharmacological tools. In the search for ERRγ agonists as probes, GSK4716 was the only identified agonist reported in the primary literature (). It was discovered via a combination of diversity screening and structure guided array synthesis. GSK4716 is >50-fold selective over the classic ERs. Although, GSK4716 can substantially potentiate the transcriptional activity of ERR with moderate potency, it is far from optimal as an probe since it suffers from poor metabolic stability likely due to the hydrolytically unstable hydrazide moiety., Additionally, the phenol group may be subject to phase two metabolism and excretion. Herein we describe our efforts to date to optimize the potency and metabolic stability of this ligand by modifications to all portions of the molecule. The synthesis of analogs described herein is shown in . Esters were treated with hydrazine to give acylhydrazides , which were then treated with aldehydes under microwave heating to provide acylhydrazones . The amide compounds were simply obtained by coupling acids with amines in the presence of EDCI and HOBt. Compounds were initially screened in a FRET-based peptide recruitment assay using α-HisSUMO-ERRγ-LBD, FITC-RIP140 peptide and a terbium-labelled α-HIS antibody. The FRET signal was measured by excitation at 340 nm and emission at 520 nm for fluorescein and 490 nm for terbium using a Perkin Elmer ViewLux ultra HTS microplate reader. The fold change over DMSO was calculated by the 520 nm/490 nm ratio. Graphs were plotted in GraphPad Prism as fold change of FRET signal for each compound over DMSO-only control. While the intent was always to find a suitable replacement for the hydrazone moiety, initial structure-activity relationship studies (SAR) began with examining both sides of the -acylhydrazone to see what groups were tolerated (, ). A couple of quick replacement analogs of the isopropylphenyl ring revealed a 7-fold boost in activity by incorporating a -butyl group at the -position of the phenyl ring (SR209906). Other substitutions indicated a need for something of size at the -position, as SR205163 was completely inactive. Naphthyl analog SR9861 was moderately potent, as was dimethylamine analog SR106447 leaving open the options for possible substitution patterns down the road.