**7. Therapeutic potential for targeting ERR**α **in NAFLD treatment**

Fatty liver and associated diseases including ASH/NASH are comorbid diseases with diabetes, particularly type 2 diabetes (T2D) which accounts for 95% of all diabetes cases [104]. Fatty liver disease occurs in 80% of T2D patients [87, 90]. While simple fatty liver is readily reversible, NASH can progress to more morbid forms of liver pathologies including fibrosis/cirrhosis and even liver cancer. Currently, there is no therapy besides caloric restriction for the treatment of fatty liver disease [105]. A potential therapy is under development in clinical trials that activates FXR, a bile acid receptor. In a completed clinical trial (NCT01265498), FXR ligand obeticholic acid treatment led to NAFLD resolution in 21% of the subjects vs. 13% in placebo treated subjects after 72 weeks of treatment. While these clinical trials are underway, studies exploring molecules that play roles in liver lipid dysfunction have the potential to discover novel therapies. In mouse models, caloric restriction is capable of curing fatty liver disease [105].

The function of ERRs as master regulators for metabolism made them interesting targets for the treatment of T2D, as activating ERRs has the potential to improve overall mitochondrial respiratory function and suppress hepatic glucose output. Earlier studies, tethered with the elucidation of LBD structures, have focused on developing agonists or antagonists. These studies show that ERRs are constitutively active and identified several estrogen-related hydrophilic molecules that can bind and block the establishment of active conformations. These molecules such as the diethylstilbestrol (DES) and 4-hydroxytamoxifen (4-OHT) have been described as antagonists for ERRs, where DES binds to all three ERRs, and 4-OHT does not bind to ERRα [106, 107].

Due to the lack of endogenous ligand, XCT-790 was identified as a potent inverse agonist for ERRα and is used in many studies as inhibitors of ERR functions [108]. Using the NIH compound library in a couple high throughput screening studies, it was found that several pesticides contain ERRβ genes promoting activities, and these compounds may act as potential "ligands" for ERRs [109, 110]. The activity of ERRα has been reported to be antagonized by the organochlorine pesticides chlordane and toxaphene [111, 112]. Screening approaches also identified small molecules with the ability to alter ERR transcriptional activity. An example is the discovery of compound 11 that potently inhibits ERRα's transcriptional activity by preventing binding of ERRα to PGC-1α and suppressing the proliferation of different cancer cell lines [113].

Troglitazone also interferes with binding of ERRα and γ to PGC-1α. Troglitazone was recently discovered to be an inverse agonist for ERRα and γ [114]. Troglitazone is an FDA approved therapy for T2D. In hepatocytes, troglitazone inhibits oleic acid induced liquid buildup. Thus, activity of troglitazone towards ERRα likely plays a role in this effect. In addition, pharmacological inhibition of either ERRα or ERRγ has led to improved insulin response and better tolerance to diet induced metabolic changes [79, 80].

Beyond identification of ligand-like molecules that can serve as either antagonist or agonist for ERR, effort was put into blocking its binding to DNA. Pyrrole-imidazole (Py-Im) polyamides are a class of synthetic ligands for the sequence-specific recognition in double-helical DNA minor groove [84]. Polyamides targeted at the ERRE (ERR-PA) were designed to block binding of ERR onto the promoters of genes regulated by it. An *in vitro* study showed that there was over a 50% reduction in basal and maximal respiration with 0.2 μM ERR-PA treatment and around 70% reduction with 1 μM, emphasizing the dose dependency of the polyamide molecule [84]. ERR-PA was highly effective at reducing liver steatosis in multiple NAFLD models, including one with deletion of *Pten*, the negative regulator of
