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  • Given that dimethylamine substitution was tolerated


    Given that dimethylamine substitution was tolerated as in SR106447 (), we investigated patupilone australia amine substitutions as replacements for the -butyl group (). Cyclic amines (SR19880, 19882) or simple mono-substituted anilines (SR20037) were not active, nor was an acetylated version (SR20039). The slightly bulkier diethylamine variant (SR19881) is twice as potent as the -butyl varient (SR19797) and the most potent full agonist identified in our SAR studies. Increasing the size of the alkyl substituents on the nitrogen led to some analogs with modest potency (SR20038, 19883, 20042, 20007), but too big, and potency was lost (SR20043, 20044). The isobutyl ethylamine substituted analog SR20041 was very potent, but as a partial agonist. Incorporation of a halogen ortho to the -amine group (SR20737, 20738) was not tolerated and led to a 10-fold drop in potency. The lead hydrazone GSK4716 was reported to be an ERRβ/γ-selective agonist with no activity at ERRα. To confirm selectivity was maintained in this series, a few best-in-class molecules were counterscreened in a FRET-based peptide recruitment assay using either α-HisSUMO-ERR α-LBD or α-HisSUMO-ERRβ-LBD, FITC-RIP140 peptide and a terbium-labelled α-HIS antibody (data not shown). All analogs tested (GSK4716, SR19797, SR19881, SR19894, SR20041, SR20043) showed no activity vs ERRα. Activity vs ERRβ was more interesting. GSK4716 is 5-fold less potent on ERRβ than on ERRγ (ERRβ EC=3.0 μM). SR19881 and SR19894 show a slight preference for ERRγ vs ERRβ (SR19881 ERRβ EC=0.63 μM; SR19894 ERRβ EC=2.7 μM). Other analogs showed increased selectivity (7–8-fold) for ERRβ vs ERRγ (SR19797 ERRβ EC=0.090 μM; SR20239 ERRβ EC=0.045 μM). Finally, SR20011 and SR20012 showed 30–40-fold selectivity for ERRβ vs ERRγ (SR20011 ERRβ EC=0.020 μM; SR20012 ERRβ EC=0.031 μM). These data may pave the way to designing truly (>100-fold) ERRγ-selective and ERRβ-selective agonists. To further confirm pharmacology of the compounds , GSK4716 and SR19797 were screened in HEK293T cells in a Gal4-ERRγ::UAS-Luc reporter assay with counterscreening against Gal4-VP16::UAS-Luc. Concentration-response curves (CRCs) are shown in . Both compounds present as agonists in this cell-based assay, but GSK4716 exhibits some non-specific activation of VP16 which might be contributing to its activity whereas SR19797 does not. To better understand the structural basis of binding of lead SR19797 and the diethylamine analog SR19881 to ERRγ, we performed differential hydrogen/deuterium exchange (HDX) mass spectrometry () using purified ERRγ ligand binding domain (LBD). HDX data show a clear difference in structural perturbations between the apo receptor and the liganded complexs indicating that both SR19797 and SR19881 bind to the LBD. HDX revealed that helix 11 and helix 12 regions show increased protection from solvent exchange (interpreted as stabilization, in green) with both ERRγ ligands tested, suggesting common sites of interaction within the ligand-binding pocket (LBP) of ERRγ. Moreover, presence of the diethylamine substituent (H-bond acceptor) in SR19881 but not the parent compound (SR19797) showed additional protection in helix 2 and β-sheet regions indicating ancillary hydrogen bond interactions that likely contributes to its increased binding potency. Selected compounds were tested for stability in human, rat and mouse liver microsomes (). All analogs inlcuding GSK4716 had short half-lives. Nonetheless, we have successfully replaced the acylhydrazone linkage with a saturated amide and maintained potency and comparable metabolic stability to the lead GSK4716. The phenethyl linkage is likely a metabolic soft spot in the saturated analogs. We will continue to explore substitutions here to improve stability. While not necessarily important to microsomal stability, the phenol group will likely be subject to phase 2 metabolism. Future SAR efforts will continue to search for suitable replacements for the phenol group to reduce this liability.