Exploring the binding mode and thermodynamics of inverse agonists against estrogen-related receptor alpha

Since estrogen-related receptor alpha (ERRα), one of three estrogen-related receptors, displays constitutively active transcriptional activities and important implications in both physiological and pathological processes of breast cancers, ERRα was recently recognized as a new target to fight breast cancers, and regulating the activity of ERRα with inverse agonists has thus become a promising new therapeutic strategy. A few inverse agonists cyclohexylmethyl-(1-p-tolyl-1H-indol-3-ylmethyl)-amine (compound 1), thiadiazoacrylamide (XCT790), and 1-(2,5-diethoxy-benzyl)-3-phenyl-area analogues (compounds 2 and 3) were reported to be capable of targeting ERRα. However, the detailed mechanism by which the inverse agonists deactivate ERRα remains unclear, especially in the aspects of quantitative binding and hot spot residues. Therefore, to gain insights into the interaction modes between inverse agonists and ERRα ligand binding domain, all-atom molecular dynamics (MD) simulations were firstly carried out for the complexes of inverse agonists and ERRα. The binding free energies were then calculated with MM-PBSA method to quantitatively discuss the binding of the inverse agonists with ERRα. The binding affinities were finally decomposed to per-residue contributions to identify the hot spot residues as well as assess their role in the binding mechanism. MD simulations show that the inverse agonists stretch downwards into the ERRα ligand binding pocket (LBP) formed by H3 and H11 helices, and upon the binding H12 adopts a well-defined position in the coactivator groove, where PGC-1α binds to ERRα. Binding energy analysis indicates that compound 3 and XCT790 bind more tightly to ERRα than compounds 1 and 2, and the energy difference mainly results from the contribution of van der Waals interaction. Both binding mode analysis and affinity decomposition per-residue indicate that compound 1, XCT790, and compound 3 have similar binding spectra to ERRα, primarily interacting with the residues of H3, H5, H6/H7 loop, and H11 helix, while compound 2 lacks a significant interaction with the H5 region. The hot spot residues significantly binding to the three inverse agonists in common include Leu324, Phe328, Phe382, Leu398, Phe495, and Leu500. It is essential for an effective inverse agonist to strongly bind with the aromatic ring cluster consisting of Phe328(H3), Phe495(H11), and Phe382(H5/H6 loop) as well as Leu500.