By Cameron Cohen
Originally published in the Center for Structural Biology’s Weekly Coordinates newsletter
Nuclear receptors are a group of transcription factors that control gene expression in response to endogenous metabolites and synthetic ligands. Ligands bind the C-terminal ligand-binding domain of NRs, which is widely believed to alternate between transcriptionally active and repressive states and be stabilized upon ligand binding. Various X-ray crystallography and cryo-electron microscopy studies have determined the structures of NRs bound to ligands, coregulators, and DNA, but these methods fail to capture the dynamic conformational shifts of the LBD. Further, previous work has largely focused on compounds of a single pharmacological type, such as agonists, instead of focusing on the full pharmacological spectrum.
In a new study published in Nature Communications, the Doug Kojetin and Zhongyue Yang labs use peroxisome proliferator-activated receptor gamma—more commonly known as PPAR—as a model system to understand the functional shifts of nuclear receptor LBDs.
The researchers first designed a series of inverse agonists using 2-chloro-5-nitrobenzamide as a scaffold, solely altering the amine R group. Previous structures have shown that in the transcriptionally active conformation, helix 12 of PPAR is solvent exposed and facilitates co-activator binding. Conversely, in the repressive conformation, helix 12 occupies the orthosteric ligand-binding pocket and leaves surface regions exposed for corepressor binding. Additionally, a pi-stacking interaction is observed in the repressive structure between the inverse agonist and residues in PPAR to form an aromatic triad.
With this knowledge in mind, potential inverse agonists were identified from the ZINC database or synthesized. A combination of a time-resolved fluorescence resonance energy transfer corepressor peptide interaction assay and a cell-based transcriptional reporter assay determined that each of the identified compounds displayed repressive activity. The authors then characterized a ligand series of twenty 2-chloro-5-nitrobenzamide analogs and found that the compounds spanned a pharmacological range from inverse agonist to neutral antagonist to agonist. Interestingly, all inverse agonists contained a polar pyridyl aromatic ring while most agonists had a hydrophobic non-polar phenyl ring.
In order to more specifically ascertain the structural basis of inverse agonism, crystal structures of PPAR bound to the most effectively repressive compounds were determined. In all of the structures, helix 12 of PPAR adopted the solvent-occluded conformation, and the aromatic triad residues interacted with the inverse agonist R groups. Finally, nuclear magnetic resonance spectroscopy was utilized to determine the effect of inverse agonists on PPAR dynamics. The inverse agonists were all found to shift the LBD of PPAR towards a repressive conformation and, excitingly, there was a strong correlation between a compound’s repressive activity and the strength of the repressive NMR peak in PPAR.
Overall, this study has not only identified key structural features of inverse agonism and constructed a more comprehensive ligand series for future studies, but has provided key insights into the functional regulation of NRs. Future studies, and in particular drug design studies, will benefit greatly from a focus on the dynamic nature of PPAR.
Be sure to check out the full story of NR regulation in Nature Communications!