Shifting the PPARγ conformational ensemble towards a transcriptionally repressive state improves covalent inhibitor efficacy
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eLife Assessment
This manuscript presents a fundamental advance in our understanding of nuclear receptor pharmacology by expanding on previous work demonstrating dual ligand occupancy in the peroxisome proliferator-activated receptor-gamma (PPARγ). Using a compelling combination of biophysical, structural, and cellular approaches, the authors show that covalent inhibitors with inverse agonist activities modulate receptor conformation to permit co-binding with additional ligands, leading to a finely tuned transcriptional response. The data support a model of proximal, bi-directional allostery that challenges traditional views of nuclear receptor regulation. These findings will be of broad interest to researchers in structural biology, transcriptional control, and drug discovery.
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Abstract
Abstract
The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARy) regulates transcription in response to ligand binding at an orthosteric pocket within the ligand-binding domain (LBD). We previously showed that two covalent ligands, T0070907 and GW9662-extensively used as PPARγ inhibitors to assess off-target activity-weaken but do not completely block ligand binding via an allosteric mechanism associated with pharmacological inverse agonism (Shang et al., 2024). These covalent inhibitors shift the LBD towards a repressive conformation, where the activation function-2 (AF-2) helix 12 occupies the orthosteric pocket, competing with orthosteric ligand binding. Here, we provide additional support for this allosteric mechanism using two covalent inverse agonists, SR33065 and SR36708, which better stabilize the repressive LBD conformation and are more effective inhibitors of-but also do not completely inhibit-ligand cobinding. Furthermore, we show that ligand cobinding can occur with a previously reported PPARγ dual-site covalent inhibitor, SR16832, which appears to weaken ligand binding through a direct mechanism independent of the allosteric mechanism. These findings underscore the complex nature of the PPARγ LBD conformational ensemble and highlight the need to develop alternative methods for designing more effective covalent inhibitors.
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eLife Assessment
This manuscript presents a fundamental advance in our understanding of nuclear receptor pharmacology by expanding on previous work demonstrating dual ligand occupancy in the peroxisome proliferator-activated receptor-gamma (PPARγ). Using a compelling combination of biophysical, structural, and cellular approaches, the authors show that covalent inhibitors with inverse agonist activities modulate receptor conformation to permit co-binding with additional ligands, leading to a finely tuned transcriptional response. The data support a model of proximal, bi-directional allostery that challenges traditional views of nuclear receptor regulation. These findings will be of broad interest to researchers in structural biology, transcriptional control, and drug discovery.
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Reviewer #1 (Public review):
Summary:
This paper focuses on understanding how covalent inhibitors of peroxisome proliferator-activated receptor-gamma (PPARg) show improved inverse agonist activities. This work is important because PPARg plays essential roles in metabolic regulation, insulin sensitization, and adipogenesis. Like other nuclear receptors, PPARg, is a ligand-responsive transcriptional regulator. Its important role, coupled with its ligand-sensitive transcriptional activities, makes it an attractive therapeutic target for diabetes, inflammation, fibrosis, and cancer. Traditional non-covalent ligands like thiazolininediones (TZDs) show clinical benefit in metabolic diseases, but utility is limited by off-target effects and transient receptor engagement. In previous studies, the authors characterized and developed covalent …
Reviewer #1 (Public review):
Summary:
This paper focuses on understanding how covalent inhibitors of peroxisome proliferator-activated receptor-gamma (PPARg) show improved inverse agonist activities. This work is important because PPARg plays essential roles in metabolic regulation, insulin sensitization, and adipogenesis. Like other nuclear receptors, PPARg, is a ligand-responsive transcriptional regulator. Its important role, coupled with its ligand-sensitive transcriptional activities, makes it an attractive therapeutic target for diabetes, inflammation, fibrosis, and cancer. Traditional non-covalent ligands like thiazolininediones (TZDs) show clinical benefit in metabolic diseases, but utility is limited by off-target effects and transient receptor engagement. In previous studies, the authors characterized and developed covalent PPARg inhibitors with improved inverse agonist activities. They also showed that these molecules engage unique PPARg ligand binding domain (LBD) conformations whereby the c-terminal helix 12 penetrates into the orthosteric binding pocket to stabilize a repressive state. In the nuclear receptor superclass of proteins, helix 12 is an allosteric switch that governs pharmacologic responses, and this new conformation was highly novel. In this study, the authors did a more thorough analysis of how two covalent inhibitors, SR33065 and SR36708 influence the structural dynamics of PPARg LBD.
Strengths:
(1) The authors employed a compelling integrated biochemical and biophysical approach.
(2) The cobinding studies are unique for the field of nuclear receptor structural biology, and I'm not aware of any similar structural mechanism described for this class of proteins.
(3) Overall, the results support their conclusions.
(4) The results open up exciting possibilities for the development of new ligands that exploit the potential bidirectional relationship between the covalent versus non-covalent ligands studied here.
Weaknesses:
(1) The major weakness in this work is that it is hard to appreciate what these shifting allosteric ensembles actually look like on the protein structure. Additional graphical representations would really help convey the exciting results of this study.
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Reviewer #2 (Public review):
Summary:
The authors use ligands (inverse agonists, partial agonists) for PPAR, and coactivators and corepressors, to investigate how ligands and cofactors interact in a complex manner to achieve functional outcomes (repressive vs. activating).
Strengths:
The data (mostly biophysical data) are compelling from well-designed experiments. Figures are clearly illustrated. The conclusions are supported by these compelling data. These results contribute to our fundamental understanding of the complex ligand-cofactor-receptor interactions.Weaknesses:
This is not the weakness of this particular paper, but the general limitation in using simplified models to study a complex system.
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