Structural Mechanisms Reveal Unexpected Cobinding of Covalent Inhibitors and Synthetic Ligands to the Nuclear Receptor PPARγ

Core Concepts

Covalent inhibitors GW9662 and T0070907 do not prevent binding of synthetic ligands to PPARγ; instead, they allosterically stabilize a repressive conformation that reduces the binding affinity of synthetic ligands, which can still adopt orthosteric or alternate binding modes.

Abstract

The study investigates the structural mechanisms underlying the cobinding of covalent inhibitors and synthetic ligands to the nuclear receptor PPARγ. Key findings:

Contrary to the prevailing view, covalent inhibitors GW9662 and T0070907 do not completely block the binding of synthetic ligands to PPARγ. Instead, they allosterically stabilize a repressive conformation of the receptor that reduces the binding affinity of synthetic ligands.

NMR data show that the covalent inhibitors, especially the repressive inverse agonist T0070907, shift the conformational ensemble of PPARγ towards a transcriptionally repressive state, which clashes with and weakens the binding of synthetic ligands.

Crystal structures reveal that the synthetic ligands can still bind to PPARγ in the presence of covalent inhibitors, adopting either their native orthosteric binding mode or alternate site binding modes. The specific cobinding mechanism depends on the size and binding affinity of the synthetic ligand.

The covalent inhibitors can also adopt different binding poses within the orthosteric pocket to accommodate the cobinding of synthetic ligands, highlighting the flexibility and malleability of the PPARγ ligand-binding domain.

These findings have important implications for the use of GW9662 and T0070907 as chemical tools to study PPARγ ligand binding specificity, as they do not completely block the binding of other synthetic ligands as previously assumed.

Stats

The TR-FRET ligand displacement assay data show the Ki values for the synthetic ligands binding to PPARγ LBD (Figure 1B).
The TR-FRET coregulator interaction assay data show the effects of the synthetic ligands on the binding of NCoR1 corepressor and TRAP220 coactivator peptides to PPARγ LBD, both in the absence and presence of the covalent inhibitors GW9662 and T0070907 (Figure 2).

Quotes

"GW9662 and T0070907 should not be used as reliable chemical tools to inhibit the binding of other ligands to PPARγ."
"Our findings not only highlight the significant flexibility of the PPARγ orthosteric pocket and its ability to accommodate multiple ligands simultaneously, but also demonstrate that GW9662 and T0070907 should not be used as reliable chemical tools to inhibit the binding of other ligands to PPARγ."

Key Insights Distilled From

Unanticipated mechanisms of covalent inhibitor and synthetic ligand cobinding to PPARγ

by Shang,J., Ko... at www.biorxiv.org 05-15-2024

https://www.biorxiv.org/content/10.1101/2024.05.15.594037v1

Deeper Inquiries

How might the insights from this study on PPARγ ligand binding mechanisms be applied to develop more selective and potent PPARγ modulators for therapeutic applications

The insights gained from this study on PPARγ ligand binding mechanisms can be instrumental in the development of more selective and potent PPARγ modulators for therapeutic applications. By understanding the structural mechanisms of ligand binding, researchers can design ligands that specifically target certain conformations of the receptor associated with desired functional outcomes. For example, the identification of unique binding poses for different ligands, as observed in the study, can guide the design of ligands that interact with specific regions of the PPARγ ligand-binding domain to modulate its activity in a controlled manner. This knowledge can be leveraged to develop ligands with enhanced selectivity and efficacy, minimizing off-target effects and improving therapeutic outcomes. Additionally, the study highlights the importance of considering the dynamic nature of the receptor-ligand interactions, which can inform the design of ligands that can stabilize or disrupt specific receptor conformations to achieve the desired pharmacological effects.

What other nuclear receptors or ligand-binding proteins might exhibit similar flexibility and unexpected ligand cobinding behaviors, and how could this knowledge be leveraged for drug discovery

Other nuclear receptors or ligand-binding proteins that exhibit similar flexibility and unexpected ligand cobinding behaviors may include other members of the nuclear receptor superfamily, such as the retinoic acid receptors (RARs), liver X receptors (LXRs), and farnesoid X receptor (FXR). Additionally, ligand-binding proteins like the estrogen receptor (ER) and androgen receptor (AR) may also display similar characteristics. Understanding the malleability and alternate binding modes of these receptors can provide valuable insights for drug discovery efforts. By studying the ligand binding mechanisms of these receptors, researchers can identify novel ligand-binding sites, allosteric modulation sites, and conformational changes that can be targeted for the development of selective modulators. Leveraging this knowledge, researchers can design ligands that exhibit unique binding profiles and functional outcomes, leading to the discovery of more effective and specific therapeutics for various diseases and conditions.

Given the malleability of the PPARγ ligand-binding domain observed in this study, what computational or experimental approaches could be used to better predict and design ligands that can selectively target specific conformational states of the receptor

Given the malleability of the PPARγ ligand-binding domain observed in this study, computational and experimental approaches can be employed to better predict and design ligands that selectively target specific conformational states of the receptor. Computational methods such as molecular docking, molecular dynamics simulations, and structure-based drug design can be utilized to explore the binding interactions between ligands and the receptor in different conformations. By simulating the dynamics of ligand binding and receptor conformational changes, researchers can identify key binding sites, allosteric pockets, and structural features that influence ligand selectivity and potency. Experimental techniques such as NMR spectroscopy, X-ray crystallography, and biophysical assays can complement computational studies by providing detailed structural information on ligand-receptor interactions in real-time. Integrating computational and experimental approaches can enhance the understanding of PPARγ ligand binding dynamics and facilitate the rational design of novel ligands with improved pharmacological properties.