Structural Basis of Antagonist Selectivity in Endothelin Receptors

Medicinal Chemistry Chemistry Biochemistry Cardiovascular System Life Sciences Immunology Medicine Cell Biology
antagonist endothelin receptors structural biology drug design vascular homeostasis

Deciphering Antagonist Selectivity of Endothelin Receptors: Insights from Cryo-EM-Based Structural Studies

Academic Background

Endothelin (ET) is a potent vasoconstrictor peptide that plays a significant role in regulating cardiovascular functions. The endothelin family includes ET-1, ET-2, and ET-3, which regulate vascular tone and cardiovascular homeostasis by binding to endothelin receptors (ETRs). ETRs primarily include two subtypes, ETA and ETB. Although these receptors share 63% sequence homology, they show substantial differences in ligand affinity and function: ETA preferentially binds ET-1 and ET-2 to induce potent vasoconstriction, whereas ETB binds all three endothelin isoforms with similar affinity, primarily leading to vasodilation by releasing nitric oxide and facilitating ET-1 clearance.

Since ETRs play crucial roles in various cardiovascular diseases, they have become therapeutic targets, particularly for the treatment of pulmonary arterial hypertension (PAH). Currently, ETA antagonists (e.g., Macitentan, Ambrisentan) and ETA/ETB dual antagonists (e.g., Bosentan) have been approved for clinical use. However, despite extensive research into ETB receptor binding and activation mechanisms, there is insufficient understanding of the structural selectivity of ETA antagonists, limiting the development of selective antagonists. This study aims to address these unanswered questions by revealing, for the first time, the molecular mechanisms of key ETA antagonist binding and exploring the possibility of antibody-mediated regulation of ETA activity.

Research Source

The study, conducted in collaboration with Zhongshan Hospital affiliated with Fudan University, the iHuman Institute of ShanghaiTech University, and other institutes, was published in 2024 in the internationally renowned journal, Cell Discovery. The research team includes Junyi Hou, Shenhui Liu, Xiaodan Zhang, and several others, hailing from fields such as cardiac intensive care, life sciences, and technology research.

Research Design and Procedures

1. Innovative Strategy for Cryo-EM Studies:
The researchers used cryo-electron microscopy (Cryo-EM) to elucidate the complex structures of ETA with three antagonists: Macitentan, Ambrisentan, and Zibotentan. To address the challenge of determining the inactive-state structures of G-protein coupled receptors (GPCRs), the team developed an optimized strategy, including introducing a thermostable Bril protein into the intracellular loop of ETA to enhance the stability of the complex. Additionally, they introduced an antibody (Fab301) specifically targeting ETA, which significantly improved the quality of Cryo-EM data.

2. Role of Antibody Fab301:
Fab301 not only helped stabilize the ETA complex for structural determination but also exhibited antagonist effects against ET-1-induced ETA signaling. The resolutions of the ETA complexes combined with Fab301 reached between 3.1 to 3.2 Å.

3. Experimental Workflow:
The research comprised the following key steps: - Using Cryo-EM to determine the high-resolution structures of ETA in complex with the three antagonists (Macitentan, Ambrisentan, and Zibotentan). - Comparing the different structural features of ETA and ETB receptors when bound to agonists or antagonists. - Further validating the effects of key amino acids on ligand binding and selectivity using molecular dynamics simulations and site-directed mutagenesis.

Key Findings

1. Molecular Mechanisms of ETA Antagonist Binding:
- Macitentan:
As a derivative of the dual antagonist Bosentan, Macitentan demonstrates high ETA selectivity. Its key sulfonamide group forms hydrogen bonds and ionic interactions with residues such as R326, K166, and K255, significantly enhancing binding stability. Additionally, the bromophenyl group of Macitentan is embedded in the receptor’s hydrophobic core, forming various hydrophobic interactions with TM5 and TM6.

  • Ambrisentan:
    Ambrisentan, a propionic acid derivative, has a smaller molecular weight but shows extremely high affinity for ETA. Its carboxyl group forms ionic bonds with K166 and R326, and hydrogen bonds with Q165, ensuring its stable insertion into the binding site. Moreover, its benzene ring is tightly packed with the hydrophobic residues of TM3 and TM4, enhancing binding selectivity.

  • Zibotentan:
    This sulfonamide-based antagonist adopts a unique “chair-like” conformation when binding to ETA. Its sulfonamide group forms electrostatic interactions with Q165, K166, and R326, while its pyridine ring engages in critical polar interactions, contributing to selectivity for ETA.

2. Key Residues for ETA Selectivity:
The residues F1613.28 and Y1292.53 of ETA play significant roles in forming a compact binding pocket and establishing crucial interaction networks. In contrast, ETB has smaller residues, V1773.28 and H1502.53, at these positions, accommodating bulkier ligands like Bosentan.

3. Mechanism of Antibody Fab301:
Fab301 antagonizes ETA by interacting with key residues in the extracellular loop 2 (ECL2) of ETA, such as R232 and G233. The binding of Fab301 prevents ET-1 from interacting with ETA and changes the conformation of ECL2, obstructing receptor activation.

Study Significance

This study, through Cryo-EM analysis, has revealed for the first time the molecular basis of antagonist selectivity for the ETA receptor. These findings provide a theoretical basis for designing highly selective and efficient ETA antagonists and antibodies, which is of great importance for the precision treatment of PAH and other cardiovascular-related diseases. Furthermore, Fab301, as a novel antibody, provides a new model for receptor-selective regulation and opens new pathways for the development of antibody drugs.

Highlights and Future Prospects

  1. Deep Insights into Molecular Mechanisms:
    The study sheds light on the role of critical residues in the ETA antagonist binding pocket, validated by molecular simulations and mutational analyses of their biological significance.

  2. Innovative Research Strategy:
    The innovative approach of using Fab301 to stabilize the complex and assist in Cryo-EM analysis provides a valuable reference for future studies.

  3. Clinical Application Prospects:
    The findings provide a robust structural foundation for optimizing PAH therapeutic drugs, while the application of Fab301 offers a new direction for drug development.

The researchers have not only furthered the understanding of the relationship between GPCR structure and function but also provided crucial scientific support for the precise treatment of cardiovascular diseases.