Cyclic Peptide Inhibitors Function as Molecular Glues to Stabilize Gq/11 Heterotrimers

Chemistry Medicinal Chemistry Biophysics Biochemistry Life Sciences Medicine
cyclic peptide inhibitors molecular glues Gq/11 heterotrimers X-ray crystallography BRET biosensors

Academic Background

G protein-coupled receptors (GPCRs) are the largest family of membrane proteins in the human body, transmitting extracellular signals through heterotrimeric G proteins (composed of Gα and Gβγ subunits). G proteins function as molecular switches, with their activity states regulated by the GTP/GDP cycle:
- Inactive state: Gα binds GDP and forms a stable complex with Gβγ
- Active state: GPCRs catalyze GDP release, allowing Gα to bind GTP and dissociate from Gβγ

For a long time, tools for specifically inhibiting G protein signaling have been scarce. Although the natural cyclic peptides FR900359 (FR) and YM-254890 (YM) can effectively inhibit the Gq/11 subfamily, their molecular mechanisms remain incompletely understood. The traditional view holds that they solely prevent GDP release by “wedging” between the GTPase domain and the α-helical domain of Gα (i.e., GDI function). This study aims to reveal whether these inhibitors exert broader regulatory effects by stabilizing the Gα-Gβγ interface.

Source of the Paper

This research was a collaborative effort by an international team, with corresponding authors Gebhard Schertler (Paul Scherrer Institute, Switzerland), Evi Kostenis (University of Bonn, Germany), and Xavier Deupi (Swiss Institute of Bioinformatics). It was published in PNAS (Vol. 122, No. 19) in May 2025, DOI: 10.1073/pnas.2418398122.

Research Process and Results

1. High-Resolution Crystal Structure Determination

Study Subjects:
- Engineered soluble Gα11β1γ2 heterotrimer (G11in3-s)
- Crystals in complex with FR or YM

Methodological Innovations:
- Used N-terminal GFP fusion tags to enhance protein solubility
- Obtained crystals via vapor diffusion at resolutions of 1.43 Å (FR complex) and 1.70 Å (YM complex) (the highest-resolution structures of G protein heterotrimers to date)

Key Findings:
- Anchor 1 region: The alkyl chains of FR/YM insert into the Gα-Gβ interface, forming 4 hydrogen bonds (FR) or 3 hydrogen bonds (YM) with Arg96 of Gβ
- Nucleotide-binding pocket: The phenyl ring of the inhibitor directly contacts Ser53 of Gα (a conserved P-loop residue), fixing its side-chain conformation and thereby hindering GDP release
- Linker stabilization: Interaction with Tyr67 in Linker 1 enhances the rigidity of the α-helical domain

Structural Differences:
- FR exhibits more stable Arg96/Gβ conformation due to an additional propionyl group
- YM’s β-hydroxyleucine displays dual conformations, weakening interactions with Gβ

2. Biophysical Validation Experiments

Thermal Stability Analysis:
- Wild-type (WT) G11in3-s: FR/YM increased the melting temperature ™ from 51.4°C/57.6°C (biphasic) to 60.9°C (monophasic)
- Gβ-R96A mutant: Inhibitor stabilization effect reduced by 1.4–1.7°C

Kinetic Binding Assays (GCI technology):
- WT binding affinity: FR (KD = 2.1 nM) > YM (KD = 5.8 nM)
- R96A mutation reduced affinity by 2–3-fold

3. Functional Validation at the Cellular Level

BRET Biosensor System:
- Experimental Design: Monitored competitive binding between Gβγ and membrane-anchored GRK3ct to quantify heterotrimer stability
- Key Findings:
- 10 μM FR/YM reduced WT G11 signaling by 35%/25%
- Gβ-R96A/D118A double mutation completely abolished stabilization
- Pathogenic mutant Gβ-R96L (spontaneously enhances Gα binding) was insensitive to inhibitors

Signal Inhibition Assays:
- All mutants retained the ability to inhibit M3R activation, confirming that Gα binding is sufficient for inhibition

Research Conclusions and Significance

  1. Mechanistic Innovation:
    FR/YM are the first G protein inhibitors confirmed to function as “molecular glues,” simultaneously binding Gα (inhibiting nucleotide exchange) and Gβ (enhancing subunit interface stability) to achieve full heterotrimer locking.

  2. Taxonomic Implications:
    Proposed a new classification standard for G protein inhibitors:

    • Type I (e.g., GD20): Inhibit Gα but compete with Gβγ binding
    • Type II (FR/YM): Synergistically enhance Gα-Gβγ interactions

  3. Translational Value:

    • Provides a precision-targeting strategy for Gq/11-related diseases (e.g., uveal melanoma)
    • The conserved Arg96/Gβ target can be extended to inhibitor design for other G protein subfamilies

Research Highlights

  • Technical Breakthrough: 1.43 Å resolution reveals the regulatory role of water networks in the binding pocket
  • Conceptual Innovation: First proposal of an “active heterotrimer stabilization” mechanism
  • Structural Guidance: The polar pocket in the Anchor 1 region offers new directions for derivative design

Additional Value

  • Methodological Contribution: Established a BRET-GRK3ct detection system for real-time monitoring of G protein conformational dynamics in living cells
  • Evolutionary Insight: Pathogen toxins (e.g., pertussis toxin) and FR/YM employ similar strategies to regulate G proteins but target different sites