Lanthanide GTP Probes for Fusion-Free GPCR Activation Detection

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Solution Overview

Problem

Existing energy transfer techniques for detecting molecules that modulate G protein-coupled receptor activation are restrictive, requiring fusion proteins and limiting high-throughput screening, and do not allow study of endogenously expressed GPCRs and G proteins.

Innovation Solution

Development of GTP analogs coupled to lanthanide complexes, represented by formula (I), which can bind to G proteins and facilitate detection through energy transfer techniques, allowing identification of molecules modulating GPCR activation.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If energy transfer techniques use donor conjugated to GPCR and acceptor conjugated to G protein, then interaction between GPCR and G protein can be detected, but the technique requires preparation of fusion proteins and does not allow study of endogenously expressed GPCRs and G proteins

Engineering Contradiction:

Improvedetection capabilityVSAvoidpreparation complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The patent uses an acceptor-conjugated antibody as an intermediary to detect G protein activation without requiring fusion proteins. The antibody specifically binds to the G protein alpha subunit, serving as a mediator that bridges the detection system to the endogenously expressed G proteins while maintaining measurement precision and eliminating the need for complex fusion protein preparation

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent employs a fluorescent GTP analog that copies the natural GTP binding properties of the G protein alpha subunit. This analog retains the ability to bind to the active conformation of the G protein, allowing detection of endogenously expressed G proteins without modification, thus simplifying the system while maintaining detection capability

Inventive Principle:

Principle #26Copying

2Measurement precision

If affinity tests use radiolabeled ligands to measure ligand affinity for GPCR, then affinity measurement is achieved, but the tests are difficult to carry out and require membrane filtration stages that limit high-throughput screening

Engineering Contradiction:

Improveaffinity measurementVSAvoidhigh-throughput screening capability

Core Design Contradiction:

Measurement precisionVSProductivity

Solution Approach 1:

The patent replaces the mechanical membrane filtration step with a fluorescence-based detection system. By using a fluorescent GTP analog that binds to activated G proteins and an acceptor-conjugated antibody, the system achieves affinity measurement through fluorescence intensity or FRET signals, eliminating the need for filtration and enabling high-throughput screening while maintaining measurement precision

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

3Adaptability or versatility

If energy transfer techniques require preparation of multiple membrane samples for different G alpha protein subtypes, then discrimination between subtypes is achieved, but the technique becomes more complex and time-consuming

Engineering Contradiction:

Improvesubtype discrimination capabilityVSAvoidsample preparation complexity

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The patent employs a universal acceptor-conjugated antibody that can detect multiple G alpha protein subtypes (Gαs, Gαi, Gαq) through a single detection system. The antibody recognizes conserved epitopes on the G protein alpha subunits, allowing discrimination and detection of different subtypes without requiring separate membrane preparations for each subtype, thus reducing complexity while maintaining versatility

Inventive Principle:

Principle #6Universality (Multi-functionality)

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

Enables efficient detection and identification of molecules modulating G protein-coupled receptor activation using FRET principles, overcoming limitations of existing methods by binding to G proteins and providing a high-throughput screening capability.

Implementation Method 1

These tests are based in particular on energy transfer techniques (RET—Resonance Energy Transfer), such as FRET (Fluorescence Resonance Energy Transfer)

Methodology Applied

Scientific EffectFluorescence Resonance Energy Transfer (FRET):

Implementation Method 2

Mention may be made, for example, of the energy transfer techniques demonstrating the interaction between a GPCR and the G protein by using either a donor conjugated to the GPCR and an acceptor conjugated to the G protein

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Data Source

PatentUS20250346611A1Fluorescent GTP analogues and use

Publication Date: 2025.11.13 CISBIO BIOASSAYS

AI summary

The invention relates to compounds of formula:in which X, Y, L and Ln3+ are as defined in the description.