Second Harmonic Generation Probe for Allosteric Modulator Detection
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Solution Overview
Problem
Current methods for identifying allosteric modulators of target proteins are limited by low throughput and sensitivity, particularly for 'undruggable' cancer targets, as conventional techniques like X-ray crystallography and NMR are not suitable for rapid or high-throughput detection of conformational changes.
Innovation Solution
The use of second harmonic generation technology to label target proteins with specific moieties, allowing for real-time detection of conformational changes when allosteric modulators bind, using surface-selective techniques to generate detectable signals.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional techniques like X-ray crystallography or NMR are used to obtain structural information, then measurement precision is improved, but productivity deteriorates due to low throughput
Solution Approach 1:
The patent replaces conventional structural biology techniques (X-ray crystallography, NMR) with a fluorescence-based optical detection system. This substitution enables real-time monitoring of conformational changes through fluorescence anisotropy measurements, achieving both high precision in detecting structural changes and high throughput in screening allosteric modulators
Solution Approach 2:
The patent changes the detection parameter from static structural snapshots (obtained through crystallography or NMR) to dynamic fluorescence anisotropy signals that report real-time conformational changes. This parameter change enables continuous monitoring of protein conformational states during allosteric modulator binding, simultaneously improving measurement precision for conformational detection and productivity through rapid screening
2Measurement precision
If conventional techniques are used for detecting conformational changes, then measurement precision is improved, but loss of time increases due to inability to perform real-time detection
Solution Approach 1:
The patent implements continuous real-time monitoring of conformational changes through continuous fluorescence anisotropy measurements. This allows uninterrupted observation of protein conformational dynamics during allosteric modulator binding, eliminating the time loss associated with discrete sampling or post-experiment analysis while maintaining high measurement precision
3Ease of manufacture
If competitive binding to active site is used to inhibit protein function, then ease of manufacture is improved, but object-affected harmful factors increase due to unintended clinical side effects
Solution Approach 1:
The patent employs an intermediary approach by using the fluorescent probe as a mediator to detect conformational changes induced by allosteric modulators. This indirect detection method enables identification of compounds that bind to allosteric sites rather than competing for the active site, thereby reducing unintended clinical side effects while maintaining drug development feasibility
Solution Approach 2:
The patent substitutes competitive active site binding with allosteric modulation detected through fluorescence anisotropy. This substitution allows identification of therapeutics that modulate protein function through conformational changes at allosteric sites, avoiding the harmful effects of active site competition while preserving ease of drug development through the high-throughput screening capability
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 rapid and high-throughput identification of allosteric modulators, overcoming the limitations of traditional methods by providing sensitive and real-time detection of conformational changes in target proteins, facilitating the development of therapeutic agents for previously 'undruggable' cancer targets.
Implementation Method 1
the target protein is labeled with a second harmonic-active moiety (such as, a label) having a net orientation at an interface, wherein a detectable signal is generated by the second harmonic-active moiety (such as, a label) using a surface selective technique
Data Source
AI summary
The present invention discloses, inter alia, methods for labeling a target protein with an SHG-active probe for detection by second harmonic or sum-frequency generation in order to identify agents which bind to an allosteric site on the target protein thereby altering its structural conformation.