Real-Time Cellular Thermal Shift Assay for Drug Discovery
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Solution Overview
Problem
The Cellular Thermal Shift Assay (CETSA) is low-throughput, time-consuming, and limited to single temperature or compound concentrations, with traditional methods unable to generate full aggregation profiles for multiple samples in parallel due to the low melting temperature of nanoLuciferase.
Innovation Solution
Development of protein constructs comprising a target protein, a first peptide linker, and a reporter region with a thermally stable LgBiT and HiBiT fragment, along with a biological vector for expression in cells, and an analytical device for real-time luminescence detection across a temperature gradient.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If nanoLuciferase is used as a reporter in CETSA, then luminescent signal detection is enabled, but the low melting temperature of nanoLuciferase prevents full CETSA temperature ramp analysis
Solution Approach 1:
The nanoLuciferase reporter is segmented into two separate fragments (LgBiT and HiBiT) that are incorporated into different fusion proteins. These fragments only complement each other and generate luminescence when the target proteins are in close proximity, enabling detection without requiring the reporter itself to remain stable at high temperatures.
Solution Approach 2:
The patent introduces complementary peptide fragments (LgBiT and HiBiT) as intermediaries that mediate the detection process. These fragments act as molecular spies that report on protein-protein interactions or conformational changes through luminescence without being affected by the thermal conditions that denature full-length nanoLuciferase.
2Measurement precision
If traditional CETSA methods are used, then thermal stability assessment is possible, but the method is low-throughput and time-consuming with single temperature or compound concentration analysis
Solution Approach 1:
The patent merges multiple CETSA experiments into a single high-throughput format by combining temperature ramping with parallel processing of multiple samples containing different compound concentrations. The split-luciferase system enables simultaneous monitoring of multiple conditions, transforming low-throughput sequential analysis into high-throughput parallel analysis.
Solution Approach 2:
The patent implements periodic temperature ramping cycles that systematically progress through increasing temperatures while continuously monitoring luminescence signals. This periodic heating protocol, combined with high-throughput detection, enables comprehensive thermal stability profiling across multiple samples and conditions in a single experiment.
3Measurement precision
If western blot detection is used in traditional CETSA, then target protein detection is possible, but the method is time-consuming and requires substantial optimization
Solution Approach 1:
The patent replaces the mechanical and chemical processes of western blotting (protein separation, transfer, blocking, antibody incubation, and chemiluminescent detection) with a direct luminescent reporter system. The split-luciferase fusion proteins provide built-in detection capability that eliminates multiple manual steps, reducing detection time from hours to minutes while maintaining or improving sensitivity.
Solution Approach 2:
The target proteins are engineered to self-report their thermal stability and conformational state through the luminescent properties of the incorporated split-nanoLuciferase fragments. This self-reporting mechanism eliminates the need for external detection reagents and complex processing steps required by western blotting, enabling rapid and straightforward analysis.
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 real-time thermal shift data collection for multiple samples, allowing for full aggregation profiles and improved detection of target protein-ligand interactions, overcoming the limitations of traditional CETSA methods.
Implementation Method 1
the reporter region comprises, consists of, or consists essentially of, from N-Terminus to C-Terminus, a LgBiT fragment, a second peptide linker, and a HiBiT fragment
Implementation Method 2
exposing the cells to an increasing temperature gradient while detecting the change in luminescence of the sample in real time
Data Source
AI summary
The disclosure provides methods for carrying out Real Time Cellular Thermal Shift Assays (RT-CETSA). Also provided are molecular constructs and protein constructs for use in such assays and devices suitable for carrying out such assays.