An isothermal shift assay for proteome scale drug-target identification

Area of Science:

• Biochemistry
• Proteomics
• Pharmacology

Background:

• * Small molecule drugs modulate protein function through physical interactions.
• * Identifying specific drug targets within the human proteome is crucial but challenging for drug discovery.
• * Current target identification methods are often complex and have low throughput.

Purpose of the Study:

• * To introduce the isothermal shift assay (iTSA), a novel mass spectrometry-based method for proteome-wide drug target identification.
• * To present iTSA as a simplified and more powerful alternative to existing thermal proteome profiling techniques.
• * To demonstrate the application of iTSA in identifying drug targets in both cell lysates and living cells.

Main Methods:

• * Development and application of the isothermal shift assay (iTSA), a mass spectrometry technique.
• * Utilizing thermal stability shifts induced by small molecule binding to identify drug targets.
• * Comparative analysis of iTSA performance against established thermal proteome profiling methods.

Main Results:

• * iTSA successfully identified known targets of the pan-kinase inhibitor staurosporine in cell lysates and living cells.
• * The assay demonstrated improved performance and statistical power compared to existing methods.
• * iTSA identified known and novel candidate targets for the kinase inhibitor harmine in cell and tissue lysates.

Conclusions:

• * The isothermal shift assay (iTSA) offers a simplified and powerful approach for proteome-wide drug target identification.
• * iTSA facilitates efficient discovery of drug-target interactions in complex biological systems.
• * This method has significant implications for advancing drug discovery and development pipelines.