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Published on: March 14, 2019
Thermal Dissociation Assay for Time-Resolved Fluorescence Detection of Protein Post-Translational Modifications
Ville Eskonen1, Natalia Tong-Ochoa1, Salla Valtonen1
1Materials Chemistry and Chemical Analysis, Department of Chemistry, University of Turku, Vatselankatu 2, FI-20014 Turku, Finland.
Abstract:
Post-translational modifications (PTMs) of proteins provide an important mechanism for cell signal transduction control. Impaired PTM control is a key feature in multiple different disease states, and thus the enzyme-controlling PTMs have drawn attention as highly promising drug targets. Due to the importance of PTMs, various methods to monitor PTM enzyme activity have been developed, but universal high-throughput screening (HTS), a compatible method for different PTMs, remains elusive. Here, we present a homogeneous single-label thermal dissociation assay for the detection of enzymatic PTM removal. The developed method allows the use of micromolar concentration of substrate peptide, which is expected to be beneficial when monitoring enzymes with low activity and peptide binding affinity. We prove the thermal dissociation concept functionality using peptides for dephosphorylation, deacetylation, and demethylation and demonstrate the HTS-compatible flash isothermal method for PTM enzyme activity monitoring. Using specific inhibitors, we detected literature-comparable IC50 values and Z' factors from 0.61 to 0.72, proving the HTS compatibility of the thermal peptide-break technology.
Insights
A new thermal dissociation assay enables high-throughput screening of enzymes controlling protein post-translational modifications (PTMs). This method efficiently detects enzymatic PTM removal, offering a universal platform for drug discovery targeting PTM-related diseases.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- Protein post-translational modifications (PTMs) are crucial for cellular signaling.
- Dysregulation of PTMs is linked to various diseases, highlighting PTM-modifying enzymes as drug targets.
- Existing methods for monitoring PTM enzyme activity lack universal high-throughput screening (HTS) compatibility.
Purpose of the Study:
- To develop a homogeneous, single-label thermal dissociation assay for detecting enzymatic PTM removal.
- To establish a universal HTS-compatible method for monitoring diverse PTM enzyme activities.
- To enable the use of micromolar substrate peptide concentrations for enzymes with low activity or binding affinity.
Main Methods:
- Development of a homogeneous single-label thermal dissociation assay.
- Application of the assay to monitor dephosphorylation, deacetylation, and demethylation activities.
- Demonstration of HTS-compatible flash isothermal monitoring.
- Validation using specific inhibitors to determine IC50 values and Z' factors.
Main Results:
- The thermal dissociation assay successfully detected enzymatic PTM removal across different modification types.
- The method demonstrated HTS compatibility with Z' factors ranging from 0.61 to 0.72.
- Literature-comparable IC50 values were obtained using specific enzyme inhibitors.
- The assay supports micromolar substrate peptide concentrations, beneficial for low-activity enzymes.
Conclusions:
- A novel, HTS-compatible thermal peptide-break technology has been established for monitoring PTM enzyme activity.
- This assay provides a universal platform for screening PTM-modifying enzymes, advancing drug discovery efforts.
- The method's efficiency and compatibility with low-affinity substrates offer significant advantages for PTM research.
