A "clickable" MTX reagent as a practical tool for profiling small-molecule-intracellular target interactions via
Martijn D P Risseeuw1, Dries J H De Clercq, Sam Lievens
1Laboratory for Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, 9000 Gent, Belgium.
Chemmedchem
|January 24, 2013
Summary
Researchers developed a new reagent for creating drug-protein fusion compounds. This method enhances the identification of intracellular protein targets using the MASPIT system, improving sensitivity and drug discovery.
Area of Science:
- Chemical Biology
- Molecular Biology
- Drug Discovery
Background:
- Identifying intracellular protein targets of small molecules is crucial for understanding drug mechanisms.
- Existing methods for target identification can be limited in sensitivity and scope.
- The Mammalian three-hybrid system (Maspit) is a powerful tool for discovering protein-small molecule interactions.
Purpose of the Study:
- To develop a versatile reagent for synthesizing Methotrexate fusion compounds (MFCs) with an azide ligation handle.
- To demonstrate the utility of these MFCs in the MASPIT system for identifying mammalian cytosolic protein targets.
- To improve the sensitivity and efficiency of target identification compared to previous methods.
Main Methods:
- Scalable synthesis of a novel Methotrexate (MTX) reagent featuring an azide ligation handle.
- Copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction for γ-selective conjugation of drug baits to the MTX reagent.
- Application of the resulting MTX fusion compounds (MFCs) in the MASPIT system for target identification screens.
Main Results:
- Successful synthesis of MFCs using diverse drug molecules (tamoxifen, reversine, FK506), confirming reagent versatility.
- Maspit system demonstrated concentration-dependent reporter signals with the new MTX reagent.
- Enhanced sensitivity was observed with the new MTX reagent compared to previous non-regiomeric conjugates.
- FK506 MFC successfully identified FKBP12 as a target protein in a cellular array screen of nearly 2000 human ORFs.
Conclusions:
- The newly developed MTX reagent enables straightforward and efficient generation of MFCs.
- This synthetic strategy significantly enhances the capabilities of the MASPIT system for uncovering novel intracellular targets.
- The improved sensitivity and applicability make this approach a promising tool for drug discovery and target validation.
