Deciphering the Cellular Targets of Bioactive Compounds Using a Chloroalkane Capture Tag

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Phenotypic screening is crucial for drug discovery but identifying cellular targets remains challenging.
• Existing methods for target identification often fail with low-affinity or low-abundance targets and are incompatible with living cells.
• Verifying target engagement in a cellular context is essential for drug development.

Purpose of the Study:

• To develop a novel capture tag for identifying compound-interacting proteins in living cells.
• To overcome limitations of existing target identification methods, particularly for low-affinity interactions.
• To validate the identified targets using a method compatible with cellular phenotypes.

Main Methods:

• Development of a novel chloroalkane capture tag that minimally impacts compound potency.
• Utilizing HaloTag protein for immobilization and subsequent isolation of tagged protein complexes.
• Employing resonance energy transfer for direct binding verification of identified targets.
• Applying the method to study the interaction of Vorinostat (SAHA) with histone deacetylases (HDACs).

Main Results:

• The chloroalkane tag effectively captured low-affinity and low-abundance targets in cultured cells.
• All known HDAC targets of SAHA were identified, along with two novel targets: ADO and CPPED1.
• Direct binding of SAHA to identified targets was verified using resonance energy transfer.
• The method successfully identified and validated targets under physiologically relevant conditions.

Conclusions:

• The novel chloroalkane capture tag facilitates robust target identification and validation in phenotypic drug discovery.
• This method enhances the ability to discover novel drug targets and understand drug mechanisms of action.
• The identification of ADO as a SAHA target may offer insights into SAHA's role in Huntington's disease.