Stability-Based Proteomic Methods Add Value to Activity-Based Protein Profiling Studies

Area of Science:

• Proteomics
• Chemical Biology
• Drug Discovery

Background:

• Covalent inhibitors offer therapeutic benefits but need thorough profiling for on- and off-target effects.
• Identifying off-target interactions is crucial for understanding drug mechanisms and potential toxicities.

Purpose of the Study:

• To evaluate stability-based proteomic methods (SPROX and TPP) for identifying protein targets of covalent inhibitors.
• To characterize the off-target interactions of the KRAS G12C inhibitor ARS-1620.

Main Methods:

• Utilized stability of proteins from rates of oxidation (SPROX) and thermal protein profiling (TPP) on H358 cell lysates.
• Employed mass spectrometry-based covalent site mapping and enzymatic assays.
• Performed covalent docking and functional knockdown experiments.

Main Results:

• Both SPROX and TPP identified KRAS as the on-target and revealed multiple off-targets.
• Aldehyde dehydrogenase 1A3 (ALDH1A3) was identified as a reproducible off-target missed by previous methods.
• ARS-1620 covalently binds to C314 on ALDH1A3, inhibiting its enzymatic activity and contributing to ARS-1620's cell-killing effects.

Conclusions:

• Stability-based proteomics is a powerful strategy for covalent drug target identification.
• ALDH1A3 plays a significant role in the mode of action of ARS-1620.
• This study expands the understanding of ARS-1620's biological activity and off-target profile.