Abstract

Understanding both the on- and off-target protein binding interactions of small molecules is an essential part of the drug discovery process.  A large proportion of toxicity findings in non-clinical or clinical studies are precipitated by the parent drug or a metabolite binding an off-target protein target, such as an enzyme or receptor, modifying its function resulting in cellular dysfunction and toxicity. 

Capture Compound Mass Spectrometry (CCMS) is an unbiased, proteome-wide approach for the identification of specific-binding protein targets for small molecules and peptides. The technology combines medicinal chemistry and in vitro pharmacology, coupled to high resolution proteomics mass spectrometry to isolate and identify target proteins that are responsible for an observed biological response. Thus, through in vitro investigation in target tissues of interest, the candidate proteins and pathways causing in vivo toxicity can be elucidated.

An overview of CCMS technology and its application in identification of off-target compound activity is presented.  The CCMS technology has been used to determine on- and off-target interactions of the catechol-O-methyl transferase (COMT) inhibitor tolcapone in a human liver cancer cell line (HepG2). A comprehensive interaction profile was generated, revealing both on- and off-target binding proteins. Differential profiles of tolcapone which causes liver toxicity and entacopone that does not were elucidated and highlighted 3-hydroxyisobutyrly-CoA hydrolase (HIBCH) as a candidate target mediating toxicity. Medicinal chemistry was then initiated focusing on molecules without HIBCH activity resulting in ‘tolcapone-like’ molecules with reduced toxicity profiles that could lead the way to the development of improved COMT inhibitors.