PROTEOMICS-AIDED INSIGHT TO DISCLOSE NATURAL PRODUCTS CELLULAR TARGETS

Actually, the identification of the potential primary cellular targets of bioactive natural products (NPs), along with their off-targets, is an important concern. Currently, affinity purification targets identification methods based on mass spectrometry, called AP-MS, are the most reliable ones [1]. However, they are limited by the NPs chemical modification: to overcome this constraint, a simple, universally applicable approach based on the direct binding of an unmodified compound to its targets, termed DARTS (drug affinity responsive target stability), can be performed. DARTS takes advantage of a reduction in the protease susceptibility of the target protein upon drug binding. This phenomenon allows the drug target(s) to be revealed by a gel-based approach, useful for protein bands visualization showing different protease accessibility, and proteomics identification [2]. Then, LiP-MRM approach couples limited proteolysis (LiP) with targeted mass spectrometric tools, exploiting the sensitivity and background filtering capabilities of multiple reaction monitoring (MRM) experiments [3]. As previously described for DARTS, LiP involves the use of a broadspecificity protease, such as subtilisin, under controlled conditions such that primary cleavage sites are dictated by the structural conformation of the protein which can be modified by drug interaction. Altered LiP patterns can be measured directly in a complex proteome matrix pointing out which protein regions are masked by NPs. Several examples will be discussed.