Discovery and characterization of protein-modifying natural products by MALDI mass spectrometry reveal potent SIRT1 and p300 inhibitors.

Natural products are a rich resource for the development of chemical probes, functional foods (nutraceuticals), and pharmaceuticals. The remarkable structural diversity of natural product or biology-oriented compound libraries in particular argues for their use in applications such as drug screenings. Enzymes that can posttranslationally modify proteins or nucleic acids are attractive drug targets and include kinases and phosphatases, methyltransferases and acetyltransferases, and deacetylases. Recently, histone-modifying enzymes like the deacetylase sirtuin 1 (SIRT1) were suggested as drug targets for treating a variety of age-related disorders such as neuropathogenic diseases, metabolic diseases, and cancer. Compound screenings for SIRT1 modulators have revealed promising enzymatic activators such as the natural product resveratrol and the synthetic compound SRT1720. However, these findings are still highly controversial due to reported assay artifacts. The employed screening assays were based on fluorescence-labeled peptide substrates and resulted in the purported but artificial enzymatic activation of SIRT1. These findings have misled many researchers over the years. Besides the generation of artifacts as observed in SIRT1 assays, there is a second general drawback of fluorescencebased assays which is broadly underestimated: Autofluorescence of compound libraries and in particular natural product libraries interferes with widely applied optical analyses such as time-resolved fluorescence resonance energy transfer (TRFRET). By contrast, mass spectrometry (MS) represents an attractive alternative as substrates are detected directly. Peptides are ionized and detected according to their mass to charge ratios (m/z). For example, deacetylation of a substrate peptide can be directly detected as a 42 Da mass peak shift (Figure 1).