Comparative profiling of analog targets: a case study on resveratrol for mouse melanoma metastasis suppression

Abstract

Many plant-specialized metabolites have remedial properties and provide an endless chemical resource for drug discovery. However, most of these metabolites have promiscuous binding targets in mammalian cells and elicit a series of responses that collectively change the physiology of the cells. To explore the potential of these multi-functional and multi-targeted drugs, it is critical to understand the direct relationships between their key chemical features, the corresponding binding targets and the relevant biological effects, which is a prerequisite for future drug modification and optimization.Methods: We introduced and demonstrated a general workflow, called Comparative Profiling of Analog Targets (CPAT), to connect specific biological effects with defined chemical structures of drugs. Using resveratrol (RSV) as an example, we have synthesized and characterized a series of partial functional analogs of RSV. An analog (named RSVN) that specifically lost the inhibitory effect of RSV in cell migration was identified. The binding targets of RSVN and RSV was profiled and compared.Results: Comparative profiling of the RSV and RSVN binding targets showed that, unlike RSV, RSVN failed to target specific components involved in DNA methylation (histone deacetylase 1 [HDAC1] and DNA methyltransferase 3 alpha [DNMT3a]), suggesting that RSV suppresses cell migration through epigenetic regulation. Indeed, RSV treatment recruited HDAC1 and DNMT3a to the promoter region of the focal adhesion kinase (FAK), a key factor involved in cell adhesion, enhanced the promoter methylation, and thus attenuated the protein expression. The inhibitory effect of RSV in cell migration was diminished once FAK expression was restored. Thus, the mechanism of RSV in inhibiting cell migration could be largely accounted to epigenetically control of FAK expression.Conclusion: Our results showed that even though RSV exhibits promiscuous binding, its inhibitory effect on cell migration can be mechanistically understood. First, the presence of 4'-hydroxystilbene within the RSV structure is essential for this activity. Second, it inhibits cell migration through epigenetically based downregulation of FAK expression. Taken together, we propose that CPAT might also be adapted to delineate the specific function of other natural products (NPs) that exhibit binding promiscuity.

Extracellular signal regulated kinase 5 promotes cell migration, invasion and lung metastasis in a FAK-dependent manner

“…Our lab has previously reported that nucleotides from −170 to +43 constitute the minimal mouse FAK promoter that is responsible for basal FAK transcription (Chen et al, 2018 ). After P-Match analysis, which is a new method for identifying transcription factor (TF) binding sites in DNA sequences, we found that the core promoter region of FAK might contain four USF binding sites, three NFκB/Rel binding sites, two cEts-1/PEA3 binding sites, one GATA1 binding site and one Pax-3 binding site (Supplementary file 2: Fig.…”

Quantitative Chemical Proteomics Reveals Resveratrol Inhibition of A549 Cell Migration Through Binding Multiple Targets to Regulate Cytoskeletal Remodeling and Suppress EMT

“…To elucidate the mechanism of action of RSV’s antimigration effect on A549 cells, a quantitative chemical proteomics approach was applied to identify the protein targets of RSV in A549 cells, as illustrated in Figure 2 . Briefly, based on the structure-activity relationship of RSV ( Chen et al, 2018 ), we first synthesized an RSV probe (RSV-P) derived from RSV ( Figure 3A ). The structure of RSV-P was validated with NMR and MS ( Supplementary Figures S3, S4 ).…”

Target identification of natural medicine with chemical proteomics approach: probe synthesis, target fishing and protein identification

“…As a result, we identified histone deacetylase I (HDAC1) as the protein target of resveratrol in mouse melanoma cells and revealed an epigenetic regulation pathway of focal adhesion kinase. 41 Other excellent examples are listed in Fig. 3b.…”

“…With in vivo and in vitro experiments, we confirmed that resveratrol inhibits focal adhesion kinase (FAK) expression by interacting with HDAC1. 41 Although the method has been widely used in protein identification, [112][113][114] it has two disadvantages: (i) some low-abundance but vital target proteins are still invisible after Coomassie brilliant blue staining or even silver staining, resulting in target loss; and (ii) some distinct bands or spots, especially bands, contain more than one protein, so the nonspecific binding in these bands or spots may also be identified.…”

“…[35][36][37] Numerous pharmacological studies have applied chemical proteomics to identify drug targets and study their MOA, [38][39][40] especially in the last few years. 41 Hence, these studies provides us with a unique background to summarize the recent achievements in this field.…”