Chemoproteomics reveals proteome-wide covalent and non-covalent targets of withaferin A

Withaferin A (WA), a natural product used in traditional medicine, has recently garnered attention because of its diverse pharmacological effects. However, the direct targets responsible for these effects remain elusive. The discovery of targets is usually serendipitous and research has predominantly concentrated on covalent interactions, overlooking non-covalent targets. The unbiased and proteome-wide mapping of WA-interacting proteins in living cells remains largely unexplored. We have developed a chemical proteomics platform that enabled profiling of the covalent/non-covalent interactome and target occupancy in disease-related cells, which was used to reveal the landscape of the targets of WA in triple-negative breast cancer (TNBC) cells. Analysis of the discovered high-occupancy targets suggested that WA was substantially involved in the RNA metabolism pathway, in addition to other biological processes. Moreover, we biochemically validated a selection of previously unknown high-occupancy targets from various important biological pathways, including the non-covalent target MVK and covalent targets HNRNPF and CKAP4, which all play critical roles in TNBC. Collectively, these findings provided a target map for comprehensive understanding of the anti-TNBC activity of WA, and present WA-targetable proteins as new avenues for pharmacological intervention in TNBC. We anticipate that this platform will be applicable for the unbiased profiling of the targets of WA in various other disease-related cell models, as well as for other bioactive electrophilic natural products in different pathophysiological systems.