Proteomic characterization of Withaferin A-targeted protein networks for the treatment of monoclonal myeloma gammopathies

Abstract

Multifunctional antitumor compounds are of great potential since they reduce the risk of multidrug resistance in chemotherapy. Unfortunately, characterization of all protein targets of a multifunctional compound is lacking. Therefore, we optimized an SILAC quantitative chemoproteomics workflow to identify the potential protein targets of Withaferin A (WA), a natural multifunctional compound with promising antitumor properties. To further understand the antitumor mechanisms of WA, we performed a differential protein expression analysis and combined the altered expression data with chemoproteome WA target data in the highly curated Ingenuity Pathway database. We provide a first global overview on how WA kills multiple myeloma cancer cells and serve as a starting point for further in depth experiments. Furthermore, the combined approach can be used for other types of cancer and/or other promising multifunctional compounds, thereby increasing the potential development of new antitumor therapies.

Covalent Cysteine Targeting of Bruton’s Tyrosine Kinase (BTK) Family by Withaferin-A Reduces Survival of Glucocorticoid-Resistant Multiple Myeloma MM1 Cells

“…Finally, WA-mediated inhibition of BTK kinase activity was also accompanied by a time-dependent decrease in BTK mRNA and protein expression, not observed for IBR, suggesting that WA targets BTK hyperactivation at multiple levels [ 62 ]. For example, WA may indirectly decrease BTK activity by reducing kinase protein levels by inhibition of Sp1-dependent transcription [ 78 ], by post-transcriptional microRNA silencing mechanisms [ 53 , 79 ], or by decreasing kinase stability via heat shock chaperone proteins [ 45 , 47 ].…”

“…Indeed, in addition to BTK, BCR signaling is fine-tuned by Ser/Thr kinases (for example, IKK2, PKCβ) [ 15 , 88 ] and phosphatases (i.e., SHIP1, PTEN) [ 89 , 90 ]. Finally, chemoproteomic strategies have identified additional WA non-kinase target proteins, which may further strengthen the potential cancer therapeutic efficacy of WA [ 45 , 91 ]. Therefore, functional silencing approaches with shRNA libraries will be informative to further distinguish druggable key cancer targets from adverse off-targets to defeat therapy resistance in MM by WA [ 87 , 92 ].…”

“…WA reveals broad-spectrum therapeutic activities in several (drug-resistant) cancer cell types [ 44 ], including B-cell lymphoma and MM [ 45 , 46 , 47 ]. Of particular interest, some of WA’s antitumor effects have been attributed to its ability to covalently target kinase activity [ 48 , 49 , 50 , 51 , 52 ].…”

Tackling Chronic Inflammation with Withanolide Phytochemicals—A Withaferin A Perspective

“…This is especially true for neurodegenerative diseases where astrocyte reactivity is often associated with increased expression and polymerization of IF [ 53 ]. Affinity purification (using biotinylated WA) and LC-MS/MS analysis confirmed the interaction of WA with IF and even identified the WA binding sites of these proteins [ 129 , 130 , 131 , 132 ]. Through this covalent interaction with IF, WA perturbs structural organization of IF by lowering the amount of filaments and causing the formation of short aggregates [ 53 , 129 , 132 ].…”

Withaferin A inhibits LFA-1-stimulated ZAP70 activity and T-cell motility

“…P434, V435, S436, C560, C564, P565, P566 and L600. DTT is known to reverse WFA-mediated suppression of kinase activity by blocking alkylation of thiol-sensitive redox pathways (10). Given that DTT-treated human primary T-cells did not show WFA-Biotin and ZAP70 interaction after immunoprecipitation, the most probable thiol donors in the docking complex were either C560, C564 or both residues.…”