Hsp72 Is an Intracellular Target of the α,β-Unsaturated Sesquiterpene Lactone, Parthenolide

Abstract

The electrophilic natural product parthenolide has generated significant interest as a model for potential chemotherapeutics. Similar to other α,β-unsaturated carbonyl electrophiles, parthenolide induces the heat shock response in leukemia cells, potentially through covalent adduction of heat shock proteins. Other thiol-reactive electrophiles have also been shown to induce the heat shock response as well as to covalently adduct members of the heat shock protein family, such as heat shock protein 72 (Hsp72). To identify sites of modification of Hsp72 by parthenolide, we used high-resolution tandem mass spectrometry to detect 10 lysine, histidine, and cysteine residues of recombinant Hsp72 as modified in vitro by 10 and 100 μM parthenolide. To further ascertain that modification of Hsp72 by parthenolide occurs inside cells and not simply as an in vitro artifact, an alkyne-labeled derivative of parthenolide was synthesized to enable enrichment and detection of protein targets of parthenolide using copper-catalyzed [3 + 2] azide–alkyne cycloaddition. The alkyne-labeled parthenolide derivative displays an half maximal inhibitory concentration (IC50) in undifferentiated acute monocytic leukemia cells (THP-1) of 13.1 ± 1.1 μM, whereas parthenolide has an IC50 of 4.7 ± 1.1 μM. Concentration dependence of protein modification by the alkyne–parthenolide derivative was demonstrated, as well as in vitro adduction of Hsp72. Following treatment of THP-1 cells in culture by the alkyne–parthenolide, adducted proteins were isolated with neutravidin resin and detected by immunoblotting in the enriched protein fraction. Hsp70 proteins were detected in the enriched proteins, indicating that Hsp70 proteins were adducted intracellularly by the alkyne–parthenolide derivative.

Parthenolide Covalently Targets and Inhibits Focal Adhesion Kinase in Breast Cancer Cells

“…S1). Using a more specific, previously-reported alkyne-functionalized parthenolide probe (parthenolide-alkyne) (Shin et al, 2017), we observed multiple labeled proteins in 231MFP proteomes, of which some, but not all, targets were competed by parthenolide ( Fig. S1).…”

“…Parthenolide, a natural product found in the feverfew plant (Tanacetum parthenium), possesses myriad therapeutic activities, including anti-inflammatory and anti-cancer effects. Through covalent bond formation between its reactive a-methylene-g-butyrolactone moiety and various protein targets, multiple cellular signaling pathways are impacted (Ghantous et al, 2013;Kwok et al, 2001;Liu et al, 2018;Mathema et al, 2012;Shin et al, 2017). Moreover, this natural product belongs to the broader family of sesquiterpene lactones (estimated at > 5000 members), many members of which are also cytotoxic and have been hypothesized or shown to act through covalent mechanisms (Coricello et al, 2018;Quintana and Estévez, 2019).…”

“…Using a biotinylated parthenolide analog, previous studies by the lab of Crews established that one of the primary targets that drives the anti-inflammatory and anti-cancer activity of parthenolide is IKK-b wherein cysteine 179 (C179) is modified thus impairing IKK-b and NFkB signaling (Kwok et al, 2001). Additional studies have revealed other direct targets of parthenolide that may help to explain the therapeutic properties of this natural product, including targeting of specific cysteines within heat shock protein Hsp72 and STAT3 downstream signaling targets such as Janus kinases JAK2 (Liu et al, 2018;Shin et al, 2017). Moreover, this natural product has also been shown to affect additional cell signaling pathways including induction of oxidative stress and apoptosis, focal adhesion kinase 1 (FAK1) signaling, HIF-1a signaling, epithelial-to-mesenchymal transition, Wnt/b-catenin signaling, MAPK signaling, and mitochondrial function (Carlisi et al, 2011(Carlisi et al, , 2016Jafari et al, 2018;Kim et al, 2017;Kwok et al, 2001;Lin et al, 2017;Zhang et al, 2017).…”

“…Here, we have used ABPP-based chemoproteomic platforms to identify C427 of FAK1 as a novel ligandable and functional site targeted by the anti-cancer natural product parthenolide. This target adds further complexity to the polypharmacological landscape of parthenolide which also includes IKK-b, heat shock protein Hsp72, and thioredoxin reductase as targets (Duan et al, 2016;Kwok et al, 2001;Shin et al, 2017).…”

“…Parthenolide and dimethylaminoparthenolide were obtained from Cayman Chemicals. Synthesis of the parthenolide-alkyne probe was performed as previously reported (Shin et al, 2017). All other chemicals were obtained from Millipore-Sigma unless otherwise noted.…”