Prodrugs Bioactivated to Quinones Target NF-κB and Multiple Protein Networks: Identification of the Quinonome

Abstract

Electrophilic reactive intermediates resulting from drug metabolism have been associated with toxicity and off-target effects and in some drug discovery programs trigger NO-GO decisions. Many botanicals and dietary supplements are replete with such reactive electrophiles, notably Michael acceptors, which have been demonstrated to elicit chemo-preventive mechanisms; and Michael acceptors are gaining regulatory approval as contemporary cancer therapeutics. Identifying protein targets of these electrophiles is central to understanding potential therapeutic benefit and toxicity risk. NO-donating NSAID prodrugs (NO-NSAIDs) have been the focus of extensive clinical and preclinical studies in inflammation and cancer chemoprevention and therapy: a subset exemplified by pNO-ASA, induces chemopreventive mechanisms following bioactivation to an electrophilic quinone methide (QM) Michael acceptor. Having previously shown that these NO-independent, QM-donors activated Nrf2 via covalent modification of Keap-1, we demonstrate that components of canonical NF-κB signaling are also targets, leading to the inhibition of NF-κB signaling. Combining bio-orthogonal probes of QM-donor ASA prodrugs with mass spectrometric proteomics and pathway analysis, we proceeded to characterize the quinonome: the protein cellular targets of QM-modification by pNO-ASA and its ASA pro-drug congeners. Further comparison was made using a biorthogonal probe of the “bare-bones”, Michael acceptor, and clinical anti-inflammatory agent, dimethyl fumarate, which we have shown to inhibit NF-κB signaling. Identified quinonome pathways include post-translational protein folding, cell-death regulation, protein transport, and glycolysis; and identified proteins included multiple heat shock elements, the latter functionally confirmed by demonstrating activation of heat shock response.

Potential Adverse Effects of Resveratrol: A Literature Review

“…In the case of RE metabolism, o-quinones are formed through a hydroxylation reaction by cytochrome P450 (Figure 2), leading to the formation of piceatannol, followed by catechol oxidation to form an o-quinone product. These RE metabolites could have different effects on several biological targets [81][82][83].…”

Formation and biological targets of botanical o-quinones

“…Once formed o -quinones have a variety of biological targets (Fig. 4) (Aggarwal and Shishodia, 2006; Bolton and Dunlap, 2017; Pierce et al, 2016). Initial reaction with GSH the major non-protein thiol leads to GSH depletion either through direct alkylation and/or through oxidation generating GSSG (Fig.…”

“…Alkylation/oxidation of cysteine residues on key proteins can lead to o -quinone signaling. For example, a major protein target of o -quinones is Keap1 leading to induction of detoxification enzymes including NAD(P)H-quinone oxidoreductase 1 (NQO1), glutathione-S-transferase (GST), and heme oxygenase-1 (HO-1) (Pierce et al, 2016). Alkylation/oxidation of IκB kinase (IKK) results in modulation of NF-κB and inhibition of inflammatory pathways (Bolton and Dunlap, 2017).…”

Effect of Nucleosome Assembly on Alkylation by a Dynamic Electrophile

“…47 Still, the behavior of these histones stands in stark contrast to numerous other proteins (>300) that have been identified to form DNA conjugates with the para-QMs in vivo. 47,48 The ortho-QMs are generally considered more reactive than their para-QM isomers, 45 and the half-life of the simplest ortho-QM (6-methylene-2,4-cyclohexadien-1-one) has been measured under aqueous conditions to be ~0.14 s. 49 The half-life of the comparable para-QM (4-methylene-2,5-cyclo-hexadiene-1-one) is almost 37-fold longer (~5 s). 50 Many nucleophiles have the potential to react with the transient mono-and bisQMs generated after deprotection of their precursors (Scheme 1).…”