Helenalin Analogues Targeting NF‐κB p65: Thiol Reactivity and Cellular Potency Studies of Varied Electrophiles

Abstract

Helenalin is a pseudoguaianolide natural product that targets Cys38 within the DNA binding domain of NF-κB transcription factor p65 (RelA). Helenalin contains two Michael acceptors that covalently modify cysteines: a α-methylene-γ-butyrolactone and a cyclopentenone. We recently reported two simplified helenalin analogues that mimic the biological activity of helenalin and contain both electrophilic moieties. To determine the individual contributions of the Michael acceptors toward NF-κB inhibition, we synthesized a small library of helenalin-based analogues containing various combinations of α-methylene-γ-butyrolactones and cyclopentenones. The kinetics of thiol addition to a subset of the analogues was measured to determine the relative thiol reactivities of the embedded electrophiles. Additionally, the cellular NF-κB inhibitory activities of the analogues were determined to elucidate the contributions of each Michael acceptor to biological potency. Our studies suggest the α-methylene-γ-butyrolactone contributes most significantly to the NF-κB inhibition of our simplified helenalin analogues.

Anticancer Targets and Signaling Pathways Activated by Britannin and Related Pseudoguaianolide Sesquiterpene Lactones

“…The α-methylene-γ-lactone moiety of these molecules is a reactive entity, capable of Michael-type addition with biological nucleophiles, in particular with the sulfhydryl groups of proteins [4] (Figure 8). BRT has been shown to bind covalently to a cysteine residue (Cys-151) of Keap1 [85], and helenalin shows a high reactivity toward specific thiolcontaining proteins, such as the p65 subunit of NFκB [10,11]. Helenalin potently inhibits human telomerase, an enzyme activated in most cancer cells [130,131].…”

“…Biomedicines 2021, 9, x FOR PEER REVIEW 2 of 18 the α-methylene-γ-butyrolactone moiety [10,11]. However, the mechanism of action at the origin of the anticancer properties of BRT is multifactorial, with several protein targets implicated or at least proposed.…”

“…Another important member of the group is britannin (BRT in Figure 2), initially isolated from the plant Inula britannica L. [9]. These compounds have been characterized as inhibitors of the transcription factor NFκB, due to the targeting of a cysteine residue of the p65 subunit by the α-methylene-γ-butyrolactone moiety [10,11]. However, the mechanism of action at the origin of the anticancer properties of BRT is multifactorial, with several protein targets implicated or at least proposed.…”

Function‐Oriented and Modular (+/−)‐cis‐Pseudoguaianolide Synthesis: Discovery of New Nrf2 Activators and NF‐κB Inhibitors

“…While the majority of medicinal chemistry and drug discoverye fforts re- lated to (pseudo)guaianolides involves isolation and/or semisynthesis, [37] there are numerous total syntheses(pseudoguaianolides [12][13][14][15] and guaianolides [38][39][40][41][42][43][44][45] )m ethods/approaches, [36,[46][47][48][49][50][51] and de novo analogue syntheses. [52,53] For drug discovery purposes, it would be ideal to have am odularr oute to pseudoguaianolide-analogues that retains the structural complexityo f the natural product family,c an tempert he reactivity of the Michael acceptors, [36] and allows for significant diversification aroundt he scaffold. [54] With these goals in mind, we sought to develop af unction-oriented synthetic route [54] to structurally complexp seudoguaianolidec hemical space whereby the lactone-ringi sc leaved and a" functional group" ("FG") is installed at the C-1 position ( Figure 2B).…”

“…As such, the (pseudo)guaianolide family and their analogues have been implicated as leads in drug development. While the majority of medicinal chemistry and drug discovery efforts related to (pseudo)guaianolides involves isolation and/or semisynthesis, [37] there are numerous total syntheses(pseudoguaianolides [12–15] and guaianolides [38–45] ) methods/approaches, [36, 46–51] and de novo analogue syntheses [52, 53] . For drug discovery purposes, it would be ideal to have a modular route to pseudoguaianolide‐analogues that retains the structural complexity of the natural product family, can temper the reactivity of the Michael acceptors, [36] and allows for significant diversification around the scaffold [54] .…”