Inhibition of IκB kinase-β and IκB kinase-α by heterocyclic adamantyl arotinoids
Introduction
The adamantyl arotinoids (AdArs)1, 2 are included within the group of atypical retinoids or retinoid-related molecules (RRMs),3, 4, 5 a name that reflects the fact that they exert their anticancer activities independently of the transactivation of the retinoid receptors (RARs, subtypes α, β, and γ; and RXRs, subtypes α, β, and γ).6, 7 RARs and RXRs are members of the nuclear hormone receptor superfamily of ligand-responsive transcription factors,8 which mediate the multifarious actions of natural and synthetic retinoids in embryo and throughout life. Some AdArs, however, are known to bind to RARs, such as 1a (6-[(3-adamant-1-yl)-4-hydroxy-phenyl]-2-naphthoic acid, CD437, also called AHPN), which is a RARγ agonist.5 Other analogs of 1a, such as 2a (AHPC, Adarotene, which also binds RARγ and is a stronger apoptogenic agent than 1a), 5-Cl-AHPN 1b and 3-Cl-AHPC 2b (see Fig. 1), lack RAR transactivation activity while preserving the induction of growth arrest and apoptotic activity in a variety of cancer cell lines.9, 10, 11 Moreover, 3-Cl-AHPC 2b binds the nuclear hormone receptor small heterodimer partner (SHP) and modulates SHP interaction with the Sin3A repressor.12, 13 While still unclear, the mechanism of RRM-mediated cell death, in particular the apoptosis induced by 1a, appears to be largely dependent on cell type. Evidences for caspase-dependent and independent mechanisms14, 15, 16 via the intrinsic17, 18 and extrinsic pathways19 have been provided for these compounds.
In addition to SHP, RRMs with pro-apoptotic activity can target the IKK/NFκB signaling pathway via direct inhibition of IKKβ, which has been validated as a cancer target in vitro and in preclinical studies with very promising results, although clinical studies have not yet produced positive outcomes.20, 21 Thus, great efforts have been dedicated to develop IKK/NFκB inhibitors as potential therapies for the treatment of cancer and inflammatory diseases.22, 23, 24 We initially found that MX781 3 substantially inhibited IKK isolated from TNFα-stimulated HeLa cells, and displayed effective and consistent inhibition of IKK/NFκB activity in various cancer cell lines, thus confirming IKK as an AdAr target.25 Furthermore, a number of chalcone containing analogs with an additional substituent ortho to the carbonyl group were prepared and found to elicit enhanced inhibition of recombinant IKKβ in vitro, which paralleled increased growth inhibitory and apoptosis inducing activities in cancer cells.26 Most recently, substitution of the chalcone functionality by a heterocyclic group has demonstrated that AdArs can inhibit IKK and induce apoptosis independently of a potential Michael addition from a Cys residue to the α,β-unsaturated ketone.27 Contrasting with the inhibition of IKK/NFκB signaling by MX781 3 and its apoptogenic analogs observed by us, activation of NFκB by CD437 1a is necessary for the induction of apoptosis in prostate carcinoma cells.28, 29 Cinnamic acid derivative 2b also activates IKKα and IKKβ in breast carcinoma and leukemia cancer cells.28, 30, 31 The exact mechanism of IKK/NFκB activation by these AdArs is still undefined.13, 30, 31
In a previous study, we prepared a series of analogs of 2 that incorporate an additional Me substituent at the adamantyl-phenol end ortho to the biphenyl connection and naphthoic, cinnamic and phenylacetic acids as polar end groups (compounds 4–6).32 The compounds derived from cinnamic and naphthoic acid exhibited potent antiproliferative activities in several cancer cell lines, and this effect correlated in general with the induction of apoptosis as measured by caspase activation. However, the strong deviation of planarity of these AdArs due to the presence of the 2,2′-disubstituted biphenyl connection altered their binding profile from RAR to RXR. We were able to show that some of those analogs induced RXR activity as efficiently as 9-cis-retinoic acid in transient transfection assays, suggesting that RXR might be responsible for their observed tumor cell growth inhibitory effects.32
We now report on the activities of these analogues in the inhibition of IKKα, IKKβ and IKKε in vitro and found that none of the RXR ligands with the exception of the partial agonist 4b inhibited IKKβ by >75%. In contrast, potent growth inhibitory activity was seen with 4a, which correlated well with strong inhibition of both IKKα and IKKβ in the absence of RAR/RXR activity. We also profiled a novel series of AdArs (7–10) that incorporate a central heterocyclic ring that connects the adamantyl-phenol and the carboxylic acid at the polar termini. The MEM derivative of the phenol ortho to the adamantyl group was also included in order to reveal the role of this substituent that is present in the parent compound MX781 3, which is a potent inhibitor of IKK/NFκB signaling.26, 27 The new heterocyclic compounds elicited negligible RAR and RXR transactivation activity and only the thiazole 8b functioned as strong RAR antagonist. Furthermore, the benzoic-linked thiazoles 8a–d and methoxypyrazine 10a elicited an even greater inhibitory activity against recombinant IKKβ in vitro than compound 3, which in some cases (8a and 10a) correlated with superior anticancer activity. In general, the presence of a MEM group reduced IKKβ inhibitory activity compared to the hydroxyl group-containing analog.
Section snippets
Synthesis
The synthesis of AdArs 4-6a,b has been reported before.32 An improved preparation of 3-acetamidopropyloxy-AHPC, 3-A-AHPC 2c, an antagonist of 2a, and its methyl analog 5c has been developed, which differs from that of the original procedure in the order of steps for the formation of the bromocinnamic ester 15,11 and thus does not require protection/deprotection of the phenol. Bromination of 3-hydroxybenzaldehyde 11 was followed by the condensation of 12 with triethylphosphonoacetate and DBU11
Inhibition of IKKβ and IKKα by the novel AdArs
Following up on our previous reports that portrayed the inhibition of IKKβ by MX781 3 and derivatives,25, 26, 27 we evaluated the effect of AdArs on recombinant IKKs using a LANCE TR-FRET kinase assay. The naphthoic acid 2′-Me-5-Cl-AHPN 4a was a strong inhibitor of both IKKα and IKKβ with an IC50 of 4.75 μM against IKKβ, about 2.5 times more potent than MX781 3 (IC50 11.83 μM) (Table 1). The presence of a MEM chain in compound 4b substantially reduced the activity against IKKα, while preserving
Discussion
Adamantyl arotinoids (AdArs) have been recognized as promising anticancer therapeutic agents due to their growth inhibitory and apoptosis inducing activities. Although originally derived from RARγ/β selective retinoid-related molecules, AdArs are often classified as atypical retinoids as they exert their cancer cell growth inhibitory and apoptogenic activities independently of RAR transactivation. While certain AdArs have been shown to target IKKβ as a likely mediator of their anticancer
General
Solvents were dried according to published methods and distilled before use. All other reagents were commercial compounds of the highest purity available. All reactions were carried out under argon atmosphere, and those not involving aqueous reagents were carried out in oven-dried glassware. Analytical thin layer chromatography (TLC) was performed on aluminium plates with Merck Kieselgel 60F254 and visualized by UV irradiation (254 nm) or by staining with a solution of phosphomolibdic acid.
Acknowledgements
This work was supported by funds from the Spanish MICINN (SAF2010-17935-FEDER), Xunta de Galicia (Grant 08CSA052383PR from DXI+D+i; Consolidación 2006/15 from DXPCTSUG; INBIOMED-FEDER ‘Unha maneira de facer Europa’), EU (FP7/REGPOT-2012-2013.1] under grant agreement No. 316265, BIOCAPS), and NIH grant (CA133475, to F.J.P.).
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