Characterization of novel small-molecule NRF2 activators: Structural and biochemical validation of stereospecific KEAP1 binding.

BACKGROUND: Semi-synthetic oleanane triterpenoid antioxidant inflammation modulators (tpAIMs) are small molecules that interact with KEAP1 cysteine residue 151 (C151) and activate NRF2. Exploration of the structure-activity relationship between the tpAIMs and KEAP1 is limited by the predominantly hydrocarbon nature of the oleanane triterpenoid pentacyclic ring structure. Therefore, we used novel, chemically-tractable, synthetic antioxidant inflammation modulators (sAIMs) to probe the stereoselectivity of the ligand-protein interaction. METHODS: We measured several parameters of NRF2 activation to assess the potency of sAIM enantiomers with natural (tpAIM-like) 4(S),5(S),10(R) or unnatural 4(R),5(R),10(S) configurations. Additionally, we determined the crystal structure of the KEAP1 BTB domain in complex with two different sAIMs. RESULTS: We found that the potencies of sAIM enantiomers in the natural configuration were similar to those of the tpAIM, RTA 405. Strikingly, sAIM enantiomers in the unnatural configuration were 10- to 40-fold less potent than their natural counterparts. Crystallographic studies of sAIMs in complex with the KEAP1 BTB domain demonstrated that these ligands form a covalent bond with C151 and revealed the presence of additional hydrogen bonds, Van der Waals interactions, and pi-stacking interactions. CONCLUSIONS: Although KEAP1 C151 is required for NRF2 activation by tpAIMs and sAIMs, interactions with other KEAP1 residues are critical for the stereospecific recognition and potency of these ligands. GENERAL SIGNIFICANCE: This work demonstrates that reversible cyanoenone Michael acceptors, such as the tpAIMs and sAIMs, can be specifically tuned to regulate redox sensitive cysteine residues on key signaling molecules, an approach with significant promise for innovative drug development.

Cancer Cell Growth Is Differentially Affected by Constitutive Activation of NRF2 by KEAP1 Deletion and Pharmacological Activation of NRF2 by the Synthetic Triterpenoid, RTA 405.

Synthetic triterpenoids are antioxidant inflammation modulators (AIMs) that exhibit broad anticancer activity. AIMs bind to KEAP1 and inhibit its ability to promote NRF2 degradation. As a result, NRF2 increases transcription of genes that restore redox balance and reduce inflammation. AIMs inhibit tumor growth and metastasis by increasing NRF2 activity in the tumor microenvironment and by modulating the activity of oncogenic signaling pathways, including NF-κB, in tumor cells. Accumulating evidence suggests that KEAP1 loss or mutation--which results in high levels of sustained NRF2 activity--may promote cancer growth and increase chemoresistance. Loss of KEAP1 also increases the levels of other oncogenic proteins, including IKKß and BCL2. The apparent survival advantage provided to some tumor cells by loss of functional KEAP1 raises the question of whether pharmacological inhibition of KEAP1 could promote tumor growth. To address this issue, we characterized the basal levels of KEAP1 and NRF2 in a panel of human tumor cell lines and profiled the activity of an AIM, RTA 405. We found that in tumor cell lines with low or mutant KEAP1, and in Keap1-/- murine embryonic fibroblasts, multiple KEAP1 targets including NRF2, IKKß, and BCL2 were elevated. Keap1-/- murine embryonic fibroblasts also had higher rates of proliferation and colony formation than their wild-type counterparts. In cells with functional KEAP1, RTA 405 increased NRF2 levels, but not IKKß or BCL2 levels, and did not increase cell proliferation or survival. Moreover, RTA 405 inhibited growth at similar concentrations in cells with different basal NRF2 activity levels and in cells with wild-type or mutant KRAS. Finally, pre-treatment with RTA 405 did not protect tumor cells from doxorubicin- or cisplatin-mediated growth inhibition. Collectively, these data demonstrate that pharmacological activation of NRF2 by AIMs is distinct from genetic activation and does not provide a growth or survival advantage to tumor cells.

RTA 408, A Novel Synthetic Triterpenoid with Broad Anticancer and Anti-Inflammatory Activity.

Semi-synthetic triterpenoids are antioxidant inflammation modulator (AIM) compounds that inhibit tumor cell growth and metastasis. Compounds in the AIM class bind to Keap1 and attenuate Nrf2 degradation. In the nucleus, Nrf2 increases antioxidant gene expression and reduces pro-inflammatory gene expression. By increasing Nrf2 activity, AIMs reduce reactive oxygen species and inflammation in the tumor microenvironment, which reverses tumor-mediated immune evasion and inhibits tumor growth and metastasis. AIMs also directly inhibit tumor cell growth by modulating oncogenic signaling pathways, such as IKKß/NF-κB. Here, we characterized the in vitro antioxidant, anti-inflammatory, and anticancer activities of RTA 408, a novel AIM that is currently being evaluated in patients with advanced malignancies. At low concentrations (≤ 25 nM), RTA 408 activated Nrf2 and suppressed nitric oxide and pro-inflammatory cytokine levels in interferon-γ-stimulated RAW 264.7 macrophage cells. At higher concentrations, RTA 408 inhibited tumor cell growth (GI50 = 260 ± 74 nM) and increased caspase activity in tumor cell lines, but not in normal primary human cells. Consistent with the direct effect of AIMs on IKKß, RTA 408 inhibited NF-κB signaling and decreased cyclin D1 levels at the same concentrations that inhibited cell growth and induced apoptosis. RTA 408 also increased CDKN1A (p21) levels and JNK phosphorylation. The in vitro activity profile of RTA 408 is similar to that of bardoxolone methyl, which was well-tolerated by patients at doses that demonstrated target engagement. Taken together, these data support clinical evaluation of RTA 408 for cancer treatment.

Control of mammalian glycogen synthase by PAS kinase.

The regulation of glycogen metabolism is critical for the maintenance of glucose and energy homeostasis in mammals. Glycogen synthase, the enzyme responsible for glycogen production, is regulated by multisite phosphorylation in yeast and mammals. We have previously identified PAS kinase as a physiological regulator of glycogen synthase in Saccharomyces cerevisiae. We provide evidence here that PAS kinase is an important regulator of mammalian glycogen synthase. Glycogen synthase is efficiently phosphorylated by PAS kinase in vitro at Ser-640, a known regulatory phosphosite. Efficient phosphorylation requires a region of PAS kinase outside the catalytic domain. This region appears to mediate a direct interaction between glycogen synthase and PAS kinase, thereby targeting kinase activity to this substrate specifically. This interaction is regulated by the PAS kinase PAS domain, raising the possibility that this interaction (and phosphorylation event) is modulated by the cellular metabolic state. This mode of regulation provides a mechanism for metabolic status to impinge directly on the cellular decision of whether to store or use available energy.

Coordinate regulation of sugar flux and translation by PAS kinase.

PAS kinase is a serine/threonine kinase regulated in cis by a PAS domain. A genetic study of the two PAS kinase genes in budding yeast gave evidence of the involvement of these enzymes in the control of sugar metabolism and translation. Using a biochemical screen for PAS kinase substrates, three translation factors were identified as direct phosphorylation targets. PAS kinase was also found to phosphorylate UDP-glucose pyrophosphorylase and glycogen synthase, the enzymes catalyzing the two final steps in the glycogen biosynthetic pathway. Genetic, biochemical, and physiological data provide evidence that both of these enzymes are inhibited by PAS kinase-dependent phosphorylation, thereby downregulating carbohydrate storage. These studies provide evidence of a cell-autonomous signaling system that both controls and connects the balance of fuel consumption/storage to protein synthesis.