Hsp90 Inhibition: A Promising Therapeutic Approach for ARSACS.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a neurodegenerative disease caused by mutations in the SACS gene, encoding the 520 kDa modular protein sacsin, which comprises multiple functional sequence domains that suggest a role either as a scaffold in protein folding or in proteostasis. Cells from patients with ARSACS display a distinct phenotype including altered organisation of the intermediate filament cytoskeleton and a hyperfused mitochondrial network where mitochondrial respiration is compromised. Here, we used vimentin bundling as a biomarker of sacsin function to test the therapeutic potential of Hsp90 inhibition with the C-terminal-domain-targeted compound KU-32, which has demonstrated mitochondrial activity. This study shows that ARSACS patient cells have significantly increased vimentin bundling compared to control, and this was also present in ARSACS carriers despite them being asymptomatic. We found that KU-32 treatment significantly reduced vimentin bundling in carrier and patient cells. We also found that cells from patients with ARSACS were unable to maintain mitochondrial membrane potential upon challenge with mitotoxins, and that the electron transport chain function was restored upon KU-32 treatment. Our preliminary findings presented here suggest that targeting the heat-shock response by Hsp90 inhibition alleviates vimentin bundling and may represent a promising area for the development of therapeutics for ARSACS.

Bardoxolone methyl analog attenuates proteinuria-induced tubular damage by modulating mitochondrial function.

Multiple clinical studies have shown that bardoxolone methyl, a potent activator of nuclear factor erythroid 2-related factor 2 (Nrf2), is effective in increasing glomerular filtration rate in patients with chronic kidney disease. However, whether an Nrf2 activator can protect tubules from proteinuria-induced tubular damage via anti-inflammatory and antioxidative stress mechanisms is unknown. Using an Institute of Cancer Research-derived glomerulonephritis (ICGN) mouse model of nephrosis, we examined the effects of dihydro-CDDO-trifluoroethyl amide (dh404), a rodent-tolerable bardoxolone methyl analog, in protecting the tubulointerstitium; dh404 markedly suppressed tubular epithelial cell damage in the renal interstitium of ICGN mice. The tubular epithelial cells of ICGN mice showed a decrease in the size and number of mitochondria, as well as the breakdown of the crista structure, whereas the number and ultrastructure of mitochondria were maintained by the dh404 treatment. To further determine the effect of dh404 on mitochondrial function, we used human proximal tubular cells in vitro. Stimulation with albumin and free fatty acid increased mitochondrial reactive oxygen species (ROS). However, dh404 administration diminished mitochondrial ROS. Our data show that dh404 significantly reduced proteinuria-induced tubular cell mitochondrial damage, suggesting that improved redox balance and mitochondrial function and suppression of inflammation underlie the cytoprotective mechanism of Nrf2 activators, including bardoxolone methyl, in diabetic kidney disease.-Nagasu, H., Sogawa, Y., Kidokoro, K., Itano, S., Yamamoto, T., Satoh, M., Sasaki, T., Suzuki, T., Yamamoto, M., Wigley, W. C., Proksch, J. W., Meyer, C. J., Kashihara, N. Bardoxolone methyl analog attenuates proteinuria-induced tubular damage by modulating mitochondrial function.

A novel series of cysteine-dependent, allosteric inverse agonists of the nuclear receptor RORγt.

Inhibition of the nuclear receptor Retinoic Acid Receptor-Related Orphan Receptor γt (RORγt) is a promising strategy for the treatment of autoimmune diseases. In this paper, we describe a series of allosteric, cysteine-dependent, inverse agonists of RORγt. Site-directed mutagenesis and molecular dynamics simulations are supportive of a mechanism of action through specific binding to Cys476 on alpha helix 11 of the ligand binding domain (LBD). Representative compounds in the series selectively inhibit RORγt, potently suppress interleukin-17A (IL-17A) production by human CD4+ T cells, and inhibit T helper 17 (Th17) differentiation from human naïve CD4+ T cells. The advanced compound 13 is orally bioavailable and active at a dose of 3 mg/kg in a murine collagen-induced model of rheumatoid arthritis. Collectively, these data are supportive of the development of compound 13 in autoimmune diseases.

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.

Bardoxolone methyl analogs RTA 405 and dh404 are well tolerated and exhibit efficacy in rodent models of Type 2 diabetes and obesity.

Bardoxolone methyl and related triterpenoids are well tolerated and efficacious in numerous animal models potentially relevant to patients with Type 2 diabetes and chronic kidney disease. These agents enhance glucose control and regulate lipid accumulation in rodent models of diabetes and obesity, and improve renal function, reduce inflammation, and prevent structural injury in models of renal disease. However, a recent study in Zucker diabetic fatty (ZDF) rats noted poor tolerability with the bardoxolone methyl analog RTA 405 within 1 mo after treatment initiation, although this study was confounded in part by the use of an impure RTA 405 batch. To investigate these discordant observations, the present studies were conducted to further characterize triterpenoids in rodent models of diabetes and obesity. A follow-up study was conducted in ZDF rats with two related triterpenoids (RTA 405 and dh404) for 1.5 mo. Consistent with previous rodent experience, and in contrast to the more recent ZDF report, ZDF rats administered RTA 405 or dh404 exhibited no adverse clinical signs, had laboratory values similar to controls, and exhibited no evidence of adverse liver or kidney histopathology. Additionally, RTA 405 was well tolerated in streptozotocin-induced Type 1 diabetic rats and high-fat-diet-induced obese mice. The present results are consistent with the overall published body of data obtained with triterpenoids and provide further evidence that these molecules are well tolerated without adverse effects on hepatobiliary or renal function in rodent models of diabetes and obesity.

The synthetic triterpenoid, RTA 405, increases the glomerular filtration rate and reduces angiotensin II-induced contraction of glomerular mesangial cells.

Bardoxolone methyl, a synthetic triterpenoid, improves the estimated glomerular filtration rate (GFR) in patients with chronic kidney disease and type 2 diabetes. Since the contractile activity of mesangial cells may influence glomerular filtration, we evaluated the effect of the synthetic triterpenoid RTA 405, with structural similarity to bardoxolone methyl, on GFR in rats and on mesangial cell contractility in freshly isolated glomeruli. In rats, RTA 405 increased basal GFR, assessed by inulin clearance, and attenuated the angiotensin II-induced decline in GFR. RTA 405 increased the filtration fraction, but did not affect arterial blood pressure or renal plasma flow. Glomeruli from RTA 405-treated rats were resistant to angiotensin II-induced volume reduction ex vivo. In cultured mesangial cells, angiotensin II-stimulated contraction was attenuated by RTA 405, in a dose- and time-dependent fashion. Further, Nrf2-targeted gene transcription (regulates antioxidant, anti-inflammatory, and cytoprotective responses) in mesangial cells was associated with decreased basal and reduced angiotensin II-stimulated hydrogen peroxide and calcium ion levels. These mechanisms contribute to the GFR increase that occurs following treatment with RTA 405 in rats and may underlie the effect of bardoxolone methyl on the estimated GFR in patients.

Nrf2 regulates glucose uptake and metabolism in neurons and astrocytes.

The transcription factor Nrf2 and its repressor Keap1 mediate cell stress adaptation by inducing expression of genes regulating cellular detoxification, antioxidant defence and energy metabolism. Energy production and antioxidant defence employ NADH and NADPH respectively as essential metabolic cofactors; both are generated in distinct pathways of glucose metabolism, and both pathways are enhanced by Nrf2 activation. Here, we examined the role of Nrf2 on glucose distribution and the interrelation between NADH production in energy metabolism and NADPH homeostasis using glio-neuronal cultures isolated from wild-type, Nrf2-knockout and Keap1-knockdown mice. Employing advanced microscopy imaging of single live cells, including multiphoton fluorescence lifetime imaging microscopy (FLIM) to discriminate between NADH and NADPH, we found that Nrf2 activation increases glucose uptake into neurons and astrocytes. Glucose consumption is prioritized in brain cells for mitochondrial NADH and energy production, with a smaller contribution to NADPH synthesis in the pentose phosphate pathway for redox reactions. As Nrf2 is suppressed during neuronal development, this strategy leaves neurons reliant on astrocytic Nrf2 to maintain redox balance and energy homeostasis.

Bardoxolone methyl (BARD) ameliorates ischemic AKI and increases expression of protective genes Nrf2, PPARγ, and HO-1.

Ischemic acute kidney injury (AKI) triggers expression of adaptive (protective) and maladaptive genes. Agents that increase expression of protective genes should provide a therapeutic benefit. We now report that bardoxolone methyl (BARD) ameliorates ischemic murine AKI as assessed by both renal function and pathology. BARD may exert its beneficial effect by increasing expression of genes previously shown to protect against ischemic AKI, NF-E2-related factor 2 (Nrf2), peroxisome proliferator-activated receptor-γ (PPARγ), and heme oxygenase 1 (HO-1). Although we found that BARD alone or ischemia-reperfusion alone increased expression of these genes, the greatest increase occurred after the combination of both ischemia-reperfusion and BARD. BARD had a different mode of action than other agents that regulate PPARγ and Nrf2. Thus we report that BARD regulates PPARγ, not by acting as a ligand but by increasing the amount of PPARγ mRNA and protein. This should increase ligand-independent effects of PPARγ. Similarly, BARD increased Nrf2 mRNA; this increased Nrf2 protein by mechanisms in addition to the prolongation of Nrf2 protein half-life previously reported. Finally, we localized expression of these protective genes after ischemia and BARD treatment. Using double-immunofluorescence staining for CD31 and Nrf2 or PPARγ, we found increased Nrf2 and PPARγ on glomerular endothelia in the cortex; Nrf2 was also present on cortical peritubular capillaries. In contrast, HO-1 was localized to different cells, i.e., tubules and interstitial leukocytes. Although Nrf2-dependent increases in HO-1 have been described, our data suggest that BARD's effects on tubular and leukocyte HO-1 during ischemic AKI may be Nrf2 independent. We also found that BARD ameliorated cisplatin nephrotoxicity.

Effects of Bardoxolone Methyl on Hepatic Enzymes in Patients with Type 2 Diabetes Mellitus and Stage 4 CKD.

In a multinational placebo-controlled phase III clinical trial in 2,185 patients with type 2 diabetes mellitus and stage 4 chronic kidney disease, treatment with the Nrf2 activator bardoxolone methyl increased estimated glomerular filtration rate, a measure of kidney function, but also resulted in increases in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma glutamyl transferase. These increases in liver enzyme level(s) were maximal after 4 weeks of treatment and reversible, trending back toward baseline through week 48. Total bilirubin concentrations did not increase, and no cases met Hy's Law criteria, although two subjects had ALT concentrations that exceeded 10 × the upper limit of the population reference range leading to discontinuation of treatment. Animal and cell culture experiments suggested that the increases in ALT and AST induced by bardoxolone methyl may be related to its pharmacological activity. Bardoxolone methyl significantly induced the mRNA expression of ALT and AST isoforms in cultured cells. Expression of ALT and AST isoforms in liver and kidney also positively correlated with Nrf2 status in mice. Overall, these data suggest that the increases in ALT and AST observed clinically were, at least in part, related to the pharmacological induction of aminotransferases via Nrf2 activation, rather than to any intrinsic form of hepatotoxicity.

Hook2 contributes to aggresome formation.

BACKGROUND: Aggresomes are pericentrosomal accumulations of misfolded proteins, chaperones and proteasomes. Their positioning near the centrosome, like that of other organelles, requires active, microtubule-dependent transport. Linker proteins that can associate with the motor protein dynein, organelles, and microtubules are thought to contribute to the active maintenance of the juxtanuclear localization of many membrane bound organelles and aggresomes. Hook proteins have been proposed to serve as adaptors for the association of cargos with dynein for transport on microtubules. Hook2 was shown to localize to the centrosome, bind centriolin, and contribute to centrosomal function. RESULTS: Here we show that overexpression of hook2 promotes the accumulation of the cystic fibrosis transmembrane regulator in aggresomes without altering its biochemical properties or its steady state level. A dominant negatively acting form of hook2 that lacks the centriolin binding C-terminal inhibits aggresome formation. CONCLUSION: We propose that hook2 contributes to the establishment and maintenance of the pericentrosomal localization of aggresomes by promoting the microtubule-based delivery of protein aggregates to pericentriolar aggresomes.

Aggresome formation.

Bulk protein degradation in the cell is catalyzed by the ubiquitin-proteasome system (UPS). At the heart of the UPS is the proteasome, a large multisubunit tightly-regulated protease. The UPS performs key functions in protein quality control by monitoring and eliminating potentially toxic misfolded or damaged proteins. When the capacity of this protease system is exceeded, misfolded protein substrates aggregate and are assembled through an active and regulated process to form an aggresome. Aggresomes are dynamic structures, formed as a general response to an overload of improperly folded proteins. Assembly of aggresomes occurs at the centrosome, a perinuclear structure that also serves as a site for the recruitment and concentration of components of the UPS, including the proteasome, its regulators, and other proteins typically involved in protein quality control. Thus, in addition to other cellular activities, the centrosome may play a central role in protein quality control, sitting at the crossroads of protein folding, degradation, and aggregation.

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.

Synthesis, chemical reactivity as Michael acceptors, and biological potency of monocyclic cyanoenones, novel and highly potent anti-inflammatory and cytoprotective agents.

Novel monocyclic cyanoenones examined to date display unique features regarding chemical reactivity as Michael acceptors and biological potency. Remarkably, in some biological assays, the simple structure is more potent than pentacyclic triterpenoids (e.g., CDDO and bardoxolone methyl) and tricycles (e.g., TBE-31). Among monocyclic cyanoenones, 1 is a highly reactive Michael acceptor with thiol nucleophiles. Furthermore, an important feature of 1 is that its Michael addition is reversible. For the inhibition of NO production, 1 shows the highest potency. Notably, its potency is about three times higher than CDDO, whose methyl ester (bardoxolone methyl) is presently in phase III clinical trials. For the induction of NQO1, 1 also demonstrated the highest potency. These results suggest that the reactivity of these Michael acceptors is closely related to their biological potency. Interestingly, in LPS-stimulated macrophages, 1 causes apoptosis and inhibits secretion of TNF-α and IL-1ß with potencies that are higher than those of bardoxolone methyl and TBE-31.

A screen to identify cellular modulators of soluble levels of an amyotrophic lateral sclerosis (ALS)-causing mutant SOD1.

The molecular pathology of many protein misfolding, toxic gain-of-function diseases, such as amyotrophic lateral sclerosis (ALS), is not well understood. Although protein misfolding and aggregation are common themes in these diseases, efforts to identify cellular factors that regulate this process in an unbiased fashion and on a global scale have been lacking. Using an adapted version of an extant ß-gal-based protein solubility assay, an expression screen for cellular modulators of solubility of an ALS-causing mutant SOD1 was carried out in mammalian cells. Following fluorescence-activated cell sorting enrichment of a mouse spinal cord cDNA library for gene products that increased SOD1 solubility, high-throughput screening of the cDNA pools from this enriched fraction was employed to identify pools containing relevant modulators. Positive pools, containing approximately 10 cDNA clones each, were diluted and rescreened iteratively until individual clones that improved SOD1 folding/solubility were identified. Genes with profound effects in the solubility assay were selected for validation by independent biochemical assays. Six of 10 validated genes had a significant effect on SOD1 solubility and folding in a SOD1 promoter-driven ß-gal assay, indicating that global screening of cellular targets using such protein solubility/folding assay is viable and can be adapted for other misfolding diseases.

A protein sequence that can encode native structure by disfavoring alternate conformations.

The linear sequence of amino acids contains all the necessary information for a protein to fold into its unique three-dimensional structure. Native protein sequences are known to accomplish this by promoting the formation of stable, kinetically accessible structures. Here we describe a Pro residue in the center of the third transmembrane helix of the cystic fibrosis transmembrane conductance regulator that promotes folding by a distinct mechanism: disfavoring the formation of a misfolded structure. The generality of this mechanism is supported by genome-wide transmembrane sequence analyses. Furthermore, the results provide an explanation for the increased frequency of Pro residues in transmembrane alpha-helices. Incorporation by nature of such 'negative folding determinants', aimed at preventing the formation of off-pathway structures, represents an additional mechanism by which folding information is encoded within the evolved sequences of proteins.