Deglutarylation of glutaryl-CoA dehydrogenase by deacylating enzyme SIRT5 promotes lysine oxidation in mice.

A wide range of protein acyl modifications has been identified on enzymes across various metabolic processes; however, the impact of these modifications remains poorly understood. Protein glutarylation is a recently identified modification that can be nonenzymatically driven by glutaryl-CoA. In mammalian systems, this unique metabolite is only produced in the lysine and tryptophan oxidative pathways. To better understand the biology of protein glutarylation, we studied the relationship between enzymes within the lysine/tryptophan catabolic pathways, protein glutarylation, and regulation by the deglutarylating enzyme sirtuin 5 (SIRT5). Here, we identify glutarylation on the lysine oxidation pathway enzyme glutaryl-CoA dehydrogenase (GCDH) and show increased GCDH glutarylation when glutaryl-CoA production is stimulated by lysine catabolism. Our data reveal that glutarylation of GCDH impacts its function, ultimately decreasing lysine oxidation. We also demonstrate the ability of SIRT5 to deglutarylate GCDH, restoring its enzymatic activity. Finally, metabolomic and bioinformatic analyses indicate an expanded role for SIRT5 in regulating amino acid metabolism. Together, these data support a feedback loop model within the lysine/tryptophan oxidation pathway in which glutaryl-CoA is produced, in turn inhibiting GCDH function via glutaryl modification of GCDH lysine residues and can be relieved by SIRT5 deacylation activity.

The Lateral Metalation of Isoxazolo[3,4-d]pyridazinones towards Hit-to-Lead Development of Selective Positive Modulators of Metabotropic Glutamate Receptors.

Isoxazolo[3,4-d] pyridazinones ([3,4-d]s) were previously shown to have selective positive modulation at the metabotropic glutamate receptor (mGluR) Subtypes 2 and 4, with no functional cross-reactivity at mGluR1a, mGluR5, or mGluR8. Additional analogs were prepared to access more of the allosteric pocket and achieve higher binding affinity, as suggested by homology modeling. Two different sets of analogs were generated. One uses the fully formed [3,4-d] with an N6-aryl with and without halogens. These underwent successful selective lateral metalation and electrophilic quenching (LM&EQ) at the C3 of the isoxazole. In a second set of analogs, a phenyl group was introduced at the C4 position of the [3,4-d] ring via a condensation of 4-phenylacetyl-3-ethoxcarbonyl-5-methyl isoxazole with the corresponding hydrazine to generate the 3,4-ds 2b and 2j to 2n.

10-Alkoxy-anthracenyl-isoxazole analogs have sub-micromolar activity against a Glioblastoma multiforme cell line.

A series of 10-alkoxy-Anthryl-isoxazole-pyrrole-doubletails (RO-AIMs) were synthesized using a crown ether assisted nucleophilic aromatic substitution followed by a modified Schotten-Baumann reaction. The novel RO-AIMs described here exhibit robust growth inhibition for the human SNB19 CNS glioblastoma cell line, and biphenyl analog 8c had activity in the nanomolar regime, which represents the most efficacious compound in the AIM series to date. Computational modeling for RO-AIMs binding in a ternary complex with c-myc quadruplex DNA and its helicase DHX36 is presented which represents our current working hypothesis.

Persistent, Progressive Pulmonary Fibrosis and Epithelial Remodeling in Mice.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic interstitial lung disease with underlying mechanisms that have been primarily investigated in mice after intratracheal instillation of a single dose of bleomycin. However, the model has significant limitations, including transient fibrosis that spontaneously resolves and its failure to fully recapitulate the epithelial remodeling in the lungs of patients with IPF. Thus, there remains an unmet need for a preclinical model with features that more closely resemble the human disease. Repetitive intratracheal instillation of bleomycin has previously been shown to recapitulate some of these features, but the instillation procedure is complex, and the long-term consequences on epithelial remodeling and fibrosis persistence and progression remain poorly understood. Here, we developed a simplified repetitive bleomycin instillation strategy consisting of three bi-weekly instillations that leads to persistent and progressive pulmonary fibrosis. Lung histology demonstrates increased collagen deposition, fibroblast accumulation, loss of type I and type II alveolar epithelial cells within fibrotic areas, bronchiolization of the lung parenchyma with CCSP+ cells, remodeling of the distal lung into cysts reminiscent of simple honeycombing, and accumulation of hyperplastic transitional KRT8+ epithelial cells. Micro-computed tomographic imaging demonstrated significant traction bronchiectasis and subpleural fibrosis. Thus, the simplified repetitive bleomycin instillation strategy leads to progressive fibrosis and recapitulates the histological and radiographic characteristics of IPF. Compared with the single bleomycin instillation model, we suggest that the simplified repetitive instillation model may be better suited to address mechanistic questions about IPF pathogenesis and preclinical studies of antifibrotic drug candidates.

N-Substituted pyrrolopyrimidines and purines as p90 ribosomal S6 protein kinase-2 (RSK2) inhibitors.

The RSK2 kinase is the downstream effector of the Ras/Raf/MEK/ERK pathway, that is often aberrantly activated in acute myeloid leukemia (AML). Recently, we reported a structure-activity study for BI-D1870, the pan-RSK inhibitor, and identified pteridinones that inhibited cellular RSK2 activity that did not result in concomitant cytotoxicity. In the current study, we developed a series of pyrrolopyrimidines and purines to replace the pteridinone ring of BI-D1870, with a range of N-substituents that extend to the substrate binding site to probe complementary interactions, while retaining the 2,6-difluorophenol-4-amino group to maintain interactions with the hinge domain and the DFG motif. Several compounds inhibited cellular RSK2 activity, and we identified compounds that uncoupled cellular RSK2 inhibition from potent cytotoxicity in the MOLM-13 AML cell line. These N-substituted probes have revealed an opportunity to further examine substituents that extend from the ATP- to the substrate-binding site may confer improved RSK potency and selectivity.

4-Hydroxy-2-nonenal attenuates 8-oxoguanine DNA glycosylase 1 activity.

Elevated cellular oxidative stress and oxidative DNA damage are key contributors to impaired cardiac function in diabetes. During chronic inflammation, reactive oxygen species (ROS)-induced lipid peroxidation results in the formation of reactive aldehydes, foremost of which is 4-hydroxy-2-nonenal (4HNE). 4HNE forms covalent adducts with proteins, negatively impacting cellular protein function. During conditions of elevated oxidative stress, oxidative DNA damage such as modification by 8-hydroxydeoxyguanosine (8OHdG) is repaired by 8-oxoguanine glycosylase-1 (OGG-1). Based on these facts, we hypothesized that 4HNE forms adducts with OGG-1 inhibiting its activity, and thus, increases the levels of 8OHG in diabetic heart tissues. To test our hypothesis, we evaluated OGG-1 activity, 8OHG and 4HNE in the hearts of leptin receptor deficient db/db mice, a type-2 diabetic model. We also treated the recombinant OGG-1 with 4HNE to measure direct adduction. We found decreased OGG-1 activity (P > .05), increased 8OHG (P > .05) and increased 4HNE adducts (P > .05) along with low aldehyde dehydrogenase-2 activity (P > .05). The increased colocalization of OGG-1 and 4HNE in cardiomyocytes suggest 4HNE adduction on OGG-1. Furthermore, colocalization of 8OHG and OGG-1 with mitochondrial markers TOM 20 and aconitase, respectively, indicated significant levels of oxidatively-induced mtDNA damage and implicated a role for mitochondrial OGG-1 function. In vitro exposure of recombinant OGG-1 (rOGG-1) with increasing concentrations of 4HNE resulted in a concentration-dependent decrease in OGG-1 activity. Mass spectral analysis of trypsin digests of 4HNE-treated rOGG-1 identified 4HNE adducts on C28, C75, C163, H179, H237, C241, K249, H270, and H282. In silico molecular modeling of 4HNE-K249 OGG-1 and 4HNE-H270 OGG-1 mechanistically supported 4HNE-mediated enzymatic inhibition of OGG-1. In conclusion, these data support the hypothesis that inhibition of OGG-1 by direct modification by 4HNE contributes to decreased OGG-1 activity and increased 8OHG-modified DNA that are present in the diabetic heart.

Substituted pteridinones as p90 ribosomal S6 protein kinase (RSK) inhibitors: A structure-activity study.

The activity of p90 ribosomal S6 kinase 2 (RSK2) has emerged as an attractive target for cancer therapy due to its role in the regulation of diverse cellular processes, such as cell transformation and proliferation. Several pan-RSK inhibitors have been identified with BI-D1870 and the pseudo-analogs LJH685 and LJI308 being the most selective, potent, and frequently used small molecule inhibitors. We designed and synthesized a series of pteridinones and pyrimidines to evaluate the structural features of BI-D1870 that are required for RSK2 inhibition. We have identified inhibitors of RSK2 activity, evaluated their target engagement in cells, and measured their effect on cell viability and cytotoxicity in the MOLM-13 acute myeloid leukemia (AML) cell line. The results of our studies support that RSK2 inhibition can be achieved in MOLM-13 cells without potent cytotoxicity. The structure-activity data from this study will be used as a platform to develop novel RSK2 inhibitors.

10-N-heterocylic aryl-isoxazole-amides (AIMs) have robust anti-tumor activity against breast and brain cancer cell lines and useful fluorescence properties.

A novel series of anthracenyl-isoxazole amide (AIM) antitumor agents containing N-heterocycles in the 10 position (N-het) were synthesized using palladium cross-coupling. The unique steric environment of the N-het-AIMs required individual optimization in each case. Lanthanide mediated double activation was used to couple the dimethylamino pyrrole moiety, required for antitumor action. Robust antitumor activity was observed against breast and brain cancer cell lines. The compounds were docked with the c-myc oncogene promoter sequence, which adopts a G4 quadruplex DNA conformation, and represents the working hypothesis for biological action. The N-het-AIMs have useful fluorescence properties, allowing for observation of their distribution within tumor cells.

Compensatory Expression of Nur77 and Nurr1 Regulates NF-κB-Dependent Inflammatory Signaling in Astrocytes.

Inflammatory activation of glial cells promotes loss of dopaminergic neurons in Parkinson disease. The transcription factor nuclear factor κB (NF-κB) regulates the expression of multiple neuroinflammatory cytokines and chemokines in activated glial cells that are damaging to neurons. Thus, inhibition of NF-κB signaling in glial cells could be a promising therapeutic strategy for the prevention of neuroinflammatory injury. Nuclear orphan receptors in the NR4A family, including NR4A1 (Nur77) and NR4A2 (Nurr1), can inhibit the inflammatory effects of NF-κB, but no approved drugs target these receptors. Therefore, we postulated that a recently developed NR4A receptor ligand, 1,1bis (3'indolyl) 1(pmethoxyphenyl) methane (C-DIM5), would suppress NF-κB-dependent inflammatory gene expression in astrocytes after treatment with 1-methyl-4-phenyl 1, 2, 3, 6-tetrahydropyridine (MPTP) and the inflammatory cytokines interferon γ and tumor necrosis factor α C-DIM5 increased expression of Nur77 mRNA and suppressed expression of multiple neuroinflammatory genes. C-DIM5 also inhibited the expression of NFκB-regulated inflammatory and apoptotic genes in quantitative polymerase chain reaction array studies and effected p65 binding to unique genes in chromatin immunoprecipitation next-generation sequencing experiments but did not prevent p65 translocation to the nucleus, suggesting a nuclear-specific mechanism. C-DIM5 prevented nuclear export of Nur77 in astrocytes induced by MPTP treatment and simultaneously recruited Nurr1 to the nucleus, consistent with known transrepressive properties of this receptor. Combined RNAi knockdown of Nur77 and Nurr1 inhibited the anti-inflammatory activity of C-DIM5, demonstrating that C-DIM5 requires these receptors to inhibit NF-κB. Collectively, these data demonstrate that NR4A1/Nur77 and NR4A2/Nurr1 dynamically regulated inflammatory gene expression in glia by modulating the transcriptional activity of NF-κB.

The Nurr1 Ligand,1,1-bis(3'-Indolyl)-1-(p-Chlorophenyl)Methane, Modulates Glial Reactivity and Is Neuroprotective in MPTP-Induced Parkinsonism.

The orphan nuclear receptor Nurr1 (also called nuclear receptor-4A2) regulates inflammatory gene expression in glial cells, as well as genes associated with homeostatic and trophic function in dopaminergic neurons. Despite these known functions of Nurr1, an endogenous ligand has not been discovered. We postulated that the activation of Nurr1 would suppress the activation of glia and thereby protect against loss of dopamine (DA) neurons after subacute lesioning with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Our previous studies have shown that a synthetic Nurr1 ligand, 1,1-bis(3'-indolyl)-1-(p-chlorophenyl)methane (C-DIM12), suppresses inflammatory gene expression in primary astrocytes and induces a dopaminergic phenotype in neurons. Pharmacokinetic analysis of C-DIM12 in mice by liquid chromatography-mass spectrometry demonstrated that approximately three times more compound concentrated in the brain than in plasma. Mice treated with four doses of MPTP + probenecid over 14 days were monitored for neurobehavioral function, loss of dopaminergic neurons, and glial activation. C-DIM12 protected against the loss of DA neurons in the substantia nigra pars compacta and DA terminals in the striatum, maintained a ramified phenotype in microglia, and suppressed activation of astrocytes. In vitro reporter assays demonstrated that C-DIM12 was an effective activator of Nurr1 transcription in neuronal cell lines. Computational modeling of C-DIM12 binding to the three-dimensional structure of human Nurr1 identified a high-affinity binding interaction with Nurr1 at the coactivator domain. Taken together, these data suggest that C-DIM12 is an activator of Nurr1 that suppresses glial activation and neuronal loss in vivo after treatment with MPTP, and that this receptor could be an efficacious target for disease modification in individuals with Parkinson's disease and related disorders.

Isoxazolo[3,4-d]pyridazinones positively modulate the metabotropic glutamate subtypes 2 and 4.

Isoxazolo[3,4-d] pyridazinones ([3,4-d]s) are selective positive modulators of the metabotropic glutamate receptors (mGluRs) subtypes 2 and 4, with no functional cross reactivity at mGluR1a, mGLuR5 or mGluR8. Modest binding for two of the [3,4-d]s is observed at the allosteric fenobam mGluR5 site, but not sufficient to translate into a functional effect. The structure activity relationship (SAR) for mGluR2 and mGluR4 are distinct: the compounds which select for mGluR2 all contain fluorine on the N-6 aryl group. Furthermore, the [3,4-d]s in this study showed no significant binding at inhibitory GABAA, nor excitatory NMDA receptors, and previously we had disclosed that they lack significant activity at the System Xc-Antiporter. A homology model based on Conn's mGluR1 crystal structure was examined, and suggested explanations for a preference for allosteric over orthosteric binding, subtype selectivity, and suggested avenues for optimization of efficacy as a reasonable working hypothesis.

Investigating the Sensitivity of NAD+-dependent Sirtuin Deacylation Activities to NADH.

Protein lysine posttranslational modification by an increasing number of different acyl groups is becoming appreciated as a regulatory mechanism in cellular biology. Sirtuins are class III histone deacylases that use NAD(+)as a co-substrate during amide bond hydrolysis. Several studies have described the sirtuins as sensors of the NAD(+)/NADH ratio, but it has not been formally tested for all the mammalian sirtuinsin vitro To address this problem, we first synthesized a wide variety of peptide-based probes, which were used to identify the range of hydrolytic activities of human sirtuins. These probes included aliphatic ϵ-N-acyllysine modifications with hydrocarbon lengths ranging from formyl (C1) to palmitoyl (C16) as well as negatively charged dicarboxyl-derived modifications. In addition to the well established activities of the sirtuins, "long chain" acyllysine modifications were also shown to be prone to hydrolytic cleavage by SIRT1-3 and SIRT6, supporting recent findings. We then tested the ability of NADH, ADP-ribose, and nicotinamide to inhibit these NAD(+)-dependent deacylase activities of the sirtuins. In the commonly used 7-amino-4-methylcoumarin-coupled fluorescence-based assay, the fluorophore has significant spectral overlap with NADH and therefore cannot be used to measure inhibition by NADH. Therefore, we turned to an HPLC-MS-based assay to directly monitor the conversion of acylated peptides to their deacylated forms. All tested sirtuin deacylase activities showed sensitivity to NADH in this assay. However, the inhibitory concentrations of NADH in these assays are far greater than the predicted concentrations of NADH in cells; therefore, our data indicate that NADH is unlikely to inhibit sirtuinsin vivo These data suggest a re-evaluation of the sirtuins as direct sensors of the NAD(+)/NADH ratio.

Characterizing Sirtuin 3 Deacetylase Affinity for Aldehyde Dehydrogenase 2.

Mitochondrial aldehyde dehydrogenase (ALDH2) plays a central role in the detoxification of reactive aldehydes generated through endogenous and exogenous sources. The biochemical regulation of enzyme activity through post-translational modification provides an intricate response system regulating mitochondrial detoxification pathways. ALDH2 is a known target of lysine acetylation, which arises as a consequence of mitochondrial bioenergetic flux and sirtuin deacetylase activity. The mitochondrial deacetylase Sirtuin 3 (SIRT3) has been reported to alter ALDH2 lysine acetylation status, yet the mechanism and consequence of this interaction remain unknown. The in vitro results presented here provide a novel biochemical approach using stable-isotope dilution mass spectrometry to elucidate which lysine residues are targeted by SIRT3 for deacetylation. Furthermore, HPLC-MS/MS and computational modeling elucidate a potential role for acetyl-Lys369 on ALDH2 in perturbing normal ß-nicotinamide adenine dinucleotide (NAD+) cofactor binding.

Chronic Ethanol Metabolism Inhibits Hepatic Mitochondrial Superoxide Dismutase via Lysine Acetylation.

BACKGROUND: Chronic ethanol (EtOH) consumption is a major cause of liver disease worldwide. Oxidative stress is a known consequence of EtOH metabolism and is thought to contribute significantly to alcoholic liver disease (ALD). Therefore, elucidating pathways leading to sustained oxidative stress and downstream redox imbalances may reveal how EtOH consumption leads to ALD. Recent studies suggest that EtOH metabolism impacts mitochondrial antioxidant processes through a number of proteomic alterations, including hyperacetylation of key antioxidant proteins. METHODS: To elucidate mechanisms of EtOH-induced hepatic oxidative stress, we investigate a role for protein hyperacetylation in modulating mitochondrial superoxide dismutase (SOD2) structure and function in a 6-week Lieber-DeCarli murine model of EtOH consumption. Our experimental approach includes immunoblotting immunohistochemistry (IHC), activity assays, mass spectrometry, and in silico modeling. RESULTS: We found that EtOH metabolism significantly increased the acetylation of SOD2 at 2 functionally relevant lysine sites, K68 and K122, resulting in a 40% decrease in enzyme activity while overall SOD2 abundance was unchanged. In vitro studies also reveal which lysine residues are more susceptible to acetylation. IHC analysis demonstrates that SOD2 hyperacetylation occurs near zone 3 within the liver, which is the main EtOH-metabolizing region of the liver. CONCLUSIONS: Overall, the findings presented in this study support a role for EtOH-induced lysine acetylation as an adverse posttranslational modification within the mitochondria that directly impacts SOD2 charge state and activity. Last, the data presented here indicate that protein hyperacetylation may be a major factor contributing to an imbalance in hepatic redox homeostasis due to chronic EtOH metabolism.

Dimeric isoxazolyl-1,4-dihydropyridines have enhanced binding at the multi-drug resistance transporter.

A series of dimeric isoxazolyl-1,4-dihydropyridines (IDHPs) were prepared by click chemistry and examined for their ability to bind the multi-drug resistance transporter (MDR-1), a member of the ATP-binding cassette superfamily (ABC). Eight compounds in the present study exhibited single digit micromolar binding to this efflux transporter. One monomeric IDHP m-Br-1c, possessed submicromolar binding of 510nM at MDR-1. Three of the dimeric IDHPs possessed <1.5µM activity, and 4b and 4c were observed to have superior binding selectivity compared to their corresponding monomers verses the voltage gated calcium channel (VGCC). The dimer with the best combination of activity and selectivity for MDR-1 was analog 4c containing an m-Br phenyl moiety in the 3-position of the isoxazole, and a tether with five ethyleneoxy units, referred to herein as Isoxaquidar. Two important controls, mono-triazole 5 and pyridine 6, also were examined, indicating that the triazole - incorporated as part of the click assembly as a spacer - contributes to MDR-1 binding. Compounds were also assayed at the allosteric site of the mGluR5 receptor, as a GPCR 7TM control, indicating that the p-Br IDHPs 4d, 4e and 4f with tethers of from n=2 to 5 ethylenedioxy units, had sub-micromolar affinities with 4d being the most efficacious at 193nM at mGluR5. The results are interpreted using a docking study using a human ABC as our current working hypothesis, and suggest that the distinct SARs emerging for these three divergent classes of biomolecular targets may be tunable, and amenable to the development of further selectivity.

ROCK and Rho: Promising therapeutic targets to ameliorate pulmonary fibrosis.

This commentary highlights the article by Sisson et al, which establishes the importance of the myocardin-related transcription factor/serum response factor signaling pathway as a therapeutic target in the management of fibrotic lung disease.

Identification of 5' AMP-activated kinase as a target of reactive aldehydes during chronic ingestion of high concentrations of ethanol.

The production of reactive aldehydes including 4-hydroxy-2-nonenal (4-HNE) is a key component of the pathogenesis in a spectrum of chronic inflammatory hepatic diseases including alcoholic liver disease (ALD). One consequence of ALD is increased oxidative stress and altered ß-oxidation in hepatocytes. A major regulator of ß-oxidation is 5' AMP protein kinase (AMPK). In an in vitro cellular model, we identified AMPK as a direct target of 4-HNE adduction resulting in inhibition of both H2O2 and 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR)-induced downstream signaling. By employing biotin hydrazide capture, it was confirmed that 4-HNE treatment of cells resulted in carbonylation of AMPKα/ß, which was not observed in untreated cells. Using a murine model of alcoholic liver disease, treatment with high concentrations of ethanol resulted in an increase in phosphorylated as well as carbonylated AMPKα. Despite increased AMPK phosphorylation, there was no significant change in phosphorylation of acetyl CoA carboxylase. Mass spectrometry identified Michael addition adducts of 4-HNE on Cys(130), Cys(174), Cys(227), and Cys(304) on recombinant AMPKα and Cys(225) on recombinant AMPKß. Molecular modeling analysis of identified 4-HNE adducts on AMPKα suggest that inhibition of AMPK occurs by steric hindrance of the active site pocket and by inhibition of hydrogen peroxide induced oxidation. The observed inhibition of AMPK by 4-HNE provides a novel mechanism for altered ß-oxidation in ALD, and these data demonstrate for the first time that AMPK is subject to regulation by reactive aldehydes in vivo.

Allosteric inhibitors of the Eya2 phosphatase are selective and inhibit Eya2-mediated cell migration.

Eya proteins are essential co-activators of the Six family of transcription factors and contain a unique tyrosine phosphatase domain belonging to the haloacid dehalogenase family of phosphatases. The phosphatase activity of Eya is important for the transcription of a subset of Six1-target genes, and also directs cells to the repair rather than apoptosis pathway upon DNA damage. Furthermore, Eya phosphatase activity has been shown to mediate transformation, invasion, migration, and metastasis of breast cancer cells, making it a potential new drug target for breast cancer. We have previously identified a class of N-arylidenebenzohydrazide compounds that specifically inhibit the Eya2 phosphatase. Herein, we demonstrate that these compounds are reversible inhibitors that selectively inhibit the phosphatase activity of Eya2, but not Eya3. Our mutagenesis results suggest that this class of compounds does not bind to the active site and the binding does not require the coordination with Mg(2+). Moreover, these compounds likely bind within a site on the opposite face of the active site, and function as allosteric inhibitors. We also demonstrate that this class of compounds inhibits Eya2 phosphatase-mediated cell migration, setting the foundation for these molecules to be developed into chemical probes for understanding the specific function of the Eya2 phosphatase and to serve as a prototype for the development of Eya2 phosphatase specific anti-cancer drugs.

AIMing towards improved antitumor efficacy.

Using the structure-activity relationship emerging from previous Letter, and guided by pharmacokinetic properties, new AIMs have been prepared with both improved efficacy against human glioblastoma cells and cell permeability as determined by fluorescent confocal microscopy. We present our first unambiguous evidence for telomeric G4-forming oligonucleotide anisotropy by NMR resulting from direct interaction with AIMs, which is consistent with both our G4 melting studies by CD, and our working hypothesis. Finally, we show that AIMs induce apoptosis in SNB-19 cells.

19-substituted benzoquinone ansamycin heat shock protein-90 inhibitors: biological activity and decreased off-target toxicity.

The benzoquinone ansamycins (BQAs) are a valuable class of antitumor agents that serve as inhibitors of heat shock protein (Hsp)-90. However, clinical use of BQAs has resulted in off-target toxicities, including concerns of hepatotoxicity. Mechanisms underlying the toxicity of quinones include their ability to redox cycle and/or arylate cellular nucleophiles. We have therefore designed 19-substituted BQAs to prevent glutathione conjugation and nonspecific interactions with protein thiols to minimize off-target effects and reduce hepatotoxicity. 19-Phenyl- and 19-methyl-substituted versions of geldanamycin and its derivatives, 17-allylamino-17-demethoxygeldanamycin and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), did not react with glutathione, whereas marked reactivity was observed using parent BQAs. Importantly, although 17-DMAG induced cell death in primary and cultured mouse hepatocytes, 19-phenyl and 19-methyl DMAG showed reduced toxicity, validating the overall approach. Furthermore, our data suggest that arylation reactions, rather than redox cycling, are a major mechanism contributing to BQA hepatotoxicity. 19-Phenyl BQAs inhibited purified Hsp90 in a NAD(P)H: quinone oxidoreductase 1 (NQO1)-dependent manner, demonstrating increased efficacy of the hydroquinone ansamycin relative to its parent quinone. Molecular modeling supported increased stability of the hydroquinone form of 19-phenyl-DMAG in the active site of human Hsp90. In human breast cancer cells, 19-phenyl BQAs induced growth inhibition also dependent upon metabolism via NQO1 with decreased expression of client proteins and compensatory induction of Hsp70. These data demonstrate that 19-substituted BQAs are unreactive with thiols, display reduced hepatotoxicity, and retain Hsp90 and growth-inhibitory activity in human breast cancer cells, although with diminished potency relative to parent BQAs.