Geldanamycin remains a driver in the medicinal chemistry of heat shock protein 90 (Hsp90) inhibition, even half a century after its original isolation from nature. This Perspective focuses on the properties of the benzoquinone ring of the natural product that enable a range of functionalization reactions to take place. Therefore, inherent reactivity at C-17, where the methoxy group serves as a vinylogous ester, and at C-19 that demonstrates nucleophilic, enamide-type character toward electrophiles, and also as a conjugate acceptor to react with nucleophiles, has facilitated the synthesis of semisynthetic derivatives. Thus, a range of C-17-substituted amine derivatives has been investigated in oncology applications, with a number of compounds in this series reaching clinical trials. In contrast, the 19-position of geldanamycin has received less attention, although 19-substituted derivatives offer promise with markedly reduced toxicity compared to geldanamycin itself, while retaining Hsp90 inhibitory activity albeit with diminished potency in cellular studies.

BACKGROUND:Despite advancements in chronic myeloid leukemia (CML) therapy with tyrosine kinase inhibitors (TKIs), resistance and intolerance remain significant challenges. Leukemia stem cells (LSCs) and TKI-resistant cells rely on altered mitochondrial metabolism and oxidative phosphorylation. Targeting rewired energy metabolism and inducing non-apoptotic cell death, along with the release of damage-associated molecular patterns (DAMPs), can enhance therapeutic strategies and immunogenic therapies against CML and prevent the emergence of TKI-resistant cells and LSC persistence.METHODS:Transcriptomic analysis was conducted using datasets of CML patients' stem cells and healthy cells. DNA damage was evaluated by fluorescent microscopy and flow cytometry. Cell death was assessed by trypan blue exclusion test, fluorescent microscopy, flow cytometry, colony formation assay, and in vivo Zebrafish xenografts. Energy metabolism was determined by measuring NAD+ and NADH levels, ATP production rate by Seahorse analyzer, and intracellular ATP content. Mitochondrial fitness was estimated by measurements of mitochondrial membrane potential, ROS, and calcium accumulation by flow cytometry, and morphology was visualized by TEM. Bioinformatic analysis, real-time qPCR, western blotting, chemical reaction prediction, and molecular docking were utilized to identify the drug target. The immunogenic potential was assessed by high mobility group box (HMGB)1 ELISA assay, luciferase-based extracellular ATP assay, ectopic calreticulin expression by flow cytometry, and validated by phagocytosis assay, and in vivo vaccination assay using syngeneic C57BL/6 mice.RESULTS:Transcriptomic analysis identified metabolic alterations and DNA repair deficiency signatures in CML patients. CML patients exhibited enrichment in immune system, DNA repair, and metabolic pathways. The gene signature associated with BRCA mutated tumors was enriched in CML datasets, suggesting a deficiency in double-strand break repair pathways. Additionally, poly(ADP-ribose) polymerase (PARP)1 was significantly upregulated in CML patients' stem cells compared to healthy counterparts. Consistent with the CML patient DNA repair signature, treatment with the methylated indolequinone MAC681 induced DNA damage, mitochondrial dysfunction, calcium homeostasis disruption, metabolic catastrophe, and necroptotic-like cell death. In parallel, MAC681 led to PARP1 degradation that was prevented by 3-aminobenzamide. MAC681-treated myeloid leukemia cells released DAMPs and demonstrated the potential to generate an immunogenic vaccine in C57BL/6 mice. MAC681 and asciminib exhibited synergistic effects in killing both imatinib-sensitive and -resistant CML, opening new therapeutic opportunities.CONCLUSIONS:Overall, increasing the tumor mutational burden by PARP1 degradation and mitochondrial deregulation makes CML suitable for immunotherapy.

Alzheimer's disease (AD) is one of the most prominent neurodegenerative diseases. Results from animal and cellular models suggest that FAD-deficient forms of NAD(P)H quinone oxidoreductase 1 (NQO1) may accelerate the aggregation of Alzheimer's amyloid-β peptide (Aβ1-42). Here, we examined in vitro whether NQO1 and its FAD-deficient P187S mutation (NQO1*2) directly interact with Aβ1-42 and modify its rate of aggregation. When monitored using the fluorescence of either noncovalent thioflavin T (ThT) or HiLyte Fluor 647 (HF647) dye covalently attached to the Aβ1-42 peptide, the aggregation kinetics of Aβ1-42 were markedly more rapid in the presence of NQO1*2 than the wild-type (WT) NQO1. Experiments using apo-NQO1 indicate that this increase is linked to the inability of NQO1*2 to bind to FAD. Furthermore, dicoumarol, an NQO1 inhibitor that binds near the FAD-binding site and stabilizes NQO1*2, markedly decreased the aggregation kinetics of Aβ1-42. Imaging flow cytometry confirmed in-vitro coaggregation of NQO1 isoforms and Aβ1-42. Aβ1-42 alone forms rod-shaped fibril structures while in the presence of NQO1 isoforms, Aβ1-42 is incorporated in the middle of larger globular protein aggregates surrounded by NQO1 molecules. Isothermal titration calorimetry (ITC) analysis indicates that Aβ1-42 interacts with NQO1 isoforms with a specific stoichiometry through a hydrophobic interaction with positive enthalpy and entropy changes. These data define the kinetics, mechanism, and shape of coaggregates of Aβ1-42 and NQO1 isoforms and the potential relevance of FAD-deficient forms of NQO1 for amyloid aggregation diseases.

The stress induced protein NQO1 can participate in a wide range of biological pathways which are dependent upon the interaction of NQO1 with protein targets. Many of the protein-protein interactions involving NQO1 have been shown to be regulated by the pyridine nucleotide redox balance. NQO1 can modify its conformation as a result of redox changes in pyridine nucleotides and sites on the C-terminal and helix seven regions of NQO1 have been identified as potential areas that may be involved in redox-dependent protein-protein interactions. Since post-translational modifications can modify the functionality of proteins, we examined whether redox-dependent conformational changes induced in NQO1 would alter lysine acetylation. Recombinant NQO1 was incubated with and without NADH then acetylated non-enzymatically by acetic anhydride or S-acetylglutathione (Ac-GSH). NQO1 acetylation was determined by immunoblot and site-specific lysine acetylation was quantified by mass spectrometry (MS). NQO1 was readily acetylated by acetic anhydride and Ac-GSH. Interestingly, despite a large number of lysine residues (9%) in NQO1 only a small subset of lysines were acetylated and the majority of these were located in or near the functional C-terminal or helix seven regions. Reduction of NQO1 by NADH prior to acetylation resulted in almost complete protection of NQO1 from lysine acetylation as confirmed by immunoblot analysis and MS. Lysines located within the redox-active C-terminus and helix seven regions were readily acetylated when NQO1 was in an oxidized conformation but were protected from acetylation when NQO1 was in the reduced conformation. To investigate regulatory mechanisms of enzymatic deacetylation, NQO1 was acetylated by Ac-GSH then exposed to purified sirtuins (SIRT 1-3) or histone deacetylase 6 (HDAC6). NQO1 could be deacetylated by all sirtuin isoforms and quantitative MS analysis performed using SIRT2 revealed very robust deacetylation of NQO1, specifically at K262 and K271 in the C-terminal region. No deacetylation of NQO1 by HDAC6 was detected. These data demonstrate that the same subset of key lysine residues in the C-terminal and helix seven regions of NQO1 undergo redox dependent acetylation and are regulated by sirtuin-mediated deacetylation.

In this review, we summarize the multiple functions of NQO1, its established roles in redox processes and potential roles in redox control that are currently emerging. NQO1 has attracted interest due to its roles in cell defense and marked inducibility during cellular stress. Exogenous substrates for NQO1 include many xenobiotic quinones. Since NQO1 is highly expressed in many solid tumors, including via upregulation of Nrf2, the design of compounds activated by NQO1 and NQO1-targeted drug delivery have been active areas of research. Endogenous substrates have also been proposed and of relevance to redox stress are ubiquinone and vitamin E quinone, components of the plasma membrane redox system. Established roles for NQO1 include a superoxide reductase activity, NAD+ generation, interaction with proteins and their stabilization against proteasomal degradation, binding and regulation of mRNA translation and binding to microtubules including the mitotic spindles. We also summarize potential roles for NQO1 in regulation of glucose and insulin metabolism with relevance to diabetes and the metabolic syndrome, in Alzheimer?s disease and in aging. The conformation and molecular interactions of NQO1 can be modulated by changes in the pyridine nucleotide redox balance suggesting that NQO1 may function as a redox-dependent molecular switch.

The localization of NQO1 near acetylated microtubules has led to the hypothesis that NQO1 may work in concert with the NAD+-dependent deacetylase SIRT2 to regulate acetyl α-tubulin (K40) levels on microtubules. NQO1 catalyzes the oxidation of NADH to NAD+ and may supplement levels of NAD+ near microtubules to aid SIRT2 deacetylase activity. While HDAC6 has been shown to regulate the majority of microtubule acetylation at K40, SIRT2 is also known to modulate microtubule acetylation (K40) in the perinuclear region. In this study we examined the potential roles NQO1 may play in modulating acetyl α-tubulin levels. Knock-out or knock-down of NQO1 or SIRT2 did not change the levels of acetyl α-tubulin in 16HBE human bronchial epithelial cells and 3T3-L1 fibroblasts; however, treatment with a mechanism-based inhibitor of NQO1 (MI2321) led to a short-lived temporal increase in acetyl α-tubulin levels in both cell lines without impacting the intracellular pools of NADH or NAD+. Inactivation of NQO1 by MI2321 resulted in lower levels of NQO1 immunostaining on microtubules, consistent with redox-dependent changes in NQO1 conformation as evidenced by the use of redox-specific, anti-NQO1 antibodies in immunoprecipitation studies. Given the highly dynamic nature of acetylation-deacetylation reactions at α-tubulin K40 and the crowded protein environment surrounding this site, disruption in the binding of NQO1 to microtubules may temporally disturb the physical interactions of enzymes responsible for maintaining the microtubule acetylome.

Chronic nutrient excess leads to metabolic disorders and insulin resistance. Activation of stress-responsive pathways via Nrf2 activation contributes to energy metabolism regulation. Here, inducible activation of Nrf2 in mice and transgenesis of the Nrf2 target, NQO1, conferred protection from diet-induced metabolic defects through preservation of glucose homeostasis, insulin sensitivity, and lipid handling with improved physiological outcomes. NQO1-RNA interaction mediated the association with and inhibition of the translational machinery in skeletal muscle of NQO1 transgenic mice. NQO1-Tg mice on high-fat diet had lower adipose tissue macrophages and enhanced expression of lipogenic enzymes coincident with reduction in circulating and hepatic lipids. Metabolomics data revealed a systemic metabolic signature of improved glucose handling, cellular redox, and NAD + metabolism while label-free quantitative mass spectrometry in skeletal muscle uncovered a distinct diet- and genotype-dependent acetylation pattern of SIRT3 targets across the core of intermediary metabolism. Thus, under nutritional excess, NQO1 transgenesis preserves healthful benefits.

We studied the effects of electrical stimulation on insulin release from rat insulinoma (INS-1) cells. The anodal/cathodal biphasic stimulation (ACBPS) electrical waveform resulted in a voltage- and stimulation duration-dependent increase in insulin release. ACBPS elicited insulin release both in the presence and absence of glucose. Basal and ACBPS-induced insulin secretion could be inhibited by mitochondrial poisons and calcium channel blockers, indicating that insulin release was dependent on adenosine triphosphate (ATP) and the influx of calcium. ACBPS parameters that released insulin caused no detectable plasma membrane damage or cytotoxicity, although temporary morphological changes could be observed immediately after ACBPS. ACBPS did not alter the plasma membrane transmembrane potential but did cause pronounced uptake of MitoTracker Red into the mitochondrial membrane, indicating an increased mitochondrial membrane potential. While the ATP:ADP ratio after ACBPS did not change, the guanosine triphosphate (GTP) levels increased and increased GTP levels have previously been associated with insulin release in INS-1 cells. These results provide evidence that ACBPS can have significant biological effects on cells. In the case of INS-1 cells, ACBPS promotes insulin release without causing cytotoxicity.

Biphasic (anodal pulse followed immediately by a cathodal pulse) electrical stimulation (10Hz, 5 ms duration) of rat insulinoma (Ins‐1) cells resulted in a 2–3‐fold increase in insulin release over 30 min that was stimulation period‐ and voltage‐dependent. Electrical stimulation of tissue culture medium (in the absence of cells) did not increase insulin release when subsequently added to cells, confirming that the insulin released by electrical stimulation was not due to substances generated by the culture medium. Electrical stimulation‐induced insulin release occurred in the absence or presence of glucose. Calcium channel blockade (using nifedipine or verapamil) inhibited basal insulin secretion and insulin secretion evoked by electrical stimulation, demonstrating that electrical stimulation‐induced insulin release involved activation of voltage‐dependent calcium channels and was not a non‐specific effect of stimulation on the plasma membrane. Basal insulin secretion and the increase in insulin release induced by electrical stimulation were also inhibited by the mitochondrial poisons antimycin A or oligomycin A, suggesting a role for mitochondrial function in insulin secretion. Electrical stimulation of cells also resulted in a more pronounced uptake of MitoTracker Red by mitochondria, indicating a more negative inner mitochondrial membrane potential. The failure of cells to accumulate the fluorogenic dye Cytotox green confirmed that electrical stimulation was not significantly disrupting the plasma membrane or inducing overt toxicity to the cells during the period of stimulation. These results provide further evidence that biphasic electrical stimulation can exert significant biological effects and elicit insulin release in a manner that is dependent upon mitochondrial activity and calcium channel activation but independent of glucose. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

NQO1 is a FAD containing NAD(P)H-dependent oxidoreductase that catalyzes the reduction of quinones and related substrates. In cells, NQO1 participates in a number of binding interactions with other proteins and mRNA and these interactions may be influenced by the concentrations of reduced pyridine nucleotides. NAD(P)H can protect NQO1 from proteolytic digestion suggesting that binding of reduced pyridine nucleotides results in a change in NQO1 structure. We have used purified NQO1 to demonstrate the addition of NAD(P)H induces a change in the structure of NQO1; this results in the loss of immunoreactivity to antibodies that bind to the C-terminal domain and to helix 7 of the catalytic core domain. Under normal cellular conditions NQO1 is not immunoprecipitated by these antibodies, however, following treatment with β-lapachone which caused rapid oxidation of NAD(P)H NQO1 could be readily pulled-down. Similarly, immunostaining for NQO1 was significantly increased in cells following treatment with β-lapachone demonstrating that under non-denaturing conditions the immunoreactivity of NQO1 is reflective of the NAD(P)+/NAD(P)H ratio. In untreated human cells, regions with high intensity immunostaining for NQO1 co-localize with acetyl α-tubulin and the NAD+-dependent deacetylase Sirt2 on the centrosome(s), the mitotic spindle and midbody during cell division. These data provide evidence that during the centriole duplication cycle NQO1 may provide NAD+ for Sirt2-mediated deacetylation of microtubules. Overall, NQO1 may act as a redox-dependent switch where the protein responds to the NAD(P)+/NAD(P)H redox environment by altering its structure promoting the binding or dissociation of NQO1 with target macromolecules.

NQO1 is one of the two major quinone reductases in mammalian systems. It is highly inducible and plays multiple roles in cellular adaptation to stress. A prevalent polymorphic form of NQO1 results in an absence of NQO1 protein and activity so it is important to elucidate the specific cellular functions of NQO1. Established roles of NQO1 include its ability to prevent certain quinones from one electron redox cycling but its role in quinone detoxification is dependent on the redox stability of the hydroquinone generated by two-electron reduction. Other documented roles of NQO1 include its ability to function as a component of the plasma membrane redox system generating antioxidant forms of ubiquinone and vitamin E and at high levels, as a direct superoxide reductase. Emerging roles of NQO1 include its function as an efficient intracellular generator of NAD+ for enzymes including PARP and sirtuins which has gained particular attention with respect to metabolic syndrome. NQO1 interacts with a growing list of proteins, including intrinsically disordered proteins, protecting them from 20S proteasomal degradation. The interactions of NQO1 also extend to mRNA. Recent identification of NQO1 as a mRNA binding protein have been investigated in more detail using SERPIN1A1 (which encodes the serine protease inhibitor α-1-antitrypsin) as a target mRNA and indicate a role of NQO1 in control of translation of α-1-antitrypsin, an important modulator of COPD and obesity related metabolic syndrome. NQO1 undergoes structural changes and alterations in its ability to bind other proteins as a result of the cellular reduced/oxidized pyridine nucleotide ratio. This suggests NQO1 may act as a cellular redox switch potentially altering its interactions with other proteins and mRNA as a result of the prevailing redox environment.

NQO1 can be induced by multiple forms of stress including oxidative stress. NQO1 is a pleiotropic enzyme and consideration of some of the known endogenous substrates and functions of NQO1 may explain the potential beneficial effects of NQO1 against oxidative stress. NQO1 can reduce ubiquinone and vitamin E quinone to antioxidant forms and can reduce superoxide directly which suggests a primary protective role. However, the rate constant for superoxide reduction by NQO1 is low and this reaction is only likely to be relevant where NQO1 levels are high and superoxide dismutase levels are low. NQO1 can participate in protein-protein interactions, protecting other proteins including p53 against 20S proteasomal degradation. This suggests one important role for NQO1 when induced under stress is to maintain the stability of critical proteins such as p53 and other transcription factors. RNA-protein interactome studies have also shown that NQO1 can bind to mRNA and modulate protein levels by affecting translation. Changes in the redox balance of pyridine nucleotides, which occurs during oxidative stress, alters the conformation and resultant binding of NQO1 to macromolecules. Whether this behavior of NQO1 is relevant for protection or signaling during oxidative stress remains to be determined.

Chemical synthesis was performed to produce a series of 6-amino-1,3-disubstituted-4-phenyl-1,4-dihydro pyrano[2,3-c]pyrazole-5-carbonitrile compounds (14–57) which were characterized by 1H NMR, 13C NMR and LC/MS–MS. These compounds were assessed for their effect on the in vitro anchorage independent growth of human lung cancer cell line H2122 and IC50 values calculated. Two of the more potent compounds, BQU057 40 and BQU082 57 also displayed a dose dependent effect on RalA and RalB activity in H2122 spheroids using the common RalBP1 pull-down assay. Mouse PK and tissue distribution studies on 40 and 57 were performed and demonstrated that parent drug was present in tumor 3.0h post ip (50mg/Kg) dose.

NAD(P)H: quinone oxidoreductase (NQO1) is essential for cell defense against reactive oxidative species, cancer, and metabolic stress. Recently, NQO1 was found in ribonucleoprotein (RNP) complexes, but NQO1-interacting mRNAs and the functional impact of such interactions are not known. Here, we used ribonucleoprotein immunoprecipitation (RIP) and microarray analysis to identify comprehensively the subset of NQO1 target mRNAs in human hepatoma HepG2 cells. One of its main targets, SERPINA1 mRNA, encodes the serine protease inhibitor α-1-antitrypsin, A1AT, which is associated with disorders including obesity-related metabolic inflammation, chronic obstructive pulmonary disease (COPD), liver cirrhosis and hepatocellular carcinoma. Biotin pulldown analysis indicated that NQO1 can bind the 3' untranslated region (UTR) and the coding region (CR) of SERPINA1 mRNA. NQO1 did not affect SERPINA1 mRNA levels; instead, it enhanced the translation of SERPINA1 mRNA, as NQO1 silencing decreased the size of polysomes forming on SERPINA1 mRNA and lowered the abundance of A1AT. Luciferase reporter analysis further indicated that NQO1 regulates SERPINA1 mRNA translation through the SERPINA1 3'UTR. Accordingly, NQO1-KO mice had reduced hepatic and serum levels of A1AT and increased activity of neutrophil elastase (NE), one of the main targets of A1AT. We propose that this novel mechanism of action of NQO1 as an RNA-binding protein may help to explain its pleiotropic biological effects.

AbstractNatural quinones, often linked with cellular oxidation processes, exhibit pronounced biological activity. In particular, the structurally unique isothiazolonaphthoquinone aulosirazole, isolated from blue‐green alga, possesses selective antitumor cytotoxicity, although its mechanism of action is unknown. The first synthesis of aulosirazole uses a route centered upon a late‐stage regioselective Diels–Alder reaction. The structurally related natural product pronqodine A, an inhibitor of prostaglandin release, and analogues thereof, were also prepared for comparison. Biological evaluation of the compounds identified one potential target as the immunoregulatory enzyme indoleamine‐2,3‐dioxygenase (IDO). The isothiazoloquinones are also efficient substrates for the human quinone reductase NQO1, and undergo intracellular NQO1‐dependent redox cycling resulting in the generation of reactive oxygen species, and at lower doses have the potential to alter the ratio of intracellular oxidized to reduced pyridine nucleotides.

Abstract Benzoquinone ansamycin (BQA) Hsp90 inhibitors such as 17-DMAG and 17-AAG have off target toxicities including hepatotoxicity. Mechanisms underlying the toxicity of BQAs are a function of their ability to redox cycle and/or arylate cellular nucleophiles at the unsubstituted 19-position of the molecule. Therefore, we designed 19-substituted BQAs (19BQAs) to prevent conjugation with glutathione and protein thiols as an approach to reduce the hepatotoxicity and minimize off target effects of the BQA class of Hsp90 inhibitors. Our data demonstrated that 19BQAs had reduced toxicity to liver cells relative to their parent quinones while maintaining Hsp90 inhibitory activity, validating the overall approach. 19BQAs depend on tumor cell levels of NQO1 (NAD(P)H: quinone oxidoreductase 1) for optimal activity. We examined 19-phenyl-17-DMAG as a model compound in human isogenic breast cancer cell lines MDA468 (NQO1 null) and MDA468/NQ16 (NQO1 overexpressing) and in NQO1expressing BT474 breast cancer cells. 19-phenyl-17-DMAG induced growth inhibition, apoptosis and the molecular signature of Hsp90 inhibition including decreases in Hsp90 client protein levels and compensatory induction of Hsp70. In addition, treatment of MD468/NQ16 and BT474 cell lines with 19-phenyl-17-DMAG or 17-DMAG also caused the dissociation of Hsp90 from the co-chaperone Cdc37. In these studies we also observed that treatment with19-phenyl-17-DMAG or 17-DMAG resulted in inhibition of Cdc37 phosphorylation. A major kinase responsible for phosphorylation of Cdc37 is casein kinase 2 (CK2) and inhibitors of CK2 are currently under development as anticancer drugs. CK2 activity assays, immunoprecipitation and western blot analysis demonstrated that both 19-phenyl-17-DMAG and 17-DMAG inhibited CK2 kinase activity and reduced CK2α protein subunit expression in NQO1-expressing MD468/NQ16 and BT474 breast cancer cells. Decreased phosphorylation of Cdc37 led to dissociation of the Hsp90/Cdc37/client complex and resulted in the degradation of multiple kinase clients including Raf-1, Cdk4, Akt and HER2. These data suggest that inhibition of both Hsp90 and CK2 by Hsp90 inhibitors may play a role in their antitumor effects. (Supported by CA51210). Citation Format: Chuan-Hsin Chang, David Ross, David Siegel, Christopher J Moody, Russell Kitson. Effect of 19-substituted benzoquinone ansamycin Hsp90 inhibitors on Hsp90/Cdc37/co-chaperone complexes and casein kinase 2 (CK2) activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1732. doi:10.1158/1538-7445.AM2015-1732

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.

Abstract Benzoquinone ansamycin (BQA) Hsp90 inhibitors such as 17-DMAG and 17-AAG have off-target toxicities in clinical trials including hepatotoxicity. Mechanisms underlying the toxicity of quinones are a function of their ability to redox cycle and/or arylate cellular nucleophiles at the unsubstituted 19-position of the molecule. Therefore, we designed 19-substituted BQAs to prevent glutathione conjugation and non-specific interactions with protein thiols as an approach to reduce the hepatotoxicity and minimize off-target effects of the BQA class of Hsp90 inhibitors. In this study, the results showed that 19-substituted BQAs did not react with glutathione at the 19-position, while marked reactivity was observed using parent BQAs. Importantly, while parent 17-DMAG induced cell death in primary and cultured mouse hepatocytes, 19-phenyl and 19-methyl 17-DMAG showed reduced toxicity, validating the overall approach. There was no significant difference between the redox cycling ability of either 19-phenyl or 19-methyl 17-DMAG with their parental BQAs in both mouse and human liver microsomes. Accordingly, this suggests that arylation reactions at the unsubstituted 19-position are predominantly responsible for hepatotoxicity. 19-substituted17-DMAG inhibited purified Hsp90 ATPase activity in an NQO1-dependent manner that demonstrated increased inhibitory efficacy of the hydroquinone ansamycin relative to its parent quinone. In human breast cancer cells, 19-phenyl BQAs induced growth inhibition in an NQO1-dependent manner with molecular signatures of Hsp90 inhibition, including decreases in client proteins and compensatory induction of Hsp70. These data indicate that 19-substituted BQAs may be useful Hsp90 inhibitors with decreased off target toxicity (Supported by NCI grant CA51210) Citation Format: Chuan-Hsin Chang, Derek A. Drechsel, Russell R.A. Kitson, David Siegel, Qiang You, Donald S. Backos, Cynthia Ju, Christopher J. Moody, David Ross. 19-Substituted benzoquinone ansamycins. Hsp90 inhibitors with decreased off-target toxicity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1788. doi:10.1158/1538-7445.AM2014-1788

Abstract The 19-substituted benzoquinone ansamycin (BQA) class of Hsp90 inhibitors were developed to decrease the off-target toxicity of their parent unsubstituted BQAs, geldanamcyin, 17-AAG, and 17-DMAG. We have shown that 19-BQAs do not react with thiols and show decreased toxicity to liver cell systems relative to parent BQAs. As a class, Hsp90 inhibitors exhibit anti-cancer activity by decreasing the levels of Hsp90 client proteins critical to cell growth and survival. Some Hsp90 inhibitors also disrupt interaction with Hsp90 co-chaperones, including cdc37, contributing to inhibited growth. Upon inhibition, the transcription factor heat shock factor 1 (Hsf1) dissociates from Hsp90 leading to a compensatory induction of other heat shock proteins, including Hsp70 which is commonly used as a molecular biomarker of cellular Hsp90 inhibition. We undertook a detailed study of the mechanism of action of 19-BQAs in HER2-positive breast cancer cells (BT474) using 19-phenyl- and 19-methyl-DMAG as model compounds. Their ability to disrupt association of Hsp90 with the co-chaperones p23 and cdc37, the kinetics of decreased Hsp90-Hsf1 levels, nuclear Hsf1 accumulation and increases in Hsp70 levels were defined. Both 19-phenyl- and 19-methyl -DMAG disrupted Hsp90-p23 association, confirming binding of the compounds at the N-terminal ATPase site of Hsp90. 19-Phenyl-DMAG and 19-methyl-DMAG also disrupted association of Hsp90 with cdc37, although 19-phenyl-DMAG was more potent. A detailed time course of decreased levels of Hsp90 client proteins and Hsp induction showed that increased Hsp70 was a more sensitive cellular marker as it could be detected at lower drug concentrations. Increases in Hsp70 did not appear to be a generalized stress response, since 19-substituted-BQAs caused dissociation of the Hsp90-Hsf1 dimer, nuclear translocation of Hsf-1, and subsequent increases in Hsp70 levels in a temporal manner. These data indicate that 19-substituted BQAs may exhibit their growth inhibitory effects in breast cancer cells through disruption of Hsp90 and its co-chaperones, particularly cdc37. The N-terminal ATPase site binding and Hsp90 inhibitory activity of these compounds was confirmed through disruption of Hsp90-p23 and Hsp90-Hsf1 associations, respectively (Supported by NCI grant CA51210). Citation Format: Derek A. Drechsel, Chuan-Hsin Chang, Russell Kitson, David Siegel, Christopher J. Moody, David Ross. 19-Substituted benzoquinone ansamycin Hsp90 inhibitors: Effects on Hsp90 co-chaperones and Hsp90-Hsf1 complexes in cellular systems. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1791. doi:10.1158/1538-7445.AM2014-1791