Benzoquinone Ansamycin Research Articles

A Short Review on Introduction and Researches on Anticancerous Activity of Geldanamycin

Geldanamycin (GA) bind heat-shock protein-90 (HSP-90) and destabilize its client proteins including v-Src, Bcr-Abl, RAF-1, Erb-B2, some growth factor receptors and steroid receptors. As a result, several oncoproteins are subjected to ubiquitination and proteasomal destruction by HSP-90 active compounds. HSP-90 active substances can either stop apoptosis from occurring or promote growth arrest, differentiation, and apoptosis depending on the cellular environment. Numerous preclinical models and clinical trials have demonstrated anticancer activity for a number of HSP-90 inhibitors. The well-known HSP-90 inhibitor geldanamycin’s clinical development was hampered by its hepatic toxicity. Geldanamycin at low doses can sensitize Bcr/Abl-expressing leukemia cells to death in the presence of inadequate doxorubicin concentrations by activating caspase. In another example, 17AAG in combination with taxol shows enhanced cytotoxic effects on taxol-resistant Erb-B2 overexpressing breast cancer cells. The benzoquinone ansamycin geldanamycin selectively binds to GRP94 and HSP-90 both in vivo and in vitro. When cells are treated with geldanamycin, HSP-90’s molecular chaperone function is changed. This prevents some cytosolic proteins from maturing, reduces their activity, and/or modifies their stability. On the other hand, nothing is known about GRP94’s function in protein folding or how geldanamycin affects this endoplasmic reticulum (ER) homologue of HSP-90. In this work, we show that geldanamycin is a strong inducer of the cellular stress response in the ER, leading to the transcriptional up-regulation of ER chaperones and production of the gadd153/CHOP transcription factor in a range of cell lines. Here we mention the anticancerous activity of HSP-90 (Heat Shock Protein 90) Inhibitor geldanamycin and some researches in field of anticancerous activity of Geldanamycin.

Synthesis of novel geldanamycin derivatives

The toxicity associated with the geldanamycin family of benzoquinone ansamycins when used as heat shock protein-90 inhibitor molecular therapeutics is ameliorated by substitution at the 19-position. The resulting 19-substituted derivatives have greater potential for success in oncology clinical trials and for other medicinal purposes such as the treatment of neurodegenerative conditions. Having overcome hurdles associated with the sensitivity and complexity of these molecules, through a variety of synthetic approaches, the synthesis of a series of 19-substituted geldanamycin derivatives is reported herein using optimised Stille and Suzuki coupling reactions. Further compounds were accessible via copper-mediated coupling and nucleophilic addition reactions The new compounds are of significant medicinal interest, in view of their significantly reduced toxicity previously observed for this class of substrate compared to their 19-unsubstituted counterparts that have been evaluated in the clinic.

Total synthesis based modification of benzoquinone ansamycin antibiotics: C8 diversification of C5-C15 fragments

Benzoquinone ansamycins are known to be potent Heat shock protein (Hsp90) inhibitors, and total-synthesis-based structure modification of this natural product family may lead to the discovery of novel cancer chemotherapeutics. Described in this paper is a unified synthetic route that gives access to both natural and C-8-modified C5-C15 fragments of this natural product family.

The complete genome sequence of Streptomyces autolyticus CGMCC 0516, the producer of geldanamycin, autolytimycin, reblastatin and elaiophylin

Streptomyces autolyticus CGMCC 0516 produces the anti-tumor benzoquinone ansamycins geldanamycin, autolytimycin, and reblastatin and the 16-membered macrodiolide elaiophylin. Here, we report the complete genome sequence of S. autolyticus CGMCC 0516, which consists of a 10,029,028bp linear chromosome and seven circular plasmids. Fifty-seven putative biosynthetic gene clusters for secondary metabolites were found. The geldanamycin, autolytimycin, and reblastatin biosynthetic gene clusters were located on the left arm (2.06–2.15Mb) of the chromosome, and the elaiophylin gene cluster was located on the right arm (9.45–9.53Mb). Twenty-one putative gene clusters with high or moderate similarity to important antibiotic biosynthetic gene clusters were found, including the antitumor agents echoside, bafilomycin, hygrocin, and toxoflavin; the antibacterial/antifungal agents nigericin, skyllamycin, kanamycin, naphthomycin, eco-02301, and bottromycin A2; the immunosuppressants meridamycin and brasilicardin A; the anti-inflammatory agent cyclooctatin; and the acute iron poisoning medication desferrioxamine B. The genome sequence reported here will enable us to study the biosynthetic mechanism of these important antibiotics and will facilitate the discovery of novel secondary metabolites with potential applications to human health.

Histone deacetylase activity mediates acquired resistance towards structurally diverse HSP90 inhibitors.

Heat shock protein 90 (HSP90) regulates multiple signalling pathways critical for tumour growth. As such, HSP90 inhibitors have been shown to act as effective anticancer agents in preclinical studies but, for a number of reasons, the same effect has not been observed in the clinical trials to date. One potential reason for this may be the presence of de novo or acquired resistance within the tumours. To investigate mechanisms of resistance, we generated resistant cell lines through gradual dose escalation of the HSP90 inhibitor 17‐allylamino‐17‐demethoxygeldanamycin (17‐AAG). The resultant resistant cell lines maintained their respective levels of resistance (7–240×) in the absence of 17‐AAG and were also cross‐resistant with other benzoquinone ansamycin HSP90 inhibitors. Expression of members of the histone deacetylase family (HDAC 1, 5, 6) was altered in the resistant cells. To determine whether HDAC activity contributed to resistance, pan‐HDAC inhibitors (TSA and LBH589) and the class II HDAC‐specific inhibitor SNDX275 were found to resensitize resistant cells towards 17‐AAG and 17‐dimethylaminoethylamino‐17‐demethoxygeldanamycin. Most significantly, resistant cells were also identified as cross‐resistant towards structurally distinct HSP90 inhibitors such as radicicol and the second‐generation HSP90 inhibitors CCT018159, VER50589 and AUY922. HDAC inhibition also resensitized resistant cells towards these classes of HSP90 inhibitors. In conclusion, we report that prolonged 17‐AAG treatment results in acquired resistance of cancer cells towards not just 17‐AAG but also to a spectrum of structurally distinct HSP90 inhibitors. This acquired resistance can be inhibited using clinically relevant HDAC inhibitors. This work supports the potential benefit of using HSP90 and HDAC inhibitors in combination within the clinical setting.

C15-methoxyphenylated 18-deoxy-herbimycin A analogues, their in vitro anticancer activity and heat shock protein 90 binding affinity

Benzoquinone ansamycins are important leads for the discovery of novel inhibitors of heat shock protein 90 (Hsp90), a promising target of cancer chemotherapeutics. Intrinsic hepatotoxicity caused by the benzoquinone moiety appeared to be a serious limitation to the development of these compounds. To solve this problem by rational structure optimization, a short series of C18-deoxy analogues of herbimycin A were designed based on putative interactions between the compound and the protein. Chemical synthesis of the target molecules were attempted by following the established synthetic route to the natural product, but resulted in the isolation of four serendipitous C15 phenylated final products. In vitro antiproliferative activity and Hsp90 binding affinity of the compounds were determined, suggesting the C18-oxygen of herbimycin A is removable and bulky lipophilic groups can be accommodated at C15 without loss of activity.

Acquired resistance to HSP90 inhibitor 17-AAG and increased metastatic potential are associated with MUC1 expression in colon carcinoma cells.

Heat shock protein 90 (HSP90) is a molecular chaperone required for the stability and function of many proteins. The chaperoning of oncoproteins by HSP90 enhances the survival, growth, and invasive potential of cancer cells. HSP90 inhibitors are promising new anticancer agents, in which the benzoquinone ansamycin 17-allylamino-17-demethoxygeldanamycin (17-AAG) is currently in clinical evaluation. However, the implications of acquired resistance to this class of drug remain largely unexplored. In the present study, we have generated isogenic human colon cancer cell lines that are resistant to 17-AAG by continued culturing in the compound. Cross-resistance was found with another HSP90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin. The resistant cells showed obvious morphology changes with a metastatic phenotype and significant increases in migration and adhesion to collagens. Western blotting analysis of epithelial-mesenchymal transition molecular markers found that expression of E-cadherin downregulated, whereas expression of N-cadherin and β-catenin upregulated in the resistant cells. Mucin 1 (MUC1) has been reported to mediate metastasis as well as chemical resistance in many cancers. Here, we found that MUC1 expression was significantly elevated in the acquired drug resistance cells. 17-AAG treatment could decrease MUC1 more in parental cells than in acquired 17-AAG-resistant cells. Further study found that knockdown of MUC1 expression by small interfering RNA could obviously re-sensitize the resistant cells to 17-AAG treatment, and decrease the cell migration and adhesion. These were coupled with a downregulation in N-cadherin and β-catenin. The results indicate that HSP90 inhibitor therapies in colon carcinomas could generate resistance and increase metastatic potential that might mediated by upregulation of MUC1 expression. Findings from this study further our understanding of the potential clinical effects of HSP90-directed therapies in colon carcinomas.

A Novel Hsp90 Inhibitor Activates Compensatory Heat Shock Protein Responses and Autophagy and Alleviates Mutant A53Tα-Synuclein Toxicity

A potential cause of neurodegenerative diseases, including Parkinson's disease (PD), is protein misfolding and aggregation that in turn leads to neurotoxicity. Targeting Hsp90 is an attractive strategy to halt neurodegenerative diseases, and benzoquinone ansamycin (BQA) Hsp90 inhibitors such as geldanamycin (GA) and 17-(allylamino)-17-demethoxygeldanamycin have been shown to be beneficial in mutant A53T α-synuclein PD models. However, current BQA inhibitors result in off-target toxicities via redox cycling and/or arylation of nucleophiles at the C19 position. We developed novel 19-substituted BQA (19BQA) as a means to prevent arylation. In this study, our data demonstrated that 19-phenyl-GA, a lead 19BQA in the GA series, was redox stable and exhibited little toxicity relative to its parent quinone GA in human dopaminergic SH-SY5Y cells as examined by oxygen consumption, trypan blue, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), and apoptosis assays. Meanwhile, 19-phenyl-GA retained the ability to induce autophagy and potentially protective heat shock proteins (HSPs) such as Hsp70 and Hsp27. We found that transduction of A53T, but not wild type (WT) α-synuclein, induced toxicity in SH-SY5Y cells. 19-Phenyl-GA decreased oligomer formation and toxicity of A53T α-synuclein in transduced cells. Mechanistic studies indicated that mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase signaling was activated by A53T but not WT α-synuclein, and 19-phenyl-GA decreased mTOR activation that may be associated with A53T α-synuclein toxicity. In summary, our results indicate that 19BQAs such as 19-phenyl-GA may provide a means to modulate protein-handling systems including HSPs and autophagy, thereby reducing the aggregation and toxicity of proteins such as mutant A53T α-synuclein.

Abstract 1732: Effect of 19-substituted benzoquinone ansamycin Hsp90 inhibitors on Hsp90/Cdc37/co-chaperone complexes and casein kinase 2 (CK2) activity

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

Reaction design, discovery, and development as a foundation to function-oriented synthesis.

Convergent C-C bond-forming reactions define the fabric of organic synthesis and, when applied in complex molecule synthesis, can have a profound impact on efficiency by decreasing the longest linear sequence of transformations required to convert simple starting materials to complex targets. Despite their well-appreciated strategic significance, campaigns in natural product synthesis typically embrace only a small suite of reactivity to achieve such bond construction (i.e., nucleophilic addition to polarized π-bonds, nucleophilic substitution, cycloaddition, and metal-catalyzed "cross-coupling"), therefore limiting the sites at which convergent coupling chemistry can be strategically employed. In our opinion, it is far too often that triumphs in the field are defined by chemical sequences that do not address the challenges associated with discovery, development, and production of natural product-inspired agents. We speculated that advancing an area of chemical reactivity not represented in the few well-established strategies for convergent C-C bond formation may lead to powerful new retrosynthetic relationships that could simplify approaches to the syntheses of a variety of different classes of natural products. Our studies ultimately embraced the pursuit of strategies to control the course of metallacycle-mediated "cross-coupling" between substrates containing sites of simple π-unsaturation (ubiquitous functionality in organic chemistry including alkenes, alkynes, allenes, aldehydes, and imines, among others). In just eight years since our initial publication in this area, we have defined over 20 stereoselective intermolecular C-C bond-forming reactions that provide access to structural motifs of relevance for the synthesis of polyketides, fatty acids, alkaloids, and terpenes, while doing so in a direct and stereoselective fashion. These achievements continue to serve as the foundation of my group's activity in natural product and function-oriented synthesis, where our achievements in reaction development are challenged in the context of complex targets. Among our early efforts, we achieved the most concise synthesis of a benzoquinone ansamycin ever described (macbecin I), and moved beyond this achievement to explore the role of our chemistry in function-oriented synthesis targeting the discovery of natural product-inspired Hsp90 inhibitors. These later efforts have led to the discovery of a uniquely selective benzoquinone ansamycin-inspired Hsp90 inhibitor that lacks the problematic quinone present in the natural series. This achievement was made possible by a concise chemical synthesis pathway that had at its core the application of metallacycle-mediated cross-coupling chemistry.

Cks1 proteasomal degradation is induced by inhibiting Hsp90-mediated chaperoning in cancer cells.

Cks1, a conformationally heterogenous 9 kDa protein, is markedly overexpressed in cancer cells and contributes to tumor development. Cks1 is an essential component of the SCF-Skp2 ubiquitin ligase complex that targets the Cdk inhibitors p27(Kip1) and p21(Cip1). Cks1 is known to interact with the Hsp90-Cdc37 chaperone machinery, although whether this facilitates its conformational maturation and stability is not known. To test whether abrogating the chaperone function of Hsp90 could destabilize Cks1, we examined the effects of treating different cancer cell lines with the benzoquinone ansamycin 17-allylamino geldanamycin (17-AAG), a compound that selectively binds Hsp90 and potently inhibits its ATP-dependent chaperone activity. The effect of Hsp90 inhibition using 17-AAG on Cks1 protein and associated cell cycle proteins including Skp2, p27(Kip1), p21(Cip1), and Cdk1 in cancer cells was determined by Western blotting. Ubiquitination analysis was carried out by transfecting cells with an HA-ubiquitin plasmid and specifically immunoprecipitating Cks1 to examine polyubiquitinated species. Flow cytometry was utilized to examine the effects of Hsp90 inhibition on cell cycle profiles. Here, we demonstrate for the first time that inhibition of Hsp90 utilizing 17-AAG destabilizes Cks1 in cancer cells by promoting its ubiquitination and proteasomal degradation. 17-AAG-induced Cks1 depletion was accompanied by concomitant decreases in Skp2 and Cdk1. 17-AAG treatment also induced G2/M accumulation in MCF-7 breast carcinoma cells, and G1 accumulation in the colon carcinoma lines HCT116 and SW620. We conclude that perturbing the Hsp90 pathway could provide a useful therapeutic strategy in tumors driven by Cks1 overexpression.

ChemInform Abstract: Total Synthesis of Herbimycin A.

Benzoquinone ansamycin antibiotic herbimycin A was synthesized in 19 linear steps and 4.2% yield. Highlighted is the design of a chiral γ-lactone as the C11–C15 synthon that enabled a facile catalytic asymmetric synthesis of the challenging C8–C20 fragment of the target molecule. The easy access to the stereogenic centers and high overall yield made the strategy applicable in the molecular editing of benzoquinone ansamycins.

Abstract 1791: 19-Substituted benzoquinone ansamycin Hsp90 inhibitors: Effects on Hsp90 co-chaperones and Hsp90-Hsf1 complexes in cellular systems

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

Abstract 1788: 19-Substituted benzoquinone ansamycins. Hsp90 inhibitors with decreased off-target toxicity

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

N-acetylcysteine prevents the geldanamycin cytotoxicity by forming geldanamycin–N-acetylcysteine adduct

Geldanamycin (GDN) is a benzoquinone ansamycin antibiotic with anti-proliferative activity on tumor cells. GDN cytotoxicity has been attributed to the disruption of heat shock protein 90 (Hsp90) binding and stabilizing client proteins, and by the induction of oxidative stress with concomitant glutathione (GSH) depletion. The later mechanism of cytotoxicity can be abrogated by N-acetylcysteine (NAC). It was suggested that NAC prevents GDN cytotoxicity mainly by the restoring of glutathione (GSH) level (Clark et al., 2009).Here we argue that NAC does not protect cells from the GDN cytotoxicity by restoring the level of GSH. A detailed LC/MS/MS analysis of cell extracts indicated formation of GDN adducts with GSH. The amount of the GDN–GSH adduct is proportional to the GDN concentration and increases with incubation time. While nanomolar and low micromolar GDN concentrations induce cell death without an apparent GSH decrease, only much higher micromolar GDN concentrations cause a significant GSH decrease. Therefore, only high micromolar GDN concentrations can cause cell death which might be related to GSH depletion. Addition of NAC leads to the formation of adducts with GDN which diminish formation of GDN adducts with GSH. NAC also forms stable adducts with GDN extracellularly. Although NAC induces an increase in the GSH pool, this effect is not crucial for abrogation of GDN cytotoxicity. Indeed, the presence of NAC in the growth medium causes a rapid conversion of GDN into the GDN–NAC adduct, which is the real cause of the abrogated GDN cytotoxicity.

Total synthesis of herbimycin A.

Benzoquinone ansamycin antibiotic herbimycin A was synthesized in 19 linear steps and 4.2% yield. Highlighted is the design of a chiral γ-lactone as the C11-C15 synthon that enabled a facile catalytic asymmetric synthesis of the challenging C8-C20 fragment of the target molecule. The easy access to the stereogenic centers and high overall yield made the strategy applicable in the molecular editing of benzoquinone ansamycins.

Hsp90 Blockers Inhibit Adipocyte Differentiation and Fat Mass Accumulation

Geldanamycin derivatives are benzoquinone ansamycin antibiotics that bind to Hsp90 and alter its function. The alteration of Hsp90 activity limits some cellular hormonal responses by inhibiting nuclear receptors activation. The nuclear receptors activity, such as PPARγ, the mineralocorticoid and glucocorticoid receptors (MR and GR) play a critical role in the conversion of preadipocytes to mature adipocytes. Given the importance of these nuclear receptors for adipogenesis, we investigated the effects of geldanamycin analogues (GA) on adipocyte differentiation and function. We found that early exposure of preadipocyte cells to GA inhibited their conversion into mature adipocytes by inhibiting the adipogenic transcriptional program and lipid droplets accumulation. Furthermore, GA altered the adipokines secretion profile of mature adipocyte. The anti-adipogenic effect of GA was also confirmed in mice fed a high fat diet. Biochemical analysis revealed that anti-adipogenic effects of geldanamycin analogues may result from the simultaneous inhibition of MR, GR and PPARγ activity. Taken together, our observations lead us to propose Hsp90 as a potent target for drug development in the control of obesity and its related metabolic complications.

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 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.

Redox Properties of Benzoquinone Ansamycins in Aqueous Solutions

AbstractGeldanamycin (GM) is an inhibitor of heat shock protein 90 (Hsp90) with potent and broad anti‐cancer properties, but with unacceptable levels of hepatotoxicity. These characteristics have led to the devise of a range of less toxic GM structural analogues. Redox properties and thiol reactivity are central to the therapeutic and toxicologic effects of quinones, and the question arises as to whether the toxicity of GM and its analogues relates to the reduction potential of the quinone. Using pulse radiolysis, we have previously determined the one‐electron reduction potentials (vs. the normal hydrogen electrode [NHE]) at pH 7.0 of GM, 17‐(dimethylaminoethylamino)‐17‐demethoxygeldanamycin (17‐DMAG), and 17‐allylamino‐17‐demethoxygeldanamycin (17‐AAG) to be −62±7 mV, −194±6 mV, and −273±8 mV, respectively. These experimental results are now used to establish a predictive relationship for the reduction potential of GM analogues based on the Hammett para substituent constants. These values correlate well with the drugs effects in vivo. We show that cytotoxicity of the benzoquinone ansamycin increases as its respective reduction potential increases, even after blocking of the C19‐position from nucleophilic addition. We conclude that the cytotoxicity is directly related to the reduction potential of the quinone/semiquinone couple.

Post-translational modification and conformational state of Heat Shock Protein 90 differentially affect binding of chemically diverse small molecule inhibitors

Heat shock protein 90 (Hsp90) is an essential molecular chaperone in eukaryotes that facilitates the conformational maturation and function of a diverse protein clientele, including aberrant and/or over-expressed proteins that are involved in cancer growth and survival. A role for Hsp90 in supporting the protein homeostasis of cancer cells has buoyed interest in the utility of Hsp90 inhibitors as anti-cancer drugs. Despite the fact that all clinically evaluated Hsp90 inhibitors target an identical nucleotide-binding pocket in the N domain of the chaperone, the precise determinants that affect drug binding in the cellular environment remain unclear, and it is possible that chemically distinct inhibitors may not share similar binding preferences. Here we demonstrate that two chemically unrelated Hsp90 inhibitors, the benzoquinone ansamycin geldanamycin and the purine analog PU-H71, select for overlapping but not identical subpopulations of total cellular Hsp90, even though both inhibitors bind to an amino terminal nucleotide pocket and prevent N domain dimerization. Our data also suggest that PU-H71 is able to access a broader range of N domain undimerized Hsp90 conformations than is geldanamycin and is less affected by Hsp90 phosphorylation, consistent with its broader and more potent anti-tumor activity. A more complete understanding of the impact of the cellular milieu on small molecule inhibitor binding to Hsp90 should facilitate their more effective use in the clinic.