Geldanamycin Analogues Research Articles

Microbial biotransformation of water-insoluble herbimycin A to 11-hydroxy-(11-demethoxy)-herbimycin C by Eupenicillium sp. SD017

Herbimycin A, a typically water-insoluble anti-tumor drug and an analog of geldanamycin, was transformed by fungus Eupenicillium sp. SD017. The water solubility and anti-tumor activity of converted products were evaluated after separation, purification, and structure elucidation. The results showed that herbimycin A can be converted into herbimycin C 1 and 11-hydroxy-(11-demethoxy)-herbimycin C 2 with water solubility increased. The latter is a novel compound, and its IC 50 to A549, MCF-7, Ehrlich and HeLa cells is 152.9, 0.46, 10.7 and 109.3 μg/ml, respectively, which are nearly in the range of those between herbimycin A and herbimycin C. It is suggested that an O-demethylase or/and methyltransferase may play an important role on biotransformation in this case. This study will firstly serve as a fundamental methodology for further structural alteration and functional improvement of the water-insoluble anti-tumor drugs using microbial resources.

Three-kinase inhibitor combination recreates multipathway effects of a geldanamycin analogue on hepatocellular carcinoma cell death

Multitarget compounds that act on a diverse set of regulatory pathways are emerging as a therapeutic approach for a variety of cancers. Toward a more specified use of this approach, we hypothesize that the desired efficacy can be recreated in terms of a particular combination of relatively more specific (i.e., ostensibly single target) compounds. We test this hypothesis for the geldanamycin analogue 17-Allylamino-17-demethoxygeldanamycin (17AAG) in hepatocellular carcinoma cells, measuring critical phosphorylation levels that indicate the kinase pathway effects correlating with apoptotic responsiveness of the Hep3B cell line in contrast to the apoptotic resistance of the Huh7 cell line. A principal components analysis (PCA) constructed from time course measurements of seven phosphoprotein signaling levels identified modulation of the AKT, IkappaB kinase, and signal transducer and activator of transcription 3 pathways by 17AAG treatment as most important for distinguishing these cell-specific death responses. The analysis correctly suggested from 17AAG-induced effects on these phosphoprotein levels that the FOCUS cell line would show apoptotic responsiveness similarly to Hep3B. The PCA also guided the inhibition of three critical pathways and rendered Huh7 cells responsive to 17AAG. Strikingly, in all three hepatocellular carcinoma lines, the three-inhibitor combination alone exhibited similar or greater efficacy to 17AAG. We conclude that (a) the PCA captures and clusters the multipathway phosphoprotein time courses with respect to their 17AAG-induced apoptotic responsiveness and (b) we can recreate, in a more specified manner, the cellular responses of a prospective multitarget cancer therapeutic.

Co-treatment with heat shock protein 90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin (DMAG) and vorinostat: a highly active combination against human Mantle Cell Lymphoma (MCL) cells

Heat shock protein (hsp) 90 inhibitors promote proteasomal degradation of pro-growth and pro- survival hsp90 client proteins, including CDK4, c-RAF and AKT, and induce apoptosis of human lymphoma cells. The pan-histone deacetylase inhibitor vorinostat has also been shown to induce growth arrest and apoptosis of lymphoma cells. Here, we determined the effects of the more soluble, orally bio-available, geldanamycin analogue 17-NN-dimethyl ethylenediamine geldanamycin (DMAG, Kosan Biosciences Inc) and/or vorinostat in cultured and primary human MCL cells. While vorinostat induced accumulation in the G1 phase, treatment with DMAG arrested MCL cells in the G2/M phase of the cell cycle. Both agents dose-dependently induced apoptosis of MCL cells. Vorinostat also induced hyperacetylation of hsp90 and disrupted the association of hsp90 with its co-chaperones p23 and cdc37, as well as with its client proteins CDK4 and c-RAF. Treatment of MCL cells with vorinostat or 17-DMAG was associated with the induction of p21 and p27, as well as with depletion of c-Myc, c-RAF, AKT and CDK4. Compared to treatment with either agent alone, co-treatment with DMAG and vorinostat markedly attenuated the levels of cyclin D1 and CDK4, as well as of c-Myc, c-RAF and AKT. Combined treatment with DMAG and vorinostat synergistically induced apoptosis of the cultured MCL cells, as well as induced more apoptosis of primary MCL cells than either agent alone. Therefore, these findings support the rationale to determine the in vivo efficacy of co- treatment with vorinostat and DMAG against human MCL cells.

Rational Biosynthetic Engineering for Optimization of Geldanamycin Analogues

Tailor made: We report the rational biosynthesis of C15 hydroxylated non-quinone geldanamycin analogues by site-directed mutagenesis of the geldanamycin polyketide synthase (PKS), together with a combination of post-PKS tailoring genes. Rational biosynthetic engineering allowed the generation of geldanamycin derivatives, such as DHQ3 illustrated in the figure, which had superior pharmacological properties in comparison to the parent compound. A rational biosynthetic engineering approach was applied to the optimization of the pharmacological properties of the benzoquinone ansamycin, geldanamycin. Geldanamycin and its natural or semisynthetic derivatives have the potential to serve as anticancer chemotherapeutic agents. However, these first-generation Hsp90 inhibitors share an unfavorable structural feature that causes both reduced efficacy and toxicity during clinical evaluation. We report the rationally designed biosynthesis of C15 hydroxylated non-quinone geldanamycin analogues by site-directed mutagenesis of the geldanamycin polyketide synthase (PKS), together with a combination of post-PKS tailoring genes. A 15-hydroxyl-17-demethoxy non-quinone analogue, DHQ3, exhibited stronger inhibition of Hsp90 ATPase activity (4.6-fold) than geldanamycin. Taken together, the results of the present study indicate that rational biosynthetic engineering allows the generation of derivatives of geldanamycin with superior pharmacological properties.

DURABILITY OF RESPONSE: THE ACHILLES HEEL OF SALVAGE COMBINATION IMMUNOTHERAPY WITH INTRAVESICAL BACILLUS CALMETTE-GUERIN AND INTERFERON-ALPHA 2B IN BLADDER CANCER

complex in cancer cell. Combination of Geldanamycin and HER2 monoclonal antibody enhanced the anticancer effects to breast cancer (Cancer Res2004), we hypothesized combination of Geldanamycin and IF7 has potential to improve the anticancer effects. Here we report that the cancer targeting chemotherapy using IF7 and GA to prostate tumor and other cancers. METHODS: Anxa1 binding peptide (IF7) was identified from peptide displaying phage library. In vivo phage targeting was determined by recovery of IF7 displaying phage. To confirm peptide binding in vitro, affinity of Anxa1 and Alexa 488 conjugated with IF7 (IF7-A488) were measured. For in vivo binding assay, IF7-A488 was visualized by dorsal skinfold window chamber and its signal was inhibited by anti-Anxa1 Antibody. Reverse sequence peptide (RQ7-A488) was used for control. For cancer treatment, IF7 was conjugated with Geldanamycin analogue; 17GMB-APA-GA (GA) and product (IF7-GA) was purified by silica gel chromatography. Drug effects were evaluated by PC3, MDA-MB-231, LLC and B16F1. Mice were divided 4 groups, Control, IF7, GA, and IF7GA. Drug toxicity study was performed by C57BL/6 mice. RESULTS: IF7 peptide showed strong specificity and affinity to Anxa1 in vitro and in vivo. Tumor sizes of IF7-GA group were significant small (p<0.05) and tumor apoptosis were increased compare to other groups. There was no major side effect in IF7-GA group. CONCLUSIONS: Our results show that intravenously injected IF7GA suppresses tumor growth in the mouse by inducing apoptosis. These results suggest the potential of annexin binding peptide for therapies

Targeting heat shock protein 90 with non-quinone inhibitors: A novel chemotherapeutic approach in human hepatocellular carcinoma #

The inhibition of heat shock protein 90 (Hsp90) has emerged as a promising antineoplastic strategy in diverse human malignancies. Hsp90 has been predicted to be involved in hepatocellular carcinoma (HCC) development; however, its role in hepatocarcinogenesis remains elusive. Using chemically distinctive Hsp90 inhibitors, we show that Hsp90 capacitates the aberrant expression and activity of crucial hepatocarcinogenesis-driving factors (e.g., insulin-like growth factor receptor 1, hepatocyte growth factor receptor, protein kinase B, v-raf-1 murine leukemia viral oncogene homolog 1, and cyclin-dependent kinase 4). In vitro, Hsp90 inhibition with both geldanamycin analogs (17-allylamino-17-desmethoxygeldanamycin (17-AAG) and 17-dimethylaminoethylamino-17-desmethoxygeldanamycin (17-DMAG)) and the non-quinone compound 8-(6-iodobenzo[d][1,3]dioxol-5-ylthio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine (PU-H71) reduced the viability of various HCC cell lines, induced the simultaneous degradation of numerous hepatocarcinogenic factors, and caused substantial cell cycle arrest and apoptosis. In contrast, nontumorigenic hepatocytes were less susceptible to Hsp90 inhibition. Because conventional geldanamycin-derivate Hsp90 inhibitors induce dose-limiting liver toxicity, we tested whether novel Hsp90 inhibitors lacking the benzoquinone moiety, which has been deemed responsible for hepatotoxicity, can elicit antineoplastic activity without causing significant liver damage. In HCC xenograft mouse models, PU-H71 was retained in tumors at pharmacologically relevant concentrations while being rapidly cleared from nontumorous liver. PU-H71 showed potent and prolonged in vivo Hsp90 inhibitory activity and reduced tumor growth without causing toxicity. Hsp90 constitutes a promising therapeutic target in HCC. Non-quinone Hsp90 inhibitors exhibit tumor-specific accumulation and exert potent antineoplastic activity without causing significant hepatotoxicity.

Heat shock protein inhibitors and vaccines as new agents in cancer treatment

Background: Heat shock proteins (HSP) play an essential role as molecular chaperones by assisting correct holding and folding in human cells. At the same time they present implications in tumor cell proliferation, differentiation, matrix invasion, angiogenesis, metastasis and cell death. They also possess the ability to present tumor molecules to the immune system and elicit an immune response. New agents targeting HSP, as anticancer treatment, are under clinical evaluation. Objective: The aim of this review is to explore the role of HSP90 inhibitors as anticancer agents as well as to evaluate the benefit from the use of autologous HSP vaccines in both the adjuvant setting and first-line treatment. Methods: The latest evidence regarding the use of geldanamycin analogues or newer water-soluble and synthetics molecules that inhibit the binding of HSP90 to client proteins was reviewed. Immunization using tumor-derived HSP in several cancer types was also evaluated. Conclusions: HSP90 inhibitors represent a promising therapeutic option although further evaluation in larger clinical trials is needed. On the other hand, HSP vaccination has already an established role in our armory against cancer.

Pharmacogenomics Approach Reveals MRP1 (ABCC1)-Mediated Resistance to Geldanamycins

Geldanamycin and its analogues belong to a new class of anticancer agents that inhibit the molecular chaperone heat shock protein 90. We hypothesized that membrane transporters expressed on tumor cells may contribute at least in part to cellular sensitivity to these agents. The purpose of this study is to identify novel transporters as determinant for sensitivity and resistance to geldanamycins. To facilitate a systematic study of chemosensitivity across multiple geldanamycin analogues, we correlated mRNA expression profiles of majority of transporters with anticancer drug activities in 60 human tumor cell lines (NCI-60). We subsequently validated the gene-drug correlations using cytotoxicity and transport assays. The GA analogues displayed negative correlations with mRNA expression levels of the multidrug resistance protein 1 (MRP1, ABCC1). Suppressing MRP1 efflux using the inhibitor MK-571 and small interfering RNA in cell lines with intrinsic and acquired MRP1 overexpression (A549 and HL-60/ADR) and in cell lines stably transduced with MRP1 (MCF7/MRP1) increased intracellular drug accumulation and increased tumor cell sensitivity to geldanamycin analogues. These results suggest that elevated expression of MRP1, like the alternative efflux transporter MDR1 (ABCB1, P-glycoprotein), can significantly influence tumor cell sensitivity to geldanamycins as a potential chemoresistance factor.

A New Post-PKS Modification Process in the Carbamoyltransferase Gene Inactivation Strain of Streptomyces hygroscopicus 17997

Genetic manipulation of geldanamycin (GDM) producer Streptomyces species is a rational approach to understand biosynthesis processes and create new analogues. In this study, the carbamoyltransferase gene gdmN was inactivated by insertion of an apramycin-resistance gene aac3 (IV) into the genome of the geldanamycin-producing strain Streptomyces hygroscopicus 17997. GDM analogues produced by this mutant strain were isolated and characterized, such as new compound 4,5-dihydro-7-O-descarbamoyl-7-hydroxy-19-O-glycylgeldanamycin. This compound could be converted to compound 4,5-dihydro-19-O-glycylgeldanamycin, another new GDM analogue, by a strain of Streptomyces hygroscopicus 17997 in which the GDM-pks was inactivated. These new compounds exhibited reductions of cytotoxicity against HepG2 cancer cells, but increases of aqueous solubility. These results suggest that a new post-polyketide synthase modification was involved in this process to produce new GDM analogues.

Gamendazole, an Orally Active Indazole Carboxylic Acid Male Contraceptive Agent, Targets HSP90AB1 (HSP90BETA) and EEF1A1 (eEF1A), and Stimulates Il1a Transcription in Rat Sertoli Cells1

Gamendazole was recently identified as an orally active antispermatogenic compound with antifertility effects. The cellular mechanism(s) through which these effects occur and the molecular target(s) of gamendazole action are currently unknown. Gamendazole was recently designed as a potent orally active antispermatogenic male contraceptive agent. Here, we report the identification of binding targets and propose a testable mechanism of action for this antispermatogenic agent. Both HSP90AB1 (previously known as HSP90beta [heat shock 90-kDa protein 1, beta]) and EEF1A1 (previously known as eEF1A [eukaryotic translation elongation factor 1 alpha 1]) were identified as binding targets by biotinylated gamendazole (BT-GMZ) affinity purification from testis, Sertoli cells, and ID8 ovarian cancer cells; identification was confirmed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and Western blot analysis. BT-GMZ bound to purified yeast HSP82 (homologue to mammalian HSP90AB1) and EEF1A1, but not to TEF3 or HBS1, and was competed by unlabeled gamendazole. However, gamendazole did not inhibit nucleotide binding by EEF1A1. Gamendazole binding to purified Saccharomyces cerevisiae HSP82 inhibited luciferase refolding and was not competed by the HSP90 drugs geldanamycin or novobiocin analogue, KU-1. Gamendazole elicited degradation of the HSP90-dependent client proteins AKT1 and ERBB2 and had an antiproliferative effect in MCF-7 cells without inducing HSP90. These data suggest that gamendazole may represent a new class of selective HSP90AB1 and EEF1A1 inhibitors. Testis gene microarray analysis from gamendazole-treated rats showed a marked, rapid increase in three interleukin 1 genes and Nfkbia (NF-kappaB inhibitor alpha) 4 h after oral administration. A spike in II1a transcription was confirmed by RT-PCR in primary Sertoli cells 60 min after exposure to 100 nM gamendazole, demonstrating that Sertoli cells are a target. AKT1, NFKB, and interleukin 1 are known regulators of the Sertoli cell-spermatid junctional complexes. A current model for gamendazole action posits that this pathway links interaction with HSP90AB1 and EEF1A1 to the loss of spermatids and resulting infertility.

WT1 Interaction with the Chaperone Protein HSP90 Regulates Its Expression in Myeloid Leukemia.

Introduction: Wilms tumor protein 1 (WT1) is a transcription factor that is universally expressed in AML, and clinical studies have demonstrated a significant correlation between WT1 expression and disease progression. Heat shock proteins (HSP) are ubiquitous molecular chaperones that are involved in the folding, activation and assembly of many proteins. HSP90 may facilitate the maintenance of altered oncogenic client proteins such as flt-3 and bcr-abl in myeloid malignancies. It has been also shown that WT1 is a client protein of Hsp70, and this interaction may be important for the function of WT1. Since HSP70 and HSP90 have similar functions and often complement each other in engaging with client cellular proteins we wanted to determine if WT1 was chaperoned by HSP90 and if so could this HSP90-WT1 interaction be modulated.Materials and methods: We first looked for a potential interaction between HSP90 and WT1 by confocal microscopy colocalization and then by immunoprecipitation (IP) in the myeloid cell line K562. For confocal studies, cells were grown on coverslips, fixed and permeabilized and stained with the rabbit anti-WT1 and anti- HSP90 antibodies. Samples were examined by using a laser confocal/Zeiss axiovert microscope imaging system. This interaction was further confirmed by co-IP/Western and then confirmed using a GST-pulldown assay. A glutathione S-transferase (GST)-WT1 fusion protein was expressed in E. coli strain BL21(DE3) and immobilized onto glutathione sepharose beads. The full length Hsp90 and different deletion mutant proteins were in vitro translated and [35S] methionine-labeled; the bound proteins were eluted and then separated by SDS-PAGE. The gels were fixed, dried and subjected to fluorography.Results: Confocal microscopy revealed colocalization of HSP90 and WT1 mainly in the nucleus. Immunoprecipitation of K562 lysates with either anti-WT1 or anti-HSP90 antibody followed by Western analysis for WT1 and HSP90 demonstrated that WT1 and HSP90 could be co-immunoprecipitated. The GST pull down assay confirmed this interaction. Additional mutants of HSP90 from the C to N terminus were prepared to localize the WT1 interacting domain. These studies localized the WT1 interacting domain to the HSP90 region close to the binding pocket of the HSP inhibitor geldanamycin. Treatment of K562 with the geldanamycin analog 17-allylamino-17-demethoxygeldanamycin (17-AAG) resulted in downregulation of WT1 protein expression with an IC50 of 3μM.Conclusions: Understanding the mechanisms that regulate WT1 expression thus may lead to novel therapeutic strategies to inhibit this expression and result directly in myeloid blast cell killing. We have shown elsewhere that downmodulating WT1 gene expression with interferon or shRNA potentiates myeloid leukemic blast killing by chemotherapeutic agents. We demonstrate here that the WT1 protein interacts with the co-chaperone heat shock protein HSP90. Taken together, our findings support the hypothesis that the regulation of WT1 occurs at the protein level through an interaction with WT1-protein-HSP chaperone levels, and that pharmacologically targeting this interaction with the HSP inhibitor 17AAG results in a significant reduction in WT1 protein expression. Ongoing studies in primary AML cells will confirm these findings and define novel therapeutic strategies to manipulate WT1 protein expression levels and modulate leukemic blast survival.

Molecular Characterization and Drug-Sensitivity of Pan Histone Deacetylase Inhibitor Resistant Human Acute Myeloid Leukemia Cells.

Hydroxamic acid analogue pan-histone deacetylase (HDAC) inhibitors (HA-HDIs), e.g., vorinostat, LAQ824 and LBH589, induce in vitro growth arrest, differentiation and apoptosis of human acute leukemia cells. Continuous and protracted use of HA-HDI, as currently used in the clinic against hematologic malignancies is likely to result in the emergence of HA-HDI resistance in leukemia cells. By continuous in vitro exposure of the AML HL-60 cells to the cinnamic acid analogue HA-HDI LAQ824, we have generated an in vitro and in vivo model of HA-HDI-resistant HL-60/LR cells, which are capable of growth in high concentrations (200 nM) of LAQ824. HL-60/LR versus the parental HL-60 cells have a shorter doubling time (12 versus 24 hours), increased % of cells in the S phase of the cell cycle (62.4 versus 40.0) and exhibit shorter interval to generation of leukemia and survival in NOD/SCID mice. As compared to HL-60, HL-60/LR cells have a resistance index of 100 for LAQ824, and are cross-resistant to other antileukemia agents exhibiting resistance index for LBH589: 50; trichostatin A: 15; vorinostat: 30; sodium butyrate: 10; etoposide: 5.0; Ara-C: 3.3 and TRAIL: 31.3. As compared to HL-60, HL-60/LR cells express higher levels of Bcl-xL and XIAP but lower levels of MCL-1. HL-60/LR versus HL-60 cells also express markedly reduced levels of Bim and Bak but higher levels of Bax. Although expressing higher levels of the death receptors (DR) 4 and 5 and lower levels of c-FLIP, HL-60/LR cells lack expression of caspase-8 and show barely detectable levels of FADD. Additionally, HL-60/LR versus HL-60 cells have markedly higher levels of AKT, c-RAF, and p-STAT5. Although expressing higher levels of HDAC1, HDAC2, and HDAC4, HL-60/LR cells lack detectable expression of HDAC6, with increased expression of hyper-acetylated hsp90 and α-tubulin- two of the substrates deacetylated by HDAC6. As compared to hsp90 in HL-60 cells, hyper-acetylated hsp90 in HL-60/LR cells exhibits less binding to ATP and p23. Utilizing a polyclonal antibody generated against acetylated hsp90α, confocal immunofluorescence microscopy showed higher and mostly cell surface expression of acetylated hsp90α in HL-60/LR versus HL-60 cells. As compared to HL-60, treatment of HL-60/LR cells with LAQ824 failed to induce p21 and hsp70, or increase the levels of hyper-acetylated hsp90 and α-tubulin. Notably, although cross-resistant to several anti-leukemia drugs, HL-60/LR cells are collaterally sensitive to the hsp90-inhibiting geldanamycin analogues 17-allylamino-demothoxy geldanamycin (17-AAG) and 17-DMAG with a four and five-fold increased sensitivity to 17-AAG and 17-DMAG, respectively. This was associated with a lack of both a 17-AAG mediated induction of hsp70 and a lesser decline in the levels of AKT and c-RAF in HL-60/LR versus HL-60 cells. Taken together, these findings elucidate several notable in vitro and in vivo biologic characteristics and drug-sensitivity profile of the first fully-characterized HA-HDI-resistant human AML cells. Our findings clearly demonstrate that in vitro resistance to HA-HDIs is associated with loss of HDAC6 expression, hyperacetylation of hsp90, aggressive leukemia phenotype, but cross-sensitivity to 17-AAG. These findings also suggest that hsp90 inhibitors should be tested for overriding de novo or acquired HA-HDI resistance in AML.

Drugging the Cancer Chaperone HSP90

The molecular chaperone HSP90 has emerged as an exciting target for cancer treatment. We review the potential advantages of HSP90 inhibitors, particularly the simultaneous combinatorial depletion of multiple oncogenic "client" proteins, leading to blockade of many cancer-causing pathways and the antagonism of all of the hallmark pathological traits of malignancy. Cancer selectivity is achieved by exploiting cancer "dependencies," including oncogene addiction and the stressed state of malignant cells. The multiple downstream effects of HSP90 inhibitors should make the development of resistance more difficult than with agents having more restricted effects. We review the various classes of HSP90 inhibitor that have been developed, including the natural products geldanamycin and radicicol and also the purine scaffold and pyrazole/isoxazole class of synthetic small molecule inhibitors. A first-in-class HSP90 drug, the geldanamycin analog 17-AAG, has provided proof of concept for HSP90 inhibition in patients at well tolerated doses and therapeutic activity has been seen. Other inhibitors show promise in preclinical and clinical development. Opportunities and challenges for HSP90 inhibitors are discussed, including use in combination with other agents. Most of the current HSP90 inhibitors act by blocking the essential nucleotide binding and ATPase activity required for chaperone function. Potential new approaches are discussed, for example, interference with cochaperone binding and function in the superchaperone complex. Biomarkers for use with HSP90 inhibitors are described. We stress how basic and translational research has been mutually beneficial and indicate future directions to enhance our understanding of molecular chaperones and their exploitation in cancer and other diseases.

Cystine-Glutamate Transporter SLC7A11 Mediates Resistance to Geldanamycin but Not to 17-(Allylamino)-17-demethoxygeldanamycin

The cystine-glutamate transporter SLC7A11 has been implicated in chemoresistance, by supplying cystine to the cell for glutathione maintenance. In the NCI-60 cell panel, SLC7A11 expression shows negative correlation with growth inhibitory potency of geldanamycin but not with its analog 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), which differs in the C-17 substituent in that the the methoxy moiety of geldanamycin is replaced by an amino group. Structure and potency analysis classified 18 geldanamycin analogs into two subgroups, "17-O/H" (C-17 methoxy or unsubstituted) and "17-N" (C-17 amino), showing distinct SLC7A11 correlation. We used three 17-O/H analogs and four 17-N analogs to test the role of the 17-substituents in susceptibility to SLC7A11-mediated resistance. In A549 cells, which are resistant to geldanamycin and strongly express SLC7A11, inhibition of SLC7A11 by (S)-4-carboxyphenylglycine or small interfering RNA increased sensitivity to 17-O/H, but had no effect on 17-N analogs. Ectopic expression of SLC7A11 in HepG2 cells, which are sensitive to geldanamycin and express low SLC7A11, confers resistance to geldanamycin, but not to 17-AAG. Antioxidant N-acetylcysteine, a precursor for glutathione synthesis, completely suppressed cytotoxic effects of 17-O/H but had no effect on 17-N analogs, whereas the prooxidant ascorbic acid had the opposite effect. Compared with 17-AAG, geldanamycin led to significantly more intracellular reactive oxygen species (ROS) production, which was quenched by addition of N-acetylcysteine. We conclude that SLC7A11 confers resistance selectively to 17-O/H (e.g., geldanamycin) but not to 17-N (e.g., 17-AAG) analogs partly as a result of differential dependence on ROS for cytotoxicity. Distinct mechanisms could significantly affect antitumor response and organ toxicity of these compounds in vivo.

The geldanamycin analogue 17-allylamino-17-demethoxygeldanamycin inhibits the growth of GL261 glioma cells in vitro and in vivo

Geldanamycin is a naturally occurring benzoquinone ansamycin product of Streptomyces geldanus that binds the protein chaperone heat shock protein 90. As geldanamycin binds to heat shock protein 90 interfering with its function and heat shock protein 90 is overexpressed in many cancers, heat shock protein 90 has become a target for cancer therapy. As the geldanamycin analogue 17-allylamino-17-demethoxygeldanamycin has a favorable toxicity profile, it is being tested extensively in clinical trials in patients with advanced cancer. In this study, GL261 glioma cells from C57BL/6 mice were used to investigate the anti-tumor effect of 17-allylamino-17-demethoxygeldanamycin both in vitro and in vivo. Heat shock protein 90 inhibitors possess potent anti-proliferative activity, usually at low nanomolar ranges, owing to their pharmacological characteristics of binding tightly to heat shock protein 90, coupled with a slow dissociation rate. We found that 17-allylamino-17-demethoxygeldanamycin at doses as low as 200 nmol/l showed anti-tumor activity within 24 h of treatment. Treatment with 17-allylamino-17-demethoxygeldanamycin arrested GL261 cells in the G2 phase of the cell cycle associated with the downregulation of cyclin B1. Low doses of 17-allylamino-17-demethoxygeldanamycin significantly inhibited migration of GL261 cells within 16 h of treatment, concomitant with the downregulation of phosphorylated focal adhesion kinase and matrix metalloproteinase 2 secretion. Using an orthotopic glioma model with well-established intracranial tumors, 3 weekly cycles of 17-allylamino-17-demethoxygeldanamycin significantly reduced tumor volumes of treated animals compared with untreated controls (P=0.002). Given these promising results, clinical testing of 17-allylamino-17-demethoxygeldanamycin or other novel heat shock protein 90 inhibitors being developed should be considered for glioma patients whose tumors remain refractory to most current treatment regimens.

Chaperone activation of the hepadnaviral reverse transcriptase for template RNA binding is established by the Hsp70 and stimulated by the Hsp90 system

Hepadnaviruses are DNA viruses that replicate by protein-primed reverse transcription, employing a specialized reverse transcriptase (RT), P protein. DNA synthesis from the pregenomic RNA is initiated by binding of P to the ε signal. Using ε as template and a Tyr-residue for initiation, the RT synthesizes a DNA oligo (priming) as primer for full-length DNA. Priming strictly requires prior RT activation by chaperones. Active P–ε complexes have been reconstituted in vitro, but whether in addition to the heat-shock protein 70 (Hsp70) system the Hsp90 system is essential has been controversial. Here we quantitatively compared Hsp70 versus Hsp70 plus Hsp90 RT activation, and corroborated that the Hsp70 system alone is sufficient; however, Hsp90 as well the Hsp70 nucleotide exchange factor Bag-1 markedly stimulated activation by increasing the steady-state concentration of the activated metastable RT form P*, though by different mechanisms. Hsp90 inhibition in intact cells by geldanamycin analogs blocked hepadnavirus replication, however not completely and only at severely cytotoxic inhibitor concentrations. While compatible with a basal level of Hsp90 independent in vivo replication, unambiguous statements are precluded by the simultaneous massive upregulation of Hsp70 and Hsp90.

Proteome‐wide changes induced by the Hsp90 inhibitor, geldanamycin in anaplastic large cell lymphoma cells

The molecular chaperone heat shock protein 90 (Hsp90) affects the function of many oncogenic signaling proteins including nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) expressed in anaplastic large cell lymphoma (ALCL). While ALK-positive ALCL cells are sensitive to the Hsp90 inhibitor and the geldanamycin (GA) analog, 17-allylamino-17-demethoxygeldanamycin (17-AAG), the proteomic effects of these drugs on ALK-positive ALCL cells are unpublished. In this study, we investigated the cellular, biologic, and proteomic changes occurring in ALK-positive ALCL cells in response to GA treatment. GA induced G2/M cell cycle arrest and caspase-3-mediated apoptosis. Furthermore, quantitative proteomic changes analyzed by cleavable isotope-coded affinity tag-LC-MS/MS (cICAT-LC-MS/MS) identified 176 differentially expressed proteins. Out of these, 49 were upregulated 1.5-fold or greater and 70 were downregulated 1.5-fold or greater in GA-treated cells. Analysis of biological functions of differentially expressed proteins revealed diverse changes, including induction of proteins involved in the 26S proteasome as well as downregulation of proteins involved in signal transduction and protein and nucleic acid metabolism. Pathway analysis revealed changes in MAPK, WNT, NF-kappaB, TGFbeta, PPAR, and integrin signaling components. Our studies reveal some of the molecular and proteomic consequences of Hsp90 inhibition in ALK-positive ALCL cells and provide novel insights into the mechanisms of its diverse cellular effects.

A phase I trial of the heat shock protein 90 (HSP90) inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17- DMAG, alvespimycin) administered weekly

3568 Background: The geldanamycin analogue 17-DMAG inhibits the ATPase activity of HSP90, thus altering client protein &amp; chaperone interactions and targeting client proteins for degradation. The plethora of oncogenic HSP90 client proteins offers the potential of combinatorial blockade across multiple, cancer causing signalling pathways in cancer. Methods: 17-DMAG was administered weekly to patients with advanced, solid tumours using a dose-doubling 3 + 3 Phase I design. The pharmacokinetic (PK) and pharmacodynamic (PD) analyses undertaken were validated to comply with U.K clinical trial legislation. Results: 10 patients, 7 male and 3 female with a mean age of 60 years (range 38 - 78) have received 107 infusions (mean 10.7 weeks, range 2 - 30) at dose levels of 2.5mg/m2, 5mg/m2 &amp; 20mg/m2. No dose-limiting or drug related grade 3 or 4 toxicity has occurred in 9 patients eligible for toxicity assessment. A linear relationship exists between dose and AUC and Cmax (see table ). Hsp72 induction and CDK4 depletion (consistent with HSP90 inhibition) have been detected at doses of 20mg/m2 in peripheral blood mononuclear cells. A confirmed partial response (by PSA and RECIST) has occurred in a patient with hormone refractory prostate cancer (HRPC). Conclusions: We have shown linear PK for 17-DMAG up to 20mg/m2. No dose-limiting toxicity has been encountered. A robust biologically active dose has not been reached but evidence of the pharmacological signature of HSP90 inhibition has been detected in surrogate tissue and a confirmed PR seen in a patient with HRPC. These data support dose escalation to 40mg/m2 and paired tumor biopsies will be taken to further define the PK-PD-clinical relationships. [Table: see text] [Table: see text]

Chemogenomic Analysis Identifies Geldanamycins as Substrates and Inhibitors of ABCB1

A prerequisite for geldanamycin (GA, NSC122750) to targeting heat shock protein 90 and inhibiting tumor growth is sufficient intracellular drug accumulation. We hypothesized that membrane transporters on tumor cells determine at least in part the response to GA analogues. To facilitate a systematic study of chemosensitivity across a group of GA analogues with similar chemical structures, we correlated mRNA expression profiles of most known transporters with growth inhibitory potencies of compounds in 60 tumor cell lines (NCI-60). We subsequently validated the gene-drug correlations using cytotoxicity and transport assays. Geldanamycin analogues displayed a range of negative correlations coefficients with ABCB1 (MDR1, or P-glycoprotein) expression. Suppressing ABCB1 in multidrug resistant cells (NCI/ADR-RES and K562/DOX) and ABCB1-transfected cells (BC19) increased sensitivity to GA analogues, as expected for substrates. Moreover, ABCB1-mediated efflux of daunorubicin in K562/DOX cells could be blocked markedly by GA analogues in a dose-dependent fashion. The IC(50) values (half-maximum inhibition of daunorubicin efflux) were 5.5, 7.3 and 12 muM for macbecin II (NSC330500), 17-AAG (NSC330507) and GA, respectively. These observations demonstrate that GA analogues are substrates as well as inhibitors of ABCB1, suggesting that drug interactions between GA analogues and other agents that are ABCB1 substrates may occur via ABCB1 in normal or tumor cells.

Targeting of multiple signalling pathways by heat shock protein 90 molecular chaperone inhibitors

The last decade has seen the molecular chaperone heat shock protein 90 (HSP90) emerge as an exciting target for cancer therapy. This is because HSP90 is involved in maintaining the conformation, stability, activity and cellular localisation of several key oncogenic client proteins. These include, amongst others, ERBB2, C-RAF, CDK4, AKT/PKB, steroid hormone receptors, mutant p53, HIF-1alpha , survivin and telomerase hTERT. Therefore, modulation of this single drug target offers the prospect of simultaneously inhibiting all the multiple signalling pathways and biological processes that have been implicated in the development of the malignant phenotype. The chaperone function of HSP90 requires the formation of a multichaperone complex, which is dependent on the hydrolysis of ATP and ADP/ATP exchange. Most current inhibitors of HSP90 act as nucleotide mimetics, which block the intrinsic ATPase activity of this molecular chaperone. The first-in-class inhibitor to enter and complete phase I clinical trials was the geldanamycin analogue, 17-allylamino-17-demethoxygeldanamycin. The results of these trials have demonstrated that HSP90 is a valid drug target. Evidence of clinical activity has been seen in patients with melanoma, breast and prostate cancer. This article provides a personal perspective of the present efforts to increase our understanding of the molecular and cellular consequences of HSP90 inhibition, with examples from work in our own laboratory. We also review the discovery and development of novel small-molecule inhibitors and discuss alternative approaches to inhibit HSP90 activity, both of which offer exciting prospects for the future.